Identification of a novel series of N-phenyl-5-[(2-phenylbenzimidazol-1-yl) methyl]-1,3,4-oxadiazol-2-amines as potent antioxidants and radical scavengers

Article abstract of DOI:10.1002/ardp.201300324

In this study, some novel 5-[[2-(phenyl/p-chlorophenyl)-benzimidazol-1-yl]- methyl]-N-substituted phenyl-1,3,4-oxadiazol-2-amine derivatives (28-45) with an oxadiazole ring were synthesized. The antioxidant properties and radical scavenging activities of

Full text of DOI:10.1002/ardp.201300324

276
Arch. Pharm. Chem. Life Sci. 2014, 347, 276–282
Full Paper
Identification of a Novel Series of N-Phenyl-5-[(2-
phenylbenzimidazol-1-yl)methyl]-1,3,4-oxadiazol-2-amines as
Potent Antioxidants and Radical Scavengers
Gulgun Ayhan-Kilcigil1, Canan Kuş1, Tülay Çoban2, Elcin D. Özdamar2, and Benay Can-Eke2
1
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara University, Ankara, Turkey
Faculty of Pharmacy, Department of Toxicology, Ankara University, Ankara, Turkey
2
In this study, some novel 5-[[2-(phenyl/p-chlorophenyl)-benzimidazol-1-yl]-methyl]-N-substituted phenyl-
1,3,4-oxadiazol-2-amine derivatives (28–45) with an oxadiazole ring were synthesized. The antioxidant
properties and radical scavenging activities of the compounds were investigated employing various
in vitro systems: hepatic microsomal NADPH-dependent inhibition of lipid peroxidation levels,
scavenging of DPPH free radicals, and inhibition of microsomal ethoxyresorufin O-deethylase activity
(EROD). Compounds 34 and 41 were found to be good scavengers of DPPH radicals (76% and 84%) when
compared to BHT (90%). Almost all of the compounds examined were found to possess a good inhibitor
effect on the microsomal EROD activity. Moreover, 32 and 41 were more active analogs (97% and 98%)
on the microsomal EROD activity than caffeine (85%).
Keywords: Benzimidazoles / DPPH / EROD / Lipid peroxidation / Oxadiazoles
Received: August 25, 2013; Revised: September 29, 2013; Accepted: October 1, 2013
DOI 10.1002/ardp.201300324
Introduction
effects by scavenging radicals, binding metal ions, and
inhibiting enzymatic systems. The biological activities of
antioxidants have been reviewed many times and some have
been declared to be involved in cancer, heart diseases,
brain dysfunction, atherosclerosis, and immune system
problems [12, 13].
We have already reported the synthesis, characterization,
and antioxidant properties of some benzimidazole derivatives
that have thiadiazole, triazole, and oxadiazole [5–10]. This
report deals with the antioxidant properties of some novel
benzimidazole derivatives, 5-[[2-(phenyl/p-chlorophenyl)benzimi-
dazol-1-yl]methyl]-N-substituted phenyl-1,3,4-oxadiazol-2-amines
(28–45), having an oxadiazole ring instead of thiadiazole and
triazole rings.
Nowadays, plenty of benzimidazole derivatives are in clinical
usage, e.g., as antiparasitic, antihistaminic, angiotensin II
antagonist, proton pump inhibitor, and antipsychotic. The
position and the type of the substituents on the benzimid-
azole ring are responsible for the variety of biological
activities, including antimicrobial, antifungal, anticancer,
anthelmintic, antiallergic, antioxidant, etc. Previously, anti-
microbial [1, 2], antiparasitic [3], antihistaminic [4], and
antioxidant [5–11] activity evaluations of some benzimidazole
derivatives have been studied in our faculty.
Antioxidant systems, including superoxide dismutase,
catalase, and glutathione, should keep the oxidative processes
in balance. However, in the case of the deficiency of
nutritional antioxidants (vitamin A, C, E, the minerals Results and discussion
selenium and zinc, coenzyme Q10, lipoic acid, etc.), these
systems could be affected. Antioxidants can exert their
In this study, a series of new compounds 28–45 having
2-(substituted phenylamino)oxadiazole as a substituent at 1st
position of benzimidazole were synthesized and evaluated for
their effects on the rat liver microsomal NADPH-dependent
lipid peroxidation levels by measuring the formation of
2-thiobarbituric acid reactive substances. The interaction with
Correspondence: Dr. Canan Kuş, Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Ankara University, 06100 Ankara, Turkey.
E-mail: kus@pharmacy.ankara.edu.tr
Fax: þ90 312 2131081
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Arch. Pharm. Chem. Life Sci. 2014, 347, 276–282
N-Phenyl-5-[(2-phenylbenzimidazol-1-yl)methyl]-1,3,4-oxadiazol-2-amines
277
the stable free radical DPPH and inhibition on microsomal
ethoxyresorufin O-deethylase (EROD) activity was also exam-
ined. For the synthesis of the desired compounds, the reaction
sequences outlined in Scheme 1 were followed.
clearly showed the 1H–13C connections of 43, so this
methylene carbon was very easily designated at 40.08 Hz. In
addition to this, it was not possible for the compounds 28 and
38. Because of the fluorine atom at the 13C NMR spectrum in
DMSO-d6 and at 100 MHz of 38 and 43, highly characteristic
13C–19F couplings were observed. Three doublet resonances
centered at 116.20 and 116.42 ( J ¼ 22.24), 119.29 and
119.36 ( J ¼ 7.65), 156.08 and 153.74 ( J ¼ 235.36) with 115.42
and 115.65 ( J ¼ 23.0), 118.47 and 118.54 ( J ¼ 7.0), 156.03 and
158.39 ( J ¼ 236.0 Hz), respectively, 38 and 43, arising from
the aromatic carbons of p-fluorophenyl coupled by the
fluorine atom. Resembling 13C–19F couplings were informed
in our former articles too [2]. There were similar 1H–19F
couplings in the 1H NMR spectra of 31, 35, 38, 40, and 43. As
expected, in the mass spectra of 30, 32, 34, 36, 39, 41–44, and
45, chlorine and bromine isotope signals were seen. Some
physico-chemical properties and spectral findings of final
products are given in Table 1.
Compounds 28–45 were evaluated by their effects on the
rat liver microsomal NADPH-dependent lipid peroxidation
levels by measuring the formation of 2-thiobarbituric acid
reactive substances, and also examined to interact with
the stable free radical DPPH and to inhibit on microsomal
EROD activity (Table 2). The results seem variable and fairly
similar to our former published results [5–10].
The final compounds (28–45) having oxadiazole ring
instead of triazole or thiadiazole were synthesized according
to our previous studies [5–10]. Compounds 1–3 having
benzimidazole ring were prepared via oxidative condensation
of o-phenylenediamine/4,5-dichloro-1,2-phenylenediamine,
non-substituted/p-chloro/3,4-dimethoxy benzaldehyde, and
sodium metabisulfite [14]. Treatment of compounds 1–3
with ethyl chloroacetate in KOH/dimethylsulfoxide (DMSO)
gave the N-alkylated products (4–6) [15]. Hydrazine hydrate
and the ester (4–6) in ethanol were refluxed for 4 h to give
the desired hydrazide compounds, (2-aryl-benzimidazol-1-yl)-
acetic acid hydrazides 7–9 [16]. The thiosemicarbazides
(10–27) were obtained upon the reaction of the acid
hydrazide with aryl isothiocyanates in ethanol [17]. Cycliza-
tion of 10–27 with KI and I2 [18] resulted in the formation
of N-substituted phenyl-5-[(2-substituted phenyl benzimidazol-
1-yl)-methyl]-1,3,4-oxadiazol-2-amine
(28–45)
derivatives
(Scheme 1). For the synthesis of compound 45, appropriate
thiosemicarbazide 27 (2-[(5,6-dichloro-2-(4-chloro-phenyl)-1H-
benzimidazol-1-yl)acetyl]-N-(3,4-dimethoxyphenyl)hydrazine
carbothioamide) was used directly without further
purifications.
The structures of the synthesized compounds were
consistent with the 1H and 13C NMR spectra. In the
13C NMR spectra, signal of methylene carbon of compound
29 appears at 40.28 ppm. At the same time, the signals of
methylene carbon of compounds 28, 38, and 43 could not seen
due to being overshadowed by DMSO-d6. Thus, further
verification was obtained from the HSQC spectrum, which
The inhibition of NADPH-dependent lipid peroxidation
produced by all new compounds in the rat liver
microsomes was examined by measuring the formation
of the 2-thiobarbituric acid reactive substances for their
antioxidant capacity. Compound 42 is the most active one
that caused 42% inhibition on LP level in rat liver microsomes
at 10ꢀ3 M concentration while BHT showed 65% inhibition at
the same concentration. Compounds 33 (26%), 37 (16%),
Scheme 1. Synthesis of derivatives 28–45.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 276–282
Table 1. Physical and spectral data of compounds 28–45.
%
Yield
Mp
(°C)
MS (MþH)
No.
28
Formula
Ar
Y
R
1H and 13C NMR d (ppm) DMSO-d6
(%)
C22H17N5O
–H
–H
37
277–279 5.74 (s, 2H, –CH2–), 6.95–7.05 (m, 1H, Ar–H), 7.28–7.36
(m, 4H, Ar–H), 7.48 (d, 2H, Jo ¼ 7.60 Hz, Ar–H), 7.58–7.62
(m, 3H, Ar–H), 7.69 (d, 1H, Jo ¼ 6.40 Hz, Ar–H), 7.74 (d, 1H,
Jo ¼ 6.80 Hz, Ar–H), 7.84–7.88 (m, 2H, Ar–H), 10.48 (s, 1H,
NH); 13C NMR: 111.64, 117.68, 120.03, 122.63, 123.30, 123.72,
129.57, 129.72, 130.01, 130.14, 130.77, 136.43, 139.09,
143.20, 153.75, 156.07, 160.90
368 (100)
29
C23H19N5O
–H o-CH3
72
241–243 2.19 (s, 3H, –CH3), 5.71 (s, 2H, –CH2–), 7.00–7.04 (m, 1H,
Ar–H), 7.16–7.33 (m, 4H, Ar–H), 7.47–7.60 (m, 4H, Ar–H), 7.67
(d, 1H, Jo ¼ 7.20 Hz, Ar–H), 7.74 (d, 1H, Jo ¼ 7.20 Hz, Ar–H),
7.84–7.86 (m, 2H, Ar–H), 9.53 (s, 1H, NH); 13C NMR: 18.50
(–CH3), 40.28 (–CH2–), 111.65, 120.01, 121.93, 123.29, 123.69,
124.72, 127.11, 129.53, 129.96, 130.02, 130.15, 130.74,
131.29, 136.41, 137.13, 143.19, 153.76, 156.22, 162.19
239–241 5.68 (s, 2H, –CH2–), 7.08–7.13 (td, 1H, Ar–H), 7.27–7.34 (m,
3H, Ar–H), 7.46 (dd, 1H, Jo ¼ 8.40 Hz, Jm ¼ 1.60 Hz, Ar–H),
7.56–7.58 (m, 3H, Ar–H), 7.65 (dd, 1H, Jo ¼ 6.80 Hz, Jm ¼ 1.60
Hz, Ar–H), 7.71 (dd, 1H, Jo ¼ 6.80 Hz, Jm ¼ 1.60 Hz, Ar–H),
7.82–7.86 (m, 3H, Ar–H), 9.94 (s, 1H, NH)
382 (100)
30
31
C22H16ClN5O
–H
o-Cl
45
56
402 (100),
404 (30.3)
C22H16FN5O
–H
–H
o-F
281–283 5.72 (s, 2H, –CH2–), 7.06–7.10 (m, 1H, Ar–H), 7.17–7.35 (m,
4H, Ar–H), 7.59–7.61 (m, 3H, Ar–H), 7.67 (d, 1H, Jo ¼ 8.00 Hz,
Ar–H), 7.74 (d, 1H, Jo ¼ 7.20 Hz, Ar–H), 7.86–7.88 (m, 2H,
Ar–H), 7.94–7.98 (m, 1H, Ar–H), 10.38 (s, 1H, NH)
386 (100)
32
33
C22H16BrN5O
C23H19N5O
o-Br
48
79
239–242 5.70 (s, 2H, –CH2–), 7.07–7.11 (m, 1H, Ar–H), 7.28–7.41 (m,
3H, Ar–H), 7.58–7.76 (m, 7H, Ar–H), 7.80 (dd, 2H, Jo ¼ 7.20 Hz,
Jm ¼ 2.00 Hz, Ar–H), 9.84 (s, 1H, NH)
446 (100),
448 (96.1)
–H m-CH3
258–260 2.27 (s, 3H, –CH3), 5.75 (s, 2H, –CH2–), 6.81 (d, 1H, Jo ¼ 7.82
Hz, Ar–H), 7.17–7.21 (t, 1H, Jo ¼ 7.6, Ar–H), 7.26–7.35 (m, 4H,
Ar–H), 7.58–7.64 (m, 3H, Ar–H), 7.70 (d, 1H, Jo ¼ 7.42 Hz,
Ar–H), 7.75 (d, 1H, Jo ¼ 7.81 Hz, Ar–H), 8.34 (d, 2H, Jm ¼ 1.95
Hz, Ar–H), 10.43 (s, 1H, NH)
382 (100)
34
35
C
22H16ClN5O
–H
–H
m-Cl
m-F
38
45
247–250 5.74 (s, 2H, –CH2–), 7.03 (d, 1H, Jo ¼ 7.42 Hz, Ar–H), 7.27–7.37
(m, 4H, Ar–H), 7.58–7.73 (m, 6H, Ar–H), 7.85 (dd, 2H,
Jo ¼ 7.03 Hz, Jm ¼ 2.35 Hz, Ar–H), 10.76 (s, 1H, NH)
402 (100),
404 (37.3)
C22H16FN5O
C22H16BrN5O
C23H19N5O
274–276 5.77 (s, 2H, –CH2–), 6.82 (m, 1H, Ar–H), 7.24 (d, 1H, Ar–H),
7.31–7.36 (m, 4H, Ar–H), 7.44 (d, 1H, Ar–H), 7.61 (d, 2H,
Jm ¼ 2.4 Hz, Ar–H), 7.70 (d, 1H, Jo ¼ 7.2 Hz, Ar–H), 7.75 (d, 1H,
Jo ¼ 7.6 Hz, Ar–H), 7.86–7.87 (m, 2H, Ar–H), 10.79 (s, 1H, NH)
265–269 5.76 (s, 2H, –CH2–), 7.17 (d, 1H, Jo ¼ 7.82 Hz, Ar–H), 7.27–7.42
(m, 4H, Ar–H), 7.59–7.61 (m, 3H, Ar–H), 7.69 (d, 1H,
386 (100)
36
37
–H
m-Br
51
62
446 (79),
448 (100)
Jo ¼ 8.21 Hz, Ar–H), 7.74 (d, 1H, Jo ¼ 7.81 Hz, Ar–H), 7.81 (s,
1H, Ar–H), 7.85–7.87 (m, 2H, Ar–H), 10.75 (s, 1H, NH)
287–289 2.23 (s, 3H, –CH3), 5.73 (s, 2H, –CH2–), 7.11 (d, 2H, Jo ¼ 8.59
Hz, Ar–H), 7.30–7.38 (m, 4H, Ar–H), 7.59–7.61 (m, 3H, Ar–H),
7.68 (d, 1H, Jo ¼ 7.42 Hz, Ar–H), 7.74 (d, 1H, Jo ¼ 7.42 Hz,
Ar–H), 7.85–7.87 (m, 2H, Ar–H), 10.37 (s, 1H, NH)
–H p-CH3
382 (100)
(Continued)
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Arch. Pharm. Chem. Life Sci. 2014, 347, 276–282
Table 1. (Continued)
N-Phenyl-5-[(2-phenylbenzimidazol-1-yl)methyl]-1,3,4-oxadiazol-2-amines
279
%
Yield
Mp
(°C)
MS (MþH)
No.
38
Formula
Ar
Y
R
1H and 13C NMR d (ppm) DMSO-d6
(%)
C22H16FN5O
–H
p-F
54
284–286 5.75 (s, 2H, –CH2–), 7.16–7.20 (m, 2H, Ar–H), 7.29–7.37 (m,
2H, Ar–H), 7.50–7.53 (m, 2H, Ar–H), 7.60–7.62 (m, 3H, Ar–H),
7.69 (d, 1H, Jo ¼ 7.81 Hz, Ar–H), 7.74–7.87 (m, 3H, Ar–H),
10.54 (s, 1H, NH); 13C NMR: 111.63 [116.20 and 116.42 (d,
J ¼ 22.24)] [119.29 and 119.36 (d, J ¼ 7.65)], 120.03, 123.29,
123.72, 129.56, 130.01, 130.13, 130.77, 135.60, 136.43,
143.19, 153.74 [156.08 and 153.74 (J ¼ 235.36 Hz)], 160
286–288 5.76 (s, 2H, –CH2–), 6.98 (td, 1H, Jo ¼ 7.04, 7.42 Hz, Ar–H),
7.29–7.37 (m, 4H, Ar–H), 7.48 (d, 2H, Jo ¼ 6.81 Hz, Ar–H),
7.66–7.75 (m, 4H, Ar–H), 7.89 (d, 2H, Jo ¼ 8.60 Hz, Ar–H),
10.47 (s, 1H, NH)
386 (100)
39
C22H16ClN5O
–H
–H
42
402.1 (100),
404.1 (35.4)
40
41
C22H15ClFN5O
C22H15BrClN5O
–H
–H
o-F
48
69
270–272 5.73 (s, 2H, –CH2–), 7.18–7.34 (m, 5H, Ar–H), 7.65–7.69
(m, 3H, Ar–H), 7.73 (d, 1H, Jo ¼ 8.20 Hz, Ar–H), 7.88–7.94
(m, 3H, Ar–H), 10.35 (s, 1H, NH)
420 (MþH)
o-Br
280–283 5.73 (s, 2H, –CH2–), 7.10–7.14 (m, 1H, Ar–H), 7.29–7.36
479.9 (65.7),
(m, 3H, Ar–H), 7.48 (dd, 1H, Jo ¼ 7.60 Hz, Jm ¼ 1.20 Hz, Ar–H), 480.1 (87),
7.65–7.75 (m, 4H, Ar–H), 7.85–7.89 (m, 3H, Ar–H), 9.95
(s, 1H, NH)
482.1 (100),
482.3 (91.6)
416 (100),
418 (34.8)
42
43
C23H18ClN5O
C22H15ClFN5O
–H p-CH3
63
43
268–270 2.23 (s, 3H, –CH3), 5.74 (s, 2H, –CH2–), 7.11 (d, 2H, Jo ¼ 8.21
Hz, Ar–H), 7.31–7.37 (m, 4H, Ar–H), 7.65–7.75 (m, 4H, Ar–H),
7.89 (d, 2H, Jo ¼ 8.60 Hz, Ar–H), 10.35 (s, 1H, NH)
–H
p-F
279–281 5.75 (s, 2H, –CH2–), 7.15 (td, 2H, Jo ¼ 8.98, 8.99 Hz, Ar–H),
7.18–7.35 (m, 2H, Ar–H), 7.48–7.51 (m, 2H, Ar–H), 7.51–7.70
(m, 3H, Ar–H), 7.74 (d, 1H, Jo ¼ 7.43 Hz, Ar–H), 7.87–7.89
(m, 2H, Ar–H), 10.51 (s, 1H, NH); 13C NMR: 40.08, 110.85
[115.65 and 115.42 (J ¼ 23.0 Hz)] [118.54 and 118.47 (J ¼ 7.0
Hz)], 119.24, 122.51, 122.93, 128.77, 129.22, 129.35, 129.98,
134.75, 135.64, 142.41, 152.94, 155.32 [158.39 and 156.03
(J ¼ 236.0 Hz)], 160.11
420 (100),
422 (39.2)
44
45
C23H18ClN5O2
C24H18Cl3N5O3
–H p-OCH3
45
48
267–269 3.7 (s, 3H, –OCH3), 5.73 (s, 2H, –CH2–), 6.89 (d, 2H,
Jo ¼ 8.98 Hz, Ar–H), 7.31–7.40 (m, 4H, Ar–H), 7.65–7.75 (m,
4H, Ar–H), 7.88 (d, 2H, Jo ¼ 8.21 Hz, Ar–H), 10.25 (s, 1H, NH)
271–272 3.82 (s, 3H, –OCH3), 3.84 (s, 3H, –OCH3), 5.78 (s, 2H, –CH2–),
432 (100),
434 (37.8)
–Cl
p-Cl
530 (100),
7.15 (d, 1H, Jo ¼ 8.20 Hz, Ar–H), 7.36–7.40 (m, 4H, Ar–H), 7.52 532 (99.8),
(d, 2H, Jo ¼ 8.99 Hz, Ar–H), 8.01 (s, 1H, Ar–H), 8.11 (s, 1H,
Ar–H), 10.67 (s, 1H, NH)
534 (31.9),
536 (3.5)
41 (28%), and 45 (12%) displayed highly limited inhibitory
effects on LP and the rest of the compounds enhanced LP
levels.
caffeine. Significant inhibitory activities were also observed
for compounds 28 (77%), 29 (84%), 33 (74%), 35 (65%), 36 (81%),
43 (66%), and 44 (71%). Compounds 32 and 41 have o-Br
substituent at aniline moiety and they are the most active
analogs of this series (97% and 98%, respectively) on the
microsomal EROD activity. Both of the compounds 30 and 45
inhibited the microsomal EROD activity (89%) being similarly
better than that of the specific inhibitor caffeine at 10ꢀ3 M
concentration. The rest of the compounds inhibited
EROD activity in the range of 52–48%.
Compound 45 containing p-chlorophenylamino substitu-
ent at the second position of oxadiazole ring has allowed
us to obtain a very good EROD profile (89%). When it is
compared to mercapto substituted analog (60%), 5-[5,6-
dichloro-2-(3,4-dimethoxy)phenyl-benzimidazol-1-yl-methyl]-2-
mercapto-[1,3,4]-oxadiazol, reported before [8], it can be
Compound 44, being an oxadiazole isostere of triazole [7]
and thiadiazole [7], enhanced LP level but the other isosteres
inhibited lipid peroxidation by 55% and 85%, respectively.
Similar results, oxadiazole derivatives enhancing lipid
peroxidation levels, were obtained in another study on liver
LP levels too [8]. These results clearly indicate that triazole
and thiadiazole isosteres are more appropriate for LP
inhibition.
Nearly all of the tested compounds showed significant
inhibition of EROD activity. Compounds 30, 32, 41, and 45
decreased liver EROD activities by 89%, 97%, 98%, and 89%,
respectively, better than the specific inhibitor caffeine (85%).
Compounds 34 and 42 caused 85% inhibition being equal to
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Arch. Pharm. Chem. Life Sci. 2014, 347, 276–282
Table 2. Effects of compounds on the liver LP levels, EROD enzyme, and DPPH free radical scavenging activities in vitro.a)
LP
Percent of
control %
EROD
pmol/mg/min
Percent of
control %
DPPH %
inhibition
DPPH
IC50 (mM)
Comp.b)
nmol/mg/min
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
64.18 ꢁ 0.26
20.98 ꢁ 2.58
20.28 ꢁ 1.60
24.31 ꢁ 0.37
22.36 ꢁ 0.63
11.97 ꢁ 0.42
57.69 ꢁ 0.42
26.52 ꢁ 0.98
17.63 ꢁ 1.43
13.61 ꢁ 2.31
24.75 ꢁ 4.72
20.03 ꢁ 2.67
24.31 ꢁ 2.57
11.66 ꢁ 1.69
9.37 ꢁ 1.67
396
129
125
150
138
74
355
163
108
84
9.66 ꢁ 0.89
6.71 ꢁ 0.25
4.64 ꢁ 1.13
22.13 ꢁ 4.23
1.05 ꢁ 0.25
10.83 ꢁ 0.96
6.12 ꢁ 0.33
14.60 ꢁ 1.07
7.75 ꢁ 1.13
21.43 ꢁ 1.29
19.96 ꢁ 1.22
19.90 ꢁ 2.45
17.89 ꢁ 0.51
0.91 ꢁ 0.05
6.35 ꢁ 2.65
14.13 ꢁ 2.58
12.07 ꢁ 2.70
4.66 ꢁ 0.87
23
16
11
53
3
26
15
35
19
52
48
48
49
2
28 ꢁ 3.9
39 ꢁ 4.4
30 ꢁ 2.2
15 ꢁ 2.0
28 ꢁ 0.6
12 ꢁ 2.2
76 ꢁ 3.8
9 ꢁ 0.6
0.35
0.2
16 ꢁ 0.8
8 ꢁ 1.0
152
123
149
72
12 ꢁ 1.7
10 ꢁ 0.6
20 ꢁ 0.8
84 ꢁ 3.2
30 ꢁ 3.8
14 ꢁ 3.0
25 ꢁ ꢁ 4.4
25 ꢁ 3.5
90 ꢁ 0.6
58
15
34
29
11
17.83 ꢁ 3.65
20.53 ꢁ 3.56
14.30 ꢁ 0.63
5.68 ꢁ 0.22
110
126
88
44
45
BHT
Caffeine
Controlc)
35
6.41 ꢁ 0.36
15
100
16.25 ꢁ 1.45
100
41.53 ꢁ 0.99
a)
Each value represents mean ꢁ SD of two to four independent experiments.
Concentration in incubation medium (10ꢀ3 M).
DMSO only, control for compounds.
b)
c)
concluded that the substituted-phenylamino moiety is more
no interaction with DPPH-like oxadiazole derivatives except
34 and 41. If we compare with N-methyl and N-aryl series
reported before [5, 7], in all the series, triazole derivatives have
moderate activity and thiosemicarbazides [10] display the best
effect on DPPH.
Compounds that have pyridinyl or p-cholorophenyl
substituent at second position of benzimidazole derivatives
and thiosemicarbazides showed highest interaction with
DPPH radical, even better than BHT [7]. Compound 41 has a
p-chlorophenyl substituent at second position of benzimid-
azole ring like the compounds published in Ayhan-Kilcigil
et al. [7].
important than mercapto substituent for EROD activity.
In terms of inhibition on EROD activity, it can be said that
oxadiazole derivatives displayed better activity than thiadi-
azole and triazole derivatives. One of our studies demonstrated
that in these series of compounds, to have cyano group is
very important to improve EROD activity. Namely, some of
the 2-(p-cyanophenyl)benzimidazole derivatives [8] have better
activities (98% and 100%) than compounds 34 (85%) and 36
(81%) because they have phenyl substituent at 2nd position
of benzimidazole.
Almost all of the tested compounds displayed significant
inhibition on EROD activity in the range of 98–47%. In
these series of compounds, the most active analogs on the
microsomal EROD activity are 32 (97%) and 41 (98%).
In this study, almost all of the compounds’ DPPH free
radical scavenging activities were not enough, except for
compounds 34 and 41. IC50 values of N-(m-chlorophenyl)-5-[(2-
phenyl-1H-benzimidazol-1-yl)methyl]-2,5-dihydro-1,3,4-oxadiazol-
2-amine (34) (76%) and N-(o-bromophenyl)-5-[(2-(p-chlorophenyl)-
1H-benzimidazol-1-yl)methyl]-2,5-dihydro-1,3,4-oxadiazol-2-amine
(41) (84%) are similar, 0.35 and 0.2 mM, respectively, so it can be
said that there is a little bit difference between their scavenger
effects on DPPH radical.
It is well known that there exist two mechanisms for an
antioxidant to scavenge DPPH, which we reported before. The first
one is a direct H-atom abstraction process (Eq. 1), and the second
one is a proton concerted electron-transfer process (Eq. 2) [19]
DPPH• þ RXH ! DPPHH þ RXH•
ð1Þ
ð2Þ
DPPH• þ RXH ! DPPHꢀ þ RXHþ ! DPPHH þ RX•
DPPH-scavenging mechanism of our compounds could not
have been clarified yet but our efforts in this direction are
continuing [20].
Because of the diversity of the methods, it is quite difficult
to explain the observed variant effects of synthesized
Our other published results [5] clearly indicate that both N-
aryl and N-methyl thiadiazole derivatives were found to have
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Arch. Pharm. Chem. Life Sci. 2014, 347, 276–282
N-Phenyl-5-[(2-phenylbenzimidazol-1-yl)methyl]-1,3,4-oxadiazol-2-amines
281
compounds. Separate effects of compounds in these systems
have been noticed previously [5–10]. Therefore, the observa-
tion of distinct effects of synthetic compounds on DPPH
radical, superoxide radical, LP levels, and EROD is not
surprising since the mechanisms of production of oxidative
stress using these methods are different [12, 13, 21]. The
differences in the kinetic behavior of the radicals and
substrates should also be considered when comparing the
results of different free radical scavenging methods to
determine antioxidant capacity [22]. Therefore, it is extremely
difficult to compare the results from different assays.
As a result, data obtained from all our researches associated
with this field guide us for the development of novel
antioxidant compounds.
were expressed in terms of nmol malondialdehyde (MDA) per mg
protein. The assay was essentially derived from the methods of
Wills [24, 25] as modified by Bishayee and Balasubramanian [26].
Lipid peroxidation was determined spectrophotometrically at
532 nm as the thiobarbituric acid reactive material. Compounds
inhibit the production of MDA, and therefore the produced color
after addition of thiobarbituric acid is less intensive. A typical
optimized assay mixture contained 0.2 nM Feþþ, 90 mM KCl,
62.5 mM potassium phosphate buffer, pH 7.4, NADPH-generating
system consisting of 0.25 mM NADPþ, 2.5 mM MgCl2, 2.5 mM
glucose-6-phosphate, 1.0 U glucose-6-phosphate dehydrogenase
and 14.2 mM potassium phosphate buffer pH 7.8, and 0.2 mg
microsomal protein in a final volume of 1.0 mL.
EROD enzyme activity
EROD activity was measured by the spectrofluorometric method
of Burke et al. [27]. A typical optimized assay mixture contained
1.0 mM ethoxyresorufin, 100 mM Tris–HCl buffer pH 7.8, NADPH
generating system consisting of 0.25 mM NADPþ, 2.5 mM MgCl2,
2.5 mM glucose-6-phosphate, 1.0 U glucose-6-phosphate dehydro-
genase and 14.2 mM potassium phosphate buffer pH 7.8, and
0.2 mg liver microsomal protein in a final volume of 1.0 mL.
Experimental
Chemistry
Melting points were determined with an electrothermal melting
point apparatus and are uncorrected. 1H, 13C NMR, and HSQC
spectra were measured with a Varian Mercury 400, 100 MHz
instrument using TMS internal standard and DMSO-d6; coupling
constants (J) are reported in Hertz. All chemical shifts were
reported as d (ppm) values. ES-MS were obtained with a Waters ZQ
Micromass LC–MS spectrometer with positive electrospray
ionization method. All instrumental analyses were performed
at Ankara University, Faculty of Pharmacy. The chemical reagents
used in synthesis were purchased from E. Merck and Aldrich.
BHT and caffeine were obtained from Sigma. Analytical thin-
layer chromatography (TLC) was performed with Merck
precoated TLC plates and spots were visualized with ultraviolet
light. Compounds 1–27 were previously prepared in our
laboratory [5–8].
DPPH free radical scavenging activity
The free radical scavenging activities of these compounds were
tested by their ability to bleach the stable radical 2,2-diphenyl-1-
picrylhydrazyl (DPPH) as described by Blois [28]. This assay has
often been used to estimate the antiradical activity of anti-
oxidants. Because of its odd electron, DPPH gives a strong
absorption band at 517 nm in visible spectroscopy. DPPH was
dissolved in methanol to give a 100 mM solution. To 1.0 mL of
the methanolic solution of DPPH, 0.1 mL of the test compounds
was added and BHT dissolved in DMSO. The absorbance at
517 nm was determined after 30 min at room temperature, and
the scavenging activity was calculated as a percentage of
radical reduction. Each experiment was performed in triplicate.
DMSO was used as a control solution and BHT as a reference
compound. The radical scavenging activity was expressed as
IC50, which was determined from a calibration curve for
compounds 34 and 41.
General procedure for the preparation of N-(substituted
phenyl)-5-[(2-substituted phenyl-1H-benzimidazol-1-yl)-
methyl]-2,5-dihydro-1,3,4-oxadiazol-2-amines (28–45)
Appropriate thiosemicarbazides (1 mmol) 10–27 were dissolved in
5 mL of EtOH and 0.5 mL of 5 N NaOH. I2 solution was dropped into
the solution at room temperature until brown color appeared.
After reflux for 1–3 h, the reaction mixture was cooled and poured
into ice water. The crude product was filtered off and recrystal-
lized from ethanol to yield the desired oxadiazole derivatives. All
physical, spectral data, and structure of 28–45 are seen in Table 1.
The authors have declared no conflict of interest.
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