Biological activity and DNA binding studies of 2-substituted benzimidazo[1,2-a]quinolines bearing different amino side chains

Article abstract of DOI:10.1039/c3md00193h

This manuscript describes the synthesis and biological activity of 2-substituted benzimidazo[1,2-a]quinolines substituted with different amino side chains on the quinoline nucleus prepared by microwave assisted amination. The majority of compounds were newly synthesized and active at submicromolar IC ₅₀ concentrations, while the alkylamino substituents, either acyclic or cyclic, increased antitumor activities in comparison with previously published nitro and amino substituted benzimidazo[1,2-a]quinolines. The compound with the longest tertiary amino side chain (16) was the least active. A series of additional experiments, including DNA binding propensities, topoisomerases I and II inhibition, inhibition of recombinant green fluorescent protein in a cell-free translation system, cell cycle perturbances and cellular localization, was performed to shed more light on the mechanisms of action of the most active compounds. The DNA intercalation activity correlates with anti-proliferative effect. Several DNA intercalators (11, 20 and 21) also evidence some sequence selective DNA binding. However, only N,N-dimethylaminopropyl analogue 11 was unequivocally demonstrated to be a strong DNA-binder and intercalative agent, which efficiently targets DNA in the cells, while the activity of compound 10, with a bulky i-butylamino side chain, points to its potential antimitotic activity.

Full text of DOI:10.1039/c3md00193h

MedChemComm
View Article Online
View Journal | View Issue
CONCISE ARTICLE
Biological activity and DNA binding studies of 2-
substituted benzimidazo[1,2-a]quinolines bearing
different amino side chains
Cite this: Med. Chem. Commun., 2013,
4, 1537
a
b
c
c
ˇ
´ `
Natasa Perin, Irena Martin-Kleiner, Raja Nhili, William Laine, Marie-Helene David-
c
b
a
b
*
Cordonnier, Oliver Vugrek, Grace Karminski-Zamola, Marijeta Kralj
and Marijana Hranjec
a
*
This manuscript describes the synthesis and biological activity of 2-substituted benzimidazo[1,2-a]-
quinolines substituted with different amino side chains on the quinoline nucleus prepared by
microwave assisted amination. The majority of compounds were newly synthesized and active at
submicromolar IC50 concentrations, while the alkylamino substituents, either acyclic or cyclic, increased
antitumor activities in comparison with previously published nitro and amino substituted benzimidazo
[1,2-a]quinolines. The compound with the longest tertiary amino side chain (16) was the least active. A
series of additional experiments, including DNA binding propensities, topoisomerases I and II inhibition,
inhibition of recombinant green fluorescent protein in
a cell-free translation system, cell cycle
perturbances and cellular localization, was performed to shed more light on the mechanisms of action
of the most active compounds. The DNA intercalation activity correlates with anti-proliferative effect.
Several DNA intercalators (11, 20 and 21) also evidence some sequence selective DNA binding.
However, only N,N-dimethylaminopropyl analogue 11 was unequivocally demonstrated to be a strong
DNA-binder and intercalative agent, which efficiently targets DNA in the cells, while the activity of
compound 10, with a bulky i-butylamino side chain, points to its potential antimitotic activity.
Received 10th July 2013
Accepted 18th September 2013
DOI: 10.1039/c3md00193h
www.rsc.org/medchemcomm
development of novel, more selective anticancer agents.8 Ben-
zannulated benzimidazoles usually possess a highly conju-
Introduction
The permanent and growing interest in the synthesis of ben-
zannulated benzimidazole derivatives, one of the most exten-
sively studied classes of heterocyclic compounds, is a direct
consequence of their diverse biological properties.1–4 Since
benzimidazole exhibits structural similarity with some natu-
rally occurring compounds, their derivatives play a crucial role
in the function of some biologically important molecules or can
easily interact with biomolecules like DNA, RNA or different
proteins of the living systems.5,6 DNA still represents one of the
principal targets in drug development strategies designed to
produce novel therapeutics for diseases such as cancer.7 An
understanding of the molecular basis of interactions of small
heteroaromatic organic molecules with DNA is a promising
approach, which is of utmost importance in the rational
gated, planar chromophore, which imparts them the ability to
intercalate between adjacent DNA base pairs.9–11 Due to high
uorescence intensity and excellent spectroscopic characteris-
tics, fused benzimidazoles offer a potential application as
uorescent probes for detection of biologically important
molecules such as DNA or proteins in biomedical diagnos-
tics.12–14 Recently, as part of our continuous scientic research
in the eld of potential biologically active benzimidazoles, we
have reported on the synthesis and biological activity of several
groups of benzimidazo[1,2-a]quinolines, including positively
charged amidino-substituted benzimidazo[1,2-a]quinolines
and their heteroaromatic analogues as well as versatile benzi-
midazo[1,2-a]quinolines-6-carbonitriles.2,3,15 Biological studies
conrmed the anticancer potential of this class of compounds,
especially that of positively charged amidino-substituted
analogues of benzimidazo[1,2-a]quinolines, which intercalate
into double-stranded DNA or RNA. The most active one,
2-imidazolinyl-substituted benzimidazo[1,2-a]quinoline with
pronounced selectivity towards colon carcinoma cells, inhibi-
ted topoisomerase II and induced strong G2/M cell cycle
arrest. On the other hand, uncharged amino- and positively
charged diamino-substituted benzimidazo[1,2-a]quinolines
aDepartment of Organic Chemistry, Faculty of Chemical Engineering and Technology,
´
University of Zagreb, Marulicev trg 20, P. O. Box 177, HR-10000 Zagreb, Croatia.
E-mail: mhranjec@it.hr; Fax: +385 14597250; Tel: +385 14597245
b
ˇ
´
ˇ
Division of Molecular Medicine, Rudꢀer Boskovic Institute, Bijenicka cesta 54, P. O. Box
180, HR-10000 Zagreb, Croatia. E-mail: marijeta.kralj@irb.hr; Fax: +385 1 4561 010;
Tel: +385 1 4571 235
cINSERM U837, Jean-Pierre Aubert Research Centre (JPARC), Team “Molecular and
Cellular Targeting for Cancer Treatment”, Institut pour la Recherche sur le Cancer
´
de Lille, Universite Lille 2, IMPRT-IFR-114, France
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 1537–1550 | 1537

View Article Online
MedChemComm
Concise Article
6-(1-Piperidyl)-ethylamino-substituted derivative with longest
side chain showed the highest activity against human osteo-
genic sarcoma MG-63 in nanomolar concentration, IC50 30 nM
(Fig. 1). DNA-binding study reveals intercalation of this near-
planar chromophore.
Prompted by the results from those previously reported
studies, we set out to explore and synthesize 2-substituted
benzimidazole[1,2-a]quinolines with different amino substitu-
ents on the quinoline nucleus of the condensed tetracyclic ring.
All synthesized compounds were tested for their antiproliferative
activity in vitro against a panel of several human tumor cell lines
while compounds 10, 11, 20 and 21 were chosen for additional
biological studies. A DNA-binding study as well as top-
oisomerase I and II inhibition was performed for compounds 10,
11, 16 and 18–21.
´
Fig. 1 Amino substituted benzimidazo[1,2-c]quinazolines prepared by Brana
et al.17
weakly interact with DNA by partial intercalating or agglomer-
ating along the DNA double helix.16b A uorescence microscopy
study showed cytoplasmic distribution of the compounds,
demonstrating that DNA is not their primary target. Benzimi-
dazo[1,2-a]quinolines, substituted with piperidine, pyrrolidine
and piperazine nuclei were prepared in order to study their
spectroscopic properties, especially uorescence intensity in
the presence of CT-DNA. Results revealed signicantly
enhanced uorescence emission and thus offer potential
applications of those compounds as DNA-specic uorescent
probes.13 Another group has published the synthesis of amino-
substituted benzimidazo[1,2-c]quinazolines with different
lengths of amino side chains designed from comparison with
other intercalator patterns.16 All tested compounds showed
high cytotoxic activities against several human tumor cell lines.
Results and discussion
Chemistry
All prepared 2-substituted benzimidazo[1,2-a]quinolines 8–21
with different amino side chains were synthesized by a micro-
wave assisted amination reaction according to the main
synthetic procedure shown in Scheme 1.
Fused starting precursors 6 and 7 were prepared using a
previously described method, by
a photochemical dehy-
drohalogenation of non-fused E-2-(2-benzimidazolyl)-3-(4-hal-
ophenyl)acrylonitriles 4 and 5, which were prepared by the
conventional aldol condensation of 2-cyanomethylbenzimida-
zole 3 with 4-halobenzaldehydes 1 and 2.3,14 Amino-substituted
derivatives 8–20 were prepared in moderate to high yield
Scheme 1 Synthesis of amino substituted benzimidazo[1,2-a]quinolines 8–21.
1538 | Med. Chem. Commun., 2013, 4, 1537–1550
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Concise Article
MedChemComm
33–99%. Based on the series of experiments which were interactions of specic protons of both methyl groups, which
undertaken in order to optimize the reaction times and yields, showed only mutual NOE interaction as well as interactions
the amination reactions were conducted using 800 W power at with protons of the piperazine nucleus. Introduction of the N,N-
170 ꢀC in acetonitrile with a ve to sevenfold excess of the dimethylated piperazine nucleus as an iodide salt in compound
amine.14 Among all prepared compounds, three compounds 21 caused a downeld shi of the all aromatic protons in
were previously prepared in our scientic group,14 while one comparison to other amino-substituted derivatives 8–20.
compound was previously prepared by another group of
authors.18 The structures of all prepared compounds were
determined by NMR analysis based on H–H coupling constants
Biological results and discussion
The tested compounds showed different, but mostly prominent
antiproliferative effects on the tested panel cell lines. The length
and the type of amino side chains signicantly affected anti-
proliferative activity. Compound 8, which has the shortest
secondary methylamino side chain, showed modest activity, but
pronounced selectivity to MCF-7 cells. Although long lipophilic
alkyl substituents play an important role on cell penetration
and distribution, unexpectedly the compound with the longest
tertiary amino side chain (16) is the least active compound,
which might be due to its lower water solubility caused by its
high lipophilicity (log P ¼ 8.33, calculated by ChemDraw 6
soware), or steric hindrance. All other compounds have IC50
concentrations in the micromolar (9, 14 and 18), or sub-
micromolar range. Still, it should be pointed out that
compound 9 formed precipitates, which severely obstructed the
MTT assay, therefore, these results should not be correlated
with others (Table 2).
Structure–activity relationship (SAR) studies indicated that
a secondary amine chain is generally preferred over a tertiary
amine chain, although N,N-dipropyl-substituted compound 15
showed prominent antiproliferative activity against HCT116
and H460 cells. Compounds substituted with cyclic tertiary
amino substituents 19 and 20 also showed prominent anti-
proliferative activity with the exception of piperidinyl-
substituted derivative 18, which could be explained by the
presence of another O or N heteroatom that could contribute
as well as chemical shis (Table 1) and by mass spectrometry.
The H NMR spectra of amino-substituted benzimidazo[1,2-a]-
1
quinolines 8–20 showed a downeld shi of the aromatic
protons in comparison to 2-uoro(2-chloro)benzimidazo[1,2-a]-
quinoline-6-carbonitriles 6 and 7 and the appearance of protons
related to amino substituents in the aliphatic region of the NMR
spectra between 4.01 and 0.97 ppm. The 1H NMR spectra of
tertiary amino-substituted derivatives 13–16 showed a down-
eld shi of the H-1, H-3, H-4 and H-5 protons of the quinoline
part of the molecule. The same inuence on the chemical shis
of H-1, H-3, H-4 and H-5 protons accompanied the introduction
of cyclic amino substituents in compounds 18–20.
N,N-Dimethylated compound 21 as an iodide salt was
prepared from piperazinyl substituted benzimidazo[1,2-a]-
quinoline 20 with an excess of methyl iodide. An attempt to
prepare a monomethylated piperazinyl substituted benzimi-
dazo[1,2-a]quinoline with an equimolar amount of methyl
iodide was not successful. The structure of compound 21 was
conrmed by using 2D NMR spectroscopy based on NOE
Table 1 1H NMR chemical shifts of aromatic protons of compounds 8–21
a
Table 2 IC50 values (in mM)
Cell lines
Compound
HCT116
MCF-7
H460
8bc
9cd
10
11
12
>10
4 ꢁ 0.9
0.2 ꢁ 0.1
1 ꢁ 0.2
>10
2 ꢁ 0.2
1H NMR (d/ppm)
Comp. H-1 H-3 H-4 H-5 H-8 H-9
0.6 ꢁ 0.01
0.23 ꢁ 0.05
0.2 ꢁ 0.01
2 ꢁ 0.03
0.5 ꢁ 0.02
>10
0.4 ꢁ 0.1
0.4 ꢁ 0.1
0.3 ꢁ 0.02
2 ꢁ 1.4
0.3 ꢁ 0.05
0.3 ꢁ 0.1
0.2 ꢁ 0.09
1.6 ꢁ 0.5
0.3 ꢁ 0.01
>10
H-10
H-11
8
7.67 6.90 7.83 8.48 7.94 7.61–7.52 7.61–7.52 8.52
14c
15bc
16b
18c
19bc
20
9
7.74 6.91 7.81 8.46 7.94 7.57
7.80 6.94 7.82 8.47 7.95 7.57
7.76 6.92 7.80 8.46 7.95 7.58
7.80 6.95 7.82 8.48 7.95 7.59
7.51 7.07 7.88 8.53 7.96 7.59
7.56 7.08 7.88 8.51 7.96 7.57
7.51 7.06 7.85 8.49 7.96 7.60–7.53 7.60–7.53 8.34
7.54 7.06 7.88 8.52 7.97 7.59 7.52 8.37
7.44 6.95 7.90 8.54 7.95 7.61–7.52 7.61–7.52 8.51
7.79 7.30 7.90 8.56 7.97 7.59
7.73 7.27 7.89 8.52 7.96 7.59
7.66 7.24 7.83 8.54 7.94 7.58
7.92 7.44 8.03 8.66 8.05 7.65
7.54
7.53
7.53
7.53
7.52
7.54
8.52
8.54
8.53
8.60
8.48
8.40
2.5 ꢁ 1
10
11
12
13
14
15
16
17
18
19
20
21
39 ꢁ 13
8 ꢁ 4
2 ꢁ 0.9
5 ꢁ 0.4
0.3 ꢁ 0.09
0.06 ꢁ 0.04
0.6 ꢁ 0.04
0.2 ꢁ 0.04
0.2 ꢁ 0.001
2.4 ꢁ 1.5
0.3 ꢁ 0.05
0.2 ꢁ 0.03
2 ꢁ 0.4
21b
a
IC50; the concentration that causes 50% growth inhibition as assessed
b
by MTT assay following 72 hours of incubation. The highest tested
c
7.54
7.53
7.52
7.59
8.52
8.49
8.43
8.67
concentration was 10 mM. Compounds were dissolved in DMSO to a
stock solutions concentration of 1 ꢂ 10ꢃ3 to 4 ꢂ 10ꢃ2 M. However,
the precipitation in the cell culture medium at 10ꢃ4 M aer 72 hours
was observed. d The precipitates interfered with the MTT test.
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 1537–1550 | 1539

View Article Online
MedChemComm
Concise Article
Table 4 The effects of compound 21 at 0.5 and 5 mM concentrations on the cell
cycle distribution of HCT116 cells after 24 and 48 h treatments. The numbers
represent the percentages of cells in the respective cell cycle phase (G1, S and G2/
M), along with the percentage of cells in the subG1 (dead/apoptotic cells)
obtained by flow cytometry
to interactions with potential biological targets. In compound
21, one of the piperazine N atoms was quaternized to evaluate
the effect of positive permanent charge on the antiproliferative
activity and, based on the obtained results, it can be revealed
that the antiproliferative activity decreased. The decrease in
antiproliferative activity is probably due to the fact that the N
heteroatom has lost the ability to interact with potential bio-
logical targets because of the steric hindrance of two attached
methyl groups. Since there is in general no signicant differ-
ence in the IC50 concentrations between the activity of the
most active compounds (e.g. 10 and 11), while at the same time
compound 20 and its iodide salt 21 exert drastically different
activities, we attempted ow cytometry analysis of potential
cell cycle perturbations in HCT116 cells aer the treatment
with i-butyl substituted derivative 10, N,N-dimethylamino-
propyl substituted derivative 11, piperazinyl-substituted
derivative 20 and its N,N-dimethyl-substituted iodide salt 21
(Tables 3 and 4).
Percentage of cells (%)
Treatment
SubG0/G1
G0/G1
S
G2/M
24 h
Control
4 ꢁ 1
33 ꢁ 1
32 ꢁ 0.3
38 ꢁ 2
37 ꢁ 2
37 ꢁ 0.3
43 ꢁ 3
39 ꢁ 0.2
36 ꢁ 3
33 ꢁ 1
32 ꢁ 1
32 ꢁ 1
29 ꢁ 3
28 ꢁ 1
32 ꢁ 3
29 ꢁ 4
31 ꢁ 0.1
31 ꢁ 2
28 ꢁ 0.1
21, 0.5 mM
21, 5 mM
Control
21, 0.5 mM
21, 5 mM
6 ꢁ 1
7 ꢁ 0.3
4 ꢁ 1
48 h
6 ꢁ 2
5 ꢁ 0.1
Inuence of compounds on protein synthesis
The effect of alkylation of DNA and RNA (coding for a single
We tested the effects of various concentrations (0.1, 0.5, 1 protein) by nitrogen mustard was previously shown using
and 5 mM) aer 24 and 48 h incubation; however, except for rabbit reticulocyte lysate as a translation system in vitro.19 The
compound 21, signicant effects were seen even at the 0.1 mM, authors showed that when alkylated DNA was used as the
which are still lower than their IC50 concentrations obtained in template for coupled transcription and translation, a decreased
the MTT test. The results demonstrated that the compounds amount of luciferase protein was detected. In addition, it was
10, 11 and 20 drastically reduced the number of cells in the S demonstrated that alkylation of DNA leads to suppression of
phase and induced severe G2/M phase arrest, indicating green uorescence protein (GFP) plasmid expression in tumor
impairment in progress to mitosis. Also, the concentration- cells.20 In this study, the A549 cells were transfected with a
dependant activation of apoptosis can be observed (the SubG1 prealkylated GFP plasmid with N-methylquinolinium quinone
phase), which goes up to 40% aer treatment with 5 mM methide and a signicant suppression of GFP protein expres-
concentration of compounds (data not shown). These results sion was detected.
suggest that DNA might be the main target of compounds 10,
In present work we used a new approach to evaluate novel
11 and 20. Interestingly, compound 21 did not cause any effect compounds in regard to their reactivity and DNA damaging
up to the concentration of 5 mM and 48 h of incubation, when it capacity. To do so we used as a reporter, for the rst time to our
caused a very slight delay of the cell cycle progression in G1 knowledge, Enhanced Green Fluorescent Protein (EGFP)
phase, pointing to a completely different mechanism of action expression in a cell-free environment with a high-capacity T7
(Table 4).
promotor driven in vitro translation system. Namely, samples of
DNA encoding the EGFP gene were treated with compounds 10,
11, 20 and 21 and a range of known DNA-damaging agents, and
then subjected to coupled transcription and translation to nd
a correlation between the effect of DNA damage and the amount
of transcribed and subsequently translated gene product.
Various DNA-damaging compounds were used for comparison;
it is evident that the most pronounced inhibitory effect was
obtained with the cross-linking agent cisplatin (94% inhibition)
and a strong intercalative agent doxorubicin (79% inhibition)
(Fig. 2). Also, the alkylating agent chlorambucyl inhibits protein
expression to 45%; while on the other hand, camptothecin
(CPT) or etoposide (data not shown) did not exert any inhibitory
effect (p > 0.05). Such results were expected because CPT binds
reversibly to DNA-topoisomerase I complexes, but not to the
enzyme or DNA alone. Therefore, its toxicity is a result of the
induction of DNA-strand breaks in intact cells and in the nuclei
but not in puried DNA preparations in the absence of top-
oisomerase I.21 Other tested compounds directly damage DNA
(cross-linking, alkylation, intercalation) and thus most likely
suppress transcription and translation of protein. Similar
results, although in different cell-free systems, were obtained by
other authors.19,22,23
Table 3 The effects of compounds 10, 11 and 20 at 0.1 and 1 mM concentra-
tions on the cell cycle distribution of HCT116 cells after 24 and 48 h treatments.
The numbers represent the percentages of cells in the respective cell cycle phase
(G1, S and G2/M), along with the percentage of cells in the SubG1 (dead/
apoptotic cells) obtained by flow cytometry
Percentage of cells (%)
Treatment
24 h Control
SubG1
G1
S
G2/M
2 ꢁ 0.1
4 ꢁ 0.2
16 ꢁ 5
6 ꢁ 1
23 ꢁ 2
14 ꢁ 1
7 ꢁ 7
48 ꢁ 6
44 ꢁ 1
8 ꢁ 11
47 ꢁ 1
13 ꢁ 18
1 ꢁ 1
29 ꢁ 8
42 ꢁ 2
85 ꢁ 4
40 ꢁ 2
65 ꢁ 25
80 ꢁ 2
85 ꢁ 3
28 ꢁ 3
17 ꢁ 1
90 ꢁ 4
17 ꢁ 0.3
58 ꢁ 1
30 ꢁ 2
69 ꢁ 2
10, 0.1 mM
10, 1 mM
11, 0.1 mM
11, 1 mM
20, 0.1 mM
20, 1 mM
Control
13 ꢁ 1
22 ꢁ 8
19 ꢁ 1
15 ꢁ 3
60 ꢁ 2
62 ꢁ 5
10 ꢁ 4
60 ꢁ 0.1
41 ꢁ 1
59 ꢁ 3
30 ꢁ 2
5 ꢁ 0.5
2 ꢁ 0.3
12 ꢁ 9
5 ꢁ 2
0 ꢁ 0
48 h
12 ꢁ 5
21 ꢁ 6
0 ꢁ 0
10, 0.1 mM
10, 1 mM
11, 0.1 mM
11, 1 mM
20, 0.1 mM
20, 1 mM
6 ꢁ 3
30 ꢁ 2
4 ꢁ 1
23 ꢁ 0.3
1 ꢁ 0.1
11 ꢁ 4
1 ꢁ 0.3
8 ꢁ 0.2
11 ꢁ 1
12 ꢁ 4
1540 | Med. Chem. Commun., 2013, 4, 1537–1550
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Concise Article
MedChemComm
Fig. 2 Inhibition of EGFP protein translation. Plasmid containing EGFP gene under T7 promoter was incubated with compounds 10, 11, 20 and 21, as well as with
cisplatin (CIS), chlorambucyl (CBL), doxorubicin (DOX) and camptothecin (CPT) at 50 mM concentrations for 1 h at 24 ꢀC. Treated or untreated plasmid was then
incubated with the S30 T7 High-Yield Protein Expression System, which enabled the EGFP protein translation/synthesis. The graph represents the percentages of EGFP
fluorescence, corresponding to the amount of EGFP protein obtained during the in vitro translation procedure. The bars represent average values (ꢁsd) of three
experiments performed in duplicates. * ¼ p < 0.05, ** ¼ p < 0.005.
Fig. 3 Fluorescence microscopy images of H460 cells treated with 10 mM concentration of compounds 10 (A), 11 (B), 20 (C) and 21 (D) for 2 h. All compounds, except
11, show predominantly cytoplasmic distribution (magnification 400ꢂ).
Fig. 4 Confocal laser scanning microscopy of H460 cells treated for 2 h with 10 mM concentration of compound 11. (A) and (B) represent two optical sections taken at
different levels along the z-axis throughout the same cells, (C) and (D) demonstrate intensive staining of mitotic chromosomes. Upper panel – the fluorescence images,
excitation at lex ¼ 405 nm and detection at 424–527 nm; lower panel – the same images in the transmitted light.
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 1537–1550 | 1541

View Article Online
MedChemComm
Concise Article
Subcellular localization of compounds 10, 11, 20 and 21
To check the intracellular distribution of the compounds in the
tumor cells, we incubated the H460 cells with the tested
compound and performed a uorescence microscopy study. The
H460 cell line was used because the cells are round when
attached to the cover slide, have relatively wide cytoplasm and
therefore enable better visualization of the intracellular distri-
bution of the compound. Unexpectedly, all compounds show
cytoplasmic, mostly punctate staining, pointing to their locali-
zation within organelles in the cells and not the nucleus (Fig. 3).
Only compound 11 showed both cytoplasmic and nuclear
localization (Fig. 3B). Still, there is evidence of differences
between the compounds. For example, there is a low general
uptake of compound 21 in the cytosol but distinct and somewhat
enriched staining in the perinuclear region (Fig. 3D). Similar,
but enhanced perinuclear and reticulate uorescent image of
cells treated with compound 20 suggests location at the endo-
plasmic reticulum (ER), along with a distinctive staining of
nucleoli (Fig. 3C). Interestingly, compound 10 showed faint, but
very homogenous cytoplasmic staining (Fig. 3A).
Fig. 5 (A) Variation of DNA melting temperature. (B) UV/visible spectroscopic
analyses of the DNA binding of compounds 10, 11, 20 and 21. (B) Spectra of the
different compounds (20 mM) alone (dashed lanes) or incubated with graded
concentrations of CT-DNA (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160,
180, 200 mM; plain lanes) measured in quartz cuvettes from 230 to 530 nm
against a similar cuvette containing the same amount of CT-DNA.
Since we noted the remarkable staining pattern of 11, we also
performed laser scanning confocal microscopy to obtain better
insight of its localization (Fig. 4).
Interestingly, we observed the accumulation of uorescence
forming cytoplasmic aggregates (Fig. 4A), which could suggest
their localization in the lysosomes, or endosomes, along with
nuclear staining (Fig. 4B), while in the mitotic cells intensive
staining of mitotic chromosomes is evident, conrming its
affinity towards DNA (Fig. 4C and D). Further co-localization
studies should be performed to conrm the localization in
particular organelles.
The presented results clearly demonstrate that at DNA level,
50 mM concentrations of all tested compounds inhibit formation
of the full-length translated protein. However, the inuence of
compound 10 is subtle (17% inhibition), but non-signicant
(p ¼ 0.1), pointing to its lower DNA binding/intercalating ability.
Compounds 20 and 21 exert intermediate suppression (38% and
45% inhibition, respectively), while compound 11 strongly
inhibits EGFP translation (72% inhibition, p ¼ 0.01), correlating
with its strong DNA-binding and potential intercalative ability.
DNA binding properties
Based on cellular distribution, a selection of compounds from
this series was evaluated in vitro for DNA binding properties.
Fig. 6 Circular dichroism spectra of a fixed concentration of CT-DNA (50 mM) incubated with increasing concentrations of compounds 11, 20 or 21 (1, 5, 10, 20, 30, 40
and 50 mM) are recorded from 230 to 480 nm. Each spectrum is the average of three recordings.
1542 | Med. Chem. Commun., 2013, 4, 1537–1550
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Concise Article
MedChemComm
CT-DNA upon addition of increasing concentration of the
compounds (Fig. 6). The binding of compound 20 to CT-DNA
reveals the highest dose-dependent increase of a negative
induced-circular dichroism (ICD) within its absorption wave-
length (410–440 nm, Fig. 5B). Such negative ICD suggests an
intercalation of 20 between adjacent base pairs of the DNA
helix. An ICD with much smaller intensity was also observed
using compounds 11, 19 and 21 suggesting less potent inter-
calative process.
The ICD for compound 11 visualized at 415–440 nm is
associated with a decrease of the CD bands associated with the
DNA helix (negative peak at 245 nm and positive peak at
280 nm) and is in agreement with an intercalation process that
changes the helicity of the DNA. By contrast, compounds 10, but
also 16 and 18 used as control, did not change the CD prole of
CT-DNA, suggesting that those compounds do not intercalate
into the DNA base pairs.
Fig. 7 Topoisomerase I-induced relaxation of the DNA.
The intercalation process was conrmed using top-
oisomerase I-induced relaxation of supercoiled plasmid DNA
(Fig. 7).
The typical prole with less and less negatively supercoiled
DNA to relaxed DNA and to more and more positively super-
The evaluation of the variation of the DNA melting temperature
upon the binding of the compounds evidenced compound 10 as
a weak DNA binder and compounds 11, 20 and 21 as strong
DNA binders (Fig. 5A).
Compounds 16 and 18, which were poorly cytotoxic (>8 mM), coiled plasmid DNA is seen, suggesting the intercalation of the
did not stabilize the DNA helix. However, compound 19, which core of the different molecules between adjacent base pairs,
also failed to stabilize the DNA helix, is cytotoxic in all tested cell resulting in a constrain that favors topoisomerase I-induced
relaxation of the supercoiled plasmid to then convert the
relaxed form to positively supercoiled circular DNA struc-
tures.24,25 Only 1 mM (10, 20) or 2 mM (21) of compounds is
lines. UV/visible spectroscopic measurements were performed
using a xed concentration of compounds 10, 11, 20 and 21
incubated with increasing concentrations of CT-DNA (Fig. 5B).
The results conrm the binding of the four compounds to the required to fully relax the supercoiled circular plasmid DNA (Rel
DNA with hypochromic (compounds 10, 11, 20 and 21) and band). By contrast, compounds 10 and 19 only slightly enhance
bathochromic (compounds 11, 20 and 21) effects. The presence the topoisomerase I-induced relaxation effect on the global
of an isosbestic point suggests a single mode of binding.
This mode of binding (intercalation, groove binding) was intercalating drugs (20–50 mM) and suggesting much weaker
intercalative processes as indicated by CD spectra. Compounds
structure of the DNA helix, suggesting that they do not act as
rst investigated using circular dichroism measurements of
Fig. 8 Sequence-specificity assessed using DNase I footprinting assays. (A) DNase I footprinting gel. The 30-end radiolabeled 265 bp DNA was incubated with
increasing concentrations of compounds 11, 20 or 21 prior to addition of appropriate concentrations of DNase I enzyme. G+a tracks evidenced guanine and adenine as
stronger and weaker bands, respectively, and are obtained from DMS treatment following by hot piperidine cleavage. (B) Densitometric analyses of the gel using
compound 11 (2, 5 and 10 mM), 20 (5 and 10 mM) and 21 (2 and 5 mM). Black lanes locate the strongest footprint, both on the gel and on the densitometric spectra.
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 1537–1550 | 1543

View Article Online
MedChemComm
Concise Article
16 and 18 do not affect the global structure of the DNA helix, extent obtained by compound 19. The most efficient DNA
suggesting that they do not act as intercalating drugs. Super- intercalators (10, 20 and 21) also evidence some sequence-
coiled pUC19 plasmid DNA (Sc) was incubated with increasing selective DNA binding. The results were also not completely
concentrations of the indicated compounds (mM) or CPT as a straightforward, except for N,N-dimethylaminopropyl analogue
non-intercalative drug, inducing a nicked DNA that co-migrates 11, which was unequivocally demonstrated to be a strong DNA-
with fully relaxed DNA (Rel). Upon treatment with top- binder and intercalative agent, which efficiently targets DNA in
oisomerase I, a small amount of negatively and then positively viable cells. On the other hand, compounds 20 and 21, despite
supercoiled topoisomers (topo) are evidenced aer migration of demonstrating their DNA-binding properties in vitro (spectro-
a 1% agarose gel and post-electrophoresis staining with scopic evaluation, topoisomerase I-mediated DNA relaxation
ethidium bromide. Based on their DNA intercalation ability, the and to some extent the inhibition of protein synthesis in vitro),
compounds were evaluated for topoisomerases
I and II did not conrm the same effects in viable cells. Namely, only 20
poisoning activities. None of the compounds were able to block demonstrated nuclear staining (more specically, staining of
cleavable complexes from either topoisomerase I or II (data not the nucleoli), while the uptake of quaternary ammonium iodide
shown). We nally looked at potential sequence-selective DNA salt 21 was rather modest and mostly perinuclear staining was
binding using DNase I footprinting experiments. Only a small observed, accompanied by a poor and completely different
selectivity was obtained using the strongest DNA binders 11, 20 inuence on the cell cycle. What is more, compound 10 with a
and 21 (Fig. 8).
bulky i-butylamino side chain, although conrmed by all
The covered sections of DNA correspond to the 70-TCACTAA- methods not to be a typical intercalator, demonstrated a very
64 sequence that seems to be covered twice: rst at the 70-TCA- pronounced G2/M arrest, along with a drastic S phase reduc-
68 site and second at the 67-CTAA-64 site. A third and weaker tion, pointing to mitosis inhibition that could be achieved by
coverage portion is seen at the 110-CTAT-103 site.
both DNA intercalation and mitotic spindle-damage. This new
series of compounds clearly do not poison topoisomerases I and
II (data not shown) even if some compounds strongly bind DNA
and intercalate between adjacent base pairs of the DNA helix.
Conclusions
This work represents the synthesis of 2-substituted benzimi- However, some sequence-selective DNA binding has been evi-
dazo[1,2-a]quinolines derivatives 8–21 with different amino side denced, corroborating with the EGFP protein translation assays,
chains placed on the quinoline nucleus with potential chemo- suggesting some role of DNA targeting in the cellular effect of
therapeutic activities. Amino-substituted compounds were the compounds. Alternatively, it is possible that it interferes
synthesized by uncatalyzed microwave assisted amination from with cellular tubulin polymerization, thus leading to the inhi-
the corresponding chloro(uoro)-substituted precursor. Benzi- bition of mitotic progression. Finally, we can conclude that N,N-
midazo[1,2-a]quinolines and related benzannulated benzimid- dimethylaminopropyl analogue 11 should be further optimized
azoles, which possess highly conjugated planar chromophores, as a lead compound for novel DNA-intercalative compounds,
are well known DNA intercalators while amino chains with while the i-butylamino-substituted benzimidazo[1,2-a]quino-
different length and exibility could signicantly enhance line 10 should be tested further to corroborate its antimitotic
additional interactions with DNA bases. All compounds showed ability, i.e. to test its ability to target microtubules.
prominent antiproliferative activity against three tumor cell
lines, while the length of the secondary or tertiary amino chains
linked to the benzimidazo[1,2-a]quinoline nucleus inuenced
the antiproliferative activity. From this study it has been
Experimental section
General methods
revealed that the alkylamino substituents, either acyclic or All chemicals and solvents were purchased from commercial
cyclic, increased antitumor activities in comparison with suppliers Acros, Aldrich or Fluka. Melting points were recorded
1
previously published nitro and amino substituted benzimidazo- on SMP11 Bibby and Buchi 535 apparatus. The H and 13C NMR
¨
[1,2-a]quinolines.16 Compound 8, which has the shortest spectra were recorded on a Varian Gemini 300 or Varian Gemini
secondary amino side chain, showed modest activity, but 600 at 300, 600, 150 and 75 MHz, respectively. All NMR spectra
pronounced selectivity to MCF-7 cells, while the compound with were measured in DMSO-d6 solutions using TMS as an internal
the longest tertiary amino side chain (16) was the least active, standard. Chemical shis are reported in ppm (d) relative to
probably due to steric hindrance. Although the majority of TMS. In preparative photochemical experiments the irradiation
compounds were active at submicromolar IC50 concentrations, was performed at room temperature with a water-cooled
we performed a series of additional experiments to shed more immersion well with “Origin Hanau” 400 W high pressure
light on the mechanisms of action of the most active ones (10, mercury arc lamp using Pyrex glass as a cut-off lter of wave-
11, 20 and 21), including DNA binding propensities, top- lengths below 280 nm. All compounds were routinely checked
oisomerases I and II inhibition, cell cycle perturbances and by TLC with Merck silica gel 60F-254 glass plates. Mass spectra
cellular localization. The DNA intercalation activity (as evalu- were recorded on an Agilent 1200 series LC/6410 QQQ instru-
ated using topoisomerase I-induced DNA relaxation and ment. The electronic absorption spectra were recorded on Var-
circular dichroism) of all tested compounds correlates with the ian Cary 50 spectrometer using quartz cuvette (1 cm). Elemental
antiproliferative effect since all compounds except 16 and 18 analyses for carbon, hydrogen and nitrogen were performed on
present intercalation proles with, however, a much lesser a Perkin-Elmer 2400 elemental analyzer. Where analyses are
1544 | Med. Chem. Commun., 2013, 4, 1537–1550
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Concise Article
MedChemComm
indicated only as symbols of elements, analytical results 8.5 Hz, Harom.), 8.02 (d, 1H, J ¼ 8.3 Hz, Harom.), 7.73 (d, 1H, J ¼
obtained are within 0.4% of the theoretical value.
8.43 Hz, Harom.), 7.66–7.57 (m, 2H, Harom.); 13C NMR (150 MHz,
DMSO-d6): 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);
Synthesis
2-(2-Benzimidazolyl)-3-(2-chloro-4-uorophenyl)acrylonitrile found: C, 68.99; H, 2.74; N, 15.29. Calc. for C16H8N3Cl: C, 69.20;
4. Compound 4 was prepared from 3 (1.00 g, 6.37 mmol) and 2- H, 2.90; N, 15.13%; MS (ESI): m/z ¼ 278.1 ([M + 1]+).
chloro-4-uorobenzaldehyde 1 (1.00 g, 6.31 mmol) in absolute
ethanol (7 ml) aer reuxing for 2 h and recrystallization from
General method for preparation of compounds 8–21
ethanol to yield 1.41 g (75%) of slightly yellow crystals; mp 234–
1
ꢀ
237 C; H NMR (300 MHz, DMSO-d6): d/ppm ¼ 13.31 (s, 1H, Compounds 8–21 were prepared using microwave irradiation,
NHbenzim.), 8.46 (s, 1H, Harom.), 8.21 (dd, 1H, J1 ¼ 6.22 Hz, J2 ¼ at optimized power and reaction time, from compound 6 or 7 in
9.07 Hz, Harom.), 7.74 (dd, 1H, J1 ¼ 2.69 Hz, J2 ¼ 8.82 Hz, Harom.), acetonitrile (10 ml) with an excess of the corresponding amine.
7.66 (bs, 2H, Hbenzim.), 7.51 (dt, 1H, J1 ¼ 2.81 Hz, J2 ¼ 8.07 Hz, Aer cooling, the reaction mixture was ltered off and resulting
H
benzim.), 7.30 (bs, 1H, Hbenzim.); 13C NMR (75 MHz, DMSO-d6): product was separated by column chromatography on SiO2
d/ppm ¼ 165.28 (s), 161.92 (s), 147.05 (s), 140.69 (d), 135.62 (s), using dichloromethane–methanol as eluent.
135.32 (s), 131.93 (d), 128. 41 (s), 124.54 (d), 122.97 (d), 119.96
2-N-Methylaminobenzimidazo[1,2-a]quinoline-6-carbonitrile
(d), 117.97 (d), 115.80 (s), 115.66 (d), 112.23 (d), 107.16 (s); 8. Compound 8 was prepared using the above described method
found: C, 64.81; H, 3.04; N, 6.40. Calc. for C17H12N4: C, 64.55; H, from 6 (60 mg, 0.23 mmol) and a 33% solution of methylamine
3.05; N, 6.38%; MS (ESI): m/z ¼ 298.1 ([M + 1]+).
in ethanol (0.14 ml, 1.40 mmol) aer 4 h of irradiation to yield
2-(2-Benzimidazolyl)-3-(2,4-dichlorophenyl)acrylonitrile 5. 61 mg (96%) of yellow crystals; mp > 280 ꢀC. 1H NMR (300 MHz,
Compound 5 was prepared from 3 (1.30 g, 8.29 mmol) and 2,4- DMSO-d6): d/ppm ¼ 8.52 (d, 1H, J ¼ 7.5 Hz, H-11), 8.48 (s, 1H, H-
dichlorobenzaldehyde 1 (1.45 g, 8.29 mmol) in absolute ethanol 5), 7.94 (d, 1H, J ¼ 7.5 Hz, H-8), 7.83 (d, 1H, J1 ¼ 1.7 Hz, J2 ¼ 8.7
(10 ml) aer reuxing for 2 h and recrystallization from ethanol Hz, H-4), 7.67 (s, 1H, H-1), 7.61–7.52 (m, 2H, H-9 and H-10), 7.42
to yield 1.85 g (71%) of slightly yellow crystals; mp 257–260 ꢀC; (q, 1H, J ¼ 4.9 Hz, NH), 6.90 (dd, 1H, J1 ¼ 1.7 Hz, J2 ¼ 8.8 Hz, H-
1H NMR (300 MHz, DMSO-d6): d/ppm ¼ 13.32 (s, 1H, NHbenzim.), 3), 2.99 (d, 3H, J ¼ 4.9 Hz, CH3); 13C NMR (75 MHz, DMSO-d6): d/
8.45 (s, 1H, Harom.), 8.15 (d, 1H, J ¼ 8.46 Hz, Harom.), 7.89 (d, 1H, J ppm ¼ 155.06, 146.37, 144.68, 104.55, 138.69, 133.05, 130.87,
¼ 2.10 Hz, Harom.), 7.69 (dd, 1H, J1 ¼ 2.10 Hz, J2 ¼ 8.52 Hz, 125.17 (2C), 122.89 (2C), 119.99, 117.23, 114.94, 111.61, 92.52,
H
arom.), 7.67 (bs, 1H, Hbenzim.), 7.27 (bs, 2H, Hbenz.); 13C NMR (75 29.78; found: C, 74.90; H, 4.43; N, 20.49. Calc. for C17H12N4: C,
MHz, DMSO-d6): d/ppm ¼ 146.96 (s), 143.92 (s), 140.62 (d), 74.98; H, 4.44; N, 20.58%; MS (ESI): m/z ¼ 273.1 ([M + 1]+).
140.54 (d), 136.85 (s), 135.31 (s), 131.36 (d), 131.30 (d), 130.71 (s),
2-N-Butylaminobenzimidazo[1,2-a]quinoline-6-carbonitrile 9.
130.19 (d), 130.14 (d), 129.78 (d), 128.70 (s), 128.54 (d), 115.68 (s), Compound 9 was prepared using the above described method
107.69 (s); found: C, 61.41; H, 2.88; N, 13.40. Calc. for C17H12N4: from 6 (60 mg, 0.23 mmol) and n-butylamine (0.16 ml, 1.60
C, 61.17; H, 2.89; N, 13.38%; MS (ESI): m/z ¼ 314.1 ([M + 1]+).
mmol) aer 10 h of irradiation to yield 42 mg (62%) of yellow
6. crystals; mp 234–236 ꢀC. 1H NMR (300 MHz, DMSO-d6): d/ppm ¼
2-Fluorobenzimidazo[1,2-a]quinoline-6-carbonitrile
Compound 4 (0.50 g, 1.68 mmol) was dissolved in 4 ml of sul- 8.52 (d, 1H, J ¼ 7.7 Hz, H-11), 8.46 (s, 1H, H-5), 7.94 (d, 1H, J ¼ 7.7
folane and the reaction mixture was heated for 25 minutes at Hz, H-8), 7.81 (d, 1H, J ¼ 8.8 Hz, H-4), 7.74 (s, 1H, H-1), 7.57 (dt,
280 ꢀC. The cooled mixture was poured into water (15 ml), and 1H, J1 ¼ 1.4 Hz, J2 ¼ 8.2 Hz, H-9), 7.54 (dt, 1H, J1 ¼ 1.5 Hz, J2 ¼ 8.2
the resulting product was ltered off and recrystallized from Hz, H-10), 7.34 (t, 1H, J ¼ 5.7 Hz, NH), 6.91 (d, 1H, J ¼ 8.7 Hz, H-
ethanol (450 ml) to obtain a yellow powder (0.32 g, 72%); mp 3), 1.71–1.61 (m, 2H, CH2), 1.54–1.42 (m, 4H, CH2.), 0.97 (t, 3H, J
250–254 ꢀC; 1H NMR (300 MHz, DMSO-d6): d/ppm ¼ 8.77 (s, 1H, ¼ 7.3 Hz, CH3); 13C NMR (150 MHz, DMSO-d6): d/ppm ¼ 153.88,
H
arom.), 8.71 (d, 1H, J ¼ 8.25 Hz, Harom.), 8.52 (dd, 1H, J1 ¼ 2.10 145.91, 144.20, 140.09, 132.78, 130.39, 124.69, 122.36, 119.55
Hz, J2 ¼ 10.89 Hz, Harom.), 8.20 (dd, 1H, J1 ¼ 6.44 Hz, J2 ¼ 8.42 (2C), 116.74, 114.33 (2C), 111.13, 97.54, 91.96, 42.09, 30.49, 19.73,
Hz, Harom.), 7.99 (d, 1H, J ¼ 8.21 Hz, Harom.), 7.61 (t, 1H, J ¼ 6.82 13.70; found: C, 76.26; H, 5.78; N, 17.77. Calc. for C20H18N4: C,
Hz, Harom.), 7.58–7.51 (m, 2H, Harom.); 13C NMR (DMSO-d6) (d/ 76.41; H, 5.77; N, 17.82%; MS (ESI): m/z ¼ 315.2 ([M + 1]+).
ppm): 165.48 (s), 163.81 (s), 144.35 (s), 143.63 (s), 140.01 (d),
2-N-i-Butylaminobenzimidazo[1,2-a]quinoline-6-carbonitrile
133.71 (d), 130.22 (s), 125.28 (d), 123.70 (d), 120.13 (d), 118.14 10. Compound 10 was prepared using the above described
(s), 115.26 (s), 114.83 (d), 113.82 (d), 102.96 (d), 100.33 (s); method from 6 (60 mg, 0.23 mmol) and i-butylamine (0.11 ml,
found: C, 73.27; H, 3.10; N, 16.05. Calc. for C17H12N4: C, 73.56; 1.20 mmol) aer 12 h of irradiation to yield 34 mg (47%) of
H, 3.09; N, 16.00%; MS (ESI): m/z ¼ 262.1 ([M + 1]+).
yellow crystals; mp 220–223 ꢀC. 1H NMR (300 MHz, DMSO-d6): d/
2-Chlorobenzimidazo[1,2-a]quinoline-6-carbonitrile 7. An ppm ¼ 8.54 (dd, 1H, J1 ¼ 1.3 Hz, J2 ¼ 7.4 Hz, H-11), 8.47 (s, 1H,
ethanolic solution (400 ml) of compound 5 (0.90 g, 2.87 mmol) H-5), 7.95 (dd, 1H, J2 ¼ 1.3 Hz, J2 ¼ 7.4 Hz, H-8), 7.82 (d, 1H, J ¼
was irradiated at room temperature, with a 400 W high-pressure 8.8 Hz, H-4), 7.80 (s, 1H, H-1), 7.57 (dt, 1H, J1 ¼ 1.3 Hz, J2 ¼ 7.3
mercury lamp using a Pyrex lter for 10 h. The obtained product Hz, H-9), 7.53 (dt, 1H, J1 ¼ 1.4 Hz, J2 ¼ 7.3 Hz, H-10), 7.40 (t, 1H,
was ltered off to give 0.43 g (54%) of yellow crystals; mp 288–291 J ¼ 5.5 Hz, NH), 6.94 (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ 8.8 Hz, H-3), 3.15
ꢀC. 1H NMR (600 MHz, DMSO-d6): d/ppm ¼ 8.80 (s, 1H, Harom.), (t, 2H, J ¼ 6.2 Hz, CH2), 2.01–1.92 (m, 1H, CH), 1.03 (d, 6H, J ¼
8.73 (d, 1H, J ¼ 7.5 Hz, Harom.), 8.70 (s, 1H, Harom.), 8.16 (d, 1H, J ¼ 6.7 Hz, CH3); 13C NMR (75 MHz, DMSO-d6): d/ppm ¼ 154.55,
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 1537–1550 | 1545

View Article Online
MedChemComm
Concise Article
146.40, 144.69, 138.52, 133.30, 130.88, 125.17, 122.84, 114.80 irradiation to yield 60 mg (80%) of yellow crystals; mp 197–199
1
(2C), 111.61, 92.43, 50.58, 28.11, 20.80 (2C); found: C, 76.48; H, ꢀC. H NMR (300 MHz, DMSO-d6): d/ppm ¼ 8.51 (s, 1H, H-5),
5.75; N, 17.83. Calc. for C20H18N4: C, 76.41; H, 5.77; N, 17.82%; 8.40 (dd, 1H, J1 ¼ 1.8 Hz, J2 ¼ 7.2 Hz, H-11), 7.96 (dd, 1H, J1 ¼ 1.8
MS (ESI): m/z ¼ 315.2 ([M + 1]+).
Hz, J2 ¼ 7.2 Hz, H-8), 7.88 (d, 1H, J ¼ 9.2 Hz, H-4), 7.57 (dt, 1H, J1
2-[N-(N,N-Dimethylaminopropyl-1-amino)]benzimidazo[1,2- ¼ 2.2 Hz, J2 ¼ 7.3 Hz, H-9), 7.56 (s, 1H, H-1), 7.54 (dt, 1H, J1 ¼ 2.1
a]quinoline-6-carbonitrile 11. Compound 11 was prepared Hz, J2 ¼ 7.2 Hz, H-10), 7.08 (dd, 1H, J1 ¼ 2.3 Hz, J2 ¼ 9.1 Hz, H-3),
using the above described method from 6 (120 mg, 0.46 mmol) 3.67 (q, 4H, J ¼ 7.1 Hz, CH2), 1.28 (t, 6H, J ¼ 7.1 Hz, CH3); 13C
and N,N-dimethylaminopropyl-1-amine (0.33 ml, 2.30 mmol) NMR (150 MHz, DMSO-d6): d/ppm ¼ 151.24, 145.91, 144.21,
aer 6 h of irradiation to yield 120 mg (76%) of yellow crystals; 139.74, 138.40, 132.57, 130.29, 124.71, 122.70, 119.63, 116.69,
mp 185–187 ꢀC. 1H NMR (600 MHz, DMSO-d6): d/ppm ¼ 8.53 (d, 114.05, 110.67, 110.47, 94.78, 92.36, 44.57 (2C), 12.11 (2C);
1H, J ¼ 8.3 Hz, H-11), 8.46 (s, 1H, H-5), 7.95 (d, 1H, J ¼ 8.2 Hz, H- found: C, 76.26; H, 5.78; N, 17.75. Calc. for C20H18N4: C, 76.41;
8), 7.80 (d, 1H, J ¼ 8.8 Hz, H-4), 7.76 (s, 1H, H-1), 7.58 (dt, 1H, J1 H, 5.77; N, 17.82%; MS (ESI): m/z ¼ 315.2 ([M + 1]+).
¼ 1.1 Hz, J2 ¼ 7.5 Hz, H-9), 7.53 (dt, 1H, J1 ¼ 1.3 Hz, J2 ¼ 7.4 Hz,
2-N,N-Dipropylaminobenzimidazo[1,2-a]quinoline-6-carbon-
H-10), 7.38 (t, 1H, J ¼ 5.4 Hz, NH), 6.92 (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ itrile 15. Compound 15 was prepared using the above described
8.8 Hz, H-3), 3.37 (q, 2H, J ¼ 6.6 Hz, CH2), 2.37 (t, 2H, J ¼ 6.7 Hz, method from 6 (90 mg, 0.34 mmol) and dipropylamine (0.24 ml,
CH2), 2.18 (s, 6H, CH3), 1.82–1.78 (m, 2H, CH2); 13C NMR (75 1.70 mmol) aer 9 h of irradiation to yield 70 mg (60%) of yellow
1
ꢀ
MHz, DMSO-d6): d/ppm ¼ 164.06, 154.41, 146.41, 144.70, crystals; mp 208–211 C. H NMR (300 MHz, DMSO-d6): d/ppm
140.56, 133.21, 130.88, 125.20 (2C), 122.83 (2C), 120.03, 117.24, ¼ 8.49 (s, 1H, H-5), 8.34 (d, 1H, J ¼ 7.8 Hz, H-11), 7.96 (d, 1H, J ¼
114.86, 111.63, 92.46, 56.94 (2C), 45.74 (2C), 27.15; found: C, 7.8 Hz, H-8), 7.85 (d, 1H, J ¼ 9.1 Hz, H-4), 7.60–7.53 (m, 2H, H-9
73.66; H, 6.14; N, 20.36. Calc. for C21H21N5: C, 73.44; H, 6.16; N, and H-10), 7.51 (s, 1H, H-1), 7.06 (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ 9.1
20.39%, MS (ESI): m/z ¼ 344.2 ([M + 1]+).
Hz, H-11), 3.57 (t, 4H, J ¼ 7.6 Hz, CH2), 1.77–1.65 (m, 4H, CH2),
2-[N-(N,N-Diethylethylenediamino)]benzimidazo[1,2-a]quin- 1.00 (t, 6H, J ¼ 7.3 Hz, CH3); 13C NMR (75 MHz, DMSO-d6): d/
oline-6-carbonitrile 12. Compound 12 was prepared using the ppm ¼ 152.23, 150.06, 146.44, 144.75, 140.28, 138.82, 132.99,
above described method from 6 (60 mg, 0.23 mmol) and N,N- 130.78, 125.24, 123.07, 120.24, 117.18, 114.39, 111.17, 95.56,
diethylethylenediamine (0.20 ml, 1.40 mmol) aer 2 h of irra- 92.89, 52.80 (2C), 20.30 (2C), 11.58 (2C); found: C, 77.47; H, 6.46;
diation to yield 40 mg (44%) of yellow crystals; mp 183–186 ꢀC. N, 16.33. Calc. for C22H22N4: C, 77.16; H, 6.48; N, 16.36%; MS
1H NMR (600 MHz, DMSO-d6): d/ppm ¼ 8.60 (d, 1H, J ¼ 8.3 Hz, (ESI): m/z ¼ 343.3 ([M + 1]+).
H-11), 8.48 (s, 1H, H-5), 7.95 (d, 1H, J ¼ 8.2 Hz, H-8), 7.82 (d, 2H,
2-N,N-Dipentylaminobenzimidazo[1,2-a]quinoline-6-carbon-
J ¼ 8.7 Hz, H-4), 7.80 (s, 1H, H-1), 7.59 (dt, 1H, J1 ¼ 1.2 Hz, J2 ¼ itrile 16. Compound 16 was prepared using the above described
7.3 Hz, H-9), 7.53 (dt, 1H, J1 ¼ 1.2 Hz, J2 ¼ 7.3 Hz, H-10), 7.27 (t, method from 6 (60 mg, 0.23 mmol) and dipentylamine (0.24 ml,
1H, J ¼ 5.0 Hz, NH), 6.95 (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ 8.8 Hz, H-3), 1.20 mmol) aer 12 h of irradiation to yield 30 mg (33%) of
3.42 (q, 2H, J ¼ 6.5 Hz, CH2), 2.70 (t, 2H, J ¼ 6.6 Hz, CH2), 2.6 (q, yellow crystals; mp 131–134 ꢀC. 1H NMR (300 MHz, DMSO-d6): d/
4H, J ¼ 7.1 Hz, CH2), 1.01 (t, 6H, J ¼ 7.1 Hz, CH3); 13C NMR (150 ppm ¼ 8.52 (s, 1H, H-5), 8.37 (d, 1H, J ¼ 8.1 Hz, H-11), 7.97 (d,
MHz, DMSO-d6): d/ppm ¼ 153.80, 145.91, 144.22, 140.01, 1H, J ¼ 7.9 Hz, H-8), 7.88 (d, 1H, J ¼ 9.1 Hz, H-4), 7.59 (t, 1H, J ¼
138.13, 132.68, 130.40, 124.68, 122.28, 119.52, 116.69, 114.46, 7.6 Hz, H-9), 7.54 (s, 1H, H-1), 7.52 (t, 1H, J ¼ 7.6 Hz, H-10), 7.06
111.24, 92.15, 51.39, 46.58 (2C), 41.15 (2C), 11.61 (2C); found: C, (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ 9.1 Hz, H-3), 3.60 (t, 4H, J ¼ 7.7 Hz,
74.07; H, 6.47; N, 19.66. Calc. for C22H23N5: C, 73.92; H, 6.49; N, CH2), 1.74–1.66 (m, 4H, CH2), 1.44–1.37 (m, 8H, CH2), 0.92 (t,
19.59%; MS (ESI): m/z ¼ 358.2 ([M + 1]+).
6H, J ¼ 6.9 Hz, CH3); 13C NMR (75 MHz, DMSO-d6): d/ppm ¼
2-N,N-Dimethylaminobenzimidazo[1,2-a]quinoline-6-carbon- 152.04, 146.43, 144.75, 140.22, 138.75, 132.95, 130.71, 125.24,
itrile 13. Compound 13 was prepared using the above described 122.79, 120.23, 117.16, 114.33, 111.14, 111.07, 95.44, 92.86,
method from 7 (50 mg, 0.18 mmol) and 33% solution of 51.20 (2C), 29.11 (2C), 26.62 (2C), 22.49 (2C), 14.52 (2C); found:
dimethylamine in ethanol (0.18 ml, 0.90 mmol) aer 16 h of C, 78.59; H, 7.57; N, 14.03. Calc. for C26H30N4: C, 78.35; H, 7.59;
irradiation to yield 30 mg (61%) of yellow crystals; mp > 280 ꢀC. N, 14.06%; MS (ESI): m/z ¼ 399.4 ([M + 1]+).
1H NMR (300 MHz, DMSO-d6): d/ppm ¼ 8.53 (s, 1H, H-5), 8.48
2-N-Pyrrolydinylbenzimidazo[1,2-a]quinoline-6-carbonitrile
(d, 1H, J ¼ 7.8 Hz, H-11), 7.96 (d, 1H, J ¼ 7.8 Hz, H-8), 7.88 (d, 17. Compound 17 was prepared using the above described
1H, J ¼ 9.1 Hz, H-4), 7.59 (dt, 1H, J1 ¼ 1.9 Hz, J2 ¼ 7.3 Hz, H-9), method from 7 (50 mg, 0.18 mmol) and pyrrolidine (0.07 ml,
7.52 (dt, 1H, J1 ¼ 1.9 Hz, J2 ¼ 7.3 Hz, H-10), 7.51 (s, 1H, H-1), 7.07 0.90 mmol) aer 6 h of irradiation to yield 51 mg (90%) of yellow
(dd, 1H, J1 ¼ 2.1 Hz, J2 ¼ 9.1 Hz, H-3), 3.26 (s, 6H, CH3); 13C NMR crystals; mp > 295 C. H NMR (600 MHz, DMSO-d6): d/ppm ¼
1
ꢀ
(150 MHz, DMSO-d6): d/ppm ¼ 153.41, 145.83, 144.16, 139.89, 8.54 (s, 1H, H-1), 8.51 (d, 1H, J ¼ 7.9 Hz, H-11), 7.95 (d, 1H, J ¼
138.02, 132.22, 130.28, 124.74, 119.54, 116.61, 114.59, 110.90, 7.9 Hz, H-8), 7.90 (d, 1H, J ¼ 9.0 Hz, H-4), 7.61–7.52 (m, 2H, H-9
110.64, 95.25, 92.76, 40.05 (2C); found: C, 75.73; H, 4.92; N, and H-10), 7.44 (s, 1H, H-1), 6.95 (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ 9.0
19.51. Calc. for C18H14N4: C, 75.50; H, 4.93; N, 19.57%; MS (ESI): Hz, H-3), 3.67 (t, 4H, J ¼ 6.7 Hz, CH2), 2.55 (t, 4H, J ¼ 6.7 Hz,
m/z ¼ 287.1 ([M + 1]+).
CH2); 13C NMR (150 MHz, DMSO-d6): d/ppm ¼ 164.63, 153.15,
2-N,N-Diethylaminobenzimidazo[1,2-a]quinoline-6-carbonitrile 147.73, 143.82, 142.99, 138.50, 133.60, 130.78, 127.76, 125.27,
14. Compound 14 was prepared using the above described 124.20, 122.07, 115.15, 113.08, 112.88, 111.76, 48.37 (2C), 25.45
method from 6 (60 mg, 0.23 mmol) and a 70% solution of (2C); found: C, 77.11; H, 5.33; N, 17.82. Calc. for C20H16N4: C,
diethylamine in ethanol (0.17 ml, 1.60 mmol) aer 19 h of 76.90; H, 5.16; N, 17.94%; MS (ESI): m/z ¼ 313.4 ([M + 1]+).
1546 | Med. Chem. Commun., 2013, 4, 1537–1550
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Concise Article
MedChemComm
5.4 Hz, CH2), 3.70 (t, 4H, J ¼ 5.5 Hz, CH2), 3.30 (s, 6H, CH3);
13C NMR (75 MHz, DMSO-d6): d/ppm ¼ 153.55, 145.95, 144.57,
140.36, 138.32, 132.92, 130.77, 125.54, 123.56, 120.45, 116.57,
115.44, 113.43, 112.87, 99.26, 96.37, 60.49 (2C), 51.01 (2C),
41.59 (2C); found: C, 54.83; H, 4.58; N, 14.46. Calc. for
2-N-Piperidinylbenzimidazo[1,2-a]quinoline-6-carbonitrile 18.
Compound 18 was prepared using the above described method
from 7 (50 mg, 0.18 mmol) and piperidine (0.09 ml, 0.90 mmol)
aer 6 h of irradiation to yield 45 mg (85%) of yellow crystals; mp
254–256 ꢀC. 1H NMR (600 MHz, DMSO-d6): d/ppm ¼ 8.56 (s, 1H,
H-5), 8.52 (d, 1H, J ¼ 8.2 Hz, H-11), 7.97 (d, 1H, J ¼ 8.2 Hz, H-8),
7.90 (d, 1H, J ¼ 9.1 Hz, H-4), 7.79 (bs, 1H, H-1), 7.59 (dt, 1H, J1 ¼
1.4 Hz, J2 ¼ 7.3 Hz, H-9), 7.54 (dt, 1H, J1 ¼ 1.5 Hz, J2 ¼ 7.3 Hz, H-
10), 7.30 (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ 9.2 Hz, H-3), 3.69–3.62 (m, 6H,
CH2), 1.67 (t, 4H, J ¼ 6.3 Hz, CH2), 1.23 (s, 2H); 13C NMR (150
MHz, DMSO-d6): d/ppm ¼ 154.54, 146.27, 144.70, 140.15, 138.82,
132.86, 130.83, 125.27, 123.34, 120.16, 116.90, 115.05, 112.99,
112.31, 97.83, 94.34, 48.57 (2C), 25.46 (2C), 24.23; found: C, 77.55;
H, 5.78; N, 17.09. Calc. for C21H18N4: C, 77.28; H, 5.56; N, 17.17%;
MS (ESI): m/z ¼ 327.2 ([M + 1]+).
C
22H22IN5: C, 54.67; H, 4.59; N, 14.49%; MS (ESI): m/z ¼ 356.4
([M ꢃ Iꢃ]+).
Antiproliferative evaluation assay
The experiments were carried out on three human cell lines,
which are derived from three cancer types. The following cell
lines were used: HCT116 (colon carcinoma), H460 (lung carci-
noma) and MCF-7 (breast carcinoma). The cells were cultured
as monolayers and maintained in Dulbecco's modied Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 2 mM L-glutamine, 100 U per ml penicillin and 100 mg
per ml streptomycin in a humidied atmosphere with 5% CO2
2-N-Morpholinylbenzimidazo[1,2-a]quinoline-6-carbonitrile 19.
Compound 19 was prepared using the above described method
from 6 (60 mg, 0.23 mmol) and morpholine (0.12 ml, 1.40 mmol)
aer 4 h of irradiation to yield 42 mg (56%) of yellow crystals; mp >
280 ꢀC. 1H NMR (300 MHz, DMSO-d6): d/ppm ¼ 8.52 (s, 1H, H-5),
8.49 (d, 1H, J ¼ 8.1 Hz, H-11), 7.96 (d, 1H, J ¼ 8.1 Hz, H-8), 7.89 (d,
1H, J ¼ 9.1 Hz, H-4), 7.73 (s, 1H, H-1), 7.59 (t, 1H, J ¼ 7.4 Hz, H-9),
7.53 (t, 1H, J ¼ 7.2 Hz, H-10), 7.27 (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ 9.0 Hz,
ꢀ
at 37 C.
The growth inhibition activity was assessed as described
previously.21 The cell lines were inoculated onto a series of
standard 96-well microtiter plates on day 0, at 3 ꢂ 104 cells per
ml (HCT116, H460) to 5 ꢂ 104 cells per ml (MCF-7), depending
on the doubling times of a specic cell line. Test agents were
then added in ten-fold dilutions (10ꢃ8 to 10ꢃ4 M) and incubated
for further 72 h. Working dilutions were freshly prepared on the
day of testing. Aer 72 h of incubation the cell growth rate was
evaluated by performing the MTT assay, which detects dehy-
drogenase activity in viable cells. The absorbance (A) was
measured using 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:
H-11), 3.85 (t, 4H, J ¼ 4.6 Hz, CH2), 3.56 (t, 4H, J ¼ 4.6 Hz, CH2); 13
C
NMR (75 MHz, DMSO-d6): d/ppm ¼ 163.16, 154.73, 144.54, 140.32,
138.43, 132.75, 130.74, 125.38, 123.43, 120.19, 116.81, 115.31,
113.20, 112.69, 98.03, 95.21, 66.34 (2C), 42.24 (2C); found: C, 73.44;
H, 4.89; N, 17.04. Calc. for C20H16N4O (326.3): C, 73.15; H, 4.91; N,
17.06%; MS (ESI): m/z ¼ 329.2 ([M + 1]+).
2-N-Piperazinylbenzimidazo[1,2-a]quinoline-6-carbonitrile 20.
Compound 20 was prepared using the above method from 7 (50
mg, 0.18 mmol) and piperazine (780 mg, 0.90 mmol) aer 10 h of
irradiation to yield 33 mg (56%) of yellow crystals; mp 192–196
ꢀC. 1H NMR (600 MHz, DMSO-d6): d/ppm ¼ 8.87 (s, 1H, NH), 8.54
(s, 1H, H-5), 8.43 (d, 1H, J ¼ 8.0 Hz, H-11), 7.94 (d, 1H, J ¼ 8.0 Hz,
H-8), 7.83 (dd, 1H, J1 ¼ 1.2 Hz, J2 ¼ 9.2 Hz, H-4), 7.66 (s, 1H, H-1),
7.58 (t, 1H, J ¼ 8.2 Hz, H-9), 7.52 (t, 1H, J ¼ 8.2 Hz, H-10), 7.24
(dd, 1H, J1 ¼ 1.3 Hz, J2 ¼ 9.2 Hz, H-3), 3.49 (t, 4H, J ¼ 4.5 Hz,
CH2), 2.93 (t, 4H, J ¼ 4.5 Hz, CH2); 13C NMR (150 MHz, DMSO-
d6): d/ppm ¼ 154.12, 145.55, 144.03, 139.66, 137.94, 132.17,
130.18, 124.79, 122.85, 119.61, 116.37, 114.67, 112.22, 112.20,
97.33, 94.24, 47.16 (2C), 44.89 (2C); found: C, 73.18; H, 5.15; N,
21.55. Calc. for C20H19N5: C, 73.37; H, 5.23; N, 21.34%; MS (ESI):
m/z ¼ 328.3 ([M + 1]+).
If (mean Atest ꢃ mean Atzero) $ 0, then PG ¼ 100
ꢂ (mean Atest ꢃ mean Atzero)/(mean Actrl ꢃ mean Atzero).
If (mean Atest ꢃ mean Atzero) < 0, then: PG ¼ 100 ꢂ (mean Atest
ꢃ 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
measurements aer the desired period of time and the mean
Actrl is the average of optical density measurements aer 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 concentration–response
curves using linear regression analysis by tting the test
concentrations that give PG values above and below the refer-
ence value (i.e. 50%). If, however, all of the tested concentrations
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.
Each test was performed in quadruplicate in at least two indi-
vidual experiments.
2-N-(4-N,N-Dimethylpiperazin-1-yl)benzimidazo[1,2-a]-quin-
oline-6-carbonitrile iodide 21. A mixture of compound 20 (50
mg, 0.15 mmol) and anhydrous potassium carbonate (21 mg,
0.15 mmol) was reuxed in acetonitrile (20 ml) with methyl
iodide (0.03 ml, 0.46 mmol) for 2 h. The reaction mixture was
concentrated under reduced pressure to a volume of 5 ml and
ltered off to yield pure compound 21 as yellow powder (50
mg, 67%); mp 270–273 ꢀC. 1H NMR (600 MHz, DMSO-d6):
d/ppm ¼ 8.67 (d, 1H, J ¼ 8.4 Hz, H-11), 8.66 (s, 1H, H-5), 8.05
(d, 1H, J ¼ 8.4 Hz, H-8), 8.03 (d, 1H, J ¼ 8.9 Hz, H-4), 7.92 (s,
1H, H-1), 7.65 (t, 1H, J ¼ 7.6 Hz, H-9), 7.59 (t, 1H, J ¼ 7.6 Hz, H-
10), 7.44 (dd, 1H, J1 ¼ 1.9 Hz, J2 ¼ 8.8 Hz, H-3), 4.01 (t, 4H, J ¼
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 1537–1550 | 1547

View Article Online
MedChemComm
Concise Article
ꢀ
mixture was incubated at 32 C for 3 h. Aerwards, GFP uo-
rescence was recorded on microtiter plates using a Fluoroskan
Ascent Microplate Fluorometer (excitation at 485 nm, emission
at 538 nm; ThermoScientic). The mean uorescence of negative
controls was subtracted from the means of tested and control
samples and percentages from control were calculated. A
minimum of three experiments were performed, and statistical
difference was calculated using Microso Excel t-test.
Cell cycle analysis
Tumor cells were seeded per well into a 6-well plate (2 ꢂ 105 per
well). Aer 24 hours the tested compounds were added at various
concentrations (as shown in the Results section). Aer the desired
length of time the attached cells were trypsinized, combined with
oating cells, washed with phosphate buffer saline (PBS), xed
ꢀ
with 70% ethanol and stored at ꢃ20 C. Immediately before the
analysis, the cells were washed with PBS and stained with 50 mg
mlꢃ1 of propidium iodide (PI) with the addition of 0.2 mg mlꢃ1 of
RNAse A. The stained cells were then analyzed with a FACScalibur
(Becton Dickenson) ow cytometer (20 000 counts were
measured). The percentage of the cells in each cell cycle phase was
determined using the ModFit LTꢀ soware (Verity Soware
House) based on the DNA histograms. The tests were performed
in duplicates and repeated at least twice.
DNA binding
DNA melting temperature studies. CT-DNA (Sigma Aldrich,
France) was dissolved and dialyzed overnight in water prior to
use. Tested compounds were dissolved in DMSO (10 mM stock
solutions), aliquoted and stored at ꢃ20 ꢀC to then be extem-
porarily diluted at 10 mM in 1 ml of BPE buffer (6 mM Na2HPO4,
2 mM NaH2PO4, 1 mM EDTA, pH 7.1) and incubated (analyzed
sample) or not (reference sample) with 20 mM of CT-DNA (drug/
DNA ratio ¼ 0.5). The absorbence of DNA was measured at
260 nm in quartz cells using an Uvikon 943 spectrophotometer
thermostatted with a Neslab RTE111 cryostat every minute over
a range of 20 to 100 ꢀC with an increment of 1 ꢀC per min. The
melting temperature (Tm) values were obtained from the
midpoint of the hyperchromic transition obtained from rst-
derivative plots. The variation of melting temperature (DTm) was
measured by subtracting the melting temperature measure-
ment of 20 mM of CT-DNA incubated alone (control Tm) from
that obtained with DNA incubated with increasing concentra-
tions of the various tested compounds (DTm values ¼ Tm[Drug+DNA]
ꢃ Tm[DNA alone]).
Intracellular distribution of compounds
H460 cells were seeded on round microscopic cover slips placed
in 24-well-plates (10 000 cells per well) and grown at 37 ꢀC for 24
h in DMEM, as described above. Cells were then incubated with
compounds 10, 11, 20 and 21 at 10 mM nal concentrations for 2
h. Cover slips were rinsed twice with PBS, placed on the
microscopic slides and immediately analyzed. The uptake and
intracellular distribution of tested chemicals were analyzed
under a uorescence microscope (Olympus BX51) and recorded
with an Olympus DP70 Digital Camera. Also, confocal laser
scanning microscopy was performed using a Leica TCS SP2
AOBS confocal microscope equipped with an HCX PL APO
l-Blue 63 ꢂ /1.4 objective (Leica Microsystems). Fluorescence
and transmission images were acquired simultaneously. For
illumination, the 405 nm line from an argon-ion gas laser was
used and uorescence was collected in the 424–527 nm range.
Confocal uorescence images were taken using the pinhole size
of 114 mm (1 Airy unit).
UV/visible and circular dichroism (CD) spectrometries
The UV/visible spectra were recorded from 230 nm to 430 nm in
a quartz cuvette of 10 mm path length using an Uvikon XL
spectrophotometer and referenced against a cuvette containing
BPE. Increasing concentrations of CT-DNA (from 10 to 100 mM
with 10 mM steps and then from 100 to 200 mM with steps of 20
mM of base pairs) were added in both the drug (20 mM in BPE
buffer) and the reference (BPE buffer alone) cuvettes and the
spectra were recorded stepwise. For CD spectra, CT-DNA (50
mM) was incubated with or without (control) a increasing
concentrations of the tested drugs (from 1 to 50 mM) in BPE in a
quartz cell of 10 mm path length. The CD spectra were collected
from 480 to 230 nm using a J-810 Jasco spectropolarimeter at a
Inhibition of in vitro translation
The compounds 10, 11, 20 and 21 were tested for inhibitory
effect on translation of Enhanced Green Fluorescence Protein
(EGFP) using an E. coli derived cell-free protein synthesis system
(S30 T7 High-Yield Protein Expression System, Promega, USA). It
can produce high levels of recombinant proteins if supple-
mented with an appropriate expression plasmid, T7 RNA poly-
merase for transcription, and other necessary components for
translation such as amino acids. Briey, 500 ng of pEGFP–C1
plasmid (Clontech, USA) was incubated with the tested
ꢀ
controlled temperature of 20 C xed by a PTC-424S/L peltier
type cell changer (Jasco) essentially as described previously.26
Topoisomerase I-mediated DNA relaxation and
topoisomerases cleavage assays
ꢀ
compounds (at 50 mM concentration) for 1 h at 24 C. Positive
controls contained the plasmid in sterile water and negative
controls did not contain the DNA template (plasmid), respec- Topoisomerase I-mediated DNA relaxation experiments were
tively. Along with the tested compounds, commercial DNA- performed as previously described27 with graded concentrations
damage-inducing anticancer drugs doxorubicine, camptothecin, of the tested compounds incubated with supercoiled pUC19
cisplatin and chlorambucyl (50 mM concentration, Sigma) were plasmid prior to the addition of human topoisomerase I
also used for comparison. Aer that, the protein expression (4 units, Topogen, USA) for 45 min at 37 ꢀC in relaxation buffer.
system was deployed according to the manufacturer’s recom- Addition of SDS (0.25%) and proteinase K (250 mg mlꢃ1) for a 30
ꢀ
mendations and protocol with slight modication of incubation min incubation at 50 C stopped the reaction. The DNA forms
temperature to ensure optimal GFP folding. Instead of 37 ꢀC the were separated on a 1% agarose gel without ethidium bromide
1548 | Med. Chem. Commun., 2013, 4, 1537–1550
This journal is ª The Royal Society of Chemistry 2013

View Article Online
Concise Article
MedChemComm
for 2 h at 120 V in TBE buffer. Gels were stained post-electro- Nhili and the IRCL for technical expertise (Sabine Depauw) and
phoresis in an ethidium bromide containing-bath, washed and post-doctoral fellowship (Raja Nhili).
photographed under UV light. Topoisomerase I DNA cleavage
assays were performed in the same condition but samples being
loaded on a 1% agarose gel containing ethidium bromide.
References
Topoisomerase II DNA cleavage assays were performed as
described.16 Camptothecin (CPT, 20 mM) and etoposide (50 mM)
were used as poisons for topoisomerase I and II, respectively.
1 S. Demirayak, U. Abu Mohsen and A. Cagri Karaburun, Eur.
J. Med. Chem., 2002, 37, 255–260.
ˇ
´
2 M. Hranjec, M. Kralj, I. Piantanida, M. Sedic, L. Suman,
´
K. Pavelic and G. Karminski-Zamola, J. Med. Chem., 2007,
50, 5696–5711.
DNase I footprinting
3 Y. Bansal and O. Silakari, Bioorg. Med. Chem., 2012, 20, 6208–
6236.
The 265 bp DNA fragment was obtained from EcoRI and PvuII
double digestion of the pBluscript plasmid (Stratagene, La Jolla,
CA) followed by 30-end labeled at the EcoRI site upon incorpo-
ration of a-[32P]-dATP (3000 Ci mmolꢃ1, PerkinElmer, France)
by Klenow enzyme for 30 min at 37 ꢀC. Aer separation on a 6%
native polyacrylamide gel, the portion of the gel containing the
265 bp radio-labeled DNA fragment was cut off from the gel,
crushed, dialyzed overnight and ltered through a 0.22 mm
membrane (Millipore). DNA fragments were nally precipitated
from ethanol, dried and dissolved in an appropriate amount of
MQ water. DNase I footprinting experiments were performed
essentially as previously described.27 The 265 bp radio-labeled
DNA fragment was incubated with increasing concentrations of
the tested compounds (15 min at 37 ꢀC) prior to mild digestion
for 4 min with DNase I (0.002 unit per ml) in reaction buffer
(20 mM NaCl, 2 mM MgCl2, 2 mM MnCl2, pH 7.3). The reaction
was stopped by freeze-drying, lyophilisation and subsequent
dissolution in 4 ml of denaturing loading buffer (80% form-
amide solution containing tracking dyes). The DNA samples
were then heated at 90 ꢀC for 4 min, chilled on ice for another 4
min and separated on denaturing 8% polyacrylamide gels
containing 8 M urea for 90 min at 65 W in TBE buffer (89 mM
Tris base, 89 mM boric acid, 2.5 mM Na2EDTA, pH 8.3). Aer
migration, gels were soaked in 10% acetic acid, transfꢀerred to
Whatman 3 MM paper and dried under vacuum at 80 C to be
exposed on a storage screen, and the results were collected
using a Molecular Dynamics STORM 860. Each base was local-
ized from comparison with the bands from dimethyl-sulfate
(DMS) and piperidine treatment revealing purines with a pref-
erence for guanines (strong cut) over adenines (weaker cuts)
and is referred to on gels as G+a-track.
4 L. W. Deady, T. Rodemann, G. J. Finlay, B. C. Baguley and
W. A. Denny, Anti-Cancer Drug Des., 2000, 15, 339–346.
5 M. Demeunynck, C. Bailly and W. D. Wilson, in D.N.A. and
R.N.A. Binders, Wiley-VCH, Weinheim, 2002.
6 W. D. Wilson, B. Nguyen, F. A. Tanious, A. Mathis, J. E. Hall,
C. E. Stephens and D. W. Boykin, Curr. Med. Chem.: Anti-
Cancer Agents, 2005, 5, 389–408.
7 R. B. Silverman, The Organic Chemistry of Drug Design and
Drug Action, Elsevier Academic Press, 2nd edn, 2004.
´
´
´
8 R. Martınez and L. Chacon-Garcıa, Curr. Med. Chem., 2005,
12, 127–151.
´
´
´
´
´
9 P. Vivas-Mejıa, J. L. Rodrıguez-Caban, M. Dıaz-Velazquez,
´
´
M. G. Hernandez-Perez, O. Cox and F. A. Gonzalez, Mol.
Cell. Biochem., 1997, 177, 69–77.
10 C. Bailly, Curr. Med. Chem., 2000, 7, 39–58.
11 M. F. Brana, M. Cacho, A. Gradillas, B. de Pascual-Teresa and
A. Ramos, Curr. Pharm. Des., 2001, 7, 1745–1780.
12 K. Hirano, Y. Oderaotoshi, S. Minataka and M. Komatsu,
Chem. Lett., 2001, 1262–1263.
13 V. B. Kovalska, D. V. Kryvorotenko, A. O. Balanda,
M. Y. Losytsky, V. P. Tokar and S. M. Yarmoluk, Dyes
Pigm., 2005, 67, 47–54.
´
14 N. Perin, M. Hranjec, G. Pavlovic and G. Karminski-Zamola,
Dyes Pigm., 2011, 91, 79–88.
15 M. Hranjec, I. Piantanida, M. Kralj, L. Suman, K. Pavelic and
ˇ
´
G. Karminski-Zamola, J. Med. Chem., 2008, 51, 4899–4910.
´
´
16 (a) M. Hranjec, G. Pavlovic, M. Marjanovic, M. Kralj and
G. Karminski-Zamola, Eur. J. Med. Chem., 2010, 45, 2405–
2417; (b) N. Perin, L. Uzelac, I. Piantanida, G. Karminski-
Zamola, M. Kralj and M. Hranjec, Bioorg. Med. Chem.,
2011, 19, 6329–6339.
´
17 M. F. Brana, J. M. Castellano, G. Keilhaurer, A. Machuca,
Acknowledgements
Y. Martin, C. Redondo, E. Schlick and N. Walker, Anti-
Cancer Drug Des., 1994, 9, 527–538.
We greatly appreciate the nancial support of the Croatian
Ministry of Science Education and Sports (Projects 125-0982464- 18 D. Gunther and R. Erckel, DE 2640 549 A1.
1356, 098-0982464-2514) and the bilateral Hubert Curien part- 19 A. Masta, P. J. Gray and D. R. Phillips, Nucleic Acids Res.,
nership between Croatian and French institutions (Cogito
program) as the Egide Project no. 24765PH. We are thankful to 20 Q. Zhou, Y. Qu, J. B. Mangrum and X. Wang, Chem. Res.
Dr Igor Weber for help with confocal microscopy experiments. Toxicol., 2011, 24, 402–411.
M. H. David-Cordonnier is grateful to the Ligue Nationale 21 R. P. Hertzberg, M. J. Caranfa and S. M. Hecht, Biochemistry,
1995, 23, 3508–3515.
´
Contre le Cancer (Comite du Pas-de-Calais, Septentrion), the
Association Laurette Fugain and the Association pour la 22 K. A. Heminger, S. D. Hartson, J. Rogers and R. L. Matts,
Recherche sur le Cancer for grants; the Institut pour la Arch. Biochem. Biophys., 1997, 344, 200–207.
Recherche sur le Cancer de Lille (IRCL), the CHRU de Lille and 23 R. L. Momparler, M. Karon, S. E. Siegel and F. Avila, Cancer
1989, 28, 4629–4638.
´
the Region Nord/Pas-de-Calais for a PhD fellowship to Raja
Res., 1976, 36, 2891–2895.
This journal is ª The Royal Society of Chemistry 2013
Med. Chem. Commun., 2013, 4, 1537–1550 | 1549

View Article Online
MedChemComm
Concise Article
24 P. Peixoto, C. Bailly and M. H. David-Cordonnier, Methods 26 T. Lemster, U. Pindur, S. Depauw, G. Lenglet, C. Dassi and
Mol. Biol., 2010, 613, 235–256.
25 A. Lansiaux, L. Dassonneville, M. Facompre, A. Kumar,
M. H. David-Cordonnier, Eur. J. Med. Chem., 2009, 44,
3235–3252.
´
C. E. Stephens, M. Bajic, F. Tanious, W. D. Wilson, 27 P. Peixoto, Y. Liu, S. Depauw, M. P. Hildebrand,
D. W. Boykin and C. Bailly, J. Med. Chem., 2002, 45, 1994–
D. W. Boykin, C. Bailly, W. D. Wilson and M. H. David-
2002.
Cordonnier, Nucleic Acids Res., 2008, 36, 3341–3353.
1550 | Med. Chem. Commun., 2013, 4, 1537–1550
This journal is ª The Royal Society of Chemistry 2013