Novel cyano- and amidino-substituted derivatives of styryl-2-benzimidazoles and benzimidazo[1,2-a]quinolines. Synthesis, photochemical synthesis, DNA binding, and antitumor evaluation, part 3

Article abstract of DOI:10.1021/jm070876h

Synthesis of novel cyano- and amidino-substituted styryl-2-benzimidazoles and benzimidazo[1,2-a]quinolines by condensation reactions and photochemical dehydrocyclization and dehydrohalogenation cyclization is described. Thermal denaturation experiments re

Full text of DOI:10.1021/jm070876h

5696
J. Med. Chem. 2007, 50, 5696-5711
Novel Cyano- and Amidino-Substituted Derivatives of Styryl-2-Benzimidazoles and
Benzimidazo[1,2-a]quinolines. Synthesis, Photochemical Synthesis, DNA Binding, and Antitumor
Evaluation, Part 3
Marijana Hranjec,† Marijeta Kralj,*,‡ Ivo Piantanida,§ Mirela Sedic´,‡ Lidija Sˇuman,‡ Kresˇimir Pavelic´,‡ and
Grace Karminski-Zamola*,†
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, UniVersity of Zagreb, Marulic´eV trg 20,
Post Office Box 177, HR-10000 Zagreb, Croatia, DiVision of Molecular Medicine, “Ruder BosˇkoVic´” Institute, Bijenicˇka cesta 54,
Ä
Post Office Box 180, HR-10000 Zagreb, Croatia, and DiVision of Organic Chemistry and Biochemistry, “Ruder BosˇkoVic´” Institute,
Ä
Post Office Box 180, HR-10002 Zagreb, Croatia
ReceiVed July 20, 2007
Synthesis of novel cyano- and amidino-substituted styryl-2-benzimidazoles and benzimidazo[1,2-a]quinolines
by condensation reactions and photochemical dehydrocyclization and dehydrohalogenation cyclization is
described. Thermal denaturation experiments reveal that cyclic derivatives considerably stabilize DNA double
helix, while the effect of their acyclic analogues is negligible. According to the spectroscopic study of the
interaction of cyclic derivative 19, we propose intercalation of benzimidazo[1,2-a]quinoline moiety into
ct-DNA as a dominant interaction underlying biologically relevant effects of this compound, whereas for its
acyclic derivative 11, we propose binding into the minor groove of DNA. All compounds show noticeable
antiproliferative effect. Morpholino- and chloro-substituted compound 9 is the most active among all acyclic
derivatives. All cyclic compounds were 2- to 10-fold more potent, which is correlated with their property
to intercalate into DNA. The most active imidazolyl-substituted compound 19 inhibits topoisomerase II and
induces strong G2/M cell cycle arrest, pointing to the impairment in mitotic progression. Its pronounced
selectivity toward colon carcinoma cells encourages further development of this compound as a lead.
Introduction
toxicity against a panel of human and murine cell lines, and
showed that the most active compound bound to DNA as an
intercalator.14 The authors also showed intercalation ability of
compounds having nearly planar chromophores. Moreover, a
series of benzimidazo[2,1-a]isoquinolines with carboxamide side
chains were prepared to study the biological effects induced by
the variation in the side-chain position in this tetracyclic series.15
It was shown that 6-carboxamides with the side chain attached
to one of the central rings were not active, while 1- and 11-
carboxamides with the side chain attached to one of the terminal
rings of the chromophore short axis exerted reasonable cyto-
toxicity in vitro and in vivo.
One of the most important goals in medicinal chemistry is
the development of new heterocyclic compounds with antitumor
activity. The most used classes of chemotherapeutic agents in
cancer therapy comprise molecules that interact with DNA, such
as groove binders, DNA alkylating agents, or intercalators.
Intercalators in most cases consist of two or more planar fused
aromatic or heteroaromatic rings able to insert between adjacent
base pairs of a DNA molecule without disturbing the overall
stacking pattern.1,2 This binding mode is characterized by low
selectivity toward different DNA or RNA sequences as well as
low or nonselective bioactivity.2 However, the activity of many
antitumor drugs is based on intercalation, but selectivity could
be improved by introduction of specific substituents on the
intercalative aromatic core.2,3 Several antibiotics are DNA
intercalators and are recognized as a new, promising class of
antitumor agents.4
Substituted benzimidazoles and their azino-fused cyclic
derivatives have drawn considerable attention of medicinal and
organic chemists due to a wide range of biological activities
exerted by this class of compounds.5-13 Also, the corresponding
benzo-annulated analogues, such as benzimidazo[2,1-a]iso-
quinolines, benzimidazo[1,2-c]quinazolines, or benzimidazo[3,2-
a]quinolinium hydrochloride salts showed interesting biological
properties. Bran˜a et al. prepared amino- and amido-substituted
benzimidazo[1,2-c]quinazolines, evaluated their in vitro cyto-
Several methylated derivatives of benzimidazo[1,2-b]iso-
quinolines had the activity similar to ellipticine as mammalian
topoisomerase II inhibitors and were also highly active in vitro,
whereby they inhibited the growth of several human tumor cell
lines.16 The group of authors prepared a series of benzimidazo-
[3,2-a]quinolinium hydrochloride salts17-19 and evaluated their
biological activity as well as interaction with DNA and inhibition
of topoisomerase II activity.20,21 All compounds exhibited
moderate antitumor activity in vitro, and the results suggested
that their primary mechanism of action was inhibition of the
enzymes implicated in DNA functionality rather than DNA
binding, whereby substitutions in the benzimidazole moiety had
striking effects on the biological activity of examined com-
pounds.
Benzimidazo[1,2-a]quinolines have remained unexplored for
their biological activity most likely because of the lack of general
synthetic methods starting from easily accessible precursors.
Unsubstituted benzimidazo[1,2-a]quinoline was first prepared
by the classical method from 2-aminoquinoline and picric acid22
and later also in reaction of photochemical dehydrocyclization
by UV irradiation of methanolic solution of styryl-2-benzimi-
* To whom correspondence should be addressed. Tel.: +385 1 4571
235 (M.K.); ++38514597215 (G.K.-Z.). Fax: +3851 4561 010 (M.K.);
++38514597250 (G.K.-Z.). E-mail: mhorvat@irb.hr, marijeta.kralj@irb.hr
(M.K.); gzamola@pierre.fkit.hr (G.K.-Z.).
† University of Zagreb.
‡ Division of Molecular Medicine, “Ruder Bosˇkovic´” Institute.
Ä
§ Division of Organic Chemistry and Biochemistry, “Ruder Bosˇkovic´”
Institute.
Ä
10.1021/jm070876h CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/13/2007

Synthesis of Styryl-2-benzimidazole DerViatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5697
Figure 1. Cyano and amidino-substituted derivatives of 2-styrylbenzimidazoles (2-17, 24, 25, and 30) and benzimidazo[1,2-a]quinolines (18-21,
26, 27, and 31-35).
Scheme 1. Synthesis of Amidino-Substituted Derivatives of E-
and Z-Styryl-2-benzimidazoles 2-17 and
Benzimidazo[1,2-a]quinolines 18-21
dazole.23 Recently, Venkatesh has developed a new efficient
palladium-catalyzed synthetic method for preparing substituted
benzimidazo[1,2-a]quinolines starting from phenylamino-
substituted benzimidazoles.24 Other substituted benzimidazo-
[1,2-a]quinolines were prepared by thermal cyclization of
corresponding o-substituted 2-styrylbenzimidazoles. Because of
their optical properties, they can be used as fluorescent optical
whiteners and disperse dyes.25-27
These considerations prompted us to explore a new series of
cyano- and amidino-substituted styryl-2-benzimidazoles (2-17,
24, 25, 30) as well as their cylic, fused derivatives, benzimidazo-
[1,2-a]quinolines (18-21, 26, 27, 31-35) with substituents on
different positions of the condensed rings (Figure 1), prepared
by reaction of photochemical dehydrocyclization and photo-
chemical dehydrohalogenation cyclization. Full details about the
synthesis, evaluation of antitumor activity, and DNA-binding
properties are reported herein.
Chemistry
All compounds shown in Figure 1 were prepared according
to Schemes 1-3 using different methods for the preparation of
benzimidazole nuclei.28 Novel N-amidino-substituted E-2-styryl-
1H-benzimidazoles 2-9 were prepared by the condensation
reaction29-32 of o-substituted-3-phenyl-propenales 1a,b with
corresponding, earlier-prepared, 4-N-amidino-substituted 1,2-
phenylenediamines and p-benzoquinone in 49-74% yield. 4-N-
amidino-substituted 1,2-o-phenylenediamines were prepared
from cyano derivatives in the Pinner reaction using earlier
described methods.33,34 Cyano-substituted E-2-styryl-1H-benz-
1H-benzimidazoles 2-9.23 Reaction of photochemical isomer-
ization was followed by UV/vis spectroscopy according to the
imidazoles 24 and 25 were prepared from o-substituted ben-
zaldehydes and 6-cyano-2-methylbenzimidazole.35 Novel N-
amidino-substituted Z-2-styryl-1H-benzimidazoles 10-17 were
prepared by photochemical isomerization of ethanolic solution
(c ) 1.3 × 10-2 mol dm-3) of N-amidino-substituted E-2-styryl-
Figure 2.
All compounds were obtained in 60-78% yield. N-amidino-
substituted benzimidazo[1,2-a] quinolines 18-21 and cyano-

5698 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Hranjec et al.
Scheme 2. Synthesis of Cyano-Substituted Derivatives of E-Styryl-2-benzimidazoles 24-25 and Benzimidazo[1,2-a]quinolines 26-27
Scheme 3. Cyano- and Amidino-Substituted Benzimidazo[1,2-a]quinolines 31-35
substituted benzimidazo[1,2-a]quinolines 26, 27, and 31 were
prepared by photochemical dehydrocyclization, while com-
pounds 18-21 were also prepared by photochemical dehydro-
halogenation cyclization. There was no significant difference
in the time of irradiation, as well as, in the yield of photode-
hydrocyclization or photodehydrohalogenation reactions ac-
cording to the Table 1.
Taking into account the simplicity and mutual similarity of
the studied structures, we decided to perform the fast screening
by thermal denaturation experiments (Table 3).
Obtained results reveal that thermal stabilization of ct-DNA
by addition of acyclic derivatives 2-5 and 10-13 is negligible
(close to the error of the method), while their cyclic analogues
strongly stabilize DNA double helix.
These reactions were carried out in ethanolic solution (c )
1.3 × 10-3 mol dm-3) of N-amidino-substituted E-2-styryl-1H-
benzimidazoles 2-9 and compounds 18-21 were obtained in
31-40% yield as a mixture of two unseparable structure
isomers. 2-amidino-benzimidazo[1,2-a]quinolines 32-35 were
prepared by the Pinner reaction from its cyano derivative 31.
The UV/vis electronic absorption data of prepared compounds,
recorded at a concentration of ∼2 × 10-5 mol dm-3, are
presented in the Table 2. Cyclization of 2-9 into 18-21 and
30 into 31, respectively, resulted in pronounced bathochromic
shifts of the absorption maxima of acyclic compounds.
Because studied cyclic compounds 18-21 and 32-35 are
related analogues that stabilize DNA within the same order of
magnitude, we chose 19 as a representative for more detailed
studies on DNA binding by spectrophotometric titrations and
compared it with its acyclic analogue 11.
Absorbencies of aqueous solutions of 19 and 11 were
proportional to their concentrations up to 1 × 10-4 mol dm-3
,
indicating the lack of significant intermolecular stacking that
might give rise to hypochromicity effects. Aqueous solutions
of 19 and 11 were stable over longer periods and also under
the condition of increased temperature for shorter periods (few
hours at up to 100 °C). Compounds 19 and 11 exhibit
fluorescence emissions with maxima at about 415 and 383 nm,
respectively. Therefore, excitation spectra fit in well with the
corresponding absorption spectra. Fluorescence intensities of
both compounds were found to be linearly dependent on their
Interaction of Acyclic Derivatives 2-5; 10-13 and their
Cyclic Analogues 18-21; 32-35 with Double-Stranded
DNA. Among all compounds presented in this study, represen-
tatives of acyclic derivatives 2-5 and 10-13 along with their
cyclic analogues 18-21 and 32-35 were chosen for preliminary
screening of their noncovalent interactions with ct-DNA. The
aim of fast screening was to estimate the probability that
biological activity of compounds (addressed in Biological
Results and Discussion) is at least partially the consequence of
DNA binding.
concentrations up to 2 × 10-6 mol dm-3
.
Addition of ct-DNA to compound 19 (c ) 4.0 × 10-5 mol
dm-3) resulted in a strong hypochromic effect (39%) and a
batochromic shift of maxima for ∆λmax ) +5 nm, accompanied
with pronounced broadening of the UV/vis spectrum, while

Synthesis of Styryl-2-benzimidazole DerViatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5699
Figure 2. (A) Reaction of photochemical isomerization of compound 9 into compound 17 monitored by UV/vis spectroscopy (ethanol, c(sample)
) 3.5 × 10-5 mol dm-3); and (B) dependence of absorbance intensity on reaction time.
Table 1. Reaction Times and Yields of Reactions of Photochemical
Dehydrocyclization and Dehydrohalogenation Cyclization of Compounds
2-9 To Give 18-21
spectroscopic changes appeared already at excess of studied
compounds over ct-DNA, thus hampering the accurate calcula-
tion of the binding constant by means of Scatchard equation.36
18
19
20
21
Nevertheless, strong fluorescence of aqueous solutions of 19
and 11 allowed fluorimetric titrations at about 100-times lower
concentrations than were used in UV/vis experiments (Figure
3). Addition of ct-DNA strongly quenched emission of 19 (75%)
and moderately decreased emission of 11 (37%) and also yielded
photochemical
dehydrocyclization
photochemical
yield (%)
time (h)
yield (%)
time (h)
37
7
31
7
33
6
39
7
40
7
40
6
42
6
33
6
dehydrohalogenation
a hypochromic shift of maximum of compound 11 (∆λmax
)
Table 2. Electronic Absorption Data of Compounds 2-21 and 30-35a
+8 nm). Processing of the fluorimetric titration data by means
of Scatchard equation gave binding constant log Ks ) 6.2 and
ratio n[bound 19]/[ct-DNA] ) 0.19 for compound 19, and log Ks )
6.2 and ratio n[bound 11]/[ct-DNA] ) 0.12 for compound 11.
λmax
ꢀ × 103
λmax
ꢀ × 103
(nm)
(dm3 mol-1 cm-1
)
(nm)
(dm3 mol-1 cm-1
)
2
335
268
222
325
254
334
267
223
335
270
220
36.4
17.6
25.0
30.8
25.2
36.0
17.8
25.7
37.7
17.4
20.7
15
313
222
15.6
29.8
CD Experiments. To further examine the interactions of 19
and 11 with ct-DNA, we carried out CD experiments. Addition
of 11 resulted in a marked decrease of the CD bands of ct-
DNA (Figure 4C), and in addition, a strong induced CD (ICD)
band appeared in the region between λ ) 300 and 400 nm.
Because 11 does not have any intrinsic CD spectrum, ICD band
must result from the interaction of 11 with ds-DNA, corre-
sponding well with the UV/vis spectrum of 11/ct-DNA complex
collected under the same conditions (Figure 4D).37 On the other
hand, addition of 19 resulted in a decrease of CD bands of ct-
DNA at 223 and 245 nm but also yielded an increase of CD
band at λ ) 275 nm (Figure 4A). In addition, very weak negative
ICD spectrum in the range λ ) 300-400 nm was observed
(Figure 4A). Comparison of UV/vis spectrum and CD spectrum
of 19/ct-DNA complex under the same conditions (Figure 4B)
pointed that both increase of CD band at λ ) 275 nm as well
as weak ICD band at λ ) 300-400 nm correspond well with
the UV/vis spectrum of 19.
3
4
16
17
315
220
317
221
14.8
29.3
14.9
25.9
5
18
367
350
331
255
367
334
261
367
351
331
257
368
351
332
258
351
7.0
10.1
11.7
30.6
7.1
10.1
25.3
5.7
7.6
8.8
21.6
6.3
8.7
6
7
334
267
219
327
255
34.5
18.2
30.4
32.4
21.9
19
20
8
334
270
218
34.0
18.7
28.7
21
10.0
24.5
30.7
9
335
269
220
316
223
34.5
17.6
25.6
17.8
32.7
30
31
In conclusion, although binding constants calculated from
fluorimetric titrations for cyclic derivative 19 and its acyclic
analogue 11 are comparable, cyclic derivative 19 more signifi-
cantly stabilized the ct-DNA double helix than the acyclic
analogue (Table 3). In addition, changes of the UV/vis spectrum
of cyclic derivative 19 upon addition of ct-DNA are significantly
stronger than those observed for its acyclic analogue 11.
Aforementioned results obtained for 11 are in good agreement
with the strong positive ICD band,38 thus speaking in favor of
the acyclic derivative binding to the ct-DNA minor groove. On
the other hand, results obtained for cyclic derivative 19 together
with weak negative ICD band38 point toward intercalative
binding mode.
10
373
354
267
243
363
347
264
242
357
267
242
347
266
242
350
266
243
9.8
10.7
25.4
28.7
8.3
10.1
28.7
32.1
6.2
21.5
24.6
10.6
30.7
34.9
10.0
29.8
32.2
11
312
225
18.9
30.9
32
12
13
14
317
224
15.9
26.6
33
34
35
317
223
20.0
36.7
315
221
14.4
28.2
Because structures of other studied cyclic analogues (18, 20,
21, 32, 33, 34, and 35) are closely related to 19 and all cyclic
analogues show similar thermal stabilization effect (Table 3),
we propose intercalation of benzimidazo[1,2-a]quinoline moiety
of all studied compounds into ct-DNA. Therefore, interaction
of cyclic compounds with cellular DNA could be responsible
for the observed antiproliferative effects.
a Sodium cacodilate/HCl buffer, I ) 0.05 mol dm-3, pH ) 7.0.
changes in the UV/vis spectrum of 11 (c ) 2.0 × 10-5 mol
dm-3) upon addition of ct-DNA were much weaker (hypochro-
mic effect 10%, no shift of maximum). However, most of the

5700 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Hranjec et al.
Table 3. ∆Tm Valuesa of ct-DNA upon Addition of Acyclic 2-5, 10-13, Cyclic 18-21, and 32-35b
cmpd
2
3
4
5
10
11
12
13
18
19
20
21
32
33
34
35
∆Tm/°C
1.1
1.4
0.7
0.1
1.2
1.0
-0.5
1.3
3.7
4.9
3.6
3.2
5.0
3.6
5.2
3.2
a Error in ∆Tm: (0.5 °C. b Derivatives at r[compound]/[polynucleotide] ) 0.3; pH ) 7.0; Buffer, sodium cacodilate, I ) 0.05 mol dm-3
.
Figure 3. (A) Changes in the fluorescence spectra of 19; (B) dependence of fluorescence intensity of 19 at λ ) 415 nm on a c(ct-DNA); (C)
changes in the fluorescence spectra of 11 upon the addition of ct-DNA; and (D) dependence of fluorescence intensity of 11 at λ ) 383 nm on a
c(ct-DNA). All experiments were done at pH ) 7.0 (buffer sodium cacodylate I ) 0.05 mol dm-3).
Acyclic analogues related to 11 do not stabilize ct-DNA
significantly under the conditions used in this fast screening
experiment (Table 3). Although these fast screening results do
not explicitly prove that all acyclic analogues bind into the minor
groove of ct-DNA, like compound 11, it is most likely that the
more potent biological effects of cyclic analogues results from
intercalation into cellular DNA, while other modes of interac-
tions possible for acyclic analogues yield lower antiproliferative
activity.
was slightly potentiated by introducing a chloro-substitutent.
The most active among all acyclic derivatives proved to be
morpholino- and chloro-substituted compound 9, although it is
least selective in regard to normal cells.
From the results presented in Table 4 and Figure 5, it is
evident that IC50 concentrations of all cyclic compounds (18-
21, 32-35) are 2-10-fold lower than those of their acyclic
analogues (2-17). Moreover, all cyclic compounds showed
similar activity toward all tested cell lines, having low micro-
molar IC50 concentrations (1-17 µM), except for 19, which
strongly inhibited the growth of the colon carcinoma cells,
SW620 (IC50 ) 0.4 µM). This is opposite to the previously
published results, which demonstrated that corresponding ami-
dino-substituted cyclic benzo[b]thieno[2,3-c]quinolones39 in-
hibited tumor cell growth differently and selectively in com-
parison with normal cells, although the aromatic surface and
the position of amidine substituents are similar to benzimidazo-
[1,2-a]quinolines presented in this study. This implies the
importance of the finely tuned structural and also most likely
electronic properties of condensed hetereoaromatic surface with
respect to the selectivity between tumor and normal cells.
However, the only exception was compound 20, which
weakly inhibited the growth of normal fibroblasts, thus being
the most selective cyclic compound.
Biological Results and Discussion
Compounds 2-36 were tested for their potential antiprolif-
erative effects on a panel of seven human cell lines, six of which
were derived from different cancer types, including HeLa
(cervical carcinoma), MCF-7 (breast carcinoma), SW620 (colon
carcinoma), MiaPaCa-2 (pancreatic carcinoma), Hep-2 (laryn-
geal carcinoma), H460 (lung carcinoma), and one from normal
diploid fibroblasts, WI 38.
All tested compounds showed noticeable antiproliferative
effect (Table 4). Acyclic trans-analogues (2-9) and their
corresponding cis-isomers (10-17) had comparable effects on
the cell proliferation. Moreover, all acyclic compounds exerted
the most pronounced effect on HeLa and MCF-7 cells. While
morpholino-substituted acyclic compounds (5, 9, 13 and 17)
had substantial cytostatic effects on normal human fibroblasts
(WI 38), other acyclic derivatives did not influence the growth
of this cell line. This finding points to the highly differential
(selective) effect of the aforementioned compounds. The activity
Acyclic compounds and the standard chemotheraputic agent
etoposide have comparable effects, whereas the cyclic ones have
significantly stronger activity than etoposide, but lower activity
in comparison with doxorubicin.

Synthesis of Styryl-2-benzimidazole DerViatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5701
Figure 4. (A) CD spectra of the free ct-DNA (4 × 10-5 mol dm-3) and of 19/ct-DNA complex at ratio r[19]/[ct-DNA] ) 0.2; (B) comparison of
UV/vis spectrum and CD spectrum of 19/ct-DNA complex at ratio r[19]/[ct-DNA] ) 0.2; (C) CD spectra of the free ct-DNA (4 × 10-5 mol dm-3) and
of 11 /ct-DNA complex at ratios r[19]/[ct-DNA] ) 0.2, 0.5, and 1; and (D) comparison of UV/vis spectrum and CD spectrum of 11 /ct-DNA complex
at ratio r[19]/[ct-DNA] ) 0.5. All experiments were done at pH ) 7 (buffer sodium cacodylate I ) 0.05 mol dm-3).
Table 4. In Vitro Inhibition of Compounds 2-36 on the Growth of Tumor Cells and Normal Human Fibroblasts (WI 38)
IC50a (µM)
cmpd
Hep-2
HeLa
MiaPaCa-2
SW620
MCF-7
WI 38
2
g100
11 ( 15
4 ( 3
>100
>100
9 ( 8
>100
3
16 ( 2
11 ( 0.1
13 ( 0.5
70 ( 33
16 ( 0.2
23 ( 5
10 ( 6
81 ( 9
27 ( 5
g100
19 ( 1
>100
16 ( 2
15 ( 5
12 ( 1
66 ( 22
14 ( 2
>100
7 ( 3
21 ( 2
>100
4
2 ( 1
29 ( 18
14 ( 1
5
6 ( 6
16 ( 1
48 ( 22
17 ( 1
>100
20 ( 2
>100
6
10 ( 2
4 ( 1
16 ( 2
7
5 ( 0.01
22 ( 4
42 ( 13
>100
8
7.5 ( 3
3 ( 2
9
5 ( 1
4 ( 1
4 ( 4
7 ( 9
10
11
12
13
14
15
16
17
18
19
20
21
32
33
34
35
36
DOXb
Etob
23 ( 4
35 ( 20
5.4 ( 4
13 ( 2
19 ( 5
11 ( 5
20 ( 2
7 ( 4
84 ( 20
25 ( 3
>100
70 ( 1
45 ( 7
78 ( 25
22 ( 3
29 ( 10
46 ( 18
>100
33 ( 25
17 ( 5
>100
99 ( 35
>100
36 ( 16
25 ( 9
61 ( 40
30 ( 1
g100
47 ( 23
28 ( 2
62 ( 23
85 ( 19
15 ( 3
3 ( 0.3
1 ( 0.2
13 ( 1
2 ( 0.01
8.1 ( 8
5.6 ( 0.5
4.5 ( 3.6
10 ( 0.3
18 ( 6
0.02 ( 0.01
15 ( 14
22 ( 2
>100
35 ( 0.5
23 ( 2
>100
29 ( 6
38 ( 6
>100
9 ( 2
15 ( 2
3 ( 1
0.4 ( 0.01
17 ( 0.2
2 ( 1
1.6 ( 1.2
1.4 ( 0.1
2.9 ( 0.9
1.9 ( 1
23 ( 9
0.02 ( 0.02
20 ( 3
14 ( 0.2
4 ( 2
21 ( 2
10 ( 7
2 ( 1
4 ( 0
4 ( 2
2 ( 0.2
5 ( 3
3 ( 0.7
1.7 ( 0.3
7 ( 3.6
4.9 ( 5.5
7 ( 5
2 ( 0.02
13 ( 4
2 ( 1
2 ( 1
9 ( 0.4
4 ( 0.3
2.2 ( 0.2
5.3 ( 3
4 ( 1
74 ( 6
4 ( 0.01
2.7 ( 0.7
7.8 ( 4.7
6.7 ( 7
17.7 ( 5
14 ( 3
0.1 ( 0.01
N.T.
1.6 ( 0.06
2.4 ( 0.6
4 ( 1.4
4.7 ( 3
15 ( 3
0.04 ( 0.01
N.T.c
6.7 ( 4.8
16 ( 0.9
0.04 ( 0.01
50 ( 30
12 ( 0.1
0.04 ( 0.01
3 ( 1
a IC50: the concentration that causes a 50% reduction of the cell growth. b DOX, doxorubicin; Eto, etoposide. c N.T.: not tested.
Amidino-substituted cyclic compounds showed significantly
stronger activity than unsubstituted analogue benzimidazo[1,2-
a]quinoline 3623 (Figure 6). This finding unravels the important
role of amidino-group for the growth inhibition activity, whereby
the imidazole substituent exerted inhibitory effects particularly
toward the colon carcinoma cells (19 and 33).
There was no statistical difference between cyclic derivatives
bearing an amidino substituent on either the quinoline (32-
35) or the benzimidazole (18-21) side.
Interestingly, when comparing the benzimidazole compounds
bearing a cyano- instead of a amidino- substituent on the
benzimidazole side (Table 5), it could be noticed that a cyano

5702 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Hranjec et al.
Figure 5. Concentration-response profiles for the representative acyclic (16 and 11) and cyclic (20 and 19) analogues tested on various human
cell lines in vitro. The cells were treated with the compounds at different concentrations and PG was calculated. Each point represents a mean value
of four replicates in three individual experiments.
Table 6. Flow Cytometric Analysis of HeLa Cells Treated with 19 and
21
HeLa
cell cycle phaseb
24 h
Controla
3 ( 2
56 ( 1
28 ( 3
16 ( 4
48 h
72 h
SubG1
G0/G1
S
2 ( 0.2
56 ( 1
29 ( 3
15 ( 1
3 ( 0.5
54 ( 0.1
35 ( 1
12 ( 1
G2/M
Figure 6. Unsubstituted benzimidazo[1,2-a]quinoline 36.
Treated with 19a
Table 5. In Vitro Inhibition of Cyano-Substituted Compounds on the
Growth of Tumor Cells and Normal Human Fibroblasts (WI 38)
SubG1
G0/G1
S
2 ( 0.25
32 ( 0.1c
42 ( 2.4c
26 ( 2c
2 ( 0.3
34 ( 1c
36 ( 4c
32 ( 9c
3 ( 0.8
28 ( 3c
32 ( 9
IC50a (µM)
G2/M
40 ( 12c
cmpd H460
24 3 ( 0.7 2 ( 0.2
26 7 ( 4 4 ( 0.8
27 13 ( 1.8 0.05 ( 0.08
30 20 ( 8 19 ( 4
HeLa
MiaPaCa-2 SW620 MCF-7 WI 38
Treated with 21a
4 ( 0.9
7 ( 0.6
15 ( 2
36 ( 3
2.5 ( 0.3 4 ( 2
14 ( 8
5 ( 1
SubG1
G0/G1
S
3 ( 1
2 ( 0.5
36 ( 1c
37 ( 3
27 ( 4c
2 ( 0.2
25 ( 12c
39 ( 21
35 ( 9c
36 ( 0.4c
30 ( 5c
34 ( 5c
2 ( 0.2
8 ( 2
21 ( 8
18 ( 0.6 14 ( 7
23 ( 7
14 ( 16 N.T.b
G2/M
a IC50: the concentration that causes a 50% reduction of the cell growth.
b N.T.: not tested.
a c ) 5 µM. b The results are shown as percentages of cell population in
each cell cycle phase. c Statistically significant at p < 0.05.
substituent in general strongly enhances the cytotoxic activity
of acyclic compounds, but diminishes the activity of the cyclic
ones. The exception to this rule is the extremely pronounced
activity of cyclic compound 27 (with cyano group on the
benzimidazole side) on the HeLa cell line (IC50 ) 0.05 µM).
Such an extreme selectivity of heterocyclic cyano molecules
on HeLa cells was previously observed by our group for
carboxanilides bearing a cyano substituent on either the anilide
or the benzothiophene part of the molecule,39 acyclic cyano
derivatives of thiophene-2-carboxamides,40 as well as for
methyl-2-cyano-naphtho[2,1-b]thiophen-5-carboxylate and 4-ethyl-
7-cyano-thieno[2,3-e]-benzo[b]furan.41
All aforementioned HeLa selective molecules fall into two
structurally discrete groups: acyclic39,40 and cyclic.41 These
groups significantly differ in affinity and type of interactions
with DNA, but nevertheless, all show HeLa-specific selectivity.
Therefore, it seems reasonable to believe that the molecular
target that contributes to this biological selectivity is not the
DNA itself, but probably a set of proteins specifically expressed
in HeLa cells. In addition, published as well as herein presented
results point to the exceptionally well-defined binding site of a
putative macromolecule, because a number of analogues with
a differently positioned cyano group are not equally selective.
These results again prove that finely tuned interplay between
the general structure of the small molecule and the distribution
of interacting substituents precisely control selectivity. Further
studies on elucidating this phenomenon are underway.
Cell Cycle Perturbations. To shed more light on the
mechanisms underlying the antiproliferative activity of some
of the most active compounds, we selected the cyclic compounds
19 and 21 to investigate their influence on the HeLa cell cycle
after 24, 48, and 72 hour treatment periods (Table 6). Obtained
results clearly showed that, at a concentration of 5 × 10-6
M
(∼IC50), both compounds induced statistically significant reduc-
tion of the G1-phase cells over all three treatment periods. This
event was accompanied by an apparent accumulation of HeLa
cells in S and G2/M phases of the cell cycle. Interestingly,

Synthesis of Styryl-2-benzimidazole DerViatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5703
Figure 7. Flow cytometric DNA histograms of SW620 cells treated with compounds 11 and 19 (c ) 5 × 10-6 M) after different time points. aThe
results are shown as percentages of cell population in each cell cycle phase. *Statistically significant at p < 0.05.
compound 19 has a similar effect on the cell cycle-phase
distribution at lower concentration as well (10-6 M) throughout
all three time points (data not shown). On the other hand,
compounds 11 and 17 (acyclic analogues of 19 and 21) did not
have significant influence on the cell cycle distribution of HeLa
cells at the tested concentration, which is five times lower than
the IC50 concentrations for these compounds (data not shown).
Because the compound 19 exerted the most pronounced
growth inhibition effect against colon carcinoma cells (SW620),
we performed additional cell cycle analyses of this cell line upon
treatment with the aforementioned substance (Figure 7). Our
results clearly showed that cells treated with this compound at
a concentration of 5 × 10-6 M were strongly arrested in G2/M
phase after 24 h, which was accompanied by significant
reduction in G1 cells. This G2/M delay persisted during the
next 48 h, which consequently led to the decrease in the G1
and S-phase cells. Interestingly, lower concentrations of this
compound (10-6 M and 5 × 10-7 M) also showed a tendency
to increase the G2/M population. A marked rise in the sub-G0/
G1 population upon treatment with 5 × 10-6 M concentration
indicates cell death (apoptosis) induced by the compound 19.
On the other hand, the compound 11, an acyclic analogue of
19, had a significant influence on the cell cycle progression
only after a 24-hour treatment, particularly at a higher-tested
concentration (5 × 10-6 M), at which it induced S-phase arrest
and concomitant decrease in G1 and G2/M cells. Interestingly,
it seems that this pattern of the cell cycle phase distribution
was maintained after 48 h as well, while the cells were
apparently arrested in the G2/M phase after 72 h.
In Vitro Topoisomerase II Inhibitor Screening. Topoi-
somerase II plays an essential role in DNA replication,
transcription, chromosome formation, and separation of sister
chromatids. As this enzyme is particularly active during the G2
and M phases of the cell cycle, we investigated whether our
cyclic compounds interfere with mitotic progression via the
inhibition of topoisomerase II. For this purpose, we applied an
in vitro topo II inhibitor screening system. Figure 8 shows the
effects of selected compounds 19 and 11 on the relaxation
activity of topoisomerase II, whereby etoposide was used as a
control drug. Topo II incubated with pRYG DNA substrate in
the assay buffer was used as the positive control (lane 2) and
this reaction resulted in the formation of a series of topoisomers
(relaxed DNA forms differing in linking number) that could be
detected as the ladder between supercoiled and open circular
DNA (lane 2). The reaction mixture containing etoposide
resulted in the conversion of supercoiled pRYG DNA into
relaxed DNA forms and in an increased intensity of the open
circular DNA band (cleavage product). This result is in
concordance with a well-established role of etoposide in topo
II inhibition as a compound that stimulates formation of
cleavable complexes (i.e., topo II poison). More interestingly,
topo II relaxation products were absent from the reaction
mixtures containing test compounds 19 and 11 (lanes 4 and 5,
respectively), which implies an antagonizing effect of these
substances on topo II action.
Figure 9 shows the influence of test compounds on the
formation of the DNA cleavage products. Relaxed DNA bands
could be detected in the reaction mixtures containing topoi-
somerase II incubated without any compound (lane 2) and in
the presence of etoposide (lane 3). As expected, etoposide
stimulated formation of cleavable complexes, linear DNA
species and open circular DNA (lane 3). However, test
compounds 19 and 11 (lanes 4 and 5, respectively) did not give
Altogether, flow cytometric data showed that compounds 19
and 21 induced G2/M arrest, which points to the impairment
of mitotic progression. These findings augmented by the DNA
binding results provoked the conclusion that the cyclic com-
pounds presented in this study act as topoisomerase inhibitors.

5704 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Hranjec et al.
Figure 8. Influence of selected test compounds on topoisomerase II relaxation activity. Topo II was incubated with supercoiled pRYG DNA
substrate in the assay buffer alone (lane 2) or in combination with control drug etoposide (lane 3) and selected test compounds, 19 and 11 (lanes
4 and 5, respectively). Supercoiled and linear DNA markers are contained in lanes 1 and 6, respectively.
Figure 9. Influence of selected test compounds on the formation of cleavable complexes. Topo II was incubated with supercoiled pRYG DNA
substrate in the cleavage buffer alone (lane 2) or in combination with control drug etoposide (lane 3) and selected test compounds, 19 and 11 (lanes
4 and 5, respectively). Supercoiled and linear DNA markers are contained in lanes 1 and 6, respectively.
rise to the formation of linear DNA, but inhibited conversion
of supercoiled DNA substrate into relaxed DNA. Altogether,
our results suggest that compounds 19 and 11 are catalytic
inhibitors of topoisomerase II.
of transcription). Observed ability of compound 11 to bind to
the DNA minor groove might additionally account for differ-
ences in cytostatic effects between these two compounds.
Cells respond to DNA damage by activating a checkpoint
that induces G2/M arrest, thus allowing the cells to repair their
DNA before entry into mitosis. Strong G2/M arrest observed
in HeLa and SW620 cells could thus result from either the
cellular response to DNA damage caused by intercalation into
DNA or from topo II inhibition, whereby both molecular events
prevent the cells from proceeding through mitosis. Our results
are in good agreement with other reports, confirming that
catalytic inhibitors can induce G2/M arrest. For example,
Anderson et al. found that merbarone, IGRF-187, and aclaru-
bicin can induce G2 arrest or significant G2 delay in a dose-
dependent manner.44
Topo II inhibitors are among the most effective anticancer
drugs for many different malignant diseases due to the essential
role topoisomerase II plays in the replication and the cell cycle
in highly proliferating cancer cells. Unlike topo II poisons (e.g.,
etoposide and doxorubicin) whose cytotoxicity generally cor-
relates with DNA damage after stabilization of DNA-topo II
cleavable complexes, the molecular events by which catalytic
inhibitors (e.g., aclarubicin, merbarone and the bisdioxopipera-
zine derivatives) cause cell death are not fully understood.42 In
spite of an inveterate opinion that catalytic inhibitors of
topoisomerase II are non-DNA-damaging agents, Wang et al.
showed that two common catalytic inhibitors, namely, merbar-
one and ICRF-187, induced strong dose-dependent genotoxic
effects in cultured cells and observed similar clastogenic effects
in vivo in merbarone-treated mice.43 Our DNA binding study
substantiated the studies of Wang et al., as we showed the
compound 19 to be the DNA intercalator similarly to aclarubicin,
which supports the idea that catalytic inhibitors might damage
DNA as well. However, lower antiproliferative activity of the
compound 11 in comparison with 19 might be explained by
the potential ability of the latter to induce conformational
changes leading to additional DNA damage uncoupled from topo
II inhibition, and to other mechanistic outcomes (e.g., inhibition
Conclusions
We have shown that cyclic (benzimidazo[1,2-a]quinolines),
positively charged analogues intercalate into ds DNA, which
might account for their significant antiproliferative effects (IC50
concentrations are within low micromolar range). On the other
hand, the acyclic, either positively charged or neutral molecules,
show a different interaction with DNA, correlating well with
inferior biological activity (except for 24). However, their
selectivity toward tumor cells in regard to normal cells is more
pronounced. Because selectivity is very important for biological

Synthesis of Styryl-2-benzimidazole DerViatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5705
1
287 °C; IR (KBr, νmax/cm-1) 3423, 2913, 1697, 1613; H NMR
use, this phenomenon should be corroborated in in vivo
experiments and, therefore, warrants further evaluation.
Furthermore, our results support the role of tested compounds
as catalytic inhibitors of topoisomerase II, at least for two
structurally different imidazolyl-substituted analogues 11 and
19. Strong G2/M cell cycle arrest, particularly observed in
SW620 cells treated with imidazolyl-substituted compound 19,
could be either a response to DNA damage produced by its
intercalation into DNA or a direct consequence of topo II
inhibition, whereby both processes prevent mitotic progression.
Its acyclic analogue 11, which is a minor grove binder, exerts
significantly lower cytostatic effect, possibly due to a different
DNA interaction mode.
(DMSO-d6, δ/ppm) 13.64 (bs, 1H, NHbenzimidazole), 9.52 (bs, 1H,
NHamidine), 9.42 (bs, 1H, NHamidine), 9.08 (bs, 1H, NHamidine), 8.02
(s, 1H, Harom), 7.90 (d, 1H, J ) 16.81 Hz, Hethenyl), 7.72 (d, 1H, J
) 8.32 Hz, Harom), 7.70 (d, 1H, J ) 8.21 Hz, Harom), 7.68 (d, 1H,
J ) 7.20 Hz, Harom), 7.57 (d, 1H, J ) 7.2 Hz, Harom), 7.49-7.40
(m, 3H, Harom), 7.30 (d, 1H, J ) 16.55 Hz, Hethenyl), 4.14 (m, 1H,
CH(CH3)2), 1.31 (d, 6H, J ) 5.79 Hz, CH(CH3)2); 13C NMR
(DMSO-d6, δ/ppm) 162.27 (s), 149.65 (s), 143.22 (s), 135.41 (s),
129.14 (d), 128.92 (d, 2C), 127.17 (d, 2C), 122.25 (d), 121.53 (s),
117.07 (d), 115.53 (d, 2C), 111.26 (d), 44.92 (d), 21.25 (q, 2C);
MS (m/z) 305 (M+1 (- HCl)); Anal. (C19H21N4Cl) C, H, N.
E-2-(2-Styryl-3H-benzimidazol-5(6)-yl)-4,5-dihydro-3H-imi-
dazol-1-yl Hydrochloride (3). Compound 3 was prepared using
the general method described for the preparation of 2-9; a mixture
of 4-(2-imidazolinyl)-1,2-phenylenediamine (0.500 g, 2.35 mmol),
3-phenyl-propenal (0.311 g, 2.35 mmol), and p-benzoquinone (0.254
g, 2.35 mmol) in absolute ethanol (20 mL) was refluxed under
nitrogen for 3 h, and then the reaction mixture was worked up as
it is described to give 0.450 g (59%) of light violet powder; mp >
Additionally, one cyano-substituted compound (27) showed
intriguingly strong and selective antiproliferative activity on
HeLa tumor cell line.
Finally, stronger cytostatic activity of amidino-substituted
benzimidazo[1,2-a]quinolines in comparison with the com-
mercial chemotherapeutic agent etoposide warrants their further
optimization as novel intercalators and topoisomerase II inhibi-
tors. In particular, imidazolyl-substituted benzimidazo[1,2-a]-
quinoline deserves additional consideration as a putative agent
against colon carcinoma. Our further studies on the mechanism
of action of this compound are underway.
1
300 °C; IR (KBr, νmax/cm-1) 3381, 3107, 1606, 1514; H NMR
(DMSO-d6, δ/ppm) 13.50 (bs, 1H, NHbenzimidazole), 10.60 (bs, 2H,
NHamidine), 8.74 (d, 1H, J ) 16.40 Hz, Hethenyl), 7.97 (d, 1H, J )
8.31 Hz, Harom), 7.92 (s, 1H, Harom), 7.73 (d, 1H, J ) 8.25 Hz,
H
H
H
arom), 7.70 (d, 1H, J ) 7.20 Hz, Harom), 7.58 (d, 1H, J ) 7.10 Hz,
arom), 7.53-7.37 (m, 3H, Harom), 7.30 (d, 1H, J ) 16.50 Hz,
ethenyl), 4.20 (bs, 4H, 2CH2imidazole); 13C NMR (DMSO-d6, δ/ppm)
165.82 (s), 165.78 (s), 135.86 (s), 135.84 (s), 131.24 (d), 130.53
(d), 129.82 (d), 129.69 (s), 129.54 (d), 129.50 (d), 129.31 (d), 128.58
(d), 127.79 (d), 127.52 (d), 117.29 (d), 115.62 (s); MS (m/z) 289
(M+1 (- HCl)); Anal. (C18H17N4Cl) C, H, N.
Experimental Section
Chemistry. Melting points were determined on a Koffler hot
stage microscope and are uncorrected. IR spectra were recorded
on a Nicolet magna 760, a Perkin-Elmer 297, and a Perkin-Elmer
Spectrum 1 spectrophotometers with KBr disks. 1H and 13C NMR
spectra were recorded on Varian Gemini 300, Bruker Avance DPX
300 and Bruker Avance DRX 500 spectrometers using TMS as an
internal standard in DMSOa-d6. Mass sprectra were recorded on
an Agilent 1100 series LC/MSD Trap SL. Elemental analysis for
carbon, hydrogen, and nitrogen were performed on a Perkin-Elmer
2400 elemental analyzer and a Perkin-Elmer, Series II, CHNS
Analyzer 2400. Where analyses are indicated only as symbols of
elements, analytical results obtained are within 0.4% of the
theoretical value. In preparative photochemical experiments, the
irradiation was performed at room temperature with a water-cooled
immersion well with “Origin Hanau”, 400 W, high-pressure,
mercury arc lamp using Pyrex glass as a filter. All compounds were
routinely checked by TLC with Merck silica gel 60F-254 glass
plates.
General Method for the Synthesis of 5(6)-N-Amidino-
substituted E-2-Styryl-1H-benzimidazoles (2-5) and 5(6)-N-
Amidino-substituted E-2-[2-(2-Chloro-phenyl)-vinyl]-1H-ben-
zimidazoles (6-9). A mixture of corresponding 4-N-substituted-
1,2-phenylenediamines, 3-phenyl-propenal or 3-(2-chloro-phenyl)-
propenal, and p-benzoquinone in absolute ethanol (10 mL) was
stirred at reflux for 2.5-3 h under a nitrogen atmosphere. The
reaction mixture was cooled to room temperature and diethylether
was added. The resulting product was filtered off and washed with
diethylether. After recrystallization from ethanol/diethylether or
ethanol/acetone, light powders were obtained in very good yields.
E-N-Isopropyl-2-styryl-3H-benzimidazole-5(6)-carboxami-
dine Hydrochloride (2). Compound 2 was prepared using the
general method described for the preparation of 2-9; a mixture of
4-N-isopropylamidino-1,2-phenylenediamine (0.400 g, 1.75 mmol),
3-phenyl-propenal (0.231 g, 1.75 mmol), and p-benzoquinone (0.189
g, 1.75 mmol) in absolute ethanol (10 mL) was refluxed under
nitrogen for 2 h and then the reaction mixture was worked up as it
is described to give 0.355 g (60%) of gray powder; mp 285-
E-2-Styryl-3H-benzimidazole-5(6)-carboxamidine Hydrochlo-
ride (4). Compound 4 was prepared using the general method
described for the preparation of 2-9; a mixture of 4-N-amidino-
1,2-phenylenediamine (0.500 g, 2.68 mmol), 3-phenyl-propenal
(0.354 g, 2.68 mmol), and p-benzoquinone (0.284 g, 2.68 mmol)
in absolute ethanol (15 mL) was refluxed under nitrogen for 2 h,
and then the reaction mixture was worked up as it is described to
give 0.560 g (70%) of gray powder; mp 289-291 °C; IR (KBr,
ν
max/cm-1) 3080, 1670, 1640, 1621; 1H NMR (DMSO-d6, δ/ppm)
9.40 (bs, 2H, NHamidine), 9.15 (bs, 2H, NHamidine), 8.14 (s, 1H, Harom),
7.90 (d, 1H, J ) 16.53 Hz, Hethenyl), 7.70 (d, 1H, J ) 8.45 Hz,
H
arom), 7.69 (d, 1H, J ) 8.44 Hz, Harom), 7.68 (d, 1H, J ) 7.20 Hz,
arom), 7.65 (d, 1H, J ) 7.05 Hz, Harom), 7.54-7.36 (m, 3H, Harom),
H
7.30 (d, 1H, J ) 16.5 Hz, Hethenyl); 13C NMR (DMSO-d6, δ/ppm)
166.08 (s), 154.04 (s), 136.66 (d), 135.42 (s), 130.06 (d), 129.34
(d), 129.04 (d, 2C), 127.32 (d, 2C), 122.04 (d), 121.10 (s), 116.82
(d), 115.64 (d); MS (m/z) 263 (M+1 (- HCl)); Anal. (C19H20N4-
Cl2‚H2O) C, H, N.
E-N-Morpholin-4-yl-2-styryl-3H-benzimidazole-5(6)-carboxa-
midine Hydrochloride (5). Compound 5 was prepared using the
general method described for the preparation of 2-9; a mixture of
4-N-morpholinylamidino-1,2-phenylenediamine (0.440 g, 1.62 mmol),
3-phenyl-propenal (0.214 g, 1.62 mmol), and p-benzoquinone (0.175
g, 1.62 mmol) in absolute ethanol (10 mL) was refluxed under
nitrogen for 2 h, and then the reaction mixture was worked up as
it is described to give 0.300 g (49%) of light violet powder; mp
1
291-293 °C; IR (KBr, νmax/cm-1) 3380, 2926, 1645, 1612; H
NMR (DMSO-d6, δ/ppm) 13.60 (bs, 1H, NHbenzimidazole), 11.23 (bs,
1H, NHamidine), 9.77 (s, 1H, NHamidine), 9.07 (bs, 1H, NHamidine), 8.10
(s, 1H, Harom), 7.87 (d, 1H, J ) 16.42 Hz, Hethenyl), 7.70 (d, 1H, J
) 8.11 Hz, Harom), 7.62 (d, 1H, J ) 8.09 Hz, Harom), 7.48 (d, 1H,
J ) 7.56 Hz, Harom), 7.43 (d, 1H, J ) 7.60 Hz, Harom), 7.41-7.38
(m, 3H, Harom), 7.30 (d, 1H, J ) 16.54 Hz, Hethenyl), 3.78 (bs, 4H,
2CH2morpholine), 2.96 (bs, 4H, 2CH2morphiline); 13C NMR (DMSO-d6,
δ/ppm) 162.31(s), 149.72 (s), 136.49 (d), 135.45 (s), 129.19 (d),
128.95 (d, 2C), 127.21 (d, 2C), 122.08 (d), 119.11 (s), 116.96 (d),
115.59 (d, 2C), 65.52 (t, 2C), 53.86 (t, 2C); MS (m/z) 348 (M+1
(- HCl)); Anal. (C20H22N5OCl‚H2O) C, H, N.
a Abbreviations: DMEM, Dulbecco’s modified Eagle medium; DMSO,
dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tet-
razolium bromide; OD, optical density; PG, percentage of growth; PDT,
cell population doubling time.
E-2-[2-(2-Chloro-phenyl)-vinyl]-N-isopropyl-3H-benzimida-
zole-5(6)-carboxamidine Hydrochloride (6). Compound 6 was

5706 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Hranjec et al.
prepared using the general method described for the preparation
of 2-9; a mixture of 4-N-isopropylamidino-1,2-phenylenediamine
(0.618 g, 2.7 mmol), 3-(2-chloro-phenyl)-propenal (0.450 g, 2.7
mmol), and p-benzoquinone (0.292 g, 2.7 mmol) in absolute ethanol
(10 mL) was refluxed under nitrogen for 2.5 h, and then the reaction
mixture was worked up as it is described to give 0.650 g (64%) of
gray powder; mp 281-282 °C; IR (KBr, νmax/cm-1) 3423, 3068,
3.71 (bs, 4H, 2CH2morpholine), 2.90 (bs, 4H, 2CH2morpholine); 13C NMR
(DMSO-d6, δ/ppm) 162.27 (s), 133.28 (s), 132.92 (s), 131.34 (d),
130.56 (d), 129.91 (d, 2C), 127.72 (d, 2C), 127.35 (d, 2C), 119.98
(d), 119.34 (s), 65.51 (t, 2C), 53.85 (t, 2C); MS (m/z) 382 (M+1 (-
HCl)); Anal. (C20H21N5OCl2‚H2O) C, H, N.
General Method for the Synthesis of 5(6)-N-Amidino-
substituted Z-2-Styryl-1H-benzimidazoles (10-13) and 5(6)-N-
Amidino-substituted Z-2-[2-(2-Chloro-phenyl)-vinyl]-1H-benz-
imidazoles (14-17). Solutions of the corresponding 5(6)-N-
amidino-substituted E-2-styryl-1H-benzimidazoles 2-5 (0.47 mmol)
and 5(6)-N-amidino-substituted E-2-[2-(2-chloro-phenyl)-vinyl]-1H-
benzimidazoles 6-9 in ethanol (c ) 1.3 × 10-2 mol dm-3) were
irradiated at room temperature with 400 W, high-pressure, mercury
lamp using a Pyrex filter for about 4 h, when UV spectra shown
that the isomerization is finished. The solutions were concentrated,
diethylether was added, and resulting products were filtered off
and washed with diethylether. After precipitating from ethanol/
diethylether, light powders were obtained.
1
1665, 1613, 1518; H NMR (DMSO-d6, δ/ppm) 13.70 (bs, 1H,
NHbenzimidazole), 9.48 (bs, 1H, NHamidine), 9.38 (bs, 1H, NHamidine),
9.07 (bs, 1H, NHamidine), 8.05 (s, 1H, Harom), 8.03 (d, 1H, J ) 15.64
Hz, Hethenyl), 7.90 (d, 1H, J ) 8.26 Hz, Harom), 7.60 (d, 1H, J )
8.41 Hz, Harom), 7.49 (d, 2H, J ) 7.23 Hz, Harom), 7.39-7.34 (m,
2H, Harom), 7.30 (d, 1H, J ) 16.20 Hz, Hethenyl), 4.10 (m, 1H, CH-
(CH3)2), 1.23 (d, 6H, J ) 5.78 Hz, CH(CH3)2); 13C NMR (DMSO-
d6, δ/ppm) 162.31 (s), 133.33 (s), 132.94 (s), 131.17 (d), 130.58
(d), 129.95 (d, 2C), 127.77 (d, 2C), 127.37 (d, 2C), 122.66 (s),
120.11 (d), 45.03 (d), 21.32 (q, 2C); MS (m/z) 339 (M+1 (- HCl));
Anal. (C19H20N4Cl2‚H2O) C, H, N.
E-2-{2-[2-(2-Chloro-phenyl)-vinyl]-3H-benzimidazo-5(6)-yl}-
4,5-dihydro-3H-imidazol-1-yl Hydrochloride (7). Compound 7
was prepared using the general method described for the preparation
of 2-9; a mixture of 4-(2-imidazolinyl)-1,2-phenylenediamine
(0.500 g, 2.35 mmol), 3-(2-chloro-phenyl)-propenal (0.392 g, 2.35
mmol), and p-benzoquinone (0.254 g, 2.35 mmol) in absolute
ethanol (15 mL) was refluxed under nitrogen for 2.5 h, and then
the reaction mixture was worked up as it is described to give 0.630
Z-N-Isopropyl-2-styryl-3H-benzimidazole-5(6)-carboxami-
dine Hydrochloride (10). Compound 10 was prepared using the
general method described for the preparation of 10-17; a solution
of 2 (0.150 g, 0.44 mmol) in ethanol (35 mL) was irradiated for
4 h and then worked up as it is described to give 0.100 g (67%) of
gray powder; mp 190-192 °C; IR (KBr, νmax/cm-1) 3369, 3066,
g (70%) of gray powder; mp 253-255 °C; IR (KBr, νmax/cm-1
)
1665, 1613; H NMR (DMSO-d6, δ/ppm) 13.50 (bs, 1H, NHbenz
-
1
1
3406, 3130, 1607, 1514; H NMR (DMSO-d6, δ/ppm) 13.70 (bs,
1H, NHbenzimidole), 10.90 (bs, 2H, NHamidine), 8.38 (d, 1H, J ) 16.50
Hz, Hethenyl), 8.11 (d, 1H, J ) 8.40 Hz, Harom), 7.97 (s, 1H, Harom),
7.85 (d, 1H, J ) 8.40 Hz, Harom), 7.74 (d, 1H, J ) 7.50 Hz, Harom),
7.55 (d, 1H, J ) 7.50 Hz, Harom), 7.43-7.40 (m, 2H, Harom), 7.35
(d, 1H, J ) 16.70 Hz, Hethenyl), 4.35 (bs, 4H, 2CH2imidazole); 13C
NMR (DMSO-d6, δ/ppm) 165.32 (s), 154.65 (s), 133.24 (s), 133.05
(s), 131.61 (d), 131.21 (d), 130.78 (d, 2C), 127.85 (d, 2C), 127.45
(d, 2C), 119.91 (s), 115.45 (d), 44.24 (t, 2C); MS (m/z) 323 (M+1
(- HCl)); Anal. (C18H16N4Cl2) C, H, N.
imidazole), 9.80 (bs, 1H, NHamidine), 9.51 (bs, 1H, NHamidine), 9.09 (s,
1H, NHamidine), 8.01 (s, 1H, Harom), 7.99 (d, 1H, J ) 8.22 Hz, Harom),
7.71 (d, 1H, J ) 8.20 Hz, Harom), 7.57 (d, 1H, J ) 7.66 Hz, Harom),
7.49 (d, 1H, J ) 7.80 Hz, Harom), 7.46-7.37 (m, 3H, Harom), 7.00
(d, 1H, J ) 13.22 Hz, Hethenyl), 6.70 (d, 1H, J ) 13.05 Hz, Hethenyl),
3.95-3.82 (m, 1H, CH(CH3)2), 1.30 (d, 6H, J ) 6.00 Hz, CH-
(CH3)2); 13C NMR (DMSO-d6, δ/ppm) 162.41 (s), 152.16 (s),
137.22 (d), 135.46 (s), 130.09 (d, 2C), 129.09 (d), 128.85 (d), 128.18
(d, 2C), 127.37 (d), 122.71 (s), 117.03 (d), 116.80 (d), 45.03 (d),
21.36 (q, 2C); MS (m/z) 305 (M+1 (- HCl)); Anal. (C19H21N4Cl‚
2H2O) C, H, N.
E-2-[2-(2-Chloro-phenyl)-vinyl]-3H-benzimidazole-5(6)-car-
boxamidine Hydrochloride (8). Compound 8 was prepared using
the general method described for the preparation of 2-9; a mixture
of 4-N-amidino-1,2-phenylenediamine (0.575 g, 3.1 mmol), 3-(2-
chloro-phenyl)-propenal (0.513 g, 3.1 mmol), and p-benzoquinone
(0.335 g, 3.1 mmol) in absolute ethanol (15 mL) was refluxed under
nitrogen for 2.5 h, and then the reaction mixture was worked up as
it is described to give 0.760 g (74%) of light gray powder; mp
Z-2-(2-Styryl-3H-benzimidazol-5(6)-yl)-4,5-dihydro-3H-imi-
dazol-1-yl Hydrochloride (11). Compound 11 was prepared using
the general method described for the preparation of 10-17; a
solution of 3 (0.150 g, 0.46 mmol) in ethanol (36 mL) was irradiated
for 4 h and then worked up as it is described to give 0.115 g (78%)
of light violet powder; mp 220-222 °C; IR (KBr, νmax/cm-1) 3381,
1
225-227 °C; IR (KBr, νmax/cm-1) 3310, 3183, 1659, 1622; H
1
3106, 1605, 1513; H NMR (DMSO-d6, δ/ppm) 13.40 (bs, 1H,
NMR (DMSO-d6, δ/ppm) 9.42 (s, 2H, NHamidine), 9.19 (bs, 2H,
NHamidine), 8.17 (s, 1H, Harom), 8.13 (d, 1H, J ) 16.50 Hz, Hethenyl),
7.97 (d, 1H, J ) 9.00 Hz, Harom), 7.76 (d, 1H, J ) 8.78 Hz, Harom),
7.70 (d, 1H, J ) 7.48 Hz, Harom), 7.55 (d, 1H, J ) 7.48 Hz, Harom),
NHbenzimidazole), 10.68 (bs, 2H, NHamidine), 8.45 (s, 1H, Harom), 8.10
(d, 1H, J ) 8.40 Hz, Harom), 7.99 (d, 1H, J ) 8.40 Hz, Harom), 7.72
(d, 1H, J ) 7.85 Hz, Harom), 7.52-7.24 (m, 3H, Harom), 7.29 (d,
1H, J ) 7.70 Hz, Harom), 7.05 (d, 1H, J ) 12.90 Hz, Hethenyl), 6.64
(d, 1H, J ) 12.90 Hz, Hethenyl), 4.10 (bs, 4H, 2CH2imidazole); 13C
NMR (DMSO-d6, δ/ppm) 165.41 (s), 152.41 (s), 143.36 (s), 137.49
(d), 135.50 (s), 130.19 (d, 2C), 129.63 (d), 129.46 (d), 128.36 (d),
128.23 (d, 2C), 127.44 (d), 116.96 (d), 115.24 (s), 44.63 (t, 2C);
MS (m/z) 289 (M+1 (- HCl)); Anal. (C18H17N4Cl‚H2O) C, H, N.
7.45-7.39 (m, 2H, Harom), 7.36 (d, 1H, J ) 16.50 Hz, Hethenyl); 13
C
NMR (DMSO-d6, δ/ppm) 166.49 (s), 153.85 (s), 133.65 (s), 133.41
(s), 131.78 (d), 131.10 (d), 130.39 (d), 128.21 (d, 2C), 127.81 (d,
2C), 122.61 (d), 121.71 (s), 120.28 (d); MS (m/z) 297 (M+1 (-
HCl)); Anal. (C16H14N4Cl2‚H2O) C, H, N.
E-2-[2-(2-Chloro-phenyl)-vinyl]-N-morpholin-4-yl-3H-benz-
imidazole-5(6)-carboxamidine Hydrochloride (9). Compound 9
was prepared using the general method described for the preparation
of 2-9; a mixture of 4-N-morpholinylamidino-1,2-phenylenedi-
amine (1.000 g, 3.68 mmol), 3-(2-chloro-phenyl)-propenal (0.613
g, 3.68 mmol), and p-benzoquinone (0.397 g, 3.68 mmol) in
absolute ethanol (15 mL) was refluxed under nitrogen for 2.5 h,
and then the reaction mixture was worked up as it is described to
give 1.100 g (72%) of light gray powder; mp 296-298 °C; IR (KBr,
Z-2-Styryl-3H-benzimidazole-5(6)-carboxamidine Hydrochlo-
ride (12). Compound 12 was prepared using the general method
described for the preparation of 10-17; a solution of 4 (0.140 g,
0.47 mmol) in ethanol (36 mL) was irradiated for 4 h and then
worked up as it is described to give 0.098 g (71%) of light violet
powder; mp 188-189 °C; IR (KBr, νmax/cm-1) 3084, 1672, 1529;
1H NMR (DMSO-d6, δ/ppm) 13.30 (bs, 1H, NHbenzimidazole), 9.31
(bs, 2H, NHamidine), 9.10 (bs, 2H, NHamidine), 8.08 (s, 1H, Harom),
7.90 (d, 2H, J ) 8.44 Hz, Harom), 7.68-7.62 (m, 2H, Harom), 7.35-
7.25 (m, 3H, Harom), 6.93 (d, 1H, J ) 13.26 Hz, Hethenyl), 6.60 (d,
1H, J ) 13.28 Hz, Hethenyl); 13C NMR (DMSO-d6, δ/ppm) 166.06
(s), 152.25 (s), 137.26 (d), 135.34 (s), 129.93 (d, 2C), 128.95 (d),
128.69 (d), 128.03 (d, 2C), 127.26 (d), 121.93 (d), 121.07 (s), 116.83
(d).
ν
max/cm-1) 3361, 2922, 1649, 1606; 1H NMR (DMSO-d6, δ/ppm)
13.73 (bs, 1H, NHbenzimidazole), 11.22 (bs, 1H, NHamidine), 9.77 (s,
1H, NHamidine), 9.02 (bs, 1H, NHamidine), 8.08 (s, 1H, Harom), 8.06
(d, 1H, J ) 16.22 Hz, Hethenyl), 7.90 (dd, 1H, J ) 9.00 Hz, J )
7.80 Hz, Harom), 7.70 (d, 1H, J ) 8.46 Hz, Harom), 7.60 (d, 1H, J )
8.45 Hz, Harom), 7.48 (dd, 1H, J ) 9.00 Hz, J ) 7.84 Hz, Harom),
7.38-7.34 (m, 2H, Harom), 7.30 (d, 1H, J ) 16.20 Hz, Hethenyl),

Synthesis of Styryl-2-benzimidazole DerViatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5707
Z-N-Morpholin-4-yl-2-styryl-3H-benzimidazole-5(6)-carboxa-
midine Hydrochloride (13). Compound 13 was prepared using
the general method described for the preparation of 10-17; a
solution of 5 (0.075 g, 0.20 mmol) in ethanol (15 mL) was irradiated
for 4 h and then worked up as it is described to give 0.048 g (63%)
of light gray powder; mp 197-199 °C; IR (KBr, νmax/cm-1) 3394,
17 was prepared using the general method described for the
preparation of 10-17; a solution of 9 (0.150 g, 0.45 mmol) in
ethanol (35 mL) was irradiated for 4 h and then worked up as it is
described to give 0.114 (76%) of light violet powder; mp 188-
1
190 °C; H NMR (DMSO-d6, δ/ppm) 11.21 (bs, 1H, NHamidine),
9.86 (s, 1H, NHamidine), 9.06 (bs, 1H, NHamidine), 8.04 (bs, 1H, Harom),
7.73 (d, 1H, J ) 8.70 Hz, Harom), 7.65 (d, 1H, J ) 8.40 Hz, Harom),
7.58-7.50 (m, 2H, Harom), 7.40 (t, 1H, J ) 7.60 Hz, Harom), 7.28
(t, 1H, J ) 7.10 Hz, Harom), 7.09 (d, 1H, J ) 12.60 Hz, Hethenyl),
6.87 (d, 1H, J ) 12.60 Hz, Hethenyl), 3.91 (bs, 4H, 2CH2morpholine),
3.00 (bs, 4H, 2CH2morpholine); 13C NMR (DMSO-d6, δ/ppm) 163.25
(s), 137.93 (d), 136.45 (s), 135.80 (s), 132.01 (d, 2C), 130.19 (s),
129.13 (d), 128.93 (d), 128.57 (d, 2C), 127.02 (d), 123.44 (d),
119.98 (s), 65.10 (t, 2C), 53.79 (t, 2C); MS (m/z) 382 (M+1 (-
HCl)); Anal. (C20H21N5OCl2) C, H, N.
General Method for the Synthesis of N-Amidino-Substituted
Benzimidazo[1,2-a]quinolines (18-21). A solution of correspond-
ing 5(6)-N-amidino-substituted E-2-styryl-1H-benzimidazoles (2-
5) and a small amount of iodine (5%), in ethanol (c ) 1.3 × 10-3
mol dm-3), were irradiated at room temperature with 400 W, high-
pressure, mercury lamp, using a Pyrex filter for about 6-10 h, until
the UV spectra showed that the reaction of dehydrocyclization was
finished. The air was bubbled through the solution. The solutions
were concentrated under reduced pressure, diethylether was added,
and the resulting products were filtered off and washed with
diethylether. After precipitating from ethanol/diethylether and
recrystallization from acetone/water, light powders were obtained
in 37-42% yields. Compounds 18-21 were prepared as a mixture
of 9(10)-structural isomers.
Compounds 18-21 were also prepared by reaction of photo-
chemical dehydrohalogenation cyclization of chloro-substituted
5(6)-N-amidino-substituted E-2-styryl-1H-benzimidazoles 6-9. A
solutions of corresponding chloro-substituted 5(6)-N-amidino-
substituted E-2-styryl-1H-benzimidazoles (6-9) in ethanol (c )
1.2 × 10-3 mol dm-3) were irradiated at room temperature with a
400 W, high-pressure, mercury lamp using a Pyrex filter for about
6-10 h, until the UV spectra showed that the reaction of
dehydrohalogenation was completed. The solutions were concen-
trated under reduced pressure, diethyl ether was added and resulting
precipitates were filtered off and washed with diethylether. After
precipitating from ethanol/diethylether and recrystallization from
acetone/water, light powders were obtained in 31-40% yields.
N-Isopropyl-benzimidazo[1,2-a]quinoline-(9)10-carboxami-
dine Hydrochloride (18). Compound 18 was prepared using the
general method described for the preparation of 18-21: (a) a
solution of 2 (0.100 g, 0.29 mmol) and iodine (8 mg) in ethanol
(220 mL) was irradiated for 6 h and then worked up as it is
described to give 0.030 g (31%) of yellow powder; (b) a solution
of 6 (0.095 g, 0.25mmol) in ethanol (220 mL) was irradiated for
7 h and then worked up as it is described to give 0.035 g (37%) of
yellow powder; mp 219-221 °C; IR (KBr, υ/cm-1) 3435, 3105,
1676, 1613, 1537; MS (m/z) 303 (M+1 (- HCl)); Anal. (C19H19N4-
Cl) C, H, N.
1
2924, 1652, 1643, 1607; H NMR (DMSO-d6, δ/ppm) 13.33 (bs,
1H, NHbenzimidazole), 11.18 (bs, 1H, NHamidine), 9.74 (s, 1H, NHamidine),
9.01 (bs, 1H, NHamidine), 7.94 (s, 1H, Harom), 7.67 (d, 1H, J ) 8.20
Hz, Harom), 7.59 (d, 1H, J ) 8.08 Hz, Harom), 7.40 (t, 1H, J ) 7.20
Hz, Harom), 7.32-7.22 (m, 4H, Harom), 6.93 (d, 1H, J ) 13.20 Hz,
H
ethenyl), 6.62 (d, 1H, J ) 12.60 Hz, Hethenyl), 3.71 (bs, 4H,
2CH2morpholine), 2.90 (bs, 4H, 2CH2morpholine); 13C NMR (DMSO-d6,
δ/ppm) 162.25 (s), 137.13 (d), 135.44 (s), 135.40 (s), 130.01 (d,
2C), 129.19 (d), 128.93 (d), 128.70 (d), 128.01 (d, 2C), 127.20
(d), 119.13 (s), 116.93 (d), 65.20 (t, 2C), 53.85 (t, 2C); MS (m/z)
382 (M+1); Anal. (C20H22N5OCl‚2H2O) C, H, N.
Z-2-[2-(2-Chloro-phenyl)-vinyl]-N-isopropyl-3H-benzimida-
zole-5(6)-carboxamidine Hydrochloride (14). Compound 14 was
prepared using the general method described for the preparation
of 10-17; a solution of 6 (0.100 g, 0.27 mmol) in ethanol (20 mL)
was irradiated for 4 h and then worked up as it is described to give
0.060 g (66%) of light gray powder; mp 191-193 °C; IR (KBr,
ν
max/cm-1) 3359, 2979, 1667, 1614; 1H NMR (DMSO-d6, δ/ppm)
9.60 (bs, 1H, NHamidine), 9.58 (bs, 1H, NHamidine), 9.49 (s, 1H,
NHamidine), 7.93 (s, 1H, Harom), 7.74 (d, 1H, J ) 8.20 Hz, Harom),
7.64 (d, 1H, J ) 8.40 Hz, Harom), 7.56 (d, 1H, J ) 7.80 Hz, Harom),
7.51 (d, 1H, J ) 7.80 Hz, Harom), 7.39 (t, 1H, J ) 7.44 Hz, Harom),
7.27 (t, 1H, J ) 7.28 Hz, Harom), 7.08 (d, 1H, J ) 12.30 Hz, Hethenyl),
6.87 (d, 1H, J ) 12.60 Hz, Hethenyl), 4.16-4.09 (m, 1H, CH(CH3)2),
1.27 (d, 6H, J ) 6.01 Hz, CH(CH3)2); 13C NMR (DMSO-d6, δ/ppm)
162.21(s), 151.33 (s), 141.22 (s), 134.16 (s), 133.41 (d), 132.72
(s), 131.17 (d), 129.99 (d), 129.11 (d), 127.80 (d), 127.43 (d), 126.62
(d), 125.43 (s), 122.68 (s), 122.26 (d), 119.63 (d), 45.01 (d), 21.30
(q, 2C); MS (m/z) 339 (M+1 (- HCl)); Anal. (C19H20N4Cl2‚H2O)
C, H, N.
Z-2-{2-[2-(2-Chloro-phenyl)-vinyl]-3H-benzimidazo-5(6)-yl}-
4,5-dihydro-3H-imidazol-1-yl Hydrochloride (15). Compound 15
was prepared using the general method described for the preparation
of 10-17; a solution of 7 (0.160 g, 0.45 mmol) in ethanol (35 mL)
was irradiated for 4 h and then worked up as it is described to give
0.110 g (66%) of light brown powder; mp 285-287 °C; IR (KBr,
ν
max/cm-1) 3389, 3105, 1606, 1511; 1H NMR (DMSO-d6, δ/ppm)
10.61 (bs, 2H, NHamidine), 8.25 (s, 1H, Harom), 7.89 (d, 1H, J )
8.40 Hz, Harom), 7.68 (d, 1H, J ) 8.40 Hz, Harom), 7.52 (d, 1H, J )
7.80 Hz, Harom), 7.42-7.34 (m, 2H, Harom), 7.27 (d, 1H, J ) 7.60
Hz, Harom), 7.11 (d, 1H, J ) 12.60 Hz, Hethenyl), 6.84 (d, 1H, J )
2.40 Hz Hethenyl), 4.00 (t, 4H, J ) 6.20 Hz, 2CH2imidazole); 13C NMR
(DMSO-d6, δ/ppm) 165.28 (s), 151.85 (s), 133.98 (s), 133.76 (d),
133.11 (s), 132.65 (s), 131.73 (d), 130.62 (d), 129.90 (d), 128.99
(d), 127.66 (d), 126.47 (d), 122.40 (d), 119.36 (d), 115.30 (s), 44.13
(t, 2C); MS (m/z) 323 (M+1 (- HCl)); Anal. (C18H16N4Cl2) C, H,
N.
Z-2-[2-(2-Chloro-phenyl)-vinyl]-3H-benzimidazole-5(6)-car-
boxamidine Hydrochloride (16). Compound 16 was prepared
using the general method described for the preparation of 10-17;
a solution of 8 (0.200 g, 0.48 mmol) in ethanol (36 mL) was
irradiated for 4 h and then worked up as it is described to give
N-Isopropyl-benzimidazo[1,2-a]quinoline-10-carboxami-
dine Hydrochloride (60%). 1H NMR (DMSO-d6, δ/ppm) 9.76 (bs,
2H, NHamidine), 9.26 (bs, 1H, NHamidine), 9.09 (s, 1H, Harom), 9.02
(d, 1H, J ) 8.46 Hz, Harom), 8.12 (d, 1H, J ) 9.75 Hz, Hquinoline),
8.10 (d, 1H, J ) 7.70 Hz, Harom), 8.07 (d, 1H, J ) 8.70 Hz, Harom),
7.98-7.86 (m, 2H, Harom), 7.74 (d, 1H, J ) 9.48 Hz, Hquinoline),
7.67 (t, 1H, J ) 7.42 Hz, Harom), 4.22-4.18 (m, 1H, CH(CH3)2),
1.38 (d, 6H, J ) 6.00 Hz, CH(CH3)2); 13C NMR (DMSO-d6, δ/ppm)
161.99 (s), 150.13 (s), 147.42 (s), 134.52 (s), 133.53 (d), 130.75
(d), 129.97 (d), 129.76 (s), 125.19 (d), 124.95 (s), 124.38 (d), 123.09
(s), 120.19 (d), 119.60 (d), 117.05 (d), 115.46 (d), 45.18 (d), 21.30
(q, 2C).
0.110 (60%) of light gray powder; mp 198-200 °C; IR (KBr, νmax
/
cm-1) 3100, 1670, 1525; H NMR (DMSO-d6, δ/ppm) 9.31 (bs,
2H, NHamidine), 9.10 (bs, 2H, NHamidine), 8.00 (s, 1H, Harom), 7.63
(d, 1H, J ) 7.80 Hz, Harom) 7.59 (bs, 2H, Harom), 7.49 (d, 1H, J )
7.80 Hz, Harom), 7.32 (t, 1H, J ) 7.60 Hz, Harom), 7.20 (t, 1H, J )
7.60 Hz, Harom), 7.05 (d, 1H, J ) 12.00 Hz, Hethenyl), 6.78 (d, 1H,
J ) 12.60 Hz, Hethenyl); 13C NMR (DMSO-d6, δ/ppm) 166.01 (s),
151.49 (s), 140.99 (s), 134.04 (s), 133.68 (d), 132.70 (s), 131.04
(d, 2C), 129.98 (d), 129.08 (d, 2C), 126.60 (d), 125.65 (s), 122.00
(d), 121.19 (s), 119.44 (d); MS (m/z) 297 (M+1 (- HCl)); Anal.
(C19H20N4Cl2‚H2O) C, H, N.
1
N-Isopropyl-benzimidazo[1,2-a]quinoline-9-carboxamidine Hy-
1
drochloride (40%). H NMR (DMSO-d6, δ/ppm) 9.80 (bs, 1H,
NHamidine), 9.62 (bs, 1H, NHamidine), 9.22 (bs, 1H, NHamidine), 8.95
(d, 1H, J ) 8.60 Hz, Harom), 8.90 (d, 1H, J ) 8.52 Hz, Harom), 8.38
(s, 1H), 8.09 (d, 1H, J ) 7.40 Hz, Harom), 8.05 (d, 1H, J ) 9.60
Hz, Hquinoline), 7.93-7.88 (m, 2H), 7.73 (d, 1H, J ) 9.51 Hz,
Z-2-[2-(2-Chloro-phenyl)-vinyl]-N-morpholin-4-yl-3H-benz-
imidazole-5(6)-carboxamidine Hydrochloride (17). Compound

5708 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Hranjec et al.
Hquinoline), 7.65 (t, 1H, J ) 7.35 Hz, Harom), 4.22-4.18 (m, 1H, CH-
(CH3)2), 1.36 (d, 6H, J ) 6.00 Hz, CH(CH3)2); 13C NMR (DMSO-
d6, δ/ppm) 161.94 (s), 149.44 (s), 143.69 (s), 134.58 (s), 133.88
(d), 133.04 (d), 130.70 (d), 129.99 (s), 128.90 (d), 124.60 (s), 122.46
(s), 122.01 (d), 117.17 (d), 116.29 (d), 115.92 (d), 114.99 (d), 45.17
(d), 21.27 (q, 2C).
(220 mL) was irradiated for 6 h and then worked up as it is
described to give 0.048 g (40%) of yellow powder; (b) a solution
of 9 (0.110 g, 0.26mmol) in ethanol (225 mL) was irradiated for
6 h and then worked up as it is described to give 0.035 g (32%) of
yellow powder; mp 232-234 °C; IR (KBr, υ/cm-1) 3429, 3177,
1634, 1611; MS (m/z) 346 (M+1 (- HCl)); Anal. (C20H20N5OCl)
C, H, N.
N-Morpholin-4-yl-benzimidazo[1,2-a]quinoline-10-carboxa-
midine Hydrochloride (60%). 1H NMR (DMSO-d6, δ/ppm) 11.44
(s, 1H, NHamidine), 10.03 (s, 1H, NHamidine), 9.23 (s, 1H, NHamidine),
9.18 (s, 1H, Harom), 9.05 (d, 1H, J ) 8.55 Hz, Harom), 8.16 (d, 1H,
J ) 9.57 Hz, Hquinoline), 8.14 (d, 1H, J ) 7.44 Hz, Harom), 8.09 (d,
1H, J ) 8.70 Hz, Harom), 7.99-7.90 (m, 2H, Harom), 7.77 (d, 1H, J
) 9.51 Hz, Hquinoline), 7.66 (t, 1H, J ) 7.46 Hz, Harom), 3.70 (bs,
4H, 2CH2morpholine), 3.12 (bs, 4H, 2CH2morpholine); 13C NMR (DMSO-
d6, δ/ppm) 162.00 (s), 150.25 (s), 147.63 (s), 134.53 (s), 133.89
(d), 133.35 (d), 130.77 (d), 130.04 (d), 129.83 (s), 125.35 (d), 124.30
(d), 123.12 (s), 120.25 (d), 119.79 (d), 119.26 (s), 117.08 (d), 65.65
(t, 2C), 54.03 (t, 2C).
2-Benzimidazo[1,2-a]quinolin-10(9)-yl-4,5-dihydro-3H-imida-
zol-1-yl Hydrochloride (19). Compound 19 was prepared using
the general method described for the preparation of 18-21; (a) a
solution of 3 (0.095 g, 0.29 mmol) and iodine (8 mg) in ethanol
(220 mL) was irradiated for 6 h and then worked up as it is
described to give 0.031 g (33%) of light violet powder; (b) a
solution of 7 (0.105 g, 0.29 mmol) in ethanol (220 mL) was
irradiated for 7 h and then worked up as it is described to give
0.040 g (39%) of light violet powder; mp > 300 °C; IR (KBr,
υ/cm-1) 3400, 3200, 3138, 1634, 1602, 1561; MS (m/z) 287 (M+1
(- HCl)); Anal. (C18H15N4Cl) C, H, N.
2-Benzimidazo[1,2-a]quinolin-10-yl-4,5-dihydro-3H-imidazol-
1
1-yl Hydrochloride (60%). H NMR (DMSO-d6, δ/ppm) 11.09
(bs, 2H, Hamidine), 9.40 (s, 1H, Harom), 9.16 (d, 1H, J ) 8.31 Hz,
N-Morpholin-4-yl-benzimidazo[1,2-a]quinoline-9-carboxami-
1
H
arom), 8.15 (d, 1H, J ) 9.24 Hz, Hquinoline), 8.09 (d, 1H, J ) 8.40
dine Hydrochloride (40%). H NMR (DMSO-d6, δ/ppm) 11.31
Hz, Harom), 8.04 (d, 1H, J ) 7.70 Hz, Harom), 8.00-7.91 (m, 2H,
(s, 1H, NHamidine), 9.47 (s, 1H, NHamidine), 9.22 (s, 1H, NHamidine),
9.00 (d, 1H, J ) 8.88 Hz, Harom), 8.92 (d, 1H, J ) 8.58 Hz, Harom),
8.45 (s, 1H), 8.15 (d, 1H, J ) 7.80 Hz, Harom), 8.11 (d, 1H, J )
9.60 Hz, Hquinoline), 7.96-7.92 (m, 2H), 7.75 (d, 1H, J ) 9.57 Hz,
H
arom), 7.75 (d, 1H, J ) 9.48 Hz, Hquinoline), 7.66 (t, 1H, J ) 7.32
Hz, Harom), 4.08 (bs, 4H, CH2imidaz.); 13C NMR (DMSO-d6, δ/ppm)
162. 46 (s),151.28 (s), 148.03 (s), 135.13 (s), 133.98 (d), 133.74
(d), 130.95 (d), 130.16 (d), 129.97 (s), 125.84 (d), 124.24 (d), 123.41
(s), 121.01 (d), 120.13 (d), 119.46 (s), 117.54 (d), 46.03 (t, 2C).
2-Benzimidazo[1,2-a]quinolin-9-yl-4,5-dihydro-3H-imidazol-
H
quinoline), 7.65 (t, 1H, J ) 7.58 Hz, Harom), 3.69 (bs, 2H,
4CH2morpholine), 3.10 (bs, 4H, 2CH2morpholine); 13C NMR (DMSO-d6,
δ/ppm) 161.88 (s), 149.23 (s), 144.09 (s), 134.45 (s), 133.31 (s),
133.00 (d), 130.06 (d), 130.04 (s), 128.85 (d), 123.12 (d), 121.96
(d), 120.26 (s), 118.87 (d), 116.98 (d), 116.32 (d), 115.64 (d), 65.50
(t, 2C), 53.94 (t, 2C).
1
1-yl Hydrochloride (40%). H NMR (DMSO-d6, δ/ppm) 10.83
(bs, 2H, NHamidine), 9.00 (d, 1H, J ) 8.91 Hz, Harom), 8.89 (d, 1H,
J ) 8.43 Hz, Harom), 8.65 (s, 1H), 8.18 (d, 1H, J ) 7.60 Hz, Harom),
8.09 (d, 1H, J ) 9.52 Hz, Hquinoline), 7.97-7.94 (m, 2H), 7.77 (d,
1H, J ) 9,50 Hz, Hquinoline), 7.68 (t, 1H, J ) 7,65 Hz, Harom), 4.06
(bs, 4H, CH2imidaz.); 13C NMR (DMSO-d6, δ/ppm) 161.999 (s),
150.33 (s), 144.09 (s), 134.87 (s), 134.18 (d), 133.00 (d), 131.25
(d), 130.22 (s), 129.19 (d), 124.98 (s), 123.03 (s), 122.24 (d), 118.00
(d), 116.66 (d), 115.99 (d), 115.09 (d), 45.99 (t), 21.27.
Benzimidazo[1,2-a]-9(10)-carboxamidine Hydrochloride (20).
Compound 20 was prepared using the general method described
for the preparation of 18-21; (a) a solution of 4 (0.085 g, 0.29
mmol) and iodine (7 mg) in ethanol (220 mL) was irradiated for 7
h and then worked up as it is described to give 0.035 g (40%) of
light violet powder; (b) a solution of 8 (0.105 g, 0.35mmol) in
ethanol (230 mL) was irradiated for 6 h and then worked up as it
is described to give 0.041 g (40%) of light violet powder; mp 232-
234 °C; IR (KBr, υ/cm-1) 3383, 3122, 1639, 1609, 1534; MS (m/
z) 261 (M+ (- HCl)); Anal. (C16H13N4Cl) C, H, N.
Benzimidazo[1,2-a]quinoline-10-carboxamidine Hydrochlo-
ride (60%). 1H NMR (DMSO-d6, δ/ppm) 9.65 (bs, 2H, NHamidine),
9.31 (bs, 2H, NHamidine), 9.17 (s, 1H, Harom), 9.06 (d, 1H, J ) 8.49
Hz, Harom), 8.13 (d, 1H, J ) 9.21 Hz, Hquinoline), 8.06 (d, 1H, J )
8.70 Hz, Harom), 8.04 (d, 1H, J ) 7.80 Hz, Harom), 7.99-7.91 (m,
2H, Harom), 7.76 (d, 1H, J ) 9.48 Hz, Hquinoline), 7.66 (t, 1H, J )
7.42 Hz, Harom); 13C NMR (DMSO-d6, δ/ppm) 162.55 (s), 150.76
(s), 147.45 (s), 135.05 (s), 133.70 (d), 130.98 (d), 130.97 (d), 129.86
(s), 126.99 (d), 126.08 (d), 124.56 (s), 120.86 (d), 119.09 (s), 119.70
(d), 115.99 (d), 115.38 (d).
Benzimidazo[1,2-a]quinoline-9-carboxamidine Hydrochloride
(40%). 1H NMR (DMSO-d6, δ/ppm) 9.57 (bs, 2H, NHamidine), 9.30
(bs, 2H, NHamidine), 8.97 (d, 1H, J ) 8.79 Hz, Harom), 8.78 (d, 1H,
J ) 8.46 Hz, Harom), 8.49 (s, 1H), 8.08 (d, 1H, J ) 7.50 Hz, Harom),
8.03 (d, 1H, J ) 9.48 Hz, Hquinoline), 7.98-7.96 (m, 2H), 7.73 (d,
1H, J ) 9,51 Hz, Hquinoline), 7.64 (t, 1H, J ) 7,60 Hz, Harom); 13C
NMR (DMSO-d6, δ/ppm) 162.33 (s), 149.88 (s), 143.97 (s), 134.02
(s), 133.99 (d), 133.20 (d), 131.09 (d), 130.11 (s), 129.22 (d), 124.33
(s), 121.99 (s), 121.89 (d), 118.03 (d), 117.67 (d), 116.70 (d), 115.10
(d).
General Method for the Synthesis of E-5(6)-Cyano-substituted-
E-2-styryl-1H-benzimidazole (24) and E-5(6)-Cyano-substituted-
E-2-[2-(2-chloro-phenyl)-vinyl]-1H-benzimidazole (25). A mix-
ture of corresponding benzaldehydes 22a,b and 5(6)-cyano-2-
methylbenzimidazole 23 in acetic anhydride was stirred at refluxed
for 3 h. The reaction mixture was cooled to room temperature and
24 mL of i-propanole and a solution of 1.434 g of oxalic acid in 9
mL of i-propanole were added, and the resulting product was filtered
off and washed with water. Crude product was suspended in water,
and aqueous sodium hydroxide was added until pH ) 10. The
resulting product was filtered off and washed with water. After
recrystallization from ethanol, a light yellow powder was obtained.
2-Styryl-3H-benzimidazole-6-carbonitrile (24). From 3.6 mL
(35.70 mmol) of 22a and 1.50 g (9.50 mmol) of 23 in 10 mL of
acetic anhydride was obtained 1.16 g (48%) of light yellow powder;
mp 107-108 °C; IR (KBr, υ/cm-1) 3193, 2224, 1646, 1614, 1518;
1H NMR (DMSO-d6, δ/ppm) 13.10 (bs, 1H, NHbenzimidazole), 8.06
(s, 1H, Harom), 7.77 (d, 1H, J ) 16.53 Hz, Hethenyl), 7.71 (d, 2H, J
) 8.19 Hz, Harom), 7.70 (d, 1H, J ) 8.29 Hz, Harom), 7.56 (dd, 1H,
J ) 8.31 Hz, J ) 8.34 Hz, Harom), 7.46-7.39 (m, 3H, Harom), 7.30
(d, 1H, J ) 16.50 Hz, Hethenyl); 13C NMR (DMSO-d6, δ/ppm) 162.15
(s), 150.71 (s), 139.20 (s), 133.90 (s), 133.26 (s), 130.84 (d), 130.39
(d), 130.19 (d), 128.37 (d, 2C), 127.62 (d, 2C), 123.08 (d), 120.74
(d), 115.43 (d); MS (m/z) 246 (M+1); Anal. (C16H11N3) C, H, N.
2-[2-(2-Chloro-phenyl)-vinyl]-3H-benzimidazole-5(6)-carbo-
nitrile (25). From 3.1 mL (35.72 mmol) of 22b and 1.50 g (9.50
mmol) of 23 in 10 mL acetic anhydride, to obtained 1.01 g (49%)
of gray powder; mp 117-119 °C; IR (KBr, υ/cm-1) 3194, 2222,
1
1645, 1617, 1520; H NMR (DMSO-d6, δ/ppm) 13.18 (bs, 1H,
NHbenzimidazole), 8.16 (d, 1H, J ) 16.47 Hz, Hethenyl), 7.96 (s, 1H,
Harom), 7.87 (dd, 1H, J ) 8.38 Hz, J ) 8.54 Hz, Harom), 7.67 (bs,
1H, Harom), 7.60 (dd, 1H, J ) 8.46 Hz, J ) 8.31 Hz, Harom), 7.50-
7.47 (m, 1H, Harom), 7.40-7.36 (m, 2H, Harom), 7.20 (d, 1H, J )
16.53 Hz, Hethenyl); 13C NMR (DMSO-d6, δ/ppm) 162.35 (s), 150.58
(s), 138.23 (s), 135.32 (s), 135.30 (d), 129.04 (d), 128.96 (d, 2C),
127.10 (d, 2C), 122.51 (d), 116.74 (d), 113.85 (d); MS (m/z) 280
(M+1); Anal. (C16H10N3Cl) C, H, N.
N-Morpholin-4-yl-benzimidazo[1,2-a]quinoline-9(10)-carbox-
amidine Hydrochloride (21). Compound 21 was prepared using
the general method described for the preparation of 18-21; (a) a
solution of 5 (0.110 g, 0.29 mmol) and iodine (9 mg) in ethanol
Cyano-Substituted-Benzimidazo[1,2-a]quinolines (26-27). A
solution of 5(6)-cyano-substituted E-2-styryl-1H-benzimidazole 24
and a small amount of iodine (5%) in ethanol (c ) 1.3 × 10-3 mol

Synthesis of Styryl-2-benzimidazole DerViatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5709
dm-3) and a solution of chloro-substituted E-5(6)-cyano-2-styryl-
1H-benzimidazole 25 in ethanol (c ) 1.3 × 10-3 mol dm-3) were
irradiated at room temperature with 400 W, high-pressure, mercury
lamp using a Pyrex filter for about 6-10 h, until the UV spectra
showed that the reactions of photochemical dehydrocyclization and
dehydrohalogenation were completed. The air was bubbled through
the solution. The solutions were concentrated under reduced
pressure, and the mixture of 9(10)-structural isomers was obtained.
Isomers 26 and 27 were separated by column chromatography.
1-3 days at room temperature or reflux. The crude product was
then filtered off and washed with diethylether.
N-Isopropyl-benzimidazo[1,2-a]quinoline-2-carboxamidine Hy-
drochloride (32). Compound 32 was prepared using the general
method described for the preparation of 32-35; from 0.25 g (1.00
mmol) 31 and 0.25 mL (3.07 mmol) of iso-propylamine to give
0.16 g (46%) of green powder; mp 261-263 °C; IR (KBr, υ/cm-1
)
1
3400, 3344, 3011, 1670, 1640, 1618, 1543; H NMR (DMSO-d6,
δ/ppm) 9.92 (bs, 1H, NHamidine), 9.80 (bs, 1H, NHamidine), 9.36 (bs,
1H, NHamidine), 8.98 (s, 1H, Harom), 8.81 (d, 1H, J ) 9.00 Hz, Harom),
8.28 (d, 1H, J ) 8.22 Hz, Harom), 8.07 (d, 1H, J ) 9.57 Hz,
Benzimidazo[1,2-a]quinoline-10-carbonitrile (26). Yield 0.09
g (20%) of yellow powder after separation by column chromatog-
raphy (petrol ether/ethyl acetate ) 1:1); mp 184-185 °C; IR (KBr,
Hquinoline), 8.00 (d, 1H, J ) 9.0 Hz, Harom), 7.89 (d, 1H, J ) 8.60
1
υ/cm-1) 3446, 2224, 1637, 1609, 1595, 1542; H NMR (DMSO-
Hz, Harom), 7.84 (d, 1H, J ) 9.51 Hz, Hquinoline), 7.64-7.59 (m,
2H, Harom), 4.20-4.16 (m, 1H, CH(CH3)2), 1.38 (d, 6H, J ) 6.39
Hz, CH(CH3)2); 13C NMR (DMSO-d6, δ/ppm) 161.90 (s), 147.89
(s), 144.74 (s), 134.67 (s), 131.23 (d), 130.67 (s), 130.43 (s), 130.37
(d), 126.57 (s), 125.31 (d), 124.60 (d), 123.63 (d), 120.57 (d), 120.46
(d), 116.11 (d), 115.66 (d), 45.87 (d), 21.77 (d, 2C); MS (m/z) 303
(M+1 (- HCl)); Anal. (C19H19N4Cl) C, H, N.
d6, δ/ppm) 9.26 (s, 1H), 8.88 (d, 1H, J ) 8.46 Hz, Harom), 8.06 (d,
1H, J ) 9.42 Hz, Hquinoline), 8.05 (d, 1H, J ) 7.62 Hz, Harom), 7.98
(d, 1H, J ) 8.44 Hz, Harom), 7.84 (dd, 1H, J ) 8.40 Hz, J ) 8.42
Hz, Harom), 7.80 (t, 1H, J ) 7.20 Hz, Harom), 7.66 (d, 1H, J ) 9.42
Hz, Hquinoline), 7.58 (t, 1H, J ) 7.28 Hz, Harom); 13C NMR (DMSO-
d6, δ/ppm) 150.28 (s), 147.05 (s), 134.35 (s), 133.90 (d), 130.90
(d), 129.92 (d), 129.86 (s), 127.59 (d), 125.20 (d), 123.03 (s), 120.59
(d), 119.88 (s), 119.70 (d), 116.90 (d), 116.30 (d), 103.92 (s); MS
(m/z) 244 (M+1); Anal. (C16H9N3) C, H, N.
2-Benzimidazo[1,2-a]quinolin-2-yl-4,5-dihydro-3H-imidazol-
1-yl Hydrochloride (33). Compound 33 was prepared using the
general method described for the preparation of 32-35; from 0.25
g (1.00 mmol) 31 and 0.24 mL (3.50 mmol) of ethylenediamine to
give 0.07 g (21%) of yellow powder; mp 225-227 °C; IR (KBr,
Benzimidazo[1,2-a]quinoline-9-carbonitrile (27). Yield 0.06 g
(12%) of yellow powder after separation by column chromatography
(petrol ether/ethyl acetate ) 1:1); mp 188-189 °C; IR (KBr,
υ/cm-1) 3442, 2220, 1630, 1605, 1595; 1H NMR (DMSO-d6,
δ/ppm) 8.83 (d, 1H, J ) 8.56 Hz, Harom), 8.45 (d, 1H, J ) 8.42
Hz, Harom), 8.38 (s, 1H), 8.01 (d, 1H, J ) 7.62 Hz, Harom), 7.98 (d,
1H, J ) 9.48 Hz, Hquinoline), 7.81 (d, 1H, J ) 7.64 Hz, Harom), 7.79
(t, 1H, J ) 7.40 Hz, Harom), 7.62 (d, 1H, J ) 9.36 Hz, Hquinoline),
7.57 (t, 1H, J ) 7.38 Hz, Harom); MS (m/z) 244 (M+1); Anal.
(C16H9N3) C, H, N.
4-[2-(1H-Benzimidazol-2-yl]-benzonitrile (30). Heating a mix-
ture of equimolar amounts (37.81 mmol) of 4-cyanobenzaldehyde
28 and 2-methylbenzimidazole 29 in a tube at 200 °C and
recrystallization from methanol gave 6.60 g (71%) of yellow
powder; mp 220-221 °C; IR (KBr, υ/cm-1) 3311, 2227, 1645; 1H
NMR (DMSO-d6, δ/ppm) 12.77 (s, 1H, NHbenzimidazole), 7.91 (d, 2H,
J ) 7.80 Hz, Harom), 7.89 (bs, 2H, Harom), 7.72 (d, 1H, J ) 16.44
Hz, Hethenyl), 7.64 (d, 1H, J ) 7.20 Hz, Harom), 7.52 (d, 1H, J )
7.10 Hz, Harom), 7.42 (d, 1H, J ) 16.47 Hz, Hethenyl), 7.22-7.19
(m, 2H, Harom); 13C NMR (DMSO-d6, δ/ppm) 161.43 (s), 150.66
(s), 140.90 (s), 133.28 (d, 2C), 132.82 (d), 131.15 (s), 130.45 (s),
128.17 (d, 2C), 123.42 (d), 122.29 (d), 121.71 (d), 119.38 (d),
119.32 (s), 114.75 (d); MS (m/z) 246 (M+1); Anal. (C16H11N3) C,
H, N.
1
υ/cm-1) 3434, 3188, 1667, 1621, 1597, 1539; H NMR (DMSO-
d6, δ/ppm) 10.46 (bs, 2H, NHamidine), 8.96 (s, 1H, Harom), 8.86 (d,
1H, J ) 8.65 Hz, Harom), 8.26 (d, 1H, J ) 8.25 Hz, Harom), 8.02 (d,
1H, J ) 9.36 Hz, Hquinoline), 8.03-7.95 (m, 2H, Harom), 7.82 (d,
1H, J ) 9.45 Hz, Hquinoline), 7.60 (d, 1H, J ) 7.62 Hz, Harom), 7.58
(d, 1H, J ) 7.47 Hz, Harom), 4.10 (bs, 2H, CH2), 3.06 (bs, 2H,
CH2); 13C NMR (DMSO-d6, δ/ppm) 164.97 (s), 147.80 (s), 144.50
(s), 134.90 (s), 131.24 (d), 131.02 (d), 130.51 (s), 127.65 (s), 127.65
(s), 125.53 (d), 124.22 (d), 123.87 (d), 123.260 (s), 121.03 (d),
120.52 (d), 115.61 (d), 115.56 (d), 45.11 (t), 36.91 (t); MS (m/z)
287 (M+1 (- HCl)); Anal. (C18H15N4Cl) C, H, N.
Benzimidazo[1,2-a]quinoline-2-carboxamidine Hydrochloride
(34). Compound 34 was prepared using the general method
described for the preparation of 32-35; from 0.25 g (1.00 mmol)
31 and NH3(g) to give 0.11 g (36%) of light yellow powder; mp
276-278 °C; IR (KBr, υ/cm-1) 3130, 1691, 1628, 1517; 1H NMR
(DMSO-d6, δ/ppm) 9.20 (bs, 2H, NHamidine), 9.16 (bs, 2H, NHamidine),
8.93 (s, 1H, Harom), 8.72 (d, 1H, J ) 8.94 Hz, Harom), 8.19 (d, 1H,
J ) 8.10 Hz, Harom), 7.97 (d, 1H, J ) 9.48 Hz, Hquinoline), 7.90 (d,
1H, J ) 8.24 Hz, Harom), 7.87 (d, 1H, J ) 8.76 Hz, Harom), 7.84 (d,
1H, J ) 9.42 Hz, Hquinoline), 7.53-7.50 (m, 2H, Harom); 13C NMR
(DMSO-d6, δ/ppm) 164.86 (s), 147.41 (s), 144.27 (s), 134.38 (s),
130.69 (d), 130.21 (s), 130.14 (s), 129.99 (d), 126.27 (s), 124.75
(d), 123.52 (d), 123.07 (d), 120.05 (d), 119.97 (d), 115.13 (d),
115.00 (d); MS (m/z) 261 (M+1 (- HCl)); Anal. (C16H13N4Cl) C,
H, N.
N-Morpholin-4-yl-benzimidazo[1,2-a]quinoline-2-carboxami-
dine Hydrochloride (35). Compound 35 was prepared using the
general method described for the preparation of 32-35; from 0.25
g (1,00 mmol) 31 and 0.27 mL (2.80 mmol) of 4-N-aminomor-
pholine to give 0.14 g (37%) of light green powder; mp 268-
270 °C; IR (KBr, υ/cm-1) 3421, 2866, 1670, 1632, 1619, 1525;
1H NMR (DMSO-d6, δ/ppm) 11.61 (bs, 1H, NHamidine), 9.90 (bs,
1H, NHamidine), 9.23 (bs, 1H, NHamidine), 9.03 (s, 1H, Harom), 8.85
(d, 1H, J ) 8.92 Hz, Harom), 8.30 (d, 1H, J ) 8.22 Hz, Harom), 8.06
(d, 1H, J ) 9.60 Hz, Hquinoline), 7.97 (m, 1H, Harom), 7.90 (d, 1H, J
) 8.16 Hz, Harom), 7.84 (d, 1H, J ) 9.51 Hz, Hquinoline), 7.62-7.58
(m, 2H, Harom); 13C NMR (DMSO-d6, δ/ppm) 162.03 (s), 147.85
(s), 144.70 (s), 134.72 (s), 131.22 (d), 130.64 (s), 130.49 (d), 127.67
(s), 126.99 (s), 125.37 (d), 124.52 (d), 123.63 (d), 120.71 (d), 120.59
(d), 116.27 (d), 115.69 (d); MS (m/z) 346 (M+1 (- HCl)); Anal.
(C20H20N5OCl) C, H, N.
Benzimidazo[1,2-a]quinoline-2-carbonitrile (31). Irradiation of
a solution of 0.23 g (0.94 mmol) 30 in ethanol (c ) 1.3 × 10-3
mol dm-3) with a 400 W, high-pressure, mercury lamp using a
Pyrex filter, at room temperature for 12 h, gave 0.14 g (61%) of
yellow powder; mp 210-212 °C; IR (KBr, υ/cm-1) 3438, 2231,
1631, 1609, 1545; 1H NMR (DMSO-d6, δ/ppm) 9.18 (s, 1H, Harom),
8.90 (d, 1H, J ) 8.55 Hz, Harom), 8.25 (d, 1H, J ) 8.20 Hz, Harom),
8.04 (d, 1H, J ) 9.54 Hz, Hquinoline), 7.98 (d, 1H, J ) 8.01 Hz,
Harom), 7.95 (d, 1H, J ) 8.80 Hz, Harom), 7.84 (d, 1H, J ) 9.54 Hz,
H
quinoline), 7.60-7.54 (m, 2H, Harom); 13C NMR (DMSO-d6, δ/ppm)
153.26 (s), 145.59 (s), 132.38 (d), 131.09 (d), 129.43 (d), 127.93
(d), 126.83 (s), 125.20 (d), 123.87 (d), 120.52 (d), 119.68 (d), 119.22
(s), 118.97 (s), 115.52 (d), 112.40 (s), 109.52 (s); MS (m/z) 244
(M+1); Anal. (C16H9N3) C, H, N.
General Method for Preparation 2-Amidino-Substituted
Benzimidazo[1,2-a]quinolines (32-35). A stirred suspension of
31 in absolute ethanol 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 3 days until
the nitrile band disappeared (monitored by IR analysis at 2200
cm-1). The reaction mixture was purged with N2 gas and diluted
with diethylether (50 mL). The crude imidate was filtered off and
was immediately suspended in absolute ethanol (10 mL). The
corresponding amine was added, and the mixture was stirred for
Interactions with DNA. The electronic absorption spectra were
recorded on Varian Cary 100 Bio spectrometer, CD spectra were
recorded on Jasco J815, and fluorescence emission spectra were
recorded on Varian Eclipse fluorimeter in all cases using quartz
cuvettes (1 cm).

5710 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Hranjec et al.
The calf thymus DNA (ct-DNA) was purchased from Aldrich,
dissolved in the sodium cacodylate buffer, I ) 0.05 mol dm-3, pH
) 7.0, additionally sonicated and filtered through a 0.45 µm filter,
and the concentration of the corresponding solution determined
spectroscopically as the concentration of phosphates.45 The mea-
surements were performed in the aqueous buffer solution (pH )
7.0; sodium cacodylate buffer, I ) 0.05 mol dm-3). Under the
experimental conditions used, the absorbance and fluorescence
intensities of studied compounds were proportional to their
concentrations, while none of studied compounds showed CD
spectrum. In fluorimetric titrations, an excitation wavelength of λ
) 355 nm was used to avoid inner filter effects caused by absorption
of excitation light by added polynucleotide and changes of
fluorescence emission were monitored at 400 nm. Spectroscopic
titrations were performed by adding portions of polynucleotide
solution into the solution of the studied compound.
The stability constant (Ks) and [bound compound]/[polynucle-
otide phosphate] ratio (n) were calculated according to the Scatchard
equation by nonlinear least-square fitting,36 giving excellent cor-
relation coefficients (>0.999) for obtained values for Ks and n.
Thermal melting 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.45b 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
method. Given ∆Tm values were calculated subtracting Tm of the
free nucleic acid from Tm of complex. Every ∆Tm value here
reported was the average of at least two measurements, the error
in ∆Tm is (0.5 °C.
Antitumor Activity Assays. Antiproliferative Activity. The
HeLa (cervical carcinoma), Hep-2 (laryngeal carcinoma), MCF-7
(breast carcinoma), SW620 (colon carcinoma), MiaPaCa-2 (pan-
creatic carcinoma), and H460 (lung carcinoma) cells obtained from
American Type Culture Collection (ATCC, Rockville, MD) were
cultured as monolayers and maintained in Dulbecco’s modified
Eagle’s medium (DMEM), supplemented with 10% fetal bovine
serum (FBS), 2 mM l-glutamine, 100 U/mL penicillin, and 100
µg/mL streptomycin in a humidified atmosphere with 5% CO2 at
37 °C. The growth inhibition activity was assessed as described
previously, according to the slightly modified procedure of the
NationalCancerInstitute,DevelopmentalTherapeuticsProgram.39-41,46
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): 1 × 104/mL for HeLa, Hep-
2, H460, MiaPaCa-2, and SW620 cell lines (PDT ) 20-24 h) and
2 × 104/mL for MCF-7 cell lines (PDT ) 33 h). Test agents were
then added in five 10-fold dilutions (10-8 to 10-4 mol/L) and
incubated for an additional 72 h. Working dilutions were freshly
prepared on the day of testing. After 72 h of incubation, the cell
growth rate was evaluated by performing the MTT assay, which
detects dehydrogenase activity in viable cells. The absorbance (OD,
optical density) was measured on a microplate reader at 570 nm.
The percentage of growth (PG) of the cell lines was calculated
according to one or the other of the following two expressions:
If (mean ODtest - mean ODtzero) g 0, then PG ) 100 × (mean
ODtest - mean ODtzero)/(mean ODctrl - mean ODtzero).
If, however, for a given cell line all of the tested concentrations
produce PGs exceeding the respective reference level of effect (e.g.,
PG value of 50), then the highest tested concentration is assigned
as the default value, which is preceded by a “>” sign. Each result
is a mean value from three separate experiments.
Cell Cycle Analysis. Cells (2 × 105) were seeded per well in a
6-well plate. After overnight incubation, tested compounds were
added. After the desired length of time, the attached cells were
trypsinized, combined with floating cells, washed with phosphate-
buffered saline (PBS), and fixed with 70% ethanol. Immediately
before the analysis, cells were washed with PBS and incubated with
0.1 µg/µl RNAse A at 37 °C for 15 min. Subsequently, cells were
stained with 1 µg/mL of propidium iodide (PI) and analyzed by
Becton Dickinson FACScalibur flow cytometer. For each analysis,
20 000 events were measured and obtained results were processed
using WinMDI 2.8 (The Scripps Institute, U.S.A.) and Cylchred
(Cardiff University, U.K.). Measurements were performed in
duplicate for two independent experiments.
In Vitro Topoisomerase II Inhibitor Screening. A topoi-
somerase II drug screening kit (TopoGEN, U.S.A.) was used to
check selected compounds for their inhibitory effect on topo II,
whereby two possible modes of inhibition can be detected:
stabilization of cleavage complexes that prevents religation of the
broken DNA strands and catalytic inhibition (topo II antagonists).
For evaluation of the cleavage complex formation, the reaction
mixture contained 2 µL 10× cleavage buffer, 1 µL pRYG DNA
substrate (supercoiled), 2 µL of 1 mM test compound or control
inhibitor (etoposide), 8 units of purified topoisomerase II, and mQ
H2O (variable volume, up to 20 µL). The mixture was incubated
at 37 °C for 30 min, and the reaction was terminated by the addition
of 2 µL of 10% SDS, followed by proteinase K (50 µg/mL)
digestion at 37 °C for 15 min. After extraction with a mixture of
chloroform and isoamyl alcohol (24:1), samples were loaded onto
1% agarose gel containing 0.5 µg/mL ethidium bromide along with
the marker DNAs (linear and supercoiled pRYG DNAs). Gels were
run at 40 V constant voltage in horizontal electrophoresis system
(BIO-RAD, U.S.A.). Formation of cleavage products (linear and
open circular DNA) was monitored under the UV light at 254 nm
(Image Master VDS, Pharmacia Biotech, Sweden). For establishing
the effect of test compounds on the topo II relaxation activity, we
used the same reaction system and conditions as described above,
except that the cleavage buffer was replaced with 10× assay buffer
and that ethidium bromide was omitted from the gel. Instead, gels
were stained with ethidium bromide (0,5 µg/mL) for 30 min once
electrophoresis was completed. Monitoring of the supercoiled DNA
conversion into relaxed DNA forms was possible in the assay
reaction.
Acknowledgment. Support for this study by the Ministry
of Science, Education and Sport of Croatia is gratefully
acknowledged (Projects 125-0982464-1356, 098-0982464-2514,
98-0982914-2918, 098-0982464-2393).
Supporting Information Available: Elemental analyses. This
material is available free of charge via the Internet at http://
pubs.acs.org.
If (mean ODtest - mean ODtzero) < 0, then PG ) 100 × (mean
ODtest - mean ODtzero)/ODtzero, where mean ODtzero ) the average
of optical density measurements before exposure of cells to the
test compound; mean ODtest ) the average of optical density
measurements after the desired period; and mean ODctrl ) the
average of optical density measurements after the desired period
with no exposure of cells to the test compound.
Each test was performed in quadruplicate in 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 concentration-response curves
using linear regression analysis by fitting the test concentrations
that give PG values above and below the reference value (i.e., 50%).
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