Novel aminated benzimidazo[1,2-a]quinolines as potential fluorescent probes for DNA detection: Microwave-assisted synthesis, spectroscopic characterization and crystal structure determination

Article abstract of DOI:10.1016/j.dyepig.2011.02.003

A synthesis of novel benzimidazo[1,2-a]quinolines, substituted with piperidine, pyrrolidine and piperazine nuclei has been accomplished using an uncatalyzed amination protocol under microwave heating. All compounds were characterized by means of ¹H, ¹³C NMR, IR, UV/Vis and fluorescence spectroscopy. Crystal and molecular structures of 2-chloro- and 2-piperidinyl-benzimidazo[1,2-a]quinoline-6-carbonitrile were determined by X-ray diffractometry. These molecules are essentially planar. Their molecular assembly is characterized by the existence of weak intermolecular hydrogen bonds of C-HN type and π-π aromatic interactions. The π-π aromatic stacking observed in the solid state structures of the 2-chloro- and 2-piperidinyl- derivatives are not analogous. The spectroscopic properties of these amino substituted benzimidazo[1,2-a]quinolines as their hydrochloride salts in the presence of ct-DNA were studied by means of fluorescence spectroscopy. Comparison of binding properties of amino substituted benzimidazo[1,2-a]quinoline hydrochloride salts to ct-DNA revealed significantly enhanced fluorescence emission intensity and thus offer the potential applications as a DNA-specific fluorescent probes.

Full text of DOI:10.1016/j.dyepig.2011.02.003

Dyes and Pigments 91 (2011) 79e88
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Novel aminated benzimidazo[1,2-a]quinolines as potential fluorescent
probes for DNA detection: Microwave-assisted synthesis, spectroscopic
characterization and crystal structure determination
a
a
,
a,
ꢁ b
*
**
ꢀ
Natasa Perin , Marijana Hranjec , Gordana Pavlovic , Grace Karminski-Zamola
a
ꢁ
Department of Organic Chemistry, University of Zagreb, Marulicev trg 20, P.O. Box 177, HR-10000 Zagreb, Croatia
Department of Applied Chemistry, University of Zagreb, Prilaz baruna Filipovica 28a, HR-10000 Zagreb, Croatia
b
ꢁ
a r t i c l e i n f o
a b s t r a c t
Article history:
A synthesis of novel benzimidazo[1,2-a]quinolines, substituted with piperidine, pyrrolidine and pipera-
zine nuclei has been accomplished using an uncatalyzed amination protocol under microwave heating. All
compounds were characterized by means of 1H, 13C NMR, IR, UV/Vis and fluorescence spectroscopy. Crystal
and molecular structures of 2-chloro- and 2-piperidinyl-benzimidazo[1,2-a]quinoline-6-carbonitrile were
determined by X-ray diffractometry. These molecules are essentially planar. Their molecular assembly is
Received 5 November 2010
Received in revised form
2 February 2011
Accepted 3 February 2011
Available online 2 April 2011
characterized by the existence of weak intermolecular hydrogen bonds of CeH,,,N type and
pep
aromatic interactions. The aromatic stacking observed in the solid state structures of the 2-chloro-
pep
Keywords:
and 2-piperidinyl- derivatives are not analogous. The spectroscopic properties of these amino substituted
benzimidazo[1,2-a]quinolines as their hydrochloride salts in the presence of ct-DNA were studied by
means of fluorescence spectroscopy. Comparison of binding properties of amino substituted benzimidazo
[1,2-a]quinoline hydrochloride salts to ct-DNA revealed significantly enhanced fluorescence emission
intensity and thus offer the potential applications as a DNA-specific fluorescent probes.
Ó 2011 Elsevier Ltd. All rights reserved.
Benzimidazoles
Benzimidazo[1,2-a]quinolines
Microwave synthesis
Amination
Crystal structure determination
Interaction with ct-DNA
1. Introduction
much attention, due to the notable progress in fluorescence
instrumentation as well as in the synthesis of different fluo-
In recent years substituted benzimidazoles and their azino fused
derivatives have been one of the most extensively studied classes of
heterocyclic compounds. The permanent and growing interest in
their synthesis is a direct consequence of their well known bio-
logical activities [1e5]. Besides the fact that benzimidazoles are
widely incorporated in the structure of numerous important
medical and biochemical agents, they exhibit structural similarity
with some naturally occurring compounds such as purine and,
therefore, can easily interact with biomolecules [6,7]. Due to their
planar structure, they have ability to intercalate between adjacent
base pairs of a DNA molecule and thus offer a potential application
as fluorescent probes in homogeneous assays of biological mole-
cules [8e10]. Nowadays, optical biomarkers are widely used in
biochemical and medicinal studies for the detection of biologically
important molecules such as DNA, RNA, proteins and enzymes.
Detection of biomolecules based upon fluorescence has received
rophores. Since benzannulated benzimidazoles usually posses
a highly conjugated planar chromophore, other important appli-
cation areas of this class of compounds are in optoelectronics,
optical lasers, organic luminophores or fluorescent dyes in tradi-
tional textile and polymer fields [11e16]. Microwave assisted
chemistry has greatly expanded over the last few years and has
been used for the rapid synthesis of a variety of organic compounds
[17e20]. Microwave assisted organic reactions have been proven to
be much quicker than those relying upon conventional heating
[21,22]. Moreover, microwave assisted amination of arylhalides has
become an important and frequently required reaction in the
synthesis of N-aryl substituted organic molecules in many research
fields such as medicinal and pharmaceutical chemistry, dyes,
optoelectronics and electronic materials [23e26].
All of the above mentioned considerations prompted us to
synthesize novel aminated benzimidazo[1,2-a]quinolines using an
uncatalyzed microwave assisted amination. The investigation is the
part of our continuing research on the synthesis of benzimidazole
derivatives. The amination reaction was carried out upon the
common precursor 4 which was prepared photochemically from its
non-fused benzimidazole derivative 3 which in turn was derived
* Corresponding author. Tel.: þ385 14597245; fax: þ385 14597250.
** Corresponding author. Tel.: þ385 14597215; fax: þ385 14597224.
E-mail addresses: mhranjec@fkit.hr (M. Hranjec), gzamola@fkit.hr (G. Karminski-
Zamola).
0143-7208/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2011.02.003

80
N. Perin et al. / Dyes and Pigments 91 (2011) 79e88
Table 1
from the condensation between cyanomethylbenzimidazole 1 and
aldehyde 2. Although the one-pot synthesis of chloro-substituted
benzimidazo[1,2-a]quinoline 4 has recently been reported by
a copper-catalyzed cascade reactions [27], we have rather used
a photochemical environmentally friendly approach, using ethanol
as a less toxic solvent in comparison with solvents used in the
above mentioned synthesis (DMF, DMSO) and without any addi-
tionally necessary catalysts or ligands.
General and crystal data and summary of intensity data collection and structure
refinement for compounds 4 and 5.
4
5
Formula
Mr
Crystal system, colour
and habit
C16H8ClN3
277.70
Monoclinic, yellow,
prism
P 21/n
0.29 ꢂ 0.17 ꢂ 0.09
C21H18N4
326.39
Triclinic, yellow,
needle
P 1
Space group
Crystal dimensions (mm3)
Unit cell parameters:
a (Å)
0.21 ꢂ 0.08 ꢂ 0.01
2. Experimental
9.86860(10)
7.23480(10)
17.7606(2)
7.4971(8)
10.7027(9)
11.0809(15)
83.13(1)
71.14(1)
75.95(1)
815.42(16)
2
1.329
0.081
344
3e25
b (Å)
c (Å)
2.1. General methods
ꢀ
ꢀ
a
/
b
/
102.87(1)
All chemicals and solvents were purchased from commercial
suppliers. Melting points were recorded on an Original Keller Mik-
roheitztisch apparatus (Reichert, Wien) and Büchi 535. 1H and 13C
NMR spectra were recorded on a Varian Gemini 300 or Varian Gemini
600 at 300, 600 and 150 and 75 MHz, respectively. All NMR spectra
were measured in DMSO-d6 solutions using TMS as an internal stan-
ꢀ
/
g
V (Å3)
1236.22(3)
4
1.492
2.654
568
Z
Dc (g cmꢁ3
)
m
(mmꢁ1
)
F(000)
q
range for data collection (ꢀ)a
6e75
dard. Chemical shifts are reported in ppm (d) relative to TMS. In
h,k,l range
ꢁ11 to 12
ꢁ9 to 6
ꢁ20 to 22
u
ꢁ8 to 8
preparative photochemical experiments the irradiation was per-
formed at room temperature with a water-cooled immersion well
with an “Origin Hanau” 400-W high-pressure mercury arc lamp using
Pyrex glass as a cut-off filter of wavelengths below 280 nm. Micro-
wave-assisted synthesis was performed in a Milestone start S micro-
wave oven using quartz cuvettes under the pressure of 40 bar. All
compounds were routinely checked by TLC with Merck silica gel 60F-
254glass plates. Elemental analysis forcarbon, hydrogen and nitrogen
were performed on a PerkineElmer 2400 elemental analyzer.
ꢁ12 to 11
ꢁ12 to 13
u
Scan type
No. measured reflections
No. independent reflections (Rint.
6134
2479 (0.065)
181/0
9973
2825 (0.0356)
226/0
)
No. refined parameters/Restraints
No. observed reflections, I ꢃ 2
s
(I)
2151
1535
g1, g2 in w
0.0830, 0.1569
0.0463, 0.1285
0.0540, 0.1218
1.06
0.1241, 0.5214
0.0838, 0.2861
0.1370, 0.2655
1.16
R, wR [I ꢃ 2
s(I)]
R, wR [all data]
Goodness of fit on F2, S
Min. and max. electron
ꢁ0.29, 0.30
ꢁ0.29, 0.20
density (e Åꢁ3
)
2.2. Synthesis
Maximum
D
/
s
0.001
<0.001
2.2.1. 2-Chloro-benzimidazo[1,2-a]quinoline-6-carbonitrile 427
An ethanolic solution (400 mL) of compound 3 (0.900 g,
2.87 mmol) was irradiated at room temperature, with 400 W high-
pressure mercury lamp, using a Pyrex filter for 10 h [28,29]. The
obtained product was filtered off to give 0.432 g (54%) of yellow
a
The data collection for structures 4 and 5 were carried with Oxford Xcalibur
(CuK
) ¼ 1.54184 Å (compound
4) and Oxford Xcalibur PX diffractometer with graphite-monocromated
Nova diffractometer with graphite-monocromated
l
a
l
(MoK
using
a
u
) ¼ 0.71073 Å (compound 5) at ambient temperature with CCD detectors
scan mode.
crystals; m.p. 288e291 ꢀC. IR (diamond):
n
/cmꢁ1 ¼ 2361, 2235,
J ¼ 6.34 Hz, CH2); 13C NMR (DMSO-d6, 150 MHz):
/ppm ¼ 154.54 (s),
d
1598, 1535; 1H NMR (DMSO-d6, 600 MHz):
d/ppm ¼ 8.80 (s, 1H,
146.27 (s),144.70 (s),140.15 (d),138.82 (s),132.86 (d),130.83 (s),125.27
(d), 123.34 (d), 120.16 (d), 116.90 (s), 115.05 (d), 112.99 (d), 112.31 (s),
97.83 (d), 94.34 (8s), 48.57 (t, 2C), 25.46 (t, 2C), 24.23 (t); Anal. Calcd for
C21H18N4 (326.4): C, 77.28; H, 5.56; N, 17.17. Found: C, 77.55; H, 5.78;
N,17.09.
Harom.), 8.73 (d,1H, J ¼ 7.5 Hz, Harom.), 8.70 (s,1H, Harom.), 8.16 (d,1H,
J ¼ 8.5 Hz, Harom.), 8.02 (d, 1H, J ¼ 8.3 Hz, Harom.), 7.73 (d, 1H,
J ¼ 8.43 Hz, Harom.), 7.66e7.57 (m, 2H, Harom.); 13C NMR (DMSO-d6,
150 MHz):
d
/ppm ¼ 145.04 (s), 142.74 (s), 142.69 (d), 141.02 (s),
139.12 (s), 135.43 (d), 133.05 (s), 128.28 (d), 128.14 (d), 126.74 (d),
122.98 (d), 122.82 (s), 118.06 (d), 117.94 (s), 117.71 (d), 104.19 (s);
Anal. Calcd for C16H8N3Cl (277.5): C, 69.20; H, 2.90; N, 15.13. Found:
C, 68.99; H, 2.74; N,15.29.
2.2.2.2. 2-Pyrrolidinylbenzimidazo[1,2-a]quinoline-6-carbonitrile
6. Compound 6was prepared using the above method from 4(0.050 g,
0.18 mmol) and pyrrolidine (0.07 mL, 0.9 mmol) to yield 0.051 g (90%)
of yellow crystals; m.p. >295 ꢀC. IR (diamond):
n
/cmꢁ1 ¼ 2848, 2210,
2.2.2. General method for preparation of compounds 5e7
1608,1402; 1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.54 (s,1H, Harom.),
Compounds 5e7 were prepared using microwave irradiation, at
optimized power and reaction time, from compound 4 in acetonitrile
(10 mL) with a fivefold excess of the corresponding amine. After
cooling, the reaction mixture was filtered off to yield pure
compounds 5e7 as yellow crystals. The details of reaction conditions
as well as obtained yield for compound 5 are presented in the Table 2.
Compounds 6e7 are prepared using similar reaction conditions.
8.51 (d, 1H, J ¼ 7.92 Hz, Harom.), 7.95 (d, 1H, J ¼ 7.71 Hz, Harom.), 7.90 (d,
1H, J ¼ 9.03 Hz, Harom.), 7.61e7.52 (m, 2H, Harom.), 7.44 (s, 1H, Harom.),
6.95 (d,1H, J ¼ 8.88 Hz, Harom.), 3.67 (t, 4H, J ¼ 6.68 Hz, CH2), 2.55 (t, 4H,
J ¼ 6.68 Hz, CH2); 13C NMR (DMSO-d6, 150 MHz):
/ppm ¼ 164.63 (s),
d
153.15 (s),147.73 (s),143.82 (s),142.99 (s),138.50 (d),133.60 (d),130.78
(s), 127.76 (d), 125.27 (d), 124.20 (s), 122.07 (d), 115.15 (d), 113.08 (d),
112.88 (s), 111.76 (d), 48.37 (t, 2C), 25.45 (8t, 2C); Anal. Calcd for
C20H16N4 (312.3): C, 76.90; H, 5.16; N, 17.94. Found: C, 77.11; H, 5.33;
N,17.82.
2.2.2.1. 2-Piperidinylbenzimidazo[1,2-a]quinoline-6-carbonitrile
5. Compound 5was prepared using the above method from 4(0.050 g,
0.18 mmol) and piperidine (0.09 mL, 0.9 mmol) to yield 0.045 g (85%)
Table 2
of yellow crystals; m.p. 254e256 ꢀC. IR (diamond):
n
/cmꢁ1 ¼ 2933,
The effect of reaction time on yield of 5 by using a constant power and temperature.
2218, 1608, 1402; 1H NMR (DMSO-d6, 600 MHz):
d/ppm ¼ 8.56 (s, 1H,
Reaction conditions
Compound 5
Harom.), 8.52 (d, 1H, J ¼ 8.22 Hz, Harom.), 7.97 (d,1H, J ¼ 8.23 Hz, Harom.),
7.90 (d, 1H, J ¼ 9.09 Hz, Harom.), 7.79 (bs, 1H, Harom.), 7.63e7.52 (m, 2H,
Harom.), 7.30 (d, 1H, J ¼ 9.16 Hz, Harom.), 3.69e3.62 (m, 6H), 1.67 (t, 4H,
Time (min)
Yield (%)
10
35
25
46
60
54
120
66
180
75
240
80
360
85

N. Perin et al. / Dyes and Pigments 91 (2011) 79e88
81
2.2.2.3. 2-Piperazinylbenzimidazo[1,2-a]quinoline-6-carbonitrile
7. Compound 7was prepared using the above method from 4(0.050 g,
0.18 mmol) and piperazine (0.78 g, 0.9 mmol) to yield 0.033 g (56%) of
119.56 (d), 116.43 (s), 115.63 (d), 113.61 (s), 113.46 (d), 98.74 (d),
94.93 (s), 44.35 (t, 2C), 42.78 (t, 2C); Anal. Calcd for C20H18ClN5
(363.1): C, 66.02; H, 4.99; N,19.25. Found: C, 66.20; H, 5.12; N,19.45.
yellow crystals; m.p. 192e196 ꢀC. IR (diamond):
n
/cmꢁ1 ¼ 2920, 2220,
1618, 1454; 1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.87 (s, 1H, NH),
2.3. Spectroscopic characterization
8.48 (s, 1H, Harom.), 8.43 (d, 1H, J ¼ 8.04 Hz, Harom.), 7.94 (d, 1H,
J ¼ 8.04 Hz, Harom.), 7.83 (d, 1H, J ¼ 8.04 Hz, Harom.), 7.66 (s, 1H, Harom.),
7.58 (t,1H, J ¼ 8.20 Hz, Harom.), 7.52 (t, 1H, J ¼ 8.22 Hz, Harom.), 7.24 (dd,
1H, J1 ¼9.8 Hz, J2 ¼1.45 Hz, Harom.), 3.49 (t, 4H, J¼ 4.52 Hz, CH2), 2.93 (t,
UV absorption spectra were recorded, against the solvent, at
(25 ꢄ 0.1) ꢀC, using a Varian Cary 50 spectrophotometer operated
in double-beam mode. The wavelength range covered was
200e450 nm. Quartz cells of 1-cm path length were used
throughout and absorbancies were sampled at 0.1 nm intervals.
Fluorescence measurements were carried out on a Varian Cary
Eclipse fluorescence spectrophotometer at 25 ꢀC using 1-cm path
quartz cells. Excitation maxima were determined from excitation
spectra covering the range of 200e450 nm. Emission spectra were
recorded from 400 to 600 nm and corrected for the effects of time-
and wavelength-dependent light-source fluctuations using a stan-
dard of rhodamine 101, a diffuser provided with the fluorimeter and
the software supplied with the instrument. The measurements were
performed in ethanol (HPLC grade). Relative fluorescence quantum
yields were determined according to Miller [28] using Eq. (1)
4H, J ¼ 4.52 Hz, CH2); 13C NMR (DMSO-d6, 150 MHz):
/ppm ¼ 154.12
d
(s), 145.55 (s), 144.03 (s), 139.66 (d), 137.94 (s), 132.17 (d), 130.18 (s),
124.79 (d),122.85 (d),119.61 (d),116.37 (s),114.67 (d),112.22 (d),112.20
(s), 97.33 (d), 94.24 (s), 47.16 (t, 2C), 44.89 (8t, 2C); Anal. Calcd for
C20H19N5 (327.2): C, 73.37; H, 5.23; N, 21.34. Found: C, 73.18; H, 5.15; N,
21.55.
2.2.3. General method for preparation of compounds 8e10
A stirred amount of 5e7 (0.15 mmol, 0.11 mmol) in absolute
ethanol (10 mL) was cooled in an ice-salt bath and was saturated
with HCl gas. The reaction mixture was stirred at the room
temperature for 24 h and the obtained product was filtered off and
washed with small amount of diethyl-ether (10 mL) to yield
powdered products.
fx
¼
fs ꢂ AsDxn2x =AxDsn2s
(1)
wherein
f is the emission quantum yield, A is the absorbance at the
2.2.3.1. 2-Piperidinylbenzimidazo[1,2-a]quinoline-6-carbonitrile
hydrochloride 8. Compound 8 was prepared using the above
method from 5 (0.050 g, 0.15 mmol) to yield 0.045 g (80%) of yellow
excitation wavelength, D is the area under the corrected emission
curve and n is the refractive index of the solvents used. The subscripts
s and x refer to the standard and to the unknown, respectively. The
standard employed was quinine sulfate with a published fluores-
cence quantum yield of 0.54 [29]. All samples were purged with
argon to displace oxygen. The reproducibility (difference between
the largest and the smallest value in a series of three independent
measurements, divided by their arithmetic mean) of quantum yield
measurements was better than 10%.
powder; m.p. 207e211 ꢀC. IR (diamond):
1598, 1359: 1H NMR (DMSO-d6, 600 MHz):
n
/cmꢁ1 ¼ 3149, 2245,
d/ppm ¼ 8.96 (s, 1H,
Harom.), 8.71 (d, 1H, J ¼ 8.07 Hz, Harom.), 8.11 (d, 1H, J ¼ 7.77 Hz,
Harom.), 8.01 (d, 1H, J ¼ 9.18 Hz, Harom.), 7.95 (bs, 1H, NHþ), 7.91 (s,
1H, Harom.), 7.78e7.67 (m, 2H, Harom.), 7.51 (d, 1H, J ¼ 8.82 Hz,
Harom.), 3.84e3.81 (m, 6H, CH2), 1.72 (t, 4H, J ¼ 6.30 Hz, CH2); 13C
NMR (DMSO-d6, 150 MHz):
d
/ppm ¼ 165.63 (s), 163.48 (s), 154.26
(s), 143.36 (s), 138.35 (d), 137.28 (s), 133.44 (d, 2C), 128.20 (s), 127.69
(d), 125.42 (d), 117.96 (s), 116.40 (d), 115.44 (d), 114.08 (s), 48.84 (t,
2C), 25.42 (t, 2C), 24.02 (t); Anal. Calcd for C21H19ClN4 (362.9): C,
69.51; H, 5.28; N, 15.44. Found: C, 69.72; H, 5.45; N, 15.21.
2.4. Single-crystal X-ray diffraction experiment
Selected crystallographic and refinement data for structures 4
and 5 obtained by the single-crystal X-ray diffraction experiments
are reported in Table 1. Data collection for both structures has been
performed by applying the CrysAlis Software system, Version
1.171.33.31 [30]. The Lorentz-polarization effect was corrected and
the intensity data reduced by the CrysAlis RED application of the
CrysAlis Software system, Version 1.171.33.31 [30]. The diffraction
data have been scaled for absorption effects by the multi-scanning
method. Structures were solved by direct methods and refined on F2
by weighted full-matrix least-squares. Programs SHELXS97 and
SHELXL97 [30] integrated in the WinGX v.1.70 [31] software system
were used to solve and refine structures. All non-hydrogen atoms
were refined anisotropically. Hydrogen atoms belonging to Csp2
carbon atoms were placed in geometrically idealized positions
[Csp2-H 0.93 Å with Uiso(H) ¼ 1.2 Ueq(C)] and they were constrained
to ride on their parent atoms by using the appropriate SHELXL97
HFIX instructions. The molecular geometry calculations and
graphics were obtained using ORTEP-3 [32] integrated in the WinGX
software system, PLATON [33] programme and Mercury [34].
2.2.3.2. 2-Pyrrolidinylbenzimidazo[1,2-a]quinoline-6-carbonitrile
hydrochloride 9. Compound 9 was prepared using the above
method from 6 (0.035 g, 0.11 mmol) to yield 0.034 g (87%) of orange
powder; m.p. 264e268 ꢀC. IR (diamond):
n
/cmꢁ1 ¼ 3149, 2234,
1594, 1332; 1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.83 (s, 1H,
Harom.), 8.57 (d, 1H, J ¼ 7.80 Hz, Harom.), 8.06 (d, 1H, J ¼ 7.65 Hz,
Harom.), 8.04 (bs,1H, NHþ), 7.95 (d,1H, J ¼ 8.88 Hz, Harom.), 7.70e7.60
(m, 2H, Harom.), 7.48 (s, 1H, Harom.), 7.03 (d, 1H, J ¼ 7.89 Hz, Harom.),
3.60 (t, 4H, J ¼ 6.67 Hz, CH2), 2.13 (t, 4H, J ¼ 6.69 Hz, CH2); 13C NMR
(DMSO-d6, 150 MHz):
d
/ppm ¼ 165.73 (s), 151.64 (s), 148.72 (s),
144.82 (s), 144.12 (s), 138.24 (d), 137.82 (s), 133.34 (d), 127.50 (d),
126.57 (d), 124.72 (s), 124.20 (d), 116.19 (d), 116.07 (d), 114.13 (d),
112.76 (s), 48.45 (t, 2C), 25.37 (t, 2C); Anal. Calcd for C20H17ClN4
(312.3): C, 68.86; H, 4.19; N, 16.06. Found: C, 69.10; H, 4.36; N, 16.20.
2.2.3.3. 2-Piperazinylbenzimidazo[1,2-a]quinoline-6-carbonitrile
hydrochloride 10. Compound 10 was prepared using the above
method from 7 (0.050 g, 0.15 mmol) to yield 0.050 g (90%) of yellow
powder; m.p. > 280 ꢀC. IR (diamond):
1400; 1H NMR (DMSO-d6, 600 MHz):
n
/cmꢁ1 ¼ 3214, 2230, 1604,
2.5. Interaction with ct-DNA
d/ppm ¼ 9.65 (brs, 2H, NH,
NHþ), 8.59 (s, 1H, Harom.), 8.53 (d, 1H, J ¼ 7.74 Hz, Harom.), 7.95 (t, 2H,
J ¼ 7.14 Hz, Harom.), 7.74 (s,1H, Harom.), 7.62 (t,1H, J ¼ 7.20 Hz, Harom.),
7.55 (t, 1H, J ¼ 7.26 Hz, Harom.), 7.34 (d, 1H, J ¼ 8.37 Hz, Harom.), 4.14
(t, 4H, J ¼ 4.50 Hz, CH2), 3.32 (t, 4H, J ¼ 4.50 Hz, CH2); 13C NMR
The calf thymus DNA (ct-DNA) was purchased from Aldrich,
dissolved in sodium cacodylate buffer, I ¼ 0.05 mol dmꢁ3, pH ¼ 7,
additionally sonicated and filtered through a 0.45 mm filter and the
concentration of corresponding solution determined spectroscop-
ically as the concentration of phosphates. The measurements were
performed at room temperature in the aqueous buffer solution
(DMSO-d6, 150 MHz):
d/ppm ¼ 153.81 (s), 145.48 (s), 142.99 (s),
140.80 (d), 138.10 (s), 132.96 (d), 130.36 (s), 125.64 (d), 123.82 (d),

82
N. Perin et al. / Dyes and Pigments 91 (2011) 79e88
H
N
Cl
Cl
Cl
Cl
CN
EtOHabs.
H
N
+
CH2CN
CHO
N
3
N
2
1
EtOH
h , I , O
ν
2
2
Cl
R
R
× HCl
amine
EtOHabs.
HCl(g)
Acetonitrile
MW
N
N
N
40 bar
N
NC
N
N
NC
NC
4
6-7
8-10
5, 8 R=
N
N
6 ,9 R=
7 ,10 R=
N
NH
Scheme 1. Synthesis of compounds 3e10.
(pH ¼ 7.0; sodium cacodylate buffer, I ¼ 0.05 mol dmꢁ3). Under the
experimental conditions used the absorbance and fluorescence
intensities of studied compounds were proportional to their
concentrations. Obtained data were corrected for dilution. Spec-
troscopic titrations were performed by adding portions of poly-
nucleotide solution into the solution of the compounds 8e10.
described method [35], by photochemical dehydrohalogenation of
compound 3.
Benzimidazo[1,2-a]quinolines 5e7, substituted with piperidine,
pyrrolidine and piperazine nuclei, were prepared by using
a microwave irradiation in relative high yields 56e90%. A series of
experiments were undertaken in order to optimise the amination
reactions of substrate 4 (Table 2). Based on these experiments it
was concluded that the optimal yield of 5 (85%, HPLC/MS) was
obtained by using 800 W power and 170 ꢀC in MeCN with a fivefold
excess of the amine. Compounds 6 and 7 were prepared using
identical reaction conditions.
3. Results and discussion
3.1. Synthesis
The uncatalyzed amination of compound 4 conducted by
conventional heating in EtOH, MeCN or DMF as solvents, after
several days, gave compounds 5e7 only in very low yields (<10%).
Compounds 3e10 were successfully synthesized according to
the Scheme 1. Fused compound 4 was prepared by a previously
3
800
700
600
500
400
300
200
100
0
3
0.8
4
4
5
5
6
6
7
7
0.6
0.4
0.2
0.0
8
8
9
9
10
10
250
300
350
400
450
500
450
500
550
600
λ/nm
λ/nm
Fig. 1. UV/Vis spectra of compounds 3e10.
Fig. 2. Fluorescence emission spectra of compounds 3e10.

N. Perin et al. / Dyes and Pigments 91 (2011) 79e88
83
Table 3
Electronic absorption and fluorescence emission data of compounds 3e10 in ethanol.
Comp.
3
4
5
6
7
8
9
10
lmax/nm
388
350
276
248
432
414
272
252
436
414
300
289
272
423
404
295
272
249
11.7
11.9
9.8
422
406
292
272
425
402
290
270
250
419
399
293
270
249
355
283
3
ꢂ 103 (dm3 molꢁ1 cmꢁ1
)
7.5
38.0
24.2
27.5
12.8
14.5
20.8
7.6
9.0
31.2
28.5
35.7
40.4
33.8
19.9
14.8
14.8
19.8
27.6
25.3
31.7
13.3
14.7
17.3
17.1
15.0
24.1
20.5
16.1
13.3
lemiss/nm
e
e
478
78.8
464
750.1
460
458
459
246.4
456
277.4
445
469
202.7
172.6
Rel. Fluo. Int (arb. un.)
399.5
13.8
f
e
e
0.545
202
0.543
192
0.529
188
0.756
186
0.497
187
0.473
206
0.607
175
Stokes shift (nm)
The results lead us to conclude that the microwave assisted ami-
nation provided shorter reaction time, highly increased yield as well
as simple product isolation procedure. Hydrochlorides 8e10 were
prepared to improve the poor solubility of compounds 5e7 in water.
The 1H NMR spectrum of thefused benzimidazo[1,2-a]quinoline 4
showed no signal for the NH group of benzimidazole nucleus of
compound 3, thus confirmingcyclic structure formation. The 1H NMR
spectra of amino substituted benzimidazo[1,2-a]quinolines 5e7
showed a downfield shift of the aromatic protons in comparison to
2-chloro-benzimidazo[1,2-a]quinoline-6-carbonitrile 4 and appear-
ance of protons related to piperidine, pyrrolidine or piperazine
nuclei. Protonation of compounds 5e7 lead to the appearance of
signal related to NHþ group in the 1H NMR spectra of compounds
8e10 as well as a downfield shift of protons related to corresponding
cyclic amine substituent.
undertaken in ethanol. The stock solutions of compounds 3e10
were prepared in ethanol by concentrations as it follows: c
(3) ¼ 4.46 ꢂ 10ꢁ3 mol dmꢁ3; c(4) ¼ 1.8 ꢂ 10ꢁ4 mol dmꢁ3; c
(5) ¼ 9 ꢂ 10ꢁ5 mol dmꢁ3; c(6) ¼ 4 ꢂ 10ꢁ5 mol dmꢁ3; c
(7) ¼ 2.14 ꢂ 10ꢁ3 mol dmꢁ3; c(8) ¼ 1.52 ꢂ 10ꢁ3 mol dmꢁ3; c
(9) ¼ 3.8 ꢂ 10ꢁ4 mol dmꢁ3; c(10) ¼ 2.0 ꢂ 10ꢁ4 mol dmꢁ3; UV/Vis
spectra of prepared compounds were recorded at the same
concentration of 2.0 ꢂ 10ꢁ5 mol dmꢁ3 and can be visualized in
Fig. 1. Precursor 3 showed one intense main absorption band at
355 nm while compound 4 showed several absorption bands,
especially intense in the region from 250 to 280 nm with high
extinction coefficients. Most of these electronic transitions are
made from
p to p p-system.
* orbitals of tetracyclic conjugated
3.2. Spectroscopic characterization
In order to study the spectroscopic properties of prepared
compounds 3e10, UV/Vis and fluorescence emission spectra was
Fig. 3. ORTEP drawing of molecular structure of
4
with atom labeling scheme.
Fig. 4. ORTEP drawing of molecular structure of
5 with atom labeling scheme.
The displacement ellipsoids are drawn at the 50% probability level at 296 K.
The displacement ellipsoids are drawn at the 50% probability level at 296 K.

84
N. Perin et al. / Dyes and Pigments 91 (2011) 79e88
Conversion of chlorine into the piperidine, pyrrolidine or pipera-
zine substituents resulted in strong bathochromic shift of w50 nm
and an increasing in the molar extinction coefficient. Heteroatoms
with non-bonding electrons, such as nitrogen, can absorb UV
4 and Stokes shift values are summarised in Table 3. Excitation
spectra of all studied compounds are in a good agreement with
their absorption spectra. The difference in energy between the
absorbed and emitted radiation is known as the Stokes shift. All
benzimidazo[1,2-a]quinolines 4e10 presented large Stokes shift
values (175e206 nm) which could be generally attributed to
a different charge distribution (or geometry) in the excited state of
molecules compared to the ground state.
radiation when the electrons make a transition from n orbitals to p
*
orbitals. The non-bonding electrons of nitrogen can take part in the
resonance of the tetracyclic aromatic structure which causes
a bathochromic shift in the absorbance maximum.
Compared with 6e7 and 9e10, with the pyrrolidine and piper-
azine substituents, 5 and 8 with the piperidine substituent showed
a hyperchromic effect. This may be explained by the fact that
N-atom in cyclic amines approximates a planar arrangement during
the facile interconversion between ring forms, especially in more
polar solvents. The interconversion is more rapid for piperidine
derivatives than other cyclic amines, a feature which is also sug-
gested in some previous studies [36].
3.3. Crystal structure study of compounds 4 and 5
ORTEP images (50% of atomic displacement parameters) of
compounds 4 and 5 are depicted in Fig. 3 (compound 4) and Fig. 4
(compound 5), while crystal packing structures are shown in Figs. 5
and 6 for compounds 4 and 5, respectively. The selected molecular
geometry is listed in Table 4 and hydrogen bonding geometry and
Fluorescence emission spectra were recorded at the same
concentration of 5.0 ꢂ 10ꢁ8 mol dmꢁ3 and can be visualized in Fig. 2.
Fluorescence excitation was performed at the wavelength of
maximum absorption. Compounds 4e9 exhibit characteristic fluo-
rescence emission with maxima at about 470 nm and show single
emission band while compound 10 showed two emission bands.
Compound 3 did not show any emission band under the applied
conditions. Hydrochloride salts 8e10 showed a hypsochromic shift
and lower fluorescence intensity than compounds 5e7.
p
,,,p interactions in Tables 5 and 6, respectively. The molecules of
4 and 5 are essentially planar (except piperidine substituent at C13
atom in 5). The dihedral angles calculated between the two planes
defined by atoms N1 N2 C1 C2 C3 C4 C5 C6 and C7 (one plane) and
N1 C1 C8 C9 C10 C11 C12 C13 C14 and C15 (another plane) amount
2.0(1)ꢀ and 2.9(2)ꢀ for 4 and 5, respectively. These values are
comparable for similar benzimidazo[1,2-a]quinoline molecular
systems substituted at position 2 with the angle range 0.65(6)o
[29]e4.27(4)ꢀ [7]. The piperidine substituent at C13 in 5 is rotated
around C13-N4 single bond (Table 4) by 14.0(3)ꢀ implicating that
the molecule as a whole is not planar. The benzimidazo[1,2-a]
Electronic absorption and fluorescence data of compounds 3e10
recorded at the same concentration in ethanol, quantum yield data
Fig. 5. a) Mercury drawing of crystal structure of 4 viewed down a axis. The intermolecular hydrogen bonds of the CeH,,,N type are shown by dashed lines. Imidazole N2 nitrogen
atom participates into CeH,,,N hydrogen bond which links molecules into discrete dimers spreading along b axis, while cyano N3 nitrogen atoms forms another CeH,,,N
hydrogen bond which further joins dimers along c axis. In such way, the layers are formed.

N. Perin et al. / Dyes and Pigments 91 (2011) 79e88
85
Table 5
Hydrogen bond geometry (Å, ꢀ) for compounds 4 and 5.
DeH,,,A
DeH H,,,A D,,,A
:DeH,,,A Symmetry code
4
C6eH6,,,N3
C3eH3,,,N2
0.93 2.540 3.376(3) 150
0.93 2.649 3.571(2) 171
1/2 þ x,1/2 ꢁ y,1/2 þ z
x þ 1/2,ꢁy þ 1/2,þz
þ 1/2
5
C20eH20A,,,N3 0.97 2.670 3.591(9) 159
C3eH3,,,N2 0.93 2.645 3.561(8) 168
ꢁx þ 1,ꢁy þ 1,ꢁz
ꢁx þ 1,ꢁy þ 1,ꢁz
3.4. Interaction with ct-DNA
Interaction of compounds 8e10 as hydrochloride salts with ct-
DNA was studied using fluorescence spectroscopy. Compounds
8e10 could interact with ct-DNA resulting in an increase in fluo-
rescence intensity. In such a way 8e10 could be used for a possible
application as fluorescent probes for DNA detection. Stock solutions
of compounds
8
and
9
were prepared in DMSO,
c
Fig. 6. Mercury drawing of crystal structure of 5. The intermolecular hydrogen bonds
of the CeH,,,N type are shown by dashed lines. The molecules are mutually con-
nected by two different types of rings which alternate thus forming the chains of rings.
(8) ¼ 1.65 mol dmꢁ3; c(9) ¼ 4.8 ꢂ 10ꢁ4 mol dmꢁ3 while stock
solution of compound 10 was prepared in H2O,
c
(10) ¼ 1.38 ꢂ 10ꢁ4 mol dmꢁ3. Stock solutions of compounds 8e10
were stable over longer periods (60 days).
quinoline fragment in 4 and 5 contains sp3 hybridized N1 atom
Small aliquots of DMSO stock solutions were added into the
aqueous medium for compounds 8 and 9 providing that DMSO
content in experiments was less than 1%.
The interaction with ct-DNA was performed in aqueous buffer at
pH 7 at compound concentrations of 5.0 ꢂ 10ꢁ7 mol dmꢁ3 [7,42,43].
The effects on emission maxima of 8e10 upon the addition of ct-
DNA are presented in Fig. 7.
Interactions of compound 8e10 with ct-DNA were measured at
different ratios r ((compound)/(ct-DNA)) followed by increasing
concentration of added ct-DNA up to high excess of ct-DNA. The
changes of fluorescence intensity on emission maxima for
compounds 8e10 upon the addition of corresponding amounts of
ct-DNA are presented in Table 7.
which lone pair electrons are delocalized within the
p-system of
fused rings. The CeN bond distances with the sp3 hybridized N1
atom (C15eN21, C1eN1 and C7eN1) are significantly longer then
CeN bonds formed by the sp2 hybridized N2 atom (shorter C1eN2
and longer C2eN2 in imidazolyl ring), which are dominantly
p in
character (Table 4), as it is found in analogous benzimidazo[1,2-a]
quinolines [7,37].
The intermolecular contacts are characterized by weak
CeH,,,N hydrogen bonds formed between phenyl eCH groups and
imidazole sp2 hybridized N2 atom or the N3 atom of the cyano
group (Table 5). The C3eH3,,,N2 intermolecular hydrogen bond is
found in both structures 4 and 5, while in 4 the proton acceptor
cyano N3 atom participates in hydrogen bond formation with
phenyl eCH group, and in 5 with less acidic methylene eCH2 group
of piperidine ring. The analogous hydrogen bonding arrangement
has been observed in the crystal structures of benzimidazo[1,2-a]
Table 6
Geometrical parameters of p,,,p
interactionsa (Å, ꢀ) for compounds 4 and 5.
quinolines [7,37]. The
are not analogous. The
p
,,,
p
aromatic stacking [38e41] in 4 and 5
aromatic stacking interactions are
Interactionb CgꢁCg distance Cg,,,P1b Cg,,,P2c ad
4
be
Slippage
p,,,
p
found between the imidazole and phenyl ring of quinoline frag-
ment as well as between imidazole and N1, C1, C8, C9, C10 and C15
atoms of quinoline ring in 4, while in 5 the condensed benzimi-
dazolyl heterocyclic rings are included (see Table 6 for ring atoms).
Cg3,,,Cg4i 3.634(1)
Cg3,,,Cg6i 3.601(1)
Cg3,,,Cg6ii 3.725(1)
Cg4,,,Cg4i 3.564(1)
Cg4,,,Cg6ii 3.651(1)
5
ꢁ3.351(1) ꢁ3.338(1) 1.09(8) 23.30 1.437
ꢁ3.353(1) ꢁ3.375(1) 1.07(8) 20.42 1.256
3.394(1)
3.403(1) 1.07(8) 24.00 1.515
ꢁ3.351(1) ꢁ3.351(1)
0
19.91 1.214
3.383(1) 3.386(1) 0.32(7) 21.96 1.365
Cg6,,,Cg7iii 3.746(3)
Cg7,,,Cg7iv 4.029(3)
ꢁ3.513(2) ꢁ3.527(2) 3.2(2) 19.71 1.263
3.522(2) 3.521(2) 29.07 1.958
Table 4
0
Selected interatomic distances (Å) and valence and torsion angles (ꢀ) for the
a
compounds 4 and 5.
Those possible interactions for which perpendicular distance between two
planes is longer than 3.8 Å (3.3e3.8 Å) (Cg,,,P1 and Cg,,,P2; the perpendicular
distance of corresponding centroid to a plane) and dihedral angles (between P1 and
P2) larger than 20ꢀ are not taken into account. The calculated slippage on the basis of
Cg,,,Cg distances as well as the value of the angle between Cg,,,Cg vector and
vertical line on corresponding plane has been taken into account as one of the
Selected bond distances
4
5
Cl1eC13 (C13eN4 in 5)
C1eN2
C2eN2
C1eN1
C15eN1
1.7334(17)
1.305(2)
1.384(2)
1.403(2)
1.407(2)
1.396(2)
1.144(2)
1.398(5)
1.307(6)
1.394(6)
1.398(5)
1.400(6)
1.400(5)
1.127(6)
geometrical criteria, too (approx. 1.5 Å for p,,,p stacking interactions).
b
Rings Cg3, Cg4 and Cg6 are defined by the atoms N1, C1, N2, C2 and C7; N1, C1,
C8, C9 C10 and C15; C10, C11, C12, C14, C14 and C15 in 4, respectively. Rings Cg6
and Cg7 are defined by the atoms N1, C1, N2, C2, C3, C4, C5, C6 and C7; N1, C7, C2,
N2, C1, C8, C9, C10 and C15 in 5, respectively.
C7eN1
C16eN3
c
Cg,,,P1 (or P2) is the perpendicular distance of corresponding centroid to
a plane. Planes P1 or P2 are defined by the atoms, which define the corresponding
Bond angles
127.4(1)
104.8(1)
132.8(1)
178.2(2)
centroids.
C8eC1eN2
C1eN2eC2
C15eN1eC7
C8eC16eN3
127.1(4)
104.4(4)
133.2(3)
178.7(7)
d
Dihedral angles between P1 and P2.
e
b
is the angle between Cg,,,Cg vector and vertical line on corresponding plane.
x,ꢁy,ꢁz, ii x,1 y,ꢁz, iii x,1 y,ꢁz;
Symmetry codes:
iv ¼ 1 ꢁ x,1 ꢁ y,ꢁz.
i
¼
2
ꢁ
¼
2
ꢁ
ꢁ
¼
ꢁ

86
N. Perin et al. / Dyes and Pigments 91 (2011) 79e88
A
B
600
8
500
400
300
200
100
0
r = 0.1
r = 0.05
r = 0.01
r = 0.005
550
7
500
r = 0.1
450
r = 0.05
r = 0.01
400
r = 0.005
350
increasing ct-DNA
300
increasing ct-DNA
250
200
150
100
50
7
0
450
475
500
525
550
575
10
600
450
500
550
600
λ/ nm
λ/ nm
C
r = 0.1
600
r = 0.05
r = 0.01
r = 0.001
500
400
300
200
100
0
incresaing ct-DNA
450
500
550
600
λ/ nm
Fig. 7. Fluorescence emission spectra of compounds 8 (A), 9 (B) and 10 (C) in the presence of different ratios r of ct-DNA at compounds concentration of c ¼ 5 ꢂ 10ꢁ7 mol dmꢁ3
excited on the wavelength of absorption maxima.
In aqueous buffered solution, significant changes in the emis-
sion spectra of compounds 8e9 occurred during the addition of
small aliquots of ct-DNA. These changes resulted in a strong
hyperchromic shift with fluorescence enhancement and without
any changes in the wavelength of emission maxima. The addition of
ct-DNA to compounds 8 and 9 with piperidine and pyrrolidine
substituents increased the fluorescence intensity by ca. 3.38
and 1.47 times, respectively. Thus, both compound 8 and 9 offer
possible applications for DNA detection in biological systems as
fluorescence probes since they exhibit high binding affinity to DNA
resulted in fluorescence enhancement as well as intense fluores-
cence. As it can be viewed in fluorescence emission spectra which
represents interaction of compound 10 with ct-DNA (Fig. 7C),
compound 10 showed strong hypochromic shift by increasing
concentration of ct-DNA as well as a slight bathochromic shift of
5 nm. Above mentioned results are in good agreement with the
previously published ones. Namely, our previously published
results have shown that positively charged benzimidazo[1,2-a]
quinolines intercalate into ds DNA since this chromophore posses
a planar, highly conjugated tetracyclic aromatic structure which has
the ability to intercalate into double helix of DNA molecule. Addi-
tional positively charged groups attached to this chromophore, like
cyclic amine substituents in compounds 8e10, would be expected
to form additional binding interactions.
Table 7
Fluorescence intensity changes of compounds 8e10 upon the addition of ct-DNA at
different ratio
r
((compound)/(ct-DNA)) at pH
¼
7.0 (Buffer Na-Cacodylate,
I ¼ 0.05 mol dmꢁ3).
ra
Re. Fluo. Int/(a.u.)
D
Int/(a.u.)
F/F0
b
8
0
0.1
0.05
0.01
0.005
0
138.2
167.6
202.3
384.8
467.0
375.7
392.2
409.2
441.3
552.6
588.2
245.1
179.0
165.2
165.0
e
e
29.4
1.21
1.46
2.78
3.38
e
Based on obtained experimental results, together with previ-
ously published ones, we can suggest that the possible mechanism
of interaction between the fluorescent probes 8e10 and ct-DNA
could be intercalation.
64.1
246.6
328.8
e
9
0.1
16.5
33.5
65.6
176.9
e
1.04
1.09
1.17
1.47
e
0.05
0.01
0.005
0
4. Conclusions
10
The main aim of this study was to prepare amino substituted
benzimidazo[1,2-a]quinolines 5e7 in good yields. Their hydro-
chloride salts 8e10 were prepared by protonation with HCl gas to
enhance their solubility for spectroscopic characterization of their
interaction with ct-DNA. The benzimidazo[1,2-a]quinolines 4e10
showed interesting spectroscopic properties with several absorp-
tion bands. In comparison with compound 4, aminated compounds
0.1
343.1
409.2
423.0
423.2
2.40
3.29
3.56
3.56
0.05
0.01
0.005
a
r¼(compound)/(ct-DNA).
b
F ¼ fluorescence of compound-DNA complex; F0 ¼ fluorescence of compound
without ct-DNA addition.

N. Perin et al. / Dyes and Pigments 91 (2011) 79e88
87
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