Synthesis and antioxidant capacities of some new benzimidazole derivatives

Article abstract of DOI:10.1002/ardp.200700088

In this study, we prepared some new oxadiazolyl benzimidazole derivatives and investigated their antioxidant properties by determination of microsomal NADPH-dependent inhibition of lipid peroxidation levels (LP assay) and microsomal ethoxyresorufin O-deet

Full text of DOI:10.1002/ardp.200700088

Arch. Pharm. Chem. Life Sci. 2007, 340, 607–611
G. Ayhan-Kılcıgil et al.
607
Full Paper
Synthesis and Antioxidant Capacities of Some New
Benzimidazole Derivatives
1
1
2
2
2
˙
Gꢀlgꢀn Ayhan-Kılcıgil , Canan Kus˛ , Elꢁin D. ꢂzdamar , Benay Can-Eke , and Mꢀmtaz Is˛can
1
ˇ
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara,
Turkey
2
ˇ
Department of Toxicology, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey
In this study, we prepared some new oxadiazolyl benzimidazole derivatives and investigated
their antioxidant properties by determination of microsomal NADPH-dependent inhibition of
lipid peroxidation levels (LP assay) and microsomal ethoxyresorufin O-deethylase activity (EROD
assay). Some of these compounds 20, 23 had slightly inhibitory effects (28%) on the lipid peroxi-
dation levels at 10– 3 M concentration lower than standard BHT (65%). 5-[2-(Phenyl)-benzimidazol-
1-yl-methyl]-2-mercapto-[1,3,4]-oxadiazole 16 was found to be more active than caffeine on the
ethoxyresorufin O-deethylase activity with an IC50 value of 2.0 6 10– 4 M.
Keywords: Antioxidant activity / Benzimidazoles / Oxadiazoles /
Received: April 28, 2007; accepted: August 21, 2007
DOI 10.1002/ardp.200700088
Introduction
Many reports indicate that benzimidazoles have a variety
of biological activities such as antimicrobial, antituber-
cular, anticancer, antihelmintic, antiallergic, and antiox-
idant. In our previous papers, we have already reported
some antioxidant benzimidazole derivatives (Fig. 1). Fur-
thermore, we have found that some compounds show
potent antioxidant properties in various in-vitro systems.
In this study, we synthesized some new compounds by
modifying the triazole / thiadiazole ring to oxadiazole
Figure 1. Structures of previously synthesized benzimidazole
compounds.
either at the N1 position or at the C5 position of the benzi-
midazole ring [1–5].
Results and discussion
For the synthesis of the target compounds (16–20, 23) the
reaction sequences outlined in Scheme 1 and Scheme 2,
are the following. 1H-Benzimidazole derivatives 1–5
were prepared via oxidative condensation of o-phenylene-
diamine/4,5-dichloro-1,2-phenylenediamine, appropriate
benzaldehyde derivatives, and sodium metabisulfite [6].
Treatment of 2-phenyl/substituted phenyl/pyridinyl-1H-
benzimidazole with ethyl chloroacetate in KOH/DMSO
Correspondence: Gꢀlgꢀn Ayhan-Kılcıgil, Department of Pharmaceutical
ˇ
Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandogan,
Ankara, Turkey.
E-mail: kilcigil@pharmacy.ankara.edu.tr
Fax: +90 312 213-1081
Abbreviations: lipid peroxidation levels (LP assay); ethoxyresorufin O-
deethylase activity (EROD assay); thiobarbituric acid reactant substances
(TBARS)
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G. Ayhan-Kılcıgil et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 607–611
Reagents: (a) Na2S2O3 adduct of the related benzaldehyde or pyridinecarboxaldehyde/DMF; (b) Ethyl chloroacetate/KOH;
(c) Hydrazine/EtOH; (d) CS2/KOH.
Scheme 1. Synthesis of compounds 1–20.
Reagents: (a) MeOH/H+; (b) p-Chlorobenzylamine; (c) H2, Pd/C; (d) Na2S2O3 adduct of benzaldehyde/DMF; (e) Hydrazine/EtOH; (f) CS2/KOH.
Scheme 2. Synthesis of compounds 21–23.
gave the N-alkylated product, (2-aryl-benzimidazol-1-yl)- benzoic acid. Nucleophilic displacement of the chlorine
acetic acid ethyl esters 6–10 [7]. Hydrazine hydrate and atom of methyl 3-nitro-4-chlorobenzoate by reaction
the related esters 6–10 in ethanol were refluxed for 4 h with p-chlorobenzylamine in DMF gave methyl 4-(p-chlor-
to give the desired hydrazide compounds, (2-aryl-benzi- obenzylamino)-3-nitro-benzoate. Its reduction with H2
midazol-1-yl)-acetic acid hydrazides 11–15, in the range and Pd/C produced a 3-amino derivative. Condensation of
of 81-94% yields [8]. Cyclization of 11–15 with CS2 and methy 3-amino-4-(p-chlorobenzylamino)benzoate with
KOH [9] resulted to the formation of 5-[2-(phenyl/substi- the Na2S2O5 adduct of benzaldehyde gave 21. Subsequent
tuted phenyl/pyridinyl)-benzimidazol-1-yl-methyl]-2-mer-
capto-[1,3,4]-oxadiazoles 16–20.
reaction steps were carried out by the same route as 16–
20 (Scheme 2).
5-[1-(4-Chlorobenzyl)-2-phenyl-benzimidazol-1-yl-
We synthesized and analyzed the antioxidant proper-
methyl]-2-mercapto-[1,3,4]-oxadiazole 23 was also obtain- ties of six new benzimidazole derivatives carrying 5-mer-
ed in the analogous way, starting from 3-nitro-4-chloro- capto oxadiazole ring at the N-1 or C-5 position of benzi-
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Arch. Pharm. Chem. Life Sci. 2007, 340, 607–611
Antioxidant Benzimidazole Derivatives
609
Table 1. The in-vitro effects of some compounds on liver LP lev-
of benzimidazole ring exhibited good activities. Among
the compounds bearing the oxadiazole ring at the N-1
position, the best activity was observed with compound
16 which has a non-substituted phenyl at the C-2 position
of the benzimidazole ring. In addition, it appears that
compounds 18 and 19 contain electron-donating sub-
stituents which allowed us to obtain the good EROD pro-
file compared to compounds bearing electron-accepting
groups (compound 17 and 20).
elsa).
Compoundsb)
Protein (nmol/mg)
% of Control
16
17
18
19
20
23
16.57 l 0.18
16.57 l 0.07
15.69 l 0.05
17.00 l 1.30
11.78 l 0.85
11.78 l 0.73
16.25 l 1.45
5.68 l 0.22
102
102
97
105
72
72
100
35
Controlc)
BHT
The authors have declared no conflict of interest.
a)
Each value represents the mean l S.D. of 2–4 independent
experiments.
Concentration in incubation medium 10– 3 M.
Dimethylsulfoxide only, control for compounds and BHT.
b)
c)
Experimental
Chemistry
Table 2. The in-vitro effects of some compounds and caffeine
Melting points were determined with Bꢀchi SMP-20 (Bꢀchi
Labortechnik, Flawil, Switzerland) and Electrothermal 9100
capillary melting point apparatus (Electrothermal, Essex, U.K.)
on EROD activity in the livera).
Compounds
EROD ((pmol/mg)/min) % of Control
and are uncorrected. H- and 13C-NMR spectra were measured
1
with a Varian Mercury 400, 400 MHz instrument (Varian Inc.,
Palo Alto, CA, USA) using TMS internal standard and DMSO-d6,
coupling constants (J) are reported in Hertz. All chemical shifts
were reported as d (ppm) values. ESMS were obtained with a
Waters ZQ Micromass LC-MS spectrometer (Waters Corporation,
Milford, MA, USA) with Positive Electrospray Ionization Method.
Elemental analyses (C, H, N, S) were determined on a Leco CHNS
932 instrument (Leco-932, St.Joseph, MI, USA), and were within
l 0.4% of the theoretical values. All instrumental analyses were
performed at Ankara University, Faculty of Pharmacy. The chem-
ical reagents used in synthesis were purchased from Aldrich
(Steinheim, Germany), Merck (Darmstadt, Germany) or Fluka
(Germany). BHT and caffeine were obtained from Sigma (Sigma-
Aldrich). Analytical thin layer chromatography was performed
with Merck precoated TLC plates and spots were visualized with
ultraviolet light. Compounds 1–3, 5–8, 10–13, and 15 were syn-
thesized according to the literature [1, 2, 4]. Methyl-[3-amino-4-
(p-chlorobenzylamino)]benzoate (mp: 1058C) was synthesized
according to the literature [10].
16
17
18
19
20
23
4.31 l 0.36
25.05 l 1.80
17.87 l 0.90
16.61 l 0.73
27.41 l 2.10
24.14 l 1.91
41.53 l 0.99
8.31 l 0.42
10
60
43
40
66
58
100
20
Controlb)
Caffeine
a)
Each value represents the mean l S.D. of 2–4 independent
experiments.
Dimethylsulfoxide only, control for compounds and caffeine.
b)
midazole ring. The in-vitro effects of compounds 16–20,
23 and BHT on rat liver lipid peroxidation (LP) levels are
shown in Table 1. BHT being a classical antioxidant com-
pound was used as a positive control for comparison
with benzimidazoles. Both compound 20 and 23 showed
slightly inhibitory effects on LP levels at 10– 3 M (28%).
The inhibitory effect of BHT on LP was found to be
approximately two-fold stronger (65%) than these com-
pounds 20, 23 (Table 1).
5,6-Dichloro-2-[(3,4-dimethoxy)phenyl]-1H-
benzimidazole 4
The mixture of 4,5-dichloro-o-phenylendiamine (1 mmol) and
sodium metabisulfite adduct of 3,4-dimethoxybenzaldehyde
(1.25 mmol) in DMF were heated at 1108C for 5 h. Water was
added, solid product was collected by filtration, washed with
water, and crystallized from EtOH. Mp: 2698C, C15H12Cl2N2O2, MS
(ESI+) m/z (%) 323 (100) [M+H], 325 (100) [M+2], 327 (34) [M+4], 1H-
NMR (DMSO-d6) d: 3.85 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 7.16 (s,
1H, H-69), 7.65–7.82 (m, 3H, H-4,7,59), 7.89 (s, 1H, H-29), 13.01 (s,
1H, NH).
The in-vitro effects of compounds 16–20, 23 and caf-
feine on ethoxyresorufin O-deethylase activity (EROD) are
shown in Table 2. As shown in Table 2, these compounds
displayed various degrees on EROD, with decreasing
activity in the following order: 20 A 17 A 23 A 18 X 19 A 16.
Among them, 2-(non-substituted phenyl) benzimidazole
derivative 16 was chosen for further experiments with
90% inhibition value at 10– 3 M concentration. Com-
pound 16 inhibited EROD activity with a 2.0610– 4 IC50
Ethyl 5,6-dichloro-2-(3,4-dimethoxyphenyl)-1H-
benzimidazole acetate 9
Dimethylsulfoxide (15 mL) was added to potassium hydroxide
(1.5 g, 0.027 mol; crushed pellets) and the mixture was stirred
for 15 min. 5,6-Dichloro-2-[3,4-dimethoxyphenyl]-1H-benzimida-
zole (6.7 mmol) was added and then the mixture was stirred 2 h.
value while IC50 value of caffeine was 5.2610– 4
According to these results, the compounds bearing the
oxadiazole ring at the N-1 position instead of C-5 position
.
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Arch. Pharm. Chem. Life Sci. 2007, 340, 607–611
Ethylchloroacetate (3 mL, 0.027 mol) was added and the mixture
was cooled briefly and stirred for further 2 h. Water was added
and the mixture was extracted with ether. Ether layers were
washed with water, dried, and solvent and excess of ethylchlor-
oacetate was removed under reduced pressure. The residue was
recrystallized from ethanol and gave the desired ester com-
pound 9.
C19H18Cl2N2O4, mp: 207–2088C, MS (ESI+) m/z (%) 409 (100)
[M+H], 411 (64) [M+2], 413 (42) [M+4], 1H-NMR (DMSO-d6) d: 1.13 (t,
3H, -H2C-CH3), 3.80 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 4.12 (q, 2H, -
H2C-CH3), 5.35 (s, 2H, -N-CH2), 7.16 (d, 1H, Jo = 8.2 Hz, H-59), 7.28 (d,
1H, Jo = 8.2 Hz; H-29), 7.3 (s, 1H, H-69), 7.99 (s, 1H, H-4), 8.24 (s, 1H,
H-7).
5-(2-Phenyl-benzimidazol-1-yl-methyl)-2-mercapto-
[1,3,4]-oxadiazole 16
Mp: 2288C, MS (ESI+) m/z (%) 309 (100) [M+H], 1H-NMR (DMSO-d6) d
5.7 (s, 2H, -CH2), 7.25–7.80 (m, 9H, Ar-H), Anal. (C16H12N4OS 6 0.25
C2S 6 0.2 H2O) C, H, N, S.
5-[2-(4-Chlorophenyl)-benzimidazol-1-yl-methyl]-2-
mercapto-[1,3,4]-oxadiazole 17
Mp: 2778C dec., MS (ESI+) m/z (%) 343 (100) [M+H], 345 (48) [M+2],
1H-NMR (DMSO-d6) d 5.72 (s, 2H, CH2), 7.29–7.37 (m, 2H, H-5,6),
7.65–7.75 (m, 4H, H-29,69,4,7), 7.82 (d, 2H, Jo = 8.6 Hz, H-39,59), Anal
(C16H11ClN4OS 6 0.7 H2O) C, H, N, S.
5-[2-(4-Methoxyphenyl)-benzimidazol-1-yl-methyl]-2-
Methyl 1-(p-chlorobenzyl)-2-phenyl-1H-benzimidazole-5-
acetate 21
mercapto-[1,3,4]-oxadiazole 18
Mp: 245–2478C, MS (ESI+) m/z (%) 339 (100) [M+H], 1H-NMR
(DMSO-d6) d 3,85 (s, 3H, -OCH3), 5.68 (s, 2H, -CH2), 7.14 (d, 2H, Jo =
8.99 Hz, H-39,59), 7.27–7.33 (m, 2H, H-5,6), 7.63–7.71 (m, 2H, H-
4,7), 7.74 (d, 2H, Jo = 8.99 Hz, H-29,69). 13C-NMR (DMSO-d6) 56, 111,
115, 119, 122, 123.3, 123.5, 131, 136, 142, 153, 159, 161, 178.
Anal (C17H14N4O2S) C, H, N, S.
The mixture of methyl-[3-amino-4-(p-chlorobenzylamino)]ben-
zoate (1 mmol) and sodium metabisulfite adduct of benzalde-
hyde (1.25 mmol) in DMF were heated at 1108C for 5 h. Water
was added, solid product was collected by filtration, washed
with water and crystallized from MeOH. C22H17ClN2O2, mp: 144–
1468C, MS (ESI+) m/z (%) 377 (100) [M+H], 379 (42) [M+2], 1H-NMR
(DMSO-d6) d: 3.84 (s, 3H, OCH3), 5.62 (s, 2H, CH2), 6.98–7.90 (m,
11H, Ar-H), 8.32 (d, 1H, Jm = 1.17 Hz, H-4).
5-[5,6-Dichloro-2-(3,4-dimethoxy)phenyl-benzimidazol-1-
yl-methyl]-2-mercapto-[1,3,4]-oxadiazole 19
Mp: 2508C, MS (ESI+) m/z (%) 437 (100) [M+H], 439 (56) [M+2], 441
(14) [M+4], 1H-NMR (DMSO-d6) d 3.81 (s, 3H, -OCH3), 3.83 (s,3H, -
OCH3), 5.69 (s, 2H, -CH2), 7.13 (d, 1H, Jo = 8.2 Hz, H-59), 7.29–7.32
(m, 2H, H-29,69), 7.99 (s, 1H, H-4 ), 8.09 (s, 1H, H-7), Anal
(C18H14Cl2N4O3S 6 0.1 H2O) C, H, N, S.
General procedure for the preparation of 14 and 22
Hydrazine hydrate (4 mL) and related esters 9, 21 (1.5 mmol) in
ethanol (5 mL) were refluxed for 4 h. The reaction mixture was
cooled and poured into water. The crude product was filtered off
and recrystallized from ethanol to give the desired hydrazide
compounds 14, 22.
5-[2-(4-Pyridinyl)-benzimidazol-1-yl-methyl]-2-mercapto-
[1,3,4]-oxadiazole 20
Mp: 2368C bubl., ESI (+) [M+H] 310, 1H-NMR (DMSO-d6) d 5.79 (s,
2H, -CH2), 7.33–7.42 (m, 2H, H-5,6), 7.74 (d, 1H, Jo = 7.42 Hz, H-4),
5,6-Dichloro-2-(3,4-dimethoxyphenyl)-1H-benzimidazole
acetic acid hydrazide 14
7.79 (d, 1H, Jo = 7.42 Hz, H-7), 7.82 (dd, 2H, Jo = 4.69 Hz, Jm
=
C17H16Cl2N4O3, mp: 265–2668C, MS (ESI+) m/z (%) 395 (100) [M+H],
1.56 Hz, H-39,59), 8.79 (d, 2H, Jo = 5.86 Hz, H-29,69), Anal (C15H11N5OS
6 0.6 H2O) C, H, N, S.
1
397 (66) [M+2], 399 (12) [M+4], H-NMR (DMSO-d6) d: 3.78 s, 3H,
OCH3), 3.82 (s, 3H, OCH3), 4.4 (brs, 2H, NH2), 4.87 (s, 2H, -CH2), 7.10
(d, 1H, Jo = 8.0 Hz, H-5'), 7.31–7.35 (m, 2H-H-2‘,69), 7.83 (s, 1H, H-4),
7.93 (s, 1H, H-7), 9.54 (s, 1H, NH).
5-[1-(4-Chlorobenzyl)-2-phenyl-benzimidazol-1-yl-
methyl]-2-mercapto-[1,3,4]-oxadiazole 23
Mp: 3238C, MS (ESI+) m/z (%) 419 (100) [M+H], 421 (43) [M+2], 1H-
NMR (DMSO-d6) d 5.65 (s, 2H, -CH2), 7.00 (d, 2H, Jo = 8.59 Hz, H-
399,599), 7.35 (d, 2H, Jo = 8.60 Hz, H-299,699), 7.54–7.56 (m, 3H, H-
39,49,59), 7.69–7.80 (m, 4H, H-6,7,29,69), 8.19 (s, 1H, H-4), Anal
(C22H15ClN4OS 6 0.7 H2O) C, H, N, S.
1-(p-Chlorobenzyl)-2-phenyl-1H-benzimidazole-5-acetic
acid hydrazide 22
C21H17ClN4O, mp: 230–2338C, MS (ESI+) m/z (%) 377 (100) [M+H],
379 (36) [M+2], 1H-NMR (DMSO-d6) d: 4.50 (s, 2H, NH2), 5.62 (s, 2H,
CH2), 7.00 (d, 2H, Jo = 8.21 Hz, H-399,599), 7.35 (d, 2H, Jo = 8.21 Hz, H-
299,699), 7.54–7.78 (m, 7H, H-6,7,29,39,49,59,69), 8.22 (s, 1H, H-4), 9.78
(s, 1H, NH).
Biological evaluation – Antioxidant activity studies
Lipid peroxidation assay
Male albino Wistar rats (200–225 g) were used in the experi-
ments. Animals were fed with standard laboratory rat chow and
tab water ad libitum. The animals were starved for 24 h prior to
sacrifice and then killed by decapitation under anesthesia. The
livers were removed immediately and washed in ice-cold water
and the microsomes were prepared as described previously [11].
NADPH-dependent lipid peroxidation (LP) was determined
using the optimum conditions as determined and described pre-
viously [11]. NADPH-dependency was measured spectrophoto-
metrically by estimation of thiobarbituric acid reactant substan-
General procedure for the preparation of 16–20 and 23
Corresponding hydrazide compounds (0.4 mmol) and CS2
(31 mg, 0.4 mmol) were added to a solution of KOH (22.4 mg,
0.4 mmol) in 1 mL of H2O and 1 mL of ethanol. The reaction mix-
ture was refluxed for 3 h. After evaporating under reduced pres-
sure, a solid was obtained. This was dissolved in water and acidi-
fied with conc. HCl. The precipitate was filtered, washed with
water, and recrystallized from ethanol.
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Arch. Pharm. Chem. Life Sci. 2007, 340, 607–611
Antioxidant Benzimidazole Derivatives
611
ces (TBARS). Amounts of TBARS were expressed in terms of nmol
malondialdehyde (MDA)/mg protein. The assay was essentially
derived from the methods of Wills [12, 13] as modified by Bish-
ayee [14]. A typical optimized assay mixture contained 0.2 nM
Fe++, 90 mM KCl, 62.5 mM potassium-phosphate buffer, pH 7.4,
NADPH generating system consisting of 0.25 mM NADP+,
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.
[2] G. Ayhan-Kılcıgil, C. Ku˛s, T. ꢁoban, B. Can-Eke, M. I˛scan, J.
Enzyme Inhib. Med. Chem. 2004, 19, 129–135.
[3] Z. Ate˛s-Alagꢂz, C. Ku˛s, T. ꢁoban, J. Enzyme Inhib. Med.
Chem. 2005, 20, 325–333.
[4] G. Ayhan-Kılcıgil, C. Ku˛s, T. ꢁoban, B. Can-Eke, et al., J.
Enzyme Inhib. Med. Chem. 2005, 20, 503–514.
[5] I. Kerimov, G. Ayhan-Kılcıgil, B. Can-Eke, N. Altanlar, M.
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[6] H. F. Ridley, R. G. W. Spickett, G. M. Timmis, J. Heterocycl.
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EROD assay
[7] H. Heaney, S. V. Ley, J. Chem. Soc. Perkin I, 1973, 499–500.
7-Ethoxyresorufin-O-deethylase (EROD) activity was measured by
the spectrofluorometric method of Burke et al. [15]. A typical
optimized assay mixture contained 1.0 mM ethoxyresorufin,
100 mM Tris-HCl buffer, pH 7.8, NADPH generating system con-
sisting of 0.25 mM NADP+, 2.5 mM MgCl2, 2.5 mM glucose-6-phos-
phate, 1.0 U glucose-6-phosphate dehydrogenase, and 14.2 mM
potassium phosphate buffer, pH 7.8, and 0.2 mg liver microso-
mal protein in a final volume of 1.0 mL. EROD activity of com-
pound 16 and caffeine was expressed as IC50 which determined
from a calibration curve.
[8] P. A. S. Smith in Organic Reactions, Volume III, (Eds.: R.
Adams, W. E. Bachmann, L. F. Fieser, J. R. Johnson, H. R.
Snyder) John Wiley & Sons, Inc. London, Chapman &
Hall, Ltd, 1949, pp. 337–389.
[9] N. Demirba˛s, Turk. J. Chem. 2005, 29, 125–133.
[10] H. Gꢂker, E. Tebrizli, U. Abbasoglu, Farmaco 1996, 51,
53–58.
[11] M. I˛scan, E. Arinꢃ, N. Vural, M. Y. I˛scan, Comp. Biochem.
Physiol. 1984, 77C, 177–190.
[12] E. D. Wills, Biochem. J. 1966, 99, 667–676.
[13] E. D. Wills, Biochem. J. 1969, 113, 333–341.
[14] S. Bishayee, A. S. Balasubramanian, Neurochem. 1971, 18,
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i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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