Novel amidino-substituted thienyl- and furylvinylbenzimidazole: Derivatives and their photochemical conversion into corresponding diazacyclopenta[c] fluorenes. Synthesis, interactions with DNA and RNA, and antitumor evaluation

Article abstract of DOI:10.1021/jm8000423

Synthesis of novel nonfused amidino-substituted thienyl- and furylvinylbenzimidazole: derivatives and their photochemical cyclization into corresponding diazacyclopenta[c]fluorenes is described. All studied compounds showed prominent growth inhibitory eff

Full text of DOI:10.1021/jm8000423

J. Med. Chem. 2008, 51, 4899–4910
4899
Novel Amidino-Substituted Thienyl- and Furylvinylbenzimidazole: Derivatives and Their
Photochemical Conversion into Corresponding Diazacyclopenta[c]fluorenes. Synthesis,
Interactions with DNA and RNA, and Antitumor Evaluation. 4
§
§
§
,†
Marijana Hranjec,† Ivo Piantanida,*,‡ Marijeta Kralj, Lidija Suman, Kresˇimir Pavelic´, and Grace Karminski-Zamola*
ˇ
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, UniVersity of Zagreb, Marulic´eV trg 20, P.O. Box 177,
HR-10000 Zagreb, Croatia, DiVision of Organic Chemistry and Biochemistry, “Rudjer BosˇkoVic´” Institute, P.O. Box 180, HR-10002 Zagreb,
Croatia, and DiVision of Molecular Medicine, “Rudjer BosˇkoVic´” Institute, Bijenicˇka cesta 54, P.O. Box 180, HR-10000 Zagreb, Croatia
ReceiVed January 17, 2008
Synthesis of novel nonfused amidino-substituted thienyl- and furylvinylbenzimidazole: derivatives and their
photochemical cyclization into corresponding diazacyclopenta[c]fluorenes is described. All studied compounds
showed prominent growth inhibitory effect. The fused compounds showed stronger activity than nonfused
ones, whereby imidazolyl-substituted compound 11 proved to be the most active one. Besides, it induced
strong G2/M arrest of the cell cycle followed by drastic apoptosis, which is in accordance with the DNA
intercalative binding mode determined by the spectroscopic studies. Nonfused derivatives induced strong S
phase arrest of the cell cycle followed by apoptosis that together with DNA minor groove binding mode
pointed to topoisomerase I inhibition. In addition, all nonfused compounds revealed pronounced selectivity
toward tumor cells in comparison with nontumor cells. On the basis of the presented results, both nonfused
and fused thiophene-containing imidazolyl derivatives should be considered as promising lead compounds
for further investigation.
Introduction
complex stability between the dications and the DNA through
H-bonding and electrostatic interactions.1,6 However, quite often
diarylamidines aggregate within the DNA grooves; for example,
phenylfuranbenzimidazole diamidine binds to AT-rich sequences
as a monomer while to GC-rich sequences bind as dimers.28
Pentamidine, the minor groove binding agent, has been suc-
cessfully used in the clinic treatment of human parasitic
diseases.29 Phenylthiophenebenzimidazole diamidines showed
10-fold increased affinity for the minor groove binding at AT
sequences.30
As a part of our medicinal chemistry project aimed at the
synthesis of potential anticancer agents, we have prepared
amidino-substituted styryl-2-benzimidazoles and their fused
benzimidazo[1,2-a]quinoline derivatives.31 The most active
imidazolyl-substituted fused derivative inhibited topoisomerase
II and induced strong G2/M cell cycle arrest, pointing to the
impairment in mitotic progression. Its pronounced selectivity
toward colon carcinoma cells encouraged further development
of this compound as a lead. The nonfused derivatives showed
a different interaction with DNA and pronounced selectivity
toward tumor cells in regard to normal cells. These results
prompted us to synthesize heterocyclic furyl- and thienyl-
substituted analogues of the above-mentioned compounds. In
this work we present the synthesis of E-N-substituted-2-(2-
thiophen-2-ylvinyl)-3H-benzimidazole-5(6)-carboxamidine hy-
drochlorides 2-5, E-2-(2-furan-2-ylvinyl)-N-substituted-3H-
benzimidazole-5(6)-carboxamidine hydrochlorides 6-9, and
N-substituted 3-thia-6,10b-diazacyclopenta[c]fluorene-8(9)-car-
boxamidine hydrochlorides 10-13 (Figure 1), detailed study
of their interactions with DNA/RNA, and antiproliferative
activity on a panel of tumor cell lines.
In spite of great advances in cancer therapy, there has been
tremendous growth in recent years in the number and types of
new anticancer agents, with an emphasis on creating new DNA-
active drugs.1–3 Most of the presently used anticancer drugs
interact with DNA noncovalently by two main binding modes:
(a) DNA minor groove binding through a combination of
hydrophobic, electrostatic, and hydrogen bonding interactions
and (b) intercalative binding in which a planar aromatic moiety
slides between the DNA base pairs.4–6 The interactions of
chemotherapeutics with the DNA can cause changes in the
physiological functions of DNA, and therefore, understanding
the interactions of small molecules with DNA is of significance
in the rational design of more powerful and selective anticancer
agents.7,8
The benzimidazole unit is the key building block for a variety
of derivatives that are known to play crucial roles in the
functions of a number of biologically important molecules.9 In
addition, the constant and growing interest over the past few
years for the synthesis and biological studies of substituted
benzimidazoles and their azino fused derivatives is owed partly
to their well-known biological activities such as anticancer,10–14
antimicrobial,15–17 antiviral,10,18–20 antihistaminic,21,22 and an-
tifungal activities.23,24
Furthermore, amidines are structural parts of numerous
compounds of biological interest including important medical
and biochemical agents.6,25–27 The amidine groups at the termini
of the molecule seemed to contribute significantly to the
* To whom correspondence should be addressed. For I.P.: phone, +385
1 4561 111/1306; fax, +3851 4680 195; e-mail, pianta@irb.hr. For G.K.-
Z.: phone, ++38514597215; fax, ++38514597250; e-mail, gzamola@
pierre.fkit.hr.
Synthesis
†
University of Zagreb.
Division of Organic Chemistry and Biochemistry, “Rudjer Bosˇkovic´”
Novel E-N-substituted 2-(2-thiophen-2-ylvinyl)-3H-benzimi-
dazole-5(6)-carboxamidines hydrochlorides 2-5 and E-2-(2-
furan-2-ylvinyl)-N-substituted-3H-benzimidazole-5(6)-carbox-
‡
Institute.
§
Division of Molecular Medicine, “Rudjer Bosˇkovic´” Institute.
10.1021/jm8000423 CCC: $40.75
2008 American Chemical Society
Published on Web 07/25/2008

4900 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Hranjec et al.
Figure 1. Structures of the studied compounds.
Scheme 1. Synthetic Procedures for the Amidino-Substituted
E-2-(2-Thiophen-2-ylvinyl)-3H-benzimidazoles 2-5,
E-2-(2-Furan-2-ylvinyl)-3H-benzimidazoles 6-9, and
3-Thia-6,10b-diazacyclopenta[c]fluorenes 10-13
methods. Novel N-substituted-3-thia-6,10b-diaza-cyclopenta[c]-
fluorene-8(9)-carboxamidine hydrochlorides 10-13 were pre-
pared by photochemical dehydrocyclization of ethanolic solution
(c ) 1.3 × 10-3 mol dm-3) of E-N-substituted-2-(2-thiophen-
2-ylvinyl)-3H-benzimidazole-5(6)-carboxamidines hydrochlo-
rides 2-5 in 67-80% yield as a mixture of two inseparable
structure isomers. Photochemical dehydrocyclization of amidino-
substituted E-2-(2-furan-2-ylvinyl)-3H-benzimidazoles 6-9 did
not give the desired fused products.
All structures of novel nonfused 2-9 and fused derivatives
10-13 were determined by NMR analysis, based on the analysis
1
of H-H coupling constants as well as chemical shifts. In H
NMR spectra of all nonfused derivatives 2-9, two doublets for
trans-ethylenic protons with coupling constants of ∼16 Hz can
be observed. The photocyclization reaction leads to a downfield
shift of the signal of the ethylenic proton and most other protons.
Proton of NH group of benzimidazole ring and one proton of
thiophene ring disappeared, thus confirming fused structure
formation. Doublets for two protons on fluorene ring, with
coupling constants of ∼9.5 Hz, can be observed, and these
values are characteristic for this type of fused compound.
Spectroscopic Properties of Studied Compounds
Compounds studied in this paper could be divided in two
groups: “non-fused” derivatives (Figure 1, 2-9) and their
“fused” photoproducts (Figure 1, 10-13). Because of the
structural similarity, we have chosen to study compounds 3, 4,
and 8 as representatives of the “nonfused” derivatives and
compounds 11 and 12 as representatives of “fused” derivatives.
Since for DNA binding studies of the above-mentioned com-
pounds application of spectrophotometric methods was neces-
sary, we have characterized their aqueous solutions by means
of electronic absorption (UV/vis) and fluorescence emission
spectra (Table 1).
Absorbencies of aqueous solutions of compounds are pro-
portional to their concentrations in the range 2 × 10-6 to 2 ×
10-5 mol dm-3 and also did not change significantly with a
temperature increase up to 90 °C. These observations indicated
that there is no significant intermolecular stacking, which should
give rise to hypochromicity effects. Aqueous solutions of all
studied compounds were shown to be stable over a longer
period. The “nonfused” derivatives 3, 4, and 8 exhibited rather
amidines hydrochlorides 6-9 were prepared by the condensation
reaction of 3-thiophen-2-ylpropenal and 3-furan-2-ylpropenal
1a,b with the corresponding, earlier prepared 4-N-amidino-
substituted 1,2-phenylenediamines and p-benzoquinone in
50-88% yield (Scheme 1). 4-N-Amidino-substituted 1,2-
phenylenediamines were prepared from cyano-substituted de-
rivatives in the Pinner reaction, using the earlier described

Benzimidazole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4901
Table 1. Electronic Absorption Maxima and Corresponding Molar
Extinction Coefficients of Studied Compounds in Water, Fluorescence
Emission Maxima, and Corresponding Relative Quantum Yield (Q)a
to originate from the loss of mobility around the methine bridge
connecting the quinoline and benzoxazole, respectively, and
benzothiazole moieties due to the constrictive DNA environ-
ment. In the free form, isomerization around this bridge is an
important nonradiative decay channel of the photoexcited dye
molecule, whereas upon intercalation, large amplitude motion
of the probe is strongly hindered.34 Only fluorescence of 4 was
similarly enhanced by both polyA-polyU and polyG-polyC,
while for all other compounds (3, 8, 11, 12) fluorescence change
(enhancement or quenching) was significantly more pronounced
upon addition of polyA- polyU than polyG-polyC (Table 3).
Processing of the fluorimetric titration data by Scatchard
equation33 gave binding constants (log K values) (Table 3).
Obtained binding constants depend significantly on the com-
pound structure, as well as on the base pair type in the
polynucleotide. The affinity of “fused” derivatives (11, 12)
toward ct-DNA is somewhat higher than the affinity of “non-
fused” analogues (3, 4, 8), but this preference is not obvious
for the ds-RNA polynucleotides. Furthermore, fluorimetric
titrations of some “nonfused” analogues with polyG-polyC (3)
and with both ds-RNAs (8) pointed toward simultaneous
formation of more different complexes, thus hampering the
calculation of binding constants.1,32
Thermal Denaturation Experiments. In the thermal dena-
turation experiments (Table 4) addition of “nonfused” analogues
(3, 8) yielded only weak but comparable stabilization of both
ct-DNA and polyA-polyU. On the other hand, “nonfused”
compound 4 yielded weak stabilization of exclusively ct-DNA.
The “fused” derivatives 11 (Figure 4) and 12 stabilized ct-DNA
and polyA-polyU significantly more strongly than their “non-
fused” analogues (3 and 4, respectively).
More detailed study of thermal stabilization of ct-DNA/
compound complexes revealed strongly nonlinear dependence
of ∆Tm values on the ratio r, suggesting for all compounds
saturation of binding sites at r ) 0.2-0.3, which is in good
agreement with the values of Scatchard ratio n obtained from
fluorimetric titrations (Table 3).
absorption maxima
fluorescence emission
ε × 103, dm3
compd
λ
max, nm
mol-1 cm-1
λ
max, nm
aQ
3
4
8
11
356
351
348
273
370
386
265
364
382
30.3
32.2
35.5
21.8
9.6
418
426
421
<0.1
<0.1
<0.1
437
419
0.85
7.9
12
28.4
12.8
9.5
0.84
a Relative quantum yield (Q) was determined with respect to standard
N-acetyl-L-tryptophanamide (NATA) Q ) 0.14.
weak fluorescence, while the emission of their “fused” analogues
11 and 12 was exceptionally strong, allowing measurements
even at 10 nM concentrations. Fluorescence emissions of all
studied compounds (3, 4, 8, 11, 12) were proportional to their
concentration in the range 1 × 10-7 to 5 × 10-6 mol dm-3
.
Interactions of Compounds 3, 4, 8, 11, 12 with Double
Stranded (ds-) DNA and (ds-) RNA
UV/Visible Titations. The UV/vis spectra of compounds 3,
4, 8, 11, 12 exhibited strong hypochromic changes upon titration
with ct-DNA (Figure 2A,B, Table 2), as well as during titrations
with several ds-RNAs (Figure 2C,D, Table 2). While for most
compounds addition of ct-DNA induced pronounced batochro-
mic shifts of the absorption maxima (Figure 2A,B, Table 2),
the shifts of the absorption maxima of studied compounds in
titrations with ds-RNA (∆λ, Table 2) were strongly dependent
on both the compound structure and base pair type in the
polynucleotide. It is interesting to note that polyG-polyC
exclusively induced hypsochromic shifts of absorption maxima
of some “non-fused” derivatives (3 and 4, Table 2). Such
pronounced changes of UV/vis spectra pointed toward involve-
ment of the studied compounds in strong π-π aromatic stacking
interactions, although it is not possible to distinguish between
interactions of studied compounds with ds-polynucleotide and
interactions between molecules of compound (e.g., molecule
agglomeration within the grooves of polynucleotide).
CD Experiments. So far, noncovalent interactions at 25 °C
were studied by monitoring the spectroscopic properties of
studied compound upon addition of the polynucleotides. In order
to get insight into the changes of polynucleotide properties
induced by small molecule binding, we have chosen CD
spectroscopy as a highly sensitive method toward conformational
changes in the secondary structure of polynucleotides.35 In
addition, achiral small molecules like 3, 4, 8, 11, and 12 can
eventually acquire induced CD spectrum (ICD) upon binding
to polynucleotides, from which mutual orientation of small
molecule and polynucleotide chiral axis could be derived,
consequently giving useful information about modes of interac-
tion.35
The major spectroscopic changes were observed at excess of
compound over polynucleotide. At such conditions more dif-
ferent binding modes could be present,1,32 which is strongly
supported for 3 by spectral changes in opposite directions
(Figure 2A,B). Therefore, it was not possible to process UV/
vis titration data by the Scatchard equation to obtain binding
constants.33
Fluorimetric Titrations. Strong fluorescence of the studied
compounds in aqueous media allowed titrations to be done
exclusively at high excess of DNA/RNA over compound.
Addition of any double-stranded polynucleotide strongly quenched
fluorescence of “fused” analogues 11 and 12 (Figure 3B, Table
3). It is noted that in contrast to “fused” compounds, addition
of both ds-DNA and ds-RNA compounds induced strong
fluorescence increase of “non-fused” analogues 3, 4, and 8
(Figure 3A, Table 3). That could be attributed to the increased
rigidity of the double bond of 3, 4, and 8 upon binding to the
double helix, thus forcing planar conformation of otherwise
flexible molecule, which could result in a strong increase of
fluorescence emission for the same reason as proposed for
cyanine dyes; namely, the huge enhancement of fluorescence
quantum yield of cyanine dyes upon binding to DNA is believed
Addition of “nonfused” derivatives (3, 4, 8) resulted in a
decrease of intensity of the ct-DNA CD spectrum (Figure 5).
Additionally, strong positive induced CD (ICD) band between
λ ) 350 and 400 nm appeared. Since “nonfused” analogues do
not have any intrinsic CD spectrum, ICD band most likely
resulted from the binding of 3, 4, and 8 into the minor groove
of ct-DNA.36,37
In contrast to “nonfused” compounds 3, 4, and 8, addition of
their “fused” analogues (11, 12) to ct-DNA did not yield induced
CD (ICD) band between λ ) 350 and 400 nm, thus excluding
binding into the minor groove of ct-DNA as a dominant
interaction.36,37 Surprisingly, changes of the negative (λ ) 245
nm) and positive (λ ) 278 nm) band of the ct-DNA induced
by titration with “fused” derivatives (11, 12) were different

4902 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Hranjec et al.
Figure 2. UV/vis titration of 3 (c ) 2.0 × 10-5 mol dm-3) with ct-DNA (A). Spectroscopic changes at λmax ) 356 nm as a function of ct-DNA
concentration (B). UV/vis titration of 3 (c ) 2.0 × 10-5 mol dm-3) with polyA-polyU (C). Spectroscopic changes at λmax ) 356 nm as a function
of polyA-polyU concentration (D). Experiments were done at pH 7.0, with buffer Na cacodylate, I ) 0.05 mol dm-3, ratio r ) [3]/[polynucleotide].
Table 2. Changes in the UV/Visible Spectra of 3, 4, 8, 11, 12 upon Titration with Double-Stranded DNA and RNA (pH 7, Na Cacodylate Buffer, I )
0.05 M)
3
4
8
11
12
ct-DNA
∆λ1,a nm
∆λ2,a nm
H, %
+11
0
+13
42 (348 nm)
+13
+15
39 (370 nm)
+8
+7
54 (364 nm)
c
46 (351 nm)
polyA-polyU
∆λ1,a nm
∆λ2,a nm
+4
0
0
+13
0
0
+15
H,b
%
30 (356 nm)
21 (351 nm)
23 (348 nm)
35 (370 nm)
17 (364 nm)
polyG-polyC
∆λ1,a nm
∆λ2,a nm
-5
-5
0
+13
0
0
+15
H,b
%
40 (356 nm)
30 (351 nm)
6 (348 nm)
39 (370 nm)
13 (364 nm)
a Shift of the absorbance maximum, ∆λ ) λ(complex) - λ(x - y). Absorbance maxima λ1: 8, λ ) 348 nm; 4, λ ) 351 nm; 3, λ ) 356 nm; 12, λ ) 364
nm; 11, λ ) 370 nm. Absorbance maxima λ2: 12, λ ) 382 nm; 11, λ ) 384 nm. b Hypochromic effect, H ) (Abs(compound) - Abs(complex))/Abs(compound))
× 100. Values of Abs(complex) are estimated by nonlinear extrapolation. c Absorbance changes in opposite direction (Figure 2): Abs decrease for r )
10-0.3 followed by Abs increase for r < 0.3.
(Figure 5). The intensity of the negative band (λ ) 245 nm)
substantially decreased upon each addition of 11, 12, and the
dependence of change on the ratio r[11,12]/[ct-DNA] was strongly
nonlinear, suggesting the saturation of binding sites at r )
0.2-0.3, similar to that found in the fluorimetric and thermal
denaturation experiments. On the other hand, intensity of the
positive band (λ ) 278 nm) weakly increased for r[11,12]/[ct-DNA]
) 0-0.3 and then decreased for r > 0.3, such opposite spectral
changes pointing toward presence of more than two spectro-
scopicaly active species. The experimental conditions (ratio r,
concentrations of 11, 12, and ct-DNA) of CD titrations are
comparable with UV/vis titrations, the last showing coexistence
of more binding modes. Since the strongest absorbance maxi-
mum of 11 and 12 (Table 2) is positioned close to the CD band
of ct-DNA (λ ) 278 nm), most likely different combinations
of binding modes resulted in different changes of CD spectrum
at various ratios r. Obviously, that did not influence significantly
the changes of the negative ct-DNA band (λ ) 245 nm).
The CD titrations of ds-RNA (polyA-polyU, Supporting
Information) with 3, 4, 8, 11, and 12 yielded results substantially

Benzimidazole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4903
Table 4. ∆Tm Valuesa of ct-DNA and polyA-polyU upon Addition of
Different Ratios r b of Studied Compounds at pH 7.0 (Buffer Na
Cacodylate, I ) 0.05 mol dm-3
)
∆Tm, °C
r b
3
4
8
11
12
ct-DNA
0.1
0.2
0.3
1.3
1.3
1.4
0
1.0
1.1
1.0
1.6
1.5
1.7
3.2
4.3
1.4
2.2
3.9
polyA-polyU
1.6
0.3
2.2
0
3.4
2.4
a Error in ∆Tm: (0.5 °C. b r ) [compound]/[polynucleotide].
Figure 4. Thermal denaturation curves of ct-DNA and 11 at pH 7
(sodium cacodylate buffer, I ) 0.05 mol dm-3). For measuring
conditions, see footnotes to Table 4 and the Experimental Section. The
ratio r ([11]/[ct-DNA]) values are 0.0, 0.1, 0.2, 0.3 (from bottom curve
up), and the curves are normalized.
Figure 3. Fluorimetric titration with ct-DNA of (A) “nonfused”
compound 3, c ) 8.7 × 10-7 mol dm-3, λexc ) 358 nm, λemiss ) 418
nm, tincub ) 6 min, and (B) “fused” compound 11, c ) 5.8 × 10-7 mol
dm-3, λexc ) 370 nm, λemiss ) 435 nm, tincub ) 7 min.
Viscometry. Viscometry experiments (Supporting Informa-
tion) yielded values of R(11) ) 0.92 and R(12) ) 0.74, which
differ from the value obtained for ethidium bromide (R(EB) )
0.83) by the error of the method (R ( 0.1). Obtained values
strongly support intercalation of 11 and 12 into ds-DNA as the
dominant binding mode. On the other hand, addition of
“nonfused” analogues (3, 4) resulted only in minor changes of
DNA solution viscosity (R(3,4) < 0.1), thus pointing that
intercalative binding mode is not dominant interaction of
“nonfused” compounds with ds-DNA.
Table 3. Binding Constants (log K Values) and Ratios n[bound compound]/
a
[polynucleotide phosphate] Calculated from the Fluorimetric Titrations with
Polynucleotides at pH 7.0 (Buffer Na Cacodylate, I ) 0.05 mol dm-3
)
ct-DNA
Int/Int0
polyA-polyU
polyG-polyC
b
b
b
log K
log K
Int/Int0
log K
Int/Int0
3
4
8
11
12
5.0
5.2
5.2
6.7
6.0
4
10
9
0.2
0.3
5.8
4.5
c
6.1
5.0
5.3
4.8
0.9/>10
0.8
5.6d
4.6
e
5.3
4.3
1.3
4.5
e
0
0.3
Discussion of the DNA/RNA Binding Studies of
Compounds 3, 4, 8, 11, 12
0.6
The “fused” derivatives (11 and 12) exhibited somewhat
higher affinity (log K values) and significantly stronger ∆Tm
values compared to their “nonfused” analogues (3 and 4,
respectively). All methods pointed toward saturation of binding
sites at r[11,12]/[polynucleotide] ) 0.2-0.3. The absence of a strong
positive ICD effect excluded the binding of 11 and 12 into ct-
DNA minor groove.36,37 The absence of the weak negative ICD
effect at long wavelengths, which is expected for intercalators,
can be explained in several ways, the most probable being
orientation heterogeneity of intercalated molecules.35–37 Vis-
cometry results strongly supported intercalation of 11 and 12
as a dominant interaction with ds-DNA. Taking into account
the large, planar aromatic surface, intercalation into ds-DNA
and ds-RNA is the most probable binding mode for 11 and 12.
According to the strong positive ICD bands,36,37 as well as
moderate affinity and weak thermal stabilization effects (due
to only one positive charge), the “nonfused” analogues (3, 4,
a Processing of titration data by means of Scatchard equation33 gave ratio
n[bound compound]/[polynucleotide] ) 0.3-0.1. For easier comparison, all log K values
were recalculated for fixed n ) 0.2. b Int0: starting fluorescence intensity
of compound. Int: fluorescence intensity of compound/polynucleotide
complex calculated by Scatchard equation. c Fluorimetric changes in oppo-
site direction hampered processing by Scatchard equation:33 emission
decrease for r ) 2-0.2 followed by emission increase for r < 0.2. d The
cumulative binding constant for mixed binding modes, calculated Scatchard
ratio n ) 2. e Small fluorescence changes didn’t allow accurate calculation.
different from those obtained in experiments with ct-DNA
(Figure 5). Additions of 8 did not yield any significant change
in the CD spectrum of polyA-polyU, while additions of 3, 11,
and 12 caused only a minor decrease of intensity of RNA CD
spectra. Only 4 yielded a somewhat stronger decrease of
intensity of polyA-polyU CD band at 265 nm. Quite the
opposite to the experiments with ct-DNA, in the experiments
with polyA-polyU no ICD bands at λ > 300 nm were observed.

4904 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Hranjec et al.
Table 5. In Vitro Inhibition of Compounds 2-13 on the Growth of
Tumor Cells and Normal Human Fibroblasts (WI 38)
IC50,a µM
compd Hep-2
HeLa
MiaPaCa-2 SW620 MCF-7
WI 38
2
3
4
5
6
13
7
8
9
12 ( 2
11 ( 2
9 ( 5
7 ( 5
>100
7 ( 3
7 ( 3
11 ( 6 >100 4 ( 2
>100
5 ( 0.9 10 ( 2
11 ( 0.6 3 ( 0.9 29 ( 2
4 ( 1
6 ( 3
12 ( 5
2 ( 0.08 33 ( 5
13 ( 5
62 ( 15 >100
6 ( 1 8 ( 7
9 ( 0.6 >100
3 ( 0.6 12 ( 2
20 ( 19 >100
7 ( 0.3 9 ( 5
12 ( 5 10 ( 6
>100
3 ( 0.5
50 ( 9
>100
67 ( 5 10 ( 5
65 ( 30 6 ( 2
51 ( 10
78 ( 4
19 ( 4
>100
>100
19 ( 16 4 ( 0.3 >100
17 ( 4 12 ( 0.06 16 ( 4
3 ( 0.3 3 ( 0.3 3 ( 0.7
16 ( 6 12 ( 4
10
11
12
19 ( 7 12 ( 0.2 18 ( 7
2 ( 0.4 2 ( 0.3 3 ( 0.03
4 ( 0.01 3 ( 1 9 ( 2
4 ( 2
3 ( 0.2 4 ( 0.3
a IC50: the concentration that causes a 50% reduction of the cell growth.
Figure 5. CD spectra of ct-DNA (c ) 2.0 × 10-5 mol dm-3) with 3,
4, 8, 11, and 12 at different ratios r ) [compound]/[ct-DNA] (pH 7.0,
buffer Na cacodylate, I ) 0.05 mol dm-3). Bottom right: changes of
ct-DNA CD signal at chosen wavelength with respect to ratio r. For
nonfused compounds, λ ) 275 nm, 3 (b), 4 (2), 8 (1). For fused
compounds, λ ) 245 nm, 11 ([) and 12 (9).
8) bind most likely into the minor groove of the ct-DNA.1,29,38
However, compounds 3 and 8 yielded weak thermal stabilization
of polyA-polyU, thus suggesting intercalation into ds-RNA at
least as one mode of interaction. Such a switch of binding mode
(ds-DNA minor groove binding, ds-RNA intercalation) was
earlier observed for several groups of compounds quite closely
related to the here-studied “nonfused” derivatives.38
Figure 6. Concentration-response profiles for the representative
nonfused (3) and fused (11) analogues tested on various human cell
lines in vitro. The cells were treated with the compounds at different
concentrations, and percentage of growth (PG) was calculated. Each
point represents a mean value of four replicates in three individual
experiments.
Biological Results and Discussion
Very similar effects were noticed in our previous study on
the antiproliferative action of amidino-substituted styryl-2-
benzimidazoles and benzimidazo[1,2-a]quinolines,31 whereby
the imidazolyl-substituted benzimidazoquinoline was the most
active compound and HeLa and MCF-7 cells showed greater
sensitivity.
Considering the above-mentioned DNA/RNA binding data
and similar biological activity to the previously published
results,31 we assumed that the mechanisms of action of these
compounds should also be similar (namely, noncovalent interac-
tions with cellular DNA), inducing cell cycle disturbances and
mitotic impairment. Therefore, we performed additional experi-
ments in order to gain closer analysis of the mechanisms of
tumor cell proliferation inhibition or death.
Compounds 2-13 were tested for their potential antiprolif-
erative effects on a panel of six human cell lines, five 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), and one from normal diploid fibroblasts, WI
38 (Table 5 and Figure 6).
All tested compounds showed prominent growth inhibitory
effect. There are several important points to emphasize: (i) in
general all fused compounds are more active (showing nondif-
ferential and cytotoxic effect) than nonfused ones; (ii) imida-
zolyl-substituted compound 11 proved to be the most active
one; (iii) HeLa and MCF-7 cells were the most sensitive to all
tested compounds; (iv) nonfused compounds containing the
thiophene moiety (2-5) were significantly more active than their
furan-containing analogues (6-9). Although the activities of
tiophene nonfused derivatives are overall comparable, it is
pointed out that only compound 2 did not show significant
activity toward normal cells.
Cell Cycle Perturbations and Apoptosis Induction. We
selected the fused compound 11 and its nonfused analogue
3 as the representatives of the most active compounds to
investigate their influence on the MiaPaCa-2 cell cycle after
24, 48, and 72 h treatment periods (Figure 7). Obtained results

Benzimidazole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4905
Figure 7. DNA histograms obtained by flow cytometry (see materials and methods section), and the percentages of cells in sub G1 (apoptotic
cells), G0/G1, S, and G2/M cell cycle phase, after the treatment of MiaPaCa-2 cells with 11 and 3 (both at 10 µM) for 24, 48, and 72 h.
Table 6. Percentage of Cells Undergoing Apoptosis Induced by Compounds 3, 4, 11, and 12 in MiaPaCa-2 and HeLa Cells after 72 h
apoptotic cells, %
3
4
11
12
treatment
control, 0 µM
5 µM
10 µM
5 µM
10 µM
5 µM
10 µM
5 µM
10 µM
MiaPaCa-2
HeLa
2 ( 2
8 ( 4
2 ( 0
8 ( 0
4 ( 0
14 ( 2
3 ( 2
13 ( 8
6 ( 3
17 ( 3
11 ( 2
27 ( 10
46 ( 4
47 ( 20
5 ( 3
10 ( 4
12 ( 8
27 ( 14
clearly showed that at the concentration of 10 µM, both
compounds initially induced statistically significant ac-
cumulation of cells in S phase and reduction of the G1 phase.
The nonfused compound 3 arrested the cells mostly in the S
phase throughout the entire treatment period, while fused
compound 11 induced additional drastic G2/M phase arrest
of the cell cycle that persisted over all three treatment periods
and was accompanied by the accumulation of cells in the
sub-G1 phase (apoptotic cells). There are two possible
explanations for these different outcomes: the most important
reason is certainly different DNA binding modes of 3
compared to 11, whereby 3 binds preferentially to the DNA
minor groove, while 11 intercalates into ds-DNA. Minor
groove binders, in particular benzimidazoles and terbenz-
imidazoles, were shown to be powerful DNA topoisomerase
I inhibitors.1,7,39 Moreover, topoisomerase inhibitors induce
S-phase arrest especially following prolonged exposure to
low doses of compounds,40,41 which was the case in our
experiments. On the other hand, the intercalative mode of
DNA binding along with somewhat higher concentration of
11 in regard to its IC50 concentration (3 and 10 µM for 11
and 3, respectively), induced different and/or more pro-
nounced DNA damage that induced apoptosis and conse-
quently showed more striking activity. Namely, the difference
between minor groove binders (3) and intercalators (11)
resulted in different affinity toward DNA and different
stabilization of ds-DNA helix, and in addition, only intercala-
tion could cause unwinding and extension of DNA double
helix.1 Very similar results were obtained in our previous
study with imidazolyl-substituted styryl-2-benzimidazoles and
benzimidazo[1,2-a]quinolines that could be considered as
nonfused and fused analogues to the here presented 3 and
11, respectively, except that the here-presented benzimida-
zolethiophene (3) has much more pronounced activity than
its benzimidazolestyryl analogue.31
Induction of Apoptosis. Since the sub-G1 fraction of cells
is not quite a sensitive method of apoptosis detection, a more
precise test should be used. Therefore, we assessed the annexin
V assay to confirm the induction of apoptosis (Table 6). Besides
3 and 11, we also tested additional two nonfused and fused
compound analogues (4 and 12) having a cyclohexyl substituent.
The results again confirmed that the imidazolyl-substituted

4906 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Hranjec et al.
of DNA. Being a strong intercalator, EtBr inserted itself between
the stacked base pairs and thus inhibited DNA relaxation
catalyzed by the enzyme (nonspecific, catalytic inhibitor).
Furthermore, compound 11 induced nicking of plasmid DNA,
pointing to the specific poisoning of topoisomerase I, although
to a lesser extent than CPT, but it should be noted that the
concentrations of both compounds were 100 µM, being ex-
tremely high for CMT, which is (as a specific topo I poison)
active even in much lower concentrations. Compound 3 did not
show specific topo I inhibitory effect at this concentration;
however, it strongly inhibited topo I relaxation along with
inducing topo I poisoning effect (enhanced nicking) at a
concentration of 200 µM (Figure 8b). These results are
substantiated by the analysis of the electrophoretic mobility of
plasmid DNA in the agarose gel without EtBr, which is shown
in Figure 8c, whereby the intensities of relaxed and nicked DNA
are pronounced after incubation with both compounds, although
some unwound (supercoiled) DNA is still present. On the other
hand, intercalator EtBr completely inhibited the DNA relaxation,
without causing relaxed and nicked products (see Supporting
Information, Figure S3). Taken together, these results suggest
that both compounds (3, 11) act as catalytic topoisomerse I
inhibitors and poisons, whereby compound 11 is either more
specific or inhibits the activity of topoisomerase II and
consequently induced DNA cleavage more strongly. The latter
is substantiated by the above-discussed cell cycle influence
experiments.
Conclusions
The spectrophotometric study revealed that nonfused benz-
imidazoles bind into minor groove of ds-DNA while their fused
photoproducts intercalate into double-stranded polynucleotides.
All studied compounds showed prominent growth inhibitory
effect, whereby all fused compounds were more active than
nonfused ones, most likely because of different modes of
interaction with cellular DNA.
Among nonfused compounds, those containing thiophene
moiety (2-5) were significantly more active than their furan-
containing analogues. Furthermore, nonfused derivatives induced
strong S phase arrest of the cell cycle followed by apoptosis
that together with DNA minor groove binding mode pointed to
topoisomerase I inhibition, which was additionally supported
by the results of topoisomerase I unwinding/cleavage assay. In
addition, all nonfused compounds revealed pronounced selectiv-
ity toward tumor cells in comparison with nontumor cells and
therefore should be considered as promising lead compounds
for further investigation.
On the other hand, fused analogues showed stronger but
nondifferential activity, whereby imidazolyl-substituted com-
pound 11 proved to be the most active one. Besides, it induced
strong G2/M arrest of the cell cycle followed by drastic
apoptosis, especially of HeLa cells.
Finally, it was shown that representatives of both nonfused
(3) and fused (11) derivatives act as catalytic topoisomerse I
inhibitors and poisons, whereby compound 11 as an intercalator
revealed stronger activity than its nonfused analogue (3) acting
as DNA minor groove binder. These results are in perfect
agreement with the previously published ones,31 which convinc-
ingly encourage further optimization and investigation of this
lead structure.
Figure 8. Influence of selected test compounds on relaxation of plasmid
DNA by topoisomerase I. DNA samples were separated by electro-
phoresis on an agarose gel containing ethidium bromide (a, b) or without
ethidium bromide (c). Shown are native supercoiled pCI DNA (0.4
µg) substrate in the assay buffer alone (lane pCI), incubated with
topoisomerse I (4 U) (lane Topo) or in combination with control drug
and camptothecin (100 µM) (lane CPT), ethidium bromide (10 µM)
(lane EtBr), and selected test compounds 3 (100 µM (a) and 200 µM
(b, c) and 11 (100 µM (a-c) (lanes 3 and 11).
compound 11 had most drastic influence on the apoptosis
induction in both HeLa and MiaPaCa-2 cells and was the only
compound capable of inducing apoptosis at 5 µM concentration.
HeLa cells were exceptionally sensitive also at lower (5 µM)
concentration and even after 24 h of incubation (data not shown),
which is in accordance with the growth inhibition results (Table
5). Interestingly, nonfused compounds 3 and 4 did not induce
apoptosis in MiaPaCa-2 cells, but significant proportion of HeLa
cells died by apoptosis after the treatment with 3 at 10 µM
concentration, confirming again the growth inhibition results.
Topoisomerase I Unwinding/Cleavage Assay. To ascertain
the previously mentioned assumption that tested compounds act
as topoisomerase inhibitors, we employed in vitro assay using
purified topoisomerase I from calf thymus. As described in the
Experimental Section, this assay allows discrimination between
nonspecific (due to intercalation) and specific (poisoning) effects
on topoisomerase I by separation of various forms of plasmid
DNA in agarose gels either without or containing ethidium
bromide (Figure 8). Figure 8 clearly shows that camptothecin
(CPT) as a potent poison of topoisomerase I strongly induced
nicking of plasmid DNA, which can be seen as an increased
intensity of the nicked open circular DNA band (cleavage
product). On the other hand, ethidium bromide (EtBr) inhibited
the relaxation of supercoiled DNA but did not induce nicking
Experimental Section
Chemistry. Melting points were determinate on a Koffler hot
stage microscope. IR spectra were recorded on Nicolet magna 760,

Benzimidazole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4907
Perkin-Elmer 297, and Perkin-Elmer Spectrum 1 spectrophotom-
eters, 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
DMSO-d6. 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 photochemi-
cal 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 E-N-Substituted 2-(2-
Thiophen-2-ylvinyl)-3H-benzimidazole-5(6)-carboxamidine Hy-
drochlorides (2-5). A mixture of 3-thiophen-2-ylpropenal 1a, the
corresponding 4-N-substituted-1,2-phenylenediamines, and p-ben-
zoquinone in absolute ethanol was stirred at reflux for 3 h under
nitrogen. The reaction mixture was cooled at room temperature,
diethyl ether was added, and the resulting solid was filtered off.
The products were dissolved in ethanol and precipitated with diethyl
ether, filtered off, and dried. It was repeated a few times until the
products were analytically pure.
E-N-Isopropyl-2-(2-thiophen-2-ylvinyl)-3H-benzimidazole-5(6)-
carboxamidine Hydrochloride (2). Compound 2 was prepared using
the general method described above, from 3-thiophen-2-ylpropenal
1a (0.200 g, 1.450 mmol), 4-N-isopropylamidno-1,2-phenylenedi-
amine (0.331 g, 1.450 mmol), and p-benzoquinone (0.157 g, 1.450
mmol) in absolute ethanol (10 mL) after refluxing for 2 h to obtain
0.335 g (67%) of yellow-green powder; mp 291-293 °C. Anal.
(C17H19N4ClS) C, H, N.
E-6-(4,5-Dihydro-1H-imidazol-2-yl)-2-(2-thiophen-2-ylvinyl)-3H-
benzimidazole Hydrochloride (3). Compound 3 was prepared using
the general method described above, from 3-thiophen-2-ylpropenal
1a (0.300 g, 2.171 mmol), 4-(2-imidazolinyl)-1,2-phenylenediamine
(0.461 g, 2.171 mmol), and p-benzoquinone (0.234 g, 2.171 mmol)
in absolute ethanol (15 mL) after refluxing for 3 h to obtain 0.717
g (88%) of gray powder; mp 178-180 °C. Anal. (C16H15N4ClS)
C, H, N.
E-N-Cyclohexyl-2-(2-thiophen-2-ylvinyl)-3H-benzimidazole-5(6)-
carboxamidine Hydrochloride (4). Compound 4 was prepared using
the general method described above, from 3-thiophen-2-ylpropenal
1a (0.300 g, 2.171 mmol), 4-N-cyclohexylamidno-1,2-phenylene-
diamine (0.584 g, 2.171 mmol), and p-benzoquinone (0.234 g, 2.171
mmol) in absolute ethanol (15 mL) after refluxing for 2.5 h to obtain
0.630 g (75%) of light-green powder; mp >300 °C. Anal.
(C20H23N4ClS) C, H, N.
E-N-Morpholin-4-yl-2-(2-thiophen-2-ylvinyl)-3H-benzimidazole-
5(6)-carboxamidine Hydrochloride (5). Compound 5 was prepared
using the general method described above, from 3-thiophen-2-
ylpropenal 1a (0.300 g, 2.172 mmol), 4-N-morpholinylamidno-1,2-
phenylenediamine (0.590 g, 2.172 mmol), and p-benzoquinone
(0.234 g, 2.172 mmol) in absolute ethanol (15 mL) after refluxing
for 2.5 h to obtain 0.650 g (77%) of green powder; mp >300 °C.
Anal. (C18H20N5ClOS) C, H, N.
General Method for the Synthesis of E-2-(2-Furan-2-ylvinyl)-
N-substituted-3H-benzimidazole-5(6)-carboxamidine Hydrochlo-
rides (6-9). A mixture of 3-furan-2-ylpropenal 1b, the correspond-
ing 4-N-substituted 1,2-phenylenediamines, and p-benzoquinone in
absolute ethanol was stirred at reflux for 3 h under nitrogen. The
reaction mixture was cooled at room temperature, and the resulting
solid was filtered off. The products were dissolved in water and
precipitated with acetone, filtered off, and dried. It was repeated
two times until the products were analytically pure.
E-2-(2-Furan-2-ylvinyl)-N-isopropyl-3H-benzimidazole-5(6)-car-
boxamidine Hydrochloride (6). Compound 6 was prepared using
the above described method, from 3-furan-2-ylpropenal 1b (0.534
g, 4.381 mmol), 4-N-isopropylamidno-1,2- phenylenediamine (1.000
g, 4.381 mmol), and p-benzoquinone (0.474 g, 4.381 mmol) in
absolute ethanol (20 mL) after refluxing for 2.5 h to obtain 0.700
g (50%) of gray powder; mp >300 °C. Anal. (C17H19N4OCl) C,
H, N.
6-(4,5-Dihydro-1H-imidazol-2-yl)-2-(2-furan-2-ylvinyl)-1H-ben-
zimidazole Hydrochloride (7). Compound 7 was prepared using
the above described method, from 3-furan-2-ylpropenal 1b (0.401
g, 3.290 mmol), 4-(2-imidazolinyl)-1,2-phenylenediamine (0.700
g, 3.290 mmol), and p-benzoquinone (0.356 g, 3.290 mmol) in
absolute ethanol (25 mL) after refluxing for 2 h to obtain 0.720 g
(71%) of light-brown powder; mp 215-216 °C. Anal.
(C16H15N4OCl2 ·3H2O) C, H, N.
E-N-Cyclohexyl-2-(2-furan-2-ylvinyl)-3H-benzimidazole-5-car-
boxamidine Hydrochloride (8). Compound 8 was prepared using
the above described method, from 3-furan-2-ylpropenal 1b (0.500
g, 4.100 mmol), 4-N-cyclohexylamidno-1,2- phenylenediamine
(1.100 g, 4.100 mmol), and p-benzoquinone (0.443 g, 4.100 mmol)
in absolute ethanol (25 mL) after refluxing for 2 h to obtain 0.950
g (70%) of gray powder; mp >300 °C. Anal. (C20H24N4OCl2 ·H2O)
C, H, N.
E-2-(2-Furan-2-ylvinyl)-3H-benzimidazole-5-carboxamidine Hy-
drochloride (9). Compound 9 was prepared using the above
described method, from 3-furan-2-ylpropenal 1b (0.500 g, 4.100
mmol), 4-amidino-1,2-phenylenediamine (0.764 g, 4.100 mmol),
and p-benzoquinone (0.443 g, 4.100 mmol) in absolute ethanol (30
mL) after refluxing for 2 h to obtain 0.850 g (72%) of gray powder;
mp >300 °C. Anal. (C14H13N4OCl·3H2O) C, H, N.
General Method for the Synthesis of N-Substituted 3-Thia-
6,10b-diazacyclopenta[c]fluorene-8(9)-carboxamidine Hydrochlo-
rides (10-13). A solution (c ) 1.3 × 10-3 mol/dm3) of corre-
sponding E-N-substituted 2-(2-thiophen-2-ylvinyl)-3H-benzimidazole-
5(6)-carboxamidine hydrochlorides (2-5) in ethanol was irradiated
at room temperature with a 400 W high-pressure mercury lamp
using a Pyrex filter for 6 h. The air was bubbled through the
solution. The solution was concentrated and precipitated with diethyl
ether, and the resulting solid was filtered off. After 2-fold
recrystallization from ethanol/diethyl ether, pure products in the
form of yellow powder were obtained.
N-Isopropyl-3-thia-6,10b-diazacyclopenta[c]fluorene-8(9)-car-
boxamidine Hydrochloride (10). Compound 10 was prepared as a
mixture of two regioisomers, 85% of 8-position isomer (10a) and
15% of 9-position isomer (10b), using the general method described
above, from E-N-isopropyl-2-(2-thiophen-2-ylvinyl)-3H-benzoimi-
dazole-5(6)-carboxamidine hydrochloride (2) (0.100 g, 0.287 mmol)
and iodine (0.005 g, 0.020 mmol) in ethanol (220 mL) after
irradiation for 5 h to obtain 0.080 g (80%) of yellow powder; mp
210-211 °C. Anal. (C17H17N4ClS) C, H, N.
8(9)-(1H-Imidazol-2-yl)-3-thia-6,10b-diazacyclopenta[c]fluo-
rene Hydrochloride (11). Compound 11 was prepared as a mixture
of two regioisomers, 85% of 8-position isomer (11a) and 15% of
9-position isomer (11b), using general method described above,
from 6-(4,5-dihydro-1H-imidazol-2-yl)-2-(2-thiophen-2-ylvinyl)-
3H-benzimidazole-5(6)-carboxamidine hydrochloride (3) (0.095 g,
0.291 mmol) and iodine (0.005 g, 0.020 mmol) in ethanol (220
mL) after irradiation for 6 h to obtain 0.069 g (73%) of yellow
powder; mp 120-122 °C. Anal. (C16H13N4ClS) C, H, N.
N-Cyclohexyl-3-thia-6,10b-diazacyclopenta[c]fluorene-8(9)-car-
boxamidine Hydrochloride (12). Compound 12 was prepared as a
mixture of two regioisomers, 85% of 8-position isomer (12a) and
15% of 9-position isomer (12b), using the general method described
above, from N-cyclohexyl-2-(2-thiophen-2-ylvinyl)-3H-benzimi-
dazole-5-carboxamidine hydrochloride (4) (0.100 g, 0.260 mmol)
and iodine (0.005 g, 0.020 mmol) in ethanol (220 mL) after
irradiation for 6 h to obtain 0.070 g (71%) of yellow powder; mp
233-235 °C. Anal. (C20H21N4ClS) C, H, N.
N-Morpholin-4-yl-3-thia-6,10b-diazacyclopenta[c]fluorene-8(9)-
carboxamidine Hydrochloride (13). Compound 13 was prepared
as a mixture of two regioisomers, 85% of 8-position isomer (13a)
and 15% of 9-position isomer (13b), using the general method
described above, from N-morpholin-4-yl-2-(2-thiophen-2-ylvinyl)-
3H-benzimidazole-5-carboxamidine hydrochloride (5) (0.090 g,
0.231 mmol) and iodine (0.005 g, 0.020 mmol) in ethanol (220

4908 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Hranjec et al.
mL) after irradiation for 6 h to obtain 0.060 g (67%) of yellow
powder; mp 230-232 °C. Anal. (C18H18N5ClOS) C, H, N.
Spectroscopy. The electronic absorption spectra were recorded
on a Varian Cary 100 Bio spectrometer and CD spectra on a Jasco
J815, in all cases using quartz cuvettes (1 cm). The measurements
were performed in 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 the
studied compounds were proportional to their concentrations while
none of studied compounds showed a CD spectrum. Fluorescence
emission spectra were recorded on a Varian Eclipse fluorimeter
(quartz cuvettes, 1 cm), from 350 to 550 nm and corrected for the
effects of time- and wavelength-dependent light-source fluctuations
using a standard rhodamine 101, a diffuser, and the software
provided with the instrument. The sample concentration in fluo-
rescence measurements had an optical absorbance below 0.05 at
the excitation wavelength. Relative fluorescence quantum yields
(Q) were determined according to the standard procedure used in
previous work.26 As the standard, we used N-acetyl-L-tryptopha-
namide (NATA) with published fluorescence quantum yield Q )
0.14.26
modified procedure of the National Cancer Institute, Developmental
Therapeutics Program.10,26,31 Briefly, the cells 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-1 penicillin, and 100 µg mL-1 streptomycin
in a humidified atmosphere with 5% CO2 at 37 °C. The cells were
inoculated onto a series of standard 96-well microtiter plates on
day 0, at 1 × 104 to 3 × 104 cells/mL, depending on the doubling
times of specific cell line. Test agents were then added in five or
10-fold dilutions (10-8 to 10-4 M) and incubated for a further 72 h.
Working dilutions were freshly prepared on the day of testing. The
solvent was also tested for eventual inhibitory activity by adjusting
its concentration to be the same as in the working concentrations.
The cell growth rate was evaluated by performing the MTT assay
after 72 h of incubation, which detects mitochondrial dehydrogenase
activity in viable cells.
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%).
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.
Interactions with Polynucleotides. Polynucleotides were pur-
chased from Aldrich and dissolved in the sodium cacodylate buffer
(I ) 0.05 mol dm-3, pH 7.0), and ct-DNA was additionally
sonicated and filtered through a 0.45 µm filter. The concentration
of polynucleotide solution was determined spectroscopically as the
concentration of phosphates. Spectroscopic titrations were per-
formed by adding portions of polynucleotide solution into the
solution of the studied compound.
Cell Cycle Analysis. The 2 × 105 cells were seeded per well
into a six-well plate. After 24 h the tested compounds were added
at various concentrations (as shown in the results section). After
the desired length of time the attached cells were trypsinized,
combined with floating cells, washed with phosphate buffer saline
(PBS), fixed with 70% ethanol, and stored at -20 °C. Immediately
before the analysis, the cells were washed with PBS and stained
with 1 µg/mL of the propidium iodide (PI) with the addition of 0.2
µg/µL RNase A. The stained cells were then analyzed with Becton
Dickinson FACScalibur (Becton Dickenson) flow cytometer (20 000
counts were measured). The percentage of the cells in each cell
cycle phase was determined using the ModFit LT software (Verity
Software House) based on the DNA histograms. The tests were
performed in duplicate and repeated at least twice.
Detection of Apoptosis. Annexin-V Assay. Detection and quan-
tification of apoptotic cells at the single cell level was performed
using Annexin-V-FLUOS staining kit (Roche) according to the
manufacturer’s recommendations. The 2 × 104 cells were seeded
per well into a 24-well plate. After 24 h the tested compounds
were added at various concentrations (as shown in the results
section). After the desired length of time, both floating and
attached cells were collected. The cells were then washed with
PBS, pelleted, and resuspended in staining solution (annexin-
V-fluorescein labeling reagent and propidium iodide (PI) in
Hepes buffer). The cells were then analyzed/counted under a
fluorescence microscope. Annexin-V (green fluorescent) cells
were determined to be apoptotic, and annexin-V and PI cells
were determined to be necrotic. Percentage of apoptotic cells
was expressed as a number of fluorescent cells in relation to the
total cell number (fluorescent and nonfluorescent cells), which
was expressed as 100%.43
Topoisomerase I DNA Unwinding/Cleavage Assay. In vitro assay
using purified calf thymus topoisomerase I (Invitrogen) was used
in order to check selected compounds for their inhibitory effect on
topo I according to Bailly,44 whereby two possible modes of
inhibition can be detected: nonspecific effect due to DNA intercala-
tion and specific effects resulting from the poisoning of topoi-
somerase I. Briefly, reaction mixture contained 0.8 µg of negatively
supercoiled plasmid pCI (Promega, kindly provided by Dr. Andreja
Ambriovic´ Ristov, Rudjer Bosˇkovic´ Institute, Zagreb, Croatia) and
8 U of topoisomerase I with or without tested inhibitors in 40 µL
of relaxation buffer (10 mM Tris-HCl, pH 7.5, 175 mM KCl, 5
mM MgCl2, 0.1 mM EDTA, 2.5% glycerol). The mixtures were
incubated at 37 °C for 30 min and reaction terminated by the
addition of 2.5 µL of 10% SDS, followed by proteinase K (50 µg/
The stability constant (Ks) and [bound compound]/[polynucle-
otide phosphate] ratio (n) were calculated according to the Scatchard
equation by nonlinear least-squares fitting, giving excellent cor-
relation coefficients (>0.999) for obtained values for Ks and n.
Thermal melting curves for ct-DNA and polyA-polyU and its
complexes with the studied compounds were determined as
previously described32 by following the absorption change at 260
nm as a function of temperature. The absorbance of the ligand was
subtracted from every curve, and the absorbance scale was
normalized. Obtained Tm values are the midpoints of the transition
curves, determined from the maximum of the first derivative or
graphically by a tangent method. Given ∆Tm values were calculated
by subtracting Tm of the free nucleic acid from Tm of the complex.
Every ∆Tm value here reported was the average of at least two
measurements, and the error in ∆Tm is ( 0.5 °C. Viscometry
experiments were conducted with an Ubbelohde microviscometer
system. The temperature was maintained at 25 ( 0.1 °C. Aliquots
of studied compound stock solutions were added to 5.5 mL of 5 ×
10-4 M ct-DNA solution (pH 7, buffer Na cacodylate, I ) 0.05
M), with ratio r[compound]/[ct-DNA] < 0.2. The flow times were measured
at least three times with a deviation of (0.5 s. The viscosity index
R was obtained from the flow times at varying r according to the
following equation:42
L ⁄ L0 ) [(tr - t0) ⁄ (tDNA - t0)]1⁄3 ) 1 + Rr
t0, tDNA, and tr denote the flow times of buffer, free DNA, and DNA
complex at ratio r[1]/[ct-DNA], respectively, and L/L0 is the relative
DNA lengthening. The L/L0 to r plot (Supporting Information) was
fitted to a straight line that gave slope R. The error in R is (0.1.
Under the same conditions, experiments with ethidium bromide
(EB) were performed for comparison (Supporting Information).
Antitumor Activity Assays. Antiproliferative Activity. The HeLa
(cervical carcinoma), Hep-2 (laryngeal carcinoma), MCF-7 (breast
carcinoma), SW620 (colon carcinoma), and MiaPaCa-2 (pancreatic
carcinoma) cells (obtained from American Type Culture Collection
(ATCC, Rockville, MD)) were cultured as monolayers and main-
tained in Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented 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

Benzimidazole DeriVatiVes
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4909
Delcamp, T.; Huang, S.; Wolin, R.; Bobkova, E. V.; Shaw, K. J.
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ml) digestion at 37 °C for 15 min. After extraction with a mixture
of phenol, chloroform, and isoamyl alcohol (25:24:1), aqueous
samples were removed and an amount of 4 µL of gel-loading buffer
(0.25% bromphenol blue, 0.25% xylene cyanol, 60% glycerol, 150
mM Tris, pH 7.6) was added. The samples were divided into two
and loaded onto either 1% agarose gel containing 0.5 µg/mL
ethidium bromide or 1% agarose gel without ethidium bromide.
Gels were run at 80 V constant voltage in a horizontal electro-
phoresis system (BIO-RAD). Resulting products were visualized
and documented with UV light at 254 nm (Image Master VDS,
Pharmacia Biotech, Sweden).
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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, and 098-0982464-2393).
Supporting Information Available: Elemental analysis results,
1H and 13C NMR data, viscometry experiments, additional CD
experiments, topoisomerase I inhibition assay. This material is
available free of charge via the Internet at http://pubs.acs.org.
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