Synthesis, antiproliferative activity and DNA binding properties of novel 5-Aminobenzimidazo[1,2-a]quinoline-6-carbonitriles

Article abstract of DOI:10.1016/j.ejmech.2014.04.049

The synthesis of 5-amino substituted benzimidazo[1,2-a]quinolines prepared by microwave assisted amination from halogeno substituted precursor was described. The majority of compounds were active at micromolar concentrations against colon, lung and breast carcinoma cell lines in vitro. The N,N-dimethylaminopropyl 9 and piperazinyl substituted derivative 19 showed the most pronounced activity towards all of the three tested tumor cell lines, which could be correlated to the presence of another N heteroatom and its potential interactions with biological targets. The DNA binding studies, consisting of UV/Visible absorbency, melting temperature studies, and fluorescence and circular dichroism titrations, revealed that compounds 9, 19 and 20 bind to DNA as strong intercalators. The cellular distribution analysis, based on compounds' intrinsic fluorescence, showed that compound 20 does not enter the cell, while compounds 9 and 19 do, which is in agreement with their cytotoxic effects. Compound 9 efficiently targets the nucleus whereas 19, which also showed DNA intercalating properties in vitro, was mostly localised in the cytoplasm suggesting that the antitumor mechanism of action is DNA-independent.

Full text of DOI:10.1016/j.ejmech.2014.04.049

European Journal of Medicinal Chemistry 80 (2014) 218e227
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antiproliferative activity and DNA binding properties of
novel 5-Aminobenzimidazo[1,2-a]quinoline-6-carbonitriles
a
b
c
b
a
ꢀ
Natasa Perin , Raja Nhili , Katja Ester , William Laine , Grace Karminski-Zamola ,
Marijeta Kralj c, Marie-Hélène David-Cordonnier b , Marijana Hranjec
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 19, P. O. Box 177, HR-10000 Zagreb,
,
a,
*
*
a
ꢁ
Croatia
b INSERM U837, Jean-Pierre Aubert Research Centre (JPARC), Team “Molecular and Cellular Targeting for Cancer Treatment”, Université Lille 2,
IMPRT-IFR-114, Institut pour la Recherche sur le Cancer de Lille, Place de Verdun, F-59045 Lille cedex, France
c
ꢀ
ꢁ
ꢀ
Division of Molecular Medicine, RuCer Boskovic Institute, Bijenicka cesta 54, P. O. Box 180, HR-10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of 5-amino substituted benzimidazo[1,2-a]quinolines prepared by microwave assisted
amination from halogeno substituted precursor was described. The majority of compounds were active
at micromolar concentrations against colon, lung and breast carcinoma cell lines in vitro. The N,N-
dimethylaminopropyl 9 and piperazinyl substituted derivative 19 showed the most pronounced activity
towards all of the three tested tumor cell lines, which could be correlated to the presence of another N
heteroatom and its potential interactions with biological targets. The DNA binding studies, consisting of
UV/Visible absorbency, melting temperature studies, and fluorescence and circular dichroism titrations,
revealed that compounds 9, 19 and 20 bind to DNA as strong intercalators. The cellular distribution
analysis, based on compounds’ intrinsic fluorescence, showed that compound 20 does not enter the cell,
while compounds 9 and 19 do, which is in agreement with their cytotoxic effects. Compound 9 efficiently
targets the nucleus whereas 19, which also showed DNA intercalating properties in vitro, was mostly
localised in the cytoplasm suggesting that the antitumor mechanism of action is DNA-independent.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Received 15 January 2014
Received in revised form
15 April 2014
Accepted 15 April 2014
Available online 18 April 2014
Keywords:
Benzimidazo[1,2-a]quinolines
Amino side chains
Antiproliferative activity
DNA binding properties
Cellular distribution
1. Introduction
additional interactions with potential biological targets [9e11].
High fluorescence intensity and possibility of interaction with
Benzimidazoles are important and fundamental building skel-
etons of various essential synthetic and natural pharmacological
compounds [1e3]. Heterocycles bearing a benzimidazole nucleus
possess a broad spectrum of pharmacological features like anti-
cancer [4], antibacterial [5], antifungal [6], antiviral [7], antihista-
minic [8] etc. In recent years, there is a permanent and growing
interest in the synthesis of fused, benzannulated benzimidazole
derivatives as a direct consequence of their great importance in the
natural, medicinal and environmental sciences. Usually, cyclic
benzimidazole derivatives have a highly conjugated planar chro-
mophore bearing excellent spectroscopic properties important for
their application in several other areas like optoelectronics or
chemosensors, with side chain substituents designed to enable
important biomacromolecules of the living systems offer their
potential use as fluorescent probes for detection of important
molecules as DNA or different proteins in biomedical diagnostics
[12].
A group of authors prepared amino- and amido-substituted
benzimidazo[1,2-c]quinazolines with different length of side
chains, evaluated in vitro cytotoxicity against a panel of human and
murine cell lines and showed that the most active compound was
shown to be an intercalator [13]. Moreover, some benzimidazo[2,1-
a]isoquinolines with carboxamide side chains were used for
studying biological effects induced by the variation in the side-
chain position in this tetracyclic series [14]. Lamazzi et al. have
described the antitumor potency of benzimidazo[1,2-c]quinazo-
line-6-carbonitriles which block significantly the growth of L1210
cells [15]. Volovenko and co-workers [16] have published the
synthesis of benzimidazo[1,2-a]quinoline-6-carbonitriles which
were tested by another group of authors as potential fluorescent
probes for protein structure investigation including albumin,
* Corresponding authors.
E-mail addresses: marie-helene.david@inserm.fr (M.-H. David-Cordonnier),
mhranjec@fkit.hr (M. Hranjec).
http://dx.doi.org/10.1016/j.ejmech.2014.04.049
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

N. Perin et al. / European Journal of Medicinal Chemistry 80 (2014) 218e227
219
lysozyme and papain [17]. Babichev [18] has published the syn-
thesis of amino substituted benzimidazo[1,2-c]quinazoline-6-
carbonitriles which were tested for their antimicrobial activity
against common bacterial, yeast and fungal pathogens. 6-
dialkylaminoalkylbenzimidazo[1,2-c]quinazolines were synthe-
sized as potential interferon inducers, antiviral and DNA binding
agents [19]. Recently, we have prepared various benzimidazo[1,2-a]
quinoline-6-carbonitriles and their heteroaromatic fluorene ana-
logues which exerted pronounced antiproliferative activity with
the cyano moiety important for the activity but not the selectivity
of tested compounds [20]. Some of the tested compounds signifi-
cantly enhanced fluorescence emission upon addition of CT-DNA
and thus offer the potential application as DNA-specific fluores-
cent probes. Furthermore, fluorescence microscopy study of some
amino and positively charged diamino substituted benzimidazo
[1,2-a]quinolines demonstrated that they are weak intercalators,
confirming that DNA is not the primary biological target of this
compounds [21]. Very recently, we have reported on the biological
activity of 2-aminobenzimidazo[1,2-a]quinoline-6-carbonitriles
with different lengths of the secondary or tertiary amino chains
linked to the tetracyclic skeleton which significantly influenced the
antiproliferative activity [22].
of aldol condensation of 2-chlorobenzoylchloride
1
with 2-
cyanomethylbenzimidazole 2 in absolute ethanol using piperidine
as a base in moderate yield (44%). Fused keto derivative 4 was
prepared by previously described termic cyclization reaction in
DMF using t-KOBu as a base. The main precursor for the synthesis of
targeted compounds, chloro substituted benzimidazo[1,2-a]quin-
oline 5 was prepared in the reaction of compound 4 with the POCl3
and PCl5 with yield of 70% [16,18].
Based on the series of experiments which were undertaken in
order to optimize the reaction times and yields including thermal
reactions with an access of corresponding amine in dioxane, cata-
lyzed or microwave assisted aminations, finally the amination re-
actions were conducted by microwave assisted synthesis by using
800 W power, 170 ꢀC and 40 bar in acetonitrile in order to obtain
the highest yields in the shortest reaction time. The results lead us
to conclude that the microwave assisted amination provided
shorter reaction time, highly increased yield as well as simple
product isolation procedure [11a,22].
Thus, amino-substituted benzimidazo[1,2-a]quinolines were
prepared from chloro substituted benzimidazo[1,2-a]quinoline 5 by
uncatalyzed microwave assisted nucleophilic aromatic substitution
using five or six-fold excess of the corresponding amine.
Motivated by all above-mentioned considerations and as an
extension of our previous scientific research related to benzimidazo
[1,2-a]quinolines as potential anticancer agents, we report within
this manuscript the synthesis of 5-aminobenzimidazo[1,2-a]quin-
oline-6-carbonitriles, their antiproliferative activity in vitro and the
DNA binding studies of some of the most active compounds.
All compounds were obtained in low to moderate yield (30e
74%) except the N,N-diamino substituted derivatives 12, 13 and 14
which were obtained in a very low yield 12e24% despite the
optimization of the reaction conditions. N,N-dimethylated piper-
azinyl substituted compound 20 as a iodide salt was prepared from
derivative 19 with an excess of methyl-iodide.
The structures of all prepared derivatives 6e20 were determined
by using 1H and 13C NMR spectroscopyand mass spectrometry. NMR
analysis was based on the values of HeH coupling constants and
chemical shifts in the 1H and 13C NMR spectra. The reaction of
cyclization of acyclic precursor 3 provides a downfield shift of the
aromatic protons and disappearance of one proton of the NH group
on the benzimidazole nuclei which confirmed the formation of the
benzimidazo[1,2-a]quinoline skeleton. Halogenation of the fused
2. Results and discussion
2.1. Chemistry
All compounds were prepared according to the main experi-
mental procedures shown in Scheme 1 by conventional methods of
organic synthesis. Acyclic precursor 3 was prepared in the reaction
Scheme 1. Synthesis of amino substituted benzimidazo[1,2-a]quinolines.

220
N. Perin et al. / European Journal of Medicinal Chemistry 80 (2014) 218e227
derivative 4 leads also to a downfield shift of the all aromatic protons
as well as reaction of amination. In the NMR spectra of amino-
substituted derivatives it can be observed the appearance of pro-
tons related to amino substituents in the aliphatic part both in 1H
and 13C NMR spectra (Table S1).
of compound 20 could be due to the steric hindrance of two
attached methyl groups which reduce the interaction of N hetero-
atom with potential biological targets. From the structureeactivity
relationship (SAR) studies it could be concluded that secondary
amine side chain is generally slightly preferred over tertiary amine
or cyclic amine side chain except for piperazine substituent.
2.2. Biological results and discussion
2.3. DNA binding properties
All tested compounds showed antiproliferative effect on the
tested cell lines. In general, they exert modest to prominent effect
on HCT 116 and MCF-7 cell lines, with IC50 concentrations in a
micromolar range, and modest effect on H 460 cell line (Table 1).
Compounds 9 and 19 showed the most pronounced activity to-
wards all three tested cell lines. Compounds 10 and 16 were not
tested due to significant precipitation in aqueous media.
From this new series, compounds 6e9, 11e14 and 17e20 were
evaluated for their DNA binding propensities. Measurements of the
stabilization of the DNA double helix (DTm) were performed as a
first screening. Each compound was incubated with CT-DNA at a
drug/bp ratio of 0.5 prior to be subjected to heat denaturation and
measurement of the absorbance of the DNA at 260 nm. The
hyperchromicity at 260 nm correlates with the release of single-
stranded DNA. The melting point was deduced from the midpoint
of the hyperchromicity and corresponds to the temperature for
which half of the DNA is in a single strand form and the other stays
in the double strand form. From those 12 compounds, only com-
pounds 9, 19 and 20 are able to stabilize the DNA helix with an
increase of 6.3, 2.9 and 7.3 ꢀC, respectively to achieve their melting
point from comparison with CT-DNA alone. Those compounds were
We studied the influence of amino side chains with different
type of nitrogen atom, as well as different chain lengths on the
biological activity. Compounds 6e8 bearing secondary amino side
chains showed selectivity towards MCF-7 cell lines, but without any
effect of the chain length. The introduction of additional nitrogen
heteroatom in the structure of compound 9 through the N,N-
dimethylaminopropyl side chain increased the activity towards
HTC 116 and H 460 cell lines in comparison to compounds 6e8.
Introduction of another nitrogen heteroatom in the structure of
compound 15 with 3,3ʹ-diaminodipropylamino substituent
decreased antiproliferative effect towards all cell lines. The length
of N,N-dialkylamino side chains influenced the activity of com-
pounds 11e14, whereby compounds with longest side chains (13
and 14) exerted the most pronounced activity. Compound 17 with
cyclic amino piperidine substituent showed better activity towards
MCF-7 cells while the introduction of another oxygen heteroatom
in the structure of morpholinyl substituted compound 18 decreased
the antiproliferative effects. The most active compound with cyclic
amino substituent was piperazinyl substituted derivative 19 which
showed strong antiproliferative effect towards all tested tumor cell
lines. The most pronounced antiproliferative activity of compound
19 could be explained by the presence of another N heteroatom
which could contribute to the interactions with potential biological
targets. Iodide salt of N,N-dimethylpiperazinyl substituted com-
pound 20 showed the weakest antiproliferative activity among all
tested compounds. Quaternization of piperazine N heteroatom was
performed to evaluate the effect of positive permanent charges on
the antiproliferative activity. The weakest antiproliferative activity
further analyzed at R ¼ 1, leading to an increase of the
DTm values
(Table 2).
Those three compounds were selected for further spectroscopic
analysis. UV/Visible spectroscopy of those compounds evidences
bathochromic and/or hypochromic effects upon binding to
increasing concentrations of CT-DNA (Fig. 1, upper panels). Only
UV/Visible spectroscopy performed with compound 20 evidences a
clear isosbestic point at 410 nm. The hypochromic effect was
quantified at the various CT-DNA concentrations relatively to the
absorbency of the drug alone at the indicated maximum emission
peaks (Fig. 1, bottom panels).
Both compounds were then evaluated in fluorescence spec-
troscopy upon binding to DNA (Fig. 2, upper panels) and plotted
accordingly to SterneVolmer equation (Fig. 2, bottom panels). A
more complex binding is observed using 9 with a small quenching
effect seen using the highest drug/DNA ratios, followed by a strong
enhancement of fluorescence. The fluorescence spectra only evi-
denced an enhancement of fluorescence for 19 and 20. Therefore,
the enhancing constant KSv deduced from SterneVolmer plots were
only calculated for those two compounds.
The orientation of the molecules relatively to the DNA helix
(intercalation, groove binding) was investigated using circular di-
chroism spectrometry. Of the three compounds that efficiently
binds DNA, only 19 and 20 presented a small change of the CD
spectra of CT-DNA (Fig. 3). The deduced induced CD (ICD,
embedded panels) for compounds 19 and 20 evidenced small
negative spectra at the drug absorption wavelength that are char-
acteristic for DNA intercalation between adjacent base pairs.
Such intercalation process is confirmed using topoisomerase I-
induced DNA relaxation experiments (Fig. 4). Compounds 19 and
Table 1
IC50 values (in
m
M ꢁ sd).a
a
IC50
(mM)
Compound
Cell lines
MCF-7
HTC 116
H 460
6
7
8
9
11
12
13
14
15
17
18
19
7 ꢁ 1
17 ꢁ 2
33 ꢁ 9
1.5 ꢁ 0.3
27 ꢁ 8
13 ꢁ 0.3
6 ꢁ 1
3 ꢁ 0.4
3 ꢁ 1
84 ꢁ 3
15 ꢁ 2
3 ꢁ 0.7
2 ꢁ 0.4
21 ꢁ 5
7 ꢁ 3
35 ꢁ 6
4 ꢁ 0.6
48 ꢁ 0.4
25 ꢁ 8
Table 2
4 ꢁ 1
20 ꢁ 2
Variation of the melting temperature of CT-DNA upon binding with the various
compounds. Only the active compounds are presented at the indicated drug/DNA
(bp) ratios (R).
7 ꢁ 1
6 ꢁ 3
26 ꢁ 7
8 ꢁ 2
9 ꢁ 3
12 ꢁ 0.1
19 ꢁ 7
10 ꢁ 6
37 ꢁ 2
2 ꢁ 0.2
93 ꢁ 6
2.5 ꢁ 0.6
16 ꢁ 3
2 ꢁ 0.4
15 ꢁ 7
0.02 ꢁ 0.01
1 ꢁ 0.7
Compound
D
Tm (ꢀC)
83 ꢁ 4
4 ꢁ 0.6
ꢂ100
R ¼ 0.5
R ¼ 1
20
Doxorubicin
Etoposide
0.07 ꢁ 0.02
5 ꢁ 2
0.03 ꢁ 0.01
0.1 ꢁ 0.04
9
19
20
6.3
2.9
7.3
10.2
4.4
10.7
a
IC50; the concentration that causes 50% growth inhibition.

N. Perin et al. / European Journal of Medicinal Chemistry 80 (2014) 218e227
221
Fig. 1. UV/Visible absorbency (upper panels) and percentage of hypochromicity (bottom panels) for compounds 9, 19 and 20 (20
mM) upon binding to increasing concentrations of
CT-DNA from 1 to 80 (7) or 140
compound 20.
mM (19, 20). Full arrows correspond to hypochromic (Y) and bathochromic (/) effects, respectively. Arrow head localized the isosbestic point for
Fig. 2. Fluorescence titration for drug/DNA binding. Upper panels: the fluorescence spectra were performed using 10
lanes) or with increasing concentrations (bottom to top) of CT-DNA from 0.1 to 100 (compound 9) or 200 (bold lanes)
wavelengths. Bottom panels: SterneVolmer plots at the indicated maximum emission wavelengths. Linear fitting (plain lane) of the initial points were using for compounds 19 and
18 to deduced the enhancement constant KSv
m
M of the indicated compounds incubated alone (dashed
m
M (compounds 19 and 20) at the indicated excitation
.
Fig. 3. Circular dichroism. CD spectra are presented for UV/Visible wavelengths from 230 to 430 nm using CT-DNA alone (dashed lanes) or incubated with increasing concentrations
of 9, 19 or 20 (from 1 to 70
M). Embedded panels for 19 and 20 correspond to the induced circular dichroism spectra (ICD ¼ CD[CT-DNAþcompound]-CD[CT-DNA]) using increasing
concentrations of compounds (top to bottom).
m

222
N. Perin et al. / European Journal of Medicinal Chemistry 80 (2014) 218e227
Topo I
17
20
19
14
9
Rel
Topo
Sc
Fig. 4. Topoisomerase I e induced DNA relaxation. Supercoiled circular plasmid DNA
(Sc) is treated with topoisomerase I (Topo I) in the presence of increasing concentra-
tions (mM) of the indicated compounds to evidence the presence of different top-
oisomers (Topo) or the relaxed circular DNA form (Rel) that were generated in each
DNA samples.
20, but not 17 and 14 used here as controls, facilitate the relaxation
of supercoiled (Sc) circular plasmid DNA as evidenced by the for-
mation of fully relaxed plasmid DNA forms (Rel) using 5 and 2 mM of
19 and 20, respectively, followed by the formation of positively
supercoiled plasmid DNA at higher concentrations. Such profiles
are typical for DNA intercalation as for ethidium bromide [23].
Interestingly, this functional experiment also reveals DNA interca-
lation using compound 9. Such intercalation propensity was not
evidenced using CD titration (Fig. 3) which is a strong method for
identifying DNA intercalation as a single mode of binding but less
sensitive and informative in the case of more complex binding to
the DNA helix. This is in agreement with fluorescence titration of
compound 9 evidencing a two phase binding to CT-DNA (Fig. 2).
Functionally, none of the compounds evidences topoisomerase I-
poisoning effect as part of their mechanism of action.
We then looked at potential sequence selective binding of the
five compounds to DNA but as expected from many classic DNA
intercalators, DNase I footprinting did not revealed any sequence-
specific DNA binding (data not shown).
Finally, we looked at cell entry and distribution of the various
compounds in HT-29 colon carcinoma cell line, based on their
Fig. 5. Cellular distribution in HT-29 colon carcinoma cells. Plated cells were treated
with 5 M of the various indicated compounds for 16 h prior to be labeled with
m
MitoRed dye as a cytoplasmic marker and fixed using paraformaldehyde. Cells were
analyzed using an apotome-equipped fluorescence microscope (Zeiss) at 63ꢃ (9, 17, 19)
or 40ꢃ (14, 20) enlargement. Scales are indicated as red lines (20
mm) in the first
panels. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
fluorescence properties. After 16 h of treatment with 5
mM of
compounds, cells were labeled with MitoRed for cytoplasm locali-
zation and fixed using paraformaldehyde 2% prior to be analyzed
using an apotome fluorescence microscope (Fig. 5). Interestingly,
compound 9 shows nuclear localization whereas 14, 17 and 19 are
cytoplasmic, contrasting with the less cytotoxic compound 20 that
failed to enter the cells. This results strongly suggest that i) com-
pound 9 targets the nucleus through intercalation in the DNA
(associated with an increase in the fluorescence intensity upon
binding to DNA as evidenced in vitro, Fig. 2); ii) compound 20, that
strongly binds DNA in vitro, lacks to enter the cell in agreement
with its poor cytotoxic activity; iii) compounds 14, 17 and 19 seems
not to target the DNA in cells as part of their mechanism of action
even if compounds as 17 and 19 share in vitro DNA binding potency.
For those last 3 compounds, the mechanism of action in the cyto-
plasm needs to be further evaluated.
properties and intercalates in the DNA helix but compound 9 was
the only one that entered the cell and localized in the nucleus,
whereas compound 19 enters the cell with its main localization in
the cytoplasm, suggesting that DNA is not its functional target.
Interestingly, the dimethyl-N-piperazinyl compound 20, which has
similar in vitro DNA binding properties to 9 and 19, is poorly active
on cell proliferation, which is in agreement with its inability to
enter the cell. The fluorescent compounds 14 and 17 are cytotoxic to
the evaluated cell lines and localized in the cytoplasm as compound
19. It would be of interest in the future to determine which cyto-
plasmic target is implicated in the cellular effect of compounds 14,
17, 19 and the other compounds of this series.
4. Experimental section
3. Conclusions
4.1. General methods
In this manuscript the synthesis of 5-amino substituted benzi-
midazo[1,2-a]quinolines 6e20 as potential antiproliferative and
DNA binding agents is described. In general secondary amine side
chain is associated with more pronounced antiproliferative activity
on three tumor cell lines than does tertiary amine or cyclic amine
side chain, except for piperazine substituent 19. The most active
compounds of this new series were the N,N-dimethylaminopropyl
side chain-bearing compound 9 and piperazinyl substituted de-
rivative 19. These two compounds presented strong DNA binding
All chemicals and solvents were purchased from commercial
suppliers Aldrich and Acros. Melting points were recorded on
SMP11 Bibby and Büchi 535 apparatus. All NMR spectra were
measured in DMSO-d6 solutions using TMS as an internal standard.
The 1H and 13C NMR spectra were recorded on a Varian Gemini 300
or Varian Gemini 600 at 300, 600, 150 and 75 MHz, respectively.
Chemical shifts are reported in ppm (d) relative to TMS. All com-
pounds were routinely checked by TLC with Merck silica gel 60F-

N. Perin et al. / European Journal of Medicinal Chemistry 80 (2014) 218e227
223
254 glass plates. Microwave-assisted synthesis was performed in a
Milestone start S microwave oven using quartz cuvettes under the
pressure of 40 bar. Mass spectra were recorded on an Agilent 1200
series LC/6410 QQQ instrument. The electronic absorption spectra
were recorded on Varian Cary 50 spectrometer using quartz cuvette
(1 cm). Elemental analysis for carbon, hydrogen and nitrogen were
performed on a PerkineElmer 2400 elemental analyzer. Where
analyses are indicated only as symbols of elements, analytical re-
sults obtained are within 0.4% of the theoretical value.
chromatography on SiO2 using dichloromethane/methanol as
eluent.
4.2.4.1. 5-N-methylaminobenzimidazo[1,2-a]quinoline-6-carbonitrile
6. Compound 6 was prepared using above described method from
5 (0.140 g, 0.50 mmol) and methylamine (0.26 mL, 2.50 mmol) after
1 h of irradiation to yield 0.100 g (73%) of yellow powder; m.p.
>153e156 ꢀC.
1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.63 (d, 1H, J ¼ 8.34 Hz,
H
H
arom.), 8.39 (d, 1H, J ¼ 8.22 Hz, Harom.), 8.35 (d, 1H, J ¼ 8.17 Hz,
4.2. Synthesis
arom.), 8.17 (q, 1H, J ¼ 4.74 Hz, NH), 7.89 (t, 1H, J ¼ 7.56 Hz, Harom.),
7.73 (d, 1H, J ¼ 7.86 Hz, Harom.), 7.56 (t, 1H, J ¼ 7.62 Hz, Harom.), 7.40
(t, 1H, J ¼ 7.54 Hz, Harom.), 7.32 (t, 1H, J ¼ 7.56 Hz, Harom), 3.44 (d, 3H,
4.2.1. 2-(2-Benzimidazolyl)-3-keto-(2-chloro-phenyl)acrylonitrile 3
[16]
J ¼ 4.98 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz):
/ppm ¼ 158.28 (s),
d
A solution of 1.57 g (10.00 mmol) 2-cyanomethylbenzimidazole
and 1.33 mL (10.50 mmol) 2-chlorobenzoylchloride in pyridine
(10 mL) was refluxed for 2 h. The cooled mixture was poured into
water (100 mL) and the resulting product was filtered off and
recrystallized from ethanol to obtain a brown powder (1.31 g, 44%);
m.p. >300 ꢀC.
151.64 (s), 149.57 (s), 145.22 (s), 135.44 (s), 133.33 (d), 131.33 (s),
124.74 (d), 124.61 (d), 124.33 (d), 121.76 (d), 118.87 (d), 118.16 (s),
116.96 (s), 116.67 (d), 114.05 (d), 32.54 (q); Found: C, 74.93; H, 4.53;
N, 20.54. Calc. for C17H12N4: C, 74.98; H, 4.44; N, 20.58%.
4.2.4.2. 5-N-butylaminobenzimidazo[1,2-a]quinoline-6-carbonitrile
7. Compound 7 was prepared using above described method from
5 (0.100 g, 0.36 mmol) and butylamine (0.18 mL, 1.80 mmol) after
2 h of irradiation to yield 0.070 g (62%) of white powder; m.p. 217e
220 ꢀC.
1H NMR (DMSO-d6, 300 MHz):
7.59e7.50 (m, 3H, Harom.), 7.48e7.42 (m, 3H, Harom.), 7.32e7.28 (m,
2H, Harom.); 13C NMR (DMSO-d6, 75 MHz):
d
/ppm ¼ 13.09 (bs, 2H, NH),
d
/ppm ¼ 185.01 (s),
151.04 (s), 140.93 (s), 130.88 (s, 2C), 130.80 (d), 130.02 (s), 129.91 (d),
128.91 (d), 127.51 (d), 124.09 (d, 2C), 120.29 (s), 112.71 (d, 2C), 67.62
(s).
1H NMR (DMSO-d6, 600 MHz):
arom.), 8.49 (dd, 1H, J1 ¼ 0.75, Hz, J2 ¼ 8.94 Hz, Harom.), 8.40 (d, 1H,
d
/ppm ¼ 8.64 (d, 1H, J ¼ 8.83 Hz,
H
J ¼ 8.38 Hz, Harom.), 8.17 (t, 1H, J ¼ 4.89 Hz, NH), 7.90 (dt, 1H,
J1 ¼1.12 Hz, J2 ¼ 7.20 Hz, Harom.), 7.72 (d, 1H, J ¼ 8.16 Hz, Harom.), 7.57
(t,1H, J ¼ 7.71 Hz, Harom.), 7.39 (t,1H, J ¼ 7.15 Hz, Harom.), 7.30 (dt,1H,
J1 ¼1.11 Hz, J2 ¼ 7.20 Hz, Harom.), 3.86 (q, 2H, J ¼ 6.82 Hz, CH2), 1.79e
1.74 (m, 2H, CH2), 1.46e1.40 (m, 2H, CH2), 0.94 (t, 3H, J ¼ 7.41 Hz,
CH3); 13C NMR (DMSO-d6, 75 MHz): 150.41 (s), 149.54 (s), 145.14 (s),
135.48 (s), 133.41 (d), 131.29 (s), 124.99 (d), 124.74 (d), 124.35 (d),
121.74 (d), 118.79 (d), 118.12 (s), 117.03 (s), 116.69 (d), 114.10 (d),
71.78 (s), 44.20 (t), 32.03 (t), 29.47 (t), 14.21 (q); Found: C, 76.36; H,
5.71; N, 17.93. Calc. for C20H18N4: C, 76.41; H, 5.77; N, 17.82%.
4.2.2. 5-Ketobenzimidazo[1,2-a]quinoline-6-carbonitrile 4 [16]
A solution of 0.50 g (1.69 mmol) 2-(2-benzimidazolyl)-3-keto-
(2-chloro-phenyl)acrylonitrile and 0.44 g t-KOBu in DMF (6 mL)
was refluxed for 3 h. After cooling, the reaction mixture was
evaporated under vacuum and dissolved in water (50 mL). Result-
ing product was filtered off and recrystallized from ethanol to
obtain a white powder (0.25 g, 57%); m.p. >300 ꢀC.
1H NMR (DMSO-d6, 300 MHz):
d
/ppm ¼ 8.42 (d, 1H, J ¼ 8.37 Hz,
H
arom.), 8.25 (dd, 1H, J1 ¼ 1.52 Hz, J2 ¼ 7.82 Hz, Harom.), 8.20 (d, 1H,
J ¼ 8.10 Hz, Harom.), 7.71 (dt, 1H, J1 ¼ 1.68 Hz, J2 ¼ 7.95 Hz, Harom.),
7.47 (d, 1H, J ¼ 7.83 Hz, Harom.), 7.38 (t, 1H, J ¼ 7.49 Hz, Harom.), 7.23
(t, 1H, J ¼ 7.56 Hz, Harom.), 7.10 (t, 1H, J ¼ 7.56 Hz, Harom.); 13C NMR
4.2.4.3. 5-N-i-butylaminobenzimidazo[1,2-a]quinoline-6-
carbonitrile 8. Compound 8 was prepared using above described
method from 5 (0.140 g, 0.50 mmol) and isobutylamine (0.25 mL,
2.50 mmol) after 2 h of irradiation to yield 0.092 g (58%) of yellow
crystals; m.p. 232e234 ꢀC.
(DMSO-d6, 150 MHz):
d
/ppm ¼ 172.34 (s), 153.85 (s), 145.83 (s),
136.33 (s), 131.21 (s), 130.97 (d), 126.39 (d), 124.61 (s), 122.83 (d),
122.56 (d), 120.55 (s), 118.73 (d), 116.36 (d), 114.85 (d), 112.21 (d).
1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.64 (d, 1H, J ¼ 8.40 Hz,
4.2.3. 5-Chlorobenzimidazo[1,2-a]quinoline-6-carbonitrile 5 [16]
A solution of 0.20 g (0.77 mmol) 5-ketobenzimidazo[1,2-a]
quinoline-6-carbonitrile and 0.08 g (0.39 mmol) PCl5 in POCl3
(4 mL) was refluxed for 2 h. After cooling, the reaction mixture was
evaporated under vacuum and dissolved in water (10 ml). Resulting
product was filtered off and washed with water to obtain a yellow
powder (0.15 g, 70%); m.p. >300 ꢀC.
H
arom.), 8.50 (d, 1H, J ¼ 8.28 Hz, Harom.), 8.40 (d, 1H, J ¼ 8.28 Hz,
Harom.), 8.14 (t, 1H, J ¼ 5.94 Hz, NH), 7.91 (t, 1H, J ¼ 7.74 Hz, Harom.),
7.73 (d, 1H, J ¼ 7.98 Hz, Harom.), 7.58 (t, 1H, J ¼ 7.68 Hz, Harom.), 7.40
(t, 1H, J ¼ 7.56 Hz, Harom.), 7.32 (t, 1H, J ¼ 7.71 Hz, Harom), 3.68 (t, 2H,
J ¼ 6.63 Hz, CH2), 2.23e2.15 (m, 1H, CH), 0.99 (d, 6H, J ¼ 6.60 Hz,
CH3); 13C NMR (DMSO-d6, 75 MHz):
d
/ppm ¼ 150.58 (s), 149.49 (s),
145.24 (s), 135.59 (s), 134.81 (s), 133.40 (d), 131.34 (s), 124.84 (d),
124.76 (d), 124.35 (d), 121.76 (d), 118.87 (d), 117.96 (s), 117.13 (s),
116.70 (d), 114.06 (d), 72.44 (s), 51.63 (t), 28.59 (d). 20.04 (q, 2C);
Found: C, 76.45; H, 5.65; N, 17.90. Calc. for C20H18N4: C, 76.41; H,
5.77; N, 17.82%.
1H NMR (DMSO-d6, 300 MHz):
d
/ppm ¼ 8.96 (d, 1H, J ¼ 8.37 Hz,
H
arom.), 8.79 (dd, 1H, J1 ¼ 2.16 Hz, J2 ¼ 6.46 Hz, Harom.), 8.42 (dd, 1H,
J1 ¼ 1.39 Hz, J2 ¼ 8.20 Hz, Harom.), 8.13 (dt, 1H, J1 ¼ 1.52 Hz,
J2 ¼ 7.89 Hz, Harom.), 8.06 (dd, 1H, J1 ¼ 2.18 Hz, J2 ¼ 6.10 Hz, Harom.),
7.81 (t, 1H, J ¼ 7.54 Hz, Harom.), 7.69e7.58 (m, 2H, Harom.); 13C NMR
(DMSO-d6, 150 MHz):
135.04 (d), 131.08 (s), 128.32 (d), 126.18 (d), 125.86 (d), 124.50 (d),
120.91 (d), 119.84 (s), 116.79 (d), 115.28 (d), 113.59 (s), 103.11 (s).
d
/ppm ¼ 144.64 (s), 142.98 (s), 136.17 (s),
4.2.4.4. 5-[N-(N,N-dimethylaminopropyl-1-amino)]benzimidazo[1,2-
a]quinoline-6-carbonitrile 9 [18]. Compound 9 was prepared using
above described method from 5 (0.100 g, 0.36 mmol) and N,N-
dimethylamino-propyl-1-amine (0.23 mL, 1.80 mmol) after 2 h of
irradiation to yield 0.050 g (40%) of yellow powder; m.p. 193e
195 ꢀC.
4.2.4. General method for preparation of compounds 6e19
Compounds 6e19 were prepared using microwave irradiation,
at optimized reaction time at 170 ꢀC with power 800 W and 40 bar
pressure, from compound 5 in acetonitrile (10 mL) with excess of
added corresponding amine. After cooling, the reaction mixture
was filtered off and resulting product was separated by column
1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.65 (d, 1H, J ¼ 8.28 Hz,
H
H
arom.), 8.41 (d, 1H, J ¼ 8.28 Hz, Harom.), 8.37 (d, 1H, J ¼ 8.97 Hz,
arom.), 8.32 (t, 1H, J ¼ 6.24 Hz, NH), 7.91 (t, 1H, J ¼ 7.58 Hz, Harom.),
7.73 (d,1H, J ¼ 7.58 Hz, Harom.), 7.59 (t,1H, J ¼ 7.59 Hz, Harom.), 7.41 (t,

224
N. Perin et al. / European Journal of Medicinal Chemistry 80 (2014) 218e227
1H, J ¼ 7.59 Hz, Harom.), 7.33 (t, 1H, J ¼ 7.59 Hz, Harom.), 3.94 (q, 2H,
J ¼ 6.21 Hz, CH2), 2.94 (t, 2H, J ¼ 6.21 Hz, CH2), 2.60 (s, 6H, CH3), 2.09
2.50 mmol) after 4 h of irradiation to yield 0.027 g (16%) of yellow
powder; m.p. 185e187 ꢀC.
(qvin, 2H, J ¼ 6.90 Hz, CH2); 13C NMR (DMSO-d6, 75 MHz):
d/
1H NMR (DMSO-d6, 300 MHz):
d
/ppm ¼ 8.77 (d, 1H, J ¼ 8.34 Hz,
ppm ¼ 150.50 (s), 149.36 (s), 145.10 (s), 135.48 (s), 133.48 (d), 131.26
(s), 124.85 (d), 124.70 (d), 124.41 (d), 121.85 (d), 118.86 (d), 118.17 (s),
116.97 (s), 116.75 (d), 114.13 (d), 72.14 (s), 56.05 (t), 44.24 (q), 42.87
(t), 25.94 (t); Found: C, 73.62; H, 6.10; N, 20.28. Calc. for C21H21N5: C,
73.44; H, 6.16; N, 20.39%.
H
H
H
H
arom.), 8.59 (d, 1H, J ¼ 7.80 Hz, Harom.), 8.20 (d, 1H, J ¼ 8.10 Hz,
arom.), 7.90 (t, 1H, J ¼ 7.71 Hz, Harom.), 7.88 (d, 1H, J ¼ 7.68 Hz,
arom.), 7.62 (t, 1H, J ¼ 7.61 Hz, Harom.), 7.51 (t, 1H, J ¼ 7.683 Hz,
arom.), 7.45 (t, 1H, J ¼ 7.83 Hz, Harom), 3.61 (t, 4H, J ¼ 6.99 Hz, CH2),
1.72 (m, 4H, CH2), 0.85 (t, 6H, J ¼ 7.26 Hz, CH3); 13C NMR (DMSO-d6,
75 MHz):
d
/ppm ¼ 158.56 (s), 147.30 (s), 144.82 (s), 136.80 (s),
133.62 (d), 131.08 (s), 128.47 (d), 125.08 (d), 125.03 (d), 123.07 (d),
120.93 (s), 119.87 (d), 116.93 (d), 116.93 (d), 116.34 (s), 114.75 (d),
92.54 (s), 55.25 (t, 2C), 21.14 (t, 2C), 11.70 (q, 2C); Found: C, 77.37; H,
6.56; N, 16.07. Calc. for C22H22N4: C, 77.16; H, 6.48; N, 16.36%.
4.2.4.5. 5-[N-(N,N-diethylethylenediamino)]benzimidazo[1,2-a]quin-
oline-6-carbonitrile 10 [18]. Compound 10 was prepared using
above described method from 5 (0.070 g, 0.25 mmol) and N,N-
diethylethylenediamine (0.18 mL,1.30 mmol) after 2 h of irradiation
to yield 0.042 g (47%) of yellow powder; m.p. 240e242 ꢀC.
1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.66 (d, 1H, J ¼ 8.40 Hz,
4.2.4.9. 5-N,N-dipentylaminobenzimidazo[1,2-a]quinoline-6-
carbonitrile 14. Compound 14 was prepared using above described
method from 5 (0.140 g, 0.50 mmol) and dipentylamine (0.52 mL,
2.50 mmol) after 3 h of irradiation to yield 0.048 g (24%) of yellow
powder; m.p. 149e151 ꢀC.
H
H
arom.), 8.41 (d, 1H, J ¼ 8.22 Hz, Harom.), 8.32 (d, 1H, J ¼ 8.10 Hz,
arom.), 7.91 (t, 1H, J ¼ 7.65 Hz, Harom.), 7.84 (s, 1H, NH), 7.74 (d, 1H,
J ¼ 7.92 Hz, Harom.), 7.59 (t, 1H, J ¼ 7.62 Hz, Harom.), 7.41 (t, 1H,
J ¼ 7.53 Hz, Harom.), 7.33 (t, 1H, J ¼ 7.68 Hz, Harom), 3.95 (t, 2H,
J ¼ 7.18 Hz, CH2), 2.82 (t, 2H, J ¼ 6.60 Hz, CH2), 2.57 (q, 4H,
J ¼ 7.02 Hz, CH2), 0.97 (t, 6H, J ¼ 7.05 Hz, CH3); 13C NMR (DMSO-d6,
1H NMR (DMSO-d6, 600 MHz):
d/ppm ¼ 8.79 (d, 1H, J ¼ 8.40 Hz,
H
arom.), 8.59 (d, 1H, J ¼ 8.28 Hz, Harom.), 8.19 (d, 1H, J1 ¼ 0.87 Hz,
J2 ¼ 8.49 Hz, Harom.), 7.95 (t, 1H, J1 ¼ 0.96 Hz, J2 ¼ 7.73 Hz, Harom.),
7.89 (d, 1H, J ¼ 7.86 Hz, Harom.), 7.63 (t, 1H, J ¼ 7.59 Hz, Harom.), 7.52
(t, 1H, J ¼ 7.44 Hz, Harom.), 7.46 (dt, 1H, J1 ¼ 0.95 Hz, J2 ¼ 7.72 Hz,
75 MHz):
d
/ppm ¼ 150.75 (s), 149.33 (s), 145.22 (s), 135.57 (s),
133.43 (d), 131.33 (s), 124.82 (d), 124.47 (d), 124.36 (d), 121.81 (d),
118.90 (d), 117.89 (s), 117.07 (s), 116.77 (d), 114.08 (d), 111.81 (s),
52.24 (t), 47.08 (t, 2C), 42.96 (t), 12.48 (q, 2C); Found: C, 74.02; H,
6.57; N, 19.41. Calc. for C22H23N5: C, 73.92; H, 6.49; N, 19.59%.
H
arom), 3.64 (t, 4H, J ¼ 7.14 Hz, CH2),1.64 (qvin, 4H, J ¼ 6.92 Hz, CH2),
1.29e1.20 (m, 8H), 0.79 (t, 6H, J ¼ 6.93 Hz, CH3); 13C NMR (DMSO-
d6, 75 MHz):
d
/ppm ¼ 158.39 (s), 147.27 (s), 144.91 (s), 144.37 (s),
136.82 (s), 133.58 (d), 131.15 (s), 128.36 (d), 125.02 (d, 2C), 123.08
(d), 121.10 (s), 119.93 (d), 116.87 (d), 116.17 (s), 114.73 (d), 93.52 (s),
53.61 (t, 2C), 28.96 (t, 2C), 27.53 (t, 2C), 22.21 (t, 2C), 14.86 (q, 2C);
Found: C, 78.49; H, 7.63; N, 13.88. Calc. for C26H30N4: C, 78.35; H,
7.59; N, 14.06%.
4.2.4.6. 5-N,N-dimethylaminobenzimidazo[1,2-a]quinoline-6-
carbonitrile 11. Compound 11 was prepared using above described
method from 5 (0.210 g, 0.75 mmol) and dimethylamine (0.76 mL,
3.80 mmol) after 4 h of irradiation to yield 0.090 g (41%) of yellow
powder; m.p. 185e187 ꢀC.
1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.69 (d, 1H, J ¼ 8.34 Hz,
4.2.4.10. 5-N-(3,3ʹ-diaminodipropylamino)benzimidazo[1,2-a]quino-
line-6-carbonitrile 15. Compound 15 was prepared using above
described method from 5 (0.200 g, 0.72 mmol) and 3,3ʹ-dia-
minodipropylamine (0.31 mL, 2.20 mmol) after 3 h of irradiation to
yield 0.200 g (74%) of yellow powder; m.p. 210e214 ꢀC.
H
arom.), 8.50 (d, 1H, J ¼ 8.28 Hz, Harom.), 8.18 (dd, 1H, J1 ¼ 1.11 Hz,
J2 ¼ 8.19 Hz, Harom.), 7.90 (dt, 1H, J1 ¼ 1.23 Hz, J2 ¼ 7.49 Hz, Harom.),
7.83 (d, 1H, J ¼ 7.86 Hz, Harom.), 7.58 (t, 1H, J ¼ 7.40 Hz, Harom.), 7.47
(t, 1H, J ¼ 7.41 Hz, Harom.), 7.41 (dt, 1H, J1 ¼ 1.01 Hz, J2 ¼ 7.73 Hz,
H
arom.), 3.34 (s, 6H, CH3); 13C NMR (DMSO-d6, 75 MHz):
d/
1H NMR (600 MHz, DMSO-d6):
d
/ppm ¼ 8.68 (d, 1H, J ¼ 8.40 Hz,
ppm ¼ 158.83 (s), 147.83 (s), 144.97 (s), 136.59 (s), 133.44 (d), 131.09
(s), 128.88 (d), 124.81 (d), 124.70 (d), 122.75 (d), 119.96 (s), 119.69
(d), 116.64 (d), 116.61 (s), 114.53 (d), 87.83 (s), 44.96 (q, 2C); Found:
C, 75.70; H, 4.82; N, 19.48. Calc. for C18H14N4: C, 75.50; H, 4.93; N,
19.57%.
H
H
H
H
arom.), 8.54 (d, 1H, J ¼ 8.25 Hz, Harom.), 8.43 (d, 1H, J ¼ 8.19 Hz
arom.), 7.93 (t, 1H, J ¼ 7.79 Hz, Harom.), 7.74 (d, 1H, J ¼ 7.71 Hz,
arom.), 7.61 (t, 1H, J ¼ 7.70 Hz, Harom.), 7.41 (t, 1H, J ¼ 7.55 Hz,
arom.), 7.33 (dt, 1H, J ¼ 7.65 Hz, Harom), 4.00 (t, 1H, J ¼ 6.45 Hz, NH),
2.85 (q, 4H, J ¼ 7.02 Hz, CH2), 2.78 (t, 2H, J ¼ 6.60 Hz, CH2), 2.71 (t,
2H, J ¼ 6.76 Hz, CH2), 2.62 (m, 1H, NH), 2.00 (t, 2H, J ¼ 6.60 Hz, NH2),
1.81e1.67 (m, 4H, CH2); 13C NMR (DMSO-d6, 150 MHz):
d/
4.2.4.7. 5-N,N-diethylaminobenzimidazo[1,2-a]quinoline-6-
carbonitrile 12. Compound 12 was prepared using above described
method from 5 (0.210 g, 0.75 mmol) and diethylamine (0.39 mL,
3.80 mmol) after 3 h of irradiation to yield 0.029 g (12%) of yellow
powder; m.p. 160e162 ꢀC.
ppm ¼ 157.49 (s), 150.15 (s), 149.04 (s), 144.77 (s), 135.06 (s), 132.87
(d), 130.86 (s), 124.69 (d), 124.28 (d), 123.84 (d), 121.26 (s), 118.35
(d), 117.59 (d), 116.71 (s), 116.13 (d), 113.54 (d), 46.12 (t), 46.03 (t),
42.56 (t), 37.66 (t), 28.40 (t), 26.83 (t); Found: C, 70.84; H, 6.54; N,
22.62. Calc. for C22H22IN5: C, 70.94; H, 6.44; N, 22.56%.
1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.77 (d, 1H, J ¼ 8.40 Hz,
H
H
H
H
arom.), 8.60 (d, 1H, J ¼ 8.22 Hz, Harom.), 8.20 (d, 1H, J ¼ 8.04 Hz,
arom.), 7.95 (t, 1H, J ¼ 7.68 Hz, Harom.), 7.90 (d, 1H, J ¼ 7.98 Hz,
arom.), 7.61 (t, 1H, J ¼ 7.59 Hz, Harom.), 7.52 (t, 1H, J ¼ 7.53 Hz,
arom.), 7.47 (t, 1H, J ¼ 7.65 Hz, Harom.), 3.67 (q, 4H, J ¼ 7.02 Hz, CH2),
4.2.4.11. 5-N-pyrrolydinylbenzimidazo[1,2-a]quinoline-6-carbonitrile
16. Compound 16 was prepared using above described method
from 5 (0.100 g, 0.36 mmol) and pyrrolidine (0.18 mL, 1.80 mmol)
after 2 h of irradiation to yield 0.070 g (63%) of yellow powder; m.p.
230e231 ꢀC.
1.19 (t, 6H, J ¼ 6.96 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz):
d/
ppm ¼ 157.94 (s), 148.21 (s), 144.85 (s), 136.75 (s), 133.58 (d), 131.13
(s), 128.28 (d), 125.12 (d), 125.03 (d), 123.16 (d), 121.49 (s), 120.00
(d), 116.72 (d), 116.08 (s), 114.75 (d), 113.72 (s), 47.52 (t, 2C), 13.47 (q,
2C); Found: C, 76.50; H, 5.68; N, 17.82. Calc. for C20H18N4: C, 76.41;
H, 5.77; N, 17.82%.
1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.60 (d, 1H, J ¼ 8.28 Hz,
H
H
H
H
arom.), 8.40 (d, 1H, J ¼ 8.10 Hz, Harom.), 8.30 (d, 1H, J ¼ 8.010 Hz,
arom.), 7.87 (t, 1H, J ¼ 7.62 Hz, Harom.), 7.75 (d, 1H, J ¼ 7.86 Hz,
arom.), 7.51 (t, 1H, J ¼ 7.62 Hz, Harom.), 7.42 (t, 1H, J ¼ 7.50 Hz,
arom.), 7.35 (t, 1H, J ¼ 7.26 Hz, Harom.), 4.02 (t, 4H, J ¼ 5.62 Hz, CH2),
4.2.4.8. 5-N,N-dipropylaminobenzimidazo[1,2-a]quinoline-6-
carbonitrile 13. Compound 13 was prepared using above described
method from 5 (0.140 g, 0.50 mmol) and dipropylamine (0.35 mL,
2.00 (t, 4H, J ¼ 6.37 Hz, CH2); 13C NMR (DMSO-d6, 150 MHz):
d/
ppm ¼ 154.80 (s), 149.82 (s), 145.47 (s), 135.93 (s), 132.88 (d), 131.13
(s), 128.96 (d), 124.44 (d), 123.93 (d), 121.87 (d), 119.22 (s), 119.06

N. Perin et al. / European Journal of Medicinal Chemistry 80 (2014) 218e227
225
(d), 118.13 (s), 116.16 (d), 114.08 (d), 78.47 (s), 54.67 (t, 2C), 25.72 (t,
2C); Found: C, 77.05; H, 5.30; N, 17.65. Calc. for C20H16N4: C, 76.90;
H, 5.16; N, 17.94%.
1H NMR (DMSO-d6, 300 MHz):
d/ppm ¼ 8.84 (d, 1H, J ¼ 8.40 Hz,
H
arom.), 8.65 (d, 1H, J ¼ 7.31 Hz, Harom.), 8.26 (dd, 1H, J1 ¼ 0.98 Hz,
J2 ¼ 8.15 Hz, Harom.), 8.03 (dt, 1H, J1 ¼ 1.02 Hz, J2 ¼ 7.85 Hz, Harom.),
7.95 (dd,1H, J1 ¼1.25 Hz, J2 ¼ 7.85 Hz, Harom.), 7.68 (t,1H, J ¼ 7.67 Hz,
H
arom.), 7.57 (dt, 1H, J1 ¼ 0.92 Hz, J2 ¼ 7.35 Hz, Harom.), 7.52 (dt, 1H,
4.2.4.12. 5-N-piperydinylbenzimidazo[1,2-a]quinoline-6-carbonitrile
17 [16]. Compound 17 was prepared using above described method
from 5 (0.200 g, 0.72 mmol) and piperidine (0.36 mL, 3.60 mmol)
after 2 h of irradiation to yield 0.070 g (30%) of yellow powder; m.p.
238e240 ꢀC.
J1 ¼1.34 Hz, J2 ¼ 7.49 Hz, Harom.), 4.00 (t, 4H, J ¼ 4.27 Hz, CH2), 3.80
(t, 4H, J ¼ 4.35 Hz, CH2), 3.32 (s, 6H, CH3); 13C NMR (DMSO-d6,
75 MHz):
d
/ppm ¼ 156.43 (s), 146.81 (s), 144.85 (s), 136.71 (s),
134.04 (d), 131.06 (s), 128.22 (d), 125.31 (d), 125.24 (d), 123.49 (d),
120.18 (d), 119.72 (s), 116.83 (d), 115.95 (s), 114.87 (d), 61.46 (t, 2C),
51.67 (q, 2C), 46.08 (t, 2C); Found: C, 54.55; H, 4.68; N, 14.52. Calc.
for C22H22IN5: C, 54.67; H, 4.59; N, 14.49%.
1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.73 (d, 1H, J ¼ 8.49 Hz,
H
arom.), 8.55 (d, 1H, J ¼ 8.34 Hz, Harom.), 8.13 (dd, 1H, J1 ¼ 1.44 Hz,
J2 ¼ 8.22 Hz, Harom.), 7.93 (dt, 1H, J1 ¼ 1.44 Hz, J2 ¼ 7.14 Hz, Harom.),
7.86 (d, 1H, J ¼ 8.10 Hz, Harom.), 7.62 (dt, 1H, J1 ¼ 0.84 Hz,
J2 ¼ 8.05 Hz, Harom.), 7.50 (dt, 1H, J1 ¼ 0.90 Hz, J2 ¼ 8.04 Hz, Harom.),
7.44 (dt, 1H, J1 ¼ 1.29 Hz, J2 ¼ 8.25 Hz, Harom.), 3.59 (t, 4H,
J ¼ 5.28 Hz, CH2), 1.83 (m, 4H, CH2), 1.73 (m, 2H, CH2); 13C NMR
4.3. Antiproliferative evaluation assay
The experiments were carried out on three human cell lines,
which are derived from three cancer types: HCT 116 (colon carci-
noma), H 460 (lung carcinoma) and MCF-7 (breast carcinoma). The
cells were cultured as monolayers and maintained in Dulbecco’s
modified Eagle medium(DMEM) supplemented with10%fetalbovine
(DMSO-d6, 75 MHz): (
d
/ppm) ¼ 158.02 (s), 147.06 (s), 144.29 (s),
136.01 (s), 133.02 (d), 130.49 (s), 127.38 (d), 124.54 (d), 124.90 (d),
122.44 (d), 119.61 (s), 119.29 (d), 116.23 (d), 116.03 (s), 114.16 (d),
88.52 (s), 53.61 (s, 2C), 26.01 (s, 2C), 23.55 (s); Found: C, 77.45; H,
5.58; N, 16.97. Calc. for C21H18N4: C, 77.28; H, 5.56; N, 17.17%.
serum (FBS), 2 mM
L-glutamine, 100 U/ml penicillin and 100 mg/ml
streptomycin in a humidified atmosphere with 5% CO2 at 37 ꢀC.
The growth inhibition activity was assessed as described previ-
ously [21]. The cell lines were inoculated onto a series of standard 96-
well microtiter plates on day 0, at 3 ꢃ104 cells/mL (HCT 116, H 460) to
5 ꢃ 104 cells/mL (MCF-7), depending on the doubling times of a
specific cell line. Test agents were then added in ten-fold dilutions
(10ꢄ8 to 10ꢄ4 M) and incubated for further 72 h. Working dilutions
were freshly prepared on the day of testing. After 72 h of incubation
the cell growth rate was evaluated by performing the MTT assay,
which detects dehydrogenase activity in viable cells. The absorbance
(A) was measured on a microplate reader at 570 nm. The absorbance
is directly proportional to the number of living, metabolically active
cells. The percentage of growth (PG) of the cell lines was calculated
according to one or the other of the following two expressions:
4.2.4.13. 5-N-morpholinylbenzimidazo[1,2-a]quinoline-6-
carbonitrile 18. Compound 18 was prepared using above described
method from 5 (0.050 g, 0.18 mmol) and morpholine (0.08 mL,
0.90 mmol) after 3 h of irradiation to yield 0.041 g (69%) of yellow
crystals; m.p. >300 ꢀC.
1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.79 (d, 1H, J ¼ 8.34 Hz,
H
H
H
H
arom.), 8.61 (d, 1H, J ¼ 8.28 Hz, Harom.), 8.24 (d, 1H, J ¼ 8.40 Hz,
arom.), 7.97 (t, 1H, J ¼ 7.77 Hz, Harom.), 7.91 (d, 1H, J ¼ 8.04 Hz,
arom.), 7.64 (t, 1H, J ¼ 7.62 Hz, Harom.), 7.53 (t, 1H, J ¼ 7.32 Hz,
arom.), 7.48 (t, 1H, J ¼ 7.29 Hz, Harom.), 3.92 (t, 4H, J ¼ 4.32 Hz, CH2),
3.65 (t, 4H, J ¼ 4.35 Hz, CH2); 13C NMR (DMSO-d6, 75 MHz):
d/
ppm ¼ 157.60 (s), 151.07 (s), 144.90 (s), 136.53 (s), 133.85 (d), 131.16
(s),128.16 (d),125.19 (d),125.04 (d),123.17 (d),120.00 (d),119.94 (s),
116.82 (d), 116.29 (s), 114.75 (d), 93.51 (s), 67.00 (t, 2C), 53.13 (t, 2C);
Found: C, 73.34; H, 4.93; N, 16.84. Calc. for C20H16N4O (326.3): C,
73.15; H, 4.91; N, 17.06%.
Ifðmean Atest ꢄmean AtzeroÞ ꢂ 0; then PG
¼ 100ꢃðmean Atest ꢄmean AtzeroÞ ðmean A ꢄmean AtzeroÞ:
=
ctrl
If (mean Atest ꢄ mean Atzero) < 0, then: PG ¼ 100 ꢃ (mean
Atest ꢄ mean Atzero)/Atzero, where the mean Atzero is the average of
optical density measurements before exposure of cells to the test
compound, the mean Atest is the average of optical density mea-
surements after the desired period of time and the mean Actrl is the
average of optical density measurements after the desired period of
time with no exposure of cells to the test compound. The results are
expressed as IC50, which is the concentration necessary for 50% of
inhibition. The IC50 values for each compound are calculated from
concentrationeresponse curves using linear regression analysis by
fitting the test concentrations that give PG values above and below
the reference value (i.e. 50%). If however, for all of the tested con-
centrations produce PGs exceeding the respective reference level of
effect (e.g. PG value of 50), then the highest tested concentration is
assigned as the default value, which is preceded by a “>” sign.
Minimum two individual experiments were carried out and each
test point was performed in quadruplicate.
4.2.4.14. 5-N-piperazinylbenzimidazo[1,2-a]quinoline-6-carbonitrile
19. Compound 19 was prepared using the above method from 5
(0.140 g, 0.50 mmol) and piperazine (0.260 g, 3.00 mmol) after 8 h
of irradiation to yield 0.044 g (27%) of yellow powder; m.p. 243e
245 ꢀC.
1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 8.76 (d, 1H, J ¼ 8.34 Hz,
Harom.), 8.59 (d, 1H, J ¼ 8.16 Hz, Harom.), 8.20 (d, 1H, J ¼ 8.04 Hz,
H
H
H
arom.), 7.96 (t, 1H, J ¼ 7.54 Hz, Harom.), 7.90 (d, 1H, J ¼ 7.86 Hz,
arom.), 7.63 (t, 1H, J ¼ 7.53 Hz, Harom.), 7.52 (t, 1H, J ¼ 7.41 Hz,
arom.), 7.47 (t, 1H, J ¼ 7.50 Hz, Harom.), 3.66 (t, 4H, J ¼ 4.35 Hz, CH2),
3.22 (t, 4H, J ¼ 4.55 Hz, CH2); 13C NMR (DMSO-d6, 75 MHz):
d/
ppm ¼ 160.13 (s), 157.71 (s), 144.88 (s), 136.68 (s), 133.79 (d), 131.09
(s), 128.04 (d), 125.24 (d), 125.05 (d), 123.23 (d), 120.12 (d), 116.75
(d), 116.15 (s), 114.73 (d), 90.97 (s), 52.06 (t, 2C), 45.42 (t, 2C);
Found: C, 73.28; H, 5.16; N, 21.56. Calc. for C20H19N5: C, 73.37; H,
5.23; N, 21.34%.
4.2.5. 5-N-(4-N,N-dimethylpiperazin-1-yl)benzimidazo[1,2-a]
quinoline-6-carbonitrile iodide 20
4.4. DNA binding experiments
A mixture of compound 19 (0.077 g, 0.23 mmol) and anhydrous
potassium carbonate (0.033 g, 0.23 mmol) was refluxed in aceto-
nitrile (25 mL) with methyl iodide (0.060 mL, 0.96 mmol) for 2 h.
The reaction mixture was concentrated under reduced pressure to a
volume of 5 mL and filtered off to yield pure compound 20 as
yellow powder (0.063 g, 55%); m.p. >300 ꢀC.
The tested compounds were dissolved in DMSO as 5 or 10 mM
stock solutions. CT-DNA (Sigma Aldrich, France) was prepared in
water and dialyzed overnight. Both were aliquoted and stored
at ꢄ20 ꢀC to then be freshly diluted in the appropriate aqueous
buffer.

226
N. Perin et al. / European Journal of Medicinal Chemistry 80 (2014) 218e227
4.4.1. DNA melting temperature
MitoFluor Red 588 (Mito-Red, Molecular Probes, Invitrogen) as a
cytoplasmic marker for 30 min at 37 ꢀC. After three washing of the
cells using 500 l of PBS, 2% of paraformalydehyde was added in
PBS for 15 min and cells were washed again twice and dried. The
slide was mounted on Vectashield (CliniSciences) and analyzed
with an apotome-equipped fluorescence microscope (Zeiss) with
40ꢃ or 63ꢃ immersion lens. Images were collected using AxioVi-
zion software.
CT-DNA (20 mM) was incubated or not with 10 or 20 mM of the
various tested compounds (R ¼ drug/base pair ratio of 0.5 or 1) in
1 mL BPE buffer (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA, pH
7.1). The absorbency at 260 nm was measured in quartz cells using
an Uvikon XL spectrophotometer thermostated with a peltier
cryostat every min over a range of 20e100 ꢀC with an increment of
1 ꢀC per min. The Tm values were deduced from the midpoint of the
hyperchromic transition obtained from first-derivative plots. The
m
variation of melting temperature (DTm) were obtained by sub-
Acknowledgments
tracting the melting temperature measurement of CT-DNA alone
(control Tm) to that obtained with DNA incubated with the com-
We greatly appreciate the financial support of the Croatian
Ministry of Science Education and Sports (Projects 125-0982464-
1356, 098-0982464-2514) and the bilateral Hubert Curien part-
nership between Croatian and French institutions (Cogito program)
as the Egide Project No. 24765PH. M. H. David-Cordonnier is
grateful to the Ligue Nationale Contre le Cancer (Comité du Nord,
Septentrion), the Association pour la Recherche sur le Cancer for
grants; the Institut pour la Recherche sur le Cancer de Lille (IRCL),
the CHRU de Lille and the Région Nord/Pas-de-Calais for a PhD
fellowship to Raja Nhili and the IRCL for technical expertise (Sabine
Depauw) and post-doctoral fellowship (Raja Nhili). M. H. David-
Cordonnier also thanks the IMPRT-IFR114 for giving access to the
apotome-equipped fluorescence microscope.
pounds (
D
Tm values ¼ Tm[compoundþDNA] ꢄ Tm[DNA alone]).
4.4.2. UV/Visible spectroscopy
The UV/Visible spectra were obtained in a quartz cuvette of
10 mm pathlength containing compounds 9, 19 and 20 (20 mM)
diluted in 1 mL of BPE buffer in the absence or presence of
increasing concentrations of CT-DNA (1, 2, 4, 6, 8, 10, 20, 30, 40, 50,
60, 80, 100, 120, 140
mM). Due to precipitation at higher DNA/drug
ratio, spectra were only measured up to 80
mM of 9. Each spectrum
was recorded from 240 nm to 480 nm using an Uvikon XL spec-
trophotometer and referenced against a cuvette containing DNA at
identical concentration.
4.4.3. Fluorescence spectroscopy
Appendix A. Supplementary data
Fluorescence spectra were recorded from 400 to 700 nm
essentially as described [23]. The fluorescent drugs (10
diluted in 1 mL of BPE buffer in the presence or absence of
increasing concentrations of CT-DNA (from 0.5 to 80 M for 9 and
1e200 M for 19 and 20). Excitation wavelengths were 350 nm for
mM) were
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.049.
m
m
References
compound 9 and 360 nm for compounds 19 and 20. The quenching
constant Ksv was deduced from SterneVolmer method where the
ratio of fluorescence of the compound alone (F0) over the fluores-
cence of the compound in the presence of CT-DNA (F) is presented
as a function of CT-DNA concentration. In this configuration, F0/
F ¼ 1 þ Ksv[CT-DNA]. The slope Ksv is considered as an equilibrium
constant for the static quenching process.
[1] R.B. Silverman, The Organic Chemistry of Drug Design and Drug Action, sec-
ond ed, Elsevier Academic Press, 2004.
[2] C. Bailly, Topoisomerase I poisons and suppressors as anticancer drugs, Cur-
rent Medicinal Chemistry 7 (2000) 39e58.
[3] Y. Bansal, O. Silakari, The therapeutic journey of benzimidazoles: a review,
Bioorganic & Medicinal Chemistry 20 (2012) 6208e6236.
ꢀ
ꢁ
ꢁ
[4] a) M. Hranjec, M. Kralj, I. Piantanida, M. Sedic, L. Suman, K. Pavelic,
G. Karminski-Zamola, Novel cyano- and amidino-substituted derivatives of
styryl-2-benzimidazoles and benzimidazo[1,2-a]quinolines. Synthesis,
photochemical synthesis, DNA binding and antitumor evaluation, Part 3,
Journal of Medicinal Chemistry 50 (2007) 5696e5711;
4.4.4. Circular dichroism
For circular dichroism, the various drugs (50
bated with or without (control) a fixed or increasing concentrations
of CT-DNA (from 0.1 to 100 M) in BPE. The CD spectra were
mM) were incu-
ꢀ
ꢁ
b) M. Hranjec, I. Piantanida, M. Kralj, L. Suman, K. Pavelic, G. Karminski-
Zamola, Novel amidino-substituted thienyl- and furyl-vinyl-benzimidazole
derivatives and their photochemical conversion into corresponding diaza-
cyclopenta[c]fluorenes. Synthesis, interactions with DNA and RNA and anti-
tumor evaluation, Part 4, Journal of Medicinal Chemistry 51 (2008) 4899e
4910.
m
collected in a quartz cell of 10 mm pathlength from 480 to 230 nm
using a J-810 Jasco spectropolarimeter at a controlled temperature
of 20 ꢀC fixed by a PTC-424S/L peltier type cell changer (Jasco) as
described previously [23].
[5] a) H. Göker, S. Özden, S. Yıldız, D.W. Boykin, Synthesis and potent antibacterial
activity against MRSA of some novel 1,2-disubstituted-1H-benzimidazole-N-
alkylated-5-carboxamidines, European Journal of Medicinal Chemistry 40
(2005) 1062e1069;
4.4.5. Topoisomerase I e mediated DNA relaxation
DNA intercalation evaluated by using pUC19 supercoiled
plasmid DNA and human topoisomerase I (Topogen, USA) as pre-
viously described [24].
b) Z. Ates-Alagoz, S. Yildiz, E. Buyukbingol, Antimicrobial activities of some
tetrahydronaphthalene-benzimidazole derivatives, Chemotherapy 53 (2007)
110e113.
[6] H.M. Grogan, Fungicide control of mushroom cobweb disease caused by Cla-
dobotryum strains with different benzimidazole resistance profiles, Pest
Management Science 62 (2006) 153e161.
ꢀ
ꢁ
ꢁ
[7] K. Starcevic, M. Kralj, K. Ester, I. Sabol, M. Grce, K. Pavelic, G. Karminski-
Zamola, Synthesis, antiviral and antitumor activity of 2-substituted-5-
4.4.6. DNase I footprinting
DNase I footprinting experiments were conducted essentially as
described [25]. The gels were exposed to storage screen for the
appropriated delay at room temperature. The results were collected
using a Pharos-PMI equipment (BioRad).
amidino-benzimidazoles, Bioorganic
4419e4426.
& Medicinal Chemistry 15 (2007)
[8] J. Velík, V. Baliharová, J. Fink-Gremmels, S. Bull, J. Lamka, L. Skálová, Benz-
imidazole drugs and modulation of biotransformation enzymes, Research in
Veterinary Science 76 (2004) 95e108.
[9] J. Jeong, J. Yoon, Recent progress on fluorescent chemosensors for metal ions,
Inorganica Chimica Acta 381 (2012) 2e14.
4.5. Cellular distribution
[10] M. Demeunynck, C. Bailly, W.D. Wilson, DNA and RNA Binders, Wiley-VCH,
Weinheim, 2002.
ꢁ
[11] a) N. Perin, M. Hranjec, G. Pavlovic, G. Karminski-Zamola, Novel aminated
HT-29 colon carcinoma cells (30 000 cells) were plated on Lab-
Tek glass culture chambers (Nunc) for 24 h prior to the addition of
benzimidazo[1,2-a]quinolines as potential fluorescent probes for DNA
detection: microwave-assisted synthesis, spectroscopic characterization and
crystal structure determination, Dyes and Pigments 91 (2011) 79e88;
the various tested compounds (5
mM) for 16 h. Cells were then
ꢁ
ꢁ
carefully washed twice with PBS prior to the addition of 250 nM of
b) M. Hranjec, G. Pavlovic, M. Marjanovic, M. Kralj, G. Karminski-Zamola,

N. Perin et al. / European Journal of Medicinal Chemistry 80 (2014) 218e227
227
Benzimidazole derivatives related to 2,3-acrylonitriles, benzimidazo[1,2-a]
quinolines and fluorenes: synthesis, antitumor evaluation in vitro and crystal
structure determination, European Journal of Medicinal Chemistry 45 (2010)
2405e2417.
[1,2-c]quinazolines, European Journal of Medicinal Chemistry 44 (2009)
3305e3312.
ꢁ
ꢁ
[20] M. Hranjec, G. Pavlovic, M. Marjanovic, M. Kralj, G. Karminski-Zamola,
Benzimidazole derivatives related to 2,3-acrylonitriles, benzimidazo[1,2-a]
quinolines and fluorenes: synthesis, antitumor evaluation in vitro and crystal
structure determination, European Journal of Medicinal Chemistry 45 (2010)
2405e2417.
[21] N. Perin, L. Uzelac, I. Piantanida, G. Karminski-Zamola, M. Kralj, M. Hranjec,
Novel biologically active nitro and amino substituted benzimidazo[1,2-a]
quinolines, Bioorganic & Medicinal Chemistry 19 (2011) 6329e6339.
[22] N. Perin, I. Martin-Kleiner, R. Nhili, W. Laine, M.H. David-Cordonnier,
O. Vugrek, G. Karminski-Zamola, M. Kralj, M. Hranjec, Biological activity and
DNA binding studies of 2-substituted benzimidazo[1,2-a]quinolines bearing
different amino side chains, Medicinal Chemical Communications 4 (2013)
1537e1550.
[12] V.B. Kovalska, D.V. Kryvorotenko, A.O. Balanda, M.Y. Losytskyy, V.P. Tokar,
S.M. Yarmoluk, Fluorescent homodimer styrylcyanines: synthesis and spectral
luminescent studies in nucleic acids and protein complex, Dyes and Pigments
67 (2005) 47e54.
[13] M.F. Brana, J.M. Castellano, G. Keilhauer, A. Machuca, Y. Martín, C. Redondo,
E. Schlick, N. Walker, Benzimidazo[1,2-c]quinazolines: a new class of anti-
tumor compounds, Anti-Cancer Drug Design 9 (1994) 527e538.
[14] L.W. Deady, T. Rodemann, G.J. Finlay, B.C. Baguley, W.A. Denny, Synthesis and
cytotoxic activity of carboxamide derivatives of benzimidazo[2,1-a]iso-
quinoline and pyrido[3’,2’:4,5]imidazo[2,1-a]isoquinoline, Anti-Cancer Drug
Design 15 (2000) 339e346.
[15] C. Lamazzi, S. Léonce, B. Pfeiffer, P. Renard, G. Guillaumet, C.W. Rees, T. Besson,
Expeditious synthesis and cytotoxic activity of new cyanoindolo[3,2-c]quin-
olines and benzimidazo[1,2-c]quinazolines, Bioorganic & Medicinal Chemistry
Letters 10 (2000) 2183e2185.
[16] Y.M. Volovenko, A.G. Nemazanyi, G.L. Veselskaya, S.I. Tyukhtenkoi,
F.S. Babichev, Condensed benzimidazo[1,2-a]azines with a bridgehead nitro-
gen atom, Geol. Khim. Biolog. Nauki 12 (1986) 29e32.
[17] V.L. Zima, O.M. Dyachok, N.S. Miroshnichenko, Fluorescence characteristics of
5-chloro-6-cyanobenzimidazo[1,2-a]quinoline in different solvents and dur-
ing binding with proteins, Geol. Khim. Biolog. Nauki 11 (1990) 2971e2973.
[18] F.S. Babichev, V.K. Patratii, Y.M. Volovenko, N.G. Prodanchuk, V.G. Sinchenko,
A.G. Nemazanyi, T.A. Silaeva, Antimicrobial activity of benzimidazolo[1,2-a]
quinoline derivatives, Khimiko Farmatsevticheskii Zhurnal 7 (1989) 834e836.
[19] E.A. Lyakhova, Y.A. Gusyeva, J.V. Nekhoroshkova, L.M. Shafran, S.A. Lyakhov,
Synthesis and affinity to DNA of phenylbenzoimidazoles and benzoimidazo
[23] T. Lemster, U. Pindur, S. Depauw, G. Lenglet, C. Dassi, M.H. David-Cordonnier,
Photochemical electrocyclisation of 3-vinylindoles to pyrido[2,3-a]-, pyrido
[4,3-a]- and thieno[2,3-a]-carbazoles: design, synthesis, DNA-binding and
antitumor cell cytotoxicity, European Journal of Medicinal Chemistry 44
(2009) 3235e3252.
[24] P. Peixoto, C. Bailly, M.H. David-Cordonnier, Topoisomerase I-mediated DNA
relaxation as a tool to study intercalation of small molecules into supercoiled
DNA, Methods in Molecular biology. [Methods in Molecular Biology (clifton,
N.J.)] 613 (2010) 235e256.
[25] P. Peixoto, Y. Liu, S. Depauw, M.P. Hildebrand, D.W. Boykin, C. Bailly,
W.D. Wilson, M.H. David-Cordonnier, Direct inhibition of the DNA-binding
activity of POU transcription factors Pit-1 and Brn-3 by selective binding of
a phenyl-furan-benzimidazole dication, Nucleic Acids Research 36 (2008)
3341e3353.