Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines linked by 3,4-ethylenedioxythiophene

Article abstract of DOI:10.1016/j.ejmech.2010.12.010

A series of novel 2,5-bis(amidinophenyl)-3,4-ethylenedioxythiophenes (5-10 and 15) has been synthesized. Compounds 5-10 bind to the DNA minor groove as the dominant binding site and strongly stabilize the double helix of ct-DNA. Surprisingly, the same com

Full text of DOI:10.1016/j.ejmech.2010.12.010

European Journal of Medicinal Chemistry 46 (2011) 743e755
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, DNA/RNA affinity and antitumour activity of new aromatic diamidines
linked by 3,4-ethylenedioxythiophene
,
1
,
1
c
b
,
d
b,e,
*
ꢀ a
ꢁ
ꢀ b
ꢁ
ꢁ
Ivana Stolic , Katarina Miskovic , Ivo Piantanida , Mirela Baus Loncar , Ljubica Glavas-Obrovac
,
ꢀ a
,
**
Miroslav Bajic
a Department of Chemistry and Biochemistry, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia
b Laboratory of Functional Genomics and Tissue Culture, School of Medicine, J. J. Strossmayer University of Osijek, 31000 Osijek, Croatia
c
ꢁ
ꢀ
Division of Organic Chemistry and Biochemistry, RuCer Boskovic Institute, Zagreb, Croatia
d
ꢁ
ꢀ
Division of Molecular Medicine, RuCer Boskovic Institute, Zagreb, Croatia
e Clinical Institute of Nuclear Medicine and Rad. Protection, Clinical Hospital Centre Osijek, Osijek, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of novel 2,5-bis(amidinophenyl)-3,4-ethylenedioxythiophenes (5e10 and 15) has been
Received 23 September 2010
Received in revised form
4 December 2010
Accepted 14 December 2010
Available online 21 December 2010
synthesized. Compounds 5e10 bind to the DNA minor groove as the dominant binding site and strongly
stabilize the double helix of ct-DNA. Surprisingly, the same compounds also thermally stabilize ds-RNA,
whereby most of them form stacked dimers along the RNA double helix. The only exception is compound
15 which, due to its structural features, showed no interaction with DNA or RNA. Compounds 5e10 have
shown a moderate to strong cytotoxic effect (GI50 ¼ 1.5e9.0
mM) on a panel of seven tumour cell lines.
The diimidazoline derivative 9, due to its highest inhibitory potential on the growth of all tested tumour
cell lines, was investigated in more detail by testing its ability to enter into cells and influence the cell
Keywords:
Diphenylamidine
3,4-Ethylenedioxythiophene
DNA/RNA binding
Antitumour activity
cycle. Compound 9 (5
period, followed by nuclear localization upon 90-min incubation. Significant arrest in HeLa cells in the
G2/M phase, shown by cell cycle analysis at an equitoxic (50 M) concentration, suggests interaction of
a studied compound with cellular DNA as the main mode of biological action.
Ó 2010 Elsevier Masson SAS. All rights reserved.
mM) was internalized successfully in cell cytoplasm during a 30-min incubation
m
1. Introduction
topoisomerases, has been confirmed by a number of studies per-
formed over the last few decades [10e18].
With the aging of the world’s population, changes in dietary
habits and the increasing environmental pollution, cancer has
emerged as the top threat to human life worldwide [1]. A major
goal in the current development of novel chemotherapeutics is to
maximize therapeutic responses by increasing their selectivity [2].
One of the approaches is synthesis of small organic molecules with
the ability to recognize and bind to specific DNA sequences [3].
The most studied DNA minor groove binders are aromatic dia-
midines [4e9]. The ability of this class of compounds to interfere
with processes vital to cell survival, e.g. replication and transcrip-
tion, by competing with proteins such as transcription factors and
The amidinium moiety is known to contribute to the DNA
binding of small molecules as well as to the recognition of targeted
DNA sequences by electrostatic, van der Waals and hydrogen
bonding interactions [5,8]. Although DNA binding is clearly
involved, not all diamidines are biologically active. For this reason,
it is important to understand how small changes in the structure of
a novel compound affect its biological activity.
In our search for analogues with improved DNA binding and
antitumour activity, we have focused on bisarylamidines, with an
extended thiophene core, namely 3,4-ethylenedioxythiophene or
benzo[c]thiophenes, as the central linker [10,19]. We presumed that
such a structural change could improve affinity and selectivity of
novel molecules for specific DNA sequences by increasing their
electron-donating capabilities and potential for hydrogen bonding
and van der Waals interactions, as it has been shown by studies of
Boykin and co-workers [9,20e23].
* Corresponding author. Laboratory of Functional Genomics and Tissue Culture,
School of Medicine, J. J. Strossmayer University of Osijek, 31000 Osijek, Croatia. Fax:
þ385 31 512 227.
** Corresponding author. Tel.: þ385 1 2390300; fax: þ385 1 2441390.
In our previous paper, the effect of 3,4-ethylenedioxy-extension
of thiophene core on the DNA/RNA binding properties and bio-
logical activity of bisbenzimidazole amidines was described [10].
ꢁ
E-mail addresses: glavas-obrovac.ljubica@kbo.hr (L. Glavas-Obrovac), mbajic@
ꢀ
vef.hr (M. Bajic).
1
These authors are contributed equally to this work.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.12.010

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744
Scheme 3. Reagents: (i) NH4OH, chloroform; (ii) POCl3, DMF, Py; (iii) 1. HCl(g)/
methanol, 2. ReNH2, methanol.
2,5-Bis(N-phenylamidine)-3,4-ethylenedioxythiophene dihy-
drochloride (15) was prepared from dicarboxylic acid (11) [25] as
outlined in Scheme 2. An attempt to synthesize bis(N-phenyl-
amidine) at the 2- and 5-positions on the thiophene ring, employing
the Pinner reaction from 3,4-ethylenedioxythiophene-2,5-dinitrile
(17), failed (Scheme 3), so an alternative way of synthesis was
employed. Diacid 11 was allowed to react with thionyl chloride to
give diacid chloride 12, which was converted by reaction with
aniline to the corresponding dicarboxanilide 13. Diamide was
transformed to diamidine 15 via a two-step process that involved
conversion of the former to a diimidoyl chloride derivative using
thionyl chloride, followed by reaction with anhydrous ammonia.
Scheme 1. Reagents: (i) 1. n-BuLi, THF, 2. Bu3SnCl; (ii) Pd(PPh3)4, 4-bromobenzonitrile,
THF; (iii) HCl(g)/methanol, dioxane; (iv) 1. R1eNH2, methanol, 2. HCl(g)/ethanol.
In this series of compounds, the most promising biological activity
was shown by the alkyl substituted amidine derivative. Following
these results, we report here the synthesis, DNA/RNA binding
properties and antitumour activities of 2,5-bis(4-amidinophenyl)-
3,4-ethylenedioxythiophene derivatives with alkyl substituted or
cyclic amidine at terminal positions. In addition, we have also
synthesized compound 15 with amidino moieties attached directly
to 2- and 5-positions of 3,4-ethylenedioxythiophene linker and
phenyl at terminal positions to compare the impact of the amidine
moiety position on DNA affinity and biological properties.
2.2. Spectroscopy
In order to study the spectroscopic properties of compounds
5e10 and 15, their UVeVis (Fig.1, Table 1) and fluorescence emission
spectra (Fig. 2) were studied. Linear dependence of UVeVis spectra
on the concentration of all the studied compounds in the range c
(5e10, 15) ¼ 2.3e5.5 ꢀ 10L5 mol dmꢁ3 suggested the absence of
intermolecular interactions between two or more molecules of any
of the compounds. Furthermore, the UVeVis spectra of 5e10 and 15
revealed negligible temperature dependent changes (25e90 ꢂC)
and excellent reproducibility upon cooling to 25 ꢂC. Aqueous solu-
tions of all compounds were stable for at least one month.
2. Results and discussion
2.1. Chemistry
Comparison of the UV spectra of prepared compounds indicated
that substitution of an alkyl group on the amidino nitrogen of parent
compound 5 resulted in a batochromic shift in the UVeVis spectrum
of 9. An intriguing hypsochromic shift of the absorption maximum
of compound 15 compared to compound 5 can be attributed to the
direct attachment of amidine to the thiophene core.
All the studied compounds exhibited characteristic fluorescence
emission, with maxima at 455e470 nm. Comparison of fluores-
cence spectra (Fig. 2) revealed the influence of structure on
Scheme 1 depicts the multi-step synthesis used to obtain dia-
midines 5e10. The method generally followed the established route
[24], involving the preparation of dinitrile and its subsequent
conversion into diamidines. The key intermediate, 2,5-bis(4-cya-
nophenyl)-3,4-ethylenedioxythiophene (3), was prepared in two
steps starting from 3,4-ethylenedioxythiophene (1). The first step
involved lithiation of compound 1 with n-butyllithium in THF at
room temperature, followed by addition of tributyltin chloride to
give the corresponding organotin derivative 2. In the second step,
a Stille cross-coupling reaction between compound 2 and 4-bro-
mobenzonitrile, using a catalytic amount of tetrakis(triphenyl-
phosphine) palladium, yielded dinitrile 3. The latter was
transformed, by treatment with methanolic HCl in dioxane, into the
corresponding bisimidate ester 4, which was used directly without
further purification and characterization to make the amidines. The
dicationic amidine derivatives 5e10 were obtained by reaction of
an imidate ester with appropriate amines or diamines in methanol.
Scheme 2. Reagents: (i) SOCl2, DMF, benzene; (ii) aniline, chloroform; (iii) SOCl2, DMF,
chloroform; (iv) 1. NH3, chloroform, 2. HCl(g)/ethanol.
Fig. 1. UVeVis spectra of 5e10, and 15 at c ¼ 2.28e4.61 ꢀ 10ꢁ5 mol dmꢁ3; pH 7.0,
sodium cacodylate/HCl buffer, I ¼ 0.05 mol dmꢁ3
.

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745
Table 1
Electronic absorption data of 5e10 and 15.a
lmax/nm
e
ꢀ 103/dm3 molꢁ1 cmꢁ1
5
6
7
8
9
10
15
376
371
371
372
387
372
315
45.91
49.35
47.47
46.61
59.66
57.11
22.02
a
Sodium cacodylate buffer, pH 7.0, I ¼ 0.05 mol dmꢁ3
.
emission of the compounds. It is interesting to note that alkyl
derivatives 6 and 7 exhibited stronger fluorescence than cyclo-
pentyl derivate 8 and cyclic analogues 9 and 10 compared to parent
compound 5. These data show a pronounced impact of electronic
properties of the studied molecules on fluorescence emission.
2.3. Interactions with double stranded (ds-) DNA and RNA
2.3.1. UVeVis titrations with double stranded DNA
The UVeVis spectra of the studied compounds exhibited strong
hypochromic and batochromic changes upon addition of calf
thymus (ct)-DNA under the conditions of an excess of compound
over DNA base pairs (ratio r[compd]/[ct-DNA] > 0.5 for 5 and 9; r ¼ 0.4
for 6; r ¼ 0.2 for 7, 8 and 10), while further additions of ct-DNA
(excess of DNA base pairs over compound; r << 0.2) resulted in
a hyperchromic effect and additional batochromic shift of maxima
in the UVeVis spectra (Fig. 3). Thus, the final (at high excess of
ct-DNA; r[compd]/[ct-DNA] ¼ 0.02) batochromic shifts of the absorp-
tion maxima of 5e10 at lmax > 300 nm were between þ16 and
þ25 nm. The observed changes in 5e10/ct-DNA titrations, along
with a clear deviation from the isosbestic points, supported
formation of at least two different types of complexes with ct-DNA.
Surprisingly, addition of ct-DNA did not yield any measurable
changes in the UVeVis spectrum of 15.
2.3.2. Fluorimetric titrations with ct-DNA
Addition of ct-DNA led to a high increase of fluorescence
emission of compounds 5e10 (Fig. 4). Similarly as observed in
UVeVis titration experiments, titration with ct-DNA resulted in
Fig. 3. (a) Changes in UVeVis spectrum of 7 (c ¼ 1.97 ꢀ 10ꢁ5 mol dmꢁ3) upon titration
with ct-DNA; Dependence of the absorbance of 7 at lmax ¼ 391 nm (b) and 371 nm
(c) on c(ct-DNA), at pH 7.0, sodium cacodylate buffer, I ¼ 0.05 mol dmꢁ3
.
opposite changes of fluorescence of compounds 5e10, where the
breakpoint for all titrations was at about the ratio r
[compound]/
¼ 0.2. Several different binding modes were present under
[ct-DNA]
the conditions of an excess of compound over DNA (r > 0.2, fluo-
rescence quenching), including non-specific agglomeration of
positively charged small molecules along the negatively charged
Fig. 2. Fluorescence emission spectra of 5e10 at c ¼ 3.98e4.09 ꢀ 10ꢁ6 mol dmꢁ3; pH
7.0, sodium cacodylate/HCl buffer, I ¼ 0.05 mol dmꢁ3; at lexc. ¼ 376 (5), 371 (6, 7), 372
(8, 10), 387 (9) nm.

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I. Stolic et al. / European Journal of Medicinal Chemistry 46 (2011) 743e755
746
Fig. 4. (a) Changes in the fluorescence spectrum of 7 (c ¼ 1.0 ꢀ 10ꢁ6 mol dmꢁ3
,
lexc ¼ 371 nm) upon titration with ct-DNA; (b) Dependence of fluorescence emission of 7 at
lmax ¼ 461 nm on c (ct-DNA). Done at pH 7.0, sodium cacodylate buffer, I ¼ 0.05 mol dmꢁ3
.
DNA backbone as well as binding into the minor groove of DNA. In
contrast, one dominant binding mode prevailed at an excess of DNA
over compound (r << 0.2, fluorescence increase), most likely the
mutually independent binding of the studied molecules inside the
DNA minor groove. To determine the binding affinity of 5e10
toward ct-DNA, we processed the titration data collected at a large
excess of DNA (r << 0.2, fluorescence increase) by means of the
Scatchard equation [26], which yielded comparable binding
constants for all compounds (logKs ¼ 5.5e5.8 for the Scatchard
ratio n[bound compound]/[ct-DNA] ¼ 0.2).
with the UVeVis spectrum maximum). Strong positive ICD bands of
5e8 and 10, as well as of 9 at r < 0.2, indicate binding of single
molecules within the DNA minor groove as the dominant type of
interaction [29]. However, the observed ICD bisignate exciton
bands for 9 at r > 0.2 strongly support dimer formation of 9, most
likely also within the DNA minor groove [29,31].
Additions of 5e10 to ds-RNA at the ratio r[compound]/[poly Aepoly U]
< 0.2 (excess of RNA over compound) in most cases resulted in only
a minor decrease of the RNA CD band at lmax ¼ 265 nm. No induced
(ICD) bands were observed at l > 300 nm, with the exception of the
weak positive ICD band of 9 at lmax ¼ 410 nm. However, further
2.3.3. Circular dichroism titrations
additions of most compounds (r > 0.2, excess of compound over RNA)
We chose CD spectroscopy as a highly sensitive method to
assess conformational changes in the secondary structure of poly-
nucleotides [27] in order to gain more information on the binding
mode and the structure of the complexes formed. In addition,
achiral small molecules can show induced CD signals (ICD) upon
binding to polynucleotides, giving useful information about the
modes of interaction [27,28]. The sign and magnitude of the ICD
band can depend on the binding geometry. Ligandeligand stacking
is expected to give a strong bisignate exciton CD band. Minor
groove binding to ds-DNA orientates the ligand approximately at
45ꢂ in respect to the DNA chiral axis, giving a strong positive ICD
band. Intercalation brings the aromatic moiety of the ligand in a co-
planar arrangement with the base pairs, giving only a weak ICD
band (not always but in most cases of a negative sign due to parallel
orientation of the transition vector of the ligand and longer axis of
the surrounding base pairs) [29,30].
resulted in pronounced ICD bands at l > 300 nm, with shapes
depending on the compound structure. Additions of compounds 5, 8,
9 and 10 resulted in the appearance of bisignate exciton couplets,
which suggested formation of stacked dimers uniformly oriented in
respect to the ds-RNA chiral axis [27,29], most likely bound within the
hydrophobic and narrow major groove of the RNA a-helix [32]. At
variance with other compounds, compound 6 yielded only a weak
negative ICD band at lmax ¼ 370 nm, suggesting interactions of single
molecules with RNA. Only compounds 7 and 15 produced no ICD
bands under any conditions.
It is noteworthy that 15 did not exhibit any significant change in
CD spectra of either ds-DNA or ds-RNA.
2.3.4. Thermal denaturation experiments
Interactions of compounds 5e10 and 15 with ct-DNA and with
double stranded RNA (poly Aepoly U) were studied by thermal
denaturation experiments (Table 2). Addition of 5e10 strongly
stabilized the double helix of ct-DNA, whereby the non-linear
Addition of 5e10 to the ct-DNA resulted in dramatic changes of
the corresponding CD spectrum (Fig. 5). Strongly pronounced non-
linear dependence of the changes in CD spectra on the ratio
r[compound]/[ct-DNA] (Fig. 6) suggests saturation of dominant binding
dependence of DTm values obtained for ct-DNA/5e10 on ratio r sug-
gested saturation of binding sites at about r ¼ 0.2e0.3. The obtained
sites at about r ¼ 0.2e0.4.
D
Tm values were comparable, indicating minor impact of the struc-
ture of 5e10 on the stabilization of ct-DNA. Intriguingly, the studied
compounds exerted quite a different effect on thermal denaturation
Under the conditions of higher excess of DNA over compounds
(ratio r < 0.3), addition of compounds 5e10 caused a pronounced
decrease of ct-DNA CD band at 285 nm, accompanied by a weak
induced (ICD) band at 300e310 nm (agreeing nicely with the
shoulder in the corresponding UVeVis spectra) and a strong ICD
band at about 390e400 nm (agreeing nicely with the main maxima
in the corresponding UVeVis spectra). Further additions leading to
an excess of compound over DNA (ratio r > 0.3) resulted in a further
increase of the aforementioned changes in CD spectra for
compounds 5e8 and 10, whereby isoelliptic points were largely
retained. Contrary to 5e8 and 10, an excess of compound 9 over
DNA (r > 0.2) yielded new ICD bisignate exciton bands character-
ized by a negative band at lmax ¼ 370 nm, positive band at
of ds-RNA (poly Aepoly U). Comparison of DTm values obtained for
5e8 revealed that 5, characterized by non-substituted amidine, had
the strongest effect on poly Aepoly U stabilization, while systematic
increase of the bulkiness of aliphatic substituents on amidine groups
in 6e8 series proportionally decreased the thermal stabilization
effect. Compounds 9 and 10 characterized by cyclic amidine groups
stabilized ds-RNA much more strongly compared to 6e8. Moreover,
compound 9 stabilized similarly ds-DNA and ds-RNA.
These results are in good agreement with the data published
by Wilson and co-workers for diphenylfuran diamidines [33,34].
Compounds 5, 9 and 10 had a stronger stabilization effect on the
RNA duplex than analogous amidine derivatives from the
lmax ¼ 410 nm and isoelliptic point at
l
¼ 385 nm (agreeing nicely

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I. Stolic et al. / European Journal of Medicinal Chemistry 46 (2011) 743e755
747
Fig. 5. CD titration of polynucleotides (c ¼ 2.0 ꢀ 10ꢁ5 mol dmꢁ3) with 5e10 at molar ratios r ¼ [compound]/[polynucleotide] (pH 7.0, buffer sodium cacodylate, I ¼ 0.05 mol dmꢁ3).
diphenylfuran series. A possible reason for stronger stabilization
could be 3,4-ethylenedioxy extension of the central core, which
increased hydrophobicity compared to the previously studied
furan analogues. The strongest binding effect of the imidazoline
derivative 9 could be explained by formation of an intercalation
complex with polyAepolyU at an excess of RNA over compound
(r < 0.3) [33].
Addition of 15 did not affect denaturation of ct-DNA and ds-
RNA, which is in accord with the lack of any interaction in UVeVis
and fluorimetric titrations.

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I. Stolic et al. / European Journal of Medicinal Chemistry 46 (2011) 743e755
Fig. 5. (continued).
2.3.5. Ethidium bromide displacement experiments
(EB) displacement assay (Fig. 7) as an alternative method to
estimate affinity under the conditions of binding site saturation,
at least to compare the ability of the studied molecules to
compete for binding with a classical intercalator already bound
to DNA.
The binding constants and Scatchard ratios
n could be
calculated only at a high excess of DNA due to mixed binding
modes of the studied compounds close to saturation of the
binding site of ds-DNA. We performed the ethidium bromide

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749
Fig. 6. Induced CD signals of 5e10 plotted against ligand/ct-DNA ratios.
Fig. 7. Ethidium bromide (EB) displacement assay: to ct-DNA solution (c
¼
5 ꢀ 10ꢁ5 mol dmꢁ3), ethidium bromide (c ¼ 5 ꢀ 10ꢁ6 mol dmꢁ3) was added (r [EB]/[ct-
DNA]
¼
0.1), and quenching of the EB/DNA complex fluorescence emission
(
lex ¼ 480 nm, lem ¼ 600 nm) was monitored as a function of c(EB)/c(compound). The
The obtained IC50 values (Fig. 7) suggest that affinity of 5e10
toward ct-DNA is comparable to EB affinity. This finding is in good
agreement with calculated binding constants, taking into account
that the binding constant of EB toward ct-DNA under the same
experimental conditions is logKs ¼ 6 [35].
given IC50 values represent the ratio c(EB)/c(compound) ¼ [Int(EB/DNA) ꢁ Int(EBfree)]/
2, where Int(EB/DNA) is fluorescence intensity of the EB/DNA complex and Int(EBfree
)
is fluorescence intensity of the free ethidium bromide before DNA is added.
with the dose applied and chemical structure of compound 9.
High sensitivity of the HEp-2 cells treated with compound 5
(9.3 ꢀ 10ꢁ6 mol dmꢁ3), compound 6 (9.0 ꢀ 10ꢁ6 mol dmꢁ3), and
compound 7 (9.9 ꢀ 10ꢁ6 mol dmꢁ3), respectively, was also
observed. The growth of MiaPaCa2 cells treated with 7 was more
slugged (GI50 ¼ 4.9 ꢀ 10ꢁ6 mol dmꢁ3) compared to the growth of
cells treated with compound 9 (9.0 ꢀ 10ꢁ6 mol dmꢁ3). MDCK line
was most sensitive to compound 5 (7.1 ꢀ 10ꢁ6 mol dmꢁ3), while
the inhibitory effects of other tested compounds were significantly
lower (Table 3). Compound 15 exhibited negligible inhibitory
potential against the tumour cell lines tested. TGI (total growth
inhibition) values for all compounds on all tested cell lines were
2.4. Biological results
In the present study, we investigated the cytotoxic properties
of newly synthesized compounds 5e10 and 15 and Hoechst 33258
on a panel of seven tumour cell lines of different histological
origin and Madine-Darby canine kidney (MDCK) normal cells. The
results are presented as the GI50 value, a concentration eliciting
inhibition of cell growth by 50%. The compounds displayed
different growth inhibitory effects in dependence on the dose
applied (Table 3) on the 3rd day of incubation. Compound 9
showed a significant potent antitumour effect on tested tumour
cells with the GI50 value from 1.5 ꢀ 10ꢁ6 to 9.0 ꢀ 10ꢁ6 mol dmꢁ3as
well as on normal MDCK cells (17.0 ꢀ 10ꢁ6 mol dmꢁ3). All cell
lines, except MCF-7, exposed to Hoechst 33258 exhibited GI50
from 84 ꢀ 10ꢁ6 to 191.5 ꢀ 10ꢁ6 mol dmꢁ3. Among the most
sensitive cells were MCF-7cells (GI50 ¼ 2.7 ꢀ 10ꢁ6 mol dmꢁ3) and
HeLa cells (GI50 ¼ 1.5 ꢀ 10ꢁ6 mol dmꢁ3) treated with compound 9.
A possible reason for the obtained improved sensitivity of HeLa
and MCF-7 cells could be higher cellular uptake in combination
higher than 1 ꢀ 10ꢁ4 mol dmꢁ3
.
As the chemical structure of compounds is a key determinant of
their uptake and allocation in live cells [10], we investigated the
cellular uptake of compounds 7, 8 and 9. We used Hoechst 33258 as
a reference compound since its nucleus localization is well docu-
mented [36]. As expected, after 30 min of incubation, Hoechst
33258, applied at a concentration of 1 ꢀ 10ꢁ4 mol dmꢁ3, was
observed in the nucleus of HeLa cells (Fig. 8a). Under the same
experimental conditions, compound 9 (5 ꢀ 10ꢁ6 mol dmꢁ3) pene-
trated into the cell cytoplasm (Fig. 8b) after 30 min of incubation and
60 min later, 9 was localized in the cell nucleus (Fig. 8c). Compared to
compound 9, compounds 7 and 8 at 1 ꢀ 10ꢁ4 mol dmꢁ3 concen-
tration slowly entered into the cells. After 2 h of incubation, they
were localized in the cytoplasm and nucleus of MiaPaCa2 cells
(Fig. 9a and b). Our results indicate that the differences in chemical
structure between investigated compounds led to different rates of
their entry into cells and intracellular localization.
Table 2
a
The
D
Tm values (ꢂC) of ct-DNA and poly Aepoly U upon addition of different ratios
rb of 5e10 and 15 at pH 7.0 (buffer sodium cacodylate, I ¼ 0.05 mol dmꢁ3).
ct-DNA
0.1
poly Aepoly U
rb¼
0.2
0.3
0.3
5
6
7
8
9
10
15
12.2
8.4
9.0
12.3
10.8
11.8
e
>16c
11.1
9.0
15.2
14.3
>16c
e
>16c
12.3
15.0
>16c
>16c
>16c
0
12.6
5.8
5.2
1.4
17.0
8.2
0
Cell cycle coordination, according to signals coming from their
environment is a fundamental property of cells [37]. One of the
hallmarks of carcinogenesis is deregulation of the cell cycle [1].
Some antitumour agents are capable of inducing checkpoint arrest
and apoptotic cell death by inducing DNA damage [38e40].
Numerous novel therapeutics affect cells in a specific phase of the
cell cycle [41]. Recent evidence implicates DNA repair and the so-
called genome integrity checkpoints as culprits whose defects are
a
Error in
D
Tm: ꢃ 0.5 ꢂC.
b
c
r ¼ [compound]/[polynucleotide].
D
Tm impossible to calculate since Tm is over 100 ꢂC.

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750
Table 3
Cytotoxic activity of tested compounds.
a
GI50 (1 ꢀ 10ꢁ6 mol dmꢁ3
)
Compound
5
6
7
8
9
10
8.4 ꢃ 1.2
15
Hoechst
Tumour cells
MCF-7
NCI-H358
CaCo-2
HEp-2
HeLa
25.0 ꢃ 15.6
33.0 ꢃ 12.7
13.0 ꢃ 4.2
9.3 ꢃ 1.2
67.5 ꢃ 27.6
7.3 ꢃ 0.8
7.8 ꢃ 1.2
2.7 ꢃ 1.7
4.1 ꢃ 1.0
3.1 ꢃ 1.3
3.0 ꢃ 0.8
1.5 ꢃ 0.8
4.3 ꢃ 3.8
9.0 ꢃ 0.9
129.0 ꢃ 18.4
112.0 ꢃ 8.5
134.0 ꢃ 11.3
100.3 ꢃ 17
138.5 ꢃ 3.5
113.5 ꢃ 4.9
128.5 ꢃ 21.9
5.7 ꢃ 4.5
112.7 ꢃ 9.5
76.5 ꢃ 14.8
9.0 ꢃ 0
73.3 ꢃ 2.9
42.8 ꢃ 29.9
9.9 ꢃ 0.6
62.2 ꢃ 6.3
41.0 ꢃ 26.9
61.3 ꢃ 19.9
26.7 ꢃ 11.2
45.0 ꢃ 2.6
10.9 ꢃ 5.7
98.7 ꢃ 10.0
101.3 ꢃ 15.1
97.7 ꢃ 18.1
97.5 ꢃ 10.6
81.7 ꢃ 12.6
64.7 ꢃ 25.8
91.7 ꢃ 18
84.0 ꢃ 9.8
132.5 ꢃ 7.8
115.7 ꢃ 25.5
112.0 ꢃ 12.7
191.5 ꢃ 17.7
32.3 ꢃ 12.4
72.3 ꢃ 13.3
72.3 ꢃ 18.4
13.0 ꢃ 3.5
18.8 ꢃ 9.1
59.3 ꢃ 3.8
4.9 ꢃ 7.0
AGS
116.5 ꢃ 34.6
9.3 ꢃ 1.5
MiaPaCa2
Normal cells
MDCK
7.1 ꢃ 1.6
65.3 ꢃ 4.0
59.7 ꢃ 4.2
50.0 ꢃ 5.9
17.0 ꢃ 0.9
82.0 ꢃ 17.7
79.5 ꢃ 17.7
157.5 ꢃ 20.5
Abbreviation (used cell lines): breast adenocarcinoma (MCF-7), bronchioalveolar carcinoma (NCI-H358), colon adenocarcinoma (CaCo-2), larynx carcinoma (HEp-2), cervix
adenocarcinoma (HeLa), gastric adenocarcinoma (AGS), pancreatic carcinoma (MiaPaCa) and Madine-Darby canine kidney (MDCK).
Bold values emphasized significant difference in cytotoxic efficiency of investigated compounds on tumour cells compared to normal cells.
a
GI50 valueeconcentration eliciting inhibition of cell growth by 50%. Data represent the mean GI50 value of three independent experiments ꢃ SD. Cytotoxic activity was
analyzed by the MTT assay.
largely responsible for the enhanced genetic instability of tumour
cells [42]. The G2/M checkpoint prevents a DNA damaged cell from
entering mitosis and gives more time to various repair mechanisms
to repair the damage, if possible, or directs the cell into pro-
grammed cell death. For this reason, the G2/M checkpoint is of
particular interest for novel drug design in the development of
antitumour therapy [43].
To elucidate the mechanism of cytotoxic action of compound 9,
its effect on the cell cycle of HeLa cells was monitored. Compound 9,
applied at a concentration of 5 ꢀ 10ꢁ5 mol dmꢁ3, significantly
affected the cell cycle of HeLa cells during monitored incubation
periods (24e72 h). Accumulation of cells in the G2/M phase started
24 h after application and increased up to 3 fold over incubation
time. Percentage of cells in the G2/M phase slowly increased from
Fig. 8. Fluorescence microscopy analysis of the entry and intracellular distribution of 1 ꢀ 10ꢁ4 mol dmꢁ3 Hoechst 33258 (a), 5 ꢀ 10ꢁ6 mol dmꢁ3 compound 9 (b) in HeLa cells after
30 min of incubation, and 5 ꢀ 10ꢁ6 mol dmꢁ3 compound 9 (c) after 90 min of incubation using BP 450e490 filters, LP 520. Magnification: 400ꢀ.

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751
Fig. 9. Fluorescence microscopy analysis of the entry and intracellular distribution of 1 ꢀ 10ꢁ4 mol dmꢁ3 compound 7 (a) and 1 ꢀ 10ꢁ4 mol dmꢁ3 compound 8 (b) in MiaPaCa2 cells
after 2 h of incubation using filters BP 450e490, LP 520. Magnification: 400ꢀ.
17.58% after 24 h to 21.07% after 72 h of treatment, indicating
cessation of the cell cycle in that phase. The share of G0/G1, at the
same time, decreased to a statistically significant level (Table 4).
The observed increase of the G2/M phase arrested cells during
prolonged incubation time points to DNA damage as a consequence
of compound 9 interaction with intracellular genomic components.
Weakened G2/M checkpoint in the therapeutic setting may trigger
cell death via mitotic catastrophe. Under the same experimental
conditions, Hoechst 33258, used as a binder reference compound,
stopped the cell cycle in S phase and G0/G1. The difference between
the G1 share of cells treated with compound 9 and Hoechst 33258
at 5 ꢀ 10ꢁ5 mol dmꢁ3 was very small, but statistically important in
comparison with control cells.
strength of the observed interactions with DNA and RNA. Its
successful entering into the cell cytoplasm and nucleus has been
also confirmed. In addition, an increase of the G2/M phase arrested
cells treated with compound 9 points to the mitotic cell death as
a
consequence of its interaction with intracellular genomic
components.
4. Experimental
4.1. Synthesis
Solvents were distilled from appropriate drying agents shortly
before use. TLC was carried out on DC-plastikfolien Kieselgel 60
F254, Merck. Melting points were determined on a Büchi 510
3. Conclusions
melting point apparatus and were uncorrected. IR spectra [nmax
/
cmꢁ1] were obtained on a Bruker Vertex 70 spectrophotometer. The
1H and 13C NMR spectra were recorded on a Bruker Avance
The majority of studied compounds bind strongly to both
ds-DNA and ds-RNA. The only exception is compound 15;
comparison of its structure with structures of other compounds
clearly shows that the shortening and rigidification of the core by
direct attachment of amidines to thiophene hamper its effective
interactions with DNA/RNA. Its weak cytotoxic potential for tumour
as well as normal epithelial cells has been also confirmed.
Furthermore, all applied methods suggest the DNA minor groove as
the dominant binding site of 5e10, where at an excess of DNA single
molecules are bound, while at an excess of 5, 8, 9 and 10 they form
stacked dimers, most likely also within the DNA minor groove.
Surprisingly, compounds 5e10 also thermally stabilize ds-RNA,
which is not common for typical DNA minor groove binders, and
most of them, when present in excess over RNA, form stacked
dimers along the RNA double helix, most likely within the hydro-
phobic and narrow major groove. However, intercalation of 9 at
higher excess of RNA cannot be excluded. An exception is
compound 7, whose molecules seem to moderately stabilize
ds-RNA by more or less random binding along the polynucleotide
helix, thus lacking any ICD signal. Although compounds 5e10
showed a similar binding potential to the minor groove of ds-DNA
and ds-RNA under in vitro conditions, they displayed different
cytotoxic activity against tumour and normal cells. Cytotoxic effect
of compound 9 on tumour cells is in good agreement with the
300 MHz spectrometer. Chemical shifts (d/ppm) are referred to
TMS. Mass spectra were recorded on a Waters Micromass Q-ToF
micro. Elemental analyses were performed by the Applied Labo-
ratory Research Department of Ina, d.d., Research and Development
Sector, Central Analytical Laboratory. All compounds gave C, H, N
and S analyses within ꢃ0.4% of the theoretical values.
4.1.1. 2,5-Bis(4-cyanophenyl)-3,4-ethylenedioxythiophene (3)
To a stirred solution of 3,4-ethylenedioxythiophene (6.60 g,
0.046 mol) in freshly distilled THF (230 mL) under a nitrogen
atmosphere, 2.5 M n-BuLi in hexane (40 mL, 0.1 mol) was slowly
added. The resulting solution was stirred for 1 h at room temper-
ature (RT) and 1 h at 75 ꢂC. The mixture was cooled to RT and
Bu3SnCl (26 mL, 31.2 g, 0.096 mol) was added dropwise. The
vigorously stirred mixture was refluxed for 21 h. The solvent was
removed under reduced pressure, the residue was suspended in
anhydrous hexane and the suspension was filtered off. The filtrate
was concentrated to dryness under reduced pressure and the
obtained solid was dissolved in freshly distilled THF (300 mL) under
a nitrogen atmosphere. 4-Bromobenzonitrile (17.76 g, 0.1032 mol)
and [Pd(PPh3)4] (5.126 g, 0.0094 mol) were added to the solution.
The vigorously stirred mixture was refluxed for 8 days under
a nitrogen atmosphere. The solvent was partially evaporated, dry
Table 4
Effect of compound 9 and Hoechst 33258 on cell cycle progression.a
Treatment
Phases of cell cycle
G0/G1
S
G2/M
24 h
48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
Control
62.2 ꢃ 4.4
53.3 ꢃ 6.7*
55.4 ꢃ 7.7
51.3 ꢃ 1.3*
61.2 ꢃ 2.3
44.2 ꢃ 7.2*
59.6 ꢃ 3.5
49.0 ꢃ 4.9*
63.4 ꢃ 3.2
46.4 ꢃ 6.1*
62.2 ꢃ 3.2
45.7 ꢃ 4.5*
27.7 ꢃ 4.2
29.1 ꢃ 7.0
30.2 ꢃ 2.7
41.6 ꢃ 3.5*
30.4 ꢃ 3.7
37.3 ꢃ 10.8
33.1 ꢃ 2.0
42.6 ꢃ 3.8*
28.8 ꢃ 3.2
32.5 ꢃ 10.2
25.9 ꢃ 4.6
43.1 ꢃ 7.9*
10.1 ꢃ 3.4
17.6 ꢃ 4.5*
14.4 ꢃ 6.1
7.1 ꢃ 3.1
8.4 ꢃ 3.9
7.8 ꢃ 3.1
9 (5 ꢀ 10ꢁ6 M)
9 (1 ꢀ 10ꢁ6 M)
Hoechst 33258 (5 ꢀ 10ꢁ5 M)
18.5 ꢃ 6.8*
7.3 ꢃ 2.5
8.4 ꢃ 3.5
21.1 ꢃ 6.3*
11.9 ꢃ 4.7
11.2 ꢃ 6.8
a
Data are presented as mean value ꢃ standard deviation (SD). Statistically significant differences are marked with (*) and set at p < 0.05. Experiment was performed three
times in two replicates (n ¼ 6).

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752
diethyl ether was added and the precipitate was filtered off and
recrystallized from CHCl3 to yield 6.5 g (41.7%) of yellow powder,
mp > 250 ꢂC; IR (KBr) nmax/cmꢁ1: 3403, 2926, 2223, 1601, 1521,
to the residue. The solution was stirred for 4 h at 40 ꢂC, cooled to RT
and dry diethyl ether was added. The precipitate was filtered off
and dried under vacuum at 80 ꢂC for 24 h to yield 0.312 g (43.4%) of
pale yellow powder, mp > 250 ꢂC; IR nmax/cmꢁ1: 2972, 1670, 1520,
1483, 1446, 1409, 1374, 1087, 841, 575; 1H NMR (CDCl3)
d
/ppm: 7.87
(d, 4H, J ¼ 8.80 Hz, ArH), 7.68 (d, 4H, J ¼ 8.78 Hz, ArH), 4.45 (s, 4H,
OCH2CH2O); 13C NMR (DMSO-d6)
/ppm: 141.5, 136.9, 133.4, 126.4,
1319, 1095, 818, 738, 644, 1H NMR (DMSO-d6)
d/ppm: 9.87 (s, 2H,
d
NH), 9.52 (s, 2H, NH), 9.13 (s, 2H, NH), 7.92 (d, 4H, J ¼ 8.60 Hz, ArH),
7.84 (d, 4H, J ¼ 8.60 Hz, ArH), 4.50 (s, 4H, OCH2CH2O), 3.27 (t, 4H,
J ¼ 6.56 Hz, NHCH2CH), 2.03 (m, 2H, J ¼ 6.78 Hz, CH2CH(CH3)2), 0.98
119.3, 114.8, 109.4, 65.3; Anal. calcd. for C20H12N2O2S (Mr ¼ 344.39):
C 69.75, H 3.51, N 8.13, S 9.31; found: C 70.03, H 3.80, N 8.28, S 9.19.
(d, 12H, J ¼ 6.66 Hz, CH3); 13C NMR (DMSO-d6)
d/ppm: 162.3, 140.7,
4.1.2. 2,5-Bis(4-amidinophenyl)-3,4-ethylenedioxythiophene
dihydrochloride (5)
136.51, 129.0, 126.8, 125.3, 114.1, 64.8, 49.5, 27.0, 19.9; HRMS: calcd.
for C28H35N4O2S (M þ 1)þ, 491.2481; found: 491.2453. Anal. calcd.
for C28H34N4O2S$2HCl$2H2O (Mr ¼ 599.62): C 56.09, H 6.72, N 9.34,
S 5.35; found: C 56.38, H 6.33, N 9.52, S 5.31.
Dinitrile 3 (0.513 g, 1.5 mmol) was suspended in a mixture of
anhydrous methanol (12 mL) and dry dioxane (30 mL), cooled in an
ice bath and saturated with dry HCl gas. The suspension was stirred
at RT until IR spectra indicated the absence of the cyano peak
(3 days). Anhydrous diethyl ether was added to the suspension and
the obtained solid was collected by filtration, washed with anhy-
drous diethyl ether and dried under vacuum to yield 0.668 g (92.5%)
of bis(imidate ester hydrochloride) 4. The resulting salt was used in
the next step without any additional purification.
Crude imidate ester 4 (0.668 g, 1.37 mmol) was suspended in
anhydrous methanol (80 mL) and the solution was saturated with
dry NH3 gas at ice bath temperature. The flask was sealed, and the
suspension was stirred at room temperature for 2 days. The solvent
was removed under reduced pressure and the residue was sus-
pended in anhydrous ethanol saturated with HCl(g) and stirred at
room temperature for 2 h. The reaction mixture was concentrated
and precipitated with dry diethyl ether, the solid was collected by
filtration and dried under vacuum at 80 ꢂC for 24 h. Yield: 0.115 g,
(18.3%) of pale yellow powder, mp > 250 ꢂC; IR nmax/cmꢁ1: 3407,
1660,1602,1458,1391,1089, 924, 850, 753, 689; 1H NMR (DMSO-d6)
4.1.5. 2,5-Bis(4-N-cyclopentylamidinophenyl)-
3,4-ethylenedioxythiophene dihydrochloride (8)
To a suspension of compound 4 (0.626 g, 1.28 mmol) in anhy-
drous methanol (40 mL), under a nitrogen atmosphere, cyclo-
pentylamine (0.296 g, 3.48 mmol) was added and the mixture was
stirred for 48 h at RT. The solvent was removed under reduced
pressure and methanol saturated with HCl gas (10 mL) was added
to the residue. The solution was stirred under reflux for 6 h, then
cooled to RT and dry diethyl ether was added. The precipitate was
filtered off and dried under vacuum at 80 ꢂC for 24 h to yield
0.245 g (32.6%) of pale yellow powder, mp > 250 ꢂC; IR nmax/cmꢁ1
3042, 2960, 1671, 1608, 1510, 1486, 1443, 1410, 1362, 1085, 843,
747; 1H NMR (DMSO-d6)
/ppm: 9.68 (br s, 2H, NH), 9.48 (br s, 2H,
:
d
NH), 9.10 (br s, 2H, NH), 7.91 (d, 4H, J ¼ 8.21 Hz, ArH), 7.80 (d, 4H,
J ¼ 8.33 Hz, ArH), 4.50 (s, 4H, OCH2CH2O), 4.19e4.11 (m, 2H, CH),
2.16e2.09 (m, 4H, CH2), 1.78e1.57 (m, 12H, CH2); 13C NMR (DMSO-
d6) d/ppm: 162.5, 141.2, 136.9, 129.6, 127.5, 125.8, 114.6, 65.3, 54.7,
d
/ppm: 9.31 (s, 4H, NH2), 9.07 (s, 4H, NH2), 7.94 (d, 4H, J ¼ 8.95 Hz,
31.8, 24.1; HRMS: calcd. for C30H35N4O2S (M þ 1)þ, 515.2481;
found: 515.2460. Anal. calcd. for C30H34N4O2S$2HCl$4.5H2O
(Mr ¼ 668.68): C 53.89, H 6.78, N 8.38, S 4.80; found: C 53.65,
H 6.50, N 8.11, S 5.02.
ArH), 7.90 (d, 4H, J ¼ 8.83 Hz, ArH),4.51 (s, 4H, OCH2CH2O); 13C NMR
(DMSO-d6) d/ppm: 164.8,140.9,137.1,128.9,125.8,125.4,114.3, 64.8;
HRMS: calcd. for C20H19N4O2S (M þ 1)þ, 379.1229; found: 379.1215.
Anal. calcd. for C20H18N4O2S$2HCl$0.5H2O (Mr ¼ 460.38): C 52.18, H
4.60, N 12.17, S 6.97; found: C 52.23, H 4.64, N 11.83, S 6.86.
4.1.6. 2,5-Bis[4-(2-imidazolinyl)phenyl]-
3,4-ethylenedioxythiophene dihydrochloride (9)
4.1.3. 2,5-Bis(4-N-isopropylamidinophenyl)-
3,4-ethylenedioxythiophene dihydrochloride (6)
A suspension of the imidate ester salt 4 (0.68 g, 1.4 mmol) in
30 mL anhydrous methanol and ethylenediamine (0.192 g,
3.2 mmol) was heated under reflux for 12 h under a nitrogen
atmosphere. The solvent was removed under reduced pressure and
the residue was washed with diethyl ether, dried and dissolved in
dry methanol saturated with HCl(g). The mixture was stirred under
reflux for 5 h, the solvent was partially evaporated and dry diethyl
ether was added. The resultant solid was filtered off, washed with
diethyl ether and dried under vacuum at 80 ꢂC for 24 h to yield
0.325 g (46.2%) of yellow powder, mp > 250 ꢂC; IR nmax/cmꢁ1: 3402,
3125, 1615, 1577, 1510, 1365, 1085, 1036, 845, 745, 652; 1H NMR
Freshly distilled isopropylamine (1 mL, 0.719 g, 12.1 mmol) was
added to a stirred suspension of diimidate ester 4 (0.48 g,1 mmol) in
anhydrous methanol (10 mL) and the mixture was stirred for 24 h at
RT. The solvent was removed under reduced pressure and methanol
saturated with HCl gas (10 mL) was added to the residue. The
solution was stirred for 2 h at 40 ꢂC, cooled to RT and precipitated
with dry diethyl ether. The yellow solid was filtered off and dried
under vacuum at 80 ꢂC for 24 h to yield 0.204 g (37.6%), mp > 250 ꢂC;
IR nmax/cmꢁ1: 3435, 3157, 1680, 1604, 1435, 1364, 1285, 1080, 854,
767, 555; 1H NMR (DMSO-d6)
d/ppm: 9.56 (br s, 2H, NH), 9.42 (br s,
(DMSO-d6)
d
/ppm: 10.57 (s, 4H, NH2), 8.03 (d, 4H, J ¼ 8.40 Hz, ArH),
2H, NH), 9.02 (br s, 2H, NH), 7.92 (d, 4H, J ¼ 8.47 Hz, ArH), 7.79 (d, 4H,
7.98 (d, 4H, J ¼ 8.55 Hz, ArH), 4.52 (s, 4H, OCH2CH2O), 4.03 (s, 8H,
J ¼ 8.46 Hz, ArH), 4.51 (s, 4H, OCH2CH2O), 4.04e4.02 (m, 2H, CH),
NCH2); 13C NMR (DMSO-d6)
d/ppm: 164.6, 141.8, 138.1, 129.8, 126.2,
1.29 (d, 12H, J ¼ 6.28 Hz, CH3); 13C NMR (DMSO-d6)
d/ppm: 161.6,
120.5, 115.1, 65.3, 44.8; HRMS: calcd. for C24H23N4O2S (M þ 1)þ:
431.1542; found: 431.1536. Anal. calcd. for C24H22N4O2S$2HCl$6H2O
(Mr ¼ 611.54): C 47.14, H 5.93, N 9.16, S 5.24; found: C 47.28, H 5.68,
N 8.77, S 5.24.
141.2, 136.9, 129.6, 127.5, 125.7, 114.6, 65.3, 45.6, 21.7; HRMS: calcd.
for C26H31N4O2S (M þ 1)þ, 463.2168; found: 463.2160. Anal. calcd.
for C26H30N4O2S$2HCl$3H2O (Mr ¼ 589.58): C 52.97, H 6.50, N 9.50, S
5.44; found: C 53.33, H 6.14, N 9.29, S 5.10.
4.1.7. 2,5-Bis[4-(1,4,5,6-tetrahydropyrimidin-2-yl)phenyl]-
4.1.4. 2,5-Bis(4-N-isobutylamidinophenyl)-
3,4-ethylenedioxythiophene dihydrochloride (10)
3,4-ethylenedioxythiophene dihydrochloride (7)
A suspension of the imidate ester salt 4 (0.69 g, 1.4 mmol) in
60 mL anhydrous methanol and 1,3-diaminopropane (0.89 g,
12 mmol) was heated under reflux for 3 h under a nitrogen
atmosphere. The solvent was removed under reduced pressure and
the residue was washed with diethyl ether, dried and dissolved in
dry methanol saturated with HCl(g). The mixture was stirred under
Imidate ester 4 (0.614 g, 1.26 mmol) was suspended in anhy-
drous methanol (60 mL), isobutylamine (1 mL, 0.736 g, 10 mmol)
was added under a nitrogen atmosphere and the mixture was
stirred for 18 h at RT. The solvent was removed under reduced
pressure and methanol saturated with HCl gas (10 mL) was added

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753
reflux for 2 h, the solvent was partially evaporated and dry diethyl
ether was added. The resultant solid was filtered off, washed with
diethyl ether and dried under vacuum at 80 ꢂC for 24 h to yield
0.56 g (75.1%) of yellow powder, mp > 250 ꢂC; IR nmax/cmꢁ1: 2972,
2038, 1610, 1578, 1452, 1365, 1308, 1086, 840, 744, 665; 1H NMR
4.1.10. 3,4-Ethylenedioxythiophene-2,5-dicarboxamide (16)
Ammonium hydroxide solution (140 mL) was added dropwise
to a stirred solution of diacid chloride 12 (3.52 g, 13.2 mmol) in
dichloromethane. The formed solid was filtered off, washed with
water and recrystallized from methanol to yield 2.41 g (80.2%) of
pale brown powder, mp > 250 ꢂC; IR nmax/cmꢁ1: 3426, 3185, 1680,
1615, 1484, 1456, 1412, 1354, 1129, 1092, 857, 777, 678, 568; 1H NMR
(DMSO-d6)
d
/ppm: 10.20 (s, 4H, NH), 7.92 (d, 4H, J ¼ 8.43 Hz, ArH),
7.87 (d, 4H, J ¼ 8.43 Hz, ArH), 4.50 (s, 4H, OCH2CH2O), 3.50 (t, 8H,
J ¼ 5.52 Hz, N-CH2CH2), 1.98 (q, 4H, J ¼ 5.34 Hz, N-CH2CH2); 13C
(CDCl3) d/ppm: 7.72 (s, 2H, NH), 6.81 (s, 2H, NH), 4.35 (s, 4H,
NMR (D2O)
d
/ppm: 159.3, 140.4, 137.0, 127.1, 126.0, 125.7, 114.9, 64.7,
OCH2CH2O); 13C NMR (DMSO-d6)
65.44; MS (m/z): 229.1 (M þ 1)þ.
d/ppm: 161.70, 141.04, 116.69,
38.9, 17.6; HRMS: calcd. for C26H27N4O2S (M þ 1)þ: 459.1855;
found: 459.1837. Anal. calcd. for C26H26N4O2S$2HCl$3H2O
(Mr ¼ 585.55): C 53.33, H 5.85, N 9.57, S 5.48; found: C 53.52, H 5.79,
N 9.54, S 5.37.
4.1.11. 3,4-Ethylenedioxythiophene-2,5-dicarbonitrile (17)
Dry acetonitrile (110 mL) was cooled in an ice bath and then
POCl3 (1.71 mL) and DMF (1.7 mL) were added. The resulting solution
was stirred for 1.5 h in an ice bath and 3,4-ethylenedioxythiophene-
2,5-dicarboxamide (0.94 g, 4.1 mmol) was added. Pyridine (5.6 mL)
was added after stirring for 2 h at RT and the formed suspension was
stirred for an additional 1 h at RT. The reaction mixture was extracted
with chloroform and the collected organic layers were washed with
10% HCl, saturated NaCl solution and then with water until neutral
reaction. The organic phase was dried at Na2SO4, the solvent was
removed and the residue was recrystallized from ethanol to yield
0.49 g (62.9%) of white powder, mp 133e134 ꢂC; IR nmax/cmꢁ1: 3172,
2964, 2214, 1665, 1607, 1506, 1471, 1454, 1390, 1359, 1262, 1083, 851,
4.1.8. 3,4-Ethylenedioxythiophene-2,5-dicarboxanilide (13)
3,4-Ethylenedioxythiophene-2,5-dicarboxylic acid (11) (2.465 g,
0.0107 mol) was suspended in dry benzene (120 mL), thionyl
chloride (12 mL, 19.56 g, 0.164 mol) and 12 drops of DMF were
added, and the resulting mixture was heated under reflux for 12 h.
The solvent was removed under reduced pressure and the residue
was washed with dry benzene. The formed diacid chloride was
used in the next step without further purification.
To a suspension of diacid chloride in dry chloroform (100 mL),
aniline (4.09 g, 0.044 mol) was added under a nitrogen atmosphere,
and the mixture was stirred at RT for 24 h. The solvent was evapo-
rated under reduced pressure and the residue was suspended in
water. The formed solid was filtered off and washed with 10% HCl,
10% NaHCO3 and water, and recrystallized from chloroform to yield
2.98 g (73%) of white powder, mp 240e241 ꢂC; IR nmax/cmꢁ1: 3380,
3035, 2955,1652,1601,1544,1484,1459,1434,1374,1307,1290,1240,
801, 670, 583; 1H NMR (CDCl3) /ppm: 4.43 (s, 4H); 13C NMR (CDCl3)
d
d
/ppm: 147.69, 110.45, 91.23, 65.12; MS (m/z): 193.1 (M þ 1)þ. Anal.
calcd. for C8H4N2O2S(Mr ¼ 192.20): C 49.99, H 2.10, N 14.58, S 16.68;
found: C 49.85, H 2.33, N 14.29, S 16.37.
4.2. Spectroscopic studies
1147, 1089, 878, 752, 685, 617, 576, 491; 1H NMR (CDCl3)
d/ppm: 8.53
(s, 2H, NH), 7.62 (d, 4H, J ¼ 7.67 Hz, ArH), 7.35 (t, 4H, J ¼ 8.08 Hz, ArH),
7.13 (t, 2H, J ¼ 7.41 Hz, ArH), 4.59 (s, 4H, OCH2CH2O); 13C NMR
(CHCl3): 153.02, 133.46, 132.32, 123.84, 119.33, 114.86, 60.13; MS
(m/z): 381.2 (M þ 1)þ; Anal. calcd. for C20H16N2O4S$0.25H2O
(Mr ¼ 384.93): C 62.41, H 4.32, N 7.28, S 8.33; found: C 62.35, H 4.25,
N 7.27, S 7.95.
Polynucleotides were purchased as noted: polyAepolyU
(Sigma), calf thymus (ct)-DNA (Aldrich). Polynucleotides were
dissolved in Na-cacodylate buffer, I ¼ 0.05 mol dmꢁ3, pH 7.0. Calf
thymus ct-DNA was additionally sonicated and filtered through
a 0.45
mm filter. Polynucleotide concentration was determined
spectroscopically as concentration of phosphates [35].
Electronic absorption spectrawere obtained on a Varian Cary 100
Bio spectrometer, CD spectra were collected on a Jasco J-810 spec-
trometer and fluorescence spectra were recorded on a Varian Cary
Eclipse fluorimeter; all in quartz cuvettes (1 cm). Measurements
were performed in an aqueous buffer solution (pH 7.0 e Na-caco-
dylate buffer, I ¼ 0.05 mol dmꢁ3). Absorbance and fluorescence
intensities of the studied compounds were proportional to their
concentration under the experimental conditions used. Excitation
4.1.9. 3,4-Ethylenedioxythiophene-2,5-bis(N-phenylamidine)
dihydrochloride (15)
3,4-Ethylenedioxythiophene-2,5-dicarboxanilide (13) (0.997 g,
2.6 mmol) was suspended in dry chloroform (50 mL) under
a nitrogen atmosphere, SOCl2 (2 mL) and 6 drops of dry DMF were
added, and the resulting mixture was heated under reflux for 13 h.
The solvent was removed under reduced pressure and the residue
was washed with dry benzene. Diimidoyl chloride was then sus-
pended in dry chloroform (50 mL), chilled in an ice bath and dry
NH3 gas was passed through for 4 h. The reaction mixture was
stirred at RT for 7 days. The solvent was evaporated under reduced
pressure and the residue was suspended in water, made basic to pH
10 with 1M NaOH and the precipitate was filtered off, washed with
water and dried. The solid was suspended in anhydrous methanol
saturated with HCl gas and stirred at RT for 15 h. The volume of the
solvent was reduced under reduced pressure and dry diethyl ether
was added. The solid was filtered off, washed with dry diethyl ether
and dried under vacuum at 80 ꢂC for 24 h to yield 0.9 g (76.8%) of
pale yellow powder, mp > 250 ꢂC; IR nmax/cmꢁ1: 3066, 1600, 1481,
wavelength of lexc > 360 nm was used in fluorimetric titrations to
avoid inner filter effects caused by absorption of excitation light by
added polynucleotide. The binding constant (Ks) and [bound
compound]/[polynucleotide phosphate] ratio (n) were calculated
according to the Scatchard equation by non-linear least-square
fitting, giving excellent correlation coefficients (>0.999) for the
obtained Ks and n values. Thermal melting curves for ds-poly-
nucleotides and their complexes with the compounds were deter-
mined, as previously described, by following the absorption change
at 260 nm as a function of temperature [35]. The absorbance of
a studied compound was subtracted from every curve, and the
absorbance scale was normalized. The obtained Tm values are the
midpoints of the transition curves, determined from the maximum
1357, 1083, 846, 657; 1H NMR (DMSO-d6)
d/ppm: 12.07 (br s, 2H,
NH), 9.89 (br s, 2H, NH), 9.36 (br s, 2H, NH), 7.55 (t, 4H, J ¼ 7.64 Hz,
of the first derivative or graphically by a tangent method. Given
values were calculated subtracting Tm of the free nucleic acid from
DTm
ArH), 7.45e7.41 (m, 6H, ArH), 4.46 (s, 4H, OCH2CH2O); 13C NMR
(DMSO-d6)
d
/ppm: 154.59, 144.84, 135.39, 130.37, 128.54, 125.40,
Tm of the complex. Every
at least two measurements, the error in
For the ethidium bromide (EB) displacement assay, ethidium
bromide (c ¼ 5 ꢀ 10ꢁ6 mol dmꢁ3) was added (r [EB]/[poly-
nucleotide] ¼ 0.1) to a polynucleotide solution (c ¼ 5 ꢀ 10ꢁ5
DTm value here reported was the average of
108.06, 65.61; HRMS: calcd. for C20H19N4O2S (M þ 1)þ: 379.1229;
found: 379.1226. Anal. calcd. for C20H18N4O2S$2HCl$3H2O
(Mr ¼ 505.42): C 47.53, H 5.19, N 11.09, S 6.34; found: C 47.57, H 5.14,
N 11.04, S 6.27.
D
Tm is ꢃ0.5 ꢂC.

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I. Stolic et al. / European Journal of Medicinal Chemistry 46 (2011) 743e755
754
mol dmꢁ3) and quenching of the EB/polynucleotide complex
fluorescence emission (lex ¼ 480 nm, lem ¼ 600 nm) was moni-
tored as a function of c(EB)/c(compound). The given IC50 values
solution (10ꢁ8 mol dmꢁ3) for 5 min, washed with PBS, and entry
and intracellular distribution of tested chemicals were analyzed
under the microscope.
represent the ratio c(EB)/c(compound)
¼
[Int(EB/polynucleo-
tide) ꢁ Int(EBfree)]/2, where Int(EB/polynucleotide) is the fluores-
cence intensity of EB/polynucleotide complex and Int(EBfree) is the
fluorescence intensity of free ethidium bromide before poly-
nucleotide addition.
4.3.4. Cell cycle analysis
For cell cycle analysis, HeLa cells (3 ꢀ 105 cells per well) were
seeded into 6-well plates (Greiner, Austria). Twenty-four hours
later, the tested compound 5 and Hoechst 33285 were added at
a concentration of 1 ꢀ 10ꢁ6 mol dmꢁ3 and 1 ꢀ 10ꢁ5 mol dmꢁ3
,
4.3. Biological experiments
respectively. Cells were harvested at different intervals (24, 48 and
72 h) following drug treatment. Floating and adherent cells were
collected separately, then combined, washed with phosphate buffer
saline (PBS), fixed with 70% ethanol, and stored at ꢁ20 ꢂC. Imme-
diately before the analysis, the cell pellets were washed with PBS
4.3.1. Cell culturing
Human tumour cell lines, breast adenocarcinoma (MCF-7),
bronchioalveolar carcinoma (NCI-H358), colon adenocarcinoma
(CaCo-2), larynx carcinoma (HEp-2), cervix adenocarcinoma
(HeLa), gastric adenocarcinoma (AGS), pancreatic carcinoma
(MiaPaCa2), and Madine-Darby canine kidney (MDCK) cells, were
cultured in tissue culture flasks and grown as monolayers. MCF-7,
NCI-H358 and AGS cells were grown in RPMI 1640 medium (Gibco,
EU) supplemented with 10% heat-inactivated fetal bovine ser-
umeFBS (Gibco, EU), 2 mM glutamine, 1 mM sodium pyruvate,
HEPES and 100 U/0.1 mg penicillin/streptomycin. CaCo-2, HEp-2,
HeLa, MiaPaCa and MDCK cells were cultured in Dulbecco’s Modi-
fied Eagle Medium e DMEM (Gibco, EU) supplemented with 10%
FBS 2 mM glutamine, and 100 U/0.1 mg penicillin/streptomycin. To
detach them from the flask surface, cells were trypsinized using
a 0.25% trypsin/EDTA solution. Cells were cultured in a humidified
atmosphere under the conditions of 37 ꢂC/5% of CO2 gas in a CO2
incubator (Shell Lab, Sheldon Manufacturing, USA).
and resuspended in 1 mg/mL of propidium iodide and 0.2 mg/mL of
RNase A. Stained cells were then analyzed with a Becton Dickinson
FACScalibur (Becton Dickenson) flow cytometer (20 000 cells were
analyzed). The percentage of cells in each cell cycle phase was
determined using the ModFit LTÔ software (Verity Software
House) based on DNA histograms. Tests were performed in tripli-
cates and repeated at least twice.
Acknowledgments
The authors thank Carl F. Verkoelen, PhD, Erasmus Medical
Center Rotterdam, Rotterdam, The Netherlands, for providing
ꢁ
ꢀ
MDCK cells, RuCer Boskovic Institute, Department of Molecular
Medicine, Laboratory for Systematic Biomedicine, for their help
ꢁ
with FACS analysis and Igor Bratos, PLIVA Croatia Ltd., Research &
Development, for providing high-resolution mass spectral analyses.
The Ministry of Science, Education and Sports of the Republic of
Croatia financially supported this work through Grants No: 219-
0982914-2176, 219-0982914-2179, 053-0982914-2965 and 098-
0982914-2918.
4.3.2. Cytotoxicity evaluation by the MTT assay [44]
Synthesized compounds 5e10 and 15 and Hoechst 33258 were
prepared as stock solutions in highly pure water. Working solu-
tions in a concentration range of 10ꢁ3e10ꢁ6 mol dmꢁ3 were
prepared prior to testing. Cytotoxic effects of the compounds on
tested cell lines were determined by the MTT assay. Cells were
seeded in 96 micro well flat bottom plates (Greiner, Austria) at
a concentration of 2 ꢀ 104 cells/mL and left overnight in the CO2
incubator allowing them to attach to the plate surface. Growing
medium was replaced with compound supplemented or control
medium and incubated for 72 h. Fresh medium with 5 mg/mL of
MTT was added onto cells and incubated for 4 h at 37 ꢂC. Upon
media removal, water insoluble MTT-formazan crystals formed
inside the living cells were dissolved in DMSO and the absorbance
at 570 nm proportional to the number of living cells was measured
on an Elisa Microplate Reader (Stat fax 2100, Pharmacia Biotech,
Uppsala, Sweden). All experiments were performed at least three
times in triplicates.
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