Antiproliferative potency of novel benzofuran-2-carboxamides on tumour cell lines: Cell death mechanisms and determination of crystal structure

Article abstract of DOI:10.1016/j.ejmech.2012.11.009

In this manuscript the synthesis and biological activity of novel heterocyclic derivatives of benzofuran-2-carboxamides 3a-j and 6a-f is presented. Biological evaluation in vitro revealed that only few compounds exerted concentration-depended antiproliferative effects on tumour cell lines at micromolar concentrations. In particular, 2-imidazolynyl substituted compound 6f showed selectivity on SK-BR-3 cell line while 2-N-acetamidopyridyl substituted 3h and 2-imidazolynyl substituted amide 3i showed selective concentration-dependent antiproliferative effects on SW620 cell line. Compounds 3h and 6f induced apoptosis while compound 3i acted through a cell death mechanism other than apoptosis.

Full text of DOI:10.1016/j.ejmech.2012.11.009

European Journal of Medicinal Chemistry 59 (2013) 111e119
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antiproliferative potency of novel benzofuran-2-carboxamides on tumour cell
lines: Cell death mechanisms and determination of crystal structure
,
b
a
ꢀ a
Marijana Hranjec a , Irena Sovic , Ivana Ratkaj , Gordana Pavlovic , Natasa Ilic , Linda Valjalo ,
ꢀ c
ꢁ
ꢀb
*
,
a
ꢀb
ꢀ
ꢀ b
**
ꢁ
Kresimir Pavelic , Sandra Kraljevic Pavelic
Faculty of Chemical Engineering and Technology, Department of Organic Chemistry, University of Zagreb, Marulicev trg 20, P.O. Box 177, HR-10000 Zagreb, Croatia
Department of Biotechnology, University of Rijeka, Trg brace Mazuranica 10, HR-51000 Rijeka, Croatia
Faculty of Textile Technology, Department of Applied Chemistry, University of Zagreb, Prilaz baruna Filipovica 28a, HR-10000 Zagreb, Croatia
, Grace Karminski-Zamola
a
ꢀ
b
c
ꢀ
ꢁ
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
In this manuscript the synthesis and biological activity of novel heterocyclic derivatives of benzofuran-2-
carboxamides 3aej and 6aef is presented. Biological evaluation in vitro revealed that only few
compounds exerted concentration-depended antiproliferative effects on tumour cell lines at micromolar
concentrations. In particular, 2-imidazolynyl substituted compound 6f showed selectivity on SK-BR-3 cell
line while 2-N-acetamidopyridyl substituted 3h and 2-imidazolynyl substituted amide 3i showed
selective concentration-dependent antiproliferative effects on SW620 cell line. Compounds 3h and 6f
induced apoptosis while compound 3i acted through a cell death mechanism other than apoptosis.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 28 August 2012
Received in revised form
1 November 2012
Accepted 8 November 2012
Available online 16 November 2012
Keywords:
Benzofuranes
Carboxamides
Antiproliferative activity
Apoptosis
Crystal structure determination
1. Introduction
In addition, benzofurans and their derivatives possess a broad
range of important biological activities including anticancer, anti-
Small heteroaromatic molecules still represent the mainstay in
classical chemotherapy for cancer treatment. However, the selec-
tivity issue and better activity are still major requirements in
development of novel anticancer drugs. The amide functionality is
the common backbone of numerous organic molecules and natural
products that bear diverse chemical and pharmacological features
[1,2]. Consequently, amides play a key role in important life
processes, whereas, for example, the amide bond is crucial for
protein formation. Moreover, according to the Comprehensive
Medicinal Chemistry database, amide bond is widely incorporated
in the structure of 25% well known drugs. Amides are usually stable,
neutral and have both hydrogen-bond acceptor/donator properties
which are very important for the synthesis of versatile hetero-
aromatic molecules.
bacterial, antifungal and antiviral properties. They have already
attracted considerable attention amongst organic and medicinal
chemists in the last few years [3,4]. For example, benzofuran-2-
carboxamides showed excellent oral uptake indexes and bloode
brain barrier permeability, anti-dopamine action, potent meth-
amphetamine potentiation, antiemetic action and low toxicity [5].
Another series of benzofuran-2-carboxamides proved to inhibit
mammalian 5-lipoxygenase enzyme and their usefulness in the
treatment of asthma, allergic disorders and certain cardiovascular
disorders [6]. Heteroaromatic benzofuran-2,3-dicarboxamides
were found to inhibit activated blood coagulation factor X which
is a key enzyme located in the position of extrinsic and intrinsic
coagulation cascade reactions [7]. Benzofuran-2,5-carboxamides,
structurally related to netropsin with two pyrrole-amide units as
well as amidine terminal units, have showed reduced cytotoxic
activity [8]. A group of authors has recently published cytotoxic
activity results of benzofuran-2-yl-pyridines towards human liver
cancer cell line Hep-G2 [9]. Alkyl and cykloalkyl substituted
benzofuran-2-carboxamides were found to be a selective and
directly acting H3 receptor antagonists and can be used as active
substances, particularly in the treatment or prevention of diseases
* Corresponding author. Tel.: þ385 14597245; fax: þ385 14597250.
** Corresponding author.
E-mail addresses: mhranjec@fkit.hr (M. Hranjec), sandrakp@biotech.uniri.hr
ꢀ
ꢀ
(S. Kraljevic Pavelic).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.009

112
M. Hranjec et al. / European Journal of Medicinal Chemistry 59 (2013) 111e119
which are associated with the modulation of H3 receptors [10].
Further on, 2-benzofuran derivatives containing heterocyclic nuclei
have shown antibacterial activity towards Staphylococcus aureus
and Candida albicans bacteria [11], and benzoxazole derivatives of
benzofurane-2-carboxamides showed a broad spectrum of anti-
bacterial activity as well. Some of tested compounds showed
a significant activity towards Pseudomonas aeruginosa that was
comparable to the potency of a commercial antibacterial drug
Rifampicin [12]. 5-Sulfonyl substituted versatile benzofurane
derivatives were found to act as a resistance-repellent and multi-
drug resistant retroviral HIV protease inhibitors [13]. At last, 2-
conventional methods of organic synthesis used for preparation of
similar heterocyclic amides.
Starting from benzofuran-2-carbonylchloride 1, in the reaction
with versatile substituted anilines and amino-pyridines 2aej, 2-
aminobenzothiazoles 4a, 4c and 5a and 2-aminobenzimidazoles
4b, 4d and 5b corresponding benzofuran-2-carboxamides 3aej
and 6aef were prepared. Compound 3c, as hydrochloride salt,
was prepared from compound 3b with HCl(g). Cyano and nitro
substituted 2-aminobenzazoles 4aed were prepared by earlier
described methods [17]. 2-Imidazolynyl substituted 2-
aminobenzazoles 5a,b were prepared according to Scheme 3, in
the Pinner reaction from corresponding cyano precursors 4a,b
[18,19].
The structure of all newly synthesized benzofuran-2-
carboxanilides were determined by NMR analysis, based on the
analysis of HeH coupling constants as well as chemical shifts. In 1H
NMR spectra, the presence of proton of CONH group was confirmed
by one-proton singlet at 10.30 ppm to 13.48 ppm. The molecular
and crystal structure for compound 3f is given in Supplementary
material.
(30,40,50-trimethoxybenzoyl)-benzo[b]furan
derivatives
with
electron-donating (Me, OMe or OH) or electron-withdrawing (F, Cl
and Br) substituents on the benzene ring, have shown prominent
inhibition properties on tubulin polymerization and the cell cycle
[14]. Such a large amount of evidence proves a role of benzofuran
scaffold in diverse biological activities and their therapeutic
potential. It is therefore worth an effort to further investigate this
class of compounds.
As a part of our commitment to search for novel potential anti-
cancer agents related to heterocyclic amides, we already reported
the synthesis of various cyano- or amidino-substituted hetero-
aromatic benzo[b]thiophene-2-carboxamides with strong inhibitory
activities on several human cancer cell lines (Fig. 1) [15,16]. 2-
Imidazolinyl-substituted derivatives of pyridyl-benzo[b]thiophene-
2-carboxamides exhibited strong growth inhibitory effect in the low
micromolar range. Only minor structural difference between
compounds in position of heterocyclic nitrogen in respect to the
amidine substituent had exceptionally strong impact on their bio-
logical action. We have shown that 2-pyridyl-benzo[b]thiophene-2-
carboxamide derivative binds to DNA minor groove in a form of
dimmer, while 5-pyridyl-benzo[b]thiophene-2-carboxamide deriv-
ative which had the most potent effect on the cell cycle, pointing
towards tubulin as cellular target of its biological action.
2.2. Biological activity
The in vitro screening of benzo[b]furane amides derivatives (3ae
6f) on human tumour cell lines and normal (diploid) human
fibroblasts WI38 (control cell line) revealed a strong and non-
specific cytotoxic effect of all tested compounds at higher tested
concentrations (10e100 mM) (Table 1).
Majority of tested compounds were cytotoxic to normal human
fibroblasts as well. Interestingly, SK-BR-3 cell line was less sensitive
towards all tested compounds with the exception to 2-imidazolynyl
substituted compound 6f, that showed good selectivity on this cell
line in the micromolar range. The antiproliferative activity of 2-
imidazolynyl substituted benzimidazole amide 6f is increased in
comparison to the structurally related 2-imidazolynyl substituted
benzothiazole amide 6e due to substitution of a sulphur atom
instead of nitrogen at position 1. The cell cycle analysis confirmed
a strong antiproliferative effect of compound 6f on SK-BR-3 cells
witnessed by dramatic perturbations in the cell cycle induced at
micromolar concentrations (Table 2). An increase of cells in the G1
phase ranging between 18 and 30% accompanied with an increase
of cells in the sub G1 phase in comparison to control has been
observed upon 24-h treatment. Similarly, 48-h treatment of cells
with compound 6f induced an increase of sub G1 cell population
and increased S-phase cell population by 33% at lower compound
concentration. Such strong perturbations of the cell cycle induced
Encouraged by the results from this study, we set out to explore
and synthesize novel heteroaromatic benzofuran-2-carboxamide
derivatives 3aej and 6aef (Fig. 2). All newly synthesized
compounds were tested for their antiproliferative activity on the
panel of several human tumour cell lines as well as normal human
fibroblasts.
2. Results and discussion
2.1. Chemistry
Newly prepared carboxamides shown in Fig. 2 were synthesized
according to two main procedures shown in Schemes 1 and 2, by
Cl
Cl
O
O
S
NH
R2
R1
S
NH
W
R
X
Y
+
NH2 Cl-
R1= H, COOCH3, CN, OCH3, Br,
R2= H, COOCH3, CH3, CN, OCH3, Br,
X=Y=CH; R=H; W=N
X=Y=CH; R=H; W=NH+Cl-
X=W=CH; Y=N; R=CN
Y=W=CH; X=N; R=CN
NH
H
N
X=W=CH; Y=N;
Y=W=CH; X=N;
R=
N
H+Cl-
Fig. 1. Earlier prepared heteroaromatic benzo[b]thiophene-2-carboxamides.

M. Hranjec et al. / European Journal of Medicinal Chemistry 59 (2013) 111e119
113
O
O
R
X
N
O
NH
R
O
NH
X
Y
3a-3j
6a-6e
3a X=CH, Y=CH, R=CN
3b X=CH, Y=CH, R=N(CH3)2
6a X=S, R=CN
3c X=CH, Y=CH, R=N(CH3)2×HCl
3d X=CH, Y=CH, R=NHCOCH3
3e X=CH, Y=CH, R=NO2
3f X=N, Y=CH, R=CN
6b X=NH, R=CN
6c X=S, R=NO2
6d X=NH, R=NO2
3g X=N, Y=CH, R=NO2
3h X=CH, Y=N, R=NHCOCH3
H
N
6e X=S
6f X=NH
H
R=
N
3i X=CH, Y=CH,
3j X=N, Y=CH,
N
H+Cl-
R=
N
H+Cl-
Fig. 2. Prepared heteroaromatic benzofuran-2-carboxamide derivatives 3aej and 6aef.
by very low compound concentrations confirm the strong anti-
proliferative effect previously observed in proliferation assays.
Cell death induction might be attributed to either the ability of
benzofuraneecarboxamide compounds to alkylate DNA and cause
cytotoxic effects [20] or to DNA binding properties or changes of the
cellular cytoskeleton that induce irreparable cell damage [15,16].
Current cancer chemotherapy primarily exerts the antitumour
effect by triggering apoptosis in cancer cells where proteolytic
enzymes, caspases, play a critical role in cell death execution [21].
Two major routes are known to involve different caspases in
response to anticancer chemotherapy: the extrinsic (receptor)
pathway and the mitochondria stimulated intrinsic pathway.
Analysis of caspases 3, 8 and 9 activation upon treatment of SK-BR-
3 cells with compound 6f substantiate apoptosis activation in these
cells (Fig. 3) which is consistent with antiproliferative results and
cell cycle analysis. In some tumour cells, caspase-8 is known to be
activated upon death receptor activation in quantities sufficient to
directly activate downstream effector caspase-3 as well as a mito-
chondrial amplification loop required for full activation of caspases,
inlcuding caspase 9 [22]. Our results speak in favour of this
apoptotic mechanism in SK-BR-3 cells as well.
with increased antitumour activity and selectivity in comparison to
the structurally related derivative 4-N-acetamidophenyl
substituted amide 3d. The cell cycle analysis of SW620 cells treated
with compound 3h showed increased S-phase cell population after
48-h. Similarly to compound 6f, apoptosis induction in this cell line
through death receptor activation and mitochondrial amplification
[23] was corroborated by strong activation of tested caspases 3, 8
and 9 (Fig. 3).
Further on, it seems that the imidazolynyl moiety of compound
3i added to specificity and antiproliferative properties observed on
SW620 cells. Contrary to compound 3h, a small but statistically
significant increase in G2/M cell population has been observed in
SW620 cells treated with compound 3i pointing to a cell death
mechanism in SW620 other than apoptosis, i.e. mitotic catastrophe.
Indeed, analysis of caspase activation showed no activation for
caspases 3, 8 or 9 upon treatment of cells (Fig. 3). Our previous
studies already showed that N-amidino-substituted benzimidazo
[1,2-a]quinolones exerted a similar antiproliferative effect through
mitotic catastrophe on SW620 cells as well [23]. This assumption
however should be confirmed by additional tests (Table 3, Fig. 4).
Similarly, 2-N-acetamidopyridyl substituted amide 3h and 2-
3. Conclusions
imidazolynyl substituted 3i exerted
concentration-dependent antiproliferative effects in the micro-
molar range (0.1e1 M) on SW620 cell line. In particular, pyridyl
a
highly selective
We successfully prepared a series of novel heteroaromatic
benzofuran-2-carboxamide derivatives 3aej and 6aef. In compar-
ison to our previous results obtained by benzo[b]thiophene-2-
m
moiety of 2-N-acetamidopyridyl substituted amide 3h correlated
NH2
O
O
X
TEA
+
toluene
Y
O
Cl
O
NH
R
3a-3j
1
X
Y
R
3a X=CH, Y=CH, R=CN
3b X=CH, Y=CH, R=N(CH3)2
3c X=CH, Y=CH, R=N(CH3)2×HCl
3d X=CH, Y=CH, R=NHCOCH3
3e X=CH, Y=CH, R=NO2
3f X=N, Y=CH, R=CN
2a-2j
X=Y=CH, N
EtOHaps.
HClg
3g X=N, Y=CH, R=NO2
3h X=CH, Y=N, R=NHCOCH3
H
N
3i X=CH, Y=CH,
3j X=N, Y=CH,
R=
N
H+Cl-
Scheme 1. Synthesis of benzofuran-2-carboxamide derivatives 3aej.

114
M. Hranjec et al. / European Journal of Medicinal Chemistry 59 (2013) 111e119
Table 1
R
O
N
The inhibition effects of compounds 3ae6f on the growth of selected tumour cells
in vitro. The results are given as IC50 values in M. The cell growth rate was evaluated
by performing the MTT assay: experimentally determined absorbance values were
transformed into a cell percentage growth (PG) using the formulae proposed by
National Cancer Institute and described previously in Gazivoda et al. [20].
m
H2N
+
O
X
Cl
1
4a X=S, R=CN
4b X=NH, R=CN
4c X=S, R=NO2
4d X=NH, R=NO2
a
Compound
IC50
(
m
M)
Cell lines
MCF-7
H
N
TEA
5a X=S,
5b X=NH,
SK-BR-3
SW620
MiaPaCa-2
WI38
HeLa
toluene
reflux
R=
N
3a
3b
3c
3d
3e
3i
7.6
34.2
7.4
86
27.1
>100
49.6
>100
26.6
31.5
44.7
73.1
3.1
>100
>100
>100
97.1
>100
9.3
7.9
7.6
H+Cl-
35.6
8.5
45.1
4.9
26.2
5.3
82.8
7.6
>100
21.5
63.9
50.9
>100
5.2
>100
>100
>100
>100
40.5
>100
>100
59.8
>100
>100
>100
>100
>100
>100
3.83
>100
81.2
71.2
47.6
23.3
52.4
37.8
0.4
68.7
34.9
72.9
59.2
9.1
83.7
49.1
77.3
11.4
27.5
23.9
42.4
0.9
71.9
89.6
17.5
59.8
>100
9.2
54.2
>100
27.9
6.4
69.2
76.5
1.1
67.1
>100
>100
56.4
9.2
O
R
X
N
3f
O
NH
6a-6e
3g
3h
3j
6a
6c
6e
6b
6d
6f
6a X=S, R=CN
6b X=NH, R=CN
6c X=S, R=NO2
6d X=NH, R=NO2
6.9
74.9
8.6
>100
7
91.2
68.5
H
N
a
6e X=S
6f X=N
IC50; 50% inhibitory concentration, or compound concentration required to
inhibit tumour cell proliferation by 50%. The IC50 values were calculated from dosee
response curves using linear regression analysis by fitting the mean test concen-
trations that give PG values above and below the reference value. If, however, all of
the tested concentrations produce PGs exceeding the respective reference level of
effect (e.g. PG value of 50) for a given cell line, the highest tested concentration is
assigned as the default value (>100). Each test point was performed in quadrupli-
cate in three individual experiments.
R=
N
H+Cl-
Scheme 2. Synthesis of benzofuran-2-carboxamide derivatives 6aef.
carboxamides, it seems that replacement of benzo[b]thiophene
with benzo[b]furan seems to be detrimental for the antitumour
activity. Indeed, all newly synthesized compounds were evaluated
for the antiproliferative potency in vitro on human tumour cell lines
and normal (diploid) human fibroblasts and showed non-selective
effects on all tested cells with stronger antitumour activities
detectable only in the highest tested concentrations. Obtained
results however, revealed a potential for three compounds as
selective antiproliferative compounds. In particular, 2-imidazolynyl
substituted compound 6f showed good selectivity on SK-BR-3 cell
line while 2-N-acetamidopyridyl substituted amide 3h and 2-
imidazolynyl substituted compound 3i showed selective
concentration-dependent antiproliferative effects on MiaPaCa-2
and SW620 cell lines, respectively in the micromolar range. It
seems that the antiproliferative activity of 2-imidazolynyl
substituted benzimidazole amide 6f is increased in comparison to
the structurally related 2-imidazolynyl substituted benzothiazole
amide 6e due to substitution of a sulphur atom instead of nitrogen
at position 1. In particular, pyridyl moiety of 2-N-acetamidopyridyl
substituted amide 3h correlated with increased antitumour activity
and selectivity in comparison to the structurally related derivative
4-N-acetamidophenyl substituted amide 3d. Further on, the imi-
dazolynyl moiety of compound 3i added to specificity and anti-
proliferative properties observed on SW620 cells. These
compounds exerted different antiproliferative mechanisms, i.e.
compounds 3h and 6f induced apoptosis through activation of
caspases 3, 8 and 9 that might be indicative for activation of death
receptors along with the intrinsic apoptotic pathway. Current
cancer chemotherapy indeed, aims to activate apoptosis in cancer
cells where the concomitant induction of the intrinsic apoptotic
pathway plays a crucial role in chemotherapy potency towards
tumour cells. Contrary, antiproliferative effect of compound 3i
relied on a cell death mechanism other than apoptosis, i.e. mitotic
catastrophe that should be confirmed by additional studies.
4. Experimental
4.1. Chemistry
4.1.1. General methods
All chemicals and solvents were purchased from commercial
suppliers including Aldrich, Fluka and Acros. Melting points were
recorded on an Original Keller Mikroheitztisch apparatus (Reichert,
Wien) and SMP11 Bibby apparatus. 1H and 13C NMR spectra were
recorded on a Varian Gemini 300 or Varian Gemini 600 spectro-
photometers at 300, 600, 150 and 75 MHz, respectively. All NMR
spectra were measured in DMSO-d6 solutions using TMS as an
internal standard. Chemical shifts are reported in ppm (d) relative
to TMS. UV/vis spectra were recorded on Cary Eclipse 50
HN
CN
N
N
N
H+
Cl-
1. EtOHaps., HCl(g)
2. EtOHaps.
H2N
H2N
X
X
ethylenediamine
4a X=S
4b X=NH
5a X=S
5b X=NH
Scheme 3. Synthesis of 2-imidazolinyl substituted 2-aminobenzothiazole 5a and 2-aminobenzimidazole 5b.

M. Hranjec et al. / European Journal of Medicinal Chemistry 59 (2013) 111e119
115
Table 2
Table 3
Flow cytometric analysis of the SK-BR-3 cell cycle upon 24- and 48-h treatments
Flow cytometric analysis of the SW620 cell cycle upon 24- and 48-h treatments with
compound 3i at concentrations 5 M and 10 M. Statistically significant changes
(p < 0.05) are denoted in bold and with an asterisk (*).
with compound 6f at concentrations 5
m
M and 10
m
M. Statistically significant
m
m
changes (p < 0.05) are denoted in bold and with an asterisk (*).
Compound 6f
SK-BR-3
Cell percentage (% ꢁ standard deviation)
SW620
Cell percentage (% ꢁ standard deviation)
Treatment
Sub G1
G1
S
G2/M
Compound 3i
24 h Control/untreated
Treatment
sub G1
G1
S
G2/M
6.9 ꢁ 2.1 46.3 ꢁ 1.8
7.1 ꢁ 1.7 43.4 ꢁ 2
10.4 ꢁ 0.2 45.7 ꢁ 1.9
8.5 ꢁ 1.5 51.1 ꢁ 2.5
4.9 ꢁ 0.3 46.8 ꢁ 1.8
41.1 ꢁ 0.8
43.9 ꢁ 1.2
40.9 ꢁ 1.6
39.9 ꢁ 0.5
40.7 ꢁ 3.3
12.6 ꢁ 1
24 h Control/untreated 25.1 ꢁ 0.4
33.1 ꢁ 1.2* 58.5 ꢁ 0.4* 31.5 ꢁ 1.4*
31.5 ꢁ 0.8 * 70 ꢁ 0.2* 23.6 ꢁ 0.9*
48 h Control/untreated 27.5 ꢁ 0.2
40.1 ꢁ 0.7 26.2 ꢁ 2.3 33.7 ꢁ 0.3
5
m
M
12.7 ꢁ 0.9
13.5 ꢁ 3.5
8.9 ꢁ 0.4
8.3 ꢁ 0.1
10.5 ꢁ 0.9*
5
m
M
10 ꢁ 1.1*
6.4 ꢁ 0.6*
10
mM
10 mM
48 h Control/untreated
53.3 ꢁ 1.5 35.2 ꢁ 1.8 11.6 ꢁ 0.3
5 mM
5
m
M
38.1 ꢁ 0.6* 21.8 ꢁ 1.9* 68.5 ꢁ 1.4*
9.7 ꢁ 1.4*
10
m
M
8.2 ꢁ 1.7 46.9 ꢁ 0.4* 42.6 ꢁ 0.5
10
m
M
36.3 ꢁ 0.9* 52.8 ꢁ 0.9* 31.3 ꢁ 1.1* 15.9 ꢁ 0.8*
Compound 3h
24 h Control/untreated 18.1 ꢁ 3.8 52.6 ꢁ 2.3
38.3 ꢁ 0.4
42.6 ꢁ 3.2
9.3 ꢁ 2.6
10.7 ꢁ 5
(*) Statistically significant at p < 0.05.
5
m
M
18.1 ꢁ 2.4 46.8 ꢁ 1.8
18.2 ꢁ 5.5 43.5 ꢁ 1.4
5.8 ꢁ 0.3 49.9 ꢁ 1.4
4.7 ꢁ 0.4 43.9 ꢁ 0.5
4.3 ꢁ 0.2 45.2 ꢁ 0.3
10
m
M
31.4 ꢁ 11.1 25.3 ꢁ 9.7
48 h Control/untreated
41.6 ꢁ 0.3
44.9 ꢁ 1.1
45.4 ꢁ 0.2*
8.5 ꢁ 1.6
11.2 ꢁ 0.6
9.5 ꢁ 0.5
spectrophotometer. Elemental analysis for carbon, hydrogen and
nitrogen were performed on a PerkineElmer 2400 elemental
analyzer and a PerkineElmer, Series II, CHNS Analyzer 2400. All
compounds were routinely checked by TLC with Merck silica gel
60F-254 glass plates.
5 mM
10 mM
cyanoaniline 2a (0.89 g, 7.53 mmol) in dry toluene (35 mL) fol-
lowed by the addition of Et3N (1.60 mL, 11.34 mmol). The mixture
was refluxed for 24 h and worked up as it is described to give 0.73 g
(33%) of white crystals; mp 196e197 ꢀC; 1H NMR (600 MHz, DMSO-
4.1.2. General method for the preparation of compounds 3a, 3b,
3dej and 6aef
To a solution of benzofurane-2-carbonyl chloride 1 in dry
toluene, a solution of corresponding anilines and amino-pyridines
2aej, 2-aminobenzothiazoles 4a, 4c and 5a and 2-
aminobenzimidazoles 4b, 4d and 5b in dry toluene was added
dropwise, followed by the addition of Et3N. The mixture was
refluxed for several hours. After cooling, the resulting products
were filtered off and recrystallized from methanol to obtain ben-
zofurane-2-carboxamides 3a, 3b, 3dej and 6aef.
d6):
d
¼ 10.54 (s,1H, NHamide), 8.09 (d,1H, J ¼ 7.86 Hz, Harom.), 7.95 (d,
1H, J ¼ 8.88 Hz, Harom.), 7.84 (s, 1H, Harom.), 7.78 (d, 2H, J ¼ 8.96 Hz,
Harom.), 7.70 (t, 1H, J ¼ 7.88 Hz, Harom.), 7.56 (t, 1H, J ¼ 7.78 Hz, Harom.),
6.99 (d, 2H, J ¼ 8.78 Hz, Harom.); 13C NMR (75 MHz, DMSO-d6):
d
¼ 157.7 (s),155.3 (s),148.6 (s),136.0 (s),135.3 (s),131.9 (d, 2C),131.8
(d), 128.4 (d), 127.8 (s), 124.8 (d, 2C), 123.9 (d), 118.4 (s), 112.8 (d),
112.4 (d); UV (EtOH) lmax/nm ¼ 318; elemental analysis calcd. (%) for
C16H10N2O2: C 73.27, H 3.84, N 10.68; found C 73.12, H 3.59, N 10.47.
4.1.2.1. N-(4-Cyanophenyl)benzofuran-2-carboxamide
3a.
Compound 3a was prepared using above described method, from
benzofurane-2-carbonyl chloride 1 (1.36 g, 7.53 mmol) and 4-
4.1.2.2. N-(4-N,N-Dimethylaminophenyl)benzofuran-2-carboxamide
3b. Compound3bwasprepared usingabovedescribedmethod, from
Fig. 3. Activation of caspases 3, 8 and 9 in SK-BR-3 cell line after 24 and 48 h treatments with compound 6f at a concentration of 5 mM. Control; white columns. Compound 6f; dark
grey columns.

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M. Hranjec et al. / European Journal of Medicinal Chemistry 59 (2013) 111e119
Fig. 4. Activation of caspases 3, 8 and 9 in SW620 cell line after 24 and 48 h treatments with compounds 3i and 3h at a concentration 10
dark grey columns. Compound 3h; light grey columns.
mM. Control; white columns. Compound 3i;
benzofurane-2-carbonyl chloride 1 (0.20 g, 1.10 mmol) and 4-N,N-
dimethylaminoaniline 2b (0.17 g, 1.10 mmol) in dry toluene (15 mL)
followed by the addition of Et3N (0.22 mL, 1.54 mmol). The mixture
was refluxed for 18 h and worked up as it is described to give 0.22 g
(77%) of green powder; mp 154e155 ꢀC; 1H NMR (600 MHz, DMSO-
nitroaniline 2e (0.15 g, 1.10 mmol) in dry toluene (20 mL) fol-
lowed by the addition of Et3N (0.21 mL, 1.54 mmol). The mixture
was refluxed for 16 h and worked up as it is described to give 0.17 g
(55%) of white crystals; mp 212e213 ꢀC; 1H NMR (600 MHz, DMSO-
d6):
d
¼ 11.09 (s, 1H, NHamide), 8.28 (d, 2H, J ¼ 7.20 Hz, Harom.), 8.09
d6):
d
¼ 10.30 (s, 1H, NHamide), 7.79 (d, 1H, J ¼ 7.74 Hz, Harom.), 7.69 (d,
(d, 2H, J ¼ 7.26 Hz, Harom.), 7.88 (d, 1H, J ¼ 0.72 Hz, Harom.), 7.85 (d,
1H, J ¼ 7.74 Hz, Harom.), 7.74 (dd, 1H, J1 ¼ 8.34 Hz, J2 ¼ 1.20 Hz,
Harom.), 7.37 (dt, 1H, J1 ¼ 8.38 Hz, J2 ¼ 1.12 Hz, Harom.); 13C NMR
1H, J ¼ 8.64 Hz, Harom.), 7.68 (s, 1H, Harom.), 7.60 (d, 2H, J ¼ 8.88 Hz,
Harom.), 7.47 (t, 1H, J ¼ 7.68 Hz, Harom.), 7.34 (t, 1H, J ¼ 7.56 Hz, Harom.),
6.72 (d, 2H, J ¼ 8.64 Hz, Harom.), 2.86 (s, 6H, CH3); 13C NMR (75 MHz,
(75 MHz, DMSO-d6):
d
¼ 157.6 (s), 155.1 (s), 148.5 (s), 145.2 (s), 143.2
DMSO-d6):
d
¼ 156.5 (s), 154.8 (s), 149.8 (s), 148.0 (s), 128.3 (s), 127.8
(s), 128.1 (d), 127.5 (s), 125.3 (d, 2C), 124.5 (d), 123.6 (d), 120.6 (d,
2C), 112.5 (d), 112.4 (d); UV (EtOH) lmax/nm ¼ 319; elemental
analysis calcd. (%) for C15H10N2O4: C 63.83, H 3.57, N 9.92; found C
63.95, H 3.74, N 10.13.
(s), 127.7 (d), 124.2 (d), 123.2 (d), 122.4 (d, 2C), 112.9 (d, 2C), 112.3 (d),
110.3 (d), 40.8 (q, 2C); UV (EtOH) lmax/nm ¼ 329, 273; elemental
analysis calcd. (%) for C17H16N2O2: C 72.84, H 5.75, N 9.99; found C
73.04, H 5.58, N 10.18.
4.1.2.5. N-(5-Cyanopyridin-2-yl)benzofuran-2-carboxamide
3f.
4.1.2.3. N-(4-Acetamidophenyl)benzofuran-2-carboxamide
3d.
Compound 3f was prepared using above described method, from
benzofurane-2-carbonyl chloride 1 (1.00 g, 5.54 mmol) and 2-
amino-5-cyanopyridine 2f (0.66 g, 5.54 mmol) in dry toluene
(30 mL) followed by the addition of Et3N (1.16 mL, 8.34 mmol). The
mixture was refluxed for 30 h and worked up as it is described to
give 0.41 g (28%) of light yellow crystals; mp 171e172 ꢀC; 1H NMR
Compound 3d was prepared using above described method, from
benzofurane-2-carbonyl chloride 1 (0.30 g, 1.67 mmol) and 4-
acetamidoaniline 2d (0.25 g, 1.67 mmol) in dry toluene (20 mL)
followed by the addition of Et3N (0.32 mL, 2.32 mmol). The mixture
was refluxed for 24 h and worked up as it is described to give 0.30 g
(61%) of light pink crystals; mp 252e254 ꢀC; 1H NMR (600 MHz,
(300 MHz, DMSO-d6):
d
¼ 11.42 (brs, 1H, NHamide), 8.89 (t, 1H,
DMSO-d6):
d
¼ 10.43 (s, 1H, NHamide), 9.90 (s, 1H, NH), 7.80 (d, 1H,
J ¼ 1.43 Hz, Harom.), 8.32 (t, 1H, J ¼ 1.43 Hz, Harom.), 8.10 (s, 2H,
Harom.), 7.84 (d, 1H, J ¼ 7.80 Hz, Harom.), 7.72 (d, 1H, J ¼ 8.37 Hz), 7.53
(dt, 1H, J1 ¼ 7.79 Hz, J2 ¼ 1.10 Hz, Harom.); 13C NMR (75 MHz, DMSO-
J ¼ 7.74 Hz, Harom.), 7.72 (d, 1H, J ¼ 7.92 Hz, Harom.), 7.71 (d, 2H,
J ¼ 8.98 Hz, Harom.), 7.69 (s, 1H, Harom.), 7.54 (d, 2H, J ¼ 8.88 Hz,
Harom.), 7.48 (dt, 1H, J1 ¼ 8.34 Hz, J2 ¼ 1.24 Hz, Harom.), 7.35 (dt, 1H,
J1 ¼ 8.28 Hz, J2 ¼ 1.22 Hz, Harom.), 2.03 (s, 3H, COCH3); 13C NMR
d6):
d
¼ 157.4 (s), 154.7 (s), 154.2 (s), 152.0 (d), 147.3 (s), 142.0 (d),
127.7 (d), 126.8 (s), 123.9 (d), 123.2 (d), 117.1 (s), 114.0 (d), 112.2 (d),
112.0 (d), 104.1 (s); UV (EtOH) lmax/nm ¼ 321; elemental analysis
calcd. (%) for C15H9N3O2: C 68.44, H 3.45, N 15.96; found C 68.69, H
3.56, N 15.72.
(75 MHz, DMSO-d6):
d
¼ 168.3 (s), 157.2 (s), 154.9 (s), 149.0 (s), 147.9
(s), 130.6 (s), 127.8 (d), 127.6 (s), 124.4 (d), 123.5 (d), 122.0 (d), 121.9
(d, 2C), 112.4 (d, 2C), 111.5 (d), 23.8 (q); UV (EtOH) lmax/nm ¼ 322;
elemental analysis calcd. (%) for C17H14N2O3: C 69.38, H 4.79, N
9.52; found C 69.12, H 4.96, N 9.76.
4.1.2.6. N-(5-Nitropyridin-2-yl)benzofuran-2-carboxamide
3g.
Compound 3g was prepared using above described method, from
benzofurane-2-carbonyl chloride 1 (0.20 g, 1.10 mmol) and 2-
amino-5-nitropyridine 2g (0.15 g, 1.10 mmol) in dry toluene
(20 mL) followed by the addition of Et3N (0.21 mL, 1.54 mmol). The
4.1.2.4. N-(4-Nitrophenyl)benzofuran-2-carboxamide
Compound 3e was prepared using above described method, from
benzofurane-2-carbonyl chloride 1 (0.20 g, 1.10 mmol) and 4-
3e.

M. Hranjec et al. / European Journal of Medicinal Chemistry 59 (2013) 111e119
117
mixture was refluxed for 20 h and worked up as it is described to
give 0.16 g (52%) of light grey crystals; mp 203e204 ꢀC; 1H NMR
(d), 127.6 (s), 127.2 (d), 124.1 (d), 123.2 (d), 112.2 (d), 109.8 (d), 107.8
(d), 106.0 (s), 45.6 (t, 2C); UV (EtOH) lmax/nm ¼ 322; elemental
analysis calcd. (%) for C17H15N4O2Cl: C 59.57, H 4.41, N 16.34; found
C 59.68, H 4.60, N 16.62.
(600 MHz, DMSO-d6):
d
¼ 11.72 (s, 1H, NHamide), 9.24 (d, 1H,
J ¼ 2.58 Hz, Harom.), 8.68 (dd, 1H, J1 ¼ 9.08 Hz, J2 ¼ 2.82 Hz, Harom.),
8.41 (d, 1H, J ¼ 0.72 Hz, Harom.), 8.16 (d, 1H, J ¼ 0.98 Hz, Harom.), 7.84
(d, 1H, J ¼ 7.68 Hz, Harom.), 7.74 (dd, 1H, J1 ¼ 8.40 Hz, J2 ¼ 0.86 Hz,
Harom.), 7.52 (dt, 1H, J1 ¼ 7.44 Hz, J2 ¼ 1.20 Hz, Harom.), 7.38 (dd, 1H,
J1 ¼ 7.56 Hz, J2 ¼ 1.12 Hz, Harom.); 13C NMR (75 MHz, DMSO-d6):
4.1.2.10. N-(6-Cyanobenzothiazol-2-yl)benzofuran-2-carboxamide
6a. Compound 6a was prepared using above described method,
from benzofurane-2-carbonyl chloride 1 (0.30 g, 1.67 mmol) and 2-
amino-6-cyanobenzothiazole 4a (0.29 g, 1.67 mmol) in dry toluene
(20 mL) followed by the addition of Et3N (0.32 mL, 2.32 mmol). The
mixture was refluxed for 18 h and worked up as it is described to
give 0.40 g (75%) of light white powder; mp >290 ꢀC; 1H NMR
d
¼ 157.8 (s), 154.6 (s), 149.1 (d), 146.7 (s), 143.1 (d), 128.6 (d), 127.1
(s), 123.5 (d), 123.0 (d), 116.3 (s), 115.2 (d), 113.4 (d), 111.7 (d), 103.7
(s); UV (EtOH) lmax/nm ¼ 320; elemental analysis calcd. (%) for
C14H9N3O4: C 59.37, H 3.20, N 14.84; found C 59.60, H 3.03, N 14.70.
(600 MHz, DMSO-d6):
d
¼ 13.48 (s, 1H, NHamide), 8.59 (s, 1H, Harom.),
4.1.2.7. N-(2-Acetamidopyridin-3-yl)benzofuran-2-carboxamide 3h.
Compound 3h was prepared using above described method, from
benzofurane-2-carbonyl chloride 1 (0.20 g, 1.10 mmol) and 2-
acetamido-5-aminopyridine 2h (0.17 g, 1.10 mmol) in dry toluene
(20 mL) followed by the addition of Et3N (0.21 mL, 1.54 mmol). The
mixture was refluxed for 24 h and worked up as it is described to
give 0.22 g (68%) of white crystals; mp 222e224 ꢀC; 1H NMR
8.16 (s, 1H, Harom.), 7.90 (d, 1H, J ¼ 7.56 Hz, Harom.), 7.87e7.83 (m, 2H,
Harom.), 7.74 (d, 1H, J ¼ 8.40 Hz, Harom.), 7.54 (t, 1H, J ¼ 7.98 Hz,
Harom.), 7.38 (t, 1H, J ¼ 7.78 Hz, Harom.); 13C NMR (75 MHz, DMSO-
d6):
d
¼ 164.7 (s), 157.2 (s), 155.0 (s), 148.7 (s), 135.5 (s), 129.7 (d),
129.0 (s), 128.0 (d), 127.0 (d), 126.7 (s), 124.1 (d), 123.5 (d, 2C), 119.1
(s), 113.2 (d), 112.1 (d), 105.5 (s); UV (EtOH) lmax/nm ¼ 327;
elemental analysis calcd. (%) for C17H9N3O2S: C 63.94, H 2.84, N
13.16; found C 63.82, H 3.03, N 13.30.
(600 MHz, DMSO-d6):
d
¼ 10.64 (s, 1H, NHamide), 10.43 (s, 1H, NH),
8.71 (d, 1H, J ¼ 2.58 Hz, Harom.), 8.11 (dd, 1H, J1 ¼ 8.94 Hz,
J2 ¼ 2.58 Hz, Harom.), 8.05 (d, 1H, J ¼ 8.94 Hz, Harom.), 7.81 (d, 1H,
J ¼ 7.86 Hz, Harom.), 7.74 (s, 1H, Harom.), 7.69 (d, 1H, J ¼ 8.64 Hz,
Harom.), 7.49 (dt, 1H, J1 ¼ 8.66 Hz, J2 ¼ 1.44 Hz, Harom.), 7.35 (t, 1H,
J ¼ 8.26 Hz, Harom.), 2.07 (s, 1H, COCH3); 13C NMR (75 MHz, DMSO-
4.1.2.11. N-(6-Cyanobenzimidazol-2-yl)benzofuran-2-carboxamide
6b. Compound 6b was prepared using above described method,
from benzofurane-2-carbonyl chloride 1 (0.30 g, 1.67 mmol) and 2-
amino-5(6)-cyanobenzimidazole 4b (0.26 g, 1.67 mmol) in dry
toluene (15 mL) followed by the addition of Et3N (0.32 mL,
2.32 mmol). The mixture was refluxed for 16 h and worked up as it
is described to give 0.37 g (76%) of yellow powder; mp 286e287 ꢀC;
d6):
d
¼ 169.4 (s), 157.2 (s), 154.9 (s), 148.9 (s), 148.6 (s), 140.5 (d),
131.3 (s), 130.8 (d), 127.7 (d), 127.5 (s), 124.3 (d), 123.5 (d), 113.6 (d),
112.4 (d), 111.4 (d), 24.2 (q); UV (EtOH) lmax/nm ¼ 316; elemental
analysis calcd. (%) for C16H13N3O3: C 65.08, H 4.44, N 14.23; found C
65.24, H 4.59, N 14.54.
1H NMR (600 MHz, DMSO-d6):
d
¼ 12.72 (brs, 2H, NHamide, NHben-
zimidazole), 8.02 (s, 1H, Harom.), 7.92 (s, 1H, Harom.), 7.84 (d, 1H,
J ¼ 7.86 Hz, Harom.), 7.74 (d, 1H, J ¼ 8.34 Hz, Harom.), 7.62 (d, 1H,
J ¼ 8.10 Hz, Harom.), 7.55 (dd, 1H, J1 ¼ 8.28 Hz, J2 ¼ 1.02 Hz, Harom.),
7.51 (t, 1H, J ¼ 7.92 Hz, Harom.), 7.36 (t, 1H, J ¼ 7.56 Hz, Harom.); 13C
4.1.2.8. N-[4-(2-Imidazolynyl)phenyl]benzofuran-2-carboxamide
hydrochloride 3i. Compound 3i was prepared using above
described method, from benzofurane-2-carbonyl chloride 1 (0.08 g,
0.41 mmol) and 4-(2-imidazolynyl)aniline hydrochloride 2i (0.08 g,
0.41 mmol) in dry toluene (10 mL) followed by the addition of Et3N
(0.08 mL, 0.57 mmol). The mixture was refluxed for 18 h and
worked up as it is described to give 0.11 g (78%) of white powder;
NMR (75 MHz, DMSO-d6):
134.7 (s), 132.3 (s), 128.0 (d), 127.4 (s), 126.0 (d), 124.4 (d), 123.7 (d,
2C), 120.5 (s), 112.9 (d), 112.6 (d, 2C), 103.8 (s); UV (EtOH) lmax
d
¼ 164.4 (s), 155.3 (s), 150.4 (s), 148.6 (s),
/
nm ¼ 329; elemental analysis calcd. (%) for C17H10N4O2: C 67.55, H
3.33, N 18.53; found C 67.71, H 3.14, N 18.69.
mp >290 ꢀC; 1H NMR (600 MHz, DMSO-d6):
d
¼ 10.60 (s, 1H,
NHamide), 9.94 (s, 2H, NHimidazolynyl), 8.11 (d, 2H, J ¼ 8.94 Hz, Harom.),
8.02 (d, 2H, J ¼ 8.88 Hz, Harom.), 7.95 (s, 1H, Harom.), 7.74 (d, 1H,
J ¼ 8.28 Hz, Harom.), 7.53 (d, 1H, J ¼ 8.18 Hz, Harom.), 7.37 (t, 2H,
J ¼ 8.16 Hz, Harom.), 7.23 (t, 2H, J ¼ 8.18 Hz, Harom.), 3.98 (s, 4H, CH2);
4.1.2.12. N-(6-Nitrobenzothiazol-2-yl)benzofuran-2-carboxamide 6c.
Compound 6c was prepared using above described method, from
benzofurane-2-carbonyl chloride 1 (0.30 g, 1.67 mmol) and 2-
amino-6-nitrobenzothiazole 4c (0.32 g, 1.67 mmol) in dry toluene
(20 mL) followed by the addition of Et3N (0.32 mL, 2.32 mmol). The
mixture was refluxed for 12 h and worked up as it is described to
give 0.34 g (61%) of beige powder; mp >290 ꢀC; 1H NMR (600 MHz,
13C NMR (75 MHz, DMSO-d6):
d
¼ 164.2 (s), 157.2 (s), 154.8 (s), 148.0
(s), 130.7 (d, 2C), 129.8 (d), 127.5 (d), 127.0 (s), 123.1 (d), 119.9 (d),
116.8 (s), 112.8 (d, 2C), 111.8 (d), 107.0 (s), 45.2 (t, 2C); UV (EtOH)
lmax/nm ¼ 320; elemental analysis calcd. (%) for C18H16N3O2Cl: C
63.25, H 4.72, N 12.29; found C 63.47, H 4.50, N 12.49.
DMSO-d6):
d
¼ 13.63 (s, 1H, NHamide), 9.11 (s, 1H, Harom.), 8.31 (dd,
1H, J1 ¼ 8.88 Hz, J2 ¼ 1.20 Hz, Harom.), 8.24 (s, 1H, Harom.), 7.97 (d, 1H,
J ¼ 8.76 Hz, Harom.), 7.89 (d, 1H, J ¼ 7.88 Hz, Harom.), 7.76 (d, 1H,
J ¼ 8.46 Hz, Harom.), 7.55 (t, 1H, J ¼ 7.76 Hz, Harom.), 7.39 (t, 1H,
4.1.2.9. N-[5-(2-Imidazolynyl)pyridin-2-yl]benzofuran-2-
carboxamide hydrochloride 3j. Compound 3j was prepared using
above described method, from benzofurane-2-carbonyl chloride 1
(0.09 g, 0.50 mmol) and 2-amino-5-(2-imidazolynyl)pyridine
hydrochloride 2j (0.10 g, 0.50 mmol) in dry toluene (15 mL) fol-
lowed by the addition of Et3N (0.10 mL, 0.70 mmol). The mixture
was refluxed for 22 h and worked up as it is described to give 0.12 g
(70%) of white powder; mp >290 ꢀC; 1H NMR (600 MHz, DMSO-d6):
J ¼ 7.56 Hz, Harom.); 13C NMR (150 MHz, DMSO-d6):
d
¼ 164.2 (s),
155.6 (s), 154.7 (s), 153.8 (s), 138.4 (s), 135.8 (s), 132.9 (s), 128.6 (d),
128.1 (d), 127.2 (s), 124.6 (d), 124.0 (d), 122.4 (d), 119.6 (d), 113.8 (d),
112.62 (d); UV (EtOH) lmax/nm ¼ 337; elemental analysis calcd. (%)
for C16H9N3O4S: C 56.63, H 2.67, N 12.38; found C 56.81, H 2.85, N
12.53.
d
¼ 9.94 (brs, 2H, NHimidazolynyl), 8.84 (s, 1H, NHamide), 8.61 (d, 1H,
4.1.2.13. N-(6-Nitrobenzimidazol-2-yl)benzofuran-2-carboxamide
6d. Compound 6d was prepared using above described method,
from benzofurane-2-carbonyl chloride 1 (0.30 g, 1.67 mmol) and 2-
amino-5(6)-nitrobenzimidazole 4d (0.30 g, 1.67 mmol) in dry
toluene (20 mL) followed by the addition of Et3N (0.32 mL,
2.32 mmol). The mixture was refluxed for 18 h and worked up as it
is described to give 0.36 g (68%) of light yellow powder; mp
J ¼ 2.46 Hz, Harom.), 7.94 (dd, 2H, J1 ¼ 8.88 Hz, J2 ¼ 2.58 Hz, Harom.),
7.74 (d, 1H, J ¼ 7.74 Hz, Harom.), 7.62 (dd, 1H, J1 ¼ 8.34 Hz,
J2 ¼ 0.98 Hz, Harom.), 7.53 (d, 1H, J ¼ 1.12 Hz, Harom.), 7.43 (dt, 1H,
J1 ¼ 8.46 Hz, J2 ¼ 1.32 Hz, Harom.), 7.31 (dt, 1H, J1 ¼ 7.92 Hz,
J2 ¼ 0.98 Hz, Harom.), 3.87 (s, 4H, CH2); 13C NMR (75 MHz, DMSO-d6):
d
¼ 163.7 (s), 163.5 (s), 158.8 (s), 154.6 (s), 151.5 (d), 149.6 (s), 137.0

118
M. Hranjec et al. / European Journal of Medicinal Chemistry 59 (2013) 111e119
>290 ꢀC; 1H NMR (600 MHz, DMSO-d6):
d
¼ 12.72 (brs, 2H, NHamide
,
nm ¼ 330, 273; elemental analysis calcd. (%) for C17H17N2O2Cl: C
NHbenzimidazole), 8.02 (s, 1H, Harom.), 7.92 (s, 1H, Harom.), 7.84 (d, 1H,
J ¼ 7.86 Hz, Harom.), 7.74 (d, 1H, J ¼ 8.34 Hz, Harom.), 7.62 (d, 1H,
J ¼ 8.10 Hz, Harom.), 7.55 (dd, 1H, J1 ¼ 8.28 Hz, J2 ¼ 1.02 Hz, Harom.),
7.51 (t, 1H, J ¼ 7.92 Hz, Harom.), 7.36 (t, 1H, J ¼ 7.56 Hz, Harom.); 13C
64.46, H 5.41, N 8.84; found C 64.59, H 5.27, N 8.54.
4.2. Cell culturing and cell morphology
NMR (75 MHz, DMSO-d6):
d
¼ 163.4 (s), 154.9 (s), 154.7 (s), 142.1 (s),
The cell lines HeLa (cervical carcinoma), SW620 (colorectal
adenocarcinoma, metastatic), MiaPaCa-2 (pancreatic carcinoma),
MCF-7 (breast epithelial adenocarcinoma, metastatic), SK-BR-3
(breast adenocarcinoma, metastatic) and WI38 (normal diploid
human fibroblasts) were cultured as monolayers and maintained in
Dulbecco’s modified Eagle medium (DMEM) supplemented with
133.4 (s), 130.7 (s), 127.7 (d), 126.9 (s), 123.9 (d), 123.2 (d, 2C), 117.7
(d), 112.3 (d), 112.0 (d, 2C), 104.7 (s); UV (EtOH) lmax/nm ¼ 335;
elemental analysis calcd. (%) for C16H10N4O4: C 59.63, H 3.13, N
17.38; found C 59.79, H 3.28, N 17.60.
10% foetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml peni-
4.1.2.14. N-[6-(2-Imidazolynyl)benzothiazol-2-yl]benzofuran-2-
carboxamide hydrochloride 6e. Compound 6e was prepared using
above described method, from benzofurane-2-carbonyl chloride 1
(0.06 g, 0.33 mmol) and 2-amino-6-(2-imidazolynyl)benzothiazole
hydrochloride 5a (0.09 g, 0.33 mmol) in dry toluene (15 mL) fol-
lowed by the addition of Et3N (0.07 mL, 0.46 mmol). The mixture
was refluxed for 18 h and worked up as it is described to give 0.09 g
(69%) of white powder; mp >290 ꢀC; 1H NMR (600 MHz, DMSO-
cillin and 100 mg/mL streptomycin in a humidified atmosphere with
5% CO2 at 37 ꢀC. The evaluation of cell morphology was performed
under the light invert microscope Olympus (magnification 200ꢂ).
4.3. Antitumour activity assays
The panel of monolayer tumour cell lines was inoculated into
standard 96-well microtiter plates on day 0, at 3000e6000 cells per
well depending on the doubling time of the specific cell line. Test
compounds were then added in five 10-fold dilutions (0.01e
d6):
d
¼ 12.50 (s, 1H, NHamide), 10.23 (s, 2H, NHimidazolynyl), 8.37 (d,
1H, J ¼ 1.56 Hz, Harom.), 7.85 (dd, 1H, J1 ¼ 8.52 Hz, J2 ¼ 1.74 Hz,
Harom.), 7.75 (d, 1H, J ¼ 7.86 Hz, Harom.), 7.63 (d, 1H, J ¼ 8.16 Hz,
Harom.), 7.55 (s, 1H, Harom.), 7.48 (d, 1H, J ¼ 8.52 Hz, Harom.), 7.44 (dt,
1H, J1 ¼ 8.34 Hz, J2 ¼ 1.44 Hz, Harom.), 7.32 (t, 1H, J ¼ 7.56 Hz, Harom.),
100 mM) followed by 72-h incubation. Cell viability was quantita-
tively determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) colorimetric assay. Experi-
mentally obtained absorbances were transformed into cell
percentage growth (PG) using the formulae proposed by NIH and
described previously. The IC50 values for each compound were
calculated from concentrationeresponse curves using linear
regression analysis by fitting the test concentrations that give PG
values above and below the reference value. If, however, all of the
tested concentrations produce PGs exceeding the respective refer-
ence level of effect (e.g. PG value of 50) for a given cell line, the
highest tested concentration is assigned as the default value (in the
screening data report, that default value is preceded by a “>” sign).
Each test point was performed in quadruplicate in three individual
experiments. The results were statistically analyzed (ANOVA, Tukey
post-hoc test at p < 0.05). Finally, the effects of the tested
substances were evaluated by plotting the mean percentage growth
for each cell type in comparison to control on doseeresponse
graphs.
4.05 (s, 4H, CH2); 13C NMR (150 MHz, DMSO-d6):
d
¼ 170.7 (s), 164.9
(s), 158.8 (s), 158.2 (s), 154.6 (s), 149.6 (s), 132.0 (s), 127.6 (s), 127.3
(d), 127.1 (d), 124.1 (d), 123.2 (d), 122.4 (d), 117.8 (d), 114.1 (s), 112.2
(d), 109.8 (d), 46.6 (t, 2C); UV (EtOH) lmax/nm ¼ 324; elemental
analysis calcd. (%) for C19H15N4O2ClS: C 57.21, H 3.79, N 14.05; found
C 57.47, H 3.95, N 14.30.
4.1.2.15. N-[6-(2-Imidazolynyl)benimidazol-2-yl]benzofuran-2-
carboxamide hydrochloride 6f. Compound 6f was prepared using
above described method, from benzofurane-2-carbonyl chloride 1
(0.10 g, 0.55 mmol) and 2-amino-5(6)-(2-imidazolynyl)benzimid-
azole hydrochloride 5b (0.13 g, 0.55 mmol) in dry toluene (20 mL)
followed by the addition of Et3N (0.11 mL, 0.77 mmol). The mixture
was refluxed for 18 h and worked up as it is described to give 0.18 g
(86%) of white powder; mp >290 ꢀC; 1H NMR (600 MHz, DMSO-
d6):
d
¼ 13.37 (s, 1H, NHbenzimidazole), 11.48 (s, 1H, NHamide), 10.38
(brs, 2H, NHimidazolynyl), 8.93 (s, 1H, Harom.), 8.06 (d, 1H, J ¼ 1.32 Hz,
Harom.), 7.89 (dd, 1H, J1 ¼ 8.46 Hz, J2 ¼ 1.76 Hz, Harom.), 7.75 (d, 1H,
J ¼ 7.86 Hz, Harom.), 7.63 (d, 1H, J ¼ 8.68 Hz, Harom.), 7.56 (d, 1H,
J ¼ 8.46 Hz, Harom.), 7.44 (t, 1H, J ¼ 8.48 Hz, Harom.), 7.32 (t, 1H,
J ¼ 7.74 Hz, Harom.); 4.12 (s, 4H, CH2); 13C NMR (150 MHz, DMSO-d6):
4.4. Cell cycle analysis
A total of 5 ꢂ 105 cells were seeded per petridish. After 24 h SK-
BR-3 cells were treated with the compound 6f while SW620 cells
were treated with compound 3i and 3h. The concentration of tested
compounds was 5 mM and 10 mM. After 24 h and 48 h the attached
cells were trypsinized, combined with floating cells, washed with
PBS and fixed with 70% ethanol. Immediately before the analysis,
d
¼ 164.6 (s), 158.4 (s), 154.1 (s), 152.0 (s), 149.1 (s), 134.4 (s), 130.0
(s), 127.1 (s), 126.7 (d), 124.2 (d), 123.6 (d), 122.7 (d), 116.4 (s), 111.8
(d), 111.6 (d, 2C), 109.3 (d), 45.2 (t, 2C); UV (EtOH) lmax/nm ¼ 323;
elemental analysis calcd. (%) for C19H16N5O2Cl: C 59.77, H 4.22, N
18.34; found C 59.50, H 4.17, N 18.60.
the cells were washed again with PBS and stained with 1
mg/mL of
propidium iodide (PI) with the addition of 0.2 g/mL of RNAse A.
m
4.1.3. Preparation of N-(4-N,N-dimethylaminophenyl)benzofuran-
2-carboxamide hydrochloride 3c
A stirred suspension of compound 3b (0.10 g, 0.36 mmol) in
absolute ethanol (10 mL) was saturated with HCl(g). After 24 h of
stirring small amount of diethylether was added, resulting product
was filtered off and washed with diethylether to give 0.09 g (79%) of
The stained cells were then analyzed with Becton Dickinson
FACScalibur flow cytometer (10,000 counts were measured). Each
test point was performed in duplicate. The percentage of the cells in
each cell cycle phase was based on the obtained DNA histograms
and determined using the WinMDI 2.9 and Cylchred software.
Statistical analysis was performed in Microsoft Excel by using the
ANOVA at p < 0.05.
red powder; mp >290 ꢀC; 1H NMR (600 MHz, DMSO-d6):
d
¼ 10.72
(s, 1H, NHamide), 7.88 (br s, 1H, NHþ), 7.82 (d, 2H, J ¼ 7.74 Hz, Harom.),
7.80 (s, 1H, Harom.), 7.71 (d, 2H, J ¼ 8.28 Hz, Harom.), 7.50 (t, 2H,
J ¼ 7.98 Hz, Harom.), 7.36 (t, 2H, J ¼ 7.84 Hz, Harom.), 3.06 (s, 6H, CH3);
4.5. Caspase colorimetric protease assay
13C NMR (150 MHz, DMSO-d6):
d
¼ 157.1 (s), 155.3 (s), 150.2 (s),
To determent activation status of caspase 3, 8 and 9 the Apo-
Target caspase colorimetric protease assay was used. In compli-
ances with the protocol, 6 ꢂ 106 cells were seated in petridish.
14,910 (s), 129.0 (s), 128.3 (d), 128.1 (s), 124.7 (d), 123.6 (d), 123.0 (d,
2C), 113.4 (d, 2C), 112.8 (d), 110.7 (d), 41.5 (q, 2C); UV (EtOH) lmax
/

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m
m
m
a 96-well plate 50
m
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Acknowledgements
This work was supported by the Croatian Ministry of Science,
Education and Sports (grant numbers: 125-0982464-1356, 098-
1191344-2943, 335-0982464-2393, 335-0000000-3532).
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Appendix A. Supplementary material
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evaluation of novel benzazoyl-benzamides, Heterocycles 68 (2006) 2285e
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