Synthesis, spectroscopic characterization and antiproliferative evaluation in vitro of novel Schiff bases related to benzimidazoles

Article abstract of DOI:10.1016/j.ejmech.2011.03.008

A series of novel benzimidazole substituted Schiff bases were synthesized by reaction of aromatic aldehydes with corresponding 2-aminobenzimidazoles. Their structure has been studied by ¹H and ¹³C NMR, IR and UV/Vis spectroscopy. Maj

Full text of DOI:10.1016/j.ejmech.2011.03.008

European Journal of Medicinal Chemistry 46 (2011) 2274e2279
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, spectroscopic characterization and antiproliferative evaluation in vitro
of novel Schiff bases related to benzimidazoles
,
c
Marijana Hranjec a , Kristina Starcevic , Sandra Kraljevic Pavelic , Pero Lucin , Kresimir Pavelic ,
ꢁa
ꢁ
ꢁb
ꢀ
ꢀ
ꢁb
*
ꢀ
Grace Karminski Zamola a
Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 20, P.O. Box 177, HR-10000 Zagreb, Croatia
Department of Biotechnology, University of Rijeka, Trg brace Mazuranica 10, HR-51000 Rijeka, Croatia
School of Medicine, Univesrity of Rijeka, Brace Branchetta 20, HR-51000 Rijeka, Croatia
,
**
a
ꢁ
b
ꢁ
ꢀ
ꢁ
c
ꢁ
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel benzimidazole substituted Schiff bases were synthesized by reaction of aromatic
aldehydes with corresponding 2-aminobenzimidazoles. Their structure has been studied by 1H and 13C
NMR, IR and UV/Vis spectroscopy.
Majority of prepared Schiff bases were tested on their antiproliferative activity in vitro and exerted
non-specific antiproliferative activity on the tested cell lines at the highest tested concentration.
Compounds 18 and 19 exerted the strongest non-specific antiproliferative effect on all cell lines and
a concentration-dependent effect on HeLa and MCF-7 cell lines at micromolar concentrations but
simultaneously being highly cytotoxic on human fibroblasts as well.
Received 5 November 2010
Received in revised form
1 March 2011
Accepted 1 March 2011
Available online 10 March 2011
Keywords:
Benzimidazoles
Schiff bases
Ó 2011 Elsevier Masson SAS. All rights reserved.
Antiproliferaive evaluation
UV/Vis and fluorescence spectroscopy
1. Introduction
addition, Schiff bases constitute one of the most relevant synthetic
ligand systems with importance in asymmetric catalysis [14]. The
Schiff bases represent an important class of organic compounds,
especially in the medicinal and pharmaceutical field. Thus, devel-
opment and synthesis of novel Schiff base derivatives as potential
chemotherapeutics still attracts attention of organic and medicinal
chemist [1]. Schiff bases, derived mostly from variety of heterocyclic
rings, were reported to posses a broad spectrum and a wide variety
of biological activities including antiviral [2], anticancer [3], cyto-
toxic [4], antimicrobial [5], antibacterial [6,7], anticonvulsant [8] etc.
Besides their potential use as biologically active agents, Schiff bases
and their metal complexes have been often used as chelating ligands
in the coordination chemistry of transition metals as radiophar-
maceuticals for cancer targeting and agrochemicals. They constitute
an interesting class of chelating agents capable of coordination with
metal ions which serve as models for biological systems [9e11]. The
synthesis of chemosensors for recognition and sensing of anions is
of considerable significance while anions play an important role in
a wide range of chemical and biological processes [12,13]. In
phenyl derivatives of Schiff bases are used as corrosion inhibitors
[15,16]. New optical and organic conducting materials and polymers
can be produced by these compounds [17,18].
Besides, benzimidazoles, being among most important key
building blocks for a variety of biologically important molecules,
have received considerable attention in recent years as well. The
facts that benzimidazole nuclei is very important pharmacophore
in modern drug discovery as well as in medicinal chemistry and
that benzimidazole residue is a constituent of vitamin B12, support
their potential use as therapeutics. Therefore, benzimidazoles
display diverse biological activities such as antimicroabial [19,20],
antifungal [21], antitumor [22e24], antiviral [25,26], antihistaminic
[27] etc. Benzimidazole and its derivatives have additionally found
applications in several other areas as optical laser and polymer dyes
in optoelectronics [28,29], organic luminophores, fluorescent tags
for detection of biological important molecules, as DNA, RNA or
proteins and enzymes [24,30] etc.
Above mentioned considerations prompted us to explore a new
series of benzimidazole substituted Schiff bases 8e21 with different
aromatic rings as well as different substituents on benzimidazole
nuclei. Complete report on the synthesis, spectroscopic character-
ization and antiproliferative activity in vitro evaluation are reported
herein.
* Corresponding author. Tel.: þ385 14597245; fax: þ385 14597250.
** Corresponding author. Tel.: þ385 14597215; fax: þ385 14597250.
E-mail addresses: mhranjec@fkit.hr (M. Hranjec), gzamola@fkit.hr (G. Karminski
Zamola).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.008

M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2274e2279
2275
2. Results and discussion
2.3. Antiproliferative evaluation
2.1. Chemistry
We examined the effect of fourteen Schiff bases (8e21) on cell
proliferation of human tumor cell lines and their cytotoxic activity
on normal human fibroblasts (WI38) using the MTT assay. The in
vitro screening revealed that majority of compounds exerted non-
specific antiproliferative activity on the tested panel cell lines at the
highest tested concentration (Table 1). Among them, compounds 18
and 19 showed the strongest antiproliferative effect and a strong
concentration-dependent effect on HeLa and MCF-7 cell lines at
micromolar concentrations with IC50’s ranging between 3.24 and
15.27 (Table 1). However, the highest tested concentration for
majority of tested compounds proved to be cytotoxic on control
human fibroblasts as well (WI 38) (Table 1). Lack of antiproliferative
activity on all tested cells was observed for compounds 10 and 11.
Cellular mechanisms that switch between survival and death
were further investigated for compound 18 by flow cytometry. Cell
cycle perturbations were determined for concentrations adjusted
2-aminobenzimidazoles 4e6 and Schiff bases 8e21 were
prepared according to the procedures shown in the Schemes 1 and
2. Cyano-, methyl- and nitro-substituted 2-aminobenzimidazoles
4e6 were prepared by earlier described method, from corre-
sponding o-phenylenediamines and cyanogen-bromide [31,32].
Above mentioned 2-aminobenzimidazoles 4e6, as well as
commercially available 2-aminobenzimidazole 7, were used for the
synthesis of series of novel benzimidazole substituted Schiff bases
8e21 according to the Scheme 2.
Schiff bases were prepared from 2-aminobenzimidazoles and
corresponding aromatic aldehydes in absolute ethanol for 12e24 h
at reflux. The reactions of pyridine-4-carboxaldehyde and N-
methylpyrrol-2-carboxaldehyde gave desired Schiff bases 8e9
only in the case of using 2-aminobenzimidazole 7, while reactions
with 5(6)-substituted-2-aminobenzimidazoles 4e6 failed, even
with the prolonged reaction times. The reactions of furan-2-car-
boxaldehyde and thiophene-2-carboxaldehyde gave desired Schiff
bases 10e14 in the case of using 2-amino-5(6)-cyanobenzimida-
zole 4, 2-amino-5(6)-methyl benzimidazole 5 and 2-amino-5(6)-
nitrobenzimidazole 6 only with thiophene-2-carboxaldehyde,
while reactions with 2-aminobenzimidazole 7 failed. Reaction of
4-N,N-dimethylamino-benzaldehyde gave Schiff bases 15e16 with
5(6)-substituted-2-aminobenzimidazoles 4e6 while reaction of 4-
N,N-diethylamino-2-hydroxybenzaldehyde gave Schiff bases
17e21 with all 2-aminobenzimidazoles 4e7. In order to get the
better yields of some of the prepared Schiff bases, the reactions
were conducted under the prolonged time of reflux, up to 3e4
days, but the yields did not change significantly. Some of the
reactions which did not give the desired Schiff bases were also
performed using microwave irradiation in absolute ethanol or
without solvent but these reactions also failed [33].
to IC50 values (10 and 5 mM) after 24 and 48-h treatments of HeLa
and MCF-7 cells. Interestingly, no significant changes were
observed (data not shown) and changes of cell morphology that
points to apoptosis (shrinkage of cells, chromatin condensation)
were observed only at cytotoxic concentrations between 100 and
10 mM (data not show).
3. Conclusions
In this work, we have presented the synthesis of novel benz-
imidazole substituted Schiff bases, their spectroscopic character-
ization by means of UV/Vis and fluorescence spectroscopy as well
as antiproliferative activity in vitro.
Novel benzimidazole substituted Schiff bases 8e21 were
prepared from corresponding 2-aminobenzimidazoles, prepared by
earlier described method from o-phenylenediamines and cyanogen-
bromide. 2-aminobenzimidazoles gave in the reactionwith different
aromatic aldehydes desired Schiff bases. All prepared compounds
showed interesting spectroscopic properties. Some of prepared
compounds, 15, 17, 18 and 20 were spectroscopically characterized
by means of UV/Vis and fluorescence emission spectroscopy in
several solvents in order to estimate probability for their possible
application in optoelectronics as optical dyes or anions sensors.
In the present study, we evaluated the ability of fourteen Schiff
bases (8e21) to suppress proliferation of different human cancer
cell lines along with normal human fibroblasts. Majority of the
compounds exerted a non-specific antiproliferative activity on the
tested panel cell lines at the highest tested concentration. Among
them, compounds 18 and 19 showed the strongest antiproliferative
effect and a strong concentration-dependent effect on HeLa and
MCF-7 cell lines at micromolar concentrations but simultaneously
being highly cytotoxic on human fibroblasts as well. Further
structural optimization studies might thus represent a rationale for
further investigation.
2.2. Spectroscopy
The structure of all synthesized Schiff bases 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 protons
of C]NH group was confirmed by one-proton singlet at
9.62e9.15 ppm, while singlets for imidazole protons of NH group
can be observed at 13.22e11.84 ppm.
In order to study the spectroscopic properties of some prepared
benzimidazole Schiff bases a preliminary investigation of their UV/
Vis and fluorescence emission spectra, recorded in several solvents
including ethanol, methanol, acetonitrile and acetone, was under-
taken. Prepared Schiff bases posses interesting optical properties
and thus, have a possible application in optoelectronics or as anions
sensors. UV/Vis and fluorescence emission spectra are presented in
Supplementary Material.
4. Experimental
4.1. Chemistry
H
R
H2N
H2N
R
N
4.1.1. General methods
BrCN
H2N
All chemicals were purchased from commercial suppliers.
Melting points were recorded on Büchi 535 melting apparatus. The
1H and 13C NMR spectra were recorded on a Varian Gemini 300 or
Varian Gemini 600 at 300, 600 and 150 and 75 MHz, respectively.
All NMR spectra were measured in DMSO-d6 solutions using TMS as
an internal standard. All compounds were routinely checked by TLC
with Merck silica gel 60F-254 glass plates and the spots were
MeOH
CH3CN
N
1
2
3
R=CN
R=CH3
R=NO2
4 R=CN
5
R=CH3
R=NO2
6
Scheme 1. Synthesis of 2-aminobenzimidazoles 4e6.

2276
M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2274e2279
Ar
N
N
EtOHabs.
N
+
NH2
Ar CHO
N
H
N
H
R
R
4
5
6
7
R=CN
R=CH3
R=NO2
R=H
15 R=CN, Ar=
16 R=CH3, Ar=
17 R=NO2, Ar=
8
R=H, Ar=
R=H, Ar=
N
N(Me)2
9
N
HO
18 R=CN, Ar=
19 R=CH3, Ar=
20 R=NO2, Ar=
21 R=H, Ar=
CH3
N(Et)2
10 R=CN, Ar=
11 R=CH3, Ar=
O
12 R=CN, Ar=
13 R=CH3, Ar=
14 R=NO2, Ar=
S
Scheme 2. Synthesis of Schiff bases 8e21 related to benzimidazoles.
detected under UV light. Elemental analysis for carbon, hydrogen
and nitrogen were performed on a PerkineElmer 2400 elemental
analyzer. Where analyses are indicated only as symbols of
elements, analytical results obtained are within 0.4% of the theo-
retical value.
(DMSO-d6, 150 MHz):
d
/ppm ¼ 163.72 (d), 154.69 (s), 150.70 (d, 2C),
142.30 (s), 141.58 (s),134.35 (s),122.65 (d),122.53 (d, 2C),122.14 (d),
118.99 (d), 11.42 (d); Anal. (C13H10N4) Calcd.: C, 70.26; H, 4.54; N,
25.21; Found: C, 70.11; H, 4.66; N, 24.89.
4.1.2.2. 2-(N-methylpyrrol-2-ylideneamino)benzimidazole
9.
4.1.2. General method for preparation of Schiff bases 8e21
Solutions of equimolar amounts of corresponding 5(6)-
substituted-2-aminobenzimidazoles and aromatic aldehydes in
absolute ethanol, were refluxed for 12e24 h. After cooling, the
obtained product was filtered off and recrystallized from ethanol to
obtain yellow powder products.
Compound 9 was prepared from 2-aminobenzimidazole 7 (0.40 g,
3.00 mmol), N-methylpyrrol-2-carboxaldehyde (0.33 g, 3.00 mmol)
in absolute ethanol (10 mL) after 24 h and recrystallization from
ethanol to obtain 0.34 g (51%) of yellow powder. mp 164e166 ꢀC;
UV (EtOH) lmax/nm ¼ 366 nm; IR (diamond):
n
/cmꢁ1 ¼ 2979, 1756,
1649, 1568, 1483; 1H NMR (DMSO-d6, 300 MHz):
d
/ppm ¼ 12.36 (s,
1H, NHbenzimidazole), 9.21 (s, 1H, Harom.), 7.24 (t, 1H, J ¼ 1.98 Hz,
Hpyrrole), 7.12 (d, 1H, J ¼ 8.14 Hz, Harom), 7.08 (d, 1H, J ¼ 8.12 Hz,
Harom), 6.99 (dd, 1H, J1 ¼ 3.93 Hz, J1 ¼ 1.68 Hz, Hpyrrole), 6.86e6.82
(m, 2H, Harom.), 6.24 (dd, 1H, J1 ¼ 3.91 Hz, J1 ¼ 2.49 Hz, Hpyrrole), 4.02
4.1.2.1. 2-(Pyridin-4-ylideneamino)benzimidazole 8. Compound
8
was prepared from 2-aminobenzimidazole 7 (0.40 g, 3.00 mmol),
pyridine-4-carboxaldehyde (0.32 g, 3.00 mmol) in absolute ethanol
(10 mL) after 20 h and recrystallization from ethanol to obtain
(s, 3H, CH3); 13C NMR (DMSO-d6, 150 MHz):
d
/ppm ¼ 156.95 (s),
0.40 g (60%) of yellow powder. mp 209e211 ꢀC; UV (EtOH) lmax
/
155.23 (s), 154.19 (d), 131.92 (d), 129.49 (s), 121.33 (d), 120.61 (d),
119,32 (s), 118.95 (d), 111.49 (d), 109.70 (d), 109.68 (d), 35.99 (q);
Anal. (C13H12N4) Calcd.: C, 69.62; H, 5.39; N, 24.98; Found: C, 69.89;
H, 5.82; N, 24.77.
nm ¼ 361 nm; IR (diamond):
n
/cmꢁ1 ¼ 2971, 2630, 1736, 1598,
1550; 1H NMR (DMSO-d6, 300 MHz):
d
/ppm ¼ 12.87 (s, 1H,
NHbenzimidazole), 9.49 (s, 1H, Harom.), 8.82 (d, 1H, J ¼ 5.85 Hz, Hpyr),
7.96 (d, 1H, J ¼ 5.91 Hz, Hpyr), 7.62 (d, 1H, J ¼ 8.25 Hz, Harom), 7.48
(d, 1H, J ¼ 8.27 Hz, Harom), 7.24e7.19 (m, 2H, Harom.); 13C NMR
4.1.2.3. 2-(Furan-2-ylideneamino)-5(6)-cyanobenzimidazole 10.
Compound 10 was prepared from 2-amino-5(6)-cyanobenzimi-
dazole 5 (0.50 g, 3.12 mmol), furan-2-carboxaldehyde (0.31 g,
3.12 mmol) in absolute ethanol (15 mL) after 18 h and recrys-
tallization from ethanol to obtain 0.46 g (62%) of yellow powder.
mp 239e241 ꢀC; UV (EtOH) lmax/nm ¼ 357 nm; IR (diamond):
Table 1
Comparison of IC50 values of Schiff bases 8e21 between tested tumor cell lines and
normal human fibroblasts WI38.
IC50 (m
M)a
n
/cmꢁ1 ¼ 3068, 2223, 1743, 1610, 1550, 1474; 1H NMR (DMSO-d6,
Substance No.
Cell lines
HeLa
600 MHz):
d
/ppm ¼ 13.11 (s, 1H, NHbenzimidazole), 9.15 (s, 1H,
Harom.), 8.05 (s, 1H, Hfur), 7.98 (d, 1H, J ¼ 8.22 Hz, Harom), 7.84 (d,
MCF-7
SW620
MiaPaCa-2
WI38
0.76
0.08
>100
92.33
1.58
1H, J ¼ 8.20 Hz, Harom), 7.47 (d, 2H, J ¼ 8.21 Hz, Harom), 7.44 (d, 1H,
8
9
>100
>100
>100
>100
>100
84.63
78.57
>100
60.24
>100
4.73
>100
>100
>100
>100
62.87
>100
>100
>100
>100
>100
46.87
64.14
>100
>100
49.15
52.04
72.29
44.97
>100
>100
>100
>100
>100
>100
>100
>100
>100
99.32
27.92
22.24
87.79
67.68
J ¼ 3.24 Hz, Hfur), 6.75 (dd, 1H, J1 ¼ 3.24 Hz, J2 ¼ 1.56 Hz, Hfur); 13
C
10
11
12
13
14
15
16
17
18
19
20
21
NMR (DMSO-d6, 150 MHz):
d
/ppm ¼ 155.67 (s), 153.51 (d), 151.07
(s), 148.98 (d), 142.34 (s), 126.42 (d), 125.44 (d), 122.77 (d),
120.15 (s), 119. 98 (s), 118.95 (d), 113.50 (d); Anal. (C13H8N4O)
Calcd.: C, 66.10; H, 3.41; N, 23.72; Found: C, 66.34; H, 3.70; N,
23.56.
94.84
>100
>100
>100
>100
>100
0.08
29.79
93.41
0.54
0.84
0.96
1.67
30.55
0.91
4.1.2.4. 2-(Furan-2-ylideneamino)-5(6)-methyl benzimidazole 11.
Compound 11 was prepared from 2-amino-5(6)-methyl-
benzimidazole 5 (0.40 g, 2.70 mmol), furan-2-carboxaldehyde
(0.26 g, 2.70 mmol) in absolute ethanol (10 mL) after 12 h and
recrystallization from ethanol to obtain 0.49 g (81%) of yellow
powder. mp 170e171 ꢀC; UV (EtOH) lmax/nm ¼ 367 nm; IR
9.23
3.24
24.62
>100
15.27
81.45
66.07
a
Mean IC50 ꢁ 50% inhibitory concentration, or compound concentration required
to inhibit tumor cell proliferation by 50%.

M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2274e2279
2277
(diamond):
d6, 300 MHz):
n
/cmꢁ1 ¼ 2856, 1757, 1613, 1550, 1419; 1H NMR (DMSO-
(10 mL) after 20 h and recrystallization from ethanol to obtain
d
/ppm ¼ 12.49 (s, 1H, NHbenzimidazole), 9.17 (s, 1H,
0.75 g (35%) of yellow powder. mp 218e220 ꢀC; UV (EtOH) lmax
/
Harom.), 8.06 (d, 1H, J ¼ 1.56 Hz, Hfur), 7.41 (d, 1H, J ¼ 3.24 Hz, Hfur),
7.32 (bs, 1H, Harom), 7.20 (d, 1H, J ¼ 8.21 Hz, Harom), 7.00 (t, 1H,
J ¼ 8.19 Hz, Harom), 6.80 (t, 1H, J ¼ 8.21 Hz, Harom), 6.70 (dd, 1H,
J1 ¼ 3.24 Hz, J2 ¼ 1.56 Hz, Hfur), 2.41 (s, 3H, CH3); 13C NMR (DMSO-
nm ¼ 413; IR (diamond):
n
/cmꢁ1 ¼ 2846, 1754, 1638, 1556, 1490;
1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 12.91 (s, 1H, NHbenzimi-
dazole), 9.25 (s, 1H, Harom.), 7.92 (s, 1H, Harom), 7.88 (d, 2H,
J ¼ 8.70 Hz, Harom), 7.57 (d, 1H, J ¼ 8.22 Hz, Harom), 7.50 (d, 1H,
J ¼ 8.22 Hz, Harom), 6.83 (d, 2H, J ¼ 8.76 Hz, Harom), 3.07 (s, 6H,
d6, 75 MHz):
d/ppm ¼ 156.11 (s), 153.52 (d), 151.07 (s), 148.10 (d),
142.34 (s), 132.56 (s), 126.42 (d), 123.37 (d), 120.90 (d), 121.43 (s),
113. 31 (d), 111.02 (d), 21.32 (q); Anal. (C13H11N3O) Calcd.: C, 69.32;
H, 4.92; N, 18.66; Found: C, 69.55; H, 5.11; N, 18.50.
CH3); 13C NMR (DMSO-d6, 75 MHz):
d
/ppm ¼ 166.73 (d), 160.23
(s), 154.19 (s), 132.49 (d), 125.51 (s), 122.57 (s), 121.46 (s), 120.67
(s), 112.87 (d, 2C), 111.87 (d, 2C), 111.54 (d), 103.51 (s), 40.13 (q,
2C); Anal. (C17H15N5) Calcd.: C, 70.57; H, 5.23; N, 24.21; Found: C,
70.88; H, 5.46; N, 24.33.
4.1.2.5. 2-(Thiophen -2-ylideneamino)-5(6)-cyanobenzimidazole 12.
Compound 12 was prepared from 2-amino-5(6)-cyanobenzimida-
zole 4 (0.25 g, 1.58 mmol), thiophen-2-carboxaldehyde (0.18 g,
1.58 mmol) in absolute ethanol (10 mL) after 20 h and recrystalli-
zation from ethanol to obtain 0.40 g (63%) of yellow powder. mp
4.1.2.9. 2-[(4-N,N-dimethylamino)benz-2-ylideneamino]-5(6)-meth-
ylbenzimidazole 16. Compound 16 was prepared from 2-amino-
5(6)-methylbenzimidazole 5 (0.25 g, 1.70 mmol), 4-N,N-dimethy-
lamino-benzaldehyde (0.25 g, 1.70 mmol) in absolute ethanol
(10 mL) after 20 h and recrystallization from ethanol to obtain
230e232 ꢀC; UV (EtOH) lmax/nm ¼ 363, 302; IR (diamond):
n/
cmꢁ1 ¼ 3340, 2765, 2218, 1730, 1598, 1529; 1H NMR (DMSO-d6,
300 MHz):
d
/ppm ¼ 13.22 (s,1H, NHbenzimidazole), 9.62 (s,1H, Harom.),
0.34 g (72%) of yellow powder. mp 208e210 ꢀC; UV (EtOH) lmax
/
8.05 (d, 1H, J ¼ 4.92 Hz, Hthiop), 8.00 (d, 1H, J ¼ 3.75 Hz, Hthiop), 7.57
(d, 1H, J ¼ 8.25 Hz, Harom), 7.33 (dd, 1H, J1 ¼ 3.84 Hz, J1 ¼ 4.86 Hz,
Hthiop), 7.26 (t,1H, J ¼ 8.16 Hz, Harom), 7.20 (d,1H, J ¼ 8.04 Hz, Harom),
nm ¼ 401; IR (diamond):
n
/cmꢁ1 ¼ 2896, 1740, 1652, 1581,1518; 1H
NMR (DMSO-d6, 300 MHz):
d
/ppm ¼ 12.20 (s, 1H, NHbenzimidazole),
9.19 (s, 1H, Harom.), 7.84 (d, 2H, J ¼ 8.79 Hz, Harom), 7.67 (d, 1H,
J ¼ 8.76 Hz, Harom), 7.30 (s, 1H, Harom), 6.94 (d, 1H, J ¼ 8.74 Hz,
Harom), 6.81 (d, 2H, J ¼ 8.83 Hz, Harom), 3.05 (s, 6H, CH3), 2.89 (s, 3H,
6.85 (t, 1H, J ¼ 8.18 Hz, Harom); 13C NMR (DMSO-d6, 75 MHz):
d/
ppm ¼ 160.63 (d), 159.24 (s), 141.48 (s), 135.09 (d), 129.60 (d),
125.95 (d), 123.48 (d), 121.36 (s), 120.48 (s), 112.82 (d); Anal.
(C13H8N4S) Calcd.: C, 61.89; H, 3.20; N, 22.21; Found: C, 61.60; H,
3.09; N, 22.44.
CH3); 13C NMR (DMSO-d6, 75 MHz):
d
/ppm ¼ 164.38 (d), 153.69 (s),
143.59 (s), 133.87 (s), 132.00 (d), 131.84 (d, 2C), 123.57 (s), 123.20
(d), 123.00 (s), 118.47 (s), 112.04 (d), 111.53 (d), 40.15 (q, 2C), 39.87
(q); Anal. (C17H18N4) Calcd.: C, 73.35; H, 6.52; N, 20.13; Found: C,
73.56; H, 6.40; N, 20.31.
4.1.2.6. 2-(Thiophen-2-ylideneamino)-5(6)-methyl
benzimidazole
13. Compound 13 was prepared from 2-amino-5(6)-methyl-
benzimidazole 5 (0.40 g, 2.70 mmol), thiophen-2-carboxaldehyde
(0.30 g, 2.70 mmol) in absolute ethanol (15 mL) after 16 h and
recrystallization from ethanol to obtain 0.48 g (73%) of yellow
powder. mp 102e104 ꢀC; UV (EtOH) lmax/nm ¼ 376, 286; IR (dia-
4.1.2.10. 2-[(4-N,N-dimethylamino)benz-2-ylideneamino]-5(6)-nitro-
benzimidazole 17. Compound 17 was prepared from 2-amino-5(6)-
nitrobenzimidazole 6 (0.50 g, 2.80 mmol), 4-N,N-dimethylamino-
benzaldehyde (0.42 g, 2.80 mmol) in absolute ethanol (10 mL) after
24 h and recrystallization from ethanol to obtain 0.68 g (78%) of
orange powder. mp 240e241 ꢀC; UV (EtOH) lmax/nm ¼ 423; IR
mond):
(DMSO-d6, 300 MHz):
n
/cmꢁ1 ¼ 3290, 2823, 1744, 1636, 1528, 1434; 1H NMR
d/ppm ¼ 12.49 (s, 1H, NHbenzimidazole), 9.55 (s,
1H, Harom.), 7.96 (d, 1H, J ¼ 5.00 Hz, Hthiop), 7.93 (d, 1H, J ¼ 3.60 Hz,
Hthiop), 7.30 (dd, 1H, J1 ¼ 4.92 Hz, J2 ¼ 3.75 Hz, Hthiop), 7.20 (d, 1H,
J ¼ 8.38 Hz, Harom), 7.00 (d, 1H, J ¼ 8.42 Hz, Harom), 6.98e6.92 (m,
2H, Harom), 2.41 (s, 3H, CH3); 13C NMR (DMSO-d6, 75 MHz):
(diamond):
d6, 600 MHz):
n
/cmꢁ1 ¼ 2823, 1750, 1618, 1570, 1496; 1H NMR (DMSO-
d
/ppm ¼ 13.04 (s, 1H, NHbenzimidazole), 9.26 (s, 1H
Harom.), 8.28 (s, 1H, Harom), 8.05 (dd, 1H, J1 ¼ 8.82 Hz, J2 ¼ 2.28 Hz,
Harom), 7.89 (d, 2H, J ¼ 8.88 Hz, Harom), 7.61 (d,1H, J ¼ 8.80 Hz, Harom),
6.83 (d, 2H, J ¼ 8.88 Hz, Harom), 3.07 (s, 6H, CH3); 13C NMR (DMSO-d6,
d
/ppm ¼ 158.17 (d), 158.04 (d), 155.40 (s), 144.98 (s), 137.11 (d),
133.77 (d), 131.57 (s), 129.35 (d), 128.28 (s), 123.86 (d), 120.52 (d),
112.38 (d), 21.81 (q); Anal. (C13H11N3S) Calcd.: C, 64.70; H, 4.59; N,
17.41; Found: C, 64.98; H, 4.77; N, 17.62.
75 MHz):
d
/ppm ¼ 166.58 (d),153.80 (s),144.50 (s),134.27 (s),132.14
(d, 2C), 124.59 (s), 121.98 (s), 120.01 (s), 117.36 (d), 115.31 (d), 111.78
8d), 111.64 (d, 2C), 39.63 (q, 2C); Anal. (C16H15N5O2) Calcd.: C, 62.13;
H, 4.89; N, 22.64; Found: C, 62.04; H, 4.72; N, 22.44.
4.1.2.7. 2-(Thiophen-2-ylideneamino)-5(6)-nitromethyl
benzimid-
azole 14. Compound 14 was prepared from 2-amino-5(6)-nitro-
benzimidazole 6 (0.50 g, 2.81 mmol), thiophen-2-carboxaldehyde
(0.32 g, 2.81 mmol) in absolute ethanol (15 mL) after 24 h and
recrystallization from ethanol to obtain 0.17 g (23%) of yellow
powder. mp 255e256 ꢀC; UV (EtOH) lmax/nm ¼ 370, 262; IR (dia-
4.1.2.11. 2-[(4-N,N-diethylamino-2-hydroxy)benz-2-ylideneamino]-
5(6)-cyanobenzimidazole 18. Compound 18 was prepared from
2-amino-5(6)-cyanobenzimidazole 4 (0.12 g, 0.76 mmol), 4-N,N-
diethylamino-2-hydroxybenzaldehyde (0.15 g, 0.76 mmol) in
absolute ethanol (10 mL) after 24 h and recrystallization from
ethanol to obtain 0.11 g (49%) of yellow powder. mp 228e230 ꢀC;
mond):
n
/cmꢁ1 ¼ 3221, 2799, 1748, 1642, 1534, 1419; 1H NMR
(DMSO-d6, 300 MHz):
d
/ppm ¼ 12.77 (s, 1H, NHbenzimidazole), 9.59 (s,
1H, Harom.), 8.11 (d, 1H, J ¼ 4.94 Hz, Hthiop), 7.99 (d, 1H, J ¼ 3.78 Hz,
Hthiop), 7.54 (dd, 1H, J1 ¼ 4.96 Hz, J2 ¼ 3.76 Hz, Hthiop), 7.31 (d, 1H,
J ¼ 8.22 Hz, Harom), 6.98 (d,1H, J ¼ 8.30 Hz, Harom), 6.94e6.90 (m, 2H,
UV (EtOH) lmax/nm ¼ 449, 426; IR (diamond):
n
/cmꢁ1 ¼ 3486,
2971, 2219, 1729, 1619, 1577; 1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 12.67 (s, 1H, NHbenzimidazole), 9.34 (s, 1H, Harom.), 7.94 (s,
Harom); 13C NMR (DMSO-d6, 75 MHz):
d
/ppm ¼ 161.11 (d),159.56 (s),
1H, Harom.), 7.58 (d, 1H, J ¼ 8.22 Hz, Harom), 7.53 (d, 1H, J ¼ 8.66 Hz,
Harom), 7.51 (d, 1H, J ¼ 8.42 Hz, Harom), 6.42 (d, 1H, J ¼ 8.20 Hz,
Harom), 6.15 (s, 1H, Harom.), 3.80 (bs, 1H, OH), 3.47e3.43 (m, 4H,
141.43 (s), 138.74 (d), 135.37 (d), 131.27 (s), 129.65 (d), 129.25 (s),
119.47 (s),118.24 (d),117.19 (d),109.18 (d); Anal. (C12H8N4O2S) Calcd.:
C, 52.93; H, 2.96; N, 20.58; Found: C, 52.76; H, 3.18; N, 20.79.
CH2), 1.15 (t, 6H, J ¼ 7.11 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz):
d/
ppm ¼ 165.72 (d), 164.32 (s), 153.72 (s), 142.87 (s), 135.99 (d),
131.48 (s), 125.59 (d), 120.66 (s), 118.35 (s), 108.87 (s), 105.68 (d),
104.95 (d), 97.07 (d), 96.42 (d), 44.64 (t), 44.58 (t), 13.02 (q), 12.89
(q); Anal. (C19H19N5O) Calcd.: C, 68.45; H, 5.74; N, 21.01; Found: C,
68.11; H, 5.55; N, 20.78.
4.1.2.8. 2-[(4-N,N-dimethylamino)benz-2-ylideneamino]-5(6)-cya-
nobenzimidazole 15. Compound 15 was prepared from 2-amino-
5(6)-cyanobenzimidazole 4 (0.12 g, 0.76 mmol), 4-N,N-dimethy-
lamino-benzaldehyde (0.11 g, 0.76 mmol) in absolute ethanol

2278
M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2274e2279
4.1.2.12. 2-[(4-N,N-diethylamino-2-hydroxy)benz-2-ylideneamino]-
5(6)-methylbenzimidazole 19. Compound 19 was prepared from
2-amino-5(6)-methylbenzimidazole 5 (0.25 g, 1.70 mmol), 4-N,N-
diethylamino-2-hydroxybenzaldehyde (0.33 g, 1.70 mmol) in
absolute ethanol (10 mL) after 24 h and recrystallization from
ethanol to obtain 0.32 g (60%) of yellow powder. mp 177e179 ꢀC;
4.3. In vitro studies
4.3.1. Cell culturing
The cell lines HeLa (cervical carcinoma), SW620 (colorectal
adenocarcinoma, metastatic), MiaPaCa-2 (pancreatic carcinoma),
MCF-7 (breast epithelial adenocarcinoma, metastatic) and WI38
(normal diploid human fibroblasts), were cultured as monolayers
and maintained in Dulbecco’s modified Eagle medium (DMEM)
UV (EtOH) lmax/nm ¼ 421,346; IR (diamond):
n
/cmꢁ1 ¼ 3421, 2887,
1729, 1652, 1566, 1497; 1H NMR (DMSO-d6, 300 MHz):
d/
ppm ¼ 11.84 (s, 1H, NHbenzimidazole), 9.61 (s, 1H, Harom.), 7.41 (d, 1H,
J ¼ 8.86 Hz, Harom), 7.00 (d, 1H, J ¼ 8.38 Hz, Harom), 6.98 (s, 1H,
Harom.), 6.78 (d, 1H, J ¼ 8.80 Hz, Harom), 6.33 (d, 1H, J ¼ 8.36 Hz,
Harom), 3.85 (bs, 1H, OH), 3.48e3.42 (m, 4H, CH2), 1.17 (t, 6H,
supplemented with 10% fetal bovine serum (FBS), 2 mM
L
-gluta-
mine, 100 U/ml penicillin and 100
mg/ml streptomycin in a humid-
ified atmosphere with 5% CO2 at 37 ꢀC.
J ¼ 7.11 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz):
d/ppm ¼ 165.89 (d),
4.3.2. Antiproliferative evaluation
164.02 (s), 154.33 (s), 144.88 (s), 136.05 (d), 132.04 (s), 126.19 (d),
120.45 (s), 119.56 (s), 113.02 (s), 111.66 (d), 109.94 (d),110.07 (d),
99.42 (d), 44.61 (t), 44.57 (t), 39.40 (q), 13.1302 (q), 12.9789 (q);
Anal. (C19H22N4O) Calcd.: C, 70.78; H, 6.88; N, 17.38; Found: C,
70.96; H, 6.98; N, 17.70.
A panel of monolayer tumor 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
(0.01e100 mM) followed by a 72-h incubation. Cell viability was
quantitatively determined using a 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) colorimetric assay.
Experimentally obtained absorbance values were transformed into
a cell percentage growth (PG) using the formulas proposed by NIH
and described previously [34]. The IC50 and LC50 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 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 (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 dose response graphs.
4.1.2.13. 2-[(4-N,N-diethylamino-2-hydroxy)benz-2-ylideneamino]-
5(6)-nitrobenzimidazole 20. Compound 20 was prepared from
2-amino-5(6)-nitrobenzimidazole 6 (0.25 g, 1.40 mmol), 4-N,N-
diethylamino-2-hydroxybenzaldehyde (0.27 g, 1.40 mmol) in
absolute ethanol (10 mL) after 24 h and recrystallization from
ethanol to obtain 0.23 g (52%) of orange powder. mp 232e234 ꢀC;
UV (EtOH) lmax/nm ¼ 436, 426; IR (diamond):
n
/cmꢁ1 ¼3428, 2912,
2215, 1722, 1570, 1515; 1H NMR (DMSO-d6, 300 MHz):
d/
ppm ¼ 13.04 (s, 1H, NHbenzimidazole), 12.66 (bs, 1H, OH), 9.36 (s, 1H,
Harom.), 8.29 (s, 1H, Harom.), 8.06 (dd, 1H, J1 ¼ 8.76 Hz, J2 ¼ 2.28 Hz,
Harom.), 7.60 (d, 1H, J ¼ 8.78 Hz, Harom), 7.55 (d, 1H, J ¼ 8.80 Hz,
Harom), 6.43 (dd, 1H, J1 ¼ 8.90 Hz, J2 ¼ 2.34 Hz, Harom.), 6.16 (d, 1H,
J ¼ 2.28 Hz, Harom), 3.47e3.42 (m, 4H, CH2), 1.14 (t, 6H, J ¼ 7.06 Hz,
CH3); 13C NMR (DMSO-d6, 75 MHz):
d
/ppm ¼ 165.87 (d), 164.43 (s),
153.87 (s), 141.54 (s), 136.07 (d), 133.61 (s), 126.44 (d), 124.47 (s),
121.53 (d), 117.92 (d), 113.92 (s), 108.93 (s), 105.80 (d), 97.03 (d),
44.67 (t), 44.51 (t), 13.01 (q), 12.99 (q); Anal. (C18H19N5O3) Calcd.: C,
61.18; H, 5.42; N, 19.82; Found: C, 60.97; H, 5.11; N, 19.66.
4.3.3. Cell cycle analyses
4.1.2.14. 2-[(4-N,N-diethylamino-2-hydroxy)benz-2-ylideneamino]
benzimidazole 21. Compound 21 was prepared from 2-amino-
benzimidazole 7 (0.25 g, 1.90 mmol), 4-N,N-diethylamino-2-
hydroxybenzaldehyde (0.36 g, 1.90 mmol) in absolute ethanol
(10 mL) after 24 h and recrystallization from ethanol to obtain
A total of 5 ꢂ105 cells/well were seeded in a 6-well plate (Falcon,
USA). After 24 h HeLa and MCF-7 cells were treated with compound
18 at concentrations of 5 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, the cells were washed again with PBS and stained with
0.13 g (32%) of orange powder. mp 228e230 ꢀC; UV (EtOH) lmax
/
nm ¼ 417; IR (diamond):
n
/cmꢁ1 ¼ 3451, 2973, 1757, 1647, 1564,
1 mg/mL of propidium iodide (PI) with the addition of 0.2 mg/mL of
1515; 1H NMR (DMSO-d6, 600 MHz):
d
/ppm ¼ 12.85 (s, 1H,
RNAse A. The stained cells were then analyzed with Becton Dick-
inson FACScalibur flow cytometer (10000 counts were measured).
Each test point was performed in triplicate. 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.
NHbenzimidazole), 12.36 (bs, 1H, OH), 9.31 (s, 1H, Harom.), 7.48 (d, 2H,
J ¼ 8.88 Hz, Harom.), 7.38 (d, 1H, J ¼ 8.70 Hz, Harom), 7.12 (d, 2H,
J ¼ 8.80 Hz, Harom), 6.39 (dd, 1H, J1 ¼ 8.92 Hz, J2 ¼ 2.18 Hz, Harom.),
6.14 (d, 1H, J ¼ 2.20 Hz, Harom), 3.45e3.41 (m, 4H, CH2), 1.15 (t, 6H,
J ¼ 7.09 Hz, CH3); 13C NMR (DMSO-d6, 75 MHz):
/ppm ¼ 164.62
d
(d), 163.96 (s), 155.23 (s), 153.16 (s), 143.18 (s), 135.72 (d), 131.55
(s), 124.56 (d), 121.89 (d), 116.54 (d), 108.77 (s), 105.29 (d), 102.44
(d), 97.17 (d), 44.58 (t), 44.56 (t), 13.02 (q), 13.00 (q); Anal.
(C18H20N4O) Calcd.: C, 70.11; H, 6.54; N, 18.17; Found: C, 70.33; H,
6.82; N, 18.37.
Acknowledgments
Support for this study by the Ministry of Science, Education and
Sport of Croatia is gratefully acknowledged (Projects 125-0982464-
1356, 335-0982464-2393, 335-0000000-3532).
4.2. Spectroscopy
The electronic absorption spectra were recorded by means of
Varian Cary 50 spectrometer while fluorescence emission spectra
were recorded by means of Varian Eclipse fluorimeter, in both cases
using quartz cuvettes (1 cm). Fluorescence was excited on the
wavelength of absorption maxima.
Appendix. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.03.008.

M. Hranjec et al. / European Journal of Medicinal Chemistry 46 (2011) 2274e2279
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