Synthesis, antitumor evaluation and DNA binding studies of novel amidino-benzimidazolyl substituted derivatives of furyl-phenyl- and thienyl-phenyl-acrylates, naphthofurans and naphthothiophenes

Article abstract of DOI:10.1016/j.ejmech.2008.02.010

A series of amidino-substituted benzimidazoles, related to furyl-phenyl- and thienyl-phenyl-acrylates, naphthofurans and naphthothiophenes were prepared, their antitumor evaluation and interactions with ct-DNA have been investigated. All tested compounds show differential and strong antitumor activity without apparent difference depending on their structures. Interestingly, the MCF-7 tumor cell line is highly sensitive to all compounds. Compounds 6-9 showed noticeable selectivity in regard to normal fibroblasts (WI 38). Compounds 4-9 interact with ct-DNA by more binding modes, whose mutual distribution is dependent on the compound/DNA ratio. The acyclic 4-6 and cyclic compound 7 interact mostly within the minor groove of DNA, although partial intercalation of 6 and 7 cannot be excluded. The cyclic compounds 8 and 9 intercalate between DNA base pairs at high excess of DNA over compounds.

Full text of DOI:10.1016/j.ejmech.2008.02.010

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2877e2890
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, antitumor evaluation and DNA binding studies of novel
amidino-benzimidazolyl substituted derivatives of furyl-phenyl- and
thienyl-phenyl-acrylates, naphthofurans and naphthothiophenes
a
Marijana Hranjec , Kristina Starcevic , Ivo Piantanida , Marijeta Kralj ,
a
b
c
´
Marko Marjanovic , Merima Hasani , Gunnar Westman , Grace Karminski-Zamola
ꢀ
c
d
d
a,
*
´
a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb,
´
Marulicev trg 20, P.O. Box 177, HR-10000 Zagreb, Croatia
b Division of Organic Chemistry and Biochemistry, Laboratory of Supramolecular and Nucleoside Chemistry,
´
ꢀ
RuCer Bosˇkovic Institute, Bijenicka cesta 54, P.O. Box 180, HR-10000 Zagreb, Croatia
c
´
Division of Molecular Medicine, Laboratory of Functional Genomics, RuCer Bosˇkovic, Institute,
Bijeniꢀcka cesta 54, P.O. Box 180, HR-10000 Zagreb, Croatia
d Department of Chemistry and Bioscience, Chalmers University of Technology, S-412 96 Go¨teborg, Sweden
Received 11 October 2007; received in revised form 30 January 2008; accepted 8 February 2008
Available online 29 February 2008
Abstract
A series of amidino-substituted benzimidazoles, related to furyl-phenyl- and thienyl-phenyl-acrylates, naphthofurans and naphthothiophenes
were prepared, their antitumor evaluation and interactions with ct-DNA have been investigated. All tested compounds show differential and
strong antitumor activity without apparent difference depending on their structures. Interestingly, the MCF-7 tumor cell line is highly sensitive
to all compounds. Compounds 6e9 showed noticeable selectivity in regard to normal fibroblasts (WI 38). Compounds 4e9 interact with ct-DNA
by more binding modes, whose mutual distribution is dependent on the compound/DNA ratio. The ‘‘acyclic’’ 4e6 and ‘‘cyclic’’ compound 7
interact mostly within the minor groove of DNA, although partial intercalation of 6 and 7 cannot be excluded. The ‘‘cyclic’’ compounds 8 and 9
intercalate between DNA base pairs at high excess of DNA over compounds.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Benzimidazoles; Amidines; Photocyclization; Antitumor evaluation; DNA binding
1. Introduction
drug development strategies designed to produce next genera-
tion therapeutics for diseases such as cancer [3,4].
DNA plays a major role in the life process because it carries
heritage information and instructs the biological synthesis of
proteins and enzyme through the process of replication and
transcription of genetic information. An understanding of the
drugeDNA interactions is a promising approach to developing
novel reagents and plays a key role in pharmacology today
[1,2]. Due to interaction of many biologically active com-
pounds with biomolecules, they represent a major target in
Over the past years there is a considerable interest in the
development and pharmacology of heteroaromatic organic
compounds, such as benzimidazoles [5,6], due to their wide
range of biological activity as antitumor [7,8,9], antibacterial
[10,11], antifungal [12] or antiviral [13,14] agents. Benzimid-
azole substituted derivatives are also inhibitors of cyclin-
dependent kinase or tyrosine kinase and are useful for inhibit-
ing cell proliferation for the treatment of cancer [15,16,17].
The fact that benzimidazole residue is a constituent of vitamin
B12 supports their potential use as therapeutics. Due to the
structural similarity of benzimidazole nuclei with some natu-
rally occurring compounds such as purine, they can easily
* Corresponding author. Tel.: þ385 14597215; fax: þ385 14597250.
E-mail address: gzamola@pierre.fkit.hr (G. Karminski-Zamola).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.02.010

2878
M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
interact with biomolecules of the living systems. Bis(benzimi-
dazole) derivatives as Hoechst 33342 and 33258 (Fig. 1) also
bind in the minor groove of DNA [18,19]. Hoechst 33258 has
undergone Phase I clinical evaluation as an anticancer agent
and the inhibition of topoisomerase and DNA helicase has
been proposed as its mechanism of action [20,21].
-Cl+H2N
H2N
NH2+Cl-
NH2
O
Furamidine
Fig. 2. Chemical structure of furamidine.
On the other hand, amidines are structural parts of numer-
ous compounds of biological interest including important
medical and biochemical agents [22,23]. Some aromatic dia-
midines such as pentamidine, berenil or furamidine (Fig. 2)
are minor groove binding agents and are used in the treatment
of several protozoan diseases based on their selective binding
to AT rich sequences of DNA helix [24].
Recently we reported about the synthesis and antitumor
evaluation of some new amidino benzimidazolyl substituted
naphtho[2,1-b]furan carboxylates [25] as well as synthesis,
antitumor evaluation and DNA binding of heteroaromatic acy-
clic and cyclic amidino-substituted naphthofuran or naphtho-
thiophene carboxylates [26]. As a part of our continuing
search for potential anticancer agents, herein we present the
synthesis of acyclic and cyclic condensed heterocyclic benz-
imidazole derivatives, related to naphthofurans and naphtho-
thiophenes (Fig. 3), as well as their antitumor evaluation on
several human tumor cell lines and detailed DNA binding
studies of all synthesized compounds.
Synthesis of compounds 4 and 7 has already been published
[25].
Furyl-phenyl-acrylates can be viewed as a heterocyclic an-
alogue of substituted stilbene [29]. Heterocyclic acrylates are
photochemically active [30,31] and the expected photoreac-
tions, by analogy with stilbenes and related compounds [32],
are E/Z photoisomerization from excited singlet state and
ring-closure reaction from the E isomers to form reversibly
the dihydro trans-adduct intermediate by electrocyclic reac-
tion. Under oxidative conditions the dihydro intermediate is
converted to the desired naphtho[2,1-b]furan-5-carboxylate.
In our previous paper [33] we described experiments that
present the conceptual basis for the development of new pho-
toinduced anticancer therapy based on the photochemical
transformation of a DNA/RNA inactive into an active one. Ad-
ditional advantage of this concept is to access a large number
of already known or new molecules with selected properties
compatible with physiologically relevant conditions. In order
to obtain new systems capable to undergo the photoinduced
dehydrocyclization by visible light irradiation, we prepared
previously described benzimidazole derivatives of E-methyl-
3-(2-furyl)-2-phenyl-acrylate.
2. Results and discussion
2.1. Chemistry
All compounds 4e9 shown in Fig. 3 were prepared accord-
ing to Scheme 1. Acyclic amidino-substituted compounds 4e6
were prepared by condensation of corresponding heterocyclic
aldehydes 1e3 and 4-isopropylamidino-1,2-phenylendiamine
[27] in the yields of about 30e51% [25]. We prepared only
isopropyl-amidines because our earlier obtained results
showed that isopropylamidino-substituted compounds have
shown in most cases more pronounced biological activity
[25,26a]. Aldehydes 1e3 were prepared by earlier described
methods [26]. In the reaction of photochemical dehydrocycli-
zation [28], acyclic compounds 4e6 were converted into
their cyclic products 7e9 in the yields of about 35e59%.
2.2. Interactions of compounds 4e9 with ct-DNA
2.2.1. Spectroscopic properties of compounds 4e9
Compounds studied in this paper are divided in two groups,
‘‘acyclic’’ derivatives (Fig. 3; 4e6) and their ‘‘cyclic’’ photo-
products (Fig. 3; 7e9). Since for DNA binding studies of
above-mentioned compounds application of spectrophotomet-
ric methods was necessary, we have characterized aqueous
solutions of all compounds by means of electronic absorption
(Fig. 4, Table 1) and fluorescence emission spectra (Fig. 5).
Absorbencies of aqueous solutions of compounds are pro-
portional to their concentrations up to 2.08 ꢀ 10ꢁ5 mol dmꢁ3
(4), 1.67 ꢀ 10ꢁ5 mol dmꢁ3 (5), 2.82 ꢀ 10ꢁ5 mol dmꢁ3 (6),
1.26 ꢀ 10ꢁ5 mol dmꢁ3 (7), 5.45 ꢀ 10ꢁ5 mol dmꢁ3 (8), and
2.11 ꢀ 10ꢁ5 mol dmꢁ3 (9), indicating that there is no signifi-
cant intermolecular stacking which should give rise to hypo-
chromic effect. Aqueous solutions of all studied compounds
were shown to be stable over longer period.
HO
NH
N
Comparison of UVevis spectra of both, ‘‘acyclic’’ (4e5)
and ‘‘cyclic’’ (7e8) furan derivatives, pointed out that intro-
duction of electron-withdrawing group (eCN) on the phenyl
ring resulted in bathochromic shift of absorption maxima, as
well as hypochromic effect. The thiophene derivatives (6
and 9) are characterized by significantly lower molar absorp-
tivities (3) and absorption maxima shifted toward longer wave-
lengths in comparison to furan analogues (4 and 7).
H
N
CH3
N
N
N
H
Hoechst 33258
Fig. 1. Chemical structure of Hoechst 33258.

M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
R1
2879
R1
R3
R3
N
N
X
N
H
N
H
X
R2
R2
4 X=O, R1=H, R2=COOMe, R3=i-pr-amidine
5 X=O, R1=CN, R2=COOEt, R3=i-pr-amidine
6 X=S, R1=H, R2=COOMe, R3=i-pr-amidine
7 X=O, R1=H, R2=COOMe, R3=i-pr-amidine
8 X=O, R1=CN, R2=COOEt, R3=i-pr-amidine
9 X=S, R1=H, R2=COOMe, R3=i-pr-amidine
Fig. 3. Amidino-substituted ‘‘acyclic’’ 4e6 and ‘‘cyclic’’ 7e9 benzimidazole derivatives.
All studied compounds exhibit characteristic fluorescence
emission with maxima at 419e428 nm (Table 2). Their excita-
tion spectra are in good agreement with absorption spectra.
Comparison of fluorescence spectra (Table 2, Fig. 5)
revealed that emission of ‘‘acyclic’’ compounds 4e6 is
significantly weaker if compared to their ‘‘cyclic’’ analogues
7e9, most likely due to the more efficient conjugation of rigid,
planar, cyclic moieties, while ethene linker of ‘‘acyclic’’ com-
pounds is characterized by significantly higher motional
freedom and thus upon excitation allows more modes of
non-emissive relaxation. It should be stressed that fluorescence
emission of ‘‘acyclic’’ compounds 4e6 is stable under exper-
imental conditions used, pointing out that photoinduced cycli-
sation into ‘‘cyclic’’ analogues 7e9 does not take place;
otherways due to the significantly stronger fluorescence of
‘‘cyclic’’ analogues compared to ‘‘acyclic’’, permanent irradi-
ation in the fluorimeter should result in substantial emission
increase. It is interesting to note that furan derivatives 4 and
7 exhibit more than at least order of magnitude stronger fluo-
rescence compared to their thiophene derivatives 6 and 9. In-
triguingly, introduction of cyano group on benzene moiety
strongly decreased the fluorescence of furan derivatives 5
and 8 when compared to unsubstituted analogues 4 and 6.
That could be correlated to the strong electron-withdrawing
properties of cyano group and points out the pronounced im-
pact of the electronic properties of here presented molecules
on the ratio between non-emissive and emissive relaxation.
R1
OHC
X
R2
1 X=O, R1=H, R2=COOMe
2 X=O, R1=CN, R2=COOEt
3 X=S, R1=H, R2=COOMe
NH2
-Cl+H2N
p-benzoquinone
abs. ethanol
NH2
HN
R1
NH1+Cl-
2.2.2. UVevis titrations with ct-DNA
The UVevis spectra of all studied compounds exhibited
strong hypochromic and bathochromic changes upon addition
N
HN
X
N
H
R2
4 X=O, R1=H, R2=COOMe
5 X=O, R1=CN, R2=COOEt
6 X=S, R1=H, R2=COOMe
45000
7
40000
35000
30000
25000
20000
15000
10000
5000
0
4
hν, I2, O2
5
ethanol
9
R1
NH2+Cl-
6
N
HN
N
H
X
8
R2
7 X=O, R1=H, R2=COOMe
8 X=O, R1=CN, R2=COOEt
9 X=S, R1=H, R2=COOMe
250
300
350
400
/ nm
Scheme 1. Synthesis of amidino-substituted benzimidazole derivatives.
Fig. 4. UVevis spectra of studied compounds.

2880
M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
Table 1
Electronic absorption maxima and corresponding molar extinction coefficients
of studied compounds in water
Table 2
Fluorescence emission and excitation properties of studied compounds in
water
Compound
lmax/nm
3 ꢀ 103 dm3 molꢁ1 cmꢁ1
Compound
cs/mol dmꢁ3
lemiss/nm
lexc/nm
Rel. fluo.
int. (a.u.)
Intcyclic/
Intacyclic
4
5
6
7
366
370
374
362
260
367
263
370
225
33.6
31.7
18.4
42.2
28.8
5.0
4
7
5
8
6
9
2.4 ꢀ 10ꢁ8
2.4 ꢀ 10ꢁ8
3.6 ꢀ 10ꢁ7
3.7 ꢀ 10ꢁ7
3.8 ꢀ 10ꢁ7
3.9 ꢀ 10ꢁ7
425
419
425
426
428
424
365
360
370
368
374
370
147.1
790.1
161.4
282.2
231.8
916.1
e
5.37
e
1.75
e
3.95
8
9
14.2
24.5
27.1
over compounds (Fig. 7). Such a linear dependence is not in
accord with the UVevis titrations, which pointed toward the
end of titration at much lower ct-DNA concentrations. Such
a discrepancy could be the result of other processes monitored
by fluorescence simultaneously proceeding with titration.
Therefore it was not possible to process titration data by
means of Scatchard equation in order to obtain binding affinity
(Table 4).
Similarly as observed in the UVevis titration experiments,
titration with ct-DNA resulted in opposite changes of fluores-
cence of ‘‘closed’’ analogues 7e9 depending on the com-
pound/ct-DNA ratio r. Namely, at excess of DNA strong
fluorescence quenching was observed, while ratios r < 0.2e
0.5 yielded strong increase of 7e9 fluorescence. Excellent
fitting of the titration data at high excess of ct-DNA over com-
pounds (r ꢂ 0.1) to Scatchard equation pointed to the exis-
tence of only one dominant type of complex and allowed
calculation of binding constants (log Ks, Table 4). The
obtained results (Table 4) point toward similar affinity of all
‘‘cyclic’’ compounds toward ct-DNA, which is also compara-
ble to previously studied analogues [26,33].
of ct-DNA at conditions of excess of compound over DNA
base pairs (ratio r > 0.5) (Table 3).
However, further additions of ct-DNA leading to excess of
DNA base pairs over compound (r < 0.5) resulted in hyper-
chromic effect and additional bathochromic shift of maxima
in the UVevis spectra of all studied compounds except 5.
Therefore, only for compound 5 it was possible to process
titration data by means of Scatchard equation to obtain binding
constant log Ks ¼ 5.6 and ratio n[5]/[ct-DNA] ¼ 0.5. All com-
pounds, whose UVevis spectra exhibit changes in opposite di-
rections depending on the compound/ct-DNA ratio, obviously
form at least two different types of complexes with ct-DNA.
Interestingly, at excess of ct-DNA over compound peculiar
UVevis maximum splitting can be observed exclusively for
‘‘cyclic’’ furan derivatives 7 and 8, but not for ‘‘acyclic’’ ana-
logues 4e6 as well as ‘‘cyclic’’ thiophene derivative 9 (Fig. 6).
Rather small changes in UVevis spectra as well as the
presence of at least two different types of complexes with
ct-DNA didn’t allow calculation of binding constants (log Ks)
for most of the compounds.
2.2.4. Thermal melting experiments
2.2.3. Fluorimetric titrations with ct-DNA
Interactions of compounds 4e9 with ct-DNA were also
studied by thermal melting experiments according to Table 5.
The ‘‘acyclic’’ furan derivative 4 does not cause any ther-
mal stabilization of ct-DNA, while its ‘‘cyclic’’ analogue 7 sta-
bilized DNA double helix. However, this is not the case for 5,
6, 8 and 9, since ‘‘acyclic’’ analogues 5 and 6 also thermally
stabilize ct-DNA. That observation could be correlated to dif-
ferences in the electronic absorption and fluorescence spectra
of 4 and 7 when compared to 5, 6, 8 and 9. That points toward
significant impact of electron-withdrawing group (eCN) as
well as exchange of furan with thiophene on the electronic
properties of compounds and consequently influences their
interactions with ct-DNA.
Addition of ct-DNA yielded strong increase of fluorescence
emission of ‘‘acyclic’’ derivatives, almost linearly dependent
to the c(ct-DNA) even at extremely high excess of base pairs
1000
9
7
800
600
400
8
6
5
2.2.5. Flow linear dichroism
200
Linear dichroism (LD) is the difference in absorption of
polarized light perpendicular and parallel to an orientation
axis. Since the electronic transitions of the DNA bases are per-
pendicular to the DNA helix axis they will give rise to a nega-
tive LD. Thus, intercalating ligands will also have a negative
LD whereas minor groove binding compounds have a positive
signal.
4
0
400
450
500
550
600
/ nm
Fig. 5. Fluorescence emission spectra of studied compounds, c(4,
7) ¼ 2.4 ꢀ 10ꢁ8 mol dmꢁ3, c(5, 6, 8, 9) ¼ 3.6e3.9 ꢀ 10ꢁ7 mol dmꢁ3
.

M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
2881
Table 3
Changes of UVevis spectra upon titration with ct-DNA
4c
7c
5
8c
6c
9c
Hb/%
8% decrease
up to r ¼ 0.7,
then increases
þ7 nm
51% decrease
up to r ¼ 1,
then increases
þ8 and þ15
maximum
splitting
10% decrease
10% decrease
up to r ¼ 2,
then increases
þ7 and þ11
maximum
splitting
18% decrease
up to r ¼ 0.38,
then increases
þ8 nm
15% decrease
up to r ¼ 0.5,
then increases
þ8 nm
Dla
þ7 nm
log Ks/n
e
e
5.6/0.5fix
e
e
e
a
Shift of the absorbance maximum; Dl ¼ l (complex) ꢁ l (compound); absorbance maxima l1 (4: l ¼ 366 nm, 7: l ¼ 362 nm, 5: l ¼ 370 nm, 8: l ¼ 367 nm, 6:
l ¼ 374 nm, 9: l ¼ 370 nm).
b
Hypochromic effect. H ¼ (Abs(compound) ꢁ Abs(complex))/Abs(compound) ꢀ 100.
c
Changes in opposite direction e first decrease, then increases.
Flow linear dichroism (LD) was used to determine the ori-
entation of the molecules bound to DNA. Flow LD spectra
were measured for the dyes in presence of calf thymus DNA
(ct-DNA) at two different mixing ratio, namely, dye/base
pair, of 1:24 and 1:4 (Fig. 8).
n ¼ 0.5 e ratio too high for intercalation but agreeable to
minor groove binding), while for 4 and 6 it was not possible
to estimate due to the mixed binding modes obvious in the
UVevis titrations and inconsistency of emission changes in
fluorimetric titrations. All ‘‘acyclic’’ analogues show positive
LD signal providing strong indication of DNA minor groove
binding. However, there is distinctive difference in CD exper-
iments between 4, 5 on one side and 6 on other side. Namely, 4
and 5 showed strong positive ICD signal also supporting minor
groove binding, while 6 showed ICD signal which is negative
e positive couplet e the origin of this is not clear yet but it
could be correlated to the fact that among the ‘‘acyclic’’ ana-
logues 6 yielded by far highest thermal stabilization of ct-
DNA.
The ‘‘cyclic’’ analogues 7e9 yielded considerable thermal
stabilization of ct-DNA. The UVevis and fluorimetric titra-
tions revealed that all ‘‘cyclic’’ compounds form at least two
different types of complexes with ct-DNA. Fortunately, in
fluorimetric titrations it was possible to collect enough data
at conditions of high excess of DNA over compounds. At
such conditions one type of complex was dominant, which al-
lowed calculation of binding constants by Scatchard equation
(log Ks, Table 4). Obtained log Ks values were similar for all
studied compounds as well as comparable to previously stud-
ied analogues [26,33]. The ‘‘cyclic’’ analogues 8 and 9 showed
very weak LD signal upon addition of ct-DNA indicating
a mixture of binding modes, which agreed nicely with results
of UVevis and fluorescence titrations collected close to equi-
molar compound/DNA ratios. The CD experiments revealed
quite different effect induced by each of ‘‘cyclic’’ analogues.
The addition of ct-DNA to 8 yielded almost no ICD signal
at l > 300 nm. Compound 7 showed positive ICD signal,
whose shape was dependent on the 7/base pair ratio. Com-
pound 9 showed weak, negative ICD signal only at high excess
of ct-DNA, pointing toward intercalative binding mode. Since
all other parameters of 7e9 interactions with ct-DNA were
generally comparable (affinity toward ct-DNA, changes in
fluorescence and UVevis spectra, DTm experiments), differ-
ences in CD experiments suggest that the cyclic compound 7
seems to have a clear preference for minor groove binding
(positive LD and ICD signals) [35], which is not the case
for the other two cyclic compounds (8 and 9). Possible reason
for that may be that the oxygen in the cyclic ring formed
From the LD measurements it was found that the acyclic
derivatives 4 and 5 showed a positive LD signal indicating
minor groove binding. Compound 6 showed only a weak LD
signal. All three of them showed a higher LD signal at the
higher ratio, 1:24 dye/base pair, of DNA. At the ratio 1:4
dye/base pair they showed a weaker LD signal indicating
that the dyes are bound in a more randomized way when
they are tightly packed in the DNA. The cyclic derivatives 8
and 9 showed almost no LD signal which indicates that the
molecules are bound in a random way at this mixing ratio.
If the cyclic derivatives 8 and 9 would only bind intercala-
tively the LD spectra should show a negative LD signal. Since
this is not the case one can assume that the dye binds in a ran-
domized way. Interestingly, the cyclic derivative 7 showed LD
signal, suggesting that it binds in the groove of DNA.
2.2.6. Circular dichroism
Upon binding of achiral molecules to chiral environments
such as the grooves of DNA achiral compounds show CD sig-
nal which is characteristic of their interaction with DNA.
Compared to the intercalation site between the base pairs,
the minor groove of DNA provides a more chiral environment
affording a larger ICD of minor groove binders.
Compounds 4, 5, and 7 that showed the highest LD signals
are also the compounds that show the highest ICD signal, ver-
ifying groove binding (Fig. 9).
2.2.7. Discussion of results
In the UVevis titrations all studied compounds except 5
showed mixed binding modes toward ct-DNA, with spectro-
scopic breakpoints at about r ¼ 0.2e2. However, there is dis-
tinct difference between ‘‘acyclic’’ and ‘‘cyclic’’ analogues,
UVevis spectra of latter showing much more pronounced
bathochromic and hypochromic effect upon addition with ct-
DNA.
The ‘‘acyclic’’ analogues 4e6 yield low or no thermal sta-
bilization of ct-DNA. The affinity toward DNA was estimated
only for 5 from the UVevis titration data (log Ks ¼ 5.6,

2882
M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
a 4
0.69
0.68
0.67
0.66
0.65
0.64
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
c(ct-DNA) / moldm-3
/ nm
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.6
0.5
0.4
0.3
0.2
0.1
0.0
b 7
c(ct-DNA) / moldm-3
/ nm
0.56
0.54
0.52
0.50
0.48
0.46
0.44
0.6
0.5
0.4
0.3
0.2
0.1
0.0
c 6
c(ctDNA) / moldm-3
/ nm
0.58
0.56
0.54
0.52
0.50
0.48
0.46
0.44
d 9
0.6
0.5
0.4
0.3
0.2
0.1
0.0
c(ctDNA) / moldm-3
/ nm
Fig. 6. UVevis spectral changes of (a)
4
(c ¼ 2.04 ꢀ 10ꢁ5 mol dmꢁ3), (b)
7
(c ¼ 1.26 ꢀ 10ꢁ5 mol dmꢁ3), (c)
6
(c ¼ 2.96 10ꢁ5 mol dmꢁ3
) and 9
(c ¼ 2.34 ꢀ 10ꢁ5 mol dmꢁ3) upon the addition of ct-DNA (c ¼ 6.31 ꢀ 10ꢁ3 mol dmꢁ3); sodium cacodylate/HCl buffer, I ¼ 0.05 mol dmꢁ3, pH 7.

M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
2883
a 4
1000
800
600
400
200
0
1000
800
600
400
200
0
c (ct-DNA) / moldm-3
/ nm
180
160
140
120
100
80
b 7
180
120
60
60
40
0
/ nm
c(ct-DNA) / moldm-3
c 5
800
700
600
500
400
300
200
100
0
800
600
400
200
0
c(ctDNA) / moldm-3
/ nm
700
600
500
400
300
200
d 8
700
600
500
400
300
200
100
0
/ nm
c(ctDNA) / moldm-3
Fig. 7. Fluorescence emission of (a)
4
(c ¼ 3.7 ꢀ 10ꢁ6 mol dmꢁ3), (b)
7
(c ¼ 5.3 ꢀ 10ꢁ6 mol dmꢁ3), (c)
5
(c ¼ 3.4 ꢀ 10ꢁ7 mol dmꢁ3
) and (d) 8
(c ¼ 2.9 ꢀ 10ꢁ6 mol dmꢁ3) upon the addition of ct-DNA (c ¼ 6.31 ꢀ 10ꢁ3 mol dmꢁ3); sodium cacodylate/HCl buffer, I ¼ 0.05 mol dmꢁ3, pH 7.

2884
M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
Table 4
Binding constants (log Ks) calculated from the fluorimetric titrationsa with ct-DNA at pH ¼ 7.0 (buffer sodium cacodylate, I ¼ 0.05 mol dmꢁ3
)
No.
4
7
5
8
6
9
log Ks
na
e
4.2
0.2
e
5.0
0.2
e
4.7
0.2
e
e
e
DIntb
Linear increase
after r ¼ 0.1
70% decrease up
to r ¼ 0.16 then
increases about 6ꢀ with
0.1 M NaCl similar
Linear
increase
60% decrease
up to r ¼ 0.5
then increases
50%
Linear increase
after r ¼ 0.01
61% decrease
up to r ¼ 0.16
then increases
9e13ꢀ
a
Titration data at r[compound]/[ct-DNA] < 0.1 were processed according to the Scatchard equation; the fixed value of ratio n[bound compound]/[ct-DNA] ¼ 0.2 was chosen
for easier comparison with previously obtained log Ks values [26,33].
b
Change of fluorescence; DInt ¼ Int(complex)/Int(compound).
hydrogen bonds with amino groups on DNA bases. Namely,
our previous results [34] pointed out that two compounds
with the only difference that one had a benzothiazole substit-
uent and the other one had a benzoxale substituent exhibited
different binding modes toward DNA. The oxygen derivative,
BOXTO, interacted with DNA in a more homogeneous way
whereas the sulphur analogue, BETO, exhibited more hetero-
genic interactions. Compound 9 according to the weak nega-
tive ICD signal [35] presumably binds dominantly by
intercalation at high excess of ct-DNA, while single binding
mode of compound 8 is not clearly defined, but most likely
binds by intercalation at high excess of ct-DNA.
such as Mebendazole [38] oncodazole and methyl-2-benzimi-
dazolecarbamate (Carbendazim, FB642) have also been inves-
tigated as potential antitumor drugs. Interestingly, the
antitumor activity of FB642 against MCF-7 breast tumor
xenografts in mice was among the highest for all tumor models
studied and was also better than either paclitaxel or vinorel-
bine. Although different modes of action of various benzimid-
azoles have been described in the literature (e.g. DNA groove
binding, topoisomerase I or II inhibition, interactions with
microtubules, inhibition of tumor helicases), selective growth
inhibitory activity toward breast cancer cells could also be ex-
plained by selective induction of cytochrome P450 (CYP-1)
enzymes especially in MCF-7 line, since it is known CYP-1
inducible cell line [39]. These enzymes catalyze the initial
step in either detoxification or bioactivation of environmental
toxins and xenobiotics [40]. The transcriptional regulation of
the CYP1A1 gene by polycyclic aromatic hydrocarbons
(PAH) and halogenated aromatic hydrocarbons (HAH) is
mediated via ligand-dependent activation of the aryl hydrocar-
bon receptor (AhR), which translocates to the nucleus upon
activation, dimerizes to the aryl hydrocarbon receptor nuclear
translocator (arnt) protein and binds to the xenobiotic response
element (XRE) in the regulatory region of the CYP1A1 gene
[41]. Although benzimidazole compounds do not conform to
the structural features of AhR ligands (planar hydrophobic
aromatic compounds and aryl amines) and, accordingly, do
not bind with high specificity to the AhR, it has been shown
that several sulphur-containing benzimidazole drugs induce
cytochrome P450 in humans and rodents. Among them are
some of the above-mentioned anthelmintic drugs [42]. There-
fore, the mechanism of induction of CYP1A by the benzimid-
azoles has been under debate and the results indicate that
benzimidazoles activate the AhR complex by a novel intracel-
lular mechanism distinct from those exerted by AhR ligands
and which may have components in common with an endog-
enous activation mechanism for the AhR. It is possible that
their degradation products constitute ligands of the AhR
[42]. Similar concept has been shown concerning the discov-
ery of a class of 2-(4-aminophenyl)benzothiazoles, candidate
antitumor agents that showed remarkably similar highly selec-
tive profiles of antitumor activity. Although these compounds
acted different from all known arylamines, a series of investi-
gation proved that CYP1A1 and CYP1B1 activation leads to
their toxicity through DNA [43].
2.3. Biological results
All tested compounds showed evident antiproliferative
effect on the presented panel cell lines (Table 6 and
Fig. 10AeF), with IC50 ranging from 1e85 mM. Except 4,
which showed in general non-selective growth inhibition of
all cell lines, all other compounds show very differential and
strong antiproliferative activity without obvious difference
depending on their structures. Compounds 6e9 showed no-
ticeable selectivity in regard to normal human fibroblasts
(WI 38), being clearly less cytotoxic to normal than to tumor
cell lines. Most interesting, however, is the high sensitivity of
MCF-7 tumor cell line to all compounds. We have already no-
ticed this phenomenon in our previous work [13] in which 2-
substituted-5-amidino-benzimidazoles were tested. The
observed selectivity toward breast cancer cells should be cor-
related to benzimidazole moiety, since similar apparent selec-
tivity of various benzimidazoles to breast cancer cells has also
been previously shown by other authors [36,37]. Moreover,
several compounds of benzimidazole class that have predom-
inantly been utilized as antifungal and antihelminthic agents,
Table 5
DTm valuesa (ꢃC) of ct-DNA upon addition of different ratios rb of 4e9 at
pH ¼ 7.0 (buffer sodium cacodylate, I ¼ 0.05 mol dmꢁ3
)
rb
4
7
5
8
6
9
0.1
0.2
0.3
a
0
0
0
1.8
2.6
3.8
0.6
0.8
1.3
1.0
1.4
1.6
1.7
2.3
2.7
3.9
4.9
5.8
Error in DTm: ꢄ0.5 ꢃC.
b
r ¼ [compound]/[polynucleotide].

M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
2885
Fig. 8. Flow LD spectra of compounds 4e9 in the presence of ct-DNA at two different mixing ratios, namely, dye/base pair, of 1:24 and 1:4.
3. Conclusions
the minor groove of DNA, although partial intercalation of 6
and 7 cannot be excluded. The ‘‘cyclic’’ analogues 8 and 9
at conditions of high excess of DNA over compounds concen-
tration (best presented in fluorimetric titrations) most likely
intercalate between DNA base pairs. However, for all studied
compounds close to equimolar conditions in respect to DNA
base pairs and in excess of compound over DNA, other bind-
ing modes are observed (decrease of UVevis and fluorescence
spectra, CD and LD spectroscopy, DTm experiments), most
probably as a result of agglomeration and stacking of the
non-intercalated positively charged molecules along the nega-
tively charged polynucleotide [44].
Here-presented results do not fit in our previously reported
concept of photoinduced anticancer therapy [33] because there
is no real difference in cytotoxicity against cell lines between
the ‘‘acyclic’’ and ‘‘cyclic’’ derivatives and in addition the
irradiation time necessary for photochemical dehydrocycliza-
tion is very long. Besides, it seems that, when isopropylami-
dino substituent [33] is attached directly to the naphthofuran
All tested compounds show very differential and strong
antitumor activity without apparent difference depending on
their structures. Compounds 6e9 showed noticeable selectivity
in regard to normal human fibroblasts (WI 38). The most interest-
ing observation is the high sensitivity of MCF-7 tumor cell line to
all compounds. We can conclude that the observed selectivity
toward breast cancer cells should be correlated to benzimidazole
moiety, which selectively induce cytochrome P450 (CYP-1)
enzymes especially in MCF-7 line, since it is known CYP-1
inducible cell line. These enzymes catalyze the initial step in
either detoxification or bioactivation of environmental toxins
and xenobiotics. Similar observations in MCF-7 cells treated
with various benzimidazoles were also noticed previously.
All studied compounds interact with ct-DNA by more bind-
ing modes, whose mutual distribution is dependent on the
compound/ct-DNA ratio. The ‘‘acyclic’’ analogues 4e6 as
well as cyclic’’ analogue 7 seem to interact mostly within

2886
M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
Fig. 9. CD spectra of compounds 4e9 in the presence of ct-DNA at two different mixing ratios, namely, dye/base pair, of 1:24 and 1:4.
moiety, the small planar molecule intercalates into the DNA,
while large isopropylamidinobenzimidazolyl substituent of
compounds 4e6 and 7e9 presented in this manuscript either
hampers intercalation (7) or allows mixed binding modes aside
DNA intercalation.
as symbols of elements, analytical results obtained are within
0.4% of the theoretical value.
4.2. General method for preparation of compounds 4e6
Solution of equimolar amounts of 4-N-isopropylamidino-
1,2-phenylenediamine, corresponding aldehydes 1e3 and p-
benzoquinone in absolute ethanol was refluxed for 4e5 h.
After reaction mixture was cooled to room temperature, dieth-
ylether was added and the crude product was filtered off. The
crude products were suspended in mixture of ethanoleacetone
or watereacetone several times, until the powder products
were analytically pure.
4. Experimental protocols
4.1. Chemistry
Melting points were recorded on an Original Kofler Mikro-
1
heitztisch apparatus (Reichert, Wien). The H and 13C NMR
spectra were recorded on a Varian Gemini 300 or a Bruker
Avance DPX 300 and 500 spectrometer at 300, 500 and
75 MHz, respectively. All NMR spectra were measured in
DMSO-d6 solutions using TMS as an internal standard. All
compounds were routinely checked by TLC with Merck silica
gel 60F-254 glass plates. Elemental analysis for carbon,
hydrogen and nitrogen was performed on a PerkineElmer
2400 elemental analyzer. Where analyses are indicated only
4.2.1. Methyl-E-3-[5-(5-N-isopropylamidino-2-benzimidaz
olyl)-2-furyl]-2-phenyl-acrylate hydrochloride (4) [25]
Compound 4 was prepared from 1 (1.01 g, 4.0 mmol), 4-N-
isopropylamidino-1,2-phenylenediamine (0.90 g, 4.0 mmol)
and p-benzoquinone (0.43 g, 4.0 mmol) in absolute EtOH
(25 ml), after refluxing for 4 h, introduction of HCl gas into

M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
2887
b 5
150
150
100
50
a 4
c 6
e 8
100
50
0
0
WI 38
HeLa
H 460
H 460
WI 38
HeLa
MCF-7
MCF-7
-50
-50
-100
SW 620
MiaPaCa-2
MiaPaCa-2
SW 620
-100
-9
-9
-9
-8
-7
-6
-5
log10 concentration (M)
-4
-4
-4
-3
-3
-3
-9
-9
-9
-8
-7
-6
-5
-4
log10 concentration (M)
-3
-3
-3
150
100
50
150
100
50
d 7
0
0
WI 38
HeLa
H 460
WI 38
HeLa
H 460
MCF-7
MCF-7
-50
-100
-50
-100
SW 620
MiaPaCa-2
SW 620
MiaPaCa-2
-8
-7
-6
-5
-8
-7
-6
-5
-4
log10 concentration (M)
log10 concentration (M)
150
100
50
150
100
50
f 9
0
0
WI 38
H 460
WI 38
HeLa
H 460
HeLa
MCF-7
MCF-7
-50
-100
-50
-100
SW 620
MiaPaCa-2
SW 620
MiaPaCa-2
-8
-7
log10 concentration (M)
-6
-5
-8
-7
log10 concentration (M)
-6
-5
-4
Fig. 10. Doseeresponse profiles for the compounds 4e9 tested on various human tumor 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 parallel samples in three individual experiments.
suspension of crude product in absolute ethanol and precipita-
tion from ethanoleacetone, in 0.95 g yield (41%) as slightly
green powder; m.p. 234e236 ꢃC (Ref. [25], m.p. 235e
237 ꢃC). 1H NMR (d/ppm) (300 MHz, DMSO-d6): 13.54
(s, NHbenzimidazole, 1H), 9.60 (s, NHamidine, 1H,), 9.57 (s,
NHamidine, 1H), 9.49 (br s, NHamidine, 1H), 8.01 (s, 1H), 7.77
(d, J ¼ 8.7 Hz, Harom, 1H), 7.73 (s, 1H), 7.61 (d, J ¼ 8.8 Hz,
Harom, 1H,), 7.54e7.46 (m, 3H), 7.38 (d, J ¼ 3.8 Hz, Hfuryl
,
1H), 7.31 (d, J ¼ 7.8 Hz, Harom, 2H), 5.71 (d, J ¼ 3.8 Hz,
Hfuryl, 1H), 4.07 (m, CH, 1H), 3.90 (s, OCH3, 3H), 1.34 (d,
J ¼ 6.2 Hz, CH(CH3)2, 6H); 13C NMR (d/ppm) (300 MHz,
DMSO-d6): 166.9 (s), 162.5 (s), 152.9 (s), 145.6 (s), 144.4
(s), 135.5 (s), 134.45 (s), 132.9 (s), 129.6 (d, 2C), 129.3 (d),
129.1 (d, 2C), 127.1 (d), 124.7 (d), 124.2 (s), 119.87
(s), 116.8 (d), 116.6 (d), 116.2 (d), 115.3 (d), 53.1 (q),
45.7 (d), 21.9 (q, 2C). Elemental analysis calcd (%) for
C25H27N4O3Cl3$3H2O: C 50.71; H 5.62; N 9.43. Found: C
50.34; H 5.68; N 9.04.
Table 6
In vitro growth inhibition of various tumor cell lines
a
Compound IC50 (mM)
WI 38 HeLa
MiaPaCa-2 SW 620 MCF-7 H 460
4
5
6
7
8
9
13b
17 ꢄ 0.2 19 ꢄ 0.7
14 ꢄ 0.9 9 ꢄ 2
13 ꢄ 0.1
31 ꢄ 0.1
36 ꢄ 20 28 ꢄ 13.5 21 ꢄ 13
62 ꢄ 32
2 ꢄ 2
>100
>100
>100
30 ꢄ 28.6 13 ꢄ 5
62 ꢄ 36.9 1 ꢄ 0.2 37 ꢄ 1.6
35 ꢄ 23.8 1 ꢄ 0.6 18 ꢄ 0.2
4.2.2. Ethyl-E-3-[5-(5-N-isopropylamidino-2-benzimidaz
olyl)-2-furyl]-2-(4-cyano)phenyl-acrylate hydrochloride (5)
Compound 5 was prepared from 2 (0.20 g, 0.68 mmol), 4-
N-isopropylamidino-1,2-phenylenediamine (0.090 g, 0.68 mmol)
and p-benzoquinone (0.073 g, 0.68 mmol) in absolute EtOH
18 ꢄ 12
72 ꢄ 29
21 ꢄ 4.2
61 ꢄ 40
85 ꢄ 10
29 ꢄ 15
IC50 e the concentration that causes 50% growth inhibition.
6 ꢄ 2
>100
71 ꢄ 14 15 ꢄ 0.5 16 ꢄ 3
2 ꢄ 0.7 24 ꢄ 8
a
b
Only one experiment was performed.

2888
M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
(10 ml), after refluxing for 5 h and recrystallization from etha-
noleacetone, in 0.096 g yield (30%) as yellow powder; m.p.
210e212 ꢃC. 1H NMR (d/ppm) (300 MHz, DMSO-d6):
13.70 (s, NHbenzimidazole, 1H), 9.53 (s, NHamidine, 1H), 9.40
(s, NHamidine, 1H), 8.98 (br s, NHamidine, 1H), 8.03 (s, 1H),
7.96 (d, J ¼ 7.86 Hz, Harom, 2H), 7.76 (s, 1H), 7.68 (d, 1H,
J ¼ 8.70 Hz, Harom), 7.57 (d, J ¼ 8.60 Hz, Harom, 1H), 7.55
(d, J ¼ 7.65 Hz, 2H), 7.32 (d, J ¼ 3.76 Hz, Hfuryl, 1H), 6.13
(d, J ¼ 3.76 Hz, Hfuryl, 1H), 4.21 (q, J ¼ 6.87 Hz, OCH2CH3,
2H), 4.09e4.04 (m, CH, 1H), 1.29 (d, J ¼ 6.18 Hz,
CH(CH3)2, 6H), 1.23 (t, J ¼ 7.17 Hz, OCH2CH3, 3H). Ele-
mental analysis calcd (%) for C27H26ClN5O3: C 64.35; H
5.20; N 13.90. Found: C 64.41; H 5.12; N 13.87.
as yellow powder; m.p. 256e258 ꢃC (Ref. [25], m.p. 255e
1
257 ꢃC). H NMR (d/ppm) (300 MHz, DMSO-d6): 13.65 (s,
NHbenzimidazole, 1H), 9.62 (br s, NHamidine, 1H), 9.52 (s,
NHamidine, 1H), 9.07 (s, NHamidine, 1H), 8.88 (d, J ¼ 8.71 Hz,
Harom, 1H), 8.63 (s, 1H), 8.50 (d, J ¼ 8.40 Hz, Harom, 1H),
8.47 (d, J ¼ 7.60 Hz, Harom, 1H), 8.11 (s, 1H), 7.87e7.70
(m, 3H), 7.63 (br s, 1H), 4.46 (m, CH, 1H), 4.07 (s, OCH3,
3H), 1.24 (d, J ¼ 6.21 Hz, CH(CH3)2, 6H); 13C NMR (d/
ppm) (300 MHz, DMSO-d6): 167.5 (s), 162.7 (s), 158.9 (s),
151.2 (s), 148.7 (s), 145.6 (s), 143.8 (s), 141.7 (s), 128.3 (d),
128.1 (s), 127.5 (d), 127.0 (d), 126.3 (d), 124.9 (s), 124.3
(d), 123.7 (d), 117.3 (s), 116.3 (d), 115.7 (d), 114.4 (d), 53.1
(q), 45.6 (d), 21.9 (q, 2C). Elemental analysis calcd (%) for
C25H24N4O3Cl2$2H2O: C 56.12; H 5.27; N 10.47. Found: C
56.30; H 5.16; N 10.72.
4.2.3. Methyl-E-3-[5-(5-N-isopropylamidino-2-benzimidazo
lyl)-2-thienyl]-2-phenyl-acrylate hydrochloride (6)
Compound 6 was prepared from 3 (0.60 g, 2.2 mmol), 4-N-
isopropylamidino-1,2-phenylenediamine (0.50 g, 2.2 mmol)
and p-benzoquinone (0.24 g, 2.2 mmol) in absolute EtOH
(20 ml), after refluxing for 4 h and recrystallization from
watereacetone, in 0.52 g yield (51%) as green powder; m.p.
229e231 ꢃC. 1H NMR (d/ppm) (300 MHz, DMSO-d6):
13.98 (br s, NHbenzimidazole, 1H), 9.49 (s, NHamidine, 1H),
9.34 (s, NHamidine, 1H), 8.99 (br s, NHamidine, 1H), 8.08 (s,
1H), 7.99e7.92 (m, 2H), 7.70 (br s, 1H), 7.64 (d,
J ¼ 4.05 Hz, Hthienyl, 1H), 7.56e7.54 (m, 1H), 7.53 (d,
J ¼ 7.80 Hz, Harom, 2H), 7.51 (d, J ¼ 3.20 Hz, Hthienyl, 1H),
7.30e7.27 (m, 2H), 4.12e4.09 (m, CH, 1H), 3.30 (s, OCH3,
3H), 2.05 (d, J ¼ 6.10 Hz, CH(CH3)2, 6H); 13C NMR (d/
ppm) (300 MHz, DMSO-d6): 169.20 (s), 167.27 (s), 162.74
(s), 161.09 (s), 143.34 (s), 136.45 (s), 136.37 (d), 136.08 (s),
134.72 (s), 134.22 (s), 133.26 (d), 129.55 (s), 129.08 (d),
128.50 (d), 127.99 (d), 126.59 (d), 123.88 (d), 122.97 (d),
122.47 (s), 119.20 (d), 112.53 (d), 112.01 (d), 52.74 (q),
45.46 (d), 21.73 (q, 2C). Elemental analysis calcd (%) for
C25H25N4O2SCl: C 62.42; H 5.24; N 11.65. Found: C 62.30;
H 5.48; N 11.84; S 6.92.
4.3.2. Ethyl 2-[(5-N-isopropylamidino)benzimidazolyl]-4-
cyanonaphtho[2,1-b]furan-5-carboxylate hydrochloride (8)
Compound 8 was prepared from solution of 5 (0.07 g,
0.15 mmol) in ethanol (25 ml), after irradiation for 80 h and
recrystallization from ethanolediethylether, in 0.030 g yield
(42%) as yellow powder; m.p. 229e231 ꢃC. 1H NMR (d/
ppm) (300 MHz, DMSO-d6): 14.25 (s, NHbenzimidazole, 1H),
9.62 (br s, NHamidine, 2H), 9.44 (s, NHamidine, 1H), 9.22 (s,
1H), 9.05 (d, J ¼ 8.70 Hz, Harom, 1H), 9.03 (s, 1H), 8.64 (s,
2H), 7.99 (d, J ¼ 8.65 Hz, Harom, 1H), 7.79 (br s, 1H), 7.64
(d, J ¼ 8.00 Hz, Harom, 1H), 4.45 (q, J ¼ 7.00 Hz, OCH2CH3,
2H), 4.05e4.02 (m, CH, 1H), 3.40 (s, OCH3, 3H), 1.40 (t,
J ¼ 6.90 Hz, OCH2CH3, 3H), 1.26 (d, J ¼ 6.15 Hz,
CH(CH3)2, 6H); 13C NMR (d/ppm) (300 MHz, DMSO-d6):
166.40 (s), 162.99 (s), 162.81 (s), 151.59 (s), 149.90 (s),
143.20 (s), 131.07 (d), 128.32 (s), 128.30 (s), 128.27 (d),
128.21 (d), 128.18 (d), 127.33 (s), 126.02 (s), 125.68 (s),
125.40 (d), 121.88 (d), 119.27 (s), 119.16 (d), 119.05 (d),
110.20 (s), 109.80 (d), 62.14 (t), 53.32 (q), 45.53 (d), 27.74
(d, 2C). Elemental analysis calcd (%) for C26H22N5O3Cl: C
64.00; H 4.54; N 14.35. Found: C 64.20; H 4.36; N 14.62.
4.3. General method for the preparation of compounds
7e9
4.3.3. Methyl 2-[(5-N-isopropylamidino)benzimidazolyl]
naphtho[2,1-b]thiophen-5-carboxylate hydrochloride (9)
Compound 9 was prepared from solution of 6 (0.10 g,
0.21 mmol) in ethanol (30 ml), after irradiation for 40 h and
precipitation from ethanolediethylether, in 0.60 g yield
(59%) as yellow powder; m.p. 247e249 ꢃC. 1H NMR (d/
ppm) (300 MHz, DMSO-d6): 14.35 (s, NHbenzimidazole, 1H),
9.60 (br s, NHamidine, 1H), 9.47 (s, NHamidine, 1H), 9.32 (s,
Ethanolic solutions of compounds 4e6 and small amount
of iodine (5%) were irradiated at room temperature, with
400-W high-pressure mercury lamp, using a Pyrex filter for
about 20e80 h until the UV spectra show 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 resulting prod-
ucts were filtered off and washed with diethylether. After pre-
cipitating from ethanolediethylether or recrystallization from
acetoneewater, powder products were obtained.
NHamidine, 1H), 9.04 (s, 1H), 8.87 (d, J ¼ 8.28 Hz, Harom
,
1H), 8.82 (s, 1H), 8.51 (d, J ¼ 8.20 Hz, Harom, 1H), 8.11 (d,
J ¼ 7.60 Hz, Harom, 1H), 7.87e7.72 (m, 2H), 7.83 (s, 1H),
7.58 (br s, 1H), 4.12e4.08 (m, CH, 1H), 3.98 (s, OCH3,
3H), 1.31 (d, J ¼ 6.10 Hz, CH(CH3)2, 6H); 13C NMR (d/
ppm) (300 MHz, DMSO-d6): 167.01 (s), 162.34 (s), 162.32
(s), 154.36 (s), 147.82 (s), 139.12 (s), 136.53 (s), 136.35 (s),
133.56 (s), 129.09 (s), 128.04 (s), 127.59 (d), 127.55 (d),
126.31 (d), 125.34 (d), 124.95 (s), 123.96 (d), 121.62 (s),
119.80 (d), 118.65 (s), 117.63 (d), 115.38 (d), 52.40 (q),
45.02 (d), 21.28 (q, 2C). Elemental analysis calcd (%) for
4.3.1. Methyl 2-[(5-N-isopropylamidino)benzimidazolyl]
naphtho[2,1-b]furan-5-carboxylate hydrochloride (7) [25]
Compound 7 was prepared from solution of 4 (0.90 g,
2.0 mmol) in ethanol (140 ml), after irradiation for 20 h and
recrystallization from acetoneewater, in 0.37 g yield (35%)

M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
2889
C25H23N4O2SCl: C 62.69; H 4.84; N 11.70. Found: C 62.80; H
4.99; N 11.86.
dilutions (10ꢁ8e10ꢁ4 M) and incubated for a further 72 h.
Working dilutions were freshly prepared on the day of testing.
The solvent (DMSO) was also tested for eventual inhibitory
activity by adjusting its concentration to be the same as in
working concentrations. After 72 h of incubation the cell
growth rate was evaluated by performing the MTT assay,
which detects dehydrogenase activity in viable cells, as
described previously [23,26].
4.4. Interactions with DNA
The electronic absorption spectra were recorded on Varian
Cary 100 Bio spectrometer and fluorescence emission spectra
were recorded on Varian Eclipse fluorimeter, in both cases us-
ing quartz cuvettes (1 cm). The calf thymus DNA (ct-DNA)
was purchased from Aldrich, dissolved in the sodium cacody-
late buffer, I ¼ 0.05 mol dmꢁ3, pH ¼ 7, additionally sonicated
and filtered through a 0.45 mm filter, and the concentration of
corresponding solution was determined spectroscopically as
the concentration of phosphates [45]. The measurements
were performed in the aqueous buffer solution (pH ¼ 7.0;
sodium cacodylate buffer, I ¼ 0.05 mol dmꢁ3). Under the
experimental conditions used the absorbance and fluorescence
intensities of studied compounds were proportional to their
concentrations. Obtained data were corrected for dilution. In
fluorimetric titrations, excitation wavelengths of 365 (4), 370
(5), 374 (6), 360 (7), 368 (8) and 370 (9) nm were 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 con-
stant (Ks) and [bound compound]/[polynucleotide phosphate]
ratio (n) were calculated according to the Scatchard equation
by non-linear least-square fitting [46], giving excellent corre-
lation coefficients (>0.999) for obtained values of 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. The heating rate was 1 ꢃC/min. The absor-
bance 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 DTm values were calculated subtracting Tm of
the free nucleic acid from Tm of complex. Every DTm value
here reported was the average of at least two measurements,
the error in DTm is ꢄ0.5 ꢃC. The flow LD and CD spectra
were recorded on a JASCO-720 spectropolarimeter.
Acknowledgments
This study was supported by grants 125-0982464-1356,
098-0982464-2514, 098-0982914-2918 by the Ministry of
Science, Education and Sports of the Republic of Croatia.
References
[1] C. Bailly, Curr. Med. Chem. 7 (2007) 39e58.
[2] M. Demeunynck, C. Bailly, W.D. Wilson, DNA and RNA Binders. From
Small Molecules to Drugs, Wiley-VCH, Weinheim, 2002.
[3] R.B. Silverman, The Organic Chemistry of Drug Design and Drug
Action, second ed. Elsevier Academic Press, Burlington, San Diego,
USA, London, UK, 2004.
[4] R. Langer, Science 293 (2001) 58e59.
[5] D. Lednicer, Strategies for Organic Drug Synthesis and Design, John
Wiley & Sons, New York, 1998.
[6] A.R. Katritzky, D.O. Tymoshenko, D. Monteux, V. Vvedensky,
G. Nikonov, C.B. Cooper, M. Deshpande, J. Org. Chem. 65 (2000)
8059e8062.
´
´
[7] V. Martınez, V. Burgos, J. Alvarez-Builla, G. Fernandez, A. Domingo,
R. Garcia-Nieto, F. Gago, I. Manzanares, C. Cuevas, J.J. Vaquero, J.
Med. Chem. 47 (2004) 1136e1148.
[8] S. Demirayak, U. Abu Mohsen, A. Cagri Karaburun, Eur. J. Med. Chem.
37 (2002) 255e260.
[9] M. Boiani, M. Gonzalez, Mini Rev. Med. Chem. 5 (2005) 409e424.
¨
[10] H. Goker, S. Ozden, S. Yıldız, D.W. Boykin, Eur. J. Med. Chem. 40
¨
(2005) 1062e1069.
[11] M.A. Ismail, R. Brun, T. Wenzler, F.A. Tanious, W.D. Wilson,
D.W. Boykin, Bioorg. Med. Chem. 12 (2004) 5405e5413.
[12] H. Goker, C. Kus, D.W. Boykin, S. Yıldız, N. Altanlar, Bioorg. Med.
¨
Chem. 10 (2002) 2589e2596.
ꢀ
´
´
[13] K. Starcevic, M. Kralj, K. Ester, I. Sabol, M. Grce, K. Pavelic,
G. Karminski-Zamola, Bioorg. Med. Chem. 15 (2007) 4419e4426.
[14] T. Fonseca, B. Gigante, M.M. Marques, L.T. Gilchrist, E. De Clercq,
Bioorg. Med. Chem. 12 (2004) 103e112.
[15] D. Shugar, Pharmacol. Ther. 82 (1999) 315e335.
[16] R.J. Snow, et al., Bioorg. Med. Chem. Lett. 17 (2007) 3660e3666.
[17] G. Lemaire, C. Delescluse, M. Pralavorio, N. Ledirac, P. Lesca,
R. Rahmani, Life Sci. 74 (2004) 2265e2278.
4.5. Antitumor evaluation assays
[18] (a) P. Karlovsky, A.W. De Cock, Anal. Biochem. 194 (1991)
192e197;
The HeLa (cervical carcinoma), MiaPaCa-2 (pancreatic
carcinoma), SW 620 (colon carcinoma), MCF-7 (breast carci-
noma), H 460 (lung carcinoma), and WI 38 (normal fibro-
blasts) cells were cultured as monolayers and maintained in
Dulbecco’s modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), 2 mM L-glutamine,
100 U/ml penicillin and 100 mg/ml streptomycin in a humidi-
fied atmosphere with 5% CO2 at 37 ꢃC. The cell lines were
inoculated onto a series of 96-well microtiter plates on day
0, at 1e3 ꢀ 104 cells/ml, depending on the doubling times of
specific cell line. Test agents were then added in five 10-fold
(b) A. Abu-Daya, P.M. Brown, K.R. Fox, Nucleic Acid Res. 23 (1995)
3385e3392.
[19] S. Murata, J. Kusba, G. Piszczek, I. Gryczynski, J. Lakowicz, Biopoly-
mers 57 (2000) 306e315.
[20] T.C. Jenkins, Curr. Med. Chem. 7 (2000) 99e115.
[21] M.P. Singh, T. Joseph, S. Kumar, Y. Bathini, J.W. Lown, Chem. Res. Tox-
icol. 5 (1992) 597e607.
[22] (a) T.A. Fairley, R.R. Tidwell, I. Donkor, N.A. Naiman, K.A. Ohemeng,
R.J. Lombardy, J.A. Bentley, M. Cory, J. Med. Chem. 36 (1993) 1746e
1753;
(b) S. Wang, J.E. Hall, F.A. Tanious, W.D. Wilson, D.A. Patrick,
D.R. McCurdy, B.C. Bender, R.R. Tidwell, Eur. J. Med. Chem. 34
(1999) 215e224.

2890
M. Hranjec et al. / European Journal of Medicinal Chemistry 43 (2008) 2877e2890
ˇ
[23] I. Jarak, M. Kralj, L. Suman, G. Pavlovic, J. Dogan Koruznjak,
´
I. Piantanida, M. Zinic, K. Pavelic, G. Karminski-Zamola, J. Med.
ꢀ
[34] H.J. Karlsson, M.H. Berquist, P. Lincoln, G. Westman, Bioorg. Med.
Chem. 12 (2004) 2369e2384.
ˇ
´
´
´
[35] M. Eriksson, B. Norden, Methods Enzymol. 340 (2001) 68e98.
Chem. 48 (2005) 2346e2360.
[24] W.D. Wilson, B. Nguyen, F.A. Tanious, A. Mathis, J.E. Hall,
C.E. Stephens, D.W. Boykin, Curr. Med. Chem. Anticancer Agents 5
(2005) 389e408.
[36] A. Seaton, C. Higgins, J. Mann, A. Baron, C. Bailly, S. Neidle, H. Van
den Berg, Eur. J. Cancer 39 (2003) 2548e2555.
[37] M.M. Ramla, M.A. Omar, A.-M.M. El-Khamry, H.I. El-Diwani, Bioorg.
Med. Chem. 14 (2006) 7324e7332.
ˇ
[25] M. Hranjec, M. Grdisa, K. Pavelic, D.W. Boykin, G. Karminski-Zamola,
´
Il Farmaco 58 (2003) 1319e1324.
ˇ
[26] (a) K. Starcevic, M. Kralj, I. Piantanida, L. Suman, K. Pavelic,
[38] T. Mukhopadhyay, J. Sasaki, R. Ramesh, J.A. Roth, Clin. Cancer Res. 8
(2002) 2963e2969.
ꢀ
´
G. Karminski-Zamola, Eur. J. Med. Chem. 41 (2006) 925e939;
´
[39] M. Iwanari, M. Nakajima, R. Kizu, K. Hayakawa, T. Yokoi, Arch.
Toxicol. 76 (2002) 287e298.
´
ꢀ ´
(b) G. Karminski-Zamola, D. Palanovic, K. Jakopcic, Croat. Chem. Acta
49 (1977) 521e525.
[40] M.S. Chua, E. Kashiyama, T.D. Bradshaw, S.F. Stinson, E. Brantley,
E. Sausville, M.F.G. Stevens, Cancer Res. 60 (2000) 5196e5203.
[41] O. Hankinson, Annu. Rev. Pharmacol. Toxicol. 35 (1995) 307e340.
[42] M. Backlund, L. Weidolf, M. Ingelman-Sundberg, Eur. J. Biochem. 261
(1999) 66e71.
[27] G.V. Boyd, in: Saul Patai (Ed.), Recent Advances in the Synthesis of
Amidines, The Chemistry of Amidines and Imidates, vol. 2, John Willey
& Sons, New York, 1991, p. 339.
´
[28] M. Bajic, G. Karminski-Zamola, Croat. Chem. Acta 65 (1992) 835e
846.
[29] J.K.F. Geirsson, A.J. Kvaran, Photochem. Phothobiol. A: Chem. 144
[43] A. Monks, E. Harris, C. Hose, J. Connelly, E.A. Sausville, Mol. Pharma-
col. 63 (2003) 766e772.
(2001) 175e177.
´
[44] (a) M.K. Pall, J.K. Ghosh, Spectrochim. Acta 51 (1995) 489e498;
(b) H.W. Zimmermann, Angew. Chem., Int. Ed. Engl. 25 (1986) 115.
[45] (a) J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982)
3933e3940;
ꢀ
[30] K. Starcevic, D.W. Boykin, G. Karminski-Zamola, Heterocycl. Commun.
8 (2002) 221e226.
ˇ
´
ꢀ ´
[31] G. Karminski-Zamola, L. Fiser-Jakic, K. Jakopcic, Tetrahedron 38
ˇ
´
(1982) 1329e1335.
[32] F.B. Mallory, C.W. Malory, Org. React. 30 (1984) 1e456.
(b) B.S. Palm, I. Piantanida, M. Zinic, H.J. Schneider, J. Chem. Soc.,
Perkin Trans. 2 (2000) 385e392.
ˇ
ˇ
[33] K. Starcevic, G. Karminski-Zamola, I. Piantanida, M. Zinic, L. Suman,
ꢀ
M. Kralj, J. Am. Chem. Soc. 127 (2005) 1074e1075.
´
´
[46] (a) G. Scatchard, Ann. N.Y. Acad. Sci. 51 (1949) 660e672;
(b) J.D. McGhee, P.H. von Hippel, J. Mol. Biol. 103 (1976) 679.