Synthesis, antioxidative and antiproliferative activity of methoxy amidino substituted benzamides and benzimidazoles

Article abstract of DOI:10.1007/s00044-017-1912-z

Within this manuscript the synthesis and potential antioxidative and antiproliferative activity of novel methoxy amidino substituted bezamides 6–17 and benzimidazoles 23–28 and 32–35 is presented. Their antioxidative potency has been evaluated by in vitro spectrophotometric assays and preliminary structure–activity relationships among the synthesized compounds are discussed. The compound 28 bearing three methoxy groups on a phenyl ring and imidazolinyl amidine group on benzimidazole nuclei directly attached to the phenyl ring exhibited the most prominent reducing activity as well as free-radical scavenging activity. Furthermore all novel compounds were tested against three human cell lines: HCT 116 (colon carcinoma), H 460 (lung carcinoma) and MCF-7 (breast carcinoma). The most prominent antiproliferative effect in the single-digit micromolar range was observed for compound 33 on MCF-7 (IC₅₀ = 9 μM) cell line, comparable to the standard etoposide.

Full text of DOI:10.1007/s00044-017-1912-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1912-z
ORIGINAL RESEARCH
Synthesis, antioxidative and antiproliferative activity of methoxy
amidino substituted benzamides and benzimidazoles
Maja Cindrić1 Irena Sović1 Irena Martin-Kleiner2 Marijeta Kralj2
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Tomislav Mašek3 Marijana Hranjec1 Kristina Starčević3
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Received: 19 January 2017 / Accepted: 27 April 2017
© Springer Science+Business Media New York 2017
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Abstract Within this manuscript the synthesis and potential
antioxidative and antiproliferative activity of novel methoxy
amidino substituted bezamides 6–17 and benzimidazoles
23–28 and 32–35 is presented. Their antioxidative potency
has been evaluated by in vitro spectrophotometric assays and
preliminary structure–activity relationships among the syn-
thesized compounds are discussed. The compound 28
bearing three methoxy groups on a phenyl ring and imida-
zolinyl amidine group on benzimidazole nuclei directly
attached to the phenyl ring exhibited the most prominent
reducing activity as well as free-radical scavenging activity.
Furthermore all novel compounds were tested against three
human cell lines: HCT 116 (colon carcinoma), H 460 (lung
carcinoma) and MCF-7 (breast carcinoma). The most pro-
minent antiproliferative effect in the single-digit micromolar
Keywords Amidines Benzamides Benzimidazoles
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Methoxy groups Antioxidative activity Antiproliferative
activity
Introduction
A variety of biochemical and pathophysiological processes
in the human body could produce oxygen free radicals and
other reactive oxygen species (ROS) as by-products which
may cause oxidative damage of most important biomole-
cules such as nucleic acids, lipids and proteins (Hildeman
et al. 2003). Oxidative damage of biomolecules eventually
leads to several chronic diseases including cancer, diabetes,
atherosclerosis, aging and other degenerative diseases
(Stadtman 2004; Valko et al. 2007a, b). ROS includes non-
free radical species like ozone (O3), hydrogen peroxide
(H2O2), singlet oxygen (1O2), and free radicals including
hydroperoxyl radicals (HOO•), hydroxyl radicals (OH•),
peroxyl radicals (ROO•) and superoxide anion radicals
(O•2). Thus, ROS could be also generated in metal-
catalyzed reactions, from pollutants, by irradiation with
X-rays and ultraviolet light, in mitochondria-catalyzed
electron transport reactions or during inflammation pro-
cess. A prooxidant–antioxidant equilibrium which exists in
normal cell could be impaired due to the uncontrolled
production of ROS which is called oxidative stress. Since
antioxidants are known by their function in protecting the
human body against damage by ROS as well as by inhi-
biting ROS-relayed reactions, there has been tremendous
and constant growing interest for the searching of novel
natural or synthetic organic molecules as potential anti-
oxidants. The results obtained from many antioxidative
range was observed for compound 33 on MCF-7 (IC50
9 µM) cell line, comparable to the standard etoposide.
=
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1912-z) contains supplementary material,
which is available to authorized users.
* Marijana Hranjec
mhranjec@fkit.hr
* Kristina Starčević
kstarcevic@vef.hr
1
Department of Organic Chemistry, Faculty of Chemical
Engineering and Technology, University of Zagreb, Marulićev trg
20, P. O. Box 177, HR-10000 Zagreb, Croatia
2
Division of Molecular Medicine, Ruđer Bošković Institute,
Bijenička cesta 54, P. O. Box 180, HR-10000 Zagreb, Croatia
3
Department of Animal Husbandry, Faculty of Veterinary
Medicine, University of Zagreb, Heinzelova 55, HR-10000
Zagreb, Croatia

Med Chem Res
studies revealed that tumor cells are accompanied by a
measurable emission of ROS which might be regulated by a
proper application of antioxidants. Kus and coauthors have
described the antioxidative activity of thiosemicarbazide
benzimidazole derivatives which were found to be a good
scavenger of DPPH radical (Kus et al. 2008). The benzi-
midazole nuclei represents nowadays one of the most
important structural motifs in heterocyclic and medicinal
chemistry (Monika et al. 2014). It could be found in a
variety of bioactive natural products and numerous of
synthetic medical and biochemical agents possessing dif-
ferent chemical and pharmacological features (Bansal and
Silakari 2012). Besides their broad range of biological
activities, in last decade the antioxidative potency of ver-
satile benzimidazole derivatives has been studied. A group
of authors have studied the biological evaluation of novel 4-
methylbenzimidazole derivatives which included their
antiglycation and antioxidant activities (Taha et al. 2014).
Some of tested compounds showed excellent DPPH activ-
ities ranging 12–29 μM as well as superoxide anion
scavenging activity, better than standard antioxidant n-
propylgallate. In addition, a series of 2-arylbenzimidazole
derivatives were evaluated as potential antioxidant and
antimicrobial agents by group of Chinese researches (Zhou
et al. 2013). Derivatives bearing hydroxyl group at the 5-
position of benzimidazole nuclei had a comparable or better
antioxidant activity in comparison to standard antioxidant
tert-butylhydroquinone studied by various in vitro assays.
The above mentioned considerations prompted us to an
extension of our previous scientific research on the synth-
esis and biological evaluation of versatile benzimidazole
and benzamide amidino substituted derivatives. Here we
present the synthesis, antioxidative and antiproliferative
activity of novel methoxy and amidino substituted benza-
mides and benzimidazoles.
checked by thin layer chromatography with Merck silica gel
60F-254 glass plates.
General method for the synthesis of N-substituted
methoxybenzamides (6–11)
To a solution of corresponding benzoyl chlorides in dry
toluene, a solution of corresponding anilines in dry toluene
was added dropwise, followed by the addition of triethy-
lamine (Et3N). The reaction mixture was refluxed for sev-
eral hours. After cooling the solution was concentrated and
the obtained solid was filtered off and recrystallized from
appropriate solvent.
N-(2,4-Dimethoxyphenyl)-2-methoxybenzamide (6)
This compound was prepared using above described method
from 2-methoxybenzoyl chloride 1 (0.30 g, 1.76 mmol) and
2,4-dimethoxyaniline 4 (0.27 g, 1.76 mmol) in dry toluene
(15 mL) followed by the addition of Et3N (0.34 mL, 2.46
mmol) after refluxing for 6 h and recrystalization from
methanol. The crude product was purified by column chro-
matography with dichloromethane to obtain 0.28 g (55%) of
1
light gray crystals; mp 114–115 °C; H NMR (300 MHz,
DMSO-d6): δ = 10.45 (1H, s, NHamide), 8.35 (1H, d, J =
8.85, Hz, Aromatic CH), 8.09 (1H, dd, J = 7.80, 1.80, Hz,
Aromatic CH), 7.60–7.54 (1H, m, Aromatic CH), 7.27 (1H,
d, J = 8.19, Hz, Aromatic CH), 7.14 (1H, t, J = 7.53, Hz,
Aromatic CH), 6.69 (1H, d, J = 2.61, Hz, Aromatic CH),
6.55 (1H, dd, J = 8.88, 2.64, Hz, Aromatic CH), 4.07 (3H, s,
OCH3), 3.95 (3H, s, OCH3), 3.77 (3H, s, OCH3); 13C NMR
(150 MHz, DMSO-d6): δ = 161.6, 157.1, 156.0, 149.3,
133.3, 131.2, 121.4, 121.2, 121.1, 120.1, 112.5, 104.2, 98.7,
56.4, 56.2, 55.3; anal. calcd. for C16H17NO4: C, 67.04; H,
6.12; N, 4.65. Found: C, 66.89; H, 5.96; N, 4.88.
N-(2,4-Dimethoxyphenyl)-2,4-dimethoxybenzamide (7)
Materials and methods
Chemistry
This compound was prepared using above described
method from 2,4-dimethoxybenzoyl chloride 2 (0.30 g,
1.50 mmol) and 2,4-dimethoxyaniline 4 (0.23 g, 1.50 mmol)
in dry toluene (10 mL) followed by the addition of Et3N
(0.29 mL, 2.10 mmol) after refluxing for 5 h and recrysta-
lization from methanol to obtain 0.17 g (35%) of light
purple powder; mp 130–131 °C; 1H NMR (300 MHz,
DMSO-d6): δ = 10.32 (1H, s, NHamide), 8.33 (1H, d, J =
8.85, Hz, Aromatic CH), 8.01 (1H, d, J = 8.67, Hz, Aro-
matic CH), 6.74 (1H, d, J = 2.22, Hz, Aromatic CH), 6.70
(1H, dd, J = 8.75, 2.33, Hz, Aromatic CH), 6.66 (1H, d, J
= 2.64, Hz, Aromatic CH), 6.51 (1H, dd, J = 8.88, 2.64,
Hz, Aromatic CH), 4.05 (3H, s, OCH3), 3.92 (3H, s,
OCH3), 3.84 (3H, s, OCH3), 3.74 (3H, s, OCH3); 13C NMR
(75 MHz, DMSO-d6): δ = 163.9, 161.9, 159.0, 156.2,
Melting points were determined by SMP10 Bibby apparatus
and are uncorrected. Infrared (IR) spectra were recorded on
FTIR-ATR Vertex 70 Bruker spectrophotometer (from
4000–450 cm−1).
1H and 13C NMR spectra were recorded on Bruker AV
300 and Bruker AV 600 spectrometers using tetra-
methylsilane (TMS) as an internal standard in dimethyl
sulfoxide (DMSO)-d6. Chemical shifts are reported in ppm
(δ) relative to TMS. Elemental analyses for carbon,
hydrogen, and nitrogen were performed on a Perkin-Elmer
2400 elemental analyzer and a Perkin-Elmer, Series II,
CHNS analyzer 2400. All compounds were routinely

Med Chem Res
149.6, 133.4, 122.2, 120.2, 114.4, 106.9, 104.6, 99.3, 99.2,
57.0, 56.7, 56.1, 55.8; anal. calcd. for C17H19NO5: C,
64.55; H, 6.18; N, 4.27. Found: C, 64.34; H, 6.03; N, 4.41.
MHz, DMSO-d6): δ = 10.27 (1H, s, NHamide), 7.92 (2H, d,
J = 8.79, Hz, Aromatic CH), 7.78 (2H, d, J = 8.76, Hz,
Aromatic CH), 7.69 (1H, d, J = 8.52 Hz, Aromatic CH),
6.70 (1H, d, J = 2.19, Hz, Aromatic CH), 6.66 (1H, dd, J =
8.58, 2.28, Hz, Aromatic CH), 3.92 (3H, s, OCH3), 3.83
(3H, s, OCH3); 13C NMR (75 MHz, DMSO-d6): δ = 164.9,
163.7, 158.9, 143.7, 133.7 (2C), 132.4, 120.2 (2C), 119.6,
116.4, 106.3, 105.5, 99.1, 56.7, 56.1; anal. calcd. for
C16H14N2O3: C, 67.94; H, 4.81; N, 10.20. Found: C, 68.07;
H, 5.00; N, 9.92.
N-(2,4-Dimethoxyphenyl)-3,4,5-trimethoxybenzamide (8)
This compound was prepared using above described
method from 3,4,5-trimethoxybenzoyl chloride 3 (0.30 g,
1.31 mmol) and 2,4-dimethoxyaniline 4 (0.20 g, 1.31 mmol)
in dry toluene (15 mL) followed by the addition of Et3N
(0.26 mL, 1.83 mmol) after refluxing for 6 h and recrysta-
lization from methanol to obtain 0.11 g (24%) of light
purple powder; mp 122–124 °C; 1H NMR (300 MHz,
DMSO-d6): δ = 9.35 (1H, s, NHamide), 7.38 (1H, d, J =
8.64, Hz, Aromatic CH), 7.28 (2H, s, Aromatic CH), 6.65
(1H, d, J = 2.58, Hz, Aromatic CH), 6.54 (1H, dd, J = 8.66,
2.60, Hz, Aromatic CH), 3.85 (6H, s, OCH3), 3.79 (3H, s,
OCH3), 3.78 (3H, s, OCH3), 3.73 (3H, s, OCH3); 13C NMR
(75 MHz, DMSO-d6): δ = 165.1, 158.5, 154.3, 153.1 (2C),
130.3, 127.5, 120.1, 105.6 (2C), 104.7, 99.4, 60.6, 56.5
(2C), 56.1, 55.8; anal. calcd. for C18H21NO6: C, 62.52;
H, 6.27; N, 4.25. Found: C, 62.24; H, 6.09; N, 4.03.
N-(4-Cyanophenyl)-3,4,5-trimethoxybenzamide (11)
This compound was prepared using above described
method from 3,4,5-trimethoxybenzoyl chloride 3 (1.00 g,
4.36 mmol) and 4-cyanoaniline 5 (0.51 g, 4.36 mmol) in dry
toluene (00 mL) followed by the addition of Et3N (0.85 mL,
6.07 mmol) after refluxing for 24 h. The crude product was
washed with diluted HCl and water to obtain 0.70 g (52%)
1
of light brown powder; mp 152–155 °C; H NMR (300
MHz, DMSO-d6): δ = 10.30 (1H, s, Hamide), 7.94 (2H, d, J
= 8.85, Hz, Aromatic CH), 7.81 (2H, d, J = 8.79, Hz,
Aromatic CH), 7.71 (1H, s, Aromatic CH), 6.72 (1H, d, J =
2.19, Hz, Aromatic CH), 3.95 (3H, s, OCH3), 3.86 (3H, s,
OCH3), 3.34 (3H, s, OCH3); 13C NMR (75 MHz, DMSO-
d6): δ = 166.0, 153.1, 143.9 (2C), 133.6 (2C), 129.9, 120.8
(2C), 119.5 (2C), 106.0 (2C), 105.8, 60.6, 56.6 (2C); anal.
calcd. for C17H16N2O4: C, 65.60; H, 5.30; N, 9.18. Found:
C, 65.38; H, 5.16; N, 8.97.
N-(4-Cyanophenyl)-2-methoxybenzamide (9)
This compound was prepared using above described
method from 2-methoxybenzoyl chloride 1 (0.50 g, 2.90
mmol) and 4-cyanoaniline 5 (0.35 g, 2.90 mmol) in dry
toluene (15 mL) followed by the addition of Et3N (0.57 mL,
4.10 mmol) after refluxing for 24 h. The crude product was
washed with diluted HCl and water and recrystalized from
methanol to obtain 0.27 g (37%) of light brown powder; mp
140–141 °C; 1H NMR (300 MHz, DMSO-d6): δ = 10.51
(1H, s, NHamide), 7.91 (2H, d, J = 8.73, Hz, Aromatic CH),
7.79 (2H, d, J = 8.70, Hz, Aromatic CH), 7.58 (1H, dd, J =
7.55, 1.70, Hz, Aromatic CH), 7.51 (1H, td, J = 7.88, 1.74,
Hz, Aromatic CH), 7.18 (1H, d, J = 8.34, Hz, Aromatic
CH), 7.06 (1H, t, J = 7.44, Hz, Aromatic CH), 3.87 (3H, s,
OCH3); 13C NMR (150 MHz, DMSO-d6): δ = 165.3, 156.5,
143.2, 133.2 (2C), 132.3, 129.5, 124.7, 120.5, 119.6 (2C),
119.0, 112.0, 105.2, 55.9; anal. calcd. for C15H12N2O2: C,
71.65; H, 4.56; N, 11.28. Found: C, 71.42; H, 4.79; N,
11.10.
General method for the synthesis of amidino substituted
methoxybenzamides (12–17)
A stirred suspension of corresponding cyano substituted
methoxybenzamide in absolute ethanol was cooled in an
ice-salt bath and was saturated with dry HCl gas. The
reaction mixture was maintained at room temperature until
nitrile band disappeared (monitored by IR analysis at 2200
cm−1). The corresponding imidate was filtered off, washed
with dry diethyl ether and dried under reduced pressure.
Amidines
The corresponding imidate was suspended in absolute
ethanol and dry NH3 was bubbled into the suspension. The
mixture was stirred at room temperature for 2 days. The
crude product was then filtered off and washed with diethyl
ether.
N-(4-Cyanophenyl)-2,4-dimethoxybenzamide (10)
This compound was prepared using above described
method from 2,4-dimethoxybenzoyl chloride 2 (0.40 g,
2.00 mmol) and 4-cyanoaniline 5 (0.24 g, 2.00 mmol) in dry
toluene (15 mL) followed by the addition of Et3N (0.39 mL,
2.80 mmol) after refluxing for 24 h. The crude product was
washed with diluted HCl and water to obtain 0.36 g (64%)
2-Imidazolinylamidines
Ethylenediamine (EDA) was added to a suspension of
the corresponding imidate in absolute ethanol (5 mL) and the
1
of light purple powder; mp 178–179 °C; H NMR (300

Med Chem Res
mixture was stirred at reflux for 24 h. The crude product was
then filtered off and washed with diethyl ether.
for 5 days. The crude imidate was filtered off, suspended in
absolute ethanol (10 mL) and dry NH3 was bubbled into the
suspension. The mixture was worked up as it is described to
1
N-(4-Amidinophenyl)-2-methoxybenzamide hydrochloride
give 0.09 g (53%) of light pink powder; mp > 295 °C; H
(12)
NMR (600 MHz, DMSO-d6): δ = 10.31 (1H, s, NHamide),
8.58 (4H, bs, NHamidine), 7.97 (2H, d, J = 8.85, Hz, Aro-
matic CH), 7.87 (2H, d, J = 8.85, Hz, Aromatic CH), 7.73
(1H, s, Aromatic CH), 6.73 (1H, d, J = 2.04 Hz, Aromatic
CH), 3.96 (3H, s, OCH3), 3.85 (3H, s, OCH3), 3.33 (3H, s,
OCH3); 13C NMR (75 MHz, DMSO-d6): δ = 165.2, 164.8,
163.8, 159.0, 144.5, 129.6 (2C), 122.3, 119.7 (2C), 116.2,
106.4, 99.1, 56.7, 56.7 (2C); anal. calcd. for C17H20ClN3O4:
C, 55.66; H, 5.27; N, 11.76. Found: C, 55.82; H, 5.51; N,
11.49.
This compound was prepared using above described
method. A solution of 9 (0.06 g, 0.25 mmol) in absolute
ethanol (5 mL) was saturated with dry HCl gas and stirred
for 5 days. The crude imidate was filtered off, suspended in
absolute ethanol (5 mL) and dry NH3 was bubbled into the
suspension. The mixture was worked up as it is described to
give 0.36 g (24%) of white powder; mp 259–260 °C;
1H NMR (600 MHz, DMSO-d6): δ = 10.51 (1H, s,
NHamide), 9.14 (4H, bs, NHamidine), 7.96 (2H, d, J = 8.70,
Hz, Aromatic CH), 7.88 (2H, d, J = 8.76, Hz, Aromatic
CH), 7.64 (1H, dd, J = 7.50, 1.62, Hz, Aromatic CH),
7.55–7.52 (1H, m, Aromatic CH), 7.21 (1H, d, J = 8.25,
Hz, Aromatic CH), 7.09 (1H, t, J = 7.47, Hz, Aromatic
CH), 3.91 (3H, s, OCH3); 13C NMR (75 MHz, DMSO-d6):
δ = 165.8, 165.2, 157.0, 144.5, 132.9, 130.1, 129.7 (2C),
125.1, 122.4, 121.0, 119.6 (2C), 112.6, 56.4; anal. calcd. for
C15H16ClN3O2: C, 58.76; H, 5.40; N, 13.52. Found: C,
58.92; H, 5.27; N, 13.74.
N-[4-(2-Imidazolinyl)phenyl]-2-methoxybenzamide
hydrochloride (15)
This compound was prepared using above described
method. A solution of 9 (0.06 g, 0.25 mmol) in absolute
ethanol (5 mL) was saturated with dry HCl gas and stirred
for 5 days. The crude imidate was filtered off and suspended
in absolute ethanol. EDA (0.052 mL, 0.78 mmol) was added
and the mixture was stirred at reflux for 24 h. The mixture
was worked up as it is described to give 0.060 g (36%) of
1
N-(4-Amidinophenyl)-2,4-dimethoxybenzamide
beige powder; mp > 295 °C; H NMR (600 MHz, DMSO-
hydrochloride (13)
d6): δ = 10.57 (1H, s, NHamide), 10.09 (2H, bs, NHamidine),
7.99 (4H, s, Aromatic CH), 7.64 (1H, dd, J = 7.58, 1.73,
Hz, Aromatic CH), 7.57–7.52 (1H, m, Aromatic CH), 7.21
(1H, d, J = 8.22, Hz, Aromatic CH), 7.09 (1H, t, J = 7.46,
Hz, Aromatic CH), 3.98 (4H, s, CH2), 3.91 (3H, s, OCH3);
13C NMR (75 MHz, DMSO-d6): δ = 165.9, 164.6, 157.0,
144.8, 133.0, 130.2 (2C), 130.1, 125.0, 121.0, 119.7 (2C),
117.1, 112.6, 56.4, 44.8 (2C); anal. calcd. for
C17H18ClN3O2: C, 61.80; H, 5.33; N, 12.50. Found: C,
61.54; H, 5.47; N, 12.66.
This compound was prepared using above described
method. A solution of 10 (0.15 g, 0.53 mmol) in absolute
ethanol (7.5 mL) was saturated with dry HCl gas and stirred
for 6 days. The crude imidate was filtered off, suspended in
absolute ethanol (20 mL) and dry NH3 was bubbled into the
suspension. The mixture was worked up as it is described to
give 0.07 g (37%) of light pink powder; mp 280–281 °C;
1H NMR (600 MHz, DMSO-d6): δ = 10.29 (1H, s,
NHamide), 9.29 (2H, s, NHamidine), 9.07 (2H, s, NHamidine),
7.97 (2H, d, J = 8.82, Hz, Aromatic CH), 7.87 (2H, d, J =
8.70, Hz, Aromatic CH), 7.75 (1H, d, J = 8.64, Hz, Aro-
matic CH), 6.73 (1H, d, J = 1.86, Hz, Aromatic CH), 6.69
(1H, dd, J = 8.55, 2.07, Hz, Aromatic CH), 3.97 (3H, s,
OCH3), 3.86 (3H, s, OCH3); 13C NMR (75 MHz, DMSO-
d6): δ = 164.7, 164.3, 163.3, 158.5, 144.0, 132.0, 129.1
(2C), 121.8, 119.2 (2C), 115.7, 105.9, 98.6, 56.2, 55.6;
anal. calcd. for C16H18ClN3O3: C, 57.45; H, 5.66; N, 12.74.
Found: C, 57.23; H, 5.40; N, 12.51.
N-[4-(2-Imidazolinyl)phenyl]-2,4-dimethoxybenzamide
hydrochloride (16)
This compound was prepared using above described
method. A solution of 10 (0.15 g, 0.53 mmol) in absolute
ethanol (7.5 mL) was saturated with dry HCl gas and stirred
for 6 days. The crude imidate was filtered off and suspended
in absolute ethanol. EDA (0.096 mL, 1.44 mmol) was added
and the mixture was stirred at reflux for 24 h. The mixture
was worked up as it is described to give 0.09 g (44%) of
light pink crystals; mp > 295 °C; 1H NMR (600 MHz,
DMSO-d6): δ = 10.66 (2H, s, NHamidine), 10.35 (1H, s,
NHamide), 8.08 (2H, d, J = 8.82, Hz, Aromatic CH), 7.99
(2H, d, J = 8.82, Hz, Aromatic CH), 7.75 (1H, d, J = 8.64,
Hz, Aromatic CH), 6.73 (1H, d, J = 2.16, Hz, Aromatic
CH), 6.69 (1H, dd, J = 8.67, 2.19, Hz, Aromatic CH), 3.98
N-(4-Amidinophenyl)-3,4,5-trimethoxybenzamide
hydrochloride (14)
This compound was prepared using above described
method. A solution of 11 (0.14 g, 0.45 mmol) in absolute
ethanol (15 mL) was saturated with dry HCl gas and stirred

Med Chem Res
(4H, s, CH2), 3.97 (3H, s, OCH3), 3.86 (3H, s, OCH3); 13
C
8.34, Hz, Aromatic CH), 7.18 (1H, t, J = 7.47, Hz, Aro-
matic CH), 4.07 (3H, s, OCH3); 13C NMR (150 MHz,
DMSO-d6): δ = 166.3 (2C), 157.2, 151.6, 132.6, 130.1,
122.0, 121.5, 121.0 (2C), 116.6, 112.3 (2C), 55.9; anal.
calcd. for C15H15ClN4O: C, 59.65; H, 5.13; N, 18.72.
Found: C, 59.51; H, 4.99; N, 18.51.
NMR (75 MHz, DMSO-d6): δ = 164.4, 164.1, 163.3, 158.5,
144.5, 132.0, 129.8 (2C), 119.3 (2C), 116.1, 115.7, 105.9,
98.6, 56.2, 55.6, 44.1 (2C); anal. calcd. for C18H20ClN3O3:
C, 59.60; H, 5.44; N, 11.45. Found: C, 59.75; H, 5.57; N,
11.61.
N-[4-(2-Imidazolinyl)phenyl)-3,4,5-trimethoxybenzamide
5(6)-Amidino-2-(2,4-dimethoxyphenyl)benzimidazole
hydrochloride (17)
hydrochloride (24)
This compound was prepared using above described
method. A solution of 11 (0.14 g, 0.45 mmol) in absolute
ethanol (15 mL) was saturated with dry HCl gas and stirred
for 5 days. The crude imidate was filtered off and suspended
in absolute ethanol. EDA (0.059 mL, 0.85 mmol) was added
and the mixture was stirred at reflux for 24 h. The mixture
was worked up as it is described to give 0.07 g (41%) of
light pink crystals; mp > 295 °C; 1H NMR (600 MHz,
DMSO-d6): δ = 10.62 (2H, s, NHamidine), 10.34 (1H, s,
NHamide), 8.10 (2H, d, J = 8.78, Hz, Aromatic CH), 7.93
(2H, d, J = 8.80, Hz, Aromatic CH), 7.74 (1H, s, Aromatic
CH), 6.73 (1H, s, Aromatic CH), 3.99 (6H, s, OCH3), 3.97
(4H, s, CH2), 3.86 (3H, s, OCH3); 13C NMR (75 MHz,
DMSO-d6): δ = 164.3, 164.1, 163.3, 158.4, 144.4,
129.6 (2C), 119.3 (2C), 116.1, 115.6, 105.8, 98.6, 56.2 (2C),
55.6, 44.1 (2C); anal. calcd. for C19H22ClN3O4: C, 58.40;
H, 5.79; N, 10.52. Found: C, 58.24; H, 5.66; N, 10.72.
This compound was prepared using above described
method from 2,4-dimethoxybenzaldehyde 19 (0.15 g, 0.90
mmol) in ethanol (10 mL), p-benzoquinone (0.10 g, 0.90
mmol) and 4-amidino-1,2-phenylenediamine hydrochloride
21 (0.17 g, 0.90 mmol). The mixture was refluxed for 4 h
and worked up as it is described to give 0.210 g (76%) of
1
light gray powder; mp 244–245 °C; H NMR (300 MHz,
DMSO-d6): δ = 12.55 (1H, bs, NHbenzimidazole), 9.34 (2H, s,
NHamidine), 9.13 (2H, s, NHamidine), 8.30 (1H, d, J = 8.64,
Hz, Aromatic CH), 8.15 (1H, s, Aromatic CH), 7.80 (1H, d,
J = 8.46, Hz, Aromatic CH), 7.66 (1H, dd, J = 8.49, 1.53,
Hz, Aromatic CH), 6.80 (1H, d, J = 2.13, Hz, Aromatic
CH), 6.76 (1H, dd, J = 8.72, 2.24, Hz, Aromatic CH), 4.06
(3H, s, OCH3), 3.88 (3H, s, OCH3); 13C NMR (150 MHz,
DMSO-d6): δ = 166.4 (2C), 162.9, 158.6, 152.0, 131.3,
121.6, 121.0, 115.6, 109.7, 106.6, 98.6 (2C), 56.0, 55.6;
anal. calcd. for C16H17ClN4O2: C, 57.51; H, 5.29; N, 16.99.
Found: C, 57.75; H, 5.15; N, 16.84.
General method for the synthesis of compounds 23–28
5(6)-Amidino-2-(3,4,5-trimethoxyphenyl)benzimidazole
Solution of corresponding benzaldehyde, 4-amidino-
substituted 1,2-phenylenediamine and p-benzoquinone in
equimolar amounts in absolute ethanol was refluxed for 4 h.
After reaction mixture was cooled to room temperature, die-
thyl ether was added and the crude product was filtered off.
The crude product was suspended in mixture of ethanol/die-
thyl ether several times until the powder was analytically pure.
hydrochloride (25)
This compound was prepared using above described method
from 3,4,5-trimethoxybenzaldehyde 20 (0.15 g, 0.77 mmol)
in ethanol (15 mL), p-benzoquinone (0.08 g, 0.77 mmol) and
4-amidino-1,2-phenylenediamine hydrochloride 21 (0.14 g,
0.77 mmol). The mixture was refluxed for 4 h and worked
up as it is described to give 0.125 g (45%) of light gray
1
5(6)-Amidino-2-(2-methoxyphenyl)benzimidazole
powder; mp 239–241 °C; H NMR (300 MHz, DMSO-d6):
hydrochloride (23)
δ = 13.87 (1H, bs, NHbenzimidazole), 9.37 (2H, s, NHamidine),
9.08 (2H, s, NHamidine), 8.14 (1H, s, Aromatic CH), 7.77
(1H, d, J = 8.43, Hz, Aromatic CH), 7.67—7.65 (3H, m,
This compound was prepared using above described
method from 2-methoxybenzaldehyde 18 (0.15 g, 1.10
mmol) in ethanol (10 mL), p-benzoquinone (0.12 g, 1.10
mmol) and 4-amidino-1,2-phenylenediamine hydrochloride
21 (0.21 g, 1.10 mmol). The mixture was refluxed for 4 h
and worked up as it is described to give 0.22 g (71%) of
Aromatic CH), 3.91 (6H, s, OCH3), 3.73 (3H, s, OCH3); 13
C
NMR (75 MHz, DMSO-d6): δ = 166.6 (2C), 154.7, 153.7
(2C), 140.1, 124.8, 122.5 (2C), 121.7, 116.1, 105.0 (2C),
60.7, 56.7 (2C); anal. calcd. for C17H19ClN4O3: C, 56.48; H,
5.39; N, 15.59. Found: C, 56.28; H, 5.28; N, 15.44.
1
light brown powder; mp 232–234 °C; H NMR (300 MHz,
DMSO-d6): δ = 9.41 (2H, s, NHamidine), 9.18 (2H, s,
NHamidine), 8.37 (1H, dd, J = 7.62, 1.14, Hz, Aromatic CH),
8.23 (1H, s, Aromatic CH), 7.88 (1H, d, J = 8.46, Hz,
Aromatic CH), 7.73 (1H, dd, J = 8.45, 1.14, Hz, Aromatic
CH), 7.62–7.56 (1H, m, Aromatic CH), 7.31 (1H, d, J =
5(6)-(2-Imidazolinyl)-2-(2-methoxyphenyl)benzimidazole
hydrochloride (26)
This compound was prepared using above described
method from 2-methoxybenzaldehyde 18 (0.15 g, 1.10

Med Chem Res
mmol) in ethanol (10 mL), p-benzoquinone (0.12 g, 1.10
mmol) and 4-(2-imidazolinyl)-1,2-phenylenediamine
OCH3); 13C NMR (75 MHz, DMSO-d6): δ = 165.9, 153.7
(2C), 140.0, 124.9, 116.1, 115.7, 105.1 (2C), 97.3, 60.9,
58.7 (2C), 44.7 (2C); anal. calcd. for C19H21ClN4O3: C,
58.92; H, 5.28; N, 14.27. Found: C, 58.69; H, 5.44; N,
14.41.
hydrochloride 22 (0.23 g, 1.10 mmol). The mixture was
refluxed for 4 h and worked up as it is described to give
0.175 g (49%) of light brown powder; mp 265–267 °C;
1H NMR (300 MHz, DMSO-d6): δ = 12.72 (1H, bs,
NHbenzimidazole), 10.67 (2H, s, NHamidine), 8.39 (1H, bs,
Aromatic CH), 8.33 (1H, dd, J = 7.77, 1.68, Hz, Aromatic
CH), 7.90–7.83 (2H, m, Aromatic CH), 7.59–7.53 (1H, m,
Aromatic CH), 7.29 (1H, d, J = 8.34, Hz, Aromatic CH),
7.16 (1H, t, J = 7.49, Hz, Harom.), 4.05 (3H, s, OCH3), 4.02
(4H, s, CH2); 13C NMR (150 MHz, DMSO-d6): δ = 166.1,
164.0, 157.6, 149.7, 132.9, 130.6, 122.6, 121.5 (2C), 117.6,
116.1, 115.7, 112.8, 56.0, 44.7 (2C); anal. calcd. for
C17H17ClN4O: C, 62.35; H, 5.38; N, 16.90. Found: C,
62.10; H, 5.21; N, 17.04.
General method for the synthesis of cyano substituted N-
2-benzimidazolyl methoxybenzamides (30–31)
To a solution of corresponding benzoyl chlorides 2–3 in dry
toluene, a solution of 2-amino-5(6)-cyanobenzimidazole 29
in dry toluene was added dropwise, followed by the addi-
tion of Et3N. The reaction mixture was refluxed for several
hours. After cooling the solution was concentrated and the
obtained solid was filtered off and recrystallized from
appropriate solvent.
5(6)-(2-Imidazolinyl)-2-(2,4-dimethoxyphenyl)
N-(5(6)-Cyanobenzimidazol-2-yl)-2,4-dimethoxybenzamide
benzimidazole hydrochloride (27)
(30)
This compound was prepared using above described
method from 2,4-dimethoxybenzaldehyde 19 (0.15 g, 0.90
mmol) in ethanol (10 mL), p-benzoquinone (0.10 g, 0.90
This compound was prepared using above described method
from 2,4-dimethoxybenzoyl chloride 2 (0.19 g, 0.95 mmol)
and 2-amino-5(6)-cyanobenzimidazole 29 (0.15 g, 0.95
mmol) in dry toluene (10 mL) followed by the addition of
Et3N (0.19 mL, 1.33 mmol) after refluxing for 36 h and
recrystalization from methanol/acetone to obtain 0.11 g
mmol)
and
4-(2-imidazolinyl)-1,2-phenylenediamine
hydrochloride 22 (0.19 g, 0.90 mmol). The mixture was
refluxed for 4 h and worked up as it is described to give
0.125 g (39%) of light brown powder; mp 273–275 °C;
1H NMR (300 MHz, DMSO-d6): δ = 12.48 (1H, bs,
NHbenzimidazole), 10.64 (2H, s, NHamidine), 8.38 (1H, bs,
Aromatic CH), 8.27 (1H, d, J = 8.64, Hz, Aromatic CH),
7.87 (1H, d, J = 8.40, Hz, Aromatic CH), 7.79 (1H, d, J =
8.16, Hz, Aromatic CH), 6.79 (1H, d, J = 2.19, Hz, Aro-
matic CH), 6.75 (1H, dd, J = 8.72, 2.27, Hz, Aromatic CH),
4.05 (3H, s, OCH3), 4.02 (4H, s, CH2), 3.88 (3H, s, OCH3);
13C NMR (150 MHz, DMSO-d6): δ = 165.6, 162.7, 131.3,
121.9, 119.2, 115.6, 114.8, 110.0, 106.6, 98.9, 56.0, 55.6,
44.2 (2C); anal. calcd. for C18H19ClN4O2: C, 60.40; H,
5.52; N, 15.79. Found: C, 60.25; H, 5.34; N, 15.61.
1
(36%) of light yellow powder; mp 236–239 °C; H NMR
(300 MHz, DMSO-d6): δ = 12.71 (1H, brs, NHbenzimidazole),
11.07 (1H, s, NHamide), 7.90 (1H, d, J = 8.49, Hz, Aromatic
CH), 7.89 (1H, s, Aromatic CH), 7.62 (1H, d, J = 8.25, Hz,
Aromatic CH), 7.51 (1H, dd, J = 8.27, 1.54, Hz, Aromatic
CH), 6.77 (1H, d, J = 2.01, Hz, Aromatic CH), 6.74 (1H, dd,
J = 8.64, 2.25, Hz, Aromatic CH), 4.01 (3H, s, OCH3), 3.88
(3H, s, OCH3); 13C NMR (150 MHz, DMSO-d6): δ = 164.8,
164.4, 159.7, 133.0, 113.6, 107.0, 99.2 (2C), 57.0, 56.2;
anal. calcd. for C19H20N5O3: C, 63.52; H, 4.20; N, 17.22.
Found: C, 63.35; H, 4.38; N, 17.38.
N-(5(6)-Cyanobenzimidazol-2-yl)-3,4,5-
5(6)-(2-Imidazolinyl)-2-(3,4,5-trimethoxyphenyl)
trimethoxybenzamide (31)
benzimidazole hydrochloride (28)
This compound was prepared using above described
method from 3,4,5-trimethoxybenzoyl chloride 3 (0.50 g,
2.18 mmol) and 2-amino-5(6)-cyanobenzimidazole 29
(0.35 g, 2.18 mmol) in dry toluene (40 mL) followed by the
addition of Et3N (0.19 mL, 1.33 mmol) after refluxing for
24 h and recrystalization from methanol to obtain 0.55 g
(72%) of white powder; mp 247–250 °C; 1H NMR (300 MHz,
DMSO-d6): δ = 12.75 (1H, brs, NHbenzimidazole), 12.26 (1H,
s, NHamide), 7.94 (1H, d, J = 0.80, Hz, Aromatic CH), 7.65
(1H, d, J = 8.28, Hz, Aromatic CH), 7.54 (1H, dd, J = 8.27,
1.55, Hz, Aromatic CH), 7.51 (2H, s, Aromatic CH), 3.90
(3H, s, OCH3), 3.76 (3H, s, OCH3), 3.77 (3H, s, OCH3);
This compound was prepared using above described
method from 3,4,5-trimethoxybenzaldehyde 20 (0.15 g,
0.77 mmol) in ethanol (5 mL), p-benzoquinone (0.083 g,
0.77 mmol) and 4-(2-imidazolinyl)-1,2-phenylenediamine
hydrochloride 22 (0.16 g, 0.77 mmol). The mixture was
refluxed for 4 h and worked up as it is described to give
1
0.120 g (40%) of beige powder; mp 249–250 °C; H NMR
(300 MHz, DMSO-d6): δ = 13.95 (1H, bs, NHbenzimidazole),
10.68 (2H, s, NHamidine), 8.37 (1H, bs, Aromatic CH),
7.88–7.73 (2H, m, Aromatic CH), 7.68 (2H, s, Aromatic
CH), 4.03 (4H, s, CH2), 3.93 (6H, s, OCH3), 3.76 (3H, s,

Med Chem Res
13C NMR (150 MHz, DMSO-d6): δ = 166.2, 153.2 (2C),
150.3, 141.7, 133.7, 127.8, 125.6, 120.7 (2C), 107.1, 106.3
(3C), 103.4, 60.6, 56.9 (2C); anal. calcd. for C18H16N4O4:
C, 61.54; H, 4.76; N, 15.68. Found: C, 61.36; H, 4.58; N,
15.90.
N-(5(6)-Amidinobenzimidazol-2-yl)-3,4,5-
trimethoxybenzamide hydrochloride (33)
This compound was prepared using above described
method. A solution of 31 (0.20 g, 0.57 mmol) in absolute
ethanol (15 mL) was saturated with dry HCl gas and stirred
for 6 days. The crude imidate was filtered off, suspended in
absolute ethanol (20 mL) and dry NH3 was bubbled into the
suspension. The mixture was worked up as it is described to
General method for the synthesis of amidino substituted
N-2-benzimidazolyl methoxybenzamides (32–35)
1
A stirred suspension of 5(6)-cyanobenzimidazol-2-yl sub-
stituted methoxybenzamides 30–31 in absolute ethanol was
cooled in an ice-salt bath and was saturated with dry HCl
gas. The reaction mixture was maintained at room tem-
perature until nitrile band disappeared (monitored by IR
analysis at 2200 cm−1). The corresponding imidate was
filtered off, washed with dry diethyl ether and dried under
reduced pressure.
give 0.16 g (69%) of white powder; mp 224–226 °C; H
NMR (300 MHz, DMSO-d6): δ = 12.76 (1H, s, NH), 12.21
(2H, s, NH), 9.28 (2H, s, NHamidine), 9.02 (2H, s, NHamidine),
8.02 (1H, s, Aromatic CH), 7.68 (1H, d, J = 8.34, Hz,
Aromatic CH), 7.63 (1H, d, J = 8.46, Hz, Aromatic CH),
7.53 (2H, s, Aromatic CH), 3.91 (6H, s, OCH3), 3.77 (3H, s,
OCH3); 13C NMR (75 MHz, DMSO-d6): δ = not enough
soluble; anal. calcd. for C18H20ClN5O2: C, 53.48; H, 4.75;
N, 17.44. Found: C, 53.27; H, 4.97; N, 17.26.
Amidines
N-[5(6)-2-(Imidazolinyl)benzimidazol-2-yl]-2,4-
The corresponding imidate was suspended in absolute
ethanol and dry NH3 was bubbled into the suspension. The
mixture was stirred at room temperature for 2 days. The
crude product was then filtered off and washed with diethyl
ether.
dimethoxybenzamide hydrochloride (34)
This compound was prepared using above described
method. A solution of 30 (0.18 g, 0.54 mmol) in absolute
ethanol (20 mL) was saturated with dry HCl gas and stirred
for 10 days. The crude imidate was filtered off, suspended
in absolute ethanol (20 mL) and EDA (0.107 ml, 1.61
mmol) was added into the suspension. The mixture was
worked up as it is described to give 0.09 g (41%) of light
yellow powder; mp 273–275 °C; 1H NMR (300 MHz,
DMSO-d6): δ = 11.41 (1H, s, NHamide), 10.79 (2H, s,
NHamidine), 8.29 (1H, s, Aromatic CH), 7.97 (1H, d, J =
8.49, Hz, Aromatic CH), 7.95 (1H, dd, J = 8.46, 1.50, Hz,
Aromatic CH), 7.78 (1H, d, J = 8.49, Hz, Aromatic CH),
6.82–6.76 (2H, m, Aromatic CH), 4.02 (3H, s, OCH3), 4.01
(4H, s, CH2), 3.90 (3H, s, OCH3); 13C NMR (75 MHz,
DMSO-d6): δ = 164.9, 163.3, 159.6, 147.3, 133.1, 123.4,
116.3, 115.0, 114.1, 111.8, 107.0, 98.7, 56.8, 55.6, 44.2
(2C); anal. calcd. for C19H20ClN5O3: C, 56.95; H, 5.21; N,
17.24. Found: C, 56.79; H, 5.02; N, 17.43.
2-Imidazolinylamidines
EDA was added to a suspension of the corresponding
imidate in absolute ethanol (5 mL) and the mixture was
stirred at reflux for 24 h. The crude product was then filtered
off and washed with diethyl ether.
N-(5(6)-Amidinobenzimidazol-2-yl)-2,4-
dimethoxybenzamide hydrochloride (32)
This compound was prepared using above described
method. A solution of 30 (0.18 g, 0.54 mmol) in absolute
ethanol (20 mL) was saturated with dry HCl gas and stirred
for 10 days. The crude imidate was filtered off, suspended
in absolute ethanol (20 mL) and dry NH3 was bubbled into
the suspension. The mixture was worked up as it is
described to give 0.05 g (25%) of light yellow powder; mp
259–262 °C; 1H NMR (300 MHz, DMSO-d6): δ = 11.08
(1H, s, NHamide), 9.33 (2H, s, NHamidine), 9.17 (2H, s,
NHamidine), 8.01 (1H, s, Aromatic CH), 7.92 (1H, d, J =
8.55, Hz, Aromatic CH), 7.64 (1H, d, J = 2.55, Hz,
Aromatic CH), 6.78–6.73 (2H, m, Aromatic CH), 4.03
(3H, s, OCH3), 3.87 (3H, s, OCH3); 13C NMR (150 MHz,
DMSO-d6): δ = 166.3, 164.3, 163.8, 159.2, 148.6, 132.6
(2C), 121.4, 120.6, 120.5, 113.0, 106.6, 98.7 (2C), 56.6,
55.8; anal. calcd. for C17H18ClN5O3: C, 54.61; H, 4.65; N,
18.78. Found: C, 54.33; H, 4.83; N, 18.64.
N-[5(6)-2-(Imidazolinyl)benzimidazol-2-yl]-3,4,5-
trimethoxybenzamide hydrochloride (35)
This compound was prepared using above described
method. A solution of 31 (0.20 g, 0.57 mmol) in absolute
ethanol (15 mL) was saturated with dry HCl gas and stirred
for 6 days. The crude imidate was filtered off, suspended in
absolute ethanol (20 mL) and EDA (0.128 ml, 1.92 mmol)
was added into the suspension. The mixture was worked up
as it is described to give 0.15 g (61%) of white powder; mp
277–279 °C; 1H NMR (300 MHz, DMSO-d6): δ = 11.46
(1H, s, NHamide), 9.96 (2H, s, NHamidine), 7.96 (1H, d,

Med Chem Res
Table 1 Reducing activity % with DPPH at 30/60 min (RA%) and
reducing power mmolFe2+/mmolc using the FRAP assay of tested
compounds 6–17, 23–28 and 32–35
J = 1.08, Hz, Aromatic CH), 7.65 (1H, dd, J = 8.37, 1.53,
Hz, Aromatic CH), 7.51 (2H, s, Aromatic CH), 7.48 (1H, d,
J = 8.43, Hz, Aromatic CH), 3.89 (6H, s, OCH3), 3.76 (3H,
s, OCH3), 3.66 (4H, s, CH2); 13C NMR (75 MHz, DMSO-
d6): δ = 167.2, 165.3, 153.2, 150.4, 141.9, 138.6, 136.4,
128.9, 121.7, 115.2, 114.3, 113.7, 111.1, 106.9 (2C), 60.6,
56.7 (2C), 48.4 (2C); anal. calcd. for C20H22ClN5O4: C,
55.85; H, 5.30; N, 16.38. Found: C, 55.62; H, 5.13; N,
16.22.
2+
Comp
RA% 30′ min
250 µMa
RA% 60′ min
250 µMa
FRAP mmolFe
mmolc
/
a
6
na
4.44 0.2
3.52 1.2
na
98.18 0.27
49.12 4.20
4.42 0.26
15.35 2.09
61.88 9.97
77.33 27.58
0.32 0.46
1.27 0.44
3.96 0.42
4.14 0.50
2.43 0.55
5.28 0.17
55.65 3.67
67.11 1.56
125.66 1.69
132.22 3.89
125.70 0.67
126.14 6.68
2.53 0.12
3.21 0.32
3.15 0.15
4.52 0.21
nt
7
na
8
na
9
1.96 1.1
3.52
1
10
11
12
13
14
15
16
17
23
24
25
26
27
28
32
33
34
35
1.57
na
1
1.34 0.1
na
Antioxidative activity
6.79
6.01
na
1
1
3.67 2.2
Determination of the reducing activity (RA) of the stable
radical 1,1-diphenyl-picrylhydrazyl (DPPH)
8.88
na
1
6.40
na
1
0.062663
na
The determination of RA% was measured according to
previously reported procedure with modification for use of a
96-well microplate reader. Briefly, to solution of DPPH
(final concentration 50 µM) in absolute ethanol was added
equal volume of various concentrations of tested compounds
various concentrations dissolved in DMSO. The assay was
carried out in a 96-well microtiter plate. Ethanol and DMSO
was used as control solution according to the previously
published experimental procedure (Tireli et al. 2017).
na
na
16.84
1
18.4073 0.2
36.68 0.9
22.45 0.6
55.13 1.2
44.6475 0.1
63.45 0.2
0.22 0.03
14.10 1.1
na
31.59 2.4
17.76 0.5
53.25 1.45
45.97 1.5
61.62 0.2
0.26 0.02
12.01 0.9
na
Determination of ferric reducing/antioxidant power (FRAP
assay)
na
na
Ascorbic
acid
99 1.1
99
2
The FRAP method was performed according to previously
reported procedure with minor modifications for assay on a
96-well microplate reader (Tireli et al. 2017). All results
were then expressed as Fe2+ equivalents (Fe2+ µmol). All
tests were done in triplicate and the results were averaged
and presented in Table 1.
BHT
nt
nt
679.15 37.48
Na no activity under the reported experimental conditions. Nt not
tested
a
Values are means SD of three or three different determinations
Monomethoxy, dimethioxy and trimethoxy substituted
benzamides 6–11 were prepared in the condensation from
benzoyl chlorides 1–3 and corresponding 4-substituted
anilines 4–5 in absolute toluene and triethylamine. Using
the same synthetic procedure, 2-benzimidazolyl substituted
benzamides 30–31 were additionally prepared. In addition,
cyano substituted benzamides 9–11 and 30–31 were con-
verted in the acidic Pinner reaction into amidino 12–14 and
32–33 and 2-imidazolinyl 15–17 and 34–35 substituted
benzamides (Hranjec et al. 2013). To ensure better solubi-
lity, all amidino substituted derivatives were prepared as
hydrochloride salts.
Antiproliferative activity
The experiments were carried out on three human cell lines:
HCT 116 (colon carcinoma), H 460 (lung carcinoma) and
MCF-7 (breast carcinoma) according to the previously
published experimental procedure (Hranjec et al. 2007;
Starčević et al. 2007).
Results and discussion
Chemistry
Amidino and 2-imidazolinyl substituted benzimidazoles
23–28 were prepared according to the Scheme 2 within
the condensation of corresponding benzaldehydes 18–20
with 4-amidino/4-(2-imidazolinyl)-1,2-phenylenediamines
hydrochlorides 21–22 with p-benzoquinone as oxidizing
reagent in absolute ethanol (Hranjec et al. 2007; Starčević
All novel benzamide 6–17 and benzimidazole derivatives
23–28 and 30–35 were prepared according to the main
experimental procedures shown in Schemes 1–3 starting
from the benzoyl chlorides 1–3 and benzaldehydes 18–20
(Starčević et al. 2006; Ester et al. 2009).

Med Chem Res
Scheme 1 Synthesis of
compounds 6–17
Scheme 2 Synthesis of
compounds 23–28
et al. 2007). Due to the some synthetic issues, mono-
methoxy substituted analogs of compounds 32–35 have not
been successfully synthesized.
(Gutowski and Kowalczyk 2013). Much indirect evidence
has suggested that most products of lipid peroxidation have
mutagenic and/or carcinogenic properties and cause oxida-
tive damage of important cellular constituents (lipids, pro-
teins and DNA) and play important role in the pathogenesis
of various diseases such as cancer, atherosclerosis, rheu-
matoid arthritis and ischemic injury (Lobo et al. 2010).
Determination of the antioxidant activity of single com-
pounds could be estimated by different in vitro biological
assays. Two types of assays are usually employed: (i) assays
in which scavenging of a preformed free radical by a
hydrogen atom or electron donation is taken as a marker of
antioxidant activity and (ii) assays involving the presence of
an antioxidant during the generation of the radical.
Accordingly, the radical scavenging capacity of final
The structures of all newly prepared derivatives 6–17,
1
23–28 and 30–35 were determined by using H and 13C
NMR spectroscopy and elemental analysis. NMR analysis
was based on the values of H–H coupling constants and
1
chemical shifts in the H and 13C NMR spectra. Further-
more, IR spectroscopy was used for the monitoring of
Pinner reaction.
Antioxidative activity
Lipid peroxidation is a chain reaction initiated by reactive
many ROS such as (.OH, O2.−, L, LOO., LO., etc.)

Med Chem Res
Scheme 3 Synthesis of
compounds 30–35
compounds was measured against DPPH stable radical as
well as ferric reducing ability/antioxidant power (FRAP).
The radical scavenging method using relatively stable
organic radical DPPH is based on the reduction of alcoholic
DPPH solutions in the presence of a hydrogen donating
antioxidant. DPPH solutions show a strong absorption band
at 517 nm appearing a deep violet color. The presence of
antioxidant in solution resulting in decolorization and the
remaining DPPH measured after a certain time corresponds
inversely to the radical scavenging activity of the anti-
oxidant (Molyneux 2004). The results for the RA of the
examined compounds using the stable free radical DPPH
are depicted in (Table 1). Ascorbic acid was used as a
standard antioxidant. The RA% of compounds, which
indicates their radical scavenging ability in an iron-free
system, was measured at 50 µM (30/60 min). In the DPPH
assay, the dominant chemical reaction involved is the
reduction of the DPPH radical by electron transfer from the
antioxidant. The results obtained in this study were
expressed as RA% values with the exception of compounds
8, 11, 14, 16–17 and 34–35 which did not show reaction
under the experimental condition. Based on experimental
results presented, among all synthesized compounds, 2-
substituted benzimidazoles 23–28 showed scavenging
activity towards DPPH while benzamide derivatives 6–17
and 32–35 did show low or no activity.
group induced different antioxidative activity of compounds
23–28.
From gained experimental results the best activity was
observed for compound 28 with three methoxy group
attached at position 3, 4, and 5 of phenyl moiety. Addi-
tionally, the different amidine groups at benzimidazole
moiety contributed to antioxidative activity of compounds
as well. According to results the compounds with
imidazolinyl-amidine group attached on benzimidazole
moiety showed best results in general. The ferric reducing
power (FRAP) assay was used to determine the reducing
power of novel compounds and results are presented in
Table 1 (Benzie and Strain 1996). The FRAP values are
expressed as mmols of ferrous ion per mmol of compound.
Butylated hydroxytoluene (BHT) was used as a standard
antioxidant. In general, the compounds containing two and
three methoxy groups showed highest FRAP values among
all compounds, with the exception of compound 6 which
bears one methoxy group. The phenyl derivatives with
directly attached 5-amidino benzimidazoles showed pro-
mising reducing power ability in comparison to compounds
with inserted amide bond (25–28). The imidazolinyl-
amidine group at the 5-position of benzimidazole ring
directly attached to the phenyl derivative may have an
influence on ferric reducing power of an antioxidant.
Regarding structure–activity relationships of synthesized
benzamide and benzimidazole derivatives revealed that
insertion of amide bond between two aromatic rings,
methoxy substituted phenyl and amidino substituted phenyl/
benzimidazole, inhibited scavenging activity towards DPPH
while direct substitution of phenyl derivatives with benzi-
midazole ring causes relatively high antioxidant activity
(23–28). The different number of methoxy groups at phenyl
Antiproliferative activity
The antiproliferative activities of phenyl substituted 12–17
and 2-benzimidazolyl substituted 32–35 benzamides and
benzimidazole derivatives 23–28 bearing two types of
amidine substituents were tested on HCT 116, H460 and
MCF-7 cell lines as it is presented in Table 2. The tested
compounds showed different antiproliferative activity

Med Chem Res

Med Chem Res
towards three tumor cell lines. Etoposide was used as a
standard anticancer drug. The presented compounds were
designed in order to study the influence of the number of
methoxy groups as well as the type of aromatic nuclei
connected to the phenyl ring with amide bond (12–17 and
32–35) or without amide bond (23–28) on antiproliferative
activity. In addition, our attention was also focused on the
influence of the type of amidine substituent attached on the
aromatic nuclei, including unsubstituted or 2-imidazolinyl
amidine group on antiproliferative activity.
From the SAR study it could be concluded that mono-
methoxy, dimethoxy and trimethoxy amidino substituted
benzamides 12–14 exerted selective activity against MCF-
7. With increasing the number of methoxy groups the
antiproliferative activity was improved without significant
difference between dimethoxy and trimethoxy substituted
derivatives 13 and 14. In general, their 2-imidazolinyl
substituted monomethoxy, dimethoxy and trimethoxy ana-
logs 15–17 showed significant improvement of anti-
proliferative activity without any selectivity among tested
cell lines. Due to the replacement of phenyl ring with the
benzimidazole nuclei attached to the benzamide moiety in
the structure of compound 33, activity against MCF-7 cell
line was significantly increased. Among all tested deriva-
tives, trimethoxy substituted 2-benzimidazolyl amide 33
bearing amidino group showed the most prominent anti-
proliferative activity with selective activity against MCF-7
cell line (IC50 = 9 7 μM, Fig. 1).
It is well known that lipophilicity is prerequisite for the
penetration and crossing the cell membrane of compounds.
The definition of logP as 1-octanol/water partition coeffi-
cient is indicative for lipophilicity or hydrophilicity of
newly prepared compounds. The logP values known as
clogP values were calculated by using ChemDraw Ultra
10.0 (http://www.cambridgesoft.com). In the Table 2 the
values of clogP for the neutral forma of the prepared
compounds are also presented. From the obtained results it
was obvious that the clogP values are dependent on the
number of methoxy groups. Thus, within the increasing of
methoxy groups in the structure of novel compounds, the
clogP values decreased. Furthermore, the different types of
amidino groups attached to the phenyl or benzimidazolyl
moiety showed the significant difference in the clogP
values. Derivatives bearing the amidino group exerted
lower clogP values (1.36–1.88) in comparison to the 2-
imidazolinyl substituted analogs which displayed higher
clogP values (1.87–2.39). Moreover, it could be concluded
that amidino substituted derivatives are in general more
hydrophilic than their 2-imidazolynyl analogs being more
lipophilic. Due to the removal of amide bond between two
phenyl and/or 2-benzimidazolyl nuclei, the clogP values
increased. The exception was derivative 33 which showed
the most prominent antiproliferative activity despite the low
clogP value which is not in accordance with above men-
tioned observations.
From results observed in this research it is evident that
the compounds that showed the highest antioxidant activity
are less cytotoxic and do not inhibit the proliferation of
cancer cell lines. Namely, ROS have been implicated in
cancer promotion and progression. For this reason, low
concentrations of free radicals due to an excessive admin-
istration of antioxidants may promote the proliferation of
harmful cells in the neoplastic state, promoting the devel-
opment of cancer rather than interrupting it (Valko et al.
2007a, b). Thus therapeutic antioxidants may be used for
cancer prevention to prevent early events in tumor
Surprisingly, 2-imidazolinyl analogs 34 and 35 did not
show any activity against all tested cell lines. Due to the
exclusion of amide bond in the structure of compounds 23–28
the antiproliferative activity was decreased. Amidino sub-
stituted derivatives 23–25 did not show any activity at all while
their 2-imidazolinyl monomethoxy and dimethoxy substituted
analogs 26 and 27 showed moderate antiproliferative activity
without any expressed selectivity among tested cell lines.
Trimethoxy substituted derivative 28 was inactive which is
comparable with the inactivity of its amide 35.
Fig. 1 SAR for antiproliferative
activity

Med Chem Res
multidisciplinary approach to discover selective drugs targeting cancer
stem cells: The role of potassium transport—MultiCaST).
development, where ROS are important. Nevertheless,
cancer cells have a higher level of oxidative stress compared
to normal cells due to differences in the metabolic activity
of cancer cells and also increased levels of antioxidant
enzymes (Matés et al. 2012), suggesting that a fine balance
of intracellular ROS levels is a prerequisite for cancer cell
function. Therefore, tumor cells may be more sensitive to
compounds that generate certain amounts of ROS, or
compounds that damage the ROS scavenging capacity of
cells, leading to ROS-induced apoptosis (Reuter et al. 2010;
Liou and Storz 2010). By exploring the mechanisms and
specific apoptotic pathways involved in cell death induced
by ROS could be the way to determine the most effective
antioxidative agents.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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Herein, we present the synthesis, antioxidative and anti-
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