Novel cyano- and amidino-substituted derivatives of thieno[2,3-b]- and thieno[3,2-b]thiophene-2-carboxanilides and thieno[3′,2′:4,5]thieno- and thieno[2′,3′:4,5]thieno [2,3-c]quinolones: Synthesis, photochemical synthesis, DNA binding, and antitumor evalu

Article abstract of DOI:10.1016/j.bmc.2005.12.004

A series of cyano- and amidino-substituted derivatives of thieno[2,3-b]- and thieno[3,2-b]thiophene-2-carboxanilides and their 'cyclic' derivatives (quinolones) were synthesized. 'Cyclic' compounds displayed a rather strong and differential antiproliferat

Full text of DOI:10.1016/j.bmc.2005.12.004

Bioorganic & Medicinal Chemistry 14 (2006) 2859–2868
Novel cyano- and amidino-substituted derivatives
of thieno[2,3-b]- and thieno[3,2-b]thiophene-2-carboxanilides
and thieno[30,20:4,5]thieno- and thieno[20,30:4,5]thieno
[2,3-c]quinolones: Synthesis, photochemical synthesis, DNA
binding, and antitumor evaluation
a
b
c
b
c
ˇ
ˇ
´
Ivana Jarak, Marijeta Kralj, Ivo Piantanida, Lidija Suman, Mladen Zinic,
b
a,
*
´
Kresˇimir Pavelic and Grace Karminski-Zamola
aDepartment of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb,
Marulic´ev trg 20, PO Box 177, HR-10000 Zagreb, Croatia
b
-
Division of Molecular Medicine, Ruder Bosˇkovic´ Institute, Bijenicˇka cesta 54, PO Box 180, HR-10000 Zagreb, Croatia
Ruder Bosˇkovic´ Institute, Division of Organic Chemistry and Biochemistry, Laboratory of Supramolecular and Nucleoside Chemistry,
PO Box 180, HR-10002 Zagreb, Croatia
c
-
Received 27 October 2005; revised 29 November 2005; accepted 2 December 2005
Available online 10 January 2006
Abstract—A series of cyano- and amidino-substituted derivatives of thieno[2,3-b]- and thieno[3,2-b]thiophene-2-carboxanilides and
their ‘cyclic’ derivatives (quinolones) were synthesized. ‘Cyclic’ compounds displayed a rather strong and differential antiprolifera-
tive effect on various cell lines, while the ‘acyclic’ amidino-substituted compounds were much more active, but showing mostly non-
differential cytotoxicity, whereas cyano-substituted compounds (2a,b) produced a strikingly strong effect selectively on HeLa and
Hep-2 cell lines. Antiproliferative activity of ‘cyclic’ derivatives is very likely caused by intercalation into DNA, while their ‘acyclic’
analogues use other target(s) and/or mechanisms of action.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
and displayed selective activity against P-gp-expressing
epidermoid carcinoma of the nasopharynx.3 Indolo-, pyr-
rolo-, and benzofuro-quinolones, and anilinoindoloqui-
nolone derivatives were synthesized and evaluated
in vitro against a 3-cell line panel consisting of MCF7,
NCI-H460, and SF268. The results have shown that
cytotoxicity decreases in the order of anilinoindoloqui-
nolones > indoloquinolones > pyrroloquinolones > ben-
zofuroquinolones.4 Moreover, hydroxymethyl- and
methoxymethylfuro[2,3-h]quinolin-2(1H)-ones inhibited
topoisomerase II, leading to a moderate antiproliferative
activity in mammalian cells. The antiproliferative activity
was also tested upon UVA irradiation in mammalian
cells; all compounds showed higher activity than 8-
MOP, without mutagenicity and skin phototoxicity.5,6
Quinolone antibacterials are a class of potent broad-spec-
trum drugs that target the bacterial type II DNA topoi-
somerases; they have been developed most fully for
clinical use in human medicine.1 Besides, it was shown
that they could also inhibit mammalian topoisomerase
activity and tubulin polymerization and thus act as po-
tent antitumor drugs as well. For instance, pyranoquino-
line-2-ones were synthesized and evaluated for their
in vitro cytotoxicity against a panel of human tumor cell
lines,2 while 2-arylquinazolinones displayed significant
growth inhibitory action against tumor cell lines. Some
of them were potent inhibitors of tubulin polymerization
Keywords: Thieno-thiophene carboxanilides; Thieno-thiophene quino-
lones; Synthesis; Photochemical synthesis; DNA binding; Anticancer
activity.
As part of our continuing search for potential anticancer
drug candidates related to heterocyclic quinolones, we
have prepared new 3-dimethylaminopropyl-substituted
derivatives of thieno[30,20:4,5]thieno[2,3-c]quinolones7
*
Corresponding author. Tel.: +38514597215; fax: +38514597250;
e-mail: gzamola@pierre.fkit.hr
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.004

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I. Jarak et al. / Bioorg. Med. Chem. 14 (2006) 2859–2868
and benzo[b]thieno-quinolones,8 and observed consider-
able antitumor activity on several human cell lines.
Later on, a class of substituted benzo[b]thieno[2,3-
c]quinolones containing an isopropylamidino substitu-
ent, either on the quinolone or benzo[b]thiophene part
of the condensed benzo[b]thieno-quinolone molecule,
was prepared.9 No significant difference in the antitumor
activity regarding the position of the amidine substituent
was noted for benzo[b]thieno[2,3-c] analogues. At the
same time, benzo[b]thieno[2,3-c]quinolones showed
strong, but more selective-antitumor activity compared
or isopropylamidino substituent on the anilide or quino-
lone ring (Fig. 1). DNA binding as well as their antitu-
mor activity were evaluated.
2. Results and discussion
2.1. Chemistry
All compounds (2a–4d) shown in Figure 1 were pre-
pared according to Scheme 1, starting from 3-chlorothi-
eno[2,3-b]thiophene-2-carbonyl chloride 1a and 3-
chlorothieno[3,2-b]thiophene-2-carbonyl chloride 1b,
which were synthesized according to the literature pro-
cedure, in 46% and 11.3% overall yields from thio-
phene-2- and thiophene-3-aldehyde, respectively.10
Novel derivatives of cyano-substituted 3-chlorothie-
no[2,3-b]thiophene-2-carboxamide 2a and 3-chlorothie-
no[3,2-b]thiophene-2-carboxamide 2b were prepared in
the reaction of the corresponding carbonyl chlorides
1a and 1b with p-aminobenzonitrile in 70% and 60%
yields, respectively. Cyano-substituted carboxanilides
2a and 2b were subsequently converted into amidino-
substituted derivatives by a Pinner reaction.11 Treat-
ment of 2a and 2b with dry HCl gas in anhydrous etha-
nol or methoxy-ethanol afforded the corresponding
imidate esters, which were refluxed either in methanolic
ammonia or iso-propylamine in anhydrous ethanol, to
yield N-40-amidinophenyl carboxamide hydrochloride
3a,c (87% and 85.2%, respectively) or N-40-(N0-isopro-
pylamidino)phenyl carboxamide hydrochloride 3b,d
(59% and 50%, respectively). 2-amidino-6-oxo-5,6-dihy-
drothieno[30,20:4,5]thieno[2,3-c]quinolines 4a, 4b and 2-
amidino-6-oxo-5,6-dihydrothieno[20,30:4,5]thieno[2,3-
c]quinolines 4c, 4d were prepared by the photochemical
dehydrohalogenation reaction from the corresponding
carboxamides 3a–d. 4a and 4b were obtained after
1.5 h (4a; 48%) and 2 h 45 min (4b; 82%) irradiation with
a high-pressure mercury lamp (400 W) using a Pyrex fil-
ter. Under the same conditions, 4c and 4d were obtained
after 13 h (4c; 55%) and 19 h (4d; 70%) of irradiation.
to their ‘acyclic’ amidino-substituted precursors.9
A
spectroscopic study of the interactions of some represen-
tatives of ‘cyclic’ derivatives and their ‘acyclic’ precur-
sors with ds-DNA/RNA supported strong intercalative
binding of the former and weak non-intercalative
binding of the latter group of compounds.9
Proceeding in a systematic study of the structure–activity
relationship (SAR), we have decided to prepare
analogues of previously reported thieno[30,20:4,5]thie-
no[2,3-c]quinolones7 with amidino-substituent posi-
tioned on the quinolone side (Fig. 1). Studies of the
DNA interactions and in vitro antitumor activity of such
compounds and of their precursors, when compared with
previously observed results,7 could clarify the impact of
the position of the amidine substituent on monitored
SAR parameters. In addition, such studies could eluci-
date the impact of variation of benzene vs. thiophene
moiety if novel compounds are correlated with their
previously reported benzo[b]thieno[2,3-c] analogues.8,9
In this work, we have prepared a new group of substitut-
ed thieno[2,3-b]thiophene-2-carboxanilides 2a, 3a, and
3b and thieno[3,2-b]thiophene-2-carboxanilides 2b, 3c,
and 3d and their ‘cyclic’ derivatives, thieno[30,20:4,5]thi-
eno[2,3-c]quinolones 4a, 4b and thieno[20,30:4,5]thie-
no[2,3-c]quinolones 4c, 4d, bearing a cyano, amidino
Cl
Cl
O
O
S
2.2. Spectroscopic properties of compounds 2a–4d
R
R
HN
HN
S
S
S
Since application of spectrophotometric methods in the
DNA binding studies was necessary, we characterized
aqueous solutions of compounds by means of electronic
absorption (UV/vis) and fluorescence emission spectra
(compounds 4a–d).
2a R = CN
3a R = amidine
3b R = iso-pr-amidine
2b R = CN
3c R = amidine
3d R = iso-pr-amidine
NHR
NHR
-Cl +H2N
-Cl +H2N
Absorbancies of aqueous solutions of compounds are
propoꢀrt5ional to their concentrations up to
2 · 10 mol dmꢀ3 (2a), 3 · 10ꢀ5 mol dmꢀ3 (3a, 3c),
2.3 · 10ꢀ5 mol dmꢀ3 (3b), 1.8 · 10ꢀ5 mol dmꢀ3 (2b,
4a), 1.2 · 10ꢀ5 mol dmꢀ3 (3d), 4 · 10ꢀ5 mol dmꢀ3 (4b),
3.3 · 10ꢀ5 mol dmꢀ3 (4c), and 2.6 · 10ꢀ5 mol dmꢀ3
(4d), indicating that there is no significant intermolecu-
lar stacking that would give rise to hypochromicity
effects.
NH
NH
O
S
S
S
O
S
4a R = H
4b R = CH(CH3)2
4c R = H
4d R = CH(CH3)2
Figure 1. Substituted 3-chlorothieno[2,3-b]thiophene-2-carboxamides
(2a, 3a, and 3b), 3-chlorothieno[3,2-b]thiophene-2-carboxamide (2b,
3c, and 3d), 6-oxo-5,6-dihydrothieno[30,20:4,5]thieno[2,3-c]quinolones
4a, 4b, and 6-oxo-5,6-dihydrothieno[20,30:4,5]thieno[2,3-c]quinolones
4c, 4d.
Conversion of cyano-substituted 3-chlorothieno[2,3-
b]thiophene-2-carboxamide 2a into amidino-substitut-
ed 3a and 3b resulted in bathochromic shifts and

I. Jarak et al. / Bioorg. Med. Chem. 14 (2006) 2859–2868
2861
Cl
S
H2NX
CN
Cl
O
X
X
Y
COCl
Et3N
CN
HN
Y
S
1a X= C, Y =
S
2a X= C, Y =
S
1b X= S, Y =
C
2b X= S, Y =
C
1. HCl/gas, abs.abs.
methoxyethanol
2. NH3/gas or iso-pr-amine,
abs. abs. ethanol
1. HCl/gas, abs.abs. ethanol
2. NH3/gas or iso-pr-amine,
abs. abs. ethanol
Cl
Cl
O
O
S
NH2+ Cl-
NH2+ Cl-
HN
HN
S
S
S
NHR
NHR
3c R = H
3a R = H
3d R = CH(CH3)2
3b R = CH(CH3)2
hν/ methanol or methoxyethanol
hν/ methanol
NHR
NHR
-Cl +H2N
-Cl +H2N
NH
NH
S
S
O
S
O
S
4c R = H
4a R = H
4d R = CH(CH3)2
4b R = CH(CH3)2
Scheme 1.
increased e values, while amidino-derivatives (3c and
3d) of 3-chlorothieno[3,2-b]thiophene-2-carboxamide
2b had increased e values and lower absorption maxi-
ma than the corresponding cyano-derivative (Table 1).
Cyclization of 3a–d into 2-amidino-6-oxo-5,6-dihydro-
thieno[30,20:4,5]thieno[2,3-c]quinolines 4a, 4b and
2-amidino-6-oxo-5,6-dihydrothieno[20,30:4,5]thieno[2,3-
c]quinolines 4c, 4d resulted in pronounced bathochro-
mic shifts of the absorption maxima of ‘acyclic’ com-
pounds 3a–d (Table 2). Comparison of e values and
kmax of absorption maxima strongly suggests that
positioning of the sulfur atom within 4a–4d and their
‘acyclic’ precursors, as well as the nature of the
Table 2. Electronic absorption data of 4a–da
Compound
kmax (nm)
e · 103 (dm3molꢀ1 cmꢀ1
)
4a
346
331
327
266
250
12.8
12.5
10.1
37.4
42.2
4b
4c
4d
345
330
316
264
249
9.1
8.5
6.3
25.5
31.1
348
333
318
272
254
12.6
12.9
11.2
24.0
25.6
Table 1. Electronic absorption data of 2a,b and 3a–da
Compound
kmax (nm)
e · 103 (dm3 molꢀ1 cmꢀ1
)
2a
2b
3a
3b
3c
3d
295
321
308
308
316
315
9.9
10.4
24.6
25.7
21.6
25.3
348
333
317
271
253
18.0
18.9
16.6
35.2
38.8
a Sodium cacodylate/HCl buffer, I = 0.05 mol dmꢀ3, pH 6.2.
a Sodium cacodylate/HCl buffer, I = 0.05 mol dmꢀ3, pH 6.2.

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I. Jarak et al. / Bioorg. Med. Chem. 14 (2006) 2859–2868
substituent, have a systematic impact on electron
absorption properties of studied compounds and prob-
ably electron distribution in the systems studied.
cyano 2a–b, and amidino and isopropylamidino deriva-
tives 3a–d and their ‘cyclic’ analogues 4a–d with ctDNA.
Since ‘cyclic’ derivatives contain a large aromatic moie-
ty, they should form an intercalative complex with the
double-stranded helix of ctDNA. ‘Acyclic’ derivatives
possessing a distinctively smaller aromatic surface and
a more flexible scaffold might not be able to intercalate
the double-stranded helix of DNA. The mentioned
structural differences could therefore cause different
modes in their biological action.
Compounds 4a–d exhibit characteristic fluorescence
emission with maxima at about 391 nm (4a–b), and
372 and 391 nm (4c–d); their excitation spectra are in
good agreement with absorption spectra. Fluorescence
intensities are proportional to their concentrations up
to 5 · 10ꢀ6mol dmꢀ3 (4b, 4d) and 2 · 10ꢀ6 mol dmꢀ3
(4a, 4c).
Interactions of compounds 2a–b, 3a–d and 4a–d with
ctDNA were evaluated by thermal melting experiments
and UV/vis and fluorimetric titrations.
To have predominantly protonated amidines under the
experimental conditions used (more than 99%), we per-
formed our studies at pH 6.2. Aqueous solutions of all
studied compounds have shown to be stable over longer
periods of time.
DNA binding properties of compounds 2a–b, 3a–d, and
4a–d were evaluated by thermal melting experiments at
different ratios r([compound]/[polynucleotide phos-
phate]). Melting temperatures (Tm) did not change in
the presence of ‘acyclic’ derivatives 2a, 2b, and 3a–d at
the ratio r = 0.3 (compounds in excess over the interca-
lation binding sites). On the other hand, addition of ‘cy-
clic’ derivatives 4a–d stabilized the double helix of
ctDNA (increased Tm) at all examined ratios
r(r = 0.1–0.3), as shown in Table 3. Melting transitions
were monophasic, pointing to only one dominant bind-
ing mode (Fig. 2). All studied compounds yielded a sim-
ilar stabilization of ctDNA. Increase of DTm values was
not proportional to the increase of ratio r, suggesting
saturation of binding sites within the values of r = 0.2–
0.3.
2.3. Interactions of ‘acyclic’ derivatives 2a,b, 3a–d and
their ‘cyclic’ analogues 4a–d with double-stranded ctDNA
Two series of structurally related amidino-substituted
‘acyclic’ 3-chlorothienothiophene-2-carboxamides 3a–d
and ‘cyclic’ 6-oxo-5,6-dihydrothienothienyl[2,3-c]quino-
lines 4a–d were examined for their potential antiprolifer-
ative effects. All of them possess a certain, but non-
selective, antiproliferative effect with enhanced selectivi-
ty and activity upon introduction of a cyano substituent
(2a, 2b).
To shed more light on the structure–biological activity
relationship, we have studied interactions of ‘acyclic’
Binding of ‘cyclic’ compounds 4a–d that stabilized
ctDNA was also studied by UV/vis and fluorescence
spectroscopic titrations. Addition of ctDNA to 4a
(c = 5 · 10ꢀ5 mol dmꢀ3), 4b (c = 7.1 · 10ꢀ5 mol dmꢀ3),
4c (c = 3.2 · 10ꢀ5 mol dmꢀ3), and 4d (c = 3.5 ·
10ꢀ5 mol dmꢀ3) resulted in strong hypochromic (41%
for 4a; 46.6% for 4b; 46.8% for 4c and 42.1% for 4d)
and bathochromic effects (ca. 7 nm shift of the maxi-
mum for all compounds). The observed changes in
UV/vis spectra strongly suggest intercalation as the
dominant binding mode and high values of binding
Table 3. DTm valuesa (ꢁC) of ctDNA with 4a–d at pH 6.2 (sodium
cacodylate/HCl buffer, I = 0.05 mol dmꢀ3
)
Compound
ctDNA
rb
4a
4b
4c
4d
0.1
0.2
0.3
1.9
2.4
2.7
1.9
2.2
2.7
1.4
1.7
2.6
1.9
2.5
3.9
a Error in DTm: 0.5 ꢁC.
b r = [compound]/[polynucleotide].
ct -DNA
4b r= 0.1
4b r= 0.2
4b r= 0.3
4d r= 0.1
ct-DNA
4a r = 0.1
A
B
0,14
0,13
0,12
0,11
0,10
0,09
0,08
0,07
0,06
0,05
0,04
0,03
0,02
0,01
0,00
-0,01
-0,02
-0,03
4a r = 0.2
4a r = 0.3
4c r = 0.1
4c r = 0.2
4c r = 0.3
0,02
0,00
-0,02
-0,04
-0,06
-0,08
-0,10
-0,12
4d r= 0.2
4d r= 0.3
20
30
40
50
60
70
80
90
100
45 50 55 60 65 70 75 80 85 90 95 100 105
t / 0
C
t / 0
C
Figure 2. The normalized changes of absorbance at 260 nm ctDNA on heating in the presence of (A) 4a, c and (B) 4b, d at ratios r([compound]/[ct-
DNA]) = 0.1–0.3; sodium cacodylate/HCl buffer, I = 0.05 mol dmꢀ3, pH 6.2.

I. Jarak et al. / Bioorg. Med. Chem. 14 (2006) 2859–2868
2863
Figure 3. UV/vis spectral changes of 4b (c = 7.1 · 10ꢀ5 mol dmꢀ3) upon the addition of ctDNA (c = 6.3 · 10ꢀ3 mol dmꢀ3); sodium cacodylate/HCl
buffer, I = 0.05 mol dmꢀ3, pH 6.2.
constants, Ks > 105 Mꢀ1 (Fig. 3). However, a systematic
deviation from the isosbestic point was observed for all
compounds, pointing to the presence of more than one
type of dominant complex. Since titration with ctDNA
yielded measurable changes in absorbance only at excess
of studied compounds, except for the possible intercala-
tive mode of binding, excess of positively charged mole-
cules could be bound on the outer polynucleotide
surface. A similar effect of weaker, non-intercalative
binding at a high intercalator/DNA ratio was observed
for most of the classical intercalators.12
cess of the polynucleotide. Addition of ct-DNA strongly
quenched emission of 4b–d and caused less pronounced
quenching of the emission of 4a (Fig. 4). Increasing the
amount of ctDNA, F/F0 of 4a reached ca. 0.69. Similar
quenching was observed for 4b and 4c (F/F0 ca. 0.24 and
0.17, respectively), while the complex of 4d with ctDNA
is emission inactive.
Processing of titration data by means of the Scatchard
equation gave the binding constants (logKs) and ratios
n([bound compound]/[polynucleotide phosphate]) pre-
sented in Table 4.
To make it possible to perform titrations under condi-
tions preferable for only one type of binding, we have
used strong fluorescence of aqueous solutions of 4a–d,
enabling titrations at up to 100 times lower concentra-
tions than used in UV/vis experiments and at a high ex-
The calculated binding constants (logKs) point to some-
what higher affinity of unsubstituted amidine hydrochlo-
rides 4a and 4c toward ctDNA compared to their
isopropyl-substituted amidino analogues 4b and 4d.
Figure 4. Fluorescence emission of 4d (c = 5 · 10ꢀ7 mol dmꢀ3) upon the addition of ctDNA (c = 2.58 · 10ꢀ3 mol dmꢀ3); sodium cacodylate/HCl
buffer, I = 0.05 mol dmꢀ3, pH 6.2.

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I. Jarak et al. / Bioorg. Med. Chem. 14 (2006) 2859–2868
Table 4. Binding constants (logKs) and ratios n([bound compound]/
[polynucleotide]) calculated according to fluorimetric titrations of 4a–d
(c = 5 · 10ꢀ7 mol dmꢀ3) with ctDNAa,b
of which derived from 5 cancer types: HeLa (cervical
carcinoma), MCF-7 (breast carcinoma), SW 620 (colon
carcinoma), MiaPaCa-2 (pancreatic carcinoma), Hep-2
(laryngeal carcinoma), and WI 38 (diploid fibroblasts).
For comparison and as a control of correct functionality
of the cell lines, three reference compounds (classical
antitumor drugs) were used: cisplatin (alkylating agent),
doxorubicin (intercalator), and etoposide (topoisomer-
ase II inhibitor).
c
Compound
logKs
n
F/F0
4a
4b
4c
4d
6.04
5.53
6.51
5.73
0.12
0.3
0.69
0.24
0.17
—
0.1
0.18
a UV/vis titrations were performed at pH 6.2 (sodium cacodylate/HCl
buffer, I = 0.05 mol dmꢀ3).
b Accuracy of n 30%, consequently varying values logKs 0.5.
c F = fluorescence of compound–DNA complex; F0 = fluorescence of
compound without ctDNA addition.
All tested compounds showed a certain antiprolifera-
tive effect (Table 5). Interestingly, although the com-
pounds bearing a cyano substituent, 2a and 2b, did
not generally show discernible antiproliferative effects,
they produced strikingly strong effects selectively on
HeLa and Hep-2 cell lines (Fig. 5A). A very similar ef-
fect was reported by Jarak et al.9 using carboxanilides
bearing a cyano substituent either on the anilide or
benzothiophene part of the molecule. HeLa and
Hep-2 cell lines have similar genetic backgrounds, both
having the human papilloma virus (HPV) type 18
DNA integrated into their genomes. It has even been
shown that the Hep-2 cells are HeLa cell contami-
nants/derivatives.13 It is not clear, however, why these
molecules are especially active in these cell lines, and
further studies are needed to provide the basis for a
new strategy in the antitumor therapy of HPV-related
cancers.
Possible shielding of the positive charge of amidine in
the latter compounds at least partially hampered addi-
tional stabilizing interactions with DNA phosphates.
Position of the sulfur atom in the 6-oxo-5,6-dihydrothie-
nothieno[2,3-c]quinoline moiety showed no significant
impact on the affinity toward ctDNA. The calculated n
values are in agreement with the concentration of inter-
calative binding sites and also in good agreement with
the non-linear DTm/ratio r dependency.
2.4. Biological results and discussion
Compounds 2a–4d were tested for their potential anti-
proliferative effect on a panel of 6 human cell lines, 5
Table 5. In vitro inhibition of the growth of tumor cells and normal human fibroblasts (WI 38)
a
Compound
IC50 (lM)
Hep-2
HeLa
MiaPaCa-2
SW 620
MCF-7
WI 38
2a
2b
0.7 0.1
0.7 0.01
3.8 0.8
6.3 1.3
2.7 1.3
4.8 0.6
20 3.5
21 0.3
21 1.4
0.1 0.01
0.2 0.001
2.2 0.08
36 45
>100
15.6 7.8
76 20
1.6 0.1
5.5 1.2
1.8 0.1
2.7 1.1
>100
>100
>100
18.6 30
P100
3.4 1.5
1.8 1.5
1.8 0.5
1.8 0.3
3a
3b
1.6 0.2
3.8 0.03
1.9 0.2
3
0.1
14.5
1
9.5 3.6
1.9 0.2
6.2 9.7
11 1.5
8.7 2.8
21 19
9.7 6.5
3c
3d
1.8 0.001
3.8 3.5
40 14
4.4
34 1.9
2
4a
4b
3.6
2
28
37
14
4
9
5
27.7
30.5 0.4
4
36 10
10 6.5
4.7 0.8
4c
4d
P100
11
5
12
2
18 0.8
1.8
6.4
19 20
5
Cis
Dox
Eto
2.4 0.3
0.04 0.01
N.T.
2.9 0.6
0.04 0.01
5.4 1.6
0.02 0.01
15.4 14
4
12
6
0.02 0.02
20 3.4
0.04 0.01
50 30
0.1 0.01
2.9
1
N.T.
a IC50, the concentration that causes a 50% reduction of the cell growth.
N.T., not tested; Cis, cisplatin; Dox, doxorubicin; Eto, etoposide.
Compound 2a
A
Compound 4d
B
150
150
100
50
Hep-2
Hep-2
100
HeLa
HeLa
50
0
SW 620
WI 38
SW 620
WI 38
0
MCF-7
MCF-7
-50
-100
-50
-100
MiaPaCa-2
MiaPaCa-2
-9
-8
-7
-6
-5
-4
-3
-9
-8
-7
-6
-5
-4
-3
log10 concentration (M)
log10 concentration (M)
Figure 5. Dose–response profile for compounds 2a (A) and 4d (B) tested on various human cell lines in vitro. The cells were treated with the
compounds 2a and 4d at different concentrations, and percentage of growth (PG) was calculated. Each point represents a mean value of four parallel
samples in three individual experiments.

I. Jarak et al. / Bioorg. Med. Chem. 14 (2006) 2859–2868
2865
Similar intriguing results were also reported by Beneteau
et al.14 These authors described the synthesis of novel
benzothiazoles and dioxinobenzothiazoles, among
which the 2-cyano derivatives exhibit interesting in vitro
antitumor activity, though on a different cell model
(mouse leukemia cells, L1210). Therefore, the antiprolif-
erative effect of cyano-substituted compounds 2a and 2b
could be correlated with the similar effect of a related
heteroaromatic system (benzothiazole-2-carbonitrile
derivatives).14 It is important to stress that the authors
of the aforementioned study found that the conversion
of nitrile in a number of other neutral or negatively
charged substituents clearly inhibited antiproliferative
activity. Some of the pathways they used are also possi-
ble under in vivo conditions if catalyzed by enzymes.
This suggests that both herein studied 2a and 2b, and
their benzothiazole analogues base their biological activ-
ity on interactions of the nitrile moiety with the molec-
ular target(s), but not with the possible nitrile
metabolic products.
‘acyclic’ analogues 3a–d use some other pathway of
action.
Intriguing antiproliferative effects of cyano-substituted
compounds 2a and 2b on HeLa and Hep-2 cell lines
could, similarly to the related heteroaromatic benzo-
thiazole-2-carbonitrile derivatives,14 base their biologi-
cal activity on interactions of the nitrile moiety with
the molecular target(s). Moreover, HeLa and Hep-2 cell
lines have the human papilloma virus (HPV) type 18
DNA integrated into their genomes, and HPVs types
16 and 18 have oncogenic potential, attributed mainly
to two small proteins called E6 and E7. Repression of
E6 and E7 expression results in acquisition of the senes-
cent phenotype and in the rescue of functional p53 and
p105Rb pathways; therefore, therapies directed against
either viral protein may be beneficial.16,17 Compounds
2a and 2b could potentially inhibit the E6 and E7
expression, but further studies are needed to prove this
assumption.
In contrast, N-40-amidinophenyl carboxamide hydro-
chlorides 3a, 3c and N-40-(N0-isopropylamidino)phenyl
carboxamide hydrochlorides 3b, 3d were much more ac-
tive, but in general showing mostly ‘non-differential’
cytotoxicity at the highest concentrations tested. Thus,
the introduction of a strong positively charged moiety
resulted in a strong, but not selective (differential), anti-
proliferative activity, implying a different molecular
target and/or mechanism of action.
4. Experimental
4.1. Materials
All the solvents were of analytical grade. Melting points
were determined on a Kofler hot stage microscope and
are uncorrected. IR spectra were recorded on a Nicolet
Magna 760 spectrophotometer in KBr disks. 1H and
13C NMR spectra were recorded on either a Varian
Gemini 300 or a Bruker Avance DPX 300 spectrometer
using TMS as an internal standard in DMSO-d6. Ele-
mental analyses for carbon, hydrogen, and nitrogen
were performed on a Perkin-Elmer 2400 elemental ana-
lyzer. Where analyses are indicated only as symbols of
elements, analytical results obtained are within 0.4% of
the theoretical value. Irradiation was performed at room
temperature with a water-cooled immersion well with an
‘‘Original Hanau’’ 400 W high-pressure mercury arc
lamp using Pyrex filter. All compounds were routinely
checked by TLC with Merck silica gel 60F-254 glass
plates.
Compounds 4a, 4b, 4c, and 4d display strong (4d)
(Fig. 5B) and differential (4a–c) effects, however with a
rather strong inhibitory effect on normal fibroblasts.
As a result, in contrast to the results presented in our
previous work,9 where ‘cyclic’ analogues—benzo[b]thie-
no[2,3-c]qinolones displayed prominent and selective
activity, the corresponding selectivity of their thie-
no[30,20:4,5]- and thieno[20,30:4,5]- analogues is not pro-
nounced. Nevertheless, it has been demonstrated again
that the introduction of the amidino substituent into
the herein presented series of novel compounds led to
the best antiproliferative effect on the cell lines tested.
4.2. Synthesis
3. Conclusion
4.2.1.
N-(40-Cyanophenyl)-3-chlorothieno[2,3-b]thio-
Strong bathochromic and hypochromic effects in the
UV/vis spectra of cyclic derivatives 4a–d upon addition
of ctDNA, thermal stabilization of ctDNA, the calculat-
ed characteristic values ratio n and high binding con-
stants strongly support intercalation as a dominant
binding mode.15 Interactions of substituted amidine
derivatives (4b and 4d) with DNA are somewhat weaker
compared to analogues bearing unsubstituted amidine
(4a and 4c), very probably due to steric hindering of
the voluminous isopropyl moiety. ‘Acyclic’ precursors
2a–3d do not interact significantly with the double-
stranded helix of ctDNA.
phene-2-carboxamide (2a). To a solution of 1a (2 g,
8.4 mmol) in dry toluene (90 ml) was added dropwise a
solution of 4-aminobenzonitrile (1 g, 8.4 mmol) in dry
toluene (60 ml), followed by the addition of Et3N
(1.4 ml, 10 mmol). The mixture was refluxed for 2.5 h.
After cooling, precipitated crystals were filtered off and
washed with diluted HCl, water, and acetone to yield
1.89 g (70%) of yellow crystals: mp 197–201 ꢁC; IR
(KBr) (mmax/cmꢀ1): 3380, 3100, 2200, 1650, 1590; 1H
NMR (DMSO-d6) (d ppm): 10.61 (s, 1H, Hamide), 7.82
(d, 2H, J = 8.72 Hz, Harom.), 7.78 (d, 2H, J = 8.75 Hz,
Harom.), 7.76 (d, 1H, J = 5.37 Hz, Hthiophene), 7.30 (d,
1H, J = 5.33 Hz, Hthiophene); 13C NMR (DMSO-d6) (d
ppm): 159.8, 144.4, 143.0, 138.8, 133.9, 133.7 (2C),
132.9, 120.7 (2C), 119.4, 119.2, 118.1, 106.4. Anal. Calcd
Antiproliferative activity of ‘cyclic’ derivatives 4a–d is
very likely caused by intercalation into DNA, while their

2866
I. Jarak et al. / Bioorg. Med. Chem. 14 (2006) 2859–2868
for (C14H7ClN2OS2): C, 52.74; H, 2.21; N, 8.79. Found:
C, 52.69; H, 2.26; N, 8.72.
1H, Hamide), 9.47 (br s, 3H, Hamidine), 7.98 (d, 2H,
J = 8.15 Hz, Harom.), 7.83 (d, 1H, J = 5.095 Hz,
Hthiophene), 7.77 (d, 2H, J = 8.24 Hz, Harom.), 7.375
(d, 1H, J = 4.99 Hz, Hthiophene), 4.06 (m, 1H,
J = 5.92 Hz, CHi-Pr), 1.28 (d, 6H, J = 5.84 Hz, 2CH3i-
Pr); 13C NMR (DMSO-d6) (d ppm): 161.1, 159.4,
143.9, 142.7, 138.3, 133.6, 132.5, 129.3 (2C), 124.3,
119.7 (2C), 118.8, 117.5, 44.9, 21.3 (2C). Anal. Calcd
for (C17H17Cl2N3OS2): C, 49.27; H, 4.13; N, 10.14.
Found: C, 48.95; H, 3.88; N, 10.04.
4.2.2.
N-(40-Cyanophenyl)-3-chlorothieno[3,2-b]thio-
phene-2-carboxamide (2b). To a solution of 1b (1.48 g,
6.2 mmol) in dry toluene (50 ml) was added dropwise a
solution of 4-aminobenzonitrile (0.74 g, 6.2 mmol) in
dry toluene (50 ml), followed by the addition of Et3N
(1.3 ml, 9.3 mmol). The mixture was refluxed for 2.5 h.
After cooling, precipitated crystals were filtered off and
washed with diluted HCl and water. After recrystalliza-
tion from DMF, 1.2 g (60%) of ivory crystals was ob-
tained: mp 147–151 ꢁC; IR (KBr) (mmax/cmꢀ1): 3480,
4.2.5.
N-(40-Amidinophenyl)-3-chlorothieno[3,2-b]thio-
phene-2-carboxamide hydrochloride (3c). Into the sus-
pension of 2b (0.5 g, 1.55 mmol) in absolute methoxy-
ethanol (50 ml), dry HCl was bubbled for 4 h. The
suspension was stirred at room temperature until the
–CN band was undetectable (IR). After anhydrous
diethyl ether was added, the corresponding imido-ester
was collected by filtration. The product was suspended
in absolute ethanol (25 ml) and dry NH3 was bubbled
into the suspension. The mixture was stirred at room
temperature for 7 days. The volume was reduced by
evaporation and anhydrous diethyl ether was added.
The crude product was filtered off and washed with
hot acetone. After recrystallization from methanol,
0.265 g (43%) of white crystals was obtained: mp
+300 ꢁC; IR (KBr) (mmax/cmꢀ1): 3354, 3089, 1683,
1634; 1H NMR (DMSO-d6) (d ppm): 10.76 (s, 1H,
Hamide), 9.30 (s, 2H, Hamidine), 8.99 (s, 2H, Hamidine),
8.02 (d, 1H, J = 5.20 Hz, Hthiophene), 7.92 (d, 2H,
J = 9.14 Hz, Harom.), 7.87 (d, 2H, J = 9.08 Hz, Harom.),
7.635 (d, 1H, J = 5.23 Hz, Hthiophene); 13C NMR
(DMSO-d6) (d ppm): 165.3, 159.8, 143.7, 138.9, 133.0,
132.4, 129.7 (2C), 123.3, 122.1, 120.2 (2C), 118.7. Anal.
Calcd for (C14H11Cl2N3OS2): C, 45.17; H, 2.98; N,
11.29. Found: C, 45.35; H, 2.72; N, 11.16.
1
2200, 1650, 1600; H NMR (DMSO-d6) (d ppm): 10.66
(s, 1H, Hamide), 8.99 (d, 1H, J = 5.22 Hz, Hthiophene),
7.90 (d, 2H, J = 8.81 Hz, Harom.), 7.83 (d, 2H,
J = 8.75 Hz, Harom.), 7.61 (d, 1H, J = 5.21 Hz,
Hthiophene); 13C NMR (DMSO-d6) (d ppm): 159.8,
143.0, 138.9, 138.6, 133.7 (2C), 132.9, 132.4, 122.1,
121.1 (2C), 120.7, 119.3, 106.5. Anal. Calcd for
(C14H7ClN2OS2): C, 52.74; H, 2.21; N, 8.79. Found:
C, 52.70; H, 2.11; N, 8.91.
4.2.3.
N-(40-Amidinophenyl)-3-chlorothieno[2,3-b]thio-
phene-2-carboxamide hydrochloride (3a). Into the sus-
pension of 2a (0.5 g, 1.55 mmol) in absolute ethanol
(50 ml) dry HCl was bubbled for 4 h. The suspension
was stirred at room temperature until the –CN band
was undetectable (IR). After anhydrous diethyl ether
was added, the corresponding imido-ester was collect-
ed by filtration. The product was suspended in abso-
lute ethanol (35 ml) and dry NH3 was bubbled into
the suspension. The mixture was stirred at room tem-
perature for 3 days. The crude product was filtered off
and washed with hot acetone. After recrystallization
from methanol, 0.48 g (87%) of white crystals was ob-
tained: mp 247–251 ꢁC; IR (KBr) (mmax/cmꢀ1): 3345,
3094, 1681, 1644, 1601; 1H NMR (DMSO-d6) (d
ppm): 9.04 (br s, 4H, Hamidine), 7.87 (d, 2H, J = 8.81,
Harom.), 7.83 (d, 2H, J = 8.82, Harom.), 7.82 (d, 1H,
J = 5.3, Hthiophene), 7.34 (d, 1H, J = 5.3, Hthiophene);
13C NMR (DMSO-d6) (d ppm): 164.4, 159.5, 140.0,
143.5, 138.1, 134.0, 132.3, 128.8 (2C), 123.6, 119.8,
118.7 (2C), 117.3. Anal. Calcd for (C14H11Cl2N3OS2):
C, 45.17; H, 2.98; N, 11.29. Found: C, 45.01; H,
2.86; N, 11.43.
4.2.6. N-[40-(N0-Isopropylamidino)phenyl]-3-chlorothie-
no[3,2-b]thiophene-2-carboxamide hydrochloride (3d).
Into the suspension of 2b (0.5 g, 1.55 mmol) in absolute
methoxy-ethanol (50 ml) dry HCl was bubbled for 4 h.
The suspension was stirred at room temperature until
the –CN band was undetectable (IR). After anhydrous
diethyl ether was added, the corresponding imido-ester
was collected by filtration. The product was suspended
in absolute ethanol (25 ml) and isopropylamine (5 ml,
0.06 mol) was added. The mixture was refluxed for
18 h. After cooling, anhydrous diethyl ether was added.
The crude product was filtered off and washed with hot
acetone. After recrystallization from ethanol, 0.16 g
(26%) of white crystals was obtained: mp +300 ꢁC; IR
4.2.4. N-[40-(N0-Isopropylamidino)phenyl]-3-chlorothie-
no[2,3-b]thiophene-2-carboxamide hydrochloride (3b).
Into the suspension of 2a (0.5 g, 1.55 mmol) in abso-
lute ethanol (50 ml), dry HCl was bubbled for 4 h.
The suspension was stirred at room temperature until
the –CN band was undetectable (IR). After anhydrous
diethyl ether was added, the corresponding imido-ester
was collected by filtration. The product was suspended
in absolute ethanol (20 ml), and isopropylamine
(13.8 ml, 162 mmol) was added. The mixture was re-
fluxed for 24 h. The volume was reduced by evapora-
tion and the product was filtered off and washed
with hot acetone. After recrystallization from metha-
nol, 0.36 g (59%) of white crystals was obtained: mp
305–307 ꢁC; IR (KBr) (mmax/cmꢀ1): 3360, 3037, 1672,
1658, 1614; 1H NMR (DMSO-d6) (d ppm): 10.83 (s,
1
(KBr) (mmax/cmꢀ1): 3363, 3037, 1660, 1614; H NMR
(DMSO-d6) (d ppm): 10.14 (s, 1H, Hamide), 8.94 (d,
1H, J = 9.00 Hz, Hamidine), 8.79 (s, 1H, Hamidine), 8.41
(s, 1H, Hamidine), 7.47 (d, 1H, J = 5.32 Hz, Hthiophene),
7.37 (d, 2H, J = 8.67 Hz, Harom.), 7.215 (d, 2H,
J = 8.78 Hz, Harom.), 7.09 (d, 1H, J = 5.18 Hz,
Hthiophene), 3.47 (m, 1H, J = 6.43 Hz, CHi-Pr), 1.28 (d,
6H, J = 6.28 Hz, 2CH3i-Pr); 13C NMR (DMSO-d6) (d
ppm): 116.1, 159.3, 142.6, 138.4, 138.1, 132.4, 131.9,
129.3 (2C), 124.2, 121.6, 119.7 (2C), 118.1, 44.9, 21.3
(2C). Anal. Calcd for (C17H17Cl2N3OS2): C, 49.27; H,
4.13; N, 10.14. Found: C, 49.17; H, 4.27; N, 9.76.

I. Jarak et al. / Bioorg. Med. Chem. 14 (2006) 2859–2868
2867
4.2.7. General method for the synthesis of 2-amidino-6-
oxo-5,6-dihydrothienothienyl[2,3-c]quinoline hydrochlo-
ride (4a–d).
50.07; H, 3.00; N, 12.51. Found: C, 50.27; H, 2.67; N,
12.29.
A
solution of 40-amidino-substituted
N-phenyl-3-chlorothieno[2,3-b]thiophene-2-carbox-
amide hydrochloride (3a, b) and N-phenyl-3-chlorothie-
no[3,2-b]thiophene-2-carboxamide hydrochloride (3c, d)
in methanol or methoxy-ethanol was irradiated at room
temperature with a 400-W high-pressure mercury lamp
using Pyrex filter for 1.5–19 h. The air was bubbled
through the solution. The solution was concentrated
and the obtained solid was filtered off, washed with
hot acetone and recrystallized.
4.2.11. 2-(N0-Isopropyl)amidino-6-oxo-5,6-dihydrothie-
no[20,30:4,5]thienyl[2,3-c]quinoline hydrochloride (4d). A
solution of 3d (0.101 g, 0.24 mmol) in methanol (12 ml)
was irradiated for 19 h. The formed precipitate was fil-
tered off and recrystallized from methanol to yield
0.06 g (70%): mp +300 ꢁC; IR (KBr) (mmax/cmꢀ1): 3361,
1
3211, 3060, 1645; H NMR (DMSO-d6) (d ppm): 12.51
(s, 1H, Hquinolone), 9.79 (s, 1H, Hamidine), 9.63 (s, 1H,
Hamidine), 9.15 (s, 1H, Hamidine), 8.28 (s, 1H, Harom.),
8.245 (d, 1H, J = 6.26 Hz, Hthienyl), 7.89 (d, 1H,
J = 8.79 Hz, Harom.), 7.81 (d, 1H, J = 5.08 Hz, Hthienyl),
7.69 (d, 1H, J = 8.64 Hz, Harom.), 4.10 (s, 1H, CHi-Pr),
1.34 (d, 6H, J = 6.23 Hz, 2CH3i-Pr); 13C NMR (DMSO-
d6) (d ppm): 161.5, 158.0, 143.4, 140.6, 134.2, 134.0,
133.1, 128.9, 124.3, 123.0, 120.9, 116.6, 114.7, 45.2,
21.3 (2C). Anal. Calcd for (C17H16ClN3OS2): C, 54.03;
H, 4.27; N, 11.11. Found: C, 53.84; H, 4.44; N, 11.34.
4.2.8. 2-Amidino-6-oxo-5,6-dihydrothieno[30,20:4,5]thie-
nyl[2,3-c]quinilone hydrochloride (4a). A solution of 3a
(0.05 g, 0.13 mmol) in methoxy-ethanol (10 ml) was irra-
diated for 1.5 h. The formed precipitate was filtered off
and recrystallized from methanol to yield 0.027 g
(48%): mp +300 ꢁC; IR (KBr) (mmax/cmꢀ1): 3322, 3119,
1649, 1589; 1H NMR (DMSO-d6) (d ppm): 12.47 (s,
1H, Hquinolone), 9.50 (s, 2H, Hamidine), 9.08 (s, 2H,
Hamidine), 8.74 (d, 1H, J = 1.81 Hz, Harom), 8.30 (d,1H,
J = 5.54 Hz, Hthienyl), 8.02 (d, 1H, J = 5.42, Hthienyl),
7.97 (dd, 1H, J1 = 8.73 Hz, J2 = 1.92 Hz, Harom), 7.67
(d, 1H, J = 8.7 Hz, Harom); 13C NMR (DMSO-d6) (d
ppm):165.3, 158.3, 144.5, 141.7, 141.4, 135.3, 134.4,
132.4, 128.8, 124.9, 122.1, 122.0, 117.2, 116.5. Anal.
Calcd for (C14H10ClN3OS2): C, 50.07; H, 3.00; N,
12.51. Found: C, 50.19; H, 3.12; N, 12.75.
4.3. Interactions with DNA
The electronic absorption spectra were obtained on a
Varian Cary 100 Bio spectrometer, and fluorescence
emission spectra were recorded on a Varian Eclipse fluo-
rimeter, in both cases using quartz cuvettes (1 cm).
DNA was purchased from Fluka and used without
further purification. DNA was dissolved in the sodium
cacodylate buffer, 0.05 mol dmꢀ3, pH 7, and its concen-
tration was determined spectroscopically as the concen-
tration of phosphates. The measurements were
performed in aqueous buffer solution (pH 6.2; sodium
cacodylate/HCl buffer, I = 0.05 mol dmꢀ3). Under the
experimental conditions used, the absorbance and fluo-
rescence intensities of studied compounds were propor-
tional to their concentrations. In fluorimetric titrations,
excitation wavelengths of 317 nm (4a, b) and 319 nm
(4c, d) were used to avoid inner filter effects caused by
absorption of excitation light of added polynucleotide,
and changes of emission were monitored at 391 nm
(4a, b) and 370 nm (4c, d). The binding constants (Ks)
and [bound compound]/[polynucleotide phosphate] ra-
tio n were calculated according to the Scatchard equa-
tion by non-linear least-squares fitting. Values for Ks
given in Table 4 all have satisfying correlation coeffi-
cients (>0.999). The thermal melting curves for DNA
and its complexes were determined as previously de-
scribed by following the absorption change at 260 nm
as a function of temperature. The absorbance scale
was normalized. Tm values are the midpoints of the tran-
sition curves, determined from the maximum of the first
derivative or graphically by a tangent method. DTm val-
ues were calculated by subtracting Tm of the free nucleic
acid from Tm of the complex. Every DTm value here
reported was the average of at least two measurements,
the error in DTm is 0.5 ꢁC.
4.2.9.
2-(N0-Isopropyl)amidino-6-oxo-5,6-dihydrothie-
no[30,20:4,5]thienyl[2,3-c]quinoline hydrochloride (4b). A
solution of 3b (0.1 g, 0.24 mmol) in methanol (14 ml)
was irradiated for 2 h and 45 min. The formed precipi-
tate was filtered off and recrystallized from ethanol to
yield 0.075 g (82%): mp +300 ꢁC; IR (KBr) (mmax
/
cmꢀ1): 3394, 3109, 1652, 1622, 1584; 1H NMR
(DMSO-d6) (d ppm): 12.44 (s, 1H, Hquinolone), 9.63 (s,
3H, Hamidine), 8.69 (s, 1H, Harom), 8.23 (d, 1H,
J = 5.47 Hz, Hthienyl), 8.02 (d, 1H, J = 5.38 Hz, Hthienyl),
7.89 (d, 1H, J = 8.56 Hz, Harom), 7.67 (d, 1H,
J = 8.65 Hz, Harom), 4.17–4.09 (1H, m, J = 6.29 Hz,
CHi-Pr), 1.34 (d, 6H, J = 6.30 Hz, 2CH3i-Pr); 13C NMR
(DMSO-d6) (d ppm): 161.8, 158.3, 144.5, 141.7, 140.9,
135.2, 134.3, 132.5, 129.1, 125.0, 123.3, 122.1, 116.9,
116.3,
45.6,
21.8
(2C).
Anal.
Calcd
for
(C17H16ClN3OS2): C, 54.03; H, 4.27; N, 11.11. Found:
C, 54.24; H, 4.02; N, 11.21.
4.2.10. 2-Amidino-6-oxo-5,6-dihydrothieno[20,30:4,5]thie-
nyl[2,3-c]quinoline hydrochloride (4c). A solution of 3c
(0.099 g, 0.27 mmol) in methanol (14 ml) was irradiated
for 13 h. The formed precipitate was filtered off and
recrystallized from methanol to yield 0.049 g (55%):
mp +300 ꢁC; IR (KBr) (mmax/cmꢀ1): 3311, 3100, 1672,
1633, 1592; 1H NMR (DMSO-d6) (d ppm): 12.53 (s,
1H, Hquinolone), 9.61 (s, 2H, Hamidine), 9.26 (S, 2H,
Hamidine), 8.30 (d, 1H, J = 1.86 Hz, Harom), 8.24 (d,
1H, J = 5.27 Hz, Hthienyl), 7.96 (dd, 1H, J1 = 8.69 Hz,
J2 = 1.99 Hz, Harom), 7.79 (d, 1H, J = 5.27 Hz, Hthienyl),
7.70 (d, 1H, J = 8.68 Hz, Harom); 13C NMR (DMSO-d6)
(d ppm): 165.9, 158.6, 143.9, 141.5, 134.7, 133.6, 133.56,
122.5, 115.3. Anal. Calcd for (C14H10ClN3OS2): C,
4.4. Antitumor activity assays
The HeLa (cervical carcinoma), MCF-7 (breast carci-
noma), SW 620 (colon carcinoma), MiaPaCa-2 (pancre-

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I. Jarak et al. / Bioorg. Med. Chem. 14 (2006) 2859–2868
atic carcinoma), Hep-2 (laryngeal carcinoma), and WI
38 (diploid fibroblasts) cells were cultured as monolayers
and maintained in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum
(FBS), 2 mM L-glutamine, 100 U/ml penicillin, and
100 lg/ml streptomycin in a humidified atmosphere with
5% CO2 at 37 ꢁC.
concentrations produce PGs exceeding the respective
reference level of effect (e.g., PG value of 50), then the
highest tested concentration is assigned as the default
value, which is preceded by a ‘>’ sign. Each result is a
mean value from three separate experiments.
Acknowledgment
The growth inhibition activity was assessed according to
the slightly modified procedure performed at the
National Cancer Institute, Developmental Therapeutics
Program.9,18 The cells were inoculated onto standard
96-well microtiter plates on day 0. The cell concentra-
tions were adjusted according to the cell population
doubling time (PDT): 1 · 104/ml for HeLa, Hep-2, Mia-
PaCa-2, and SW 620 cell lines (PDT = 20–24 h), 2 · 104/
ml for MCF-7 cell lines (PDT = 33 h), and 3 · 104/ml for
WI 38 (PDT = 47 h). Test agents were then added in
five-, ten-fold dilutions (10ꢀ8–10ꢀ4 mol/L) and incubat-
ed for further 72 h. Working dilutions were freshly pre-
pared 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 concen-
trations. After 72 h of incubation, the cell growth rate
was evaluated by performing the MTT assay, which
detects dehydrogenase activity in viable cells.19 The
absorbency (OD, optical density) was measured on a
microplate reader at 570 nm. The percentage of growth
(PG) of the cell lines was calculated according to one or
the other of the following two expressions:
Support for this study by the Ministry of Science, Edu-
cation and Sport of Croatia is gratefully acknowledged
(Projects 0125005, 0098093, and 0098053).
References and notes
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Mean ODtzero = the average of optical density measure-
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´ ´
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Each test point was performed in quadruplicate in three
individual experiments. The results are expressed as
IC50, which is the concentration necessary for 50% of
inhibition. The IC50 values for each compound are cal-
culated from dose–response curves using linear regres-
sion analysis by fitting the test concentrations that give
PG values above and below the reference value (i.e.,
50%). If however, for a given cell line all of the tested