Synthesis, photochemical synthesis, DNA binding and antitumor evaluation of novel cyano- and amidino-substituted derivatives of naphtho-furans, naphtho-thiophenes, thieno-benzofurans, benzo-dithiophenes and their acyclic precursors

Article abstract of DOI:10.1016/j.ejmech.2006.03.012

As a part of the research on the improvement of an alternative to conventional photodynamic therapy by light-induced formation of intercalators, we synthesized a series of novel heterocyclic compounds and their acyclic precursors. We now report details ab

Full text of DOI:10.1016/j.ejmech.2006.03.012

European Journal of Medicinal Chemistry 41 (2006) 925–939
http://france.elsevier.com/direct/ejmech
Original article
Synthesis, photochemical synthesis, DNA binding and antitumor evaluation
of novel cyano- and amidino-substituted derivatives of naphtho-furans,
naphtho-thiophenes, thieno-benzofurans, benzo-dithiophenes
and their acyclic precursors
Kristina Starčević a, Marijeta Kralj b,*, Ivo Piantanida c, Lidija Šuman b, Krešimir Pavelić b,
Grace Karminski-Zamola a,*
a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology,
University of Zagreb, Marulićev trg 20, P.O. Box 177, HR-10000 Zagreb, Croatia
b Division of Molecular Medicine, Laboratory of Functional Genomics, Ruđer Bošković Institute, Bijenička cesta 54, P.O. Box 180, HR-10000 Zagreb, Croatia
c Division of Organic Chemistry and Biochemistry, Laboratory of Supramolecular and Nucleoside Chemistry,
Rudjer Boskovic Institute, P.O. Box 180, HR-10002 Zagreb, Croatia
Received in revised form 20 March 2006; accepted 23 March 2006
Available online 02 May 2006
Abstract
As a part of the research on the improvement of an alternative to conventional photodynamic therapy by light-induced formation of inter-
calators, we synthesized a series of novel heterocyclic compounds and their acyclic precursors. We now report details about their synthesis/
characterization in respect to their potential of photoinduced cyclization, interactions with DNA and inhibition of the tumor cell growth in vitro.
Among studied compounds only amidino-furyl-substituted phenyl acrylates were efficiently converted to the corresponding naphthofuranes,
while their thiophene analogues, all non-charged derivatives and amidino-phenyl-substituted analogues didn’t show acceptable photoconversion.
The significantly stronger antiproliferative activity of cyclic analogues could be correlated to the property of these molecules to intercalate into
DNA. The acyclic molecules did not show any interaction with DNA, correlating with the inferior biological activity, except for one cyano-
bearing molecule.
© 2006 Elsevier SAS. All rights reserved.
Keywords: Antitumor substances; Intercalators; Photocyclization; Tumor cell lines
1. Introduction
response of the tumor cells are of high interest for medicinal
chemists and molecular biologists. For example, the intercala-
tion of planar aromatic molecules into the DNA double helix
and poisoning of DNA topoisomerases I and/or II are consid-
ered to be important in the therapeutic action of many antitu-
mor agents [1,2]. The best examples are anthracyclines (e.g.
doxorubicin), anthracenediones (e. g. mitoxantrone), some ac-
ridines (e.g. acridine-4-carboxamide) and ellipticines [3].
In our previous paper [4] we reported about the photoin-
duced switch of DNA/RNA inactive amidino-substituted 3-(2-
furyl)-2-phenyl-acrylate into a classical intercalator as amidino-
substituted naphthofuran carboxylate. Acyclic precursors, as
well as cyclized compounds were also tested for the antiproli-
ferative effect on a panel of six human cell lines, five of which
The discovery of new compounds with antitumoral activity
has become one of the most important goals in medicinal
chemistry. One of the most often used classes of chemothera-
peutic agents in cancer therapy comprises molecules that inter-
act with DNA, such as groove binders, DNA alkylating sub-
stances and intercalators. Moreover, the study of the exact
mechanisms of action of these agents, as well as, DNA damage
* Corresponding author.
E-mail addresses: kstarcev@fkit.hr (K. Starčević), mhorvat@irb.hr
(M. Kralj), pianta@irb.hr (I. Piantanida), suman@irb.hr (L. Šuman),
pavelic@irb.hr (K. Pavelić), gzamola@pierre.fkit.hr (G. Karminski-Zamola).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.03.012

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K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
were derived from five cancer types. The observed results
clearly demonstrate one order of magnitude superior action of
cyclic compound to its acyclic precursor. These experiments
present the conceptual basis for development of new photoin-
duced anticancer therapy based on the photochemical transfor-
mation on a non intercalating molecule into the intercalating
one, opposed the conventional photodynamic therapy, as a no-
vel approach to selective antitumor therapy.
clinical trials [10]. The results suggest that the presence of ami-
dine terminal groups plays a role in facilitating nuclear accu-
mulation into cells, probably as a result of nucleic acid binding
[11]. For example, series of pyrrolo[2,1-c][1,4]benzodiazepine
amidines has been evaluated for in vitro DNA binding through
thermal denaturation studies. Some of these compounds cause
a significant increase in melting of calf thymus DNA [12]. In-
hibition of tumor growth and polyamine uptake by tetracyclic
amidines were described [13]. DNA binding properties and cy-
totoxic activity of novel aromatic amidines in cultured human
skin fibroblasts were examined [14]. The synthesis and antitu-
mor evaluation of some new substituted amidino benzimidazo-
lyl naphtho[2,1-b]furan carboxylates was described recently
[15].
In this paper we present the synthesis of a number of new
acyclic substances, describe their potential of photoinduced
conversion into condensed heterocyclic compounds under bio-
logically relevant conditions, the study of their interactions
with DNA and tested their antitumor activity on several human
tumor cell lines.
Searching for another planar condensed heterocyclic com-
pounds, related to naphthofurans, naphthothiophenes, thieno-
benzofurans and benzodithiophenes which could serve as
DNA intercalators and, as a consequence, could exert antitu-
mor activity, we have prepared a new group of cyano- and
amidino-substituted naphtho[2,1-b]furans (6a, 6b, 7b, 8a),
naphtho[2,1-b]thiophenes (6c, 7a), benzothienofurans (11a,
12a, 12b, 15 and 16) and benzodithiophenes (11b, 12c) from
the corresponding acyclic precursors, of cyano- or amidino-
substituted-furyl-phenyl-acrylates,
thienyl-phenyl-acrylates,
furyl-thienyl acrylates and dithienyl-acrylates (2a, 2b, 3a, 3b,
3c, 4a, 4b, 4c, 5a, 5b, 5c, 10a, 10b and 14).
The constant and growing interest in the development of
new efficient and general synthetic methods for the preparation
of fused heterocyclic systems involving furan and thiophene
subunits is justified by their well-established valuable physio-
logical and pharmacological properties [5]; however, there is
little new literature data describing the synthesis and biological
activity of these systems [1,6]. Recent technical applications of
polysubstituted benzofurans and benzothiophenes, including
numerous fluorescent dyes used as retrograde tracers, and
Ca2+ and Mg2+ fluorescent indicator conjugates, etc. [5] in-
crease their significance. The biological activity of many are-
nofurane derivatives was improved by addition of positively
charged aliphatic substituents. For example, amidine-substi-
tuted heterocycles based on furan moiety have shown a number
of intriguing modes of biological activity, like antimicrobial
[7], hallucinogenic agents [8], and some furamidines were
found to be active against diverse, highly infectious parasites
[9]; the best being selected for currently undergoing phase II
2. Results and discussion
2.1. Chemistry
All compounds (2a–16) shown in the Figs. 1–3 were pre-
pared according to the Schemes 1–3. Acyclic cyano com-
pounds (2a and 2b) were prepared by aldol condensation from
Fig. 2. Substituted thieno[3,2-e]benzo[b]furans, thieno[2,3-e]benzo[b]thio-
phenes and their acyclic precursors (10a–b, 11a–b, 12a–c).
Fig. 1. Substituted naphtho[2,1-b]furans, naphtho[2,1-b]thiophenes and their acyclic precursors (2a–b, 3a–c, 4a–c, 5a–c, 6a–b, 7a–b, 8).

K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
927
Scheme 1. Synthesis of cyano- and amidino-substituted derivatives of naphtho[2,1-b]furans, naphtho[2, 1-b]thiophenes and their acyclic precursors.
5c) were prepared in the Pinner reaction [19] from cyano-deri-
vatives 2a, 2b, 3a, 3b and isopropylamine in the second phase
of the reaction, while morpholino-substituted amidines 4b, 5b
were prepared in the same manner in the reaction with morpho-
linoamine.
Cyclized amidino-substituted naphthofurans (6a and 6b)
were prepared in the photochemical dehydrocyclization reac-
tion in ethanol and water from 5a and 5b in the yields about
35%, while 4a, 4b, 4c and 5c didn’t give corresponding photo-
chemical cyclic product. Isopropylamidino-substituted
naphthofuran (8) was prepared in Pinner reaction from corre-
sponding dicyano-naphthofuran (7b) in the yields about 10–
20%. Cyano-substituted cyclized derivatives (7a ,7b),(Fig. 1),
as well as 11a, 11b and 15 (Figs. 2 and 3), were prepared in the
photochemical dehydrocyclization reaction from corresponding
Fig. 3. Substituted thieno[2,3-e]benzo[b]furans and their acyclic precursors (14,
15, 16).
cyanophenylacetic acid and corresponding heterocyclic alde-
hydes in the yield about 30%, while compounds 3a, 3b, 3c,
10 and 14 were prepared from corresponding aldehydes (1a,
1b, 1c, 9a, 9b and 13) [16–18] in the reaction with hydroxyla-
monium hydrochloride in the yield from about 50–70%. Novel
isopropylamidino-substituted acyclic compounds (4a, 4c, 5a,

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K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
2.2. Spectroscopic properties
The UV–vis spectra of all studied compounds didn’t show
any pH dependent changes in the physiologically relevant
range (pH = 3.5–8.0), which is also in agreement with the
pKa values of ~8–9 reported for aromatic amidines [20]. There-
fore, we have performed our studies at pH = 7. Stock solutions
of positively charged compounds (5a, 6a–b, 12a, 16) were pre-
pared in water, while stock solutions of cyano-substituted com-
pounds (2a–b, 3a–c, 10a, 14) due to their low solubility in
water were prepared in DMSO. Before experiment respective
aliquots were added to the aqueous buffer, partial volume of
DMSO in water not exceeding 0.1%. We were not able to
study compounds (7a–b, 11a, 15) because of their insolubility
in water. Stock solutions (both aqueous and DMSO) of all stu-
died compounds were stable over long period.
Absorbancies of aqueous solutions of compounds are pro-
portional to their concentrations up to 6 × 10–5 mol dm–3 (2a–
b, 3a–c, 10a, 14), 8 × 10–5 mol dm–3 (5a, 6a–b, 12a), 2 × 10–4
(16) mol dm–3, indicating that there is no significant intermo-
lecular stacking which should give rise to hypochromicity ef-
fects. Obtained electronic absorption data are presented in the
Section 4.
Scheme 2. Synthesis of cyano- and amidino-substituted derivatives of thieno
[3,2-e] benzo[b]furans and thieno[2,3-e]benzo[b]thiophenes and their acyclic
precursors.
2.2.1. Interactions of cyano (2a–b, 3a–c, 10a, 14) and amidino
(5a–b) acyclic derivatives and their cyclic analogues (6a–b
12a, 16) with double stranded ct-DNA
The UV–vis titrations of buffered aqueous solutions of com-
pounds 6b, 12a and 16 (c = 10–5 mol dm–3) with ct-DNA re-
sulted by significant batochromic (Δλmax = 1–9 nm) and hypo-
chromic effects (30–50%) in the range of λ > 300 nm at which
ct-DNA does not absorb light. The appearance of isosbestic
points in all UV–vis titrations at λ > 300 nm strongly supported
the presence of only two spectroscopically active species,
namely the free compounds (6b, 12a, 16) and only one type
of the complex with ct-DNA (Fig. 4).
Scheme 3. Synthesis of cyano- and amidino-substituted derivatives of thieno
[2,3-e] benzo[b]furans and their acyclic precursors.
cyano-substituted acyclic compounds (3b, 3c, 10a, 10b and
14) in the toluene solution and in the yields about 40–70%.
Finally, isopropyl- and morpholino-substituted amidino-thie-
no-benzofurans (12a and 12b) and benzo-dithiophene deriva-
tive 12c were prepared from 11a and 11b in the Pinner reac-
tion. Compound 16 was prepared in the same reaction from 15.
It is very important to mention that only amidino-com-
pounds 5a and 5b from the furan series bearing the amidino-
substituent on the position 5 of the furan ring are successfully
photochemically cyclized into the corresponding cyclic ami-
dines 6a and 6b. The reaction was introduced in ethanol, as
well as, in water, with or without the bubbling of air through
the solution. The time of the cyclization (about one hour) and
the yields in both cases were the same. All other furan acyclic
compounds with the amidino-substituent on the benzene nuclei
couldn’t be photochemically cyclized at any conditions, while
all amidino-substituted acyclic thiophene compounds decom-
posed in attempt of photochemical reaction. Acyclic neutral
cyano-compounds were photochemically cyclized successfully
into the cyclic compounds in toluene solution.
Binding constants (Ks) and ratios n calculated according to
the Scatchard equation [21] from titrations of 6b, 12a and 16
with ct-DNA (logKs = 4.6–5.3; n ≈ 0.2) were found to be in
good agreement with values previously reported for their close
analogue 6a [4] and also are in accordance with values ob-
tained for a number of heteroaromatic molecules, which bind
to ds-DNA by intercalation [2].
Thermal denaturation experiments have shown that addition
of cyclic amidino derivatives 6b, 12a and 16 weakly stabilized
the double helix of ct-DNA at all examined ratios
r[compound]/[ct-DNA] = 0.1–0.3 at highest ΔTm(12a) = 4.0 °C, all
values comparable with previously obtained for 6a [4]. Ther-
mal denaturation transitions were monophasic, pointing toward
only one dominant binding mode.
Titrations of buffered aqueous solutions of acyclic deriva-
tives 2a–b, 3a–c, 10a and 14 with ct-DNA didn’t yield any
measurable changes in the UV–vis spectra of studied com-
pounds. In line with that, addition of same series of compounds
didn’t result in measurable stabilization of ct-DNA double he-
lix in the thermal denaturation experiments even at the ratio

K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
929
Fig. 4. UV–vis spectral changes 12a (c = 8. 7 × 10–5 mol dm–3); upon the addition of ct- DNA at pH = 7, (buffer sodium cacodylate, I = 0.05 mol dm–3).
r[compound]/[ct-DNA] = 0.3 (compounds in excess over the interca-
lation binding sites). All aforementioned suggested that studied
acyclic derivatives do not bind to the ct-DNA, what is in ac-
cordance with previously presented results for analogous 5a
[4].
All tested compounds showed a certain antiproliferative ef-
fect (Tables 1a and 1b). The compounds could be divided, for
comparison reasons, into two major subclasses: positively
charged molecules (compounds 4, 5, 6, 8, 12 and 16) and neu-
tral molecules (1, 2, 3, 7, 10, 11, 14 and 15). The IC50 values
showing the cell growth inhibiting activity of positively
charged compounds (Table 1a) reveal that among these mole-
cules the “acyclic” ones in general demonstrate significantly
lower effect (Fig. 5a) than their cyclic analogues (Fig. 5b).
Within the cyclic analogues the activity of mono-substituted
isopropylamidino derivatives (6a, 16) show more pronounced
inhibitory effect (within a micromolar range) than the morpho-
linoamindino-substituted one (6b), most likely due to the steric
hindrances of positive charge by more bulky substituent of the
latter. This difference is not observed for their bis-substituted
analogues (8, 12a, b).
The above presented results support main idea presented for
5a–6a system, former (acyclic) compound showing no interac-
tions with ds-DNA, while latter (cyclic) compound forming
stable, intercalative complex with ds-polynucleotides [4].
2.3. Biological results
Compounds 1c–16 were tested for their potential antiproli-
ferative effect on a panel of 6 human cell lines, five of which
derived from five cancer types: HeLa (cervical carcinoma),
MCF-7 (breast carcinoma), SW 620 (colon carcinoma), MiaPa-
Ca-2 (pancreatic carcinoma), Hep-2 (laryngeal carcinoma) and
WI 38 (diploid fibroblasts).
On the other hand, regarding the series of neutral molecules
direct comparison of cyclic and acyclic analogues is not suita-
ble because of rather low water solubility of the former mole-

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K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
Table 1a
In vitro growth inhibition of tumor cells and normal human fibroblasts (WI 38) by positively charged molecules and reference compounds cisplatin, doxorubicin and
etoposide
a
IC50 (μM)
Compound
Cell lines
SW 620
Hep-2
≥ 100
HeLa
≥ 100
MiaPaCa-2
> 100
MCF-7
69 22
78 29
73 35
43 10
75 26
≥ 100
WI 38
4a
> 100
83 46
53 27
37 35
4b
4c
5a
5b
5c
71 37
43 26
≥ 100
89 0.5
> 100
52 10
78 21
85 18
> 100
70
8
76 26
60 27
> 100
43
61 32
53
3
53 0.9
≥ 100
79 26
50
7
> 100
56 39
6
> 100
6a
6b
8
12a
12b
16
4.6 0.7
39 15
54 11
5
0.008
6
41
20
0.3
5
7
5
0.7
4
0.2
7
2
9
2
31 14
33
3
16 22
36 0.7
4
6
22 16
4
44 17
31
7
19 17
39 10
12
41
38
5
20 14
40
19 20
0.1 0.01
N.T.
9
1.5
3
9
1
6
7
3
1
4.3 0.3
2
3
3
8
11
29
5
3
1
2
19 11
0.3
11 0.1
0.6
0.04 0.01
1
Cisb
Doxb
Etob
a
2
3
2
6
0.04 0.01
N.T.c
0.02 0.01
15.4 14
0.02 0.02
20 3.4
0.04 0.01
50 30
3
1
IC50: the concentration that causes a 50% reduction of the cell growth.
Cis: cisplatin; Dox: doxorubicin; Eto: etoposide.
N.T.: not tested.
b
c
Table 1b
In vitro growth inhibition of tumor cells and normal human fibroblasts (WI 38) by neutral molecules
a
IC50 (μM)
Compound
Cell lines
SW 620
Hep-2
HeLa
MiaPaCa-2
35
≥ 100
MCF-7
WI 38
1c
2a
2b
3a
3b
3c
62 16
96 60
91 21
64 30
71 19
78 21
44
4
7
7
2
33
31 44
67
≥ 100
5
43
50 20
46
2
73 36
68 12
>100
33 47
68
8
7
6
60
5
62
79
4
8
66 12
>100
48 14
≥ 100
>100
87 10
62 18
0.6 0.1
83
2
57 25
≥ 100
>100
32 16
46 12
18 12
7ab
10a
10b
11ab
11bb
14
2
4
1
3
20
6
6
1
6
2
6
2
23 5
86 12
≥ 100
43 23
≥ 100
≥ 100
74
8
54 24
> 100
17 12
74 10
> 100
13 10
78 23
> 100
≥ 100
97 19
4
0.5
> 100
6
4
81
8
1
1
39
6
1
0.3
43
57
6
5
52 14
71 17
86 17
≥ 100
> 100
92 17
15b
a
IC50: the concentration that causes a 50% reduction of the cell growth.
Because of low water solubility, the suspensions were used.
b
cules, which is possibly the reason of a relatively low biologi-
cal activity (7a–b, 11a–b and 15). Nevertheless, rather pro-
nounced and selective activity of cyano-substituted 7a and 15
was observed, especially on HeLa and Hep-2 cell lines
(Fig. 5c, d), regardless of low water solubility, which actually
point to even lower inhibitory concentrations sufficient for the
growth inhibition of these two cell lines and their extreme cell-
type selectivity. Moreover, similar selective effect towards
HeLa and Hep-2 cells was reported by Jarak et al. [22] using
carboxanilides bearing cyano substituent either on anilide or
benzothiophene part of the molecule, as well as “acyclic” cya-
no derivatives of thiophene-2-carboxamides [23]. Unfortu-
nately, the reason for this interesting selectivity on HeLa and
Hep-2 cell lines are not completely clear and further studies
aimed to provide the indication of what genetic and/or structur-
al background is responsible for this motivating correlation are
underway. What is known so far is that these two cell lines
have similar genetic backgrounds, both having human papillo-
ma virus (HPV) type 18 DNA integrated into their genomes.
Since it is known that the high-risk HPV E6 and E7 genes are
major viral oncogenes, responsible for the initiation of tumor-
igenesis (e.g. cervical carcinoma) [24], it could be possible that
these compounds specifically inhibit the E6/E7 expression.
Namely, repression of E6 and E7 expression results in acquisi-
tion of the senescent phenotype and in the rescue of functional
p53 and p105Rb pathways; therefore, therapies directed against
either viral protein may be beneficial [25].
Interestingly, the acyclic cyano-substituted neutral mole-
cules (10a, b) (Fig. 5e, f), show pronounced and selective ac-
tivity; 10a being an order of magnitude more active than 10b,
most probably due to two cyano-substituents. However, both
molecules do not interact with DNA (Section 2.2.1.), and the
target(s) of cyano-substituted heteroaromatic systems are not
apparent, neither is the mechanism of antiproliferative action.

K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
931
Fig. 5. Dose–response profiles for the representative acyclic (5b, A; 10a, E; 10b, F) and cyclic compounds (6a, B; 7a, C; 15, D) tested on various human cell lines in
vitro. The cells were treated with the compounds at different concentrations, and percentage of growth (PG) was calculated. Each point represents a mean value of
four parallel samples in three individual experiments.
Nevertheless, comparable interesting in vitro activity of cyano-
substituted heteroaromatic system was reported by Bénéteau et
al. [26], indicating together with our observations that cyano-
substituted heteroaromatic systems and their correlation with
the potential biomolecular targets are of highest interest for
more detailed systematic investigations.
phenyl moiety inhibited aforementioned photochemical reac-
tion. Although the study of furo-thiophene series was initiated
with the same idea, the preparation of amidino-substituted
acyclic-precursors was possible, but the pure product could
not be obtained, thus not allowing the study of the photoin-
duced dehydrocyclization in water.
In conclusion, here presented synthetic part of the research
clearly leads to the amidine substituted furyl-phenyl acrylate
system as only efficient in photoinduced dehydrocyclization
in water. Studies of new systems capable of undergoing photo-
induced dehydrocyclizations in water and by visible light irra-
diation, for in vivo application are necessary.
3. Conclusions
The main aim of the presented study was to convert acyclic
derivatives into their cyclic analogues by photodehydrocycliza-
tion in aqueous, biologically relevant conditions, which could
serve as DNA intercalators and efficient possible antitumor
substances. Among all studied compounds only amidino-fur-
yl-substituted phenyl acrylates were efficiently converted to
the corresponding naphthofuranes. The replacement of furan
by thiophene moiety did not enable the photochemical reac-
tion, pointing to the high sensitivity of photodehydrocycliza-
tion on the electronic properties of starting material. Also,
changing the position of the amidino substituent from furyl to
Apparently the significantly stronger antiproliferative activ-
ity of cyclic, positively charged analogues (IC50 concentrations
are within a micromolar range) is to be correlated to the prop-
erty of these molecules to intercalate into DNA. Although the
cyclic neutral molecules could not be adequately tested on
DNA binding due to their low solubility in water, cyano-sub-
stituted 7a and 15 compounds show intriguing strong and se-
lective antiproliferative activity on tumor cell lines. On the

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K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
other hand, the acyclic, either positively charged or neutral mo-
lecules, except 10a, did not show any interaction with DNA,
correlating with the inferior biological activity.
4.1.3. Ethyl-E-3-(5-formyl-2-furyl)-2-(4-cyano)-phenylacrylate
(1c)
Compound 1c was prepared using the method described for
preparation of compound 1a, from phosphorus oxychloride
(1.38 ml, 8 mmol), DMF (1.2 ml, 6 mmol) and methyl-E-3-
(2-furyl)-2-(4-cyano)-phenylacrylate (3.45 mmol) in DMF
(10 ml). Orange crystals 0.168 g (53%) are obtained, m.p.
111–113 °C; IR (KBr) (υmax/cm–1): 2230, 1716, 1687, 1606;
4. Experimental protocols
4.1. Chemistry
1H NMR (500 MHz, DMSO) δ: 9.46 (s, 1H) 7.9 (d, J3",2"
5",6" = 7.5 Hz, 2H), 7.70 (s, H3, 1H), 7.50 (d, J2",3 = J6",5"
=
J
Melting points were determined on a Kofler hot stage mi-
croscope and are uncorrected. IR spectra were recorded on a
= 7.5 Hz, 2H), 7.42 (d, J4’,3’ = 3.7 Hz, 1H), 6.46 (d, J3’,4’
= 3.7 Hz, 1H) 4.21 (q, J = 7.1 Hz, COOCH2CH3, 2H), 1.22
(t, J = 7.1 Hz, COOCH2CH3, 3H); 13C NMR (125 MHz;
DMSO) δ:179.3, 166.0, 154.4, 153.3, 140.8, 133.2, 133.2
(2C), 131.1 (2C), 127.3, 124.1, 119.6, 118.8, 62.2, 14.8; ele-
mental analysis calcd (%) for C16H11O4N: C 69.15; H 4.41; N
4.75; found C 68.9; H; 4.52; N 4.91.
Nicolet Magna 760 spectrophotometer in KBr discs. 1H and 13
C
NMR spectra were recorded on either a Varian Gemini 300 or
a Bruker Avance DPX 300 i 500 spectrometer using TMS as
an internal standard in DMSO-d6. Elemental analysis for car-
bon, hydrogen and nitrogen were performed on a Perkin-Elmer
2400 elemental analyzer. 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 fitted
with a 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.
4.1.4. Ethyl-E-3-(2-furyl)-2-(4-cyano)-phenylacrylate (2a)
Ethyl ester of p-cyano phenylacetic acid (5.00 g, 26 mmol)
and furfural (2.998 g, 31.2 mmol) in a mixture of triethylamine
(4.2 ml) and acetic acid anhydride (4.2 ml) were refluxed for
3 hours. After the reaction was completed, the mixture was
cooled, acidified with hydrochloric acid and extracted with
ether. The organic layer was washed with water and dried over
anhydrous MgSO4 and evaporated in vacuum. The dark resi-
due was recrystallized from petrol ether and gave yellow crys-
tals (3.170 g, 45.6%), m.p. 125–127 °C. IR (KBr) (υmax/cm–1):
4.1.1. Methyl-E-3-(5-formyl-2-furyl)-2-phenylacrylate (1a)
Corresponding methyl-E-3-(2-furyl)-2-phenylacrylate was
formylated by Vilsmeier formylation. Phosphorus oxychloride
(22.8 ml, 250 mmol) was added drop-wise with cooling to
DMF (20 ml, 250 mmol). The reaction mixture was stirred
for 0.5 h by cooling on ice, the apparatus being protected with
a calcium chloride tube. A solution of methyl-E-3-(2-furyl)-2-
phenylacrylate (13g, 57 mmol) in DMF (10 ml) was added
drop-wise to the mixture. After the addition was completed,
the mixture was stirred for 1 h at room temperature, then
heated at 90 °C for 3 h, cooled and poured onto crushed ice
and made weakly alkaline with sodium carbonate solution, and
left over-night on ice. The solid was filtered off, washed with
water and recrystallized from methanol. Orange crystals 12.2 g
(83.5%) are obtained, m.p. 102–104 °C (Ref. [18a], m.p.
103–105 °C). IR (KBr) (υmax/cm–1):1710, 1665, 1630; 1H
NMR (300 MHz, DMSO) δ: 9.57 (s, 1H, CHO), 7.65 (s, 1H,
H3), 7.50–7.17 (m, 5H), 7.00 (d, J4′,3′ = 3.5 Hz, 1H), 5.76 (d,
J3′,4′ = 3.5 Hz, 1H), 3.78 (s, OCH3, 3H).
1
2229, 1693, 1621, 1604; H NMR (300 MHz DMSO) δ: 7.88
(dd, J3",2" = J5",6" = 8.1 Hz, J3",5" = 1.5 Hz, 2H), 7.68 (d, J5’,4’
= 1.8 Hz, 1H), 7.66 (s, 1H3), 7.46 (dd, J2",3" = J6",5" = 8.1 Hz,
J
2",6" = 1.5 Hz, 2H), 6.52 (dd, J4′,3′ = 3.3 Hz; J4′,5′ = 1.8 Hz,
1H), 6.44 (d, J3′,4′ = 3.3 Hz, 1H), 4.17 (q, J = 7.1 Hz, COO
CH2CH3, 2H), 1.24 (t, J = 7.1 Hz, COOCH2CH3, 3H); 13C
NMR (300 MHz DMSO) δ: 165.77, 149.54, 146.26, 141.01,
132.01, 130.72, 127.30, 126.63, 118.82, 117.367, 112.63,
110.55, 60.95, 14.05; elemental analysis calcd (%) for
C16H13NO3: C 71.91; H 4.87; N 4.71; found C 71.99; H
4.90; N 5.13.
4.1.5. Ethyl-E-3-(2-thienyl)-2-(4-cyano)-phenylacrylate (2b)
The compound 2b was prepared in a manner similar to the
preparation of 2a, from ethyl ester of p-cyano phenylacetic
acid (5.00 g, 26 mmol) and 2-thiophene aldehyde (2.998 g,
31.2 mmol) in a mixture of triethylamine (4.2 ml) and acetic
acid anhydride (4.2 ml). Yellow crystals (3.170 g, 15%) were
obtained; m.p. 134–135 °C; IR (KBr) (υmax/cm–1): 2223, 1703,
1621, 1602; 1H NMR (300 MHz DMSO) δ: 8.07 (s, 1H3), 7.93
(dd, J3",2" = J5",6" = 8.5 Hz, J3",5" = 1.4 Hz, 2H), 7.59 (d, J3’,4’
= 5.1 Hz, 1H), 7.50–7.46 (m, 2HPh, 1Hthioph.), 7.06 (dd, J4’,3’
= 3.6 Hz, J4’,5’ = 5.1 Hz, 1H), 4.18 (q, J = 7.1 Hz, COO
CH2CH3, 2H) 1.20 (t, J1 = 7.1 Hz, COOCH2CH3, 3H); 13C
NMR (300 MHz DMSO) δ: 165.7, 140.5, 137.02, 135.4,
133.9, 132.8 (2C), 132.3, 131.2 (2C), 127.3, 127.1, 118.7,
111.2, 60.8, 14.0; elemental analysis calcd (%) for
C16H13O2SN: C 67.84; H 4.59; N 4.95; found C 67.80; 4.61;
N 5.02.
4.1.2. Methyl-E-3-(5-formyl-2-thienyl)-2-phenylacrylate (1b)
Compound 1b was prepared using the method described for
preparation of compound 1a, from phosphorus oxychloride
(36.8 ml, 0.21 mol), DMF (42 ml, 0.21 mol) and methyl-E-3-
(2-thienyl)-2-phenylacrylate (21 g, 86 mmol) in DMF (10 ml).
Orange crystals 20.3 g (87%) are obtained, mp 121–122 °C
(Ref. [18b], m.p. 120–121 °C). IR (KBr) (υmax/cm–1): 1700,
1
1660, 1610; H NMR (300 MHz, DMSO) δ: 9.79 (s, CHO,
1H), 8.09 (s, H3, 1H), 7.88 (d, J4′,3′ = 4.1 Hz, 1H), 7.64–7.47
(m, 3H), 7.49 (d, J3′,4′ = 4.1 Hz, 1H), 7.29–7.19 (m, 2H) 3.75
(s, OCH3, 3H).

K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
933
4.1.6. Methyl-E-3-(5-cyano-2-furyl)-2-phenylacrylate (3a)
Compound 1a (2.00 g, 8.33 mmol) and hydroxyl ammo-
nium hydrochloride (0.995g, 14.3 mmol) were heated in pyri-
dine (6 ml) at 60 °C for 30 min, then acetic anhydride (4 ml)
was successively added at a temperature not exceeding 95 °C,
which was being kept for additional 2 h. The mixture was
cooled to 20 °C, poured into water (37.5 ml) and the separated
nitrile was stirred for 1 h, filtered, washed with water, dried,
and crystallized from petrol ether gave 3a (1.745 g, 82.8%
yield); m.p. 85–86 °C; IR (KBr) (υmax/cm–1): 3120, 3100,
2200, 1700, 1610, 1475, 1425, 1336, 1238, 1185, 1160,
1060, 1015; UV–vis (EtOH): λmax (lgε) = 307 (32179); 1H
NMR (300 MHz DMSO) δ: 7.61 (s, 1H3), 7.49 (d, J4’,3’ = 3.9
Hz, 1H), 7.46–7.45 (m, 3H), 7.25 (dd, J2",3" = J6",5" = 8.7 Hz,
2200 cm–1). The reaction mixture was purged with N2 gas
and diluted with ether (50 ml). The crude imidate was filtered
off and was immediately suspended in anhydrous ethanol
(5 ml). The isopropylamine (0.8 ml, 9.85 mmol) was added
and the mixture was stirred for one day at room temperature.
The reaction mixture was diluted with ether (50 ml) to give a
precipitate. The precipitate was collected and recrystallized
from ethanol ether to give pale yellow powder 0.299 g
(42.8%). m.p. 225.8–226.5 °C; IR (KBr) (υmax/cm–1): 3201,
1
3042, 2980, 1703, 1676, 1620; H NMR (300 MHz DMSO)
δ : 9.42 (bs, 3H), 7.78 (d, J3",2" = J5",6" = 8.3 Hz, 2H), 7.69 (s,
2H), 7.48 (d, J2"3" = J6",5" = 8.3 Hz, 2H) 6.53 (dd, J4’,3’ = 3.4
Hz, J4’,5’ = 1.6 Hz, 1H.), 6.31 (d, J3’,4’ = 3.4 Hz, 1H), 4.19 (q,
J = 7.1 Hz, COOCH2CH3, 2H), 4.08–4.03 (m, 1H, CH(CH3)2,
1H) 1.29 (d, J = 6.3 Hz, –CH(CH3)2, 6H), 1.20 (t, J = 7.1 Hz,
COOCH2CH3, 3H); 13C NMR (300 MHz DMSO) δ: 165.26,
153.99, 140.04, 132.22, 132.12, 130.50, 125.83, 125.75,
125.02, 118.79, 117.24, 111.31, 111.15, 61.56, 14.08; elemen-
tal analysis calcd (%) for C19H23O3N2Cl: C 62.83; H 6.33; N
7.71; found C 62.61; H; 6.58; N 7.96.
J
2",6" = 1.3 Hz, 2H), 6.16 (d, J3′,4′ = 3.9 Hz, 1H.), 3.73 (s, COO
CH3, 3H); 13C NMR (300 MHz DMSO) δ: 52.5, 111.3, 115.5,
124.8, 125.1, 125.2, 128.3, 128.4, 128.8, 134.4, 134.6, 154.5,
166.0; elemental analysis calcd (%) for C15H11O3N: C 71.14;
H 4.34; N 5.53; found C 71.45; H 4.20; N 5.23.
4.1.7. Methyl-E-3-(5-cyano-2-thienyl)-2-phenylacrylate (3b)
Compound 3b was prepared using the method described for
preparation of compound 3a, from 1b (2.00 g, 7.33 mmol) and
hydroxylammonium hydrochloride (0.0.878g, 14.3 mmol).
Yellow-orange crystals of 3b 0.998 g (50.6%) were obtained.
m.p. 120–121 °C; IR (KBr) (υmax/cm–1): 2217, 1712, 1616,
4.1.10. Ethyl-E-3-(2-thienyl)-2-(4-morpholinamidino)-
phenylacrylate hydrochloride (4b)
Compound 4b was prepared using the general method de-
scribed for the preparation of 4a, from compound 2a (0.49 g,
1.84 mmol) in anhydrous EtOH (4 ml) and aminomorpholine
(0.92 ml, 9.12 mmol). Precipitate was collected and recrystal-
lized from ethanol-ether to give yellow powder (0.290 g,
38.8%). m.p. 129–130 °C; IR (KBr) (υmax/cm–1): 3378, 3092,
2979, 2927, 2860, 1703, 1654, 1603; 1H NMR (300 MHz
DMSO) δ: 11.32 (s, 1H), 9.89 (s, 1H), 9.23 (s, 1H), 8.12 (s,
1H3), 7.93 (d, J3",2" = J5",6" = 8.16 Hz, 2H), 7.63 (d, J5’,4’ = 5.0
Hz, 1H.), 7.52–7.50 (m, 3H), 7.07 (d, J4’,5’ = 5.0 Hz, 1H), 4.17
(q, J = 7.1 Hz, COOCH2CH3, 2H), 3.79 (bs, 4H), 2.97 (bs,
4H), 1.19 (t, J = 7.1 Hz, COOCH2CH3, 3H); 13C NMR
(300 MHz DMSO) δ: 166.38, 162.08, 141.45, 137.69,
135.82, 134.33, 132.56, 131.14, 129.59, 128.0, 127.79,
126.62, 66.01, 61.32, 54.33, 14.67; elemental analysis calcd
(%) for C20H24O3N3SCl: C 56.94; H 5.69; N 9.96; found C
56.31; H 5.45; N 10.05.
1
1599; H NMR (300 MHz DMSO) δ: 8.12 (s, 1H), 7.89 (d,
J4′,3′ = 3.9, 1H), 7.64 (d, J3′,4′ = 3.9, 1H), 7.59 (m, 3H), 7.26
(m, 2H) 3.72 (s, COOCH3, 3H); 13C NMR (300 MHz DMSO)
δ: 166.8, 144.9, 138.4, 134.9, 134.3, 133.7, 132.3, 130.2 (2C),
129.9, 129.7 (2C), 114.4, 111.7, 52.9; elemental analysis calcd
(%) for C15H11O2SN: C 66.91; H 4.09; N 5.20; found C 66.84;
H 4.21; N 5.02.
4.1.8. Ethyl-E-3-(5-cyano-2-furyl)-2-(4-cyano)-phenylacrylate
(3c)
Compound 3c was prepared using the method described for
preparation of compound 3a, from 1c (0.317 g, 1.07 mmol)
and hydroxylammonium hydrochloride (0.09 g, 1.3 mmol).
Yellow crystals of 3c 0.225 g (72%) were obtained; mp
1
128–129 °C; H NMR (300 MHz DMSO) δ: 7.93 (d, J3",2"
=
J
5",6" = 8.4 Hz, 2H), 7.69 (s, H3 1H), 7.55 (d, J4’,3’ = 3.8 Hz,
4.1.11. Ethyl-E-3-(2-thienyl)-2-(4-(N-isopropyl)amidino)-
phenylacrylate hydrochloride (4c)
1H), 7.53 (d, J2",3" = J6",5" = 8.4 Hz, 2H) 6.59 (d, J3’,4’ = 3.8
Hz, 1H), 4.21 (q, J = 7.1 Hz, COOCH2CH3, 2H), 1.21 (t,
J = 7.1 Hz, COOCH2CH3, 3H); 13C NMR (300 MHz DMSO)
δ: 165.26, 153.99, 140.04, 132.22, 132.12, 130.50, 125.83,
125.75, 125.02, 118.79, 117.24, 111.31, 111.15, 61.56,
14.08; elemental analysis calcd (%) for C17H12O3N2: C
69.86; H 4.11; N 9.59; found C 69.90; H; 4.15; N 9.56.
Compound 4c was prepared using the general method de-
scribed for the preparation of 4a, from 2b (1.0 g, 3.53 mmol)
in anhydrous EtOH (10 ml) and isopropylamine (50 ml). The
precipitate was collected and recrystallized from ethanol ether
to give white powder (0.250 g 18.6%); IR (KBr) (υmax/cm–1):
3203, 3031, 2973, 1698, 1679, 1614; 1H NMR (300 MHz
DMSO) δ: 9.33 (bs, 3H), 8.11 (s, 1H3), 7.85 (d, J3",2′" = J5",6"
= 8.0 Hz, 2H), 7.63 (d, J5’,4’ = 4.9 Hz, 1H), 7.51–7.48 (m, 3H)
7.07 (dd, J4,,5, = 4.9 Hz, J4,,3’ = 3.9 Hz, 1H), 4.21–4.10 (m,
COOCH2CH3, CH(CH)3, 3H), 1.30 (d, J = 6.3 Hz,
–CH(CH3)2, 6H), 1.20 (t, 3H, J1 = 7.0 Hz, COOCH2CH3, 3H);
13C NMR (300 MHz DMSO) δ: 166.4, 161.9, 154.42, 140.8,
137.7, 135.8, 134.3, 132.6, 130.9 (2C), 129.5 (2C), 128.0,
127.8, 61.3, 45.6, 45.6, 21.7, 14.7; elemental analysis calcd
4.1.9. Ethyl-E-3-(2-furyl)-2-(4-(N-isopropylamidino)-
phenylacrylate hydrochloride (4a)
A stirred suspension of 2a (0.578 g, 2.16 mmol) in anhy-
drous EtOH (5 ml) was cooled in an ice-salt bath and was
saturated with HCl gas. The flask was then tightly stoppered
and the mixture was maintained at room temperature for 2 days,
until nitrile band disappeared (monitored by IR analysis at

934
K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
(%) for C18H23O2N2SClx1/2H2O: C 58.85; H 6.19; N 7.20;
found C 58.55; H 6.33; N 7.44.
through. Prepared solution was then irradiated with high pres-
sure mercury arc lamp during 2 h. The solvent was evaporated
and recrystallization from ethanol-ether gave 6a (0.024 g,
24.1%).
4.1.12. Methyl-E-3-(5-(N-isopropylamidino)-2-furyl)-2-
phenylacrylate hydrochloride (5a)
Compound 5a was prepared using the general method de-
scribed for the preparation of 4a, from 3a (0.499 g, 1.97 mmol)
and the isopropylamine (1 ml, 9.85mmol). The precipitate was
collected and recrystallized from ethanol ether to give white
powder 0.294 g (42.8%). m.p. 189–191 °C; IR (KBr)
(υmax/cm–1): 3150, 3000, 1700, 1650, 1600, 1420, 1330,
4.1.15.2. Method B. To aqueous solution of 5a (0.134 g, 0.384
mmol in 250 ml) I2 was added and the air was bubbled
through. Prepared solution was then irradiated with high pres-
sure mercury arc lamp during 2 h. The water was evaporated
and recrystallization from ethanol-ether gave 6a (0.040 g,
30.05%). m.p. 221–223 °C; IR (KBr) (υmax/cm–1) 3010,
2930, 1705, 1650, 1610, 1435, 1420, 1325, 1260, 1190,
1
1245, 1155, 1115; H NMR (300 MHz, DMSO) δ: 9.46 (brs,
1
2H), 9.29 (brs, 1H), 7.68 (s, 1H3), 7.67 (d, J4,3’ = 3.9 Hz, 1H),
7.50–7.48 (m, 3H), 7.28 (dd, J2",3" = J6",5" = 7.5; J2",6" = 2.1
Hz, 2H), 5.77 (d, J3’,4’ = 3.3 Hz, 1H), 4.20 (m, CH(CH3)2,
1H), 3.73 (s, COOCH3, 3H) 1.23 (d, J = 6.3 Hz, CH(CH3)2,
6H); elemental analysis calcd (%) for C18H21O3N2Clx7H2O:
C, 45.52; H, 7.37; N, 5.90 found C, 45.49; H 7.63; N 5.51.
1115, 1080, 1010; HNMR (300 MHz DMSO) δ: 10.1 (bs,
1H), 9.90 (bs, 1H), 9.41 (bs, 1H), 8.80 (d, J6,7 = 8.6 Hz, 1H),
8.79 (s, H-4, 1H), 8.41–8.39 (m, H-9, H-1, 2H), 7.88 (dd, J7,8
= 8.4 Hz, J7,9 = 1.3 Hz, 1H), 7.79 (dd, J8,7 = 8.4 Hz, J8,6 = 1.3
Hz, 1H), 4.15 (m, CH(CH3)2, 1H), 4.0 (s, COOCH3, 3H), 1.34
(d, J = 6.4 Hz, CH(CH3)2, 6H); 13C (300 MHz DMSO) δ: 21.1
(2C), 45.1, 52.8, 112.5, 115.3 , 123.9, 125.9, 126.6, 127.3,
127.4, 127.5, 128.3, 128.4, 144.6, 150.7, 151.1, 166.7; elemen-
tal analysis calcd (%) for (C18H19O3N2Ì5H2O): C, 49.60; H,
6.66; N, 6.43 found C, 49.22; H 7.60; N 6.32.
4.1.13. Methyl-E-3-(5-morpholinamidino-2-furyl)-2-
phenylacrylate hydrochloride (5b)
Compound 5b was prepared using the general method de-
scribed for the preparation of 4a, from 3a (0.467 g, 1.8 mmol)
and the 4-aminomorpholin (0.9 ml, 9.2 mmol). The precipitate
was collected and recrystallized from ethanol-ether to give
light yellow powder (0.38 g, 63%); m.p. 194–195 °C; IR
4.1.16. Methyl-(2-morpholinamidino)-naphtho[2,1-b]furan-5-
carboxylate hydrochloride (6b)
Compound 6b was prepared from 5b (0.114 g, 0.35 mmol)
which was dissolved in ethanol (250 ml) and irradiated with
high pressure mercury arc 400 W lamp during 2 h. I2 was
added into the solution and the air was bubbled through. The
solvent was evaporated recrystallization from ethanol-ether
gave 6b 0.05 g (44%); m.p.165–166 °C; IR (KBr) (
1
(KBr) (υmax/cm–1): 3120, 2990, 1710, 1680, 1600; H NMR
(300 MHz DMSO) δ: 11.23 (s, 1H), 9.70 (s, 1H), 9.14 (s,
1H), 7.68 (s, 1H3), 7.61 (d, J4′,3′ = 3.9 Hz, 1H), 7.52–7.49
(m, 3H), 7.30–7.27 (m, 2H), 5.73 (d, J3′,4′ = 3.9 Hz, 1H),
3.74 (s, 3H, COOCH3), 3.62 (s, 4H), 2.88 (s, 4H); 13C NMR
(300 MHz DMSO) δ: 166.08, 153.93, 150.29, 139.81, 134.55,
129.01, 128.75, 125.96, 120.40, 114.90, 65.50, 53.52, 52.64;
elemental analysis calcd (%) for C19H22O4N4Cl: C 69.63, H
6.72, N 12.83 found C 69.45; H 6.89; N 13.01.
υ
max/cm–1) 1H NMR (300 MHz DMSO) δ: 11.5 (bs, 1H),
10.25 (bs, 1H), 9.43 (bs, 1H), 8.89 (s, H-4, 1H), 8.79 (d, J6,7
= 8.4 Hz, 1H), 8.39–8.37 (m, H-9, H-1, 2H), 7.87 (dd, J7,8
= 8.3 Hz, J7,9 = 1.3 Hz, 1H), 7.78 (dd, J8,7 = 8.3 Hz, J8,6
= 1.3 Hz, 1H), 4.01 (s, COOCH3 ,3H), 4.01 (bs, 4H), 2.99
(bs, 4H); 13C (300 MHz DMSO) δ: 168.07, 152.67, 144.23,
130.03, 129.71, 128.79, 128.68, 127.93, 127.09, 125.32,
116.69, 114.59, 66.81, 55.16, 54.12; elemental analysis calcd
(%) for (C19H20O4N3Cl): C 70.05; H 6.15; N 12.90 found C,
70.31; H 6.25; N 13.1.
4.1.14. Methyl-E-3-(5-(N-isopropylamidino)-2-thienyl)-2-
phenylacrylate hydrochloride (5c)
Compound 5c was prepared using the general method de-
scribed for the preparation of 4a, from 3b (0.968 g, 3.6 mmol)
and the isopropylamine (1.5 ml, 18 mmol) The precipitate was
collected and recrystallized from ethanol-ether to give white
4.1.17. Methyl-2-cyano-naphtho[2,1-b]thiophen-5-carboxylate
(7a)
powder (0.396 g, 30.2%); m.p. 179–180 °C; .IR (KBr) (
υ
1
max/cm–1): 3201, 3042, 2980, 1703, 1676, 1620; H NMR
(300 MHz DMSO) δ: 9.63 (bs, 1H), 9.61 (bs, 1H), 9.16 (bs,
1H), 8.07 (s, H3, 1H), 7.73 (d, J4′,3′ = 3.9 Hz, 1H), 7.55–7.54
(m, 3H), 7.27–7.25 (m, 3H), 4.02–4.0 (m, CH(CH3)2, 1H),
3.94 (s, COOCH3, 3H), 1.18 (d, J = 6.4 Hz, –CH(CH3)2, 6H);
13C NMR (300 MHz DMSO) δ: elemental analysis calcd (%)
for C18H21O2N2SCl: C 59.25, H 5.80, N 7.68; found C 59.01,
H 5.96, N 7.55.
Compound 7a was prepared from 3b (0.303 g, 1.3 mmol) in
250 ml toluene by irradiation for 3 hours. The air was bubbled
through the solution and I2 was added. After solvent was eva-
porated the crystals were recrystallized from petrol ether. Light
yellow crystals of 7a (0.046 g, 15.3%) were obtained, m.p. 179–
180 °C; IR (KBr) (υmax/cm–1) 2216, 1728, 1509; 1H NMR
(300 MHz DMSO) δ: 9.26 (s, 1H), 8.84 (s, 1H), 8.78 (d, J6,7
= 7.8 Hz, 1H), 8.67 (d, J9,8 = 7.8 Hz, 1H) 7.83–7.76 (m, H-7,
H-8, 2H), 3.99 (s, COOCH3, 3H); 13C NMR (300 MHz DMSO)
δ: 167.34, 138.86, 137.64, 135.26, 129.67, 128.51, 128.31,
128.22, 128.11, 126.69, 125.18, 125.07, 114.96, 111.97,
53.13; elemental analysis calcd (%) for (C15H9O2SN): C
67.42; H 3.37; N 5.24 found C 67.53; H; 3.41 N 5.40.
4.1.15. Methyl-2-(N-isopropylamidino)-naphtho[2,1-b]furan-
5-carboxylate hydrochloride (6a)
4.1.15.1. Method A. To ethanolic solution of 5a (0.10 g, 0.287
mmol in 250 ml) I2 was added and the air was bubbled

K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
935
4.1.18. Ethyl-2,4-dicyano-naphto[2,1-b]furan-5-carboxylate
(7b)
Compound 7b was prepared using the method described for
the preparation of 7a, from 7b (0.102 g, 0.35 mmol). White
crushed ice and made weakly alkaline with sodium carbonate
solution, and left over-night on ice. The solid was filtered off,
washed with water and recrystallized from methanol. Yellow
crystals 0.4125 g (37.3%) was obtained, m.p. 87–88 °C; IR
(KBr) (υmax/cm−1): 1705, 1668, 1605, 1508; 1H NMR
(300 MHz, DMSO) δ: 9.96 (s, CHO, 1H), 8.19 (s, 1H3), 7.80
(d, J4’,3’ = 3.9 Hz, 1H), 7.38 (d, J2",3" = 5.06 Hz, 1H), 7.27 (d,
J3’,4’ = 3.9 Hz, 1H), 7.12 (d, J4",3" = 3.4 Hz, 1H), 7.01 (dd,
crystals of 7a 0.029
g (28.6%) were obtained, m.p.
247–250 °C; IR (KBr)/cm–1 2220, 1710, 1650, 1606; 1H
NMR (300 MHz DMSO) δ: 9.16 (s, H-4, 1H), 8.95 (d, J6,7
= 8.91 Hz, 1H), 8.89 (s, H-1, 1H), 8.67 (s, H-9, 1H), 8.08 (d,
J7,6 = 8.9 Hz, 1H), 4.35 (m, COOCH2CH3, 2H), 1.50 (t,
J = 7.1 Hz, COOCH2CH3, 3H) elemental analysis calcd (%)
for C17H8O3N2: C 70.28; H 2.75; N 9.64 found C 70.51; H
3.01; N 9.6.
J
3",4" = 3.4 Hz J4’,5’ = 5.06 Hz, 1H), 4.30 (q, J = 6.9 Hz, COO
CH2CH3, 2H), 1.21 (t, J = 6.9 Hz, COOCH2CH3, 3H); elemen-
tal analysis calcd (%) for C 57.51; H 4.14, found C 57.45; H
4.21.
4.1.22. Ethyl-Z-3-(5-cyano-2-furyl)-2-(5-cyano-2-thienyl)
acrylate (10a)
4.1.19. Ethyl-2,4-(N-isopropylamidino)-naphtho[2,1-b]furan-
5-carboxylate dihydrochloride (8)
Compound 10a was prepared in similar manner as com-
pound 3a–c; compound 9a (1.5 g, 5 mmol) and hydroxylam-
monium hydrochloride (1.2 g, 17 mmol) were heated in pyri-
dine (4 ml) at 60°Cfor 30 min, then acetic anhydride (5.5 ml)
was successively added at a temperature not exceeding 95 °C,
which was being kept for additional 2 h. The mixture was
cooled to 20 °C, poured into water (25 ml) and the separated
nitrile was stirred for 1 h, filtered, washed with water, dried
and crystallized from petrol ether gave 10a 0.500 g (33%).
Compound 8 was prepared using the general method de-
scribed for the preparation of 4a, from 7b (0.100 g, 0.34 mmol)
and the isopropylamine (0.15 ml, 1.6 mmol) The precipitate
was collected and recrystallized from ethanol-ether to give
1
white powder (0.03 g, 20.7%); m.p. 190–191 °C; H NMR
(300 MHz; DMSO) δ: 9.6 (brs, 3H), 9.17 (s, H-4, 1H), 8.97 (d,
J6,7 = 8.8 Hz, 1H), 8.89 (s, H-1, 1H), 8.68 (s, H-9, 1H), 8.1 (d,
J7,6 = 8.9 Hz, 1H), 4.45 (m, COOCH2CH3, 2H), 4.13 (m,
CH(CH3)2, 2H), 1.46 (t, J = 7.1 Hz, COOCH2CH3, 3H), 1.35
(d, J = 7.1 Hz, CH(CH3)2, 12H); 13C NMR (125 MHz; DMSO)
δ: 165.6, 152.2, 130.2, 129.3, 128.6, 128.5, 128.1, 127.8,
126.7, 125.1, 118.9, 118.5, 118.4, 111.5, 110.3, 61.8, 45.1,
42.9, 21.1, 20.3, 13.9; elemental analysis calcd (%) for
C23H30O3N4Cl2: C 57.38; H 6.28; N 11.64; found C 57.62;
H 6.12; N 11.48.
1
m.p. 59–60 °C; IR (KBr) (υmax/cm–1) 2200, 1690, 1600; H
NMR (300 MHz, DMSO) δ: 8.0 (d, J4’,3’ = 3.9 Hz, 1H), 7.78
(s, H3 1H), 7.62 (d, J4",3" = 3.6 Hz, 1H), 7.32 (d, J3",4" = 3.6
Hz, 1H), 6.08 (d, J3’,4’ = 3.9 Hz, 1H), 4.21 (q, 2H, J = 7.1 Hz,
COOCH2CH3), 1.22 (t, 3H, J = 7.1 Hz, COOCH2CH3); 13C
NMR (300 MHz, DMSO) δ: 164.77, 153.40, 142.37, 138.62,
130.39, 128.27, 126.38, 125.19, 123.80, 118.82, 114.22,
111.39, 109.95, 62.05, 14.09; elemental analysis calcd (%) for
C15H10N2O3S: C 60.39; H, 3.38; N, 9.39 found C 60.40; H
3.35; N 9.59.
4.1.20. Ethyl-Z-3-(5-formyl-2-furyl)-2-(5-formyl-2-thienyl)
acrylate (9a)
Compound 9a was prepared using the method described for
preparation of compound 1a–c, Phosphorus oxychloride
(11.1 ml, 11 mmol) was added drop-wise with cooling to
DMF (10.6 ml, 12.6 mol) and the reaction mixture was stirred
for 0.5 h. A solution of Z-ethyl-3-(2-furyl)-2-(2-thienyl)acrylate
(5 g, 24 mmol) in DMF (5 ml) was added drop-wise to the
mixture and stirred for 1 h at room temperature, then heated at
90 °C for 45 min, cooled and poured onto crushed ice and
made weakly alkaline with sodium carbonate solution, and left
over-night on ice. The solid was filtered off, washed with water
and recrystallized from methanol. Orange crystals 2.76 g (38%)
are obtained, mp 120–123 °C (Ref. [16], m.p. 120–123 °C).
4.1.23. Ethyl-Z-2-(5-cyano-2-thienyl)-3-(2-thienyl)acrylate
(10b)
Compound 10b was prepared in similar manner as com-
pound 3a–c; from compound 9b (0.4 g, 1.4 mmol)) and hydro-
xylammonium hydrochloride (0.176 g, 2.7 mmol). Yellow
crystals 10b 0.339
g
(85.8%) were obtained, m.p.
135–136 °C; IR (KBr) (υmax/cm–1) 2200, 1690, 1600; 1H
NMR (300 MHz, DMSO) δ: 8.25 (s, H3, 1H), 8.05 (d, J4’,3’
= 3.8 Hz, 1H), 7.75 (d, J3",2" = 4.9 Hz, 1H), 7.65 (d, J4’,3’
= 3.8 Hz, 1H), 7.28 (d, J3,4 = 3.8 Hz, 1H), 7.13 (dd, J3",4"
= 3.8 Hz, J3,2’ = 4.9 Hz, 1H), 4.20 (q, 2H, J = 7.1 Hz, COO
CH2CH3), 1.22 (t, 3H, J = 7.1 Hz, COOCH2CH3); 13C NMR
(300 MHz, DMSO) δ: 165.58, 143.87, 140.02, 138.66, 137.49,
137.08, 134.36, 131.06, 127.91, 118.96, 114.50, 110.82,
61.69, 14.58; elemental analysis calcd (%) for C14H11NO2S2:
C 58.11; H, 3.83; N, 4.84 found C 58.45; H 4.01; N 4.69.
4.1.21. Ethyl-Z-2-(5-formyl-2-thienyl)-3-(2-thienyl)acrylate
(9b)
Compound 9b was prepared using the method described for
preparation of compound 1a–c, 9a. Phosphorus oxychloride
(1.8 ml, 12 mmol) was added drop-wise with cooling to
DMF (1.8 ml, 9 mmol) and the reaction mixture was stirred
for 0.5 h by. A solution of Z-ethyl-2-(2-thienyl)-3-(2-thienyl)
acrylate (1.0 g, 3.78 mmol) in DMF (1 ml) was added drop-
wise to the mixture and stirred for 1 h at room temperature,
then heated at 90 °C for 75 min, cooled and poured onto
4.1.24. 4-Ethyl-2,7-dicyano-thieno[3,2-e]benzo[b]furan (11)
Compound 11a was prepared from 10a (0.298 g, 1.0 mmol)
in 400 ml toluene by irradiation for 2.5 hours. The air was
bubbled through the solution and I2 was added. After solvent

936
K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
was evaporated the crystals were recrystallized from petrol
8.45 (s, H8, 1H), 4.53–4.48 (q, J = 7.2 Hz, COOCH2CH3, 2H),
3.85 (bs, 4H), 3.67 (bs, 4H), 3.03 (bs, 4H), 2.93 (bs, 4H), 1.40
(t, J = 7.2 Hz, COOCH2CH3, 3H); 13C NMR (300 MHz
DMSO) δ: 165.7 153.83, 152.35, 151.7, 148.52, 143.40,
135.36, 133.26, 127.44, 120.61, 120.24, 112.08, 104.93,
66.52, 66.18, 61.88, 59.64, 54.81, 54.71, 14.67; 3449, 3397,
ether. Light yellow crystals of 7a 0.140 g (47%) were obtained.
1
m.p. 196–198 °C; IR (KBr) (υmax/cm–1) 2200, 2190, 1695; H
NMR (300 MHz, DMSO) δ: 8.81 (s, H5, 1H), 8.61 (s, H1, 1H),
8.55 (s, H8, 1H), 4.49 (q, 2H, J = 7.1 Hz, COOCH2CH3), 1.43
(t, 3H, J = 7.1 Hz, COOCH2CH3); 13C NMR (300 MHz,
DMSO) δ: 164.66, 153.28, 136.90, 133.54, 131.75, 129.59,
125.44, 123.56, 118.60, 114.87, 114.27, 113.64, 111.50,
62.57, 14.17; elemental analysis calcd (%) for C15H8O3N2S:
C 60.81 H 2.70 N 9.46 found C 61.13 H 2.85 N 9.33.
3297,
2963;
elemental
analysis
calcd
(%)
for
C23H30N6O5SCl2: C 55.19 H 5.64 N 16.79 found C 55.94 H
5.57 N 16.52; MS (ESI, m/z, -HCl): 501.4 (MH+), 251.4
(M2H+)2+.
4.1.25. 4-Ethyl-2-cyano-thieno[2,3-e]benzo[b]thiophene (11b)
Compound 11b was prepared from 10b (0.298 g, 1.1 mmol)
in 250 ml toluene by irradiation for 2 hours. The air was
bubbled through the solution and I2 was added. After solvent
was evaporated the crystals were recrystallized from petrol
ether. Light yellow crystals of 7a 0.230 g (80%) were obtained;
4.1.28. 4-Ethyl-2-(N-isopropylamidino)-thieno[2,3-e]benzo[b]
thiophene hydrochloride (12c)
Compound 12c was prepared using the method described
for the preparation of 4a; from 11b (0.224 g, 0.78 mmol) and
the isopropyl amine (0.231g, 3.9 mmol). The precipitate was
collected and recrystallized from ethanol–ether to give light
1
1
m.p. 205–206 °C; IR (KBr) (υmax/cm–1) 2200, 1695; H NMR
yellow crystals 0.10 g (33.4%), m.p. 210–212 °C. H NMR
(DMSO) δ: 8.98 (s, 2H), 8.34 (d, J7,8 = 4.9 Hz, 1H), 8.13 (d,
J8,7 = 4.9 Hz, 1H), 4.40 (q, J = 7.1 Hz, COOCH2CH3, 2H),
1.41 (t, J = 7.1 Hz, COOCH2CH3, 3H); 13C NMR (DMSO)
δ: 162.5, 139.19, 137.38, 135.77, 134.79, 133.67, 128.70
126.44, 123.06, 119.98, 115.20, , 111.34, 62.37, 14.64; ele-
mental analysis calcd (%) for C 58.54; H 3.14; N 4.88; found
C 58.71; H 3.21; N 5.02.
(300 MHz DMSO) δ: 9.1 (bs, 3H), 8.81 (s, H4, 1H), 8.69 (s,
H1 1H), 8.26 (d, J7,8 = 5.4 Hz, 1H), 7.95 (d, J8,7 = 5.4 Hz, 1H),
4.47 (q, 2H, J = 7.1 Hz, COOCH2CH3), 4.33 (m, 1H,
CH(CH3), 1.42 (t, 3H, J = 7.1 Hz, COOCH2CH3.), 1.35 (d,
6H, J = 6.3 Hz, CH(CH3); elemental analysis calcd (%) for
C17H19N2O2S2Cl: C53.27 H 4.96 N 7.3 found C 53.41 H
5.11 N 7.12.
4.1.26. 4-Ethyl-2,7-bis-(N-isopropylamidino)-thieno[3,2-e]
benzo[b]furan dihydrochloride (12a)
Compound 12a was prepared using the method described
for the preparation of 4a; from 11a (0.103 g, 0.34 mmol) and
the isopropyl amine (0.103 g, 0.17 mmol). The precipitate was
collected and recrystallized from ethanol-ether to give light
4.1.29. Ethyl-Z-3-(5-formyl-2-furyl)-2-(3-thienyl)acrylate (13)
Compound 13 was prepared using the method described for
preparation of compound 1a, from phosphorus oxychloride
(2.4 ml, 26 mmol), DMF (2.3 ml, 26 mmol) and the ethyl-Z-
3-(2-furyl)-2-(3-thienyl)acrylate (4.4 mmol) in DMF (2 ml)
was added drop-wise to the mixture and stirred for 1 h at room
temperature, then heated at 90 °C for 45 min, cooled and
poured onto crushed ice and made weakly alkaline with so-
dium carbonate solution, and left over-night on ice. The solid
was filtered off, washed with water and recrystallized from
methanol. Yellow orange crystals 1.159 g (96%) was obtained,
yellow crystals 0.030 g (17.8%). m.p. 251–253 °C. IR(KBr) (
1
υ
max/cm–1): 3460, 3396, 3176, 2981, 1702, 1675, 1633; H
NMR (DMSO) δ: 10.42 (d, J = 7.9 Hz, 1H), 10.25 (s, 1H),
10.15 (d, J = 7.9 Hz, 1H), 9.93 (s, 1H), 9.66 (s, 1H), 9.47 (s,
1H), 8.83 (s, H5, 1H), 8.78 (s, H1, 1H), 8.55 (s, H8, 1H), 4.52
(q, J = 7.2 Hz, COOCH2CH3, 2H), 4.26–4.16 (m, 2H,
CH(CH3)2), 1.46–1.42 (t, J = 7.2 Hz, COOCH2CH3, 3H),
1.35 (d, 6H, J = 6.3 Hz, CH(CH3)2); 13C NMR (DMSO) δ:
164.54, 155.36, 152.28, 150.26, 146.25, 135.81, 134.49,
132.18, 126.98, 126.49, 126.44, 22.90, 113.81, 112.04,
62.23, 45.57, 45.26, 21.12, 21.08, 13.97; elemental analysis
calcd (%) for (C21H30N4O4SCl2x2H2O): C 48.18, H 6.10, N
10.70 found C 48.51, H 6.07, N 10.52.
1
mp 65–67 °C. IR (KBr) (υmax/cm–1) 1680, 1650, 1600; H
NMR (300 MHz DMSO) δ: 9.5 (s, CHO, 1H); 7.63 (s, H3,
1H); 7.63 (d, J5’,4’ = 5.0 Hz, 1H); 7.56 (s, H-2’, 1H); 7.43 (d,
J4",3" = 3.5 Hz, 1H); 7.03 (d, J4",5’ = 5.0 Hz, 1H); 6.21 (d, J3",4"
= 3.5 Hz, 1H); 4.20 (q, J = 7.0 Hz, COOCH2CH3, 2H); 1.22 (t,
J = 7.0 Hz, COOCH2CH3, 3H); elemental analysis calcd (%)
for C14H12O4S: C60.86 H 4.38 found C 60.61 H 4.11
4.1.30. Ethyl-Z-3-(5-cyano-2-furyl)-2-(3-thienyl)acrylate (14)
Compound 14 was prepared using the method described for
the preparation of 3a, from 13 (1 g, 3.6 mmol) and hydroxy-
lammonium hydrochloride (0.439 g) were heated in pyridine
(3 ml) at 60 °C for 30 min, then acetic anhydride (2 ml) was
successively added at a temperature not exceeding 95 °C,
which was being kept for additional 2 h. The mixture was
cooled to 20 °C, poured into water (25 ml) and the separated
nitrile was stirred for 1 h, filtered, washed with water, dried
and crystallized from petrol ether gave 14 0.623 g (63.4%).
m.p. 105–106 °C . IR (KBr) (υmax/cm–1): 2235 , 1710, 1626.;
4.1.27. 4-Ethyl-2,7-bis-(morpholinamidino)-thieno[3,2-e]
benzo[b]furan dihydrochloride (12b)
Compound 12b was prepared using the method described
for the preparation of 4a; from 11a (0.143 g, 0.48 mmol) and
the aminomorpholin (0.6 ml, 5 mmol). The precipitate was col-
lected and recrystallized from ethanol-ether to give light yellow
crystals 0.124
g (51.3%), m.p. 231–232 °C. IR(KBr)
1
(υmax/cm–1): 2821, 1701, 1616, 1581; H NMR (DMSO) δ:
12.21 (s, 1H), 11.81 (s, 1H), 10.55 (s, 1H), 10.27 (s, 1H),
9.47 (s, 1H), 9.37 (s, 1H), 8.91(s, H5, 1H), 8.85 (s, H1, 1H),

K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
937
1H NMR (300 MHz DMSO) δ: 7.64 (d, J5’,4’ = 4.9 Hz, 1H.),
Table 2
Electronic absorption data of studied compoundsa
7.57–7.56 (m, H-3, H-2’,H-4’, 3H), 7.05 (d, J4",3" = 3.7 Hz,
1H), 6.39 (d, J3",4" = 3.7 Hz, 1H), 4.2 (q, J = 7.1 Hz, COO
CH2CH3, 2H), 1.23 (t, J = 7.1 Hz, COOCH2CH3, 3H); 13C
NMR (300 MHz DMSO) δ: 165.64, 154.57, 129.45, 125.99,
125.85, 125.09, 124.84, 115.3, 111.39, 61.23, 13.99; elemental
analysis calcd (%) for (C14H11O3NS): C 61.47, H 4.02, N 5.12
found C 61.15, H 3.91, N 4.88.
λmax (nm)
ε (dm3 mol–1 cm–1
4797
6316
7680
9862
9910
15489
16853
15571
14836
15116
11572
13192
5498
10609
10702
2572
2975
2686
)
2a
2b
3a
3b
3c
323
321
321
324
319
327
258
321
341
255
321
344
268
343
357
256
333
352
318
251
325
b5a
b6a
4.1.31. 4-ethyl-7-cyano-thieno[2,3-e]-benzo[b]furan (15)
Compound 15 was prepared using the method described for
the preparation of 7a; from 13 (304 mg, 1.1 mmol). The resi-
due was recrystallized from petrol ether afforded white crystals
15 0.144 g (47%), m.p. 113–114 °C. IR(KBr) (υmax/cm–1):
2210, 1705, 1675;1H NMR (300 MHz DMSO) δ: 8.79 (s, H5,
1H), 8.52 (s, H8, 1H), 8.20 (d, J3,2 = 5.5 Hz, 1H.), 8.02 (d, J2,3
= 5.5 Hz, 1H), 4.42 (q, J = 7.1 Hz, COOCH2CH3, 2H), 1.40 (t,
J = 7.1 Hz, COOCH2CH3, 3H); 13C NMR (300 MHz DMSO)
δ: 165.26, 151.87, 135.09, 133.49, 128.72, 128.64, 124.72,
124.44, 123.53, 118.14, 112.16, 111.56, 61.44, 14.08; elemen-
tal analysis calcd (%) for (C14H9O3NS): C 61.92 H 3.32; N
5.16. found C 62.05 H 3.61 N 4.98.
6b
10a
12a
14
16
4797
5121
3437
a
Sodium cacodylate buffer, I = 0.05 mol dm–3, pH = 7.
Previously published results added for comparison [4].
b
coefficient ε(ct-DNA) = 6600 mmol–1 cm–2. The measurements
were performed in aqueous buffer solution (pH = 7; sodium
cacodylate buffer, I = 0.05 mol dm–3). Under the experimental
conditions used, the absorbencies of studied compounds were
proportional to their concentrations. The binding constants (KS)
and [bound compound]/[polynucleotide phosphate] ratio (n)
were calculated according to the Scatchard equation by non-
linear least-squares fitting [21,29], all having satisfying corre-
lation coefficients (> 0.999). The thermal denaturation experi-
ments were performed 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 transition
curves, determined from the maximum of the first derivative
or graphically by a tangent method. ΔTm values were calcu-
lated by subtracting Tm of the free nucleic acid from Tm of
the complex. Every ΔTm value here reported was the average
of at least two measurements, the error in ΔTm is 0.5 °C.
4.1.32. 4-Ethyl-7-(N-isopropylamidino)-thieno[2,3-e]-benzo[b]
furan hydrochloride (16)
Compound 16 was prepared in similar manner as 4a, from
15 (0.116 g, 0.43 mmol) and the isopropylamine (0.103 g,
2.1 mmol). The precipitate was collected and recrystallized
from ethanol-ether afforded light yellow crystals 0.134 g
(85.3%). m.p. 180–182 °C IR (KBr) (υmax/cm–1): 3360, 3296,
1
3076, 1675, 1633; H NMR (DMSO) δ: 9.8 (bs, 3H), 8.64 (s,
H5, 1H), 8.31 (s, H8, 1H), 8.26 (d, J3,2 = 5.5 Hz, 1H), 8.02 (d,
J2,3 = 5.5 Hz, 1H), 4.45 (q, J = 7.1 Hz, COOCH2CH3, 2H), 4.2
(m, CH(CH3)2, 1H 1.40 (t, J = 7.1 Hz, COOCH2CH3, 3H),
1.19 (d, J = 6.5 Hz, CH(CH3)2, 6H); 13C NMR (DMSO) δ:
165.30, 151.28, 150.40, 145.54, 135.0, 133.68, 128.72,
128.57, 124.73, 124.71, 124.65, 124.34, 124.33, 112.05,
111.85, 61.45, 45.29, 42.89, 21.18, 20.34; elemental analysis
calcd (%) for C17H21O3N2Cl: C 56.21; H 5.78; N 7.72; found
C 56.54; H 5.91; N 7.55.
4.3. Antitumor activity assay
4.2. Interactions with DNA
The HeLa (cervical carcinoma), MCF-7 (breast carcinoma),
SW 620 (colon carcinoma), MiaPaCa-2 (pancreatic carcino-
ma), Hep-2 (laryngeal carcinoma) and WI 38 (diploid fibro-
blasts) cells were cultured as monolayers and maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U
ml–1 penicillin and 100 μg ml–1 streptomycin in a humidified
atmosphere with 5% CO2 at 37 °C.
The electronic absorption spectra were obtained on Varian
Cary 100 Bio spectrometer using quartz cuvettes (1 cm) (Ta-
ble 2).
Calf thymus (ct-) DNA was purchased from Fluka, dis-
solved in the sodium cacodylate buffer, I = 0.05 mol dm–3,
pH 7 and additionally sonicated and filtered through the 0.45
μm filter. The final concentration of the calf thymus (ct-) DNA
solution expressed as the concentration of phosphates was de-
termined spectroscopically [27,28] by measuring the absor-
bance of buffered solution at λ = 260 nm for at least five inde-
pendent additions of the DNA stock solution aliquots and
dividing the averaged absorbance value by molar extinction
The growth inhibition activity was assessed according to the
slightly modified procedure performed at the National Cancer
Institute, Developmental Therapeutics Program [30]. The cells
were inoculated onto standard 96-well microtiter plates on day

938
K. Starčević et al. / European Journal of Medicinal Chemistry 41 (2006) 925–939
[2] M. Demeunynck, C. Bailly, W.D. Wilson, D.N.A. In, R.N.A. Binders,
Wiley-VCH, Weinheim, 2002.
0. The cell concentrations were adjusted according to the cell
population doubling time (PDT): 1 × 104 ml–1 for HeLa, Hep-
2, MiaPaCa-2 and SW 620 cell lines (PDT = 20–24 hours) 2 ×
104 ml–1 for MCF-7 cell lines (PDT = 33 hours) and 3 × 104
ml–1 for WI 38 (PDT = 47 hours). Test agents were then added
in five, 10-fold dilutions (10–8–10–4 mol l–1) and incubated for
further 72 hours. Working dilutions were freshly prepared on
the day of testing. The solvent (DMSO) was also tested for
eventual inhibitory activity by adjusting its concentration to
be the same as in working concentrations. After 72 hours of
incubation, the cell growth rate was evaluated by performing
the MTT assay [31], which detects dehydrogenase activity in
viable cells. The absorbance (OD, optical density) was mea-
sured 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:
[3] a) J.M. Fortune, L. Velea, D.E. Graves, T. Utsugi, Y. Yamada, N.
Oshero, Biochemistry 38 (1999) 15580–15586.; b) C. Bailly, X.Qu, J.
B.Chaires, P.Colson, C. Houssier, M. Ohkubo, S.Nishimura, T. Yoshi-
nari, J. Med. Chem. 42 (1999) 2927–2935.; c) J.A.Wilson-Byl, J.M. For-
tune, A. Burden, J.L. Nitiss, T. Utsugi, Y. Yamada; N. Oshero, Biochem-
istry 38 (1999) 15573–15579; d) H. Minderman, C. Wrzosek, S.Cao, T.
Utsugi, T. Kobunai, Y. Yamada, Y. M. Rustum, Cancer Chemother.
Pharmacol. 45 (2000) 78–84.
[4] K. Starčević, G. Karminski-Zamola, I. Piantanida, L. Šuman, M. Kralj, J.
Am. Chem. Soc. 127 (2005) 1074–1075.
[5] a) P. Cagniant, D. Cagniant, in: A.R. Katritzky, A.J. Boulton, (Eds.), Ad-
vances in Heterocyclic Chemistry, vol. 18, Academic Press, New York,
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If (mean ODtest – mean ODtzero) ≥ 0 then
PG ¼ 100 ꢀ ðmean ODtest ꢁ mean ODtzeroÞ=ðmean ODctrl
ꢁ mean ODtzero
Þ
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Where
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Mean ODtzero = the average of optical density measurements
before exposure of cells to the test compound.
Mean ODtest = the average of optical density measurements
after the desired period of time.
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Each test point was performed in quadruplicate in three in-
dividual experiments. The results are expressed as IC50, which
is the concentration necessary for 50% of inhibition. The IC50
values for each compound are calculated from dose–response
curves using linear regression analysis by fitting the test con-
centrations that give PG values above and below the reference
value (i.e. 50%). If however, for a given cell line all of the
tested concentrations produce PGs exceeding the respective re-
ference 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
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Acknowledgements
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Support for this study by the Ministry of Science, Education
and Sport of Croatia is gratefully acknowledged (Projects
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