Benzimidazole derivatives related to 2,3-acrylonitriles, benzimidazo[1,2-a]quinolines and fluorenes: Synthesis, antitumor evaluation in vitro and crystal structure determination

Article abstract of DOI:10.1016/j.ejmech.2010.02.022

A synthesis and biological evaluation of new benzimidazole derivatives, related to 2,3-disubstituted acrylonitriles, benzimidazo[1,2-a]quinoline-6-carbonitriles and heteroaromatic fluorenes was described. The molecular and crystal structures of three comp

Full text of DOI:10.1016/j.ejmech.2010.02.022

European Journal of Medicinal Chemistry 45 (2010) 2405e2417
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Benzimidazole derivatives related to 2,3-acrylonitriles, benzimidazo[1,2-a]
quinolines and fluorenes: Synthesis, antitumor evaluation in vitro and crystal
structure determination
,
c
ꢀ b
ꢀ c
Marijana Hranjec a , Gordana Pavlovic , Marko Marjanovic , Marijeta Kralj ,
*
Grace Karminski-Zamola a
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 20, P.O. Box 177, HR-10000 Zagreb, Croatia
Faculty of Textile Technology, Department of Applied Chemistry, University of Zagreb, Prilaz baruna Filipovica 28a, HR-10000, Zagreb, Croatia
Division of Molecular Medicine, RuCer Boskovic Institute, Bijenicka cesta 54, P.O. Box 180 HR-10000 Zagreb, Croatia
,
**
a
ꢀ
b
ꢀ
c
ꢁ
ꢀ
ꢁ
a r t i c l e i n f o
a b s t r a c t
Article history:
A synthesis and biological evaluation of new benzimidazole derivatives, related to 2,3-disubstituted
acrylonitriles, benzimidazo[1,2-a]quinoline-6-carbonitriles and heteroaromatic fluorenes was described.
The molecular and crystal structures of three compounds 4, 16 and 17 reveal that non-fused fluoro
derivative, 4, deviates from planarity by 13.11(2)ꢀ, while fused methyl, 16, and fluoro, 17, derivatives are
planar within 4ꢀ exhibiting a planar aromatic surface capable to intercalate into double-stranded DNA.
Compound 4 exists as E-isomer.
Received 20 November 2009
Received in revised form
4 February 2010
Accepted 8 February 2010
Available online 13 February 2010
The crystal structures confirmed that hydrogen bonding patterns are characterized dominantly by the
weak CeH/N(F) bonds, except in the case of 4 where the presence of ethanol molecule of crystallization
resulted in the NeH/O and OeH/N hydrogen bonds formation. In the crystal structures of 16 and 17
cyano group participates in hydrogen bonding formation, while in 4 this is not the case. All compounds,
except 16 and 14 exerted pronounced antiproliferative activity on five tumor cell lines, whereby
2-benzimidazolyl-3-N-methylpyrolyl-acrylonitrile 13 and its fused analogue 23 exerted the highest
Keywords:
Benzimidazoles
Acrylonitriles
Benzimidazo[1,2-a]quinoline-6-
carbonitriles
Fluorenes
Antitumor evaluation
X-ray crystal structure determination
activity on all cell lines (IC50 ¼ 0.8e30
mM) and showed a special selectivity toward HeLa cells. There is no
major difference in the biological activity between non-fused and fused analogues. Similarly, all
compounds showed significant interaction with ct-DNA, supporting the fact that their antitumor activity
could partially be the consequence of DNA-binding. The cyano moiety is important for the activity, but
not the selectivity of tested compounds.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
benzimidazo[1,2-a]quinazolines due to their planar chromophore
have ability to become inserted between adjacent base pairs of
Since the benzimidazole unit is the key building block for
a variety of compounds which have crucial roles in the functions of
biologically important molecules, there is a constant and growing
interest over the past few years for the synthesis and biological
studies of benzimidazole derivatives [1e3]. Benzimidazoles and
their azino-fused cyclic derivatives have a wide range of well
known biological activities such as anticancer [4e7], antimicrobial
[8e10], antifungal [11], antiviral [12,13], etc. Benzoannulated
benzimidazole analogues such as benzimidazo[1,2-a]quinolines or
a DNA molecule in intercalation process, or have potential appli-
cation as fluorescent probes in homogeneous assays of biological
molecules [14e18].
Furthermore, 2,3-disubstituted acrylonitriles have also received
considerable attention because of their versatile biological activi-
ties [19e21]. For example, a group of authors has recently reported
that some 3-heteroarylacrylonitriles with triazole or benzimidazole
ring have been shown to possess cytotoxic activity on several
human cancer cell lines [22,23]. More recently, some acrylonitriles
were found to possess antibacterial and antituberculostatic activity
as well as ability to inhibit tubulin polymerization [24,25].
As a part of our medicinal chemistry project aimed at the
synthesis of potential anticancer agents related to benzimidazole
derivatives, we have recently reported synthesis and strong
* Corresponding author. Tel.: þ385 14597245; fax: þ385 14597250.
** Corresponding author. Tel.: þ385 14597215; fax: þ385 14597250.
E-mail addresses: mhranjec@fkit.hr (M. Hranjec), gzamola@pierre.fkit.hr
(G. Karminski-Zamola).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.022

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M. Hranjec et al. / European Journal of Medicinal Chemistry 45 (2010) 2405e2417
inhibitory activities on several human cell lines of various amidino-
and cyano-substituted styryl-2-benzimidazoles and benzimidazo
[1,2-a]quinolines (Fig. 1) [26,27]. The benzimidazole compounds
with cyano substituent in general strongly enhances the cytotoxic
activity. Benzimidazo[1,2-a]quinoline-9(10)-carbonitrile has shown
the extremelypronounced and selectiveactivityon the HeLa cell line
previously prepared and tested for the cytotoxic activity on tumor
cell lines by Saçzewski et al. [23] and similar growth inhibitory
results were obtained.
All compounds, except 2-methylbenzimidazo[1,2-a]quinoline-
6-carbonitrile 16 and non-fused 2-benzimidazolyl-3-(5-imida-
zolyl)-acrylonitrile 14 exerted pronounced antiproliferative
activity. In general, there are no major differences in the anti-
proliferative activity among all compounds, apart from a few
exceptions. Thus, 2-benzimidazolyl-3-N-methylpyrolyl-acryloni-
trile 13 and its fused analogue 23 exerted the highest activity on all
(IC50 ¼ 0.05
mM) [26]. Such an extreme selectivity of heterocyclic
cyano molecules on HeLa cells was previouslyobserved byour group
also for another heteroaromatic compounds like carboxanilides
bearing cyano substituent on either anilide or benzothiophene part
of the molecule as well as for methyl-2-cyano-naphtho[2,1-b]thio-
phen-5-carboxylate and 4-ethyl-7-cyano-thieno[2,3-e]-benzo[b]
furan [28,29].
cell lines (IC50 ¼ 0.8e30
m
M) and also showed a special selectivity
toward HeLa cells (IC50 ¼ 0.8
mM). Similar activity was also obtained
by dimethoxy derivatives 6 and 19, but without selectivity to HeLa
cells. The presence of acetamido group on phenyl ring in non-fused
compound 7 leads to significantly better selectivity toward HeLa
and MCF-7 cell lines, while its fused analogue 20 showed better
activity with the lack of selectivity. The attachment of thiophene
ring to 3 position of 2-benzimidazolyl acrylonitrile in compound 12
leads to selectivity toward HeLa cells, which is again lost in its fused
analogue 22. Apart from these differences, there is in general no
major difference between non-fused and fused analogues with the
exception of 3 and 16, whereby non-fused analogue 3 exerted
significantly higher activity. The explanation for this could be either
in the ability of both non-fused and fused compounds to interact
(bind) with cellular macromolecules (proteins and/or DNA) and to
induce similar response in tumor cells (e.g. DNA-damage response),
or the most responsible functionality for this activity is the cyano
moiety either in quinoline ring, or as an acrylonitrile moiety.
Namely, acrylonitrile is known to react nonenzymatically with the
cysteine thiol groups present in proteins [31], but it can also be
metabolized by P-450 isoenzyme CYP2E1, to 2-cyanoethylene
oxide, which can bind irreversibly to nucleic acids as well as
proteins. Also, a very toxic metabolite e cyanide is released.
Epoxide hydrolase and glutathione S-transferase (GST) may have
protective roles, whereby GST isoenzymes responsible for this
pathway are not completely known; the polymorphic GSTT1 and
GSTM1 are predominantly involved after massive intoxications
with alkylating agents [32]. Therefore, as was also nicely elaborated
in Saçzewski et al., like acrylonitrile, the here-presented
compounds may act by inhibiting sulfhydryl-dependent enzymes
of intermediary metabolism by cyanoethylation of sulfhydryl
groups or by liberation of cyanide and subsequent inhibition of
cytochrome c oxidase [23]. Thus, either different P-450, or GST
polymorphisms could be responsible (because of a lower, or
a higher capacity for detoxication) for divergent responses of
various tumor (and other) cells to acrylonitrile, or other cyano-
substituted compounds.
The above-mentioned results prompted us to synthesize novel
non-fused heteroaromatic 2-benzimidazolyl substituted acryloni-
triles 2e14 and fused benzimidazo[1,2-a]quinoline-6-carbonitriles
15e20 and fluorenes 21e23. Full details about the synthesis,
evaluation of antitumor activity in vitro, preliminary results of
binding to ct-DNA and crystal structure determination are repor-
ted herein.
2. Chemistry
A novel series of benzimidazole derivatives 2e23 was prepared
according to Scheme 1 and Table 1. Acyclic 2-benzimidazolyl
substituted acrylonitriles 2e14 were prepared by condensation
reaction of 2-cyanomethylbenzimidazole 1 with corresponding
heteroaromatic aldehydes in absolute ethanol by adding a few
drops of piperidine [30]. Compounds 2e14 were prepared in good
yields (50e84%). Benzimidazo[1,2-a]quinoline-6-carbonitriles
15e20 and heteroaromatic fluorenes 21e23 were prepared
photochemically, from non-fused compounds 2e14, by reaction of
dehydrocyclization. Photochemical dehydrocyclization was per-
formed in ethanolic solution with addition of small amount of
iodine, under oxidative conditions, using 400-W high-pressure
mercury lamp and Pyrex filter for 6e15 h. Compounds 9e11 did not
give desired fused products.
All structures of novel non-fused 2e14 and fused derivatives
15e23 were determined by NMR analysis, based on the analysis of
HeH coupling constants as well as chemical shifts. The photo-
cyclization reaction leads to a downfield shift of the signal of most
aromatic protons, along with a disappearing of one aromatic proton
and proton of NH group on benzimidazole ring thus confirming
fused structure formation.
3. Biological results and discussion
Saçzewski et al. stated that even though the cyano group of the
acrylonitrile moiety was not an absolute requirement for activity,
compounds lacking this group were less active compared to their
acrylonitrile counterpart. Also, replacement of the nitrile group for
hydrogen had less of an effect than replacement for a methyl group.
Although, we did not check the potential activity of analogues
lacking the cyano group, our results and the results published by
3.1. Antitumor evaluation in vitro
Compounds 3e23 were screened for their antiproliferative
ability on five tumor cell lines, derived from different tumor types:
cervical carcinoma (HeLa), pancreatic carcinoma (MiaPaCa-2),
colon carcinoma (SW 620), breast carcinoma (MCF-7) and lung
carcinoma (H 460). Three compounds (2, 11 and 12) were
X
CN
H
N
N
N
X= H, Cl
CN
N
Fig. 1. Structures of earlier prepared cyano-substituted benzimidazoles and benzimidazo[1,2-a]quinolines.

M. Hranjec et al. / European Journal of Medicinal Chemistry 45 (2010) 2405e2417
2407
Ar
H
H
N
EtOH
EtOH
I2, O2
CN
N
piperidine
CH2CN
N
Ar-CHO
hν
N
Ar
N
1
N
NC
2-14
15-23
Scheme 1. Synthesis of 2-benzimidazolyl substituted acrylonitriles 2e14, benzimidazo[1,2-a]quinoline-6-carbonitriles 15e20 and heteroaromatic fluorenes 21e23.
Saçzewski et al., still supports the requirement of the cyano moiety
for good activity of tested compounds [22,23]. However, its pres-
ence alone is not sufficient to selectively inhibit tumor cell growth,
because the substituent on position 3 of acrylonitrile modulates
this selectivity (such as the N-methylpyrolyl substituent in 13 and
23) (Table 2).
concentrations up to 8.5 ꢁ10ꢂ6 mol dmꢂ3 (3),1.22 ꢁ 10ꢂ5 mol dmꢂ3
(4), 1.23 ꢁ 10ꢂ5 mol dmꢂ3 (6), 1.07 ꢁ 10ꢂ5 mol dmꢂ3 (13),
9.34 ꢁ10ꢂ6 mol dmꢂ3
(16),
1.05 ꢁ10ꢂ5 mol dmꢂ3
(17),
1.47 ꢁ 10ꢂ5 mol dmꢂ3 (19), 2.37 ꢁ 10ꢂ5 mol dmꢂ3 (23), indicating
that there is no significant intermolecular stacking which should
give rise to hypochromicity effects.
All compounds exhibited fluorescence emissions with maxima
at 452e495 nm, respectively. Therefore, excitation spectra fitted
well within the corresponding absorption spectra. Fluorescence
intensities of all compounds were found to be linearly dependent
on their concentrations up to 1.5 ꢁ10ꢂ6 mol dmꢂ3. Results of
spectroscopic characterization of aqueous solutions, which is
necessary for DNA interactions studies, by means of UV/vis and
fluorescence spectroscopy are presented in Table S1
(Supplementary data).
The aim of the screening was to estimate the probability that
biological activity of compounds (addressed in antitumor evalua-
tion in vitro) is at least partially the consequence of ct-DNA
binding. Taking into account the simplicity and mutual similarity
of the studied structures, we decided to perform the screening by
thermal denaturation experiments with the above-mentioned
compounds, as well as by means of UV/vis and fluorescence
spectroscopy.
3.2. Interaction with ct-DNA
Among all compounds presented in this study, representatives of
non-fused 3, 4, 6 and 13 and along with their fused 16, 17, 19 and 23
derivatives were chosen for preliminary screening of their non-
covalent interactions with ct-DNA. Due to the poor solubility of
studied compounds in water, stock solutions were prepared in
DMSO (c(3) ¼ 4.25 ꢁ10ꢂ3 mol dmꢂ3; c(4) ¼ 4.56 ꢁ 10ꢂ3 mol dmꢂ3
;
c
c
c(6) ¼ 5.25 ꢁ10ꢂ3 mol dmꢂ3
;
;
;
c(13) ¼ 4.03 ꢁ 10ꢂ3 mol dmꢂ3
c(17) ¼ 4.55 ꢁ10ꢂ3 mol dmꢂ3
;
;
(16) ¼ 4.67 ꢁ 10ꢂ3 mol dmꢂ3
(19) ¼ 4.72 ꢁ10ꢂ3 mol dmꢂ3
c(23) ¼ 4.47 ꢁ 10ꢂ3 mol ꢂ3). DMSO
solutions of compounds 3, 4, 6, 13, 16, 17, 19 and 23 were stable over
longer periods. In further experiments small aliquots of DMSO stock
solutions were added into the aqueous medium, provided that
DMSO content in experiments was less than 1%. Absorbencies of
aqueous solutions of compounds are proportional to their
Table 1
Prepared 2-benzimidazolyl substituted acrylonitriles 2e14, benzimidazo[1,2-a]quinoline-6-carbonitriles 15e20 and heteroaromatic fluorenes 21e23.
Comp.
Ar
Comp.
Ar
Comp.
Ar
OCH3
(H3CH2C)2N
NC
OH
2, 15
6, 19
10
H3CO
H3COCHN
H3C
3, 16
4, 17
7, 20
8, 21
11
O
F
N
12, 22
S
N
13, 23
(H3C)2N
× HCl
Br
CH3
5, 18
9
N
14
N
H

2408
M. Hranjec et al. / European Journal of Medicinal Chemistry 45 (2010) 2405e2417
Table 2
strong hypochromic effect. Most of examined compounds showed
also small hypsochromic effect according to Table S2
(Supplementary data). These results suggested that studied
non-fused compounds showed significant interaction with ct-
DNA. On the other hand, fused compounds have precipitated
upon adding of small aliquots of ct-DNA, especially at concen-
tration of ct-DNA higher than 2.0 ꢁ 10ꢂ5 mol dmꢂ3. Compounds
16 and 19 did not yield any measurable results during the UV/vis
titrations with ct-DNA.
Stronger fluorescence of fused compounds 16, 17, 19 and 23 in
aqueous medium allowed their titrations with ct-DNA to be done at
order of magnitude lower concentrations in respect to UV/vis
experiments. Non-fused compounds 3, 4, 6 and 13 showed some-
what lower fluorescence emission compared to fused compounds.
Results of fluorescence titrations with ct-DNA as well as the
dependence of fluorescence intensity on fluorescence emission
maxima on added ct-DNA of compounds 16, 17 and 19 are pre-
sented in Fig. 3.
The addition of ct-DNA to aqueous solutions of compounds 16,
17 and 19 strongly quenched fluorescence intensity of all
compounds on their emission maxima and thus yielded strong
hypochromic effects. Fluorescence titrations of compound 23
showed a different result, the addition of ct-DNA to aqueous solu-
tion of compound 23 yielded very low increasing of fluorescence
intensity according to Table S2 (Supplementary data). It should be
noted that, in contrast to fused compounds, especially 16,17 and 19,
the addition of ct-DNA to aqueous solution of non-fused
compounds 3, 4 and 13 induced lower fluorescence intensity
increase upon adding of ct-DNA. Addition of ct-DNA to non-fused
compound 6 yielded lower decrease of fluorescence intensity.
Obtained results of UV/vis and fluorescence titrations are summa-
rized in Table S2 (Supplementary data).
Considering the above-mentioned results of preliminary ct-DNA
binding studies of selected non-fused 3, 4, 6 and 13 and fused
compounds 16, 17, 19 and 23, we can conclude that all compounds
showed significant interactions with ct-DNA. Therefore, interaction
of all tested compounds with cellular DNA could be at least partially
responsible for the observed antiproliferative effects. To confirm
the exact binding mode of these derivatives to ct-DNA additional
experiments should be done.
In vitro inhibition of compounds 3e23 on the growth of tumor cells.
Comp.
IC50
HeLa
(m
M)a
MiaPaCa-2
SW 620
MCF-7
H 460
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
cisb
doxb
20 ꢃ 15
15 ꢃ 7
9 ꢃ 3
32 ꢃ 3
18 ꢃ 4
46 ꢃ 3
0.8 ꢃ 0.7
48 ꢃ 19
57 ꢃ 38
26 ꢃ 19
3 ꢃ 0.8
28 ꢃ 5
>50
87 ꢃ 7
59 ꢃ 40
>50
8 ꢃ 2
81 ꢃ 16
43 ꢃ 1
>50
14 ꢃ 5
19 ꢃ 16
12 ꢃ 3
4 ꢃ 2
1 ꢃ 0.2
3 ꢃ 2
23 ꢃ 5
27 ꢃ 5
>100
>100
21 ꢃ 11
42 ꢃ 22
>50
9 ꢃ 6
78 ꢃ 21
>50
>50
4 ꢃ 4
3 ꢃ 2
10 ꢃ 5
2 ꢃ 1
11 ꢃ 0.3
25 ꢃ 13
8 ꢃ 1
>50
17 ꢃ 8
30 ꢃ 17
13 ꢃ 3
92 ꢃ 1
>50
>50
48 ꢃ 0.2
30 ꢃ 5
70 ꢃ 27
14 ꢃ 4
>100
>50
0.8 ꢃ 0.4
40 ꢃ 24
16 ꢃ 3
>100
4 ꢃ 2
26 ꢃ 13
30 ꢃ 8
>100
>50
23 ꢃ 4
>100
>100
70 ꢃ 20
50 ꢃ 46
37 ꢃ 24
31 ꢃ 29
5 ꢃ 4
29 ꢃ 7
20 ꢃ 1
5 ꢃ 0.3
2 ꢃ 0.9
45 ꢃ 32
19 ꢃ 1
0.7 ꢃ 0.2
3 ꢃ 0.6
0.04 ꢃ 0.009
28 ꢃ 11
22 ꢃ 6
13 ꢃ 0.5
6 ꢃ 3
26 ꢃ 4
37 ꢃ 15
14 ꢃ 9
90 ꢃ 7
57 ꢃ 40
30 ꢃ 8
22 ꢃ 2
0.3 ꢃ 0.04
N.T.c
>50
23 ꢃ 4
2 ꢃ 0.6
32 ꢃ 15
28 ꢃ 15
4 ꢃ 2
5 ꢃ 3
30 ꢃ 5
16 ꢃ 3
25 ꢃ 4
4 ꢃ 1
67 ꢃ 35
31 ꢃ 20
11 ꢃ 1
12 ꢃ 6
0.04 ꢃ 0.01
4 ꢃ 3
0.01 ꢃ 0.01
0.02 ꢃ 0.01
a
IC50; the concentration that causes a 50% reduction of the cell growth.
cis e cisplatin; dox e doxorubicin.
N.T. e not tested.
b
c
Thermal denaturation experiments are usually used to appre-
ciate the capacity of the molecules to stabilize the double helix of
ct-DNA. The fast screening results are performed at the same ratio
r ¼ 0.3 for all chosen compounds and are presented in Table 3.
Both non-fused (3, 4, and 6) and fused (16, 17 and 19)
compounds stabilized the ct-DNA under the conditions used in this
experiment, except 13. Compound 23 showed a lower stabilization
of ct-DNA than other fused derivatives. Results for fused
compounds are similar to results obtained for other intercalators
related to benzimidazolo[1,2-a]quinolines prepared earlier by our
group [26,27]. Based on our previous results, we can presume that
although
DTm values of both fused and non-fused compounds are
similar, it is not likely that they bind to DNA by the same binding
mode based on their structural differences. Namely, fused
compounds have a planar aromatic surface that can intercalate into
double-stranded DNA and cyano group which could participate in
CeH/N hydrogen bonds formation, while non-fused compounds
most likely could bind to minor groove in DNA.
In order to confirm binding of chosen compounds to ct-DNA,
their interaction with ct-DNA was also monitored by UV/vis
absorption and fluorescence emission spectra.
Spectroscopic changes obtained from UV/vis titrations with
ct-DNA as well as a dependence of the absorbance intensity at
absorbance maxima on added ct-DNA of compounds 3, 4, 6 and 13
are presented in Fig. 2.
The absorption of compounds 3, 4, 6 and 13 on the absorption
maxima decreased upon addition of ct-DNA, thus, inducing
4. Crystal structure determination
4.1. Crystal structure description of compound 4
Compound 4 crystallizes with ethanol molecule as crystalliza-
tion solvent. ORTEP drawing of molecular structure is depicted in
Fig. 4.
The molecule deviates from planarity in the crystalline state
which is described by the dihedral angle [13.11(2)ꢀ] calculated
between best planes defined by atoms of phenyl (C10eC15) and
benzimidazole ring (C1, N1, N2, C2, C3, C4, C5, C6 and C7). This is to
some extent a larger value than corresponding values found in the
structures of 2-(1H-benzimidazol-2-yl)-3-(4-cyanophenyl)-acrylo-
nitrile [5.6(1)ꢀ] and 2-(1H-benzimidazol-2-yl)-3-(4-bromophenyl)-
acrylonitrile [3.5(1)ꢀ] published earlier by our group [33]. The
rotation around two bonds, which are predominantly
s in char-
ꢂ
ꢂ
acter: C1eC8 (1.462(3) A) and C9eC10 (1.452(3) A) is described by
the torsion angles C9eC8eC1eN1 and C8eC9eC10eC11 being
Table 3
Tm valuesa (ꢀC) of ct-DNA upon addition of 3, 4, 6, 13, 16, 17, 19 and 23 at ratio rb ¼ 0.3
174.7(2)ꢀ and ꢂ9.4(4)ꢀ. The C8]C9 bond has dominantly
p-char-
at pH ¼ 7.0 (buffer sodium cacodylate, I ¼ 0.05 mol dmꢂ3).
ꢂ
acter (1.346(3) A). Considering spatial orientation of H atom and
cyano group around C8]C9 bond, the molecule exists in the
crystalline state as E-isomer.
Comp.
3
4
6
13
16
17
19
23
D
Tm
2.9
3.0
3.0
0.4
4.8
4.4
4.2
2.4
a
Error in
D
Tm: ꢃ0.5 ꢀC.
The hydrogen bonding network (Table 4) is formed between
molecules of 2-(2-benzimidazolyl)-3-(4-fluorophenyl)acrylonitrile
b
r ¼ [compound]/[polynucleotide].

M. Hranjec et al. / European Journal of Medicinal Chemistry 45 (2010) 2405e2417
2409
increasing ct-DNA
3
0.22
B
0.20
0.20
0.15
0.10
0.05
A
0.18
0.16
0.14
0.12
0.10
0.08
300
325
350
375
400
c(ctDNA) / moldm-3
λ / nm
0.210
0.205
0.200
0.195
0.190
0.185
0.180
0.175
0.170
4
C
D
0.20
0.15
0.10
0.05
increasing ct-DNA
300
325
350
375
400
λ / nm
c(ctDNA) / moldm-3
0.21
0.20
0.19
0.18
0.17
0.16
0.15
0.20
0.16
0.12
0.08
0.04
0.00
E
F
6
increasing ct-DNA
300
350
400
450
λ / nm
c(ctDNA) / moldm-3
13
0.4
0.3
0.2
0.1
0.0
increasing ct-DNA
G
H
0.40
0.36
0.32
0.28
0.24
350
400
450
λ / nm
-3
c(ctDNA) / moldm
Fig. 2. A) The UV/vis titration of 3 (c ¼ 7.1 ꢁ10ꢂ6 mol dmꢂ3) with ct-DNA; B) dependence of absorbance of 3 at lmax ¼ 353 nm on concentration of ct-DNA; C) the UV/vis titration of
4 (c ¼ 1.22 ꢁ 10ꢂ5 mol dmꢂ3) with ct-DNA; D) dependence of absorbance of 4 at lmax ¼ 345 nm on concentration of ct-DNA; E) the UV/vis titration of 6 (c ¼ 1.23 ꢁ 10ꢂ5 mol dmꢂ3
)
with ct-DNA; F) dependence of absorbance of 6 at lmax ¼ 336 nm on concentration of ct-DNA; G) the UV/vis titration of 13 (c ¼ 1.07 ꢁ 10ꢂ5 mol dmꢂ3) with ct-DNA; H) dependence
of absorbance of 6 at lmax ¼ 403 nm on concentration of ct-DNA; experiments were done at pH 7 (buffer sodium cacodylate, I ¼ 0.05 mol dmꢂ3).

2410
M. Hranjec et al. / European Journal of Medicinal Chemistry 45 (2010) 2405e2417
600
500
400
300
200
100
0
A
B
16
increasing of ct-DNA
550
500
450
400
350
300
250
200
400
450
500
550
λ / nm
c(ctDNA) / moldm-3
700
600
500
400
300
200
100
0
C
D
17
increasing ct-DNA
700
650
600
550
500
450
400
350
400
450
500
550
λ / nm
c(ctDNA) / moldm-3
500
400
300
200
100
0
E
F
19
450
400
350
300
250
200
increasing ct-DNA
450
500
550
λ / nm
c(ctDNA) / moldm-3
Fig. 3. A) Changes in the fluorescence emission spectra of 16; B) dependence of fluorescence intensity of 16 at
l
¼ 472 nm on ct-DNA concentration; C) changes in the fluorescence
emission spectra of 17; D) dependence of fluorescence intensity of 17 at
l
¼ 470 nm on ct-DNA concentration; E) changes in the fluorescence emission spectra of 19; F) dependence
of fluorescence intensity of 19 at
l
¼ 482 nm on ct-DNA concentration; experiments were done at pH 7 (buffer sodium cacodylate, I ¼ 0.05 mol dmꢂ3).
and ethanol molecules such as that oxygen atom of ethanol mole-
cule acts as proton donor (O1eH11O/N1) and as double proton
acceptor (N2eH12N/O1 and C9eH9/O1). Weak intermolecular
hydrogen bonds of the CeH/N and CeH/F type act between
molecules of compound (Table 4) [34,35]. No F/F interhalogen
contacts are observed. The participation of cyano N3 atom in
hydrogen bonding formation is not found, contrary to the
structures of 2-(1H-benzimidazol-2-yl)-3-(4-cyanophenyl)-acrylo-
nitrile and 2-(1H-benzimidazol-2-yl)-3-(4-bromophenyl)-acrylo-
nitrile [33].
The geometry of pep interactions is listed in Table 5. The p/p
aromatic stacking is found between the benzimidazole and phenyl
(C10eC15) rings. Those possible interactions for which perpen-
ꢂ
dicular distance between two planes is longer than 3.8 A

M. Hranjec et al. / European Journal of Medicinal Chemistry 45 (2010) 2405e2417
2411
Table 5
Geometrical parameters of pep
interactions (A, ꢀ) for compounds 4, 16 and 17.
ꢂ
Interactiona CgeCg distance Cg/P1b
Cg/P2b
bc
Slippage
4
Cg1/Cg3i
3.7614(15)
3.7044(9)
3.4650(11) 22.90 1.464
16
Cg1/Cg2ii
Cg1/Cg4ii
Cg1/Cg4iii
Cg2/Cg2i
Cg2/Cg4iii
3.638(3)
3.611(3)
3.731(3)
3.568(3)
3.629(3)
3.3558(5) ꢂ3.3395(5)
23.37 1.443
20.21 1.247
24.26 1.405
19.85 1.211
21.28 1.317
3.3610(5)
3.3878(5)
3.3560(5)
3.3818(5)
3.3889(6)
3.4012(6)
3.3560(5)
3.3816(6)
17
Cg1/Cg3iii
Cg1/Cg4iv
Cg2/Cg4iv
Cg4/Cg1iv
3.7300(7)
3.6415(7)
3.6728(7)
3.6414(7)
3.3459(5)
3.2918(5)
3.3099(5)
3.3913(5)
3.3856(5)
3.3914(5)
3.3592(5)
3.2919(5)
26.23 1.565
21.36 1.326
23.85 1.485
25.31 1.556
a
Rings Cg1 and Cg3 are defined by the atoms N1, C1, N2, C7, C2 and C10, C11, C12,
C13, C14 and C15 in 4, respectively. Rings Cg1, Cg2 and Cg4 are defined by the atoms
N1, C1, N2, C2, C7; N1, C1, C8, C9, C10, C15 and C10, C11, C12, C13, C14 and C15 in 16,
respectively. Rings Cg1 Cg2, Cg3 and Cg4 are defined by the atoms N11, C11, N12, C12,
C17; N11, C11, C18, C19, C110, C115; C12, C13, C14, C15, C16, C17 and C110, C111,
C112, C113, C114, C115 in 17, respectively.
Fig. 4. ORTEP drawing of 2-(2-benzimidazolyl)-3-(4-fluorophenyl)acrylonitrile 4 with
the atomic numbering scheme. The thermal ellipsoids are drawn at the 50% probability
level at 293 K.
b
Cg/P(1 or 2) is the perpendicular distance of corresponding centroid to a plane.
Planes P1 or P2 are defined by the atoms, which define the corresponding centroids.
c
ꢂ
b
is the angle between Cg/Cg vector and vertical line on corresponding plane.
codes: ii ¼ 2 ꢂ x,ꢂy,1 ꢂ z, iii ¼ 2 ꢂ x,1 ꢂ y,1 ꢂ z,
i ¼ ꢂ1 þ x,y,z,
iv ¼ 1 ꢂ x,1 ꢂ y,1 ꢂ z.
(3.3e3.8 A) (Cg/P1 and Cg/P2; the perpendicular distance of
Symmetry
corresponding centroid to a plane) and dihedral angles (between
P1 and P2) larger than 20ꢀ are not taken into account [36e39]. The
calculated slippage on the basis of Cg/Cg distances as well as the
value of the angle between Cg/Cg vector and vertical line on cor-
responding plane has been taken into account as one of the
geometrical criteria, too (approx. 1.5 A for
interactions).
In the structure of 17 two CeH/N hydrogen bonds are found
mutually connecting two independent molecules. One hydrogen
bond is formed with the cyano N13 atom and another with the
imidazole N22 atom (Table 4). The interhalogen F1/F2i
ꢂ
p/p stacking
ꢂ
(i ¼ x,ꢂ1 þ y,z) contact of 2.946(2) A is found in 17. The geometry of
p/p interactions for 16 and 17 are listed in Table 6. The same
4.2. Crystal structures descriptions of compounds 16 and 17
geometrical criterion as for compound 4 has been applied.
ORTEP drawings of molecular structures for compounds 16 and
17 are depicted in Figs. 5 (compound 16) and 6 (compound 17). The
molecular structure of 17 contains two crystallographically inde-
pendent molecules within asymmetric unit. The selected molecular
geometry is listed in Table 6.
The molecules of 16 and 17 are planar. The dihedral angles
calculated between the two planes defined by atoms N1 N2 C1 C2
C3 C4 C5 C6 and C7 (one plane) and N1 C1 C8 C9 C10 C11 C12 C13
C14 and C15 (another plane) amount 2.43(4)ꢀ and 4.27(4)ꢀ (first
molecule) and 1.49(4)ꢀ (second molecule) for 16 and 17, respec-
tively. Crystal structures of 16 and 17 are dominated by CeH/N
intermolecular hydrogen bonds as it is found in analogous deriva-
tives, benzimidazo[1,2-a]quinoline-2,6-dicarbonitrile and 2-
bromo-benzimidazo[1,2-a]quinoline-2-carbonitrile [33]. In the
structure of 16 only one intermolecular hydrogen bond is found
with the cyano N3 atom as proton acceptor (Table 4).
Table 4
Hydrogen bond geometry (A, ꢀ) for compounds 4, 16 and 17.
ꢂ
DeH/A
DeH
H/A D/A
:DeH/A Symmetry code
4
N2eH12N/O1 0.96(3) 1.84(3) 2.796(2) 172(2)
O1eH11O/N1 0.97(4) 1.90(4) 2.834(2) 162(4)
e
2 ꢂ x,ꢂ1/2 þ y,ꢂ1/2 ꢂ z
e
C9eH9/N2
0.9300 2.5800 2.958(3) 105
C18eH18B/F1 0.9600 2.4900 3.332(6) 147
7/2 ꢂ x,ꢂ1 ꢂ y,1/2 þ z
16
C6eH6/N3
0.9300 2.6200 3.463(3) 151
1/2 þ x,1/2 ꢂ y,1/2 þ z
17
Fig. 5. ORTEP drawing of 2-methylbenzimidazo[1,2-a]quinoline-6-carbonitrile 16 with
the atomic numbering scheme. The thermal ellipsoids are drawn at the 50% probability
level at 293 K.
C19eH19/N22 0.9300 2.4600 3.370(2) 165
C26eH26/N13 0.9300 2.5200 3.348(2) 149
x,y,1 þ z
1 ꢂ x,2 ꢂ y,1 ꢂ z

2412
M. Hranjec et al. / European Journal of Medicinal Chemistry 45 (2010) 2405e2417
Fig. 6. ORTEP drawing of 2-fluorobenzimidazo[1,2-a]quinoline-6-carbonitrile 17 with the atomic numbering scheme. The thermal ellipsoids are drawn at the 50% probability level
at 293 K.
5. Conclusions
point to the possibility that antitumor activity of examined
compounds could be at least partially the consequence of ct-DNA
binding.
This study represents the synthesis of novel benzimidazole
derivatives related to 2,3-disubstituted acrylonitriles, benzimidazo
[1,2-a]quinolines and fluorenes as potential antitumor agents.
All compounds, except 16 and 14 exerted pronounced anti-
proliferative activity on five tumor cell lines, whereby 3-N-meth-
ylpyrolyl-2-benzimidazolyl-acrylonitrile derivative 13 and its fused
analogue 23 exerted the highest activity on all cell lines
The crystal structures of three compounds have been deter-
mined in order to confirm molecular planarity in the crystalline
state and to reveal possible types of hydrogen bonds formation. The
crystal structures of fused derivatives 16 and 17 confirmed that
hydrogen bonding patterns are characterized dominantly by the
weak CeH/N bonds with the cyano group and the imidazole N
atom acting as proton acceptors and the aryl CeH groups as proton
donors. In the structure of 4 the cyano group does not participate in
hydrogen bonds formation but the presence of ethanol molecule
yields to NeH/O and OeH/N type of hydrogen bonds as well as
weak CeH/N(F) hydrogen bonds with the F atom and the imid-
azole NH group, which acts doubly as a proton donor and a proton
acceptor.
(IC50 ¼ 0.8e30
mM) and showed a special selectivity toward HeLa
cells. There is no major difference in the biological activity between
non-fused and fused analogues. The cyano moiety is important for
good activity of tested compounds, but its presence alone is not
sufficient to selectively inhibit tumor cell growth. Preliminary
screening of non-covalent interactions of some chosen non-fused
and fused derivatives with ct-DNA, showed significant binding
affinities for ct-DNA. Non-fused derivatives 3, 4, and 6 showed
stabilization of ct-DNA while compound 13 showed negligible
stabilization of ct-DNA under the conditions used in fast screening
thermal melting experiment. Fused compounds 16, 17 and 19
showed significant stabilization of ct-DNA. All chosen compounds
showed interactions with ct-DNA according to results obtained
from UV/vis and fluorescence emission titrations. These results
6. Experimental protocols
6.1. Chemistry
All chemicals were purchased from commercial suppliers.
Melting points were recorded on an Original Keller Mikroheitztisch
apparatus (Reichert, Wien) and Büchi 535. The 1H and 13C NMR
spectra were recorded on a Varian Gemini 300 or Varian Gemini
600 at 300, 600 and 150 and 75 MHz, respectively. All NMR spectra
were measured in DMSO-d6 solutions using TMS as an internal
standard. All compounds were routinely checked by TLC with
Merck silica gel 60F-254 glass plates. Elemental analysis for carbon,
hydrogen and nitrogen were performed on a PerkineElmer 2400
elemental analyzer. Where analyses are indicated only as symbols
of elements, analytical results obtained are within 0.4% of the
theoretical value.
Table 6
ꢀ
ꢂ
Selected interatomic distances (A) and valence angles ( ) for the compounds 16 and
17.
16
17
17
(First molecule; 1) (Second molecule; 2)
Selected bond distances
C15eN1
C7eN1
C1eN1
C1eN2
1.404(2)
1.406(2)
1.399(2) 1.4005(1)
1.306(2)
1.386(2)
1.501(2)
e
1.401(2)
1.400(1)
1.397(2)
1.403(2)
1.397(1)
1.307(2)
1.381(2)
e
1.308(2)
1.381(2)
e
C2eN2
6.2. General method for preparation of compounds 2e14
C13eC17 (in 16)
C113eF1 and
C213eF2 (both in 17)
1.347 (2)
1.349(2)
Solution of equimolar amounts of 2-cyanomethylbenzimidazole
1, corresponding heteroaromatic aldehydes and few drops of
piperidine in absolute ethanol, were refluxed for 2e4 h. After
reaction mixture was cooled to the room temperature, the crude
product was filtered off and recrystallized from ethanol.
Bond angles
C1eN1eC7
C15eN1eC1
C15eN1eC7
104.87(11) 105.23(9)
122.41(11) 122.37(9)
132.72(11) 132.37(9)
105.06(9)
122.30(10)
132.62(10)

M. Hranjec et al. / European Journal of Medicinal Chemistry 45 (2010) 2405e2417
2413
6.2.1. 2-(2-Benzimidazolyl)-3-(4-cyanophenyl)acrylonitrile 2 [30]
Compound 2 was prepared from 1 (1.60 g, 12.7 mmol) and
p-cyanobenzaldehyde (2.00 g, 12.7 mmol) in absolute ethanol
(20 ml) after refluxing for 3 h and recrystallization from ethanol to
yield 2.60 g (75%) of yellow crystals; m.p. 290e292 ꢀC, m.p. (lit)
(20 ml) after refluxing for 4 h and recrystallization from ethanol to
yield 1.37 g (70%) of yellow crystals; m.p. 234e235 ꢀC.
IR (KBr) n
/cmꢂ1: 3398, 2230, 2220,1671,1645; 1HNMR (600 MHz,
DMSO-d6):
d
¼ 13.03 (s, NHbenzimidazole, 1H), 8.51 (s, 1H), 7.73 (s, 1H),
7.68 (d, J ¼ 7.80 Hz, Harom, 1H), 7.53 (d, J ¼ 7.74 Hz, Harom, 1H), 7.28
(t, J ¼ 7.74 Hz, Harom, 1H), 7.23 (d, J ¼ 7.74 Hz, Harom, 1H), 7.17e7.14
(m, Harom, 2H), 3,90 (s, OCH3, 3H), 3,81 (s, OCH3, 3H); 13C NMR
290e291 ꢀC; IR (KBr)
(300 MHz, DMSO-d6):
n
/cmꢂ1: 3332, 2225,2214, 1641, 1619; 1H NMR
¼ 13.32 (s, NHbenzimidazole, 1H), 8.40 (s, 1H),
d
8.11 (d, J ¼ 8.64 Hz, Harom, 2H), 8.04 (d, J ¼ 8.52 Hz, Harom, 2H), 7.68
(150 MHz, DMSO-d6):
d
¼ 152,94 (s),152.58 (s),147.43 (s),143.42 (s),
(br s, Harom, 1H), 7.63 (d, J ¼ 7.70 Hz, Harom, 1H), 7.27 (br s, Harom
,
139.93 (d), 134.94 (s), 123.52 (d), 122.22 (d), 121.92 (s), 119.17 (d),
119.12 (d),116.19 (s),113.37 (d),112.92 (d),111.57 (d),102.90 (s), 56.44
(q), 55.68 (q); elementalanalysiscalcd. (%)for C18H15N3O2:C 70.81, H
4.95, N 13.76; found: C 70.54, H 4.75, N 13.39.
2H).
13C NMR (75 MHz, DMSO-d6):
d
¼ 147.28 (s), 147.27 (s), 143.68
(d), 137.44 (s), 133.48 (d, 2C), 130.42 (d, 2C), 118.79 (s), 124.55 (d),
123.18 (d), 119.20 (d), 118.79 (s), 116.07 (s), 113.58 (s), 112.09 (d),
105.99 (s); elemental analysis calcd. (%) for C17H10N4: C 75.54, H
3.73, N 20.73; found: C 75.77, H 3.98, N 20.44.
6.2.6. 2-(2-Benzimidazolyl)-3-(4-N-acetamidophenyl)acrylonitrile 7
Compound 7 was prepared from 1 (1.50 g, 9.6 mmol) and p-N-
acetamidobenzaldehyde (1.57 g, 9.6 mmol) in absolute ethanol
(30 ml) after refluxing for 4 h to yield 2.10 g (72%) of yellow
powder; m.p. > 285 ꢀC.
6.2.2. 2-(2-Benzimidazolyl)-3-(4-methylphenyl)acrylonitrile 3
Compound 3 was prepared from 1 (1.00 g, 6.4 mmol) and
p-methylbenzaldehyde (0.77 g, 6.4 mmol) in absolute ethanol
(15 ml) after refluxing for 3 h and recrystallization from ethanol to
yield 1.66 g (72%) of yellow crystal; m.p. 254e256 ꢀC.
IR (KBr)
(300 MHz, DMSO-d6):
n
/cmꢂ1: 3409, 3379, 2222, 2209, 1667, 1642; 1H NMR
d
¼ 13.01 (s, NHbenzimidazole, 1H), 10.36 (s, NH,
1H), 8.25 (s, 1H), 7.97 (d, J ¼ 8.79 Hz, Harom, 2H), 7.79 (d, J ¼ 8.76 Hz,
IR (KBr)
(600 MHz, DMSO-d6):
n
/cmꢂ1: 3354, 2227, 2210, 1653, 1634; 1H NMR
Harom, 2H), 7.67e7.56 (m, Harom, 2H), 7.25 (br s, Harom, 2H), 2.11
(s, COCH3, 3H); 13C NMR (75 MHz, DMSO-d6):
d
¼ 168.94 (s), 147.80
d
¼ 13.04 (s, NHbenzimidazole, 1H), 8.31 (s, 1H),
7,91 (d, J ¼ 8.22 Hz, Harom, 2H), 7.66e7.61 (m, Harom, 2H), 7.42
(d, J ¼ 8,10 Hz, Harom, 2H), 7.29e7.25 (m, Harom, 2H), 2.41 (s, CH3,
(s), 144.82 (d), 143.37 (s), 142.43 (s), 134.77 (s), 131.43 (d), 130.79
(d, 2C), 127.12 (s), 123.43 (d), 122.16 (d), 118.95 (d, 2C), 116.58 (s),
111.44 (d), 99.67 (s), 24.15 (q); elemental analysis calcd. (%) for
C18H14N4O: C 71.51, H 4.67, N 18.53; found: C 71.77, H 4.92, N 18.29.
3H); 13C NMR (75 MHz, DMSO-d6):
d
¼ 148.08 (s), 145.87 (d), 143.00
(s), 142.70 (s), 130.67 (d), 130.49 (s),130.45 (s), 130.39 (d, 2C),130.09
(d, 2C), 124.10 (d), 119.80 (d), 21.69 (q); elemental analysis calcd. (%)
for C17H13N3: C 78.74, H 5.05, N 16.20; found: C 78.98, H 5.28, N
16.46.
6.2.7. 2-(2-Benzimidazolyl)-3-(4-pyridyl)acrylonitrile 8
Compound 8 was prepared from 1 (1.80 g, 11.5 mmol) and
4-pyridinecarboxaldehyde (1.22 g, 11.5 mmol) in absolute ethanol
(25 ml) after refluxing for 3.5 h and recrystallization from ethanol
to yield 1.34 g (50%) of orange powder; m.p. 253e255 ꢀC.
6.2.3. 2-(2-Benzimidazolyl)-3-(4-fluorophenyl)acrylonitrile 4
Compound 4 was prepared from 1 (2.00 g, 12.7 mmol) and p-
methylbenzaldehyde (1.59 g, 12.7 mmol) in absolute ethanol
(25 ml) after refluxing for 4 h and recrystallization from ethanol to
yield 2.05 g (61%) of slightly yellow crystals; m.p. 266e267 ꢀC.
IR (KBr)
n
/cmꢂ1: 3212, 2219, 1633, 1617; 1H NMR (300 MHz,
DMSO-d6):
d
¼ 13.24 (s, NHbenzimidazole, 1H), 8.81 (d, J ¼ 6.10 Hz,
Hpyridine, 2H), 8.32 (s, 1H), 7.83 (d, J ¼ 6.10 Hz, Hpyridine, 2H), 7.71
IR (KBr)
(600 MHz, DMSO-d6):
8.08 (ABq, J1 ¼8.73 Hz, J2 ¼ 5.49 Hz, Harom, 2H), 7.47 (t, J ¼ 8.83 Hz,
n
/cmꢂ1: 3369, 2219, 2203, 1644, 1621; 1H NMR
¼ 13.08 (s, NHbenzimidazole, 1H), 8.36 (s, 1H),
(t, J ¼ 8.10 Hz, Harom., 1H), 7.58 (t, J ¼ 7.90 Hz, Harom, 1H), 7.31e7.25
d
(m, Harom, 2H); 13C NMR (75 MHz, DMSO-d6):
d
¼ 151.20 (d, 2C),
147.03 (s), 143.07 (d), 140.09 (s), 129.80 (s), 126.77 (s), 124.70 (d),
123.04 (d, 2C), 123.01 (d), 120.01 (d), 115.78 (s), 112.31 (d), 107.39
(s); elemental analysis calcd. (%) for C15H10N4: C 73.16, H 4.09, N
22.75; found: C 73.33, H 4.22, N 22.51.
Harom
,
1H), 7.35 (ABq, J1 ¼8.67 Hz, J2 ¼ 5.47 Hz, Harom
, 2H),
7.30e7.25 (m, Harom, 2H), 7,06 (t, J ¼ 8,77 Hz, Harom, 1H); 13C NMR
(75 MHz, DMSO-d6):
d
¼ 148.92 (s), 145.99 (d), 144.32 (s), 134.34 (s),
132.01 (d, 2C), 131.77 (s), 131.57 (d, 2C), 127.82 (s), 124.58 (d), 123.61
(d), 120.87 (d), 117.98 (s), 113.60 (d), 103.02 (s); elemental analysis
calcd. (%) for C16H10FN3: C 72.99, H 3.83, N 15.96; found: C 73.21, H
3.9728, N 16.12.
6.2.8. 2-(2-Benzimidazolyl)-3-(4-N-dimethylaminophenyl)
acrylonitrile hydrochloride 9
Compound 9 was prepared from 1 (0.80 g, 5.2 mmol) and p-N-
dimethylaminobenzaldehyde (0.76 g, 5.2 mmol) in absolute
ethanol (10 ml) after refluxing for 2.5 h and recrystallization from
ethanol to yield 1.06 g (72%) of orange powder. Suspension of
orange powder (0.20 g, 0.70 mmol) in absolute ethanol (10 mL) was
cooled in an ice-salt bath and was saturated with HCl gas. The flask
was then tightly stoppered and the mixture was maintained at
room temperature for 36 h. Obtained product was filtered off and
recrystallized from ethanol to yield 0.18 g (81%) of violet crystals;
m.p. 282e283 ꢀC.
6.2.4. 2-(2-Benzimidazolyl)-3-(4-bromophenyl)acrylonitrile 5
Compound 5 was prepared from 1 (1.00 g, 6.4 mmol) and
p-brombenzaldehyde (1.18 g, 6.4 mmol) in absolute ethanol (15 ml)
after refluxing for 4 h and recrystallization from ethanol to yield
0.95 g (52%) of dark yellow crystal powder; m.p. 251e252 ꢀC.
IR (KBr)
(300 MHz, DMSO-d6):
n :
/cmꢂ1 3370, 2223, 2211, 1663, 1618; 1H NMR
d
¼ 13.11 (s, NHbenzimidazole, 1H), 8.31 (s, 1H),
7.92 (d, J ¼ 8.61 Hz, Harom, 2H), 7.81 (d, J ¼ 8.61 Hz, Harom, 2H), 7.70
(d, J ¼ 7.71 Hz, Harom, 1H), 7.56 (d, J ¼ 7.68 Hz, Harom, 1H), 7.29e7.23
IR (KBr)
n
/cmꢂ1: 3400, 2889, 2232,1602,1589; 1H NMR (300 MHz,
(m, 2H, Harom); 13C NMR (150 MHz, DMSO-d6):
d
¼ 147.18 (s), 144.07
DMSO-d6):
d
¼ 12.78 (s, NH,1H), 8.13(s,1H), 7.90 (d, J ¼ 8.95 Hz, Harom
,
(d), 143.34 (s), 134.78 (s), 132.32 (d, 2C), 131.92 (s), 131.26 (d, 2C),
125.11 (s), 123.78 (d), 122.38 (d), 119.27 (d), 115.92 (s), 111.61 (d),
103.09 (s); elemental analysis calcd. (%) for C16H10BrN3: C 59.28, H
3.11, N 12.96; found: C 58.93, H 3.25, N 13.31.
2H), 7.62 (d, J ¼ 7.17 Hz, Harom, 1H), 7.50 (d, J ¼ 7.12 Hz, Harom, 1H),
7.22e7.18 (m, Harom, 2H), 6.86(d, J ¼ 8.99 Hz, Harom, 2H), 3.06 (s, 2CH3,
6H);13C NMR (75 MHz, DMSO-d6):
d
¼ 155.11 (s),151.59 (s),148.16 (d),
136.57 (s), 134.55 (d, 2C), 127.89 (s), 125.48 (d), 124.54 (d), 122.43 (s),
121.28 (d), 120.44 (s), 118.77 (s), 114.52 (d, 2C), 113.82 (d), 96.37 (s),
25.24 (q, 2C); elemental analysis calcd. (%) for C18H16N4: C 74.98, H
5.59, N 19.43; found: C 75.20, H 5.68, N 19.67.
6.2.5. 2-(2-Benzimidazolyl)-3-(2,5-dimetoyxphenyl)acrylonitrile 6
Compound 6 was prepared from 1 (1.00 g, 6.4 mmol) and 2,5-
dimethoxybenzaldehyde (1.06 g, 6.4 mmol) in absolute ethanol

2414
M. Hranjec et al. / European Journal of Medicinal Chemistry 45 (2010) 2405e2417
6.2.9. 2-(2-Benzimidazolyl)-3-(4-N-diethylamino-2-
hydroxyphenyl)acrylonitrile 10
Compound 10 was prepared from 1 (1.50 g, 9.6 mmol) and p-N-
diethylamino-2-hydroxybenzaldehyde (1.84 g, 9.6 mmol) in absolute
ethanol (20 ml) after refluxing for 4 h and recrystallization from
ethanol to yield 2.06 g (65%) of orange powder; m.p. 247e248 ꢀC; IR
6.2.13. 2-(2-Benzimidazolyl)-3-(5-imidazolyl)acrylonitrile 14
Compound 14 was prepared from 1 (1.50 g, 9.6 mmol) and
5-imidazolecarboxaldehyde (0.92 g, 9.6 mmol) in absolute ethanol
(20 ml) after refluxing for 4 h and recrystallization from ethanol to
yield 1.61 g (79%) of beige powder; m.p. > 285 ꢀC.
IR (KBr)
n
/cmꢂ1: 3420, 3235, 2233, 2221, 1655, 1632; 1H NMR
1H), 13.20
(KBr)
DMSO-d6):
(d, J ¼ 9.05 Hz, Harom
n
/cmꢂ1: 3420, 3369, 2232, 2129,1602,1589; 1H NMR (300 MHz,
(300 MHz, DMSO-d6):
d
¼ 16.03 (s, NHimidazole
,
d
¼ 12.72 (s, NHbenzimidazole, 1H), 8.44 (s, 1H), 8.19
(s, NHbenzimidazole, 1H), 8.32 (s, 1H), 7.98 (s, 1H), 7.77 (s, 1H),
,
2H), 7.66e7.58 (m, Harom 2H), 7.20
,
7.74e7.69 (m, Harom, 2H), 7.33e7.26 (m, Harom, 2H); 13C NMR
(d, J ¼ 7.50 Hz, Harom, 1H), 7.12 (d, J ¼ 7.46 Hz, Harom, 1H), 6.58
(dd, J1 ¼8.73 Hz, J2 ¼ 2.28 Hz, Harom, 1H), 6.39 (s, OH, 1H), 3.44e3.40
(m, CH2, 4H), 1.14 (t, J ¼ 6.96 Hz, CH3, 6H); 13C NMR (75 MHz, DMSO-
(75 MHz, DMSO-d6):
d
¼ 148.06 (s),143.90 (s),139.24 (d),135.62 (d),
135.29 (d), 123.84 (d), 123.45 (d), 122.47 (d), 119.76 (s), 119.25 (d),
117.22 (s), 111.85 (d), 97.28 (s); elemental analysis calcd. (%) for
C13H9N5: C 66.37, H 3.86, N 29.77; found: C 66.61, H 3.98, N 29.43.
d6):
d
¼ 158.97 (s), 152.04 (s), 149.52 (d), 137.22 (s), 135.56 (d), 135.02
(d), 128.45 (s),126.03 (d), 125.09 (d), 123.49 (s), 123.09 (s),122.94 (d),
121.67 (s), 115.65 (d), 114.43 (d), 98.63 (s), 26.76 (t, 2C), 25.24 (q, 2C);
elemental analysis calcd. (%) for C20H20N4O: C 72.77, H 6.06, N 16.86;
found: C 73.00, H 5.89, N 17.10.
6.3. General method for preparation of compounds 15e23
Ethanolic solutions of compounds 2e8 and 12e13 and small
amount of iodine (5%) were irradiated at room temperature, with
400-W high-pressure mercury lamp, using a Pyrex filter for about
6e20 h, until the UV spectra shows that the reaction of dehy-
drocyclization was finished. The air was bubbled through the
solution. The solutions were concentrated under reduced pres-
sure to a volume of 2e3 ml and resulting products were filtered.
After recrystallization from acetoneewater, products were
obtained.
6.2.10. 2-(2-Benzimidazolyl)-3-(2-furyl)acrylonitrile 11
Compound 11 was prepared from 1 (2.00 g, 12.7 mmol) and
2-furylcarboxaldehyde (1.22 g, 12.7 mmol) in absolute ethanol
(20 ml) after refluxing for 1 h and recrystallization from ethanol to
yield 2.52 g (82%) of red crystals; m.p. 192e193 ꢀC, m.p. (lit)
192e194 ꢀC.
IR (KBr)
n
/cmꢂ1: 3150, 2215, 1650, 1610; 1H NMR (300 MHz,
DMSO-d6):
d
¼ 13.01 (s, NHbenzimidazole, 1H), 8.16 (s, 1H), 8.13
(d, J ¼ 1.46 Hz, Hfurane, 1H), 7.60 (br s, Harom, 2H), 7.35 (d, J ¼ 3.54 Hz,
6.3.1. 2-Cyanobenzimidazo[1,2-a]quinoline-6-carbonitrile 15 [30]
Compound 15 was prepared from 2 (1.04 g, 3.9 mmol) in ethanol
(400 ml) after irradiation for 15 h to yield 0.42 g (40%) of yellow
crystals; m.p. >295 ꢀC, m.p. (lit) > 295 ꢀC.
Hfurane, 1H), 7.25e7.22 (m, Harom, 2H), 6.85 (dd, J1 ¼3.52 Hz,
J2 ¼ 1.66 Hz, Hfurane, 1H); 13C NMR (75 MHz, DMSO-d6):
¼ 148.95
d
(s), 147.68 (d), 147.27 (s), 143.56 (s), 134.98 (s), 130.73 (d), 123.49 (d),
122.22 (d), 119.19 (d), 119.01 (d), 115.96 (s), 113.74 (d), 111.46 (d),
97.59 (s); elemental analysis calcd. (%) for C14H9N3O: C 71.48, H
3.86, N 17.86; found: C 71.70, H 3.59, N 18.14.
IR (KBr)
(300 MHz, DMSO-d6):
n
/cmꢂ1: 3058, 2231, 2224, 1609, 1600; 1H NMR
d
¼ 8.99 (s, 1H), 8.75 (d, J ¼ 8.34 Hz, Harom
,
1H), 8.73 (s, Hquinoline, 1H), 8.17 (d, J ¼ 8.10 Hz, Harom, 1H), 7.93
(d, J ¼ 8.10 Hz, Harom, 1H), 7.89 (d, J ¼ 7.86 Hz, Harom, 1H), 7.53e7.47
6.2.11. 2-(2-Benzimidazolyl)-3-(2-thienyl)acrylonitrile 12
Compound 12 was prepared from 1 (2.00 g, 12.7 mmol) and
2-thienylcarboxaldehyde (1.48 g, 12.7 mmol) in absolute ethanol
(25 ml) after refluxing for 1 h and recrystallization from ethanol to
yield 3.02 g (84%) of yellow crystals; m.p. 217e218 ꢀC, m.p. (lit)
216e218 ꢀC.
(m, Harom, 2H); 13C NMR (150 MHz, DMSO-d6):
d
¼ 144.10 (s),143.97
(s),139.97 (d),135.80 (s), 132.51 (d), 130.83 (s), 128.47(d),126.03 (d),
124.82 (s), 124.78 (d), 120.85 (d), 119.96 (d), 118.44 (s), 115.74 (d),
115.35 (s), 115.28 (s), 104.62 (s); elemental analysis calcd. (%) for
C17H8N4: C 76.11, H 3.08, N 20.88; found: C 75.87, H 3.10, N 21.14.
IR (KBr)
DMSO-d6):
n
/cmꢂ1: 3172, 2224, 1643, 1607; 1H NMR (300 MHz,
¼ 13.02 (s, NHbenzimidazole, 1H), 8,56 (s, 1H), 8.06
6.3.2. 2-Methylbenzimidazo[1,2-a]quinoline-6-carbonitrile 16
Compound 16 was prepared from 3 (0.20 g, 0.8 mmol) in
ethanol (400 ml) after irradiation for 7 h to yield 0.11 g (56%) of
slightly yellow crystals; m.p. 277e278 ꢀC.
d
(d, J ¼ 4.80 Hz, Hthiophene, 1H), 7.84 (d, J ¼ 3.30 Hz, Hthiophene, 1H),
7.65 (br s, Harom, 1H), 7.55 (br s, Harom, 1H), 7.34 (t, J ¼ 4.37 Hz,
Hthiophene, 1H), 7.29e7.24 (m, Harom., 2H); 13C NMR (75 MHz, DMSO-
IR (KBr)
(600 MHz, DMSO-d6):
(s, 1H), 8.63 (s, 1H), 8.05 (d, J ¼ 7.98 Hz, Harom, 1H), 8.02
n
/cmꢂ1: 3122, 2224, 2215, 1623, 1608; 1H NMR
d6):
d
¼ 149.76 (s), 148.01 (d), 147.88 (s), 143.98 (s), 134.99 (s), 131.13
d
¼ 8.81 (d, J ¼ 8.16 Hz, Harom, 1H), 8.74
(d), 123.89 (d), 122.67 (d), 119.20 (d), 119.09 (d), 115.87 (s), 113.77
(d), 112.48 (d), 100.57 (s); elemental analysis calcd. (%) for
C14H9N3S: C 66.91, H 3.61, N 16.72; found: C 66.71, H 3.39, N 16.54.
(d, J ¼ 8.46 Hz, Harom
, 1H), 7.64e7.58 (m, Harom, 2H), 7.52
(d, J ¼ 7.98 Hz, Harom, 1H), 2.70 (s, CH3, 3H); 13C NMR (75 MHz,
DMSO-d6):
d
¼ 145.70 (s), 144.26 (s), 141.68 (s), 141.05 (s), 136.49 (s),
6.2.12. 2-(2-Benzimidazolyl)-3-(2-N-methylpyrolyl)acrylonitrile 13
Compound 13 was prepared from 1 (1.50 g, 9.6 mmol) and
N-methylpyrrole-2-carboxaldehyde (1.04 g, 9.6 mmol) in absolute
ethanol (20 ml) after refluxing for 3 h and recrystallization from
ethanol to yield 1.60 g (68%) of yellow powder; m.p. 279e281 ꢀC.
131.52 (d), 131.00 (s), 127.03 (d), 125.59 (d), 123.97 (d), 120.60 (d),
119.53 (s), 116.09 (d), 115.68 (d), 100.45 (s), 92.33 (s), 22.36 (q);
elemental analysis calcd. (%) for C17H11N3: C 79.36, H 4.31, N 16.33;
found: C 79.61, H 4.14, N 16.56.
IR (KBr)
(300 MHz, DMSO-d6):
n
/cmꢂ1
:
3345, 3172, 2221, 1651, 1614; 1H NMR
¼ 12.88 (s, NHbenzimidazole, 1H), 8.14 (s, 1H),
6.3.3. 2-Fluorobenzimidazo[1,2-a]quinoline-6-carbonitrile 17
Compound 17 was prepared from 4 (0.50 g, 1.9 mmol) in ethanol
(400 ml) after irradiation for 10 h to yield 0.28 g (57%) of slightly
yellow crystals; m.p. 258e259 ꢀC.
d
7.62e7.52 (m, Harom, 2H), 7.42 (dd, J1 ¼4.16 Hz, J2 ¼ 1.06 Hz, Hpyrole
,
1H), 7.28 (t, J ¼ 1.77 Hz, Hpyrole, 2H), 7.21 (br s, Harom, 2H), 6.37
(dd, J1 ¼4.02 Hz, J2 ¼ 2.58 Hz, Hpyrole, 1H), 3.85 (s, NeCH3, 3H); 13C
IR (KBr)
(600 MHz, DMSO-d6):
Harom, 1H), 7.45 (dd, J1 ¼10.14 Hz, J2 ¼ 2.34 Hz, Harom, 1H), 7.35
(d, J ¼ 7.98 Hz, Harom, 1H), 7.33 (td, J1 ¼8.43 Hz, J2 ¼ 2.38 Hz, Harom
1H), 7.13 (td, J1 ¼7.98 Hz, J2 ¼ 0.72 Hz, Harom 1H), 7.06
(d, J ¼ 8.34 Hz, Harom, 1H), 6.94 (td, J1 ¼7,23 Hz, J2 ¼ 1.02 Hz, Harom
n
/cmꢂ1
:
3111, 2228, 2209, 1630, 1612; 1H NMR
¼ 7.88 (ABq, J1 ¼8.43 Hz, J2 ¼ 6.15 Hz,
NMR (75 MHz, DMSO-d6):
d
¼ 149.13 (s), 145.76 (s), 135.72 (d),
d
132.47 (d), 130.52 (d), 127.72 (s), 122.46 (d), 120.52 (s), 118.33 (d),
118.02 (s), 115.42 (d), 111.07 (d), 110.37 (d), 93.22 (s), 34.46 (q);
elemental analysis calcd. (%) for C15H12N4: C 72.56, H 4.87, N 22.57;
found: C 72.78, H 4.99, N 22.41.
,
,

M. Hranjec et al. / European Journal of Medicinal Chemistry 45 (2010) 2405e2417
2415
1H,); 13C NMR (75 MHz, DMSO-d6):
d
¼ 162.05 (s), 142.54 (s), 142.41
6.3.8. 3-Tia-6,10b-diaza-cyclopenta[c]fluorene-6-carbonitrile 22
(s), 140.86 (s), 136.68 (s), 132.83 (d), 130.89 (s), 125.48 (d), 123.90
(d), 120.04 (d), 117.04 (s), 116.08 (s), 113.30 (d), 113.01 (d), 112.33 (d),
104.38 (d); elemental analysis calcd. (%) for C16H8FN3: C 73.56, H
3.09, N 16.08; found: C 73.82, H 3.20, N 16.39.
Compound 22 was prepared from 11 (0.40 g, 1.6 mmol) in
ethanol (400 ml) after irradiation for 7 h to yield 0.20 g (52%) of
yellow crystals; m.p. 271e273 ꢀC; IR (KBr) /cmꢂ1: 3152, 2220,
n
1622, 1601; 1H NMR (300 MHz, DMSO-d6):
d
¼ 8.96 (s, Hfluorene, 1H),
8.52 (d, J ¼ 8.30 Hz, Harom, 1H), 8.51 (d, J ¼ 5.82 Hz, Hthiophene, 1H),
8.47 (d, J ¼ 5.40 Hz, Hthiophene, 1H), 7.90 (d, J ¼ 8.10 Hz, Harom, 1H),
7.53 (t, J ¼ 7.56 Hz, Harom, 1H), 7.45 (t, J ¼ 7.60 Hz, Harom, 1H); 13C
6.3.4. 2-Bromobenzimidazo[1,2-a]quinoline-6-carbonitrile 18
Compound 18 was prepared from 5 (0.78 g, 2.4 mmol) in
ethanol (400 ml) after irradiation for 15 h to yield 0.50 g (60%) of
orange crystals; m.p. 290e291 ꢀC.
NMR (150 MHz, DMSO-d6):
d
¼ 145.10 (s), 143.27 (s), 138.62 (s),
137.62 (d), 134.05 (d), 129.03 (s), 125.47 (d), 122.64 (d), 121.84 (s),
119.62 (d), 117.09 (d), 116.03 (s), 113.68 (d), 96.08 (s); elemental
analysis calcd. (%) for C14H7N3S: C 67.45, H 2.83, N 16.86; found: C
67.13, H 2.77, N 16.59.
IR (KBr) n
/cmꢂ1: 3044, 2221, 1616, 1605; 1H NMR (300 MHz,
DMSO-d6):
d
¼ 8.72 (s, 1H), 8.71 (s, 1H), 8.57 (d, J ¼ 7.68 Hz, Harom
,
1H), 8.01 (d, J ¼ 8.40 Hz, Harom, 1H), 7.97 (d, J ¼ 7.26 Hz, Harom, 1H),
7.80 (d, J ¼ 8.37 Hz, Harom, 1H), 7.60e7.55 (m, Harom, 2H); 13C NMR
6.3.9. 3-Methyl-3H-3,6,10b-triaza-cyclopenta[c]fluorene 23
Compound 23 was prepared from 13 (0.50 g, 2.0 mmol) in
ethanol (400 ml) after irradiation for 10 h to yield 0.36 g (72%) of
green powder; m.p. 277e278 ꢀC.
(150 MHz, DMSO-d6):
d
¼ 143.83 (s),13.83 (d), 136.50 (s), 132.62 (d),
130.40 (s), 128.40 (d), 129.19 (s), 127.04 (s), 125.38 (d), 123.97 (d),
120.38 (s), 120.23 (d), 118.00 (d), 115.04 (s), 114.80 (d), 101.82 (s);
elemental analysis calcd. (%) for C16H8BrN3: C 59.65, H 2.50, N
13.04; found: C 59.89, H 2.75, N 13.33.
IR (KBr)
(600 MHz, DMSO-d6):
(d, J ¼ 7.45 Hz, Harom, 2H), 7.57 (br s, 1H), 6.96 (dd, J1 ¼4.23 Hz,
J2 ¼ 1.35 Hz, Hpyrole, 1H), 6.10 (dd, J1 ¼4.16 Hz, J2 ¼ 2.40 Hz, Hpyrole
1H), 3.80 (s, NeCH3, 3H); 13C NMR (150 MHz, DMSO-d6):
n
/cmꢂ1: 3346, 3178, 2231, 1643, 1621; 1H NMR
d
¼ 7.69 (s, 1H), 7.25e7.21 (m, Harom, 2H), 7.18
6.3.5. 1,4-Dimethoxybenzimidazo[1,2-a]quinoline-6-carbonitrile 19
Compound 19 was prepared from 6 (0.50 g, 1.4 mmol) in ethanol
(400 ml) after irradiation for 12 h to yield 0.30 g (60%) of yellow
powder; m.p. 244e245 ꢀC.
,
d
¼ 148.63
(s), 145.26 (s), 135.21 (d), 131.99 (d), 130.07 (d), 127.42 (s), 127.23 (s),
120.01 (s), 117.85 (d), 117.50 (s), 114.94 (d), 110.58 (d), 109.87 (d),
92.80 (s), 34.02 (q); elemental analysis calcd. (%) for C15H10N4: C
73.10, H 4.09, N 22.75; found: C 73.32, H 4.31, N 22.99.
IR (KBr)
n
/cmꢂ1: 3148, 2232, 1633, 1612; 1H NMR (600 MHz,
DMSO-d6):
d
¼ 8.77 (d, J ¼ 9.12 Hz, Harom, 1H), 8.70 (s, 1H), 8.66
(d, J ¼ 8.10 Hz, Harom, 1H), 8.00 (d, J ¼ 7.92 Hz, Harom, 1H), 7.70 (br
s, 1H), 7.61e7.55 (m, Harom, 3H), 3.94 (s, OCH3, 3H), 3,85 (s, OCH3,
6.4. Antitumor evaluation assays
3H); 13C NMR (150 MHz, DMSO-d6):
d
¼ 155.87 (s), 144.02 (s),
143.63 (s), 140.05 (d), 130.32 (d), 130.26 (s), 124.93 (d), 123.45 (d),
122.43 (s), 121.67 (d), 120.22 (d), 117.31 (d), 115.43 (s), 114.57 (d),
112.69 (d), 101.58 (s), 56.75 (q), 55.98 (q); elemental analysis
calcd. (%) for C17H11N3O: C 71.28, H 4.32, N 13.85; found: C 71.35,
H 4.55, N 14.15.
The experiments were carried out on 5 human cell lines, which
are derived from 5 cancer types. The following cell lines were used:
HeLa (cervical carcinoma), MiaPaCa-2 (pancreatic carcinoma),
MCF-7 (breast carcinoma), SW 620 and H 460 (lung carcinoma).
The cells were cultured as monolayers and maintained in Dulbec-
co's modified Eagle's medium (DMEM) supplemented with 10%
6.3.6. 2-N-Acetamidobenzimidazo[1,2-a]quinoline-6-carbonitrile 20
Compound 20 was prepared from 7 (0.15 g, 0.5 mmol) in ethanol
(400 ml) after irradiation for 15 h to yield 0.08 g (54%) of yellow
powder; m.p. > 285 ꢀC.
fetal bovine serum (FBS), 2 mM
L-glutamine, 100 U/ml penicillin
and 100 g/ml streptomycin in a humidified atmosphere with 5%
m
CO2 at 37 ꢀC. The growth inhibition activity was assessed according
to the slightly modified procedure performed at the National
Cancer Institute, Developmental Therapeutics Program [40]. The
cells were inoculated onto standard 96-well microtiter plates on
day 0. The cell concentrations were adjusted according to the cell
population doubling time (PDT). Test agents were then added in
five consecutive 10-fold dilutions (10ꢂ8 to 10ꢂ4 mol/L) and incu-
bated 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, as previously described
[7,12,26e29]. Each test point was performed in quadruplicate in
IR (KBr)
(600 MHz, DMSO-d6):
n
/cmꢂ1: 3367, 3178, 2221, 1644, 1623; 1H NMR
d
¼ 10.73 (s, 1H, NH), 9.28 (s, 1H), 8.68 (s,
1H), 8.02 (d, J ¼ 8.94 Hz, Harom, 1H), 7.72 (d, J ¼ 8.76 Hz, Harom, 1H),
7.64e7.59 (m, Harom, 2H), 7.27 (t, J ¼ 7.30 Hz, Harom, 1H), 7.22
(t, J ¼ 7.33 Hz, Harom, 1H), 2.22 (s, COCH3, 3H); 13C NMR (150 MHz,
DMSO-d6):
d
¼ 168.65 (s), 145.67 (s), 144.32 (s), 140.25 (d), 137.85
(s), 134.66 (d), 133.85 (s), 129.08(d), 127.95 (d), 125.12 (s), 124.87
(d), 122.41 (d), 120.62(d), 119.76 (s), 117.77 (d), 116.31 (s), 115.12
(s), 106.98 (s), 26.76 (q); elemental analysis calcd. (%) for
C18H12N4O: C 71.99, H 4.03, N 18.66; found: C 72.23, H 4.22, N
18.34.
three individual experiments. The results are expressed as IC50
,
which is the concentration necessary for 50% of inhibition. The IC50
values for each compound are calculated from doseeresponse
curves using linear regression analysis.
6.3.7. 2,7,11b-Triaza-benzo[c]fluorene-6-carbonitrile 21
Compound 21 was prepared from 8 (0.70 g, 2.9 mmol) in
ethanol (400 ml) after irradiation for 6 h to yield 0.42 g (60%) of
green powder; m.p. 130e132 ꢀC.
6.5. Interactions with ct-DNA
IR (KBr)
n
/cmꢂ1: 3231, 2245, 1656, 1619; 1H NMR (300 MHz,
The electronic absorption spectra were recorded on Varian Cary
50 and Varian Cary 100 Bio spectrometer and fluorescence emis-
sion spectra were recorded on Varian Eclipse fluorimeter, in both
cases using quartz cuvettes (1 cm). The calf thymus DNA (ct-DNA)
was purchased from Aldrich, dissolved in the sodium cacodylate
DMSO-d6):
d
¼ 10.12 (s, Hpyridine, 1H), 8.97 (s, Harom, 1H), 8.82
(d, J ¼ 5.07 Hz, Hpyridine, 1H), 8.80 (d, J ¼ 5.04 Hz, Hpyridine, 1H), 8.06
(d, J ¼ 7.90 Hz, Harom, 1H), 8.04 (d, J ¼ 7.92 Hz, Harom, 1H), 7.67e7.59
(m, 2H, Hbenzimidazole); 13C NMR (75 MHz, DMSO-d6):
d
¼ 161.10 (s),
buffer, I ¼ 0.05 mol dmꢂ3
,
pH ¼ 7, additionally sonicated and
148.99 (s), 146.23 (s), 138.67 (d), 133.44 (s), 132.31 (s), 129.87 (d),
128.56 (s), 126.78 (s), 125.76 (d), 125.47 (d), 123.32 (d), 123.11(d),
121.92 (d), 117.87 (d); elemental analysis calcd. (%) for C15H8N4: C
73.76, H 3.30, N 22.94; found: C 73.98, H 3.54, N 22.65.
filtered through a 0.45
mm filter and the concentration of corre-
sponding solution determined spectroscopically as the concentra-
tion of phosphates. The measurements were performed in the

2416
M. Hranjec et al. / European Journal of Medicinal Chemistry 45 (2010) 2405e2417
Table 7
General and crystal data and summary of intensity data collection and structure refinement for compounds 4, 16 and 17.
Compound
4
16
17
Brutto formula
Mr
C16H10FN3 ꢁ EtOH
309.34
C17H11N3
257.29
C16H8FN3
261.25
Crystal system, colour and habit
Space group
Orthorhombic, pale yellow prism
P212121
Monoclinic, pale yellow prism
P21/n
Triclinic, yellow irregular prism
P1
Crystal dimensions (mm)
0.21 ꢁ 0.16 ꢁ 0.10
0.28 ꢁ 0.17 ꢁ 0.14
0.37 ꢁ 0.29 ꢁ 0.23
Unit cell parameters:
ꢂ
a (A)
7.20020(10)
14.7320(2)
15.0894(3)
9.895(5)
7.242(5)
17.903(5)
8.7961(2)
9.0619(3)
15.6928(5)
98.694(3)
90.747(2)
102.137(2)
1207.55(6)
ꢂ
b (A)
ꢂ
c (A)
ꢀ
ꢀ
ꢀ
a/
b
/
102.363(5)
g
/
3
ꢂ
V (A )
1600.58(4)
1253.2(11)
Z
4
4
2
Dc (g cmꢂ3
T/K
)
1.284
296
0.737
1.364
296
0.656
1.437
296
0.815
m
(mmꢂ1
)
F(000)
648
4e72
536
4e75
536
2e70
2
q
Range for data collection (ꢀ)
h,k,l range
Scan type
ꢂ8 to 6; ꢂ17 to 17; ꢂ18 to 17
ꢂ12 to 12; ꢂ7 to 9; ꢂ22 to 21
ꢂ10 to 10; ꢂ10 to 11; ꢂ18 to 19
u
u
u
No. of measured reflections
No. of independent reflections (Rint
No. of observed
5117
1819 (0.0191)
1641
11,494
2564 (0.0264)
2022
11,547
4525 (0.0129)
4138
)
No. of refined parameters
g1, g2 in w
217
182
361
0.0792, 0.0806
0.0435, 0.1183
0.0392, 0.1145
1.058
0.0703, 0.1085
0.0483, 0.1202
0.0382, 0.1146
1.055
0.0753, 0.1267
0.0412, 0.1173
0.0387, 0.1145
1.044
R, wR [I ꢄ 2
s(I)]
R, wR [all data]
Goodness of fit on F2, S
ꢂꢂ3
Max., min. electron-density (e A
Maximum
)
0.190, ꢂ0.169
0.184, ꢂ0.182
0.198, ꢂ0.256
D/s
<0.001
<0.001
0.001
aqueous buffer solution (pH ¼ 7.0; sodium cacodylate buffer,
I ¼ 0.05 mol dmꢂ3). Under the experimental conditions used the
absorbance and fluorescence intensities of studied compounds
were proportional to their concentrations. Obtained data were
corrected for dilution. Spectroscopic titrations were performed by
adding portions of polynucleotide solution into the solution of the
studied compounds. Thermal melting curves for ct-DNA and its
complexes with studied compounds were determined as previ-
ously described by following the absorption change at 260 nm as
a function of temperature [41]. The heating rate was 1 ꢀC per
minute. The absorbance of the ligand was subtracted from every
curve, and the absorbance scale was normalized. Obtained Tm
values are the midpoints of the transition curves, determined from
the maximum of the first derivative or graphically by a tangent
atoms were refined by the least-squares methods based on F2 using
the SHELXL97 program [43]. The hydrogen atoms, which belong to
Csp2 carbon atoms were generated at ideal geometrical positions
2
ꢂ
[Csp eH 0.93 A, Uiso(H) ¼ 1.2 Ueq(C)] and constrained to ride on the
parent carbon atom in all structures. The imidazole ring NeH
hydrogen atom in 4 was found as a small electron-density at 0.96
ꢂ
(3) A (N2eH12N) apart from nitrogen atom and was refined freely.
The hydrogen atom which belongs to ethanol molecule in 4 is also
ꢂ
found in difference Fourier maps at distance of 0.97(4) A and was
refined freely. Since compound 4 contains only “light atoms”, no
valid conclusions on the absolute structure can be drawn (unreli-
able Flack parameter and its s.u.) even in this case when Cu
radiation is used. The Friedel pairs have been merged by using
SHELXL-97 instruction MERG 3 (1097 Friedel pairs of total 2916
unique reflections). The molecular graphics were done using
ORTEP-3 [44] and PLATON programmes [45].
method. Given
DTm values were calculated subtracting Tm of the
free nucleic acid and DMSO from Tm of complex. Every
D
Tm value
here reported was the average of at least two measurements, the
error in
D
Tm is ꢃ 0.5 ꢀC.
Acknowledgments
This study was supported by grants 125-0982464-1356, 098-
0982464-2514 and 098-1191344-2943 by the Ministry of Science,
Education and Sports of the Republic of Croatia.
6.6. X-ray crystallography
The general and crystallographic data for compounds 4, 16 and
17 are listed in Table 7.
The data collection for all three structures was carried out on
Appendix. Supplementary data
ꢂ
Oxford Xcalibur Nova with CCD detector and
l
(CuKa) ¼ 1.54184 A.
Data collections for all structures have been performed by applying
the CrysAlis Software system, Version 1.171.33.31 [42]. The Lorentz-
polarization effect was corrected and the intensity data reduced by
the CrysAlis RED application of the CrysAlis Software system,
Version 1.171.33.31. The diffraction data have been scaled for
absorption effects by the multi-scanning method.
CCDC numbers 754827e754829 for compounds 4, 16 and 17
respectively, contains the supplementary crystallographic data for
this paper. These data can be obtained free of charge at www.ccdc.
cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystal-
lographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ,
UK; fax: þ44(0)1223 336033; email: deposit@ccdc.cam.ac.uk].
Structure factors table is available from the authors. Supplementary
data associated with this article can be found in the online version,
at doi:10.1016/j.ejmech.2010.02.022.
Coordinates of the non-hydrogen atoms were determined by
direct methods with the SHELXS program [43]. The coordinates and
anisotropic thermal displacement parameters for all non-hydrogen

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