Novel biologically active nitro and amino substituted benzimidazo [1,2-a]quinolines

Article abstract of DOI:10.1016/j.bmc.2011.09.002

This manuscript described the synthesis and biological activity of novel nitro substituted E-2-styrylbenzimidazoles and E-2-(2-benzimidazolyl)-3- phenylacrylonitriles and nitro and amino substituted benzimidazo[1,2-a] quinolines (4-5, 6-11, 17-20, and 21-32). All of the compounds showed significant growth inhibitory effect towards five tumor cell lines, whereby the IC₅₀ concentrations of 11, 20, 28, 29, 30, 32 are in the low micromolar range (IC₅₀ = 2-19 μM). The DNA binding experiments did not show significant affinity of two selected compounds towards ct-DNA. The flow cytometry analysis of potential cell cycle perturbations after the treatment with compounds 9, 11, 25, and 29 demonstrated that all of the compounds (5 μM ≈ IC₅₀) significantly delayed the progression through G1 phase, as demonstrated by the accumulation of cells in G1 phase, accompanied with the reduction of the cell number in the cells in S phase, which does not point to DNA damage as the main mechanism of action. Also, fluorescence microscopy study showed cytoplasmic distribution of the compounds, demonstrating that DNA is not the primary target of compounds. Thus, considerable antiproliferative effects of studied compounds are due to interactions with other biological targets within cells.

Full text of DOI:10.1016/j.bmc.2011.09.002

Bioorganic & Medicinal Chemistry 19 (2011) 6329–6339
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel biologically active nitro and amino substituted benzimidazo
[1,2-a]quinolines
Nataša Perin a, Lidija Uzelac b, Ivo Piantanida c, Grace Karminski-Zamola a, Marijeta Kralj b,
,
⇑
Marijana Hranjec a,
⇑
a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Maruli´cev Trg 20, PO Box 177, HR-10000 Zagreb, Croatia
Division of Molecular Medicine, Laboratory of Experimental Therapy, Ruder Boškovi´c Institute, Bijenicka cesta 54, PO Box 180, HR-10000 Zagreb, Croatia
Division of Organic Chemistry and Biochemistry, Laboratory for Study of Interactions of Biomacromolecules, Ruder Boškovic Institute, Bijenicka Cesta 54, PO Box 180,
b
-
ˇ
c
-
ˇ
´
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:
Received 13 July 2011
Revised 1 September 2011
Accepted 2 September 2011
Available online 10 September 2011
This manuscript described the synthesis and biological activity of novel nitro substituted E-2-styryl-
benzimidazoles and E-2-(2-benzimidazolyl)-3-phenylacrylonitriles and nitro and amino substituted
benzimidazo[1,2-a]quinolines (4–5, 6–11, 17–20, and 21–32). All of the compounds showed significant
growth inhibitory effect towards five tumor cell lines, whereby the IC50 concentrations of 11, 20, 28,
29, 30, 32 are in the low micromolar range (IC50 = 2–19 lM). The DNA binding experiments did not show
significant affinity of two selected compounds towards ct-DNA. The flow cytometry analysis of potential
cell cycle perturbations after the treatment with compounds 9, 11, 25, and 29 demonstrated that all of the
Keywords:
Benzimidazoles
Benzimidazo[1,2-a]quinolines
Antiproliferative activity
Interaction with ct-DNA
Cytometry analysis
compounds (5
l
M ꢀ IC50) significantly delayed the progression through G1 phase, as demonstrated by
the accumulation of cells in G1 phase, accompanied with the reduction of the cell number in the cells
in S phase, which does not point to DNA damage as the main mechanism of action. Also, fluorescence
microscopy study showed cytoplasmic distribution of the compounds, demonstrating that DNA is not
the primary target of compounds. Thus, considerable antiproliferative effects of studied compounds
are due to interactions with other biological targets within cells.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
agents possessing different chemical and pharmacological features
which impart them diverse biological properties like anticancer,5–7
Since classical chemotherapy, which uses small molecules or
bioactive natural products, is still the mainstay of cancer treat-
ment, there has been a tremendous growth in recent years in the
number and types of new heterocyclic anticancer agents with an
emphasis on creating new DNA active drugs. DNA molecule is
one of the principal targets for anticancer agents designed to block
the proliferation of cancer cells, since DNA synthesis and replica-
tion are important processes for cellular growth.1 Understanding
of the molecular basis for cytotoxicity by anticancer agents is very
important for the development of novel, more selective and effi-
cient agents with greater specificity of action.2–4 The benzimid-
azole unit which has a structural similarity with purine, is also
the key building block for a variety of derivatives that are known
to play crucial roles in the functions of a number of biologically
important molecules. Besides, benzimidazoles represent the major
backbone of numerous of synthetic medicinal and biochemical
antiviral,8,9 antibacterial,10,11 antifungal,12 antihistaminic13 and
anticonvulsant activity.14
As a part of our continuing search for potential anticancer agents
related to benzimidazole derivatives, we have recently reported on
the synthesis, cytostatic evaluation, DNA/RNA interaction study and
proteomic profiling of a series of amidino-substituted heterocyclic
benzimidazoles and benzimidazo[1,2-a]quinolines.15,16 Biological
study confirmed the anticancer potential of this class of compounds,
especially that of positively charged analogs of benzimidazo[1,2-
a]quinolines which intercalate into ds-DNA or RNA. Thus, the most
promising 2-imidazolinyl amidino substituted compounds inhib-
ited tumor growth, caused severe disturbance of the cell cycle,
impairment in mitotic progression, and inhibited topoisomerases,
which was related to their high DNA binding capacity. Recent
publications also showed that nitro substituted benzazolo[3,2-
a]quinolinium derivatives are potential antineoplastic agents that
interacts with DNA by intercalation and that topoisomerase II is
an important target for their biological action.17,18 It was also con-
cluded that the cytotoxic properties of these compounds appeared
to be more dependent on their ability to inhibit topoisomerase II
than on DNA binding affinity or intercalation.19 Substituted, earlier
⇑
Corresponding authors. Tel.: +385 14597245; fax: +385 14597250 (M.H.);
tel.: +385 1 4571 235; fax: +385 1 4561 010 (M.K.).
E-mail addresses: mhorvat@irb.hr, marijeta.kralj@irb.hr (M. Kralj), mhranjec@f
kit.hr (M. Hranjec).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.002

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N. Perin et al. / Bioorg. Med. Chem. 19 (2011) 6329–6339
prepared benzimidazo[1,2-a]quinoline-6-carbonitriles exerted pro-
nounced antiproliferative activity with the cyano moiety important
for the activity but not the selectivity of tested compounds.
Some of above mentioned compounds also showed significant
interaction with ct-DNA, supporting the fact that their antitumor
activity could partially be the consequence of DNA-binding.20
Prompted by the results from the previously reported studies,
we set out to explore and synthesize novel acyclic nitro 4, 5,
17–20 and cyclic nitro 6, 7, and 21–24 and amino 8–9 and 25–28
substituted benzimidazole[1,2-a]quinolines with the substituents
on the different positions of the condensed tetracyclic ring. The
hydrochloride salts 10–11 and 29–32 were prepared to improve
their solubility in water (Fig. 1). Newly synthesized compounds
were tested for their antiproliferative activity on the panel of
several human tumor cell lines while study of interaction with
calf thymus DNA (ct-DNA) was performed for compounds 11 and
25.
[1,2-a]quinolines 10–11 and 29–32 were prepared with HCl gas
in order to ensure better solubility.
All structures of novel E-2-styryl-benzimidazoles 4–5, E-2
-(2-benzimidazolyl)-3-phenylacrylonitriles
17–20,
benzimi-
dazo[1,2-a]quinolines 6–11 and benzimidazo[1,2-a]quinolines-6-
carbonitriles 21–32 were determined by the NMR analysis based
on the analysis of H–H coupling constants as well as chemical
shifts. In 1H NMR spectra of E-2-styryl-benzimidazoles 4–5 besides
all other aromatic protons, two doublets for trans-ethylenic pro-
tons with coupling constants of 16.5 Hz can be observed. Singlet
of the NH group of benzimidazole nuclei is placed around
13 ppm. The photocyclization reaction leads to a downfield shift
of the signals of the ethylenic protons and most other aromatic
protons as well as disappearance of the NH group at the benzimid-
azole nuclei, thus confirming the cyclic structure formation. In 1H
NMR spectra doublets for two protons on quinoline ring, with cou-
pling constants 9.5 Hz, can be observed and these values are char-
acteristic for this type of fused compounds.
2. Results and discussion
2.1. Chemistry
2.2. Interaction of compounds 11 and 25 with ct-DNA in an
aqueous medium
2.2.1. UV/Vis, fluorimetric and CD titrations
All newly prepared compounds ( Fig. 1) were synthesized
according to the two main procedures shown in Scheme 1, by
the conventional methods of organic synthesis for the preparation
of similar heterocyclic compounds starting from the corresponding
o-phenylenediamines 1–2. In the cyclocondensation reaction
Since application of spectrophotometric methods is necessary for
DNA binding studies of compounds 11 and 25, their aqueous buf-
fered solutions were characterized by means of electronic absorp-
tion (UV/Vis) and fluorescence emission spectroscopy (Table 1).
Stock solution of compound 11 was prepared in water
(c(11) = 1.05 ꢁ 10ꢂ3 mol dmꢂ3) while stock solution of compound
25 was prepared, due to the poor solubility in water, in DMSO
(c(25) = 1.68 ꢁ 10ꢂ3 mol dmꢂ3). Small aliquots of DMSO stock solu-
tion of compound 25 were added into the aqueous medium, pro-
vided that DMSO content in experiments was less than 1%. Stock
solutions were stable over prolonged periods of time. Absorbencies
of aqueous buffered solutions are proportional to their concentra-
tions up to 1.52 ꢁ 10ꢂ5 mol dmꢂ3 (11) and 2.5 ꢁ 10ꢂ5 mol dmꢂ3
(25) indicating that there is no significant intermolecular stacking
which should give rise to hypochromic effects. Studied compounds
11 and 25 exhibited strong fluorescence emission with maximum
at 521 nm and 562 nm. Fluorescence emissions of 11 and 25 were
proportional to their concentration in the range from 4 ꢁ
10ꢂ8 mol dmꢂ3 to 2 ꢁ 10ꢂ6 mol dmꢂ3, and their excitation spectra
were in concordance with the corresponding absorption spectra.
Absorption maxima, corresponding molar extinction coefficients
between the E-3-(2-chloro-5-nitrophenyl)acrylic acid
3 with
o-phenylenediamines 1–2 in polyphosphoric acid (PPA), E-2
-styryl-benzimidazoles 4–5 were prepared. In the reaction be-
tween o-phenylenediamines 1–2 with 2-cyanoacetamide corre-
sponding substituted 2-cyanomethylbenzimidazoles 12–14 were
prepared. They gave in the reaction of aldol condensation with aro-
matic aldehydes 15–16 substituted E-2-(2-benzimidazolyl)-3-
phenylacrylonitriles 17–20.
Cyclic nitro substituted derivatives of benzimidazo[1,2-a]quin-
olines 6–7 and benzimidazo[1,2-a]quinolines-6-carbonitriles
21–24 were prepared by the thermal reaction using sulfolane for
dehydrohalogenation cyclization at 280 °C followed by UV/Vis
spectroscopy.21 Acyclic derivatives 5 and 18–20 with substituents
on the benzimidazole nuclei gave in the cyclization reaction two
inseparable regioisomers. Amino substituted cyclic derivatives
8–9 and 25–28 were prepared from nitro substituted compounds
by reduction with SnCl2 ꢁ 2H2O in MeOH and concentrated HCl.
The hydrochloride salts of amino substituted benzimidazo
(e) and fluorescence emission maxima are presented in Table 1.
R1
R2
H
R2
Cl R3
N
N
N
N
R3
R1
4
5
R1=NO2, R2=H, R3=H
R1=NO2, R2=NO2, R3=H
6
7
8
9
R1=NO2, R2=H, R3=H
R1=NO2, R2=NO2, R3=H
R1=NH2, R2=H, R3=H
R1=NH2, R2=NH2, R3=H
24 R1=NO2, R2=CH3, R3=CN
25 R1=NH2, R2=H, R3=CN
17 R1=NO2, R2=H, R3=CN
18 R1=H, R2=NO2, R3=CN
19 R1=NO2, R2=NO2, R3=CN
20 R1=NO2, R2=CH3, R3=CN
26 R1=H, R2=NH2, R3=CN
27 R1=NH2, R2=NH2, R3=CN
28 R1=NH2, R2=CH3, R3=CN
29 R1=NH3+Cl-, R2=H, R3=CN
30 R1=H, R2=NH3+Cl-, R3=CN
31 R1=NH3+Cl-, R2=NH3+Cl-, R3=CN
32 R1=NH3+Cl-, R2=CH3, R3=CN
10 R1=NH3+Cl-, R2=H, R3=H
11 R1=NH3+Cl-, R2=NH3+Cl-, R3=H
21 R1=NO2, R2=H, R3=CN
22 R1=H, R2=NO2, R3=CN
23 R1=NO2, R2=NO2, R3=CN
Figure 1. Prepared E-2-styryl-benzimidazoles 4–5, E-2-(2-benzimidazolyl)-3-phenylacrylonitriles 17–20 and benzimidazo[1,2-a]quinolines 6–11 and 21–32.

N. Perin et al. / Bioorg. Med. Chem. 19 (2011) 6329–6339
6331
R2
NH2
NH2
Cl
CNCH2CONH2
H
PPA
1 R2=H
2 R2=NO2
R1
COOH
R2
CN
N
3 R1=NO2
H
N
R2
Cl
N
12 R2=NO2
13 R2=H
N
14 R2=CH3
4 R1=NO2, R2=H
5 R1=NO2, R2=NO2
Cl
EtOH
piperidine
R1
sulfolane
R1
CHO
15 R1=NO2
16 R1=H
R1
R2
H
N
R2
Cl NC
N
N
N
6 R1=NO2, R2=H
7 R1=NO2, R2=NO2
17 R1=NO2, R2=H
18 R1=H, R2=NO2
19 R1=NO2, R2=NO2
20 R1=NO2, R2=CH3
R1
1. SnCl2×2H2O/MeOH, HCl
2. EtOH, HClg
R1
sulfolane
R1
R2
R2
N
N
N
N
8 R1=NH2, R2=H
9 R1=NH2, R2=NH2
CN
10 R1=NH3+Cl-, R2=H
21 R1=NO2, R2=H
22 R1=H, R2=NO2
23 R1=NO2, R2=NO2
24 R1=NO2, R2=CH3
11 R1=NH3+Cl-, R2=NH3+Cl-
1. SnCl2×2H2O/MeOH, HCl
2. EtOH, HClg
R1
R2
N
N
CN
29 R1=NH3+Cl-, R2=H
30 R1=H, R2=NH3+Cl-
25 R1=NH2, R2=H
26 R1=H, R2=NH2
27 R1=NH2, R2=NH2
28 R1=NH2, R2=CH3
31 R1=NH3+Cl-, R2=NH3+Cl-
32 R1=NH3+Cl-, R2=CH3
Scheme 1.
Addition of ct-DNA to a buffered solution of compounds 11 and
25 resulted in bathochromic and hypochromic effects of UV/Vis
spectra of studied compounds (Fig. 2). as follows: H (11, 352 nm)
Table 1
Electronic absorption maxima, molar extinction coefficients and fluorescence emis-
sion maxima of studied compounds in sodium cacodylate buffer (I = 0.05 mol dmꢂ3
,
= 44%,
D
k (11, 352 nm) = 20 nm and H (25, 347 nm) = 28%,
Dk (25,
pH 7)
347 nm) = 13 nm, H (25, 362 nm) = 17%,
D
k (25, 362 nm) = 14 nm
Compd
11
Absorption maxima
Fluorescence
emission (kmax/nm)
(H (hypochromic effect) = (Abs (compound) ꢂ Abs (complex))/Abs
kmax/nm
e
ꢁ 103/dm3 molꢂ1 cmꢂ1
(compound)) ꢁ 100; values Abs (complex) estimated by non-linear
extrapolation; Dk (shift of the absorbance maximum) = k (complex)
353
261
251
347
273
23.14
20.93
23.13
10.18
37.81
521
562
ꢂ k (compound)).
Addition of ct-DNA to a buffered compounds solution strongly
quenched fluorescence intensity of 11 while enhanced fluores-
cence intensity of 25 (Fig. 3).
25

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N. Perin et al. / Bioorg. Med. Chem. 19 (2011) 6329–6339
Affinity of compounds toward ct-DNA and density of binding
2.2.3. Discussion of interaction of compounds 11 and 25 with
ct-DNA
Viscometry results showed that compound 11 (
elongated ct-DNA similarly to referent intercalator (EB,
= 0.90 0.1), while addition of 25 yielded only negligible DNA
elongation ( = 0.4 0.1) accompanied by strongly non-linear ef-
fect of the viscometry increase. Obtained results support intercala-
tion of only charged derivative 11, while its neutral analog 25 most
likely agglomerates along DNA double helix. However, 11 does not
was calculated by processing of UV/Vis and fluorescence titration
data according to Scatchard equation (Table 2).22
a
= 0.88 0.1)
Since CD spectroscopy is a highly sensitive method toward con-
formational changes in the secondary structure of polynucleotides,
we have chosen it to get insight into the changes of polynucleotide
properties induced by small molecule binding.23,24 Studied com-
pounds are achiral and therefore do not possess intrinsic CD spec-
trum. The addition of studied compounds 11 and 25 to the ct-DNA
resulted in small increase of CD band at 275 nm, which suggested
small changes in the helical structure of DNA upon binding
(Supplementary data, Fig. S1). The absence of the measurable in-
duced (ICD) band at >300 nm pointed toward non-homogenous
orientation of bound molecules of 11 and 25 in respect to DNA
helical axis.24
a
a
show any impact on ct-DNA thermal denaturation (DTm = 0),
which is not common for intercalators. Possible explanation is that
bulky amino substituents hamper complete insertion of the large
condensed aromatic moiety between DNA basepairs, whereby
partial intercalation does elongate DNA similar to EB (viscometry)
but yields very weak ICD effect and binding constant (log Ks = 5.1)
an order of magnitude lower than one could expect from the aro-
matic surface (for instance under the same experimental condi-
tions fluorimetric titration of EB with ct-DNA gave log Ks = 6).
2.2.2. Thermal denaturation experiments and viscometry
In the thermal denaturation experiments addition of compound
11 did not yield any measurable effect on the Tm value of ct-DNA
while addition of 25 yielded weak but measurable stabilization
of the ct-DNA double helix (Table 3).
2.3. Biological results and discussion
All of the compounds showed significant growth inhibitory ef-
fect towards five tumor cell lines, four of which were derived from
solid tumors and one leukemia cell line (Table 4). Compounds 4, 5,
23, 24 exerted moderate, while 11, 20, 28, 29, 30, and 32 strong
antiproliferative activity (in the low micromolar range). There is
in general no significant difference in sensitivity between cell lines,
except compound 4, which showed interesting selectivity towards
Viscometry experiments (Supplementary data) yielded values
of
lue obtained for ethidium bromide (
the method ( 0.1). Obtained results support intercalation of only
a
(11) = 0.88 and
a(25) = 0.40, former value differing from the va-
a(EB) = 0.90) by the error of
a
charged derivative 11, while its neutral analog 25 most likely
agglomerates along DNA double helix.
11
1
2
3
4
a
b
0.20
0.2
5
6
7
8
9
0.18
0.16
0.14
0.12
10
11
12
13
14
15
16
0.0
0.10
0.0
1.0x10-4
2.0x10-4 3.0x10-4
c(ctDNA)/moldm-3
5.0x10-4
4.0x10-4
300
350
400
450
λ/nm
25
1
2
3
4
c
d
0.22
0.21
0.20
0.19
0.18
0.17
0.16
0.15
0.14
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
5
6
7
8
9
10
11
12
13
14
15
16
17
1.0x10-3
2.0x10-4 4.0x10 -4
c(ctDNA)/moldm-3
6.0x10-4
8.0x10-4
0.0
350
400
450
λ/nm
Figure 2. (a) The UV/Vis titration of 11 (c = 2.0 ꢁ 10ꢂ5 mol dmꢂ3) with ct-DNA; b) spectroscopic changes at kmax = 353 nm as a function of ct-DNA concentration; c) The UV/
Vis titration of 25 (c = 1.9 ꢁ 10ꢂ5 mol dmꢂ3) with ct-DNA; d) spectroscopic changes at kmax = 348 nm as a function of ct-DNA concentration.

N. Perin et al. / Bioorg. Med. Chem. 19 (2011) 6329–6339
6333
11
1
2
3
4
5
6
7
8
9
10
11
12
13
14
a
b
620
600
500
400
300
200
100
0
610
600
590
580
570
15
16
560
550
540
530
2.0x10-6 4.0x10-6
c(ctDNA)/moldm-3
6.0x10-6 8.0x10-6 1.0x10-5
450
500
550
600
650
0.0
λ/nm
25
1
2
3
4
5
6
7
8
380
360
340
320
300
280
260
c
d
350
9
300
250
200
150
100
50
10
11
12
13
14
15
16
17
18
19
1.0x10-4
c(ctDNA) / moldm-3
1.5x10-4
2.0x10-4
5.0x10-5
0.0
450
500
550
λ / nm
600
650
Figure 3. a) Changes in fluorescence spectrum of 11 (c = 3.3 ꢁ 10ꢂ7 mol dmꢂ3) upon titration with ct-DNA (c = 1.00 ꢁ 10ꢂ3 mol dmꢂ3); b) Dependence of 11 emission at
k = 524 nm on c(ct-DNA); c) Changes in fluorescence spectrum of 25 (c = 1.12 ꢁ 10ꢂ6 mol dmꢂ3) upon titration with ct-DNA (c = 2.00 ꢁ 10ꢂ3 mol dmꢂ3); d) Dependence of 25
emission at k = 559 nm on c(ct-DNA); All experiments were carried out at pH 7.0, (buffer sodium cacodylate I = 0.05 mol dmꢂ3).
Table 2
Binding constants (log Ks)a calculated from the UV/Vis and fluorimetric titrations with
no significant difference in the IC50 concentrations between the
activity of tested compounds, and the DNA binding experiments
did not show significant affinity of two selected compounds to-
wards ds-DNA (diamino-substituted derivative 11 as dihydrochlo-
ride salt and amino–cyano substituted derivative 25), we
attempted the flow cytometry analysis of potential cell cycle per-
turbations after the treatment with diamino-substituted derivative
9 and its dihydrochloride analog 11 and their mono-substituted
analogs (25 and 29, respectively) (Table 5). The results demon-
ct-DNA at pH 7 (buffer sodium cacodylate, I = 0.05 mol dmꢂ3
)
Compound
Fluorescence log Ks
UV/Vis log Ks
11
25
4.8
5.5
4.6
5.1
a
Processing of titration data by Scatchard equation gave Scatchard ratios
n[bound compound]/[polynucleotide] = 0.1–0.2, for better comparison binding constants
were re-calculated for fixed ratio n[bound compound]/[polynucleotide] = 0.2.
strated that all of the compounds (5
l
M ꢀ IC50) significantly de-
layed the progression through G1 phase, as demonstrated by the
accumulation of cells in G1 phase, accompanied with the reduction
of the cell number in the cells in S phase, 24 h after the addition of
the compounds. Even more drastic S phase reduction was obtained
Table 3
D
Tm valuesa (°C) of ct-DNA upon addition of different
ratios rb of 11 and 25 at pH 7.0 (buffer sodium
cacodylate, I = 0.05 mol dmꢂ3
)
rb
11
25
after the treatment with higher concentration (10 lM, data not
shown). This effect diminished after 48 h; predominantly in the
cells treated with 25 and 29, while the most pronounced effect
was obtained with the charged dihydrochloride derivative 11.
These results again suggest that the DNA is not the main target
of the tested compounds, although some affinity of the charged
compounds towards DNA was demonstrated. Namely, contrary to
here-presented results, intercalators induce strong G2/M phase de-
lay/arrest, as also demonstrated in our previously-published stud-
ies.15,16 We therefore performed fluorescence microscopy study
with the compounds 11 and 29 to check the intracellular distribu-
tion of the compounds in the tumor cells. The results confirm that
0.1
0.2
0.3
0
0
0
0.9
1.9
0.2
a
Error in
D
Tm
:
0.5 °C.
b
r = [compound]/[polynucleotide].
HCT 116 cells, while being modestly active towards other cell
lines). It should be noted though, that compounds 4–6, 20,
23–25, 29, and 30 precipitated at the maximal tested concentra-
tion, which might hamper their full effect. Since there is in general

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N. Perin et al. / Bioorg. Med. Chem. 19 (2011) 6329–6339
4. Experimental
Table 4
IC50 values (in
l
M)a,b
Compound
Cell lines
SW 620
4.1. Chemistry
MOLT-4
>100
HCT 116
0.2
21 13
MCF-7
H 460
>100
4.1.1. General methods
4
5
6
9
4
28
16
6
3
3
14
20
4
2
2
1
2
1
1
1
3
1
32 19
All chemicals and solvents were purchased from commercial
suppliers. Melting points were recorded on an Original Keller Mik-
5
3
11
5
6
3
5
19
4
2
1
13
8
3
1
0.2
1
1
2
1
5
1
2
6
4
3
3
roheitztisch apparatus (Reichert, Wien) and Büchi 535. 1H and 13
C
8
4
0.3
2
11
18
19
20
22
23
24
25c
26c
27
28
29
30
31
32
4
3
3
1
NMR spectra were recorded on a Varian Gemini 300 or Varian
Gemini 600 NMR spectrometers at 300, 600, 150 and 75 MHz,
respectively. All NMR spectra were measured in DMSO-d6 solu-
tions using TMS as an internal standard. Chemical shifts are re-
ported in ppm (d) relative to TMS. IR spectra were recorded on
FTIR-ATR and Perkin-Elmer Spectrum 1 spectrophotometers. Ele-
mental analysis for carbon, hydrogen and nitrogen were performed
on a Perkin-Elmer 2400 elemental analyzer and a Perkin-Elmer,
Series II, CHNS Analyzer 2400. All compounds were routinely
checked by TLC with Merck silica gel 60F-254 glass plates.
16
5
3
4
0.2
1
10 0.2
17 0.3
19
3
8
9
2
2
15
19
3
3
5
1
5
24 22
5
1
7
4
2
4
2
38 14
10 0.2
P100
11
5
3
4
5
1
43 25
6 3
P10
11 0.4
17 12
6
3
7
4
4
1
0.1
0.1
0.03
2
4
1
4
3
4
2
5
3
P10
3
6
1
9
2
1
11
3
4
3
0.04
1
10
6
6
2
11 0.2
9
2
2
3
5
8
0.1
2
4
4
4
3
6
2
2
2
3
2
2
4
0.2
0.3
3
4
4
0.1
2
5
3
4.1.2. General method for preparation of compounds 4–5
A mixtures of equimolar amounts of corresponding 3-(2-chloro-
5-nitrophenyl)acrylic acid 3 and 1,2-phenylenediamines 1–2 to-
gether with PPA (10 g) were heated at 180 °C for 3 h. The cooled
mixture was poured into ice-water (200 ml) and the resulting solu-
tion was treated with 25% NH3 ꢁ H2O to pH 9. The suspension was
filtered off and the crude product was washed with water to pH 7
and recrystallized from ethanol to obtain E-2-styryl-benzimidaz-
oles 4–5.
5
4
3
4
1
3
0.1
a
IC50; the concentration that causes 50% growth inhibition.
b
Compounds were dissolved in DMSO to a stock solutions concentration of
1–4 ꢁ 10ꢂ2 M. However, the precipitation in the cell culture medium at 10ꢂ4
M
after 72 h was observed for compounds 4–6, 20, 23–25, 29 and 30.
c
The highest tested concentration was 10 lM.
Table 5
The effects of 25, 29, 9, and 11 at 5
l
M on the cell cycle distribution of HCT 116 cells
after the 24- and 48-hours treatments. The numbers represent the percentages of
cells in respective cell cycle phase (G1, S, and G2/M), along with the percentage of
cells in the subG1 (dead cells) obtained by flow cytometry
4.1.2.1.
E-{2-[2-(2-Chloro-5-nitrophenyl)-ethenyl]}benzimid-
azole 4.Compound 4 was prepared using above described method,
from 3-(2-chloro-5-nitrophenyl)acrylic acid 3 (1.00 g, 4.40 mmol)
and 1,2-phenylenediamine 1 (0.48 g, 4.40 mmol) to obtain a light
Treatment
Percentage of cells (%)
SubG0/G1
G0/G1
S
G2/M
brown powder (0.67 g, 51%); mp 133–136 °C; IR (diamond)
m/
24 h
Control
25
29
9
11
Control
25
29
9
3
6
4
1
1
1
3
4
2
2
40
62
55
49
65
40
47
45
54
60
39
20
20
33
14
35
25
31
25
23
21
18
25
18
21
25
28
24
21
17
cmꢂ1: 3074, 1570, 1520; 1H NMR (600 MHz, DMSO-d6): d = 12.85
(br s, NHarom., 1H), 8.70 (d, J = 2.7 Hz, Harom., 1H), 8.18 (dd,
J1 = 2.7 Hz, J2 = 8.8 Hz, 1H), 7.97 (d, J = 16.4 Hz, Harom., 1H), 7.85
(d, J = 8.8 Hz, Harom., 1H), 7.66 (d, J = 7.8 Hz, Harom., 1H), 7.55 (br s,
48 h
H
arom., 1H), 7.52 (d, J = 16.3 Hz, Harom., 1H), 7.22 (d, J = 8.3 Hz, Har-
om., 2H); 13C NMR (75 MHz, DMSO-d6): d = 150.23 (s), 147.42 (s),
139.29 (s), 135.61 (s, 2C), 131.80 (d, 2C), 127.71 (d, 2C), 124.54
(d), 123.94 (d, 2C), 122.20 (d, 2C); elemental analysis calcd (%)
for C15H10ClN3O2: C, 60.11; H, 3.36; N, 14.02; found: C, 60.33; H,
3.50; N, 14.09.
11
the compounds are not localized in the nucleus, but are distributed
throughout the cytoplasm both after 2 and 24 h (Fig. 4 and data not
shown). Certain differences, however, can be observed between the
localization of the two compounds; compound 29 is localized more
distinctively near nucleus, pointing to possible colocalization with
endoplasmatic reticulum, while compound 11 is evenly distrib-
uted, forming small cytoplasmic aggregates, which could suggest
their localization in the lysosomes, or endosomes.
4.1.2.2. E-{2-[2-(2-Chloro-5-nitrophenyl)-ethenyl]}5(6)-nitrobe
nzimidazole 5.Compound 5 was prepared using above described
method, from 3-(2-chloro-5-nitrophenyl)acrylic acid 3 (1.00 g,
4.40 mmol) and 4-nitro-1,2-phenylenediamine
4.40 mmol) to obtain a brown powder (0.42 g, 27%); mp >300 °C;
IR (diamond)
/cmꢂ1 3358, 1613, 1520, 1338; 1H NMR
2
(0.67 g,
m
:
(300 MHz, DMSO-d6): d = 8.74 (d, J = 2.6 Hz, Harom., 1H), 8.50 (d,
J = 2.1 Hz, Harom., 1H), 8.21 (dd, J1 = 2.6 Hz, J2 = 8.8 Hz, Harom., 1H),
8.13 (dd, J1 = 2.3 Hz, J2 = 9.0 Hz, Harom., 1H), 8.08 (d, J = 16.5 Hz,
3. Conclusions
H
arom., 1H), 7.86 (d, J = 8.8 Hz, Harom., 1H), 7.77 (d, J = 8.9 Hz, Harom.,
In this work we have presented the synthesis of novel acyclic
nitro substituted E-2-styryl-benzimidazoles 4–5, E-2-(2-benzimi-
dazolyl)-3-phenylacrylonitriles 17–20 and cyclic substituted benz-
imidazole[1,2-a]quinolines 6–11 and 21–32. Detailed studies of
two chosen compounds (11 and 25) revealed that they only weakly
interact with ct-DNA, whereby 11 partially intercalates and 25 just
agglomerates along DNA double helix. Accordingly, biological stud-
ies (impact on the cell cycle and fluorescence microscopy) also
showed that DNA is not the primary target of compounds. Thus,
considerable antiproliferative effects of studied compounds are
due to interactions with other biological targets within cells.
1H), 7.60 (d, J = 16.3 Hz, Harom., 1H); 13C NMR (75 MHz, DMSO-d6):
d = 154.75 (s), 149.41 (s), 147.47 (s), 147.27 (s), 143.18 (s), 139.60
(s), 135.00 (s), 131.80 (d), 130.67 (d), 124.95 (d), 122.93 (d), 122.84
(d), 122.34 (d), 122.17 (d), 118.57 (d); elemental analysis calcd (%)
for C15H9ClN4O4: C, 52.26; H, 2.63; N, 16.25; found: C, 52.44; H,
2.89; N, 16.43.
4.1.3. General method for preparation of compounds 17–20
Solution of equimolar amounts of 2-cyanomethylbenzimidazole
derivatives 12–14, corresponding heteroaromatic aldehydes and

N. Perin et al. / Bioorg. Med. Chem. 19 (2011) 6329–6339
6335
Figure 4. Fluorescent microscopy images of H 460 cells treated with compound 29 a) 100
lM for 2 h; and compound 11 b) 100
l
M for 2 h; both showing predominately
cytoplasmic distribution of the compounds.
few drops of piperidine in absolute ethanol, were refluxed for
2–3 h. After reaction mixture was cooled to the room temperature,
the crude product was filtered off and recrystallized from ethanol.
127.39 (d), 124.74 (d), 119.26 (d), 115.63 (d), 115.13 (s), 113.61
(d), 108.80 (s); elemental analysis calcd (%) for C16H8ClN5O4: C,
51.98; H, 2.18; N, 18.94; found: C, 52.28; H, 2.22; N, 19.06.
4.1.3.1. 2-(2-Benzimidazolyl)-3-(2-chloro-5-nitrophenyl)acrylo-
nitrile 17.Compound 17 was prepared from 13 (0.69 g, 4.4 mmol)
and 2-chloro-5-nitro-benzaldehyde 15 (0.81 g, 4.4 mmol) in abso-
lute ethanol (10 ml) after refluxing for 2.5 h and recrystallization
from ethanol to yield 1.16 g (66%) of brown crystals; mp >300 °C;
4.1.3.4. 2-[2-(5(6)-Methyl)benzimidazolyl)-3-(2-chloro-5-nitro-
phenyl)acrylonitrile 20.Compound 20 was prepared from 14
(0.50 g, 2.9 mmol) and 2-chloro-5-nitro-benzaldehyde (0.54 g,
2.9 mmol) in absolute ethanol (20 ml) after refluxing for 1 h and
recrystallization from ethanol to yield 0.39 g (40%) of yellow pow-
IR (diamond)
m
/cmꢂ1
:
3271, 2235, 1603, 1514; 1H NMR
der; mp 200–203 °C; IR (diamond) m
/cmꢂ1: 2225, 1633, 1524,
(600 MHz, DMSO-d6): d = 13.41 (s, 1H, NHbenzim.), 8.99 (d, 1H,
J = 2.7 Hz), 8.50 (s, 1H, Harom.), 8.38 (dd, 1H, J1 = 2.7 Hz,
J2 = 8.9 Hz), 7.98 (d, 1H, J = 8.9 Hz, Harom.), 7.76 (d, 1H, J = 7.6 Hz,
1345; 1H NMR (300 MHz, DMSO-d6): d = 12.95 (br s, NH, 1H),
8.96 (d, J = 2.6 Hz, Harom., 1H), 8.45 (s, Harom., 1H), 8.37 (dd,
J1 = 2.6 Hz, J2 = 8.9 Hz, Harom., 1H), 7.98 (d, J = 8.9 Hz, Harom., 1H),
7.55 (d, J = 8.3 Hz, Harom., 1H), 7.44 (s, Harom., 1H), 7.12 (d,
J = 8.4 Hz, Harom., 1H); 13C NMR (75 MHz, DMSO-d6): d = 146.84
(s), 140.80 (s), 138.64 (d), 134.60 (s), 132.99 (s), 131.99 (d),
131.97 (s), 126.95 (d), 124.94 (d), 124.77 (d), 119.65 (d), 115.41
(s), 112.98 (s), 111.86 (d), 109.47 (s), 21.97 (d); elemental analysis
calcd (%) for C17H11ClN4O2: C, 60.28; H, 3.27; N, 16.54; found: C,
60.44; H, 3.56; N, 16.77.
Hbenzim.), 7.60 (d, 1H, J = 7.8 Hz, Hbenzim.), 7.35–7.27 (m, 2H, Hben-
zim.); 13C NMR (75 MHz, DMSO-d6): d = 147.20 (s), 147.17 (s),
146.93 (s), 141.15 (s), 139.59 (d), 133.25 (s), 132.33 (d, 2C),
132.17 (s), 132.09 (d), 127.37 (d, 2C), 125.12 (d, 2C), 115.68 (s),
109.72 (s); elemental analysis calcd (%) for C16H9ClN4O2: C,
59.18; H, 2.79; N, 17.25; found: C, 59.33; H, 2.93; N, 17.45.
4.1.3.2. 2-[2-(5(6)-Nitro)benzimidazolyl)-3-(2-chlorophenyl) acr
ylonitrile 18.Compound 18 was prepared from 12 (2.00 g,
9.9 mmol) and 2-chlorobenzaldehyde 16 (1.40 g, 9.9 mmol) in
absolute ethanol (10 ml) after refluxing for 2 h and recrystalliza-
tion from ethanol to yield 2.52 g (78%) of brown crystal; mp
4.1.4. General method for preparation of compounds 6–7 and
21–24
Compounds 4–5 and 17–20 were dissolved in 1–4 ml of sulfo-
lane and reaction mixture was heated for 0.5–1.5 h at 280 °C. The
cooled mixture was poured into water (25 ml) and the resulting
product was filtered off and recrystallizated from ethanol to obtain
benzimidazo[1,2-a]quinolines 4–5 and 17–20.
209–213 °C; IR (diamond)
1H NMR (300 MHz, DMSO-d6): d = 13.95 (br s, NHarom., 1H), 8.64
(s, Harom. 1H), 8.56 (s, Harom. 1H), 8.19 (dd, J1 = 8.9 Hz,
m
/cmꢂ1: 3228, 2239, 1508, 1336, 1054;
,
,
J2 = 2.24 Hz, Harom., 1H), 7.96 (d, J = 8.6 Hz, Harom., 1H), 7.83 (d,
J = 8.9 Hz, Harom., 1H), 7.71 (dd, J1 = 7.7 Hz, J2 = 2.2 Hz, Harom., 1H),
7.64–7.56 (m, Harom.
d = 155.75 (d), 151.48 (s), 144.04 (d), 143.78 (s), 136.38 (s),
134.56 (s), 133.66 (d), 133.10 (s), 131.31 (s), 130.62 (d), 130.26
(d), 128.31 (d), 119.31 (d), 115.53 (s), 113.70 (d), 106.35 (s); ele-
mental analysis calcd (%) for C16H9ClN4O2: C, 59.18; H, 2.79; N,
17.25; found: C, 59.34; H, 2.96; N, 17.01.
4.1.4.1. 3-Nitro-benzimidazo[1,2-a]quinoline 6.Compound 6 was
prepared using above described method from compound 4 (0.50 g,
1.16 mmol) in sulfolane (3 ml) after 1.5 h to obtain a brown pow-
,
2H); 13C NMR (75 MHz, DMSO-d6):
der (0.42 g, 95%); mp 276–278 °C; IR (diamond)
1545, 1506; 1H NMR (600 MHz, DMSO-d6): d = 9.05 (d, J = 2.6 Hz,
arom., 1H), 9.01 (d, J = 9.3 Hz, Harom., 1H), 8.73 (d, J = 8.6 Hz, Harom.
1H), 8.56 (dd, J1 = 2.7 Hz, J2 = 9.2 Hz, Harom., 1H), 8.19 (d, J = 9.5 Hz,
arom., 1H), 7.99 (dd, J1 = 1.7 Hz, J2 = 7.1 Hz, Harom., 1H), 7.83 (d,
m
/cmꢂ1: 1609,
H
,
H
4.1.3.3. 2-[2-(5(6)-Nitro)benzimidazolyl)-3-(2-chloro-5-nitroph
enyl)acrylonitrile 19.Compound 19 was prepared from 12
(0.92 g, 4.9 mmol) and 2-chloro-5-nitro-benzaldehyde 15 (1.00 g,
4.9 mmol) in absolute ethanol (10 ml) after refluxing for 2 h and
recrystallization from ethanol to yield 0.30 g (22%) of brown pow-
J = 9.5 Hz, Hkinol., 1H), 7.62 (dt, J1 = 1.8 Hz, J2 = 7.4 Hz, Harom., 1H),
7.59 (dt, J1 = 1.5 Hz, J2 = 7.5 Hz, Harom., 1H); 13C NMR (75 MHz,
DMSO-d6): d = 148.14 (s), 144.87 (s), 143.45 (s), 138.86 (s),
131.69 (d), 130.67 (s), 125.68 (d), 125.59 (d), 124.79 (d), 124.00
(d), 123.64 (s), 120.76 (d), 119.91 (d), 117.29 (d), 115.20 (d); ele-
mental analysis calcd (%) for C15H9N3O2: C, 68.44; H, 3.45; N,
15.96; found: C, 68.66; H, 3.70; N, 16.23.
der.; mp >300 °C; IR (diamond) m
/cmꢂ1: 2230, 1606, 1527, 1341; 1H
NMR (300 MHz, DMSO-d6): d = 9.01 (d, J = 2.6 Hz, Harom., 1H), 8.62
(s, Harom., 1H), 8.57 (d, J = 2.1 Hz, Harom., 1H), 8,42 (dd, J1 = 2.7 Hz,
J2 = 8.9 Hz, Harom., 1H), 8.18 (dd, J1 = 2.2 Hz, J2 = 8.9 Hz, Harom.
,
4.1.4.2. 3,8(9)-Dinitro-benzimidazo[1,2-a]quinoline7.Compound
7 was prepared using above described method from compound 5
(0.35 g,1.02 mmol)insulfolane(1 ml)after45 mintoobtainabrown
1H), 8.00 (d, J = 8.9 Hz, Harom., 1H), 7.83 (d, J = 8.9 Hz, Harom., 1H);
13C NMR (75 MHz, DMSO-d6): d = 151.38 (s), 146.80 (s), 141.17
(d), 143.70 (s), 143.12 (s), 139.39 (s), 132.53 (s), 132.10 (d),
powder(0.05 g, 15%);mp >300 °C;IR(diamond)m
/cmꢂ1:3065, 1614,

6336
N. Perin et al. / Bioorg. Med. Chem. 19 (2011) 6329–6339
1513,1335;1HNMR(300 MHz,DMSO-d6):d = 9.42(s,Harom.,1H),9.07
(dd,J1 = 2.4 Hz,J2 = 8.3 Hz,Harom.,2H),8.98(dd,J1 = 3.0 Hz,J2 = 9.6 Hz,
4.1.4.6. 3-Nitro-8(9)-methyl-6-cyano-benzimidazo[1,2-a]quino-
line 24.Compound 24 was prepared using above described method
from compound 20 (0.25 g, 0.74 mmol) in sulfolane (1 ml) after
50 min to obtain a green powder (0.13 g, 56%); mp 250–254 °C;
IR (diamond)
(300 MHz, DMSO-d6): d = 8.90 (d, J = 2.4 Hz, Harom., 1H), 8.88 (d,
J = 2.7 Hz, Harom., 1H), 8.74 (s, Harom., 1H), 8.69 (d, J = 8.2 Hz, Harom.,
Harom., 2H),8.89(d, J = 9.2 Hz,Harom., 1H),8.74(s, Harom., 1H),8.64(dd,
J1 = 2.5 Hz,J2 = 9.3 Hz,Harom.,1H),8.55(dd,J1 = 2.3 Hz,J2 = 8.9 Hz,Har-
om.,1H),8.45(dd,J1 = 1.6 Hz,J2 = 9.1 Hz,Harom.,1H),8.35(d,J = 9.8 Hz,
m :
/cmꢂ1 2236, 1611, 1521, 1387; 1H NMR
H
arom., 2H), 8.26 (d, J = 9.7 Hz, Harom., 1H), 8.10 (d, J = 8.9 Hz, Harom.
,
1H), 7.90 (d, J = 9.9 Hz, Harom., 1H), 7.86 (d, J = 9.8 Hz, Harom., 1H); 13
C
NMR (75 MHz, DMSO-d6): not enough soluble; elemental analysis
calcd(%)forC15H8N4O4:C,58.45;H,2.62;N,18.18;found:C,58.67;H,
2.89;N,18.43.
1H), 8.86 (d, J = 8.9 Hz, Harom., 1H), 8.66 (d, J = 8.3 Hz, Harom., 1H),
8.47–8.41 (m, Harom., 2H), 8.31 (d, J = 8.4 Hz, Harom., 1H), 8.24 (s, Har-
om., 1H), 7.78 (d, J = 8.3 Hz, Harom., 1H), 7.67 (d, J = 2.6 Hz, Harom.
,
1H), 7.36 (d, J = 8.2 Hz, Harom., 1H), 7.30 (d, J = 8.2 Hz, Harom., 1H),
2.56 (s, CH3, 3H), 2.51 (s, CH3, 3H); 13C NMR (150 MHz, DMSO-
d6): d = 144.17 (s), 143.97 (s), 143.69 (s), 142.69 (s), 142.88 (s),
142.48 (s), 139.32 (d), 139.06 (d), 138.64 (s), 138.48 (s), 135.54
(s), 134.45 (s), 130.21 (s), 130.16 (s), 128.09 (s), 127.26 (d),
126.79 (d), 126.66 (d), 126.21 (d), 126.14 (d), 125.78 (d), 121.04
(s), 120.89 (s), 120.03 (d), 119.98 (d), 116.83 (d), 116.74 (d),
114.64 (s), 114.19 (d), 114.02 (d), 103.23 (s), 103.07 (s), 21.55
(d), 21.07 (d); elemental analysis calcd (%) for C17H10N4O2: C,
67.55; H, 3.33; N, 18.53; found: C, 67.89; H, 3.56; N, 18.83.
4.1.4.3. 3-Nitro-6-cyano-benzimidazo[1,2-a]quinoline 21.Com-
pound 21 was prepared using above described method from com-
pound 17 (0.50 g, 1.54 mmol) in sulfolane (2.5 ml) after 1.5 h to
obtain a brown powder (0.43 g, 97%); mp >300 °C; IR (diamond)
m
/cmꢂ1 2239, 1510, 1334; 1H NMR (600 MHz, DMSO-d6):
:
d = 9.08 (d, J = 2.7 Hz, Harom., 1H), 8.98 (d, J = 8.9 Hz, Harom., 1H),
8.96 (s, Harom., 1H), 8.72 (d, J = 8.9 Hz, Harom., 1H), 8.63 (dd,
J1 = 2.7 Hz, J2 = 9.0 Hz, Harom., 1H), 8.08–8.05 (m, Harom., 1H), 7.67–
7.64 (m, Harom., 2H); 13C NMR (75 MHz, DMSO-d6): d = 144.52 (s),
143.79 (s), 143.23 (s), 140.04 (d), 139.09 (s), 130.41 (s), 127.12
(d), 126.47 (d), 125.90 (d), 124.48 (d), 121.34 (s), 120.69 (d),
117.23 (d), 114.85 (d), 114.82 (s), 103.42 (s); elemental analysis
calcd (%) for C16H8N4O2: C, 66.67; H, 2.80; N, 19.44; found: C,
66.89; H, 2.94; N, 19.67.
4.1.5. General method for the synthesis of amino-
benzimidazo[1,2-a]quinoline derivatives 8–9 and 25–28
Corresponding nitro-benzimidazo[1,2-a]quinoline derivatives
6–7 and 21–24 and solution of SnCl2 ꢁ 2H2O in MeOH and concen-
trated HCl were refluxed for 0.5–1.5 h. After cooling, the reaction
mixture was evaporated under vacuum and dissolved in water
(50 ml). The resulting solution was treated with 20% NaOH to pH
14. Resulting product was filtered off and washed with water to
obtain amino-benzimidazo[1,2-a]quinolines.
4.1.4.4. 8(9)-Nitro-6-cyano-benzimidazo[1,2-a]quinoline 22.Com-
pound 22 was prepared using above described method from com-
pound 18 (1.50 g, 4.62 mmol) in sulfolane (4 ml) after 1 h to obtain
a brown powder (0.28 g, 31%); mp 240–245 °C; IR (diamond) m/
cmꢂ1: 3060, 2233, 1517, 1338; 1H NMR (300 MHz, DMSO-d6):
d = 8.97 (s, NHarom., 1H), 8.93 (s, Harom., 1H), 8.93 (s, Harom., 1H), 8.85
(d, J = 8.7 Hz, Harom., 1H), 8.84 (d, J = 8.7 Hz, Harom., 1H), 8.81 (d,
J = 2.3 Hz, Harom., 1H), 8.34 (dd, J1 = 9.2 Hz, J2 = 2.3 Hz, Harom., 1H),
4.1.5.1. 3-Amino-benzimidazo[1,2-a]quinoline 8.Compound 8
was prepared using above described method from compound 6
(0.40 g, 1.52 mmol), SnCl2 ꢁ 2H2O (2.90 g, 12.62 mmol), HClconcd
(5.3 ml) and H2O (5.3 ml) to obtain a green powder (0.25 g, 71%);
m
/cmꢂ1: 3192, 1612, 1562; 1H
8.18 (d, J = 8.7 Hz, Harom., 1H), 8.17 (dd, J1 = 7.8 Hz, J2 = 1.3 Hz, Harom.
2H), 8.04 (td, J1 = 7.4 Hz, J2 = 1.4 Hz, Harom., 1H), 8.03 (td, J1 = 7.5 Hz,
J2 = 1.4 Hz, Harom., 1H), 7.75–7.70 (m, Harom., 2H), 7.62–7.56 (m, Harom.
,
mp 240–243 °C; IR (diamond)
NMR (300 MHz, DMSO-d6): d = 8.59 (d, J = 7.5 Hz, Harom., 1H), 8.52
(d, J = 8.9 Hz, Harom., 1H), 7.85 (d, J = 7.4 Hz, Harom., 1H), 7.73 (d,
J = 9.5 Hz, Harom. 1H), 7.73 (d, J = 9.5 Hz, Harom., 1H), 7.47–7.40 (m,
,
2H); 13C NMR (150 MHz, DMSO-d6): d = 147.36 (s), 144.39 (s), 143.13
(s), 142.49 (d), 137.05 (d), 134.23 (s), 134.01 (d), 132.16 (d), 132.02
(d), 131.49 (d), 130.86 (s), 130.81 (s), 130.46 (d), 130.13 (d), 129.75
(d), 127.82 (d), 127.58 (d), 126.84 (s), 126.07 (d), 121.45 (s), 117.93
(d), 116.11 (d), 115.64(d), 115.51 (d), 114.88(s), 101.05(s);elemental
analysis calcd (%) for C16H8N4O2: C, 66.67; H, 2.80; N, 19.44; found: C,
66.90; H, 2.88; N, 19.78.
Harom., 2H), 7.13 (dd, J1 = 2.5 Hz, J2 = 8.9 Hz, Harom., 1H), 7.07(d,
J = 2.5 Hz, Harom.,1H), 5.45 (br s, NH2, 2H); 13C NMR (75 MHz,
DMSO-d6): d = 147.44 (s), 146.18 (s), 144.59 (s), 131.89 (d),
130.57 (s), 126.78 (s), 124.71 (s), 124.09 (d), 122.37 (d), 120.02
(d), 118.22 (d), 117.36 (d), 116.78 (d), 114.72 (d), 111.90 (d); ele-
mental analysis calcd (%) for C15H11N3: C, 72.56; H, 4.87; N,
22.57; found: C, 72.87; H, 4.98; N, 22.66.
4.1.4.5.
3,8(9)-Dinitro-6-cyano-benzimidazo[1,2-a]quinoline
23.Compound 23 was prepared using above described method
from compound 19 (0.25 g, 0.68 mmol) in sulfolane (1 ml) after
45 min to obtain a brown powder (0.03 g, 15%); mp >300 °C; IR
4.1.5.2. 3,8(9)-Diamino-benzimidazo[1,2-a]quinoline 9.Com-
pound 9 was prepared using above described method from com-
pound 7 (0.14 g, 0.45 mmol), SnCl2 ꢁ 2H2O (1.70 g, 7.54 mmol),
HClconcd (3.1 ml) and MeOH (3.1 ml) to obtain a brown powder
(diamond)
m :
/cmꢂ1 2250, 1611, 1519, 1441, 1346; 1H NMR
(0.03 g, 28%); mp >300 °C; IR (diamond)
m
/cmꢂ1: 2917, 1633,
(300 MHz, DMSO-d6): d = 9.42 (d, J = 1.8 Hz, Harom., 1H), 9.13–9.09
(m, Harom., 3H), 9.06 (s, Harom., 1H), 9.03 (d, J = 8.8 Hz, Harom., 1H),
9.00 (d, J = 9.0 Hz, Harom., 1H), 8.90 (d, J = 9.3 Hz, Harom., 1H), 8.86
1631, 1438; 1H NMR (300 MHz, DMSO-d6): d = 8.35
(d, J = 8.9 Hz, Harom., 1H), 8.22 (d, Hz, J = 8.8 Hz, Harom., 2H), 7.68
(d, J = 1.5 Hz, Harom., 1H), 7.60 (d, J = 9.5 Hz, Harom., 1H), 7.53
(d, J = 8.7 Hz, Harom., 1H), 7.49 (d, J = 9.6 Hz, Harom., 1H), 7.38
(d, J = 8.7 Hz, Harom., 1H), 7.35 (d, J = 9.7 Hz, Harom., 1H), 7.11 (dd,
J1 = 2.7 Hz, J2 = 9.1 Hz, Harom., 1H), 7.07 (dd, J1 = 3.0 Hz, J2 = 9.4 Hz,
(d, J = 2.3 Hz, Harom., 1H), 8.72 (dd, J1 = 2.3 Hz, J2 = 9.4 Hz, Harom.
1H), 8.65 (dd, J1 = 2.60 Hz, J2 = 9.3 Hz, Harom. 1H), 8.49 (dd,
J1 = 1.8 Hz, J2 = 9.0 Hz, Harom., 1H), 8.40 (dd, J1 = 2.3 Hz, J2 = 9.2 Hz,
arom., 1H), 8.22 (d, J = 9.0 Hz, Harom., 1H); 13C NMR (150 MHz,
,
,
H
DMSO-d6): d = 159.29 (s), 148.30 (s), 147.71 (s), 144.31 (s) 144.27
(s), 143.83 (s), 143.69 (s), 142.80 (d), 142.21 (d), 138.90 (s),
138.80 (s), 134.50 (s), 131.15 (s), 129.96 (s), 128.37 (d), 127.68
(d), 127.11 (d), 127.01 (d), 125.39 (s), 122.13 (s), 122.00 (s),
121.61 (d), 121.33 (d), 119.25 (d), 118.28 (d), 117.99 (d), 116.55
(d), 116.01 (d), 114.75 (s), 111.85 (d), 103.68 (s); elemental
analysis calcd (%) for C16H7N5O4: C, 57.66; H, 2.12; N, 21.01; found:
C, 57.86; H, 2.33; N, 21.28.
H
arom., 1H), 7.03–7.00 (m, Harom., 2H), 6.91 (d, J = 2.0 Hz, Harom.
,
1H), 6.83 (dd, J1 = 1.7 Hz, J2 = 8.7 Hz, Harom. 1H), 6.77 (dd,
,
J1 = 2.0 Hz, J2 = 8.8 Hz, Harom., 1H), 5.33 (br s, NH2, 8H); 13C NMR
(150 MHz, DMSO-d6): d = 146.50 (s), 146.21 (s), 145.89 (s),
145.78 (s), 145.55 (s), 145.26 (s), 144.81 (s), 136.45 (s), 131.56
(s), 130.99 (d), 129.45 (d), 127.01 (s), 126.53 (s), 124.91 (s),
124.60 (s), 122–27 (s), 120.12 (d), 118.63 (d), 117.60 (d), 117.19
(d), 116.49 (d), 116.16 (d), 116.96 (d), 114.99 (d), 114.16 (d),

N. Perin et al. / Bioorg. Med. Chem. 19 (2011) 6329–6339
6337
112.72 (d), 112.01 (d), 111.53 (d), 101.24 (d), 97.56 (d); elemental
analysis calcd (%) for C15H12N4: C, 77.23; H, 4.75; N, 18.01; found:
C, 77.51; H, 4.98; N, 18.32.
4.1.5.6. 3-Amino-8(9)-methyl-6-cyano-benzimidazo[1,2-a]quin-
oline 28.Compound 28 was prepared using above described meth-
od from compound 24 (0.09 g, 0.30 mmol), SnCl2 ꢁ 2H2O (0.56 g,
2.49 mmol), HClconcd (1.0 ml) and MeOH (1.0 ml) to obtain a green
4.1.5.3. 3-Amino-6-cyano-benzimidazo[1,2-a]quinoline 25.Com-
pound 25 was prepared using above described method from com-
pound 21 (0.20 g, 0.69 mmol), SnCl2 ꢁ 2H2O (1.30 g, 5.76 mmol),
HClconcd (2.0 ml) and MeOH (2.0 ml) to obtain a yellow powder
powder (0.07 g, 85%); mp >300 °C; IR (diamond) m
/cmꢂ1: 2236,
1632, 1567, 1531, 1448; 1H NMR (300 MHz, DMSO-d6): d = 8.63
(s, Harom., 1H), 8.60 (s, Harom., 1H), 8.57 (d, J = 8.5 Hz, Harom., 1H),
8.52 (s, Harom., 1H), 8.48 (d, J = 8.5 Hz, Harom., 2H), 8.23 (d,
(0.12 g, 67%); mp >300 °C; IR (diamond)
1568; 1H NMR (300 MHz, DMSO-d6): d = 8.67 (d, J = 9.0 Hz, Harom.
1H), 8.64 (s, Harom., 1H), 8.61 (d, J = 9.0 Hz, Harom., 1H), 7.97 (d,
J = 7.5 Hz, Harom. 1H), 7.59–7.49 (m, Harom. 2H), 7.31 (dd,
m
/cmꢂ1: 3335, 2239,
J = 8.2 Hz, Harom., 1H), 8.05 (s, Harom., 1H), 7.82 (d, J = 8.2 Hz, Harom.,
,
1H), 7.36 (s, Harom., 1H), 7.39–7.32 (m, Harom., 3H), 7.15 (d,
J = 8.2 Hz, Harom., 1H), 5.66 (s, NH2, 2H), 5.55 (s, NH2, 2H), 2.62 (s,
CH3, 3H), 2.53 (s, CH3, 3H); 13C NMR (150 MHz, DMSO-d6):
d = 163.69 (s), 147.12 (s), 146.27 (s), 146.03 (s), 144.02 (s),
143.03 (s), 140.75 (s), 139.85 (d), 134.53 (d), 134.40 (d), 133.90
(s), 132.84 (s), 132.84 (s), 132.42 (s), 130.47 (s), 128.02 (s),
127.06 (s), 126.13 (d), 125.94 (d), 124.51 (d), 124.18 (d), 123.09
(s), 122.89 (s), 121.21 (d), 120.08 (d), 119.95 (d), 119.82 (s),
119.55 (d), 119.03 (d), 116.78 (d), 116.28 (d), 115.82 (s), 21.56
(d), 21.13 (d); elemental analysis calcd (%) for C17H12N4: C,
74.98; H, 4.44; N, 20.58; found: C, 75.32; H, 4.67; N, 20.83.
,
,
J1 = 2.5 Hz, J2 = 9.0 Hz, Harom., 1H), 7.16 (d, J = 2.6 Hz, Harom., 1H),
5.70 (ss, NH2, 2H); 13C NMR (75 MHz, DMSO-d6): d = 146.32 (s),
143.78 (s), 143.59 (s), 140.42 (d), 130.25 (s), 127.06 (s), 124.51 (d),
122.94 (d), 122.56 (s), 121.11 (d), 120.00 (d), 116.70 (d), 115.72
(s), 114.54 (d), 100.67 (s); elemental analysis calcd (%) for
C16H10N4: C, 74.40; H, 3.90; N, 21.69; found: C, 74.64; H, 4.11; N,
21.76.
4.1.5.4. 8(9)-Amino-6-cyano-benzimidazo[1,2-a]quinoline 26.
Compound 26 was prepared using above described method from
compound 22 (0.25 g, 0.87 mmol), SnCl2 ꢁ 2H2O (1.63 g,
7.20 mmol), HClconcd (3.0 ml) and MeOH (3.0 ml) to obtain a brown
4.1.6. General method for the synthesis of amino-benzimidazo
[1,2-a]quinoline hydrochlorides 10–11 and 29–32
A stirred suspension of compounds 8–9 and 25–28 in absolute
ethanol (5–10 ml) was saturated with HCl(g). After 24 h of stirring
small amount of diethylether was added, resulting product was fil-
tered off and washed with diethylether to obtain amino-benzimi-
dazo[1,2-a]quinoline hydrochlorides.
powder (0.21 g, 89%); mp 195–199 °C; IR (diamond) m
/cmꢂ1: 3211,
2229, 1504, 1448; 1H NMR (300 MHz, DMSO-d6): d = 8.72 (d,
J = 8.7 Hz, Harom., 1H), 8.69 (s, Harom., 1H), 8.57 (s, Harom., 1H), 8.54
(d, J = 8.9 Hz, Harom., 1H), 8.39 (d, J = 9.0 Hz, Harom., 1H), 8.10 (t,
J = 7.8 Hz, Harom., 1H), 8.09 (t, J = 7.9 Hz, Harom., 1H), 7.96 (t,
J = 8.4 Hz, Harom., 1H), 7.95 (t, J = 8.4 Hz, Harom., 1H), 7.94 (s, Harom.
1H), 7.70 (d, J = 8.7 Hz, Harom., 1H), 7.62 (t, J = 7.5 Hz, Harom., 1H),
7.60 (t, J = 7.5 Hz, Harom., 1H), 7.03 (d, J = 2.0 Hz, Harom., 1H), 6.97
,
4.1.6.1. 3-Amino-benzimidazo[1,2-a]quinoline hydrochloride
10.Compound 10 was prepared using above described method
from compound 8 (0.12 g, 0.52 mmol) to obtain a green powder
(dd, J1 = 8.7 Hz, J2 = 1.7 Hz, Harom.
,
1H), 6.92 (dd, J1 = 9.0 Hz,
(0.11 g, 80%); mp 285–288 °C; IR (diamond) m
/cmꢂ1: 3026, 2716,
J2 = 2.1 Hz, Harom., 1H), 5.47 (br s, NH2, 2H), 5.39 (br s, NH2, 2H);
13C NMR (75 MHz, DMSO-d6): d = 150.67 (s), 147.62 (s), 147.20
(s), 146.26 (s), 144.57 (s), 139.14 (d), 138.14 (d), 138.10 (d),
136.52 (s), 136.19 (s), 135.82 (s), 133.81 (d), 133.16 (d), 132.26
(s), 131.46 (d), 131.38 (d), 125.14 (d), 124.96 (d), 123.07 (s),
121.91 (s), 121.34 (s), 121.10 (d), 116.20 (s), 116.09 (s), 115.94
(d), 115.48 (d), 115.38 (d), 113.96 (d), 102.08 (s), 101.92 (d),
101.05 (s), 97.30 (d); elemental analysis calcd (%) for C16H10N4:
C, 74.40; H, 3.90; N, 21.69; found: C, 74.58; H, 4.14; N, 21.92.
2544, 2492, 1612, 1572; 1H NMR (300 MHz, DMSO-d6): d = 8.97
(d, J = 9.4 Hz, Harom., 1H), 8.92 (d, J = 9.8 Hz, Harom., 1H), 8.46 (d,
J = 9.5 Hz, Harom., 1H), 8.04 (d, J = 8.0 Hz, Harom., 1H), 7.92 (d,
J = 9.4 Hz, Harom.
, 1H), 7.83–7.72 (m, Harom., 2H), 7.58 (d,
J = 9.0 Hz, Harom., 1H), 7.56 (br s, Harom., 1H), 5.35 (br s, NH3+, 3H);
13C NMR (75 MHz, DMSO-d6): d = 113.18 (s), 138.48 (d), 132.88
(s), 128.85 (s), 128.80 (s), 128.11 (d), 125.68 (d), 125.56 (s),
124.38 (d), 124.36 (d), 124.34 (d), 118.85 (d), 116.84 (d), 115.34
(d), 111.91 (d); elemental analysis calcd (%) for C15H12ClN3: C,
66.79; H, 4.48; N, 15.58; found: C, 66.92; H, 4.70; N, 15.81.
4.1.5.5. 3,8(9)-Diamino-6-cyano-benzimidazo[1,2-a]quinoline
27.Compound 27 was prepared using above described method from
compound 23 (0.09 g, 0.27 mmol), SnCl2 ꢁ 2H2O (1.01 g,
4.49 mmol), HClconcd (1.9 ml) and MeOH (1.9 ml) to obtain a brown
4.1.6.2. 3,8(9)-Diamino-benzimidazo[1,2-a]quinoline hydro-
chloride 11.Compound 11 was prepared using above described
method from compound 9 (0.10 g, 0.33 mmol) to obtain a light
powder (0.08 g, 81%); mp >300 °C; IR (diamond)
m
/cmꢂ1: 2236,
brown powder (0.11 g, 86%); mp 270–273 °C; IR (diamond) m/
1633, 1564, 1446, 1360; 1H NMR (300 MHz, DMSO-d6): d = 8.46 (s,
Harom., 1H), 8.44 (d, J = 9.1 Hz, Harom., 1H), 8.36 (s, Harom., 1H), 8.29
(dd, J1 = 2.6 Hz, J2 = 8.9 Hz, Harom., 2H), 7.70 (s, Harom., 1H), 7.63 (d,
J = 8.7 Hz, Harom., 1H), 7.26 (dd, J1 = 2.5 Hz, J2 = 9.2 Hz, Harom., 1H),
7.22 (dd, J1 = 2.7 Hz, J2 = 8.9 Hz, Harom., 1H), 7.11 (s, Harom., 1H),
7.10 (s, Harom., 1H), 6.98 (s, Harom., 1H), 6.91 (dd, J1 = 1.6 Hz,
J2 = 8.8 Hz, Harom., 1H), 6.86 (dd, J1 = 2.2 Hz, J2 = 9.0 Hz, Harom., 1H),
5.56 (br s, NH2, 4H), 5.29 (br s, NH2, 4H); 13C NMR (75 MHz,
DMSO-d6): d = 147.11 (s), 146.61 (s), 146.40 (s), 146.30 (s), 146.05
(s), 143.84 (s), 141.92 (s), 138.67 (d), 138.01 (d), 136.23 (s), 131.91
(s), 127.72 (s), 127.47 (s), 123.12 (s), 128.09 (s), 122.70 (s), 121.75
(d), 120.76 (d), 120.44 (d), 116.70 (d), 116.54 (s), 116.41 (s),
116.28 (d), 115.04 (d), 113.56 (d), 112.58 (d), 112.02 (d), 101.70
(d), 101.53 (s), 101.41 (d), 100.50 (s), 97.01 (d); elemental analysis
calcd (%) for C16H101N5: C, 70.32; H, 4.06; N, 25.63; found: C,
70.56; H, 4.32; N, 25.87.
cmꢂ1: 2818, 2577, 1630, 1564, 1503; 1H NMR (300 MHz, DMSO-
d6): d = 9.00 (s, Harom., 1H), 8.91 (d, J = 8.9 Hz, Harom., 1H), 8.82 (d,
J = 9.1 Hz, Harom., 1H), 8.65 (d, J = 9.1 Hz, Harom., 1H), 8.42 (d,
J = 9.5 Hz, Harom., 1H), 8.37 (d, J = 9.6 Hz, Harom., 2H), 8.01 (d,
J = 8.7 Hz, Harom., 1H), 7.87 (d, J = 9.3 Hz, Harom., 1H), 7.86 (s, Harom.
,
1H), 7.84 (d, J = 8.7 Hz, Harom., 1H), 7.81 (d, J = 9.3 Hz, Harom., 1H),
7.78 (d, J = 1.7 Hz, Harom., 1H), 7.65 (d, J = 8.6 Hz, Harom., 1H), 7.64
(s, Harom., 1H), 7.42 (dd, J1 = 1.7 Hz, J2 = 8.6 Hz, Harom., 1H), 7.79
(br s, NH3+, 12H); 13C NMR (150 MHz, DMSO-d6): d =144.17 (s),
143.44 (s), 136.48 (d), 136.40 (d), 134.98 (s), 129.73 (s), 129.70
(s), 129.63 (s), 129.60 (s), 129.58 (s), 128.48 (s), 125.02 (d),
124.97 (d), 124.80 (s), 124.71 (s), 124.67 (s), 124.56 (s), 124.26
(d), 121.39 (d), 119.77 (d), 119.73 (d), 118.07 (d), 117.73 (d),
117.03 (d), 116.85 (d), 116.62 (d), 112.94 (d), 112.32 (d), 108.84
(d); elemental analysis calcd (%) for C15H14Cl2N4: C, 56.09; H,
4.39; N, 17.44; found: C, 56.33; H, 4.52; N, 17.48.

6338
N. Perin et al. / Bioorg. Med. Chem. 19 (2011) 6329–6339
4.1.6.3. 3-Amino-6-cyano-benzimidazo[1,2-a]quinoline hydro-
chloride 29.Compound 29 was prepared using above described
method from compound 25 (0.10 g, 0.39 mmol) to obtain a yellow
3H), 7.56 (d, J = 8.7 Hz, Harom., 1H), 7.50 (d, J = 8.2 Hz, Harom., 1H),
5.01 (br s, NH3+, 6H), 2.65 (s, CH3, 3H), 2.57 (s, CH3, 3H); 13C
NMR (150 MHz, DMSO-d6): d = 164.18 (s), 164.14 (s), 162.71 (s),
162.38 (s), 137.12 (s), 137.06 (s), 136.71 (d), 136.12 (s), 135.15
(s), 135.10 (s), 132.16 (s), 129.94 (s), 129.36 (d), 128.45 (s),
128.34 (d), 127.79 (s), 127.11 (d), 126.35 (d), 126.31 (s), 125.62
(d), 123.90 (s), 123.88 (s), 123.45 (s), 123.13 (s), 118.36 (d),
118.17 (d), 117.95 (d), 117.63 (d), 115.79 (d), 115.60 (d), 115.24
(d), 115.13 (d), 21.43 (d), 21.21 (d); elemental analysis calcd (%)
for C17H13ClN4: C, 66.13; H, 4.24; N, 18.15; found C, 66.37; H,
4.36; N, 18.42.
powder (0.05 g, 44%); mp >288 °C; IR (diamond)
m
/cmꢂ1: 3321,
3142, 3045, 2812, 2582, 2050, 1664, 1579; 1H NMR (300 MHz,
DMSO-d6): d = 9.16 (s, Harom., 1H), 9.02 (d, J = 8.6 Hz, Harom., 1H),
8.99 (s, Harom., 1H), 8.91 (d, J = 8.4 Hz, Harom., 1H), 8.12 (d,
J = 7.7 Hz, Harom., 1H), 7.76 (d, J = 7.6 Hz, Harom., 1H), 7.66–7.57
(m, Harom., 2H), 5.50 (s, NH3+, 3H); 13C NMR (75 MHz, DMSO-d6):
d = 164.67 (s), 163.19 (s), 147.59 (s), 142.17 (d), 139.81 (s),
132.61 (s), 129.12 (s), 128.44 (d), 128.24 (s), 126.99 (d), 126.02
(d), 124.61 (d), 118.68 (d), 118.11 (d), 116.09 (d), 112.97 (s); ele-
mental analysis calcd (%) for C16H11ClN4: C, 65.20; H, 3.76; N,
19.01; found: C, 65.43; H, 3.94; N, 19.32.
4.2. Spectroscopy
The electronic absorption spectra were recorded on Varian Cary
50 and Varian Cary 100 Bio spectrometer, CD spectra on Jasco J815,
in all cases using quartz cuvettes (1 cm). Fluorescence emission
spectra were recorded on Varian Eclipse fluorimeter (quartz cuv-
ettes, 1 cm), from 350 to 600 nm. The sample concentration in fluo-
rescence measurements had an optical absorbance below 0.05 at
the excitation wavelength. Under the experimental conditions
used the absorbance and fluorescence intensities of studied com-
pounds were proportional to their concentrations, while none of
studied compounds showed CD spectrum. The measurements were
performed in the aqueous buffer solution (pH 7.0; sodium cacodyl-
ate buffer, I = 0.05 mol dmꢂ3).
4.1.6.4.
8(9)-Amino-6-cyano-benzimidazo[1,2-a]quinoline
hydrochloride 30.Compound 30 was prepared using above de-
scribed method from compound 26 (0.05 g, 0.19 mmol) to obtain
a light brown powder (0.05 g, 99%); mp 271–275 °C; IR (diamond)
m
/cmꢂ1: 2786, 2564, 1976, 1675, 1502; 1H NMR (300 MHz, DMSO-
d6): d = 8.96 (s, Harom., 1H), 8.94 (d, J = 8.8 Hz, Harom., 1H), 8.89 (s,
Harom., 1H), 8.83 (d, J = 9.1 Hz, Harom., 1H), 8.78 (s, Harom., 1H),
8.68 (d, J = 8.5 Hz, Harom., 1H), 8.31 (t, J = 7.8 Hz, Harom., 1H), 8.80
(t, J = 7.7 Hz, Harom., 1H), 8.20 (s, Harom., 1H), 8.07 (d, J = 8.7 Hz,
Harom., 1H), 8.04 (d, J = 8.7 Hz, Harom., 1H), 7.76 (d, J = 8.1 Hz, Harom.,
1H), 7.74 (d, J = 8.0 Hz, Harom., 1H), 7.53 (d, J=8.7 Hz, Harom., 1H),
7.12 (d, J = 7.7 Hz, Harom., 1H), 7.36 (d, J = 7.9 Hz, Harom., 1H), 3.92
(br s, NH3+, 3H), 3.90 (br s, NH3+, 3H); 13C NMR (150 MHz,
DMSO-d6): d = 163.76 (s), 163.46 (s), 151.70 (s), 149.61 (s),
145.70 (s), 145.44 (s), 144.93 (s), 143.15 (s), 138.57 (s), 136.68
(d), 136.46 (d), 134.88 (s), 134.53 (s), 133.31 (d), 132.87 (d),
131.87 (d), 131.68 (d), 129.29 (s), 126.18 (d), 126.15 (d), 125.90
(d), 122.18 (s), 122.16 (s), 120.52 (d), 119.41 (d), 119.15 (s),
117.89 (d), 117.89 (d), 116.44 (d), 116.20 (d), 115.16 (d); elemental
analysis calcd (%) for C16H11ClN4: C, 65.20; H, 3.76; N, 19.01; found:
C, 65.43; H, 3.89; N, 19.25.
4.3. Interactions with DNA
The calf thymus DNA (ct-DNA) was purchased from Aldrich, dis-
solved in the sodium cacodylate buffer, I = 0.05 mol dmꢂ3, pH 7.0,
additionally sonicated and filtered through a 0.45 lm filter and
the concentration of corresponding solution determined spectro-
scopically as the concentration of phosphates.25 The measure-
ments were performed in the aqueous buffer solution (pH 7.0;
sodium cacodylate buffer, I = 0.05 mol dmꢂ3). Spectroscopic titra-
tions were performed by adding portions of polynucleotide solu-
tion into the solution of the studied compound. The stability
constant (Ks) and [bound compound]/[polynucleotide phosphate]
ratio (n) were calculated according to the Scatchard equation by
non-linear least-square fitting, a giving excellent correlation coef-
ficients (>0.999) for obtained values for Ks and n. Thermal denatur-
ation curves for ct-DNA and its complexes with studied
compounds were determined as previously described by following
the absorption change at 260 nm as a function of temperature. The
absorbance of the ligand was subtracted from every curve, and the
absorbance scale was normalized. Obtained Tm values are the mid-
points of the transition curves, determined from the maximum of
the first derivative or graphically by a tangent method. Given
4.1.6.5. 3,8(9)-Diamino-6-cyano-benzimidazo[1,2-a]quinoline
hydrochloride 31.Compound 31 was prepared using above de-
scribed method from compound 27 (0.05 g, 0.18 mmol) to obtain
a brown powder (0.04 g, 73%); mp >300 °C; IR (diamond)
2577, 2242, 1626, 1566, 1449; 1H NMR (300 MHz, DMSO-d6):
d = 8.94 (s, Harom., 1H), 8.85 (s, Harom., 1H), 8.79 (d, J = 8.7 Hz, Harom.
1H), 8.78 (d, J = 8.9 Hz, Harom., 1H), 8.48 (d, J = 8.8 Hz, Harom., 1H),
8.17 (s, Harom., 1H), 8.03 (d, J = 8.7 Hz, Harom., 1H), 7.99 (s, Harom.
m :
/cmꢂ1
,
,
1H), 7.84 (d, J = 8.1 Hz, Harom., 1H), 7.81 (d, J = 8.8 Hz, Harom., 2H),
7.79 (d, J = 8.0 Hz, Harom., 1H), 7.62 (d, J = 8.8 Hz, Harom., 1H), 7.57
(d, J = 8.8 Hz, Harom.
+
,
1H), 5.48 (br s, NH3
,
12H); 13C NMR
(75 MHz, DMSO-d6): d = 145.70 (s), 145.43 (s), 143.98 (s), 142.76
(s), 141.36 (d), 141.11 (d), 134.51 (s), 132.18 (s), 131.91 (s),
130.27 (s), 130.20 (s), 130.16 (s), 129.74 (s), 129.71 (s), 128.75
(s), 126.58 (d), 125.94 (d), 125.91 (d), 122.75 (s), 122.62 (s),
121.31 (d), 120.86 (d), 119.02 (d), 117.69 (d), 116.71 (d), 116.19
(d), 115.57 (s), 114.39 (d), 109.93 (d), 102.20 (s), 101.94 (s); ele-
mental analysis calcd (%) for C16H13Cl2N5: C, 55.51; H, 3.78; N,
20.23; found: C, 55.76; H, 3.93; N, 20.44.
D
Tm values were calculated subtracting Tm of the free nucleic acid
from Tm of complex. Every Tm value here reported was the aver-
D
age of at least two measurements, the error in
DTm is 0.5 °C.
4.4. Antitumor evaluation assay
The experiments were carried out on five human cell lines,
which are derived from four cancer types. The following cell lines
were used: SW 620 and HCT 116 (colon carcinoma), H 460 (lung
carcinoma), MCF-7 (breast carcinoma) and MOLT-4 (T-lympho-
blast leukemia). MCF-7, SW 620, HCT 116 and H 460 cells were
cultured as monolayers and maintained in Dulbecco’s modified
Eagle medium (DMEM) while MOLT-4 cells were cultured in sus-
pension in RPMI medium, both supplemented with 10% fetal bo-
4.1.6.6. 3-Amino-8(9)-methyl-6-cyano-benzimidazo[1,2-a]quin-
oline hydrochloride 32.Compound 32 was prepared using above
described method from compound 28 (0.04 g, 0.15 mmol) to obtain
a green powder (0.03 g, 74%); mp >300 °C; IR (diamond) m :
/cmꢂ1
2846, 2582, 2340, 1677, 1625; 1H NMR (300 MHz, DMSO-d6):
d = 9.18 (s, Harom., 1H), 9.07 (d, J = 8.4 Hz, Harom., 1H), 8.96 (d,
J = 9.0 Hz, Harom., 1H), 8.91 (d, J = 9.0 Hz, Harom., 1H), 8.76 (d,
J = 8.6 Hz, Harom., 1H), 8.71 (s, Harom., 1H), 8.21 (s, Harom., 1H), 7.96
vine serum (FBS), 2 mM
100 g/ml streptomycin in a humidified atmosphere with 5% CO2
at 37 °C. The growth inhibition activity was assessed as described
L-glutamine, 100 U/ml penicillin and
l
(d, J = 8.3 Hz, Harom., 1H), 7.87 (s, Harom., 1H), 7.76–7.70 (m, Harom.
,

N. Perin et al. / Bioorg. Med. Chem. 19 (2011) 6329–6339
6339
previously.7,8,15,16,20 The panel cell lines were inoculated onto a
for 120 min and 20
lM final concentrations for 24 h. Cover slips
series of standard 96-well microtiter plates on day 0, at 1 ꢁ 104
to 3 ꢁ 104 cells/mL, depending on the doubling times of a specific
cell line. Test agents were then added in ten-fold dilutions (10ꢂ8 to
10ꢂ4 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 dehydrogenise activity in viable cells. The absor-
bance (A) was measured on a microplate reader at 570 nm. The
absorbance is directly proportional to the number of living, meta-
bolically active cells. The percentage of growth (PG) of the cell lines
was calculated according to one or the other of the following two
expressions:
were rinsed twice with PBS, placed on the microscopic slides and
immediately analyzed. The uptake and intracellular distribution
of tested chemicals were analyzed under the fluorescence micro-
scope (Olympus BX51) and recorded with Olympus DP70 Digital
Camera.
Acknowledgment
Support for this study by the Ministry of Science, Education and
Sport of Croatia is gratefully acknowledged (Projects 125-
0982464-1356, 098-0982914-2918, 098-0982464-2514).
Supplementary data
IfðmeanAtest ꢂmeanAtzeroÞꢃ0;thenPG¼100
ꢁðmeanAtest ꢂmeanAtzeroÞ=ðmeanActrl ꢂmeanAtzero
IfðmeanAtest ꢂmeanAtzeroÞ<0;then:PG¼100
Þ
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.09.002.
ꢁðmeanAtest ꢂmeanAtzeroÞ=Atzero
;
References and notes
where the mean Atzero is the average of optical density measure-
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is the average of optical density measurements 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
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