Synthesis and Evaluation of Antioxidant Properties of Novel 2-[2-(4-chlorophenyl) benzimidazole-1-yl]-N-(2-arylmethylene amino) acetamides and 2-[2-(4-chlorophenyl) benzimidazole-1-yl]-N-(4-oxo-2-aryl-thiazolidine-3-yl) acetamides-I

Article abstract of DOI:10.1111/j.1747-0285.2012.01347.x

Our approach was to synthesize and examine the antioxidant properties of some new 2-[2-(4-chlorophenyl)benzimidazole-1-yl]-N-(2-arylmethyleneamino) acetamide (1-18) and 2-[2-(4-chlorophenyl)benzimidazole-1-yl]-N-(4-oxo-2-aryl-thiazolidine-3-yl)acetamide (

Full text of DOI:10.1111/j.1747-0285.2012.01347.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01347.x
Chem Biol Drug Des 2012; 79: 869–877
Research Letter
Synthesis and Evaluation of Antioxidant
Properties of Novel 2-[2-(4-chlorophenyl)
benzimidazole-1-yl]-N-(2-arylmethylene amino)
acetamides and 2-[2-(4-chlorophenyl)
benzimidazole-1-yl]-N-(4-oxo-2-aryl-thiazolidine-3-yl)
acetamides-I
Gu¨ lgu¨ n Ayhan-Kılcıgil1*, Selen Gu¨ rkan1,
Tu¨ lay C¸ oban , Elc¸in Deniz Ozdamar and
Benay Can-Eke2
cancer, and atherosclerosis (1,2). The interest for the protective
2
2
¨
role of antioxidants has been growing over the past 15 years. An-
tioxidants are considered as potential drugs because of their abil-
ity to delay or prevent the oxidation of cellular oxidizable
substrates by ROS ⁄ RNS. The human antioxidant protections
include enzymes such as catalase, superoxide dismutase, and glu-
tathione peroxidase as well as some water- and lipid-soluble
antioxidant vitamins such as ascorbic acid (vitamin C) and
a-tocopherol (vitamin E) (1–5).
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
University of Ankara, 06100 Tandogan, Ankara, Turkey
2Department of Pharmaceutical Toxicology, Faculty of Pharmacy,
University of Ankara, 06100 Tandogan, Ankara, Turkey
*Corresponding author: Gꢀlgꢀn Ayhan-Kılcıgil,
kilcigil@pharmacy.ankara.edu.tr
It is known that lipid peroxidation is a free radical chain reac-
tion that causes the degeneration of the cell membranes. Most
products of lipid peroxidation are known to have mutagenic
and ⁄ or carcinogenic properties. On the other hand, the antio-
xidants and antioxidant enzyme systems are major protective
systems of organisms. Therefore, drugs possessing antioxidant
and free radical scavenging properties are considered for
preventing and ⁄ or treatment of such diseases that are directly
related to the lack of antioxidant capacity of organism. Reactive
oxygen species are produced by different mechanisms such as
cytochrome P450 (CYP)-dependent enzymes that metabolize chemi-
cals and endogenous substances to produce reactive oxygen
species. In this system, CYP1A1 ⁄ 2 have to play important role in
NADPH-dependent lipid peroxidation (LP). Thus, it is important to
evaluate the effects of synthesized compounds on NADPH-depen-
dent lipid peroxidation and CYP system (5). On the other hand,
it is suggested that the DPPH assay is an accurate method
with regard to measuring the antioxidant capacity of various
compounds (6).
Our approach was to synthesize and examine the anti-
oxidant properties of some new 2-[2-(4-chlorophe-
nyl)benzimidazole-1-yl]-N-(2-arylmethyleneamino)
acetamide (1–18) and 2-[2-(4-chlorophenyl)benzimid-
azole-1-yl]-N-(4-oxo-2-aryl-thiazolidine-3-yl)acetamide
(1t–18t) derivatives. Their in vitro effects on rat liver
microsomal NADPH-dependent lipid peroxidation lev-
els (LP assay) and microsomal ethoxyresorufin O-de-
ethylase activities (EROD assay) were determined.
The free radical scavenging properties of the com-
pounds were also examined in vitro determining the
interaction of 2,2-diphenyl-1-picrylhydrazyl (DPPH)
free radical. The compounds showed significant
effects in the above tests.
Key words: antioxidant activity, benzimidazole, methyleneamino
acetamide, thiazolidine
Received 25 May 2010, revised 11 January 2012 and accepted for pub-
lication 12 January 2012
It is well known that benzimidazoles have a variety of biological
activities such as antimicrobial (7–10), anticancer (11,12), antihel-
minthic (13), antiallergic (14,15), and antioxidant (16). In addition,
the thiazolidinones display anticonvulsant (17), anticancer (18), anti-
microbial (19), and antioxidant (20) activities.
Reactive oxygen species (ROS) and reactive nitrogen species (RNS)
are generated in aerobic organisms as part of the normal physio-
logical and metabolic processes or from exogenous factors and
agents. These ROS are capable of damaging a wide range of
essential macromolecules (membrane lipids, proteins, nucleic
acids). The damages by ROS are directly or indirectly implicated
in the pathogenesis of various disorders such as cardiovascular
diseases, Alzheimer's and other neurodegenerative diseases,
Previously, we reported the synthesis, characterization, and anti-
oxidant properties of some benzimidazole derivatives containing
thiadiazole, triazole, and oxadiazole rings at the 1st position
869

Ayhan-Kılcıgil et al.
(21–28). The aim of this work was to modify the structure of
either the arylmethyleneamino (1–18) or aryl thiazolidine (1t–
18t) acetamides and to continue searching for improved ROS
and RNS scavengers.
Material and Methods
Chemistry
Melting points were determined with a Thermo Scientific Electro-
thermal melting point apparatus and are uncorrected. H-NMR and
1
13C-spectra were measured with a Varian Mercury 400 MHz instru-
ment using TMS internal standard and DMSO-d6; coupling con-
stants (J) are reported in Hertz. All chemical shifts were reported
as d (ppm) values. ES–MS were obtained with a Waters ZQ Micro-
mass LC–MS spectrometer with positive electrospray ionization
method. Elemental analyses (C, H, N, and S) were determined on a
Leco CHNS 932 instrument and were within €0.4% of the theoreti-
cal values. Compounds A, B, C, and 5-methoxy-indole-3-carboxal-
dehyde were previously prepared in our laboratory (23,29).
H
N
O
Ar
Ar
N
O
S
N
N
H
O
N
N
N
N
Cl
Cl
1-18
1t-18t
General formulas of the synthesized compounds
NH2
NH2
H
N
a
ClCH2COOEt
Cl
Cl
CH2COOEt
N
N
A
Cl
N
NH2NH2
CHO
B
CH2CONHNH2
N
Cl
N
C
HOC
R1
HOC
R1
O
N
N
H
HC
H
N
N
N
HOC
Cl
OCH3
O
CH
N
H
N
4-18
N
H
N
O
Cl
N
CH
HSCH2COOH
N
N
H
OCH3
1-2
N
R1
Cl
N
HSCH2COOH
3
S
O
N
N
H
HSCH2COOH
O
N
N
Cl
S
O
N
N
H
N
H
O
4t-18t
N
Cl
S
O
H3CO
N
N
N
1t-2t
H
O
N
N
Cl
3t
Scheme 1: Synthetic pathways to compounds 1–18 and 1t–18t.
870
Chem Biol Drug Des 2012; 79: 869–877

Synthesis and Evaluation of Antioxidant Properties
All instrumental analyses were performed at the Central Laboratory,
Ankara University, Faculty of Pharmacy. The chemical reagents used
in synthesis were purchased from E. Merck and Aldrich. Butylated
hydroxy toluene (BHT) and caffeine were obtained from Sigma. Ana-
lytical thin-layer chromatography was performed with Merck pre-
coated TLC plates, and spots were visualized with ultraviolet light.
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 liver microsomal
protein in a final volume of 1.0 mL.
2,2-Diphenyl-1-picrylhydrazyl free radical
scavenging activity
The radical scavenging assay was determined by the modified
method described by Blois (35). BHT and stock solutions of the com-
pounds were prepared at 10)2 M in DMSO. A series of solutions in
DMSO were diluted to varying concentrations in 96-well micro-
plates. Then, methanolic DPPH solution (100 lM) was added to
each well. The plate was shaken and placed in the dark. After
30 min, the optical density (OD) of the solution was read at the
517 nm. The methanolic solution of DPPH served as a control. Per-
centage inhibition was calculated using the following formula:
General procedure for the preparation of (E) ⁄ (Z)-
2-[2-(4-chlorophenyl) benzimidazole-1-yl]-N-(2-
arylmethylene amino) acetamides (1–18)
A mixture of acyl hydrazide (0.02 mol), corresponding aldehyde derivatives
(0.02 mol), and ceric ammonium nitrate (0.05 mol) in ethanol (10 mL) was
heated under reflux with stirring for 30 min. Water was added, and the
precipitated product was filtered and crystallized from ethanol.
General procedure for the preparation of 2-[2-
(4-chlorophenyl) benzimidazole-1-yl]-N-(4-oxo-2-
aryl-thiazolidine-3-yl) acetamides (1t–18t)
A mixture of the hydrazone 1–18 (0.1 mmol), thioglycolic acid
(0.2 mmol), and anhydrous sodium acetate in dry toluene was ref-
luxed for 10 h, and the mixture was then filtered while hot. The fil-
trate was evaporated under reduced pressure, and the obtained
solid was crystallized from ethanol.
% Inhibition ¼ ðODcontrol ꢀ ODsampleÞ=ODcontrol ꢁ 100
where ODcontrol is the absorbance of the control with DMSO and
ODsample is the absorbance of the sample in the presence of the
H
N
NH
O
O
Antioxidant activity
N
NH
H
N
N
N
N
Lipid peroxidation level
Cl
Cl
Male albino Wistar rats (200–225 g) used in the experiments were
fed with standard laboratory rat chow and tap water ad libitum. The
animals were starved for 24 h prior to kill and then killed by decapita-
tion under anesthesia. The livers were removed immediately and
washed in ice-cold distilled water, and the microsomes were prepared
as described previously (30). NADPH-dependent LP was determined
using the optimum conditions determined and described previously
(30) and measured spectrophotometrically by estimation of thiobarbi-
turic acid-reactive substances (TBARS). Amounts of TBARS were
expressed in terms of nmol malondialdehyde (MDA) ⁄ mg protein. The
assay was essentially derived from the methods of Wills (31,32) as
modified by Bishayee (33). Lipid peroxidation was determined spectro-
photometrically at 532 nm as the thiobarbituric acid-reactive material.
Compounds inhibit the production of malondialdehyde, and therefore,
the produced color after addition of thiobarbituric acid is less inten-
sive. A typical optimized assay mixture contained 10)3 M test com-
pound, 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 buf-
fer pH 7.8 and 0.2 mg microsomal protein in a final volume of 1.0 mL.
E
Z
Figure 1: Representation of E and Z configuration of compound 4.
Table 1: 1H-NMR data for diastereomeric imines in the (E) ⁄ (Z)
diastereoisomers
d ppm–N=CH-
Diastereoselection
Compound
(E)
(Z)
Ratio (E) ⁄ (Z)a
2
3
8.40
8.39
8.26
8.25
8.21
8.22
8.18
8.21
8.23
8.06
8.04
8.01
8.02
7.99
3 ⁄ 7
3 ⁄ 7
2 ⁄ 8
3 ⁄ 7
4 ⁄ 6
3 ⁄ 7
3 ⁄ 7
4
5
6
7
8
9
10
11
14
15
16
17
18
8.18
8.18
8.57
8.16
8.15
8.16
8.12
7.99
7.99
8.38
7.97
7.94
7.97
7.90
3 ⁄ 7
3 ⁄ 7
2 ⁄ 8
3 ⁄ 7
3 ⁄ 7
3 ⁄ 7
3 ⁄ 7
7-Ethoxyresorufin O-deethylase enzyme activity
7-Ethoxyresorufin O-deethylase (EROD) activity was measured by the
spectrofluorometric method of Burke et al. (34). A typical optimized
assay mixture contained 1.0 mM ethoxyresorufin, 10)3 M test com-
pound, 100 mM Tris–HCl buffer pH 7.8, NADPH-generating system
aData obtained by relative integration of the corresponding imine-attached
hydrogens of the (E) and (Z) diastereoisomers.
Chem Biol Drug Des 2012; 79: 869–877
871

Ayhan-Kılcıgil et al.
Table 2: Some physico-chemical properties and spectral findings of compounds 1–18
H
N
Ar
N
O
N
N
Cl
MS
M + H (%)
Compound
Ar
Formulas
M.P (ꢀC)
1H-NMR
1
C26H19ClN4O
279–282
5.10, 5.59 (2s, 2H, CH2), 7.25–8.82 (m, 16H,
Ar-H, -N=CH), 11.85, 12.06 (2s, 1H, NH)
439 (72)
441 (28)
2
3
C26H19ClN4O
270–272
217–221
5.09, 5.61 (2s, 2H, CH2), 7.28–8.40 (m, 16H,
Ar-H, -N=CH), 11.91, 12.06 (2s, 1H, NH)
3.59 (s, 3H, OCH3), 5.04, 5.57 (2s, 2H, - CH2),
6.79–8.39 (m, 13H, Ar-H,-N=CH), 11.48 (s, 1H,
NH), 11.52, 11.65 (2s, 1H, NH)
439 (71)
441 (29)
458 (73)
460 (27)
H
N
C25H20ClN5O2-1.75H2O
OCH3
4
5
C22H17ClN4O-0.25H2O
C22H16ClFN4O-0.75H2O
230–231
222
5.07, 5.55 (2s, 2H, CH2), 7.22–8.26 (m, 14H,
Ar-H, -N=CH), 11.82, 11.98 (2s, 1H, NH)
389 (75)
391 (25)
5.08, 5.56 (2s, 2H, CH2), 7.26–7.99–8.25 (m,
13H, Ar-H, -N=CH), 11.84, 12.01 (2s, 1H, NH)
407 (74)
409 (26)
F
6
C22H16Cl2N4O-1.85H2O
C22H1 6 BrClN4O
252
280–281
210–212
145 (Bubbl.)
225–228
208
5.05, 5.54 (2s, 2H, CH2), 7.27–8.21 (m, 13H,
Ar-H, -N=CH), 11.85, 12.05 (2s, 1H, NH)
423 (63)
425 (37)
Cl
7
5.06, 5.54 (2s, 2H, CH2), 7.27–8.22 (m, 13H,
Ar-H, -N=CH), 11.87, 12.05 (2s, 1H, NH)
467 (47)
469 (53)
Br
8
C23H19ClN4O2-0.75H2O
C22H16 ClN5O3-1.25H2O
C29H23 ClN4O2-0.25H2O
C22H15ClF2N4O-0.75H2O
3.79 (s, 3H, OCH3), 5.04, 5.51 (2s, 2H, -CH2),
6.98–8.18 (m, 13H, Ar-H,-N=CH), 11.68, 11.83
(2s, 1H, NH)
5.12, 5.63 (2s, 2H, CH2), 7.32–8.55 (m, 13H,
Ar-H, -N=CH), 12.06, 12.21 (2s, 1H, NH)
419 (75)
421 (25)
OCH3
9
434 (76)
436 (24)
NO2
10
11
5.04, 5.51 (2s, 2H, CH2), 5.16 (s, 2H, OCH2),
7.06–8.18 (m, 18H, Ar-H, -N=CH), 11.69, 11.85
(2s, 1H, NH)
5.04, 5.50 (2s, 2H, CH2), 7.99–7.82-8.18
(m, 11H, Ar-H, -N=CH), 11.92, 12.11 (2s, 1H,
NH, exchangeable with D2O)
495 (73)
497 (27)
O
F
425 (74)
427 (26)
F
12
13
C22H15BrClFN4O-2H2O
C22H15Cl2N5O3-3.5H2O
122
5.08, 5.59 (2s, 2H, CH2), 7.28–8.22 (m, 12H,
Ar-H, -N=CH), 11.92, 12.09 (2s, 1H, NH)
485 (37)
487 (50)
489 (13)
F
Br
Cl
130 (dec.)
5.11, 5.62 (2s, 2H, CH2), 7.27–8.72 (m, 12H,
Ar-H, -N=CH), 12.16, 12.40 (2s, 1H, NH)
468 (57)
470 (43)
NO
2
Cl
14
C22H15Cl3N4O-0.5H2O
263–265
5.08, 5.56 (2s, 2H, CH2), 7.27–8.57 (m, 12H,
Ar-H, -N=CH), 12.02, 12.24 (2s, 1H, NH)
457 (43)
459 (42)
461 (15)
Cl
872
Chem Biol Drug Des 2012; 79: 869–877

Synthesis and Evaluation of Antioxidant Properties
Table 2: Continued
MS
M + H (%)
Compound
Ar
Formulas
M.P (ꢀC)
1H-NMR
OCH3
15
C24H21ClN4O3
236–238
3.76 (s, 6H, OCH3), 5.06, 5.54 (2s, 2H, - CH2),
6.56–8.16 (m, 12H, Ar-H, -N=CH), 11.85, 11.98
(2s, 1H, NH)
449 ((77)
451 (23)
OCH3
OCH3
16
C30H25ClN4O3-0.75H2O
215
3.80 (s, 3H, OCH3), 5.06, 5.10 (2s, 2H, - CH2),
5.48 (s, 2H, OCH2), 7.00–8.15 (m, 17H, Ar-H, -
N=CH), 11.73, 11.83 (2s, 1H, NH)
525 (70)
527 (30)
O
17
18
C30H25ClN4O3-0.33H2O
C36H29ClN4O3
186–189
228–229
3.79 (s, 3H, OCH3), 5.07, 5.13 (2s, 2H, - CH2),
5.67 (s, 2H, OCH2), 7.08–8.16 (m, 17H, Ar-H, -
N=CH), 11.76, 11.86 (2s, 1H, NH)
525 (72)
527 (28)
O
OCH3
5.05, 5.44 (2s, 2H, CH2), 5.13 (s, 2H, OCH2),
5.17 (s, 2H, OCH2), 7.06–8.12 (m, 22H, Ar-H, -
N=CH), 11.73, 11.84 (2s, 1H, NH)
601 (73)
603 (27)
O
O
Table 3: Some physico-chemical properties and spectral findings of compounds 1t–18t
MS
Compd.
Formulas
M.P (ꢀC)
NMR
M + H (%)
1t
C28H21ClN4O2S-0.9H2O
285–288
3.84, 3.96 (2H, 2d, j = 17.2 Hz, CH2-thiazolidine ring
C5), 4.88 ((2H, j = 17.6 Hz, AB quartet, CH2), 6.62
(s, 1H, CH thiazolidine ring C2), 7.09–8.07 (m, 15H,
Ar-H), 11.02 (s, 1H, NH)
3.83, 3.99 (2H, 2d, j = 16.0 Hz, CH2-thiazolidine ring
C5), 4.83 ((2H, j = 17.6 Hz, AB quartet, CH2), 6.01
(s, 1H, CH thiazolidine ring C2), 7.01–8.03 (m, 15H,
Ar-H), 10.87 (s, 1H, NH)
3.74 (s, 3H, OCH3), 3.80, 3.90 (2H, 2d, j = 16.0 Hz,
CH2-thiazolidine ring C5), 4.81 (2H, j = 17.1 Hz, AB
quartet, CH2), 5.56 (s, 1H, CH thiazolidine ring C2),
6.83–7.85 (m, 12H, Ar-H), 10.72 (s, 1H, NH), 11.22
(s, 1H, NH)
513 (68) 515 (32)
513 (69) 515 (31)
532 (71) 534 (29)
2t
3t
C28H21ClN4O2S-0.8HSCH2COOH
C27H22ClN5O3S-0.8H2O
262
305
4t
5t
C24H19ClN4O2S
275–278
3.76, 3.92 (2H, 2d, j = 16.0 Hz CH2-thiazolidine ring C5),
4.87 (2H, j = 17.6 Hz AB quartet, CH2), 5.80 (s, 1H, CH
thiazolidine ring C2), 7.21–7.70 (m, 13H, Ar-H), 10.83
(s, 1H, NH)
463 (77) 465 (23)
13C: 29.8, 46.1, 62.1, 11.4, 119.8, 123.0, 123.4, 128.3,
129.2, 129.4, 129.8, 131.6, 135.4, 136.9, 138.9, 142.9,
153.0, 166.9, 169.6
C24H18ClFN4O2S-1.1H2O-0.1C2H5OH
147
3.73, 3.90 (2H, 2d, j = 16.0 Hz, CH2-thiazolidine ring
C5), 4.85 (2H, j = 16.8 Hz, AB quartet, CH2), 5.80 (s,
1H, CH thiazolidine ring C2), 7.18–7.69 (m, 12H, Ar-H),
10.78 (s, 1H, NH)
482 (70) 484 (30)
6t
7t
C24H18Cl2N4O2S-2H2O
169
145
3.77, 3.93 (2H, 2d, j = 16.0 Hz , CH2-thiazolidine ring
C5), 4.80 (2H, s, CH2), 5.83 (s, 1H, CH thiazolidine ring
C2), 7.20–7.76 (m, 12H, Ar-H), 10.83 (s, 1H, NH)
3.76, 3.82 (2H, 2d, j = 16.0 Hz CH2-thiazolidine ring C5),
4.99 (2H, j = 18.0 Hz, AB quartet, CH2), 5.92 (s, 1H,
CH thiazolidine ring C2), 7.26–7.78 (m, 12H, Ar-H),
9.88 (s, 1H, NH)
497 (60) 499 (40)
C24H18 BrClN4O2S-0.2C2H5OH-0.75H2O
541 (36) 543 (46) 545 (18)
Chem Biol Drug Des 2012; 79: 869–877
873

Ayhan-Kılcıgil et al.
Table 3: Continued
MS
Compd.
Formulas
M.P (ꢀC)
NMR
M + H (%)
8t
C25H21ClN4O3S-0.5HSCH2COOH-0.9H2O
159
178
3.76, 3.88 (2H, 2d, j = 14.40 Hz CH2-thiazolidine ring
C5), 3.81 (s, 3H, OCH3), 4.85 (2H, j = 17.20 Hz, AB
quartet, CH2), 5.76 (s, 1H, CH thiazolidine ring C2),
6.98–7.82 (m, 12H, Ar-H), 10.77 (s, 1H, NH)
3.80, 4.00 (2H, 2d, j = 16.0 Hz, CH2-thiazolidine ring
C5), 4.88 (2H, AB quartet, j = 18.8 Hz CH2), 6.02 (s,
1H, CH thiazolidine ring C2), 7.17–8.34 (m, 12H, Ar-H),
10.90 (s, 1H, NH)
3.73, 3.88 (2H, 2d, j = 16.0 Hz, CH2-thiazolidine ring
C5), 4.91 (2H, AB quartet, j = 20.4 Hz, CH2), 5.24 (s,
2H, OCH2-), 5.76 (s, 1H, CH thiazolidine ring C2),
6.98–7.70 (m, 19H, Ar-H)
3.77, 3.95 (2H, 2d, j = 15.6 Hz, CH2-thiazolidine ring
C5), 4.93 (2H, s, CH2), 5.96 (s, 1H, CH thiazolidine ring
C2), 7.20–7.74 (m, 11H, Ar-H), 10.87 (s, 1H, NH)
3.75, 3.98 (2H, 2d, j = 16.0 Hz CH2-thiazolidine ring C5),
4.89 (2H, s, CH2), 5.84 (s, 1H, CH thiazolidine ring C2),
7.22–7.86 (m, 11H, Ar-H), 10.84 (s, 1H, NH)
3.81, 3.97 (2H, 2d, j = 16.4 Hz CH2-thiazolidine ring C5),
4.93 (2H, AB quartet, j = 17.5 Hz , CH2), 6.19 (s, 1H,
CH thiazolidine ring C2), 7.18–8.25 (m, 11H, Ar-H),
11.04 (s, 1H, NH)
493 (78) 495 (22)
9t
C24H18ClN5O4S-1H2O
508 (73) 510 (27)
569 (64) 571 (36)
10t
C31H25ClN4O3S-0.6C2H5OH-1.5H2O
C24H17ClF2N4O2S-0.1H2O
220 decomp.
11t
12t
13t
307–310
128
499 (71) 501 (39)
559 (37) 561 (48) 563 (15)
542 (63) 544 (37)
C24H17BrClFN4O2S-0.5
HSCH2COOH-0.9C2H5OH
C24H17Cl2N5O4S-0.85HSCH2COOH
169
14t
15t
C24H17Cl3N4O2S-0.85H2O
297–299
287–289
3.77, 3.92 (2H, 2d, j = 15.6 Hz, CH2-thiazolidine ring
C5), 4.94 (2H, s, CH2), 6.06 (s, 1H, CH thiazolidine ring
C2), 7.19–7.74 (m, 11H, Ar-H), 10.96 (s, 1H, NH)
3.72–3.93 (m, 8H, OCH3, CH2-thiazolidine ring C5), 4.89
((2H, AB quartet, j = 16.0 Hz , CH2), 5.79 (s, 1H, CH
thiazolidine ring C2), 6.55–7.70 (m, 11H, Ar-H), 10.90
(s, 1H, NH)
531 (41) 533 (45) 535 (14)
523 (70) 525 (30)
C26H23ClN4O4S-0.9HSCH2COOH-1.3H2O
13C: 29.7, 46.2, 55.9, 62.0, 101.3, 105.9, 11.4, 119.8,
123.0, 123.4, 129.1, 129.5, 131.5, 135.4, 136.9, 141.4,
142.9, 152.9, 161.3, 166.9, 169.7
16t
17t
18t
C32H27ClN4O4S-0.3HSCH2COOH-0.45H2O
C32H27ClN4O4S-0.95HSCH2COOH
119
106–107
127
3.70–3.90 (m, 5H, OCH3,CH2-thiazolidine ring C5), 4.88
((2H, AB quartet, j = 17.2 Hz , CH2), 5.05 (s, 2H,
OCH2-), 5.81 (s, 1H, CH thiazolidine ring C2),
6.96–7.69 (m, 16H, Ar-H), 10.78 (s, 1H, NH)
3.71–3.88 (m, 5H, OCH3, CH2-thiazolidine ring C5), 4.84
((2H, AB quartet, j = 17.0 Hz, CH2), 5.11 (s, 2H,
OCH2-), 5.76 (s, 1H, CH thiazolidine ring C2),
6.87–7.67 (m, 16H, Ar-H), 10.75 (s, 1H, NH)
3.74, 3.86 (2H, 2d, j = 15.6 Hz CH2-thiazolidine ring C5),
4.86 (2H, AB quartet, j = 17.6 Hz, CH2), 5.06 (s, 2H,
OCH2-), 5.18 (s, 2H, OCH2-), 5.78 (s, 1H, CH
thiazolidine ring C2), 6.94–7.70 (m, 21H, Ar-H), 10.87
(s, 1H, NH)
599 (68) 601 (32)
599 (69) 601 (31)
675 (66) 677 (34)
C38H31ClN4O4S-0.25
HSCH2COOH-1.4H2O
compounds. A dose response curve was plotted to determine the IC50
values. IC50 is defined as the concentration sufficient to obtain 50%
of a maximum scavenging capacity. All tests and analyses were run in
triplicate and averaged. The standard used in this assay was BHT.
via oxidative condensation of o-phenylenediamine, p-chlorobenzalde-
hyde, and sodium metabisulfite. Treatment of compound A with
ethyl chloroacetate in KOH ⁄ DMSO gave the N-alkylated product B.
Hydrazine hydrate and the ester B in ethanol were refluxed for 4 h
to give the desired hydrazide compound, (2-(p-chlorophenyl)-benz-
imidazole-1-yl)-acetic acid hydrazide C. Compounds 1–18 were
obtained by condensing acyl hydrazide C with the corresponding
aldehyde derivatives in the presence of catalytic amounts of ceric
ammonium nitrate (CAN) in ethanol (36) Condensation of 1–18 with
thioglycolic acid in toluene yielded 2-[2-(4-chlorophenyl) benzimid-
azole-1-yl]-N-(4-oxo-2-aryl-thiazolidine-3-yl) acetamides (1t–18t).
Results and Discussion
Chemistry
The desired benzimidazole derivatives were synthesized according
to Scheme 1. Compound A having benzimidazole ring was prepared
874
Chem Biol Drug Des 2012; 79: 869–877

Synthesis and Evaluation of Antioxidant Properties
In the NMR spectral data, all protons were seen according to the
expected chemical shift and integral values. The aromatic protons
appeared as multiplet peaks within the range 6.56–8.82 for com-
pounds 1–18 and 6.55–8.34 for compounds 1t–18t. For the com-
pounds 1–18, the signals belonging to the benzylidene group were
observed in the aromatic region.
acetamide (1t–18t) counterparts. Among the compounds 1–18,
different aryl groups such as phenyl, 1- or 2-naphthyl or indole-3-yl
have exhibited no considerable difference. The most active com-
pound 16 inhibited EROD activity with a 0.360 mM IC50 value,
which has 4-methoxy-3-benzyloxy phenyl ring as aryl group, while
IC50 value of caffeine was 0.520 mM. Significant inhibitory activi-
ties were also observed for compounds 2 (81%), 9 (81%), 4 (84%),
6 (84%), and 14 (83%).
1
The investigation of H-NMR spectra of compounds 1–18 indicated
the presence of two singlet signals referring to (E) and (Z) geometric
isomers about imino hydrogens (CH=N) and cis ⁄ trans amide conform-
ers. Karabatsos et al. (37) describes that imine-attached hydrogen
signal of (E) diastereoisomer is downfielded by 0.2–0.3 ppm from the
corresponding hydrogen atom signal of (Z) diastereoisomer. This
result showed that the construction of an imino bond of the com-
pounds 1–18 is not a diastereoselective process. Karabatsos et al.
also suggest that if the hydrazones have an aromatic substituent, it
is conceivable that the aromatic ring interacts with the protonated
group favoring the (Z) diastereoisomer (Figure 1). In our findings, E
and Z diastereoisomers were observed at 8.12–8.57 and 7.90–
8.38 ppm, respectively, and the ratio between (E) ⁄ (Z) diastereoisom-
ers could be defined from the relative integration of imine-attached
hydrogen in the corresponding 1H-NMR spectra (Table 1).
Inhibition of NADPH-dependent lipid peroxidation (Table 4) produced
by the compounds in the rat liver microsomes was examined by
measuring the formation of the TBARS for their antioxidant capac-
Table 4: Effects of the compounds on the liver LP levels, EROD
enzyme, and DPPH free radical scavenging activities in vitroa
EROD
(pmol ⁄
LP
(nmol
control ⁄ mg ⁄ dk)
DPPH
% of
control control
% of
% of
Compoundb mg ⁄ dk)
1
5.20 € 0.27
13
19
12
16
11
16
24
28
19
36
11
41
20
17
25
5
12
13
54
43
27
46
35
76
50
39
35
27
22
31
36
37
34
32
25
45
8.12 € 0.38
50
45
399
16
68
50
109
39
114
25
44
44
84
97
28
116
66
11
25
119
–
10
17
22
16
22
9
34
23
15
20
59
21
22
26
11
35
24 € 3.8
2
7.75 € 0.75
4.93 € 0.63
6.69 € 0.08
4.49 € 0.11
6.81 € 0.11
9.95 € 0.32
12.36 € 1.31
8.03 € 3.16
14.95 € 0.44
4.60 € 6.08
17.14 € 1.86
8.49 € 0.41
7.25 € 1.80
10.36 € 6.74
2.13 € 0.11
5.06 € 0.12
5.47 € 0.20
22.51 € 1.17
17.93 € 1.11
11.25 € 0.42
19.11 € 0.25
14.60 € 1.22
31.89 € 0.50
20.66 € 0.70
16.23 € 1.03
14.35 € 2.40
11.23 € 1.61
9.25 € 1.09
12.76 € 3.34
14.91 € 1.81
16.06 € 0.01
14.22 € 3.59
13.12 € 3.25
10.47 € 0.36
18.85 € 0.79
7.29 € 2.13
64.99 € 5.27
2.63 € 0.97
11.04 € 0.95
8.12 € 0.56
17.69 € 2.02
6.33 € 2.53
18.52 € 3.21
4.06 € 0.68
7.17 € 1.35
7.17 € 5.07
13.62 € 1.56
15.77 € 3.04
4.54 € 0.23
19.00 € 6.25
10.75 € 1.02
1.80 € 0.16
4.00 € 1.36
19.17 € 1.49
nd
1.67 € 0.03
2.70 € 0.48
3.54 € 0.35
2.60 € 0.08
3.63 € 0 19
1.52 € 0.48
5.64 € 0.42
3.76 € 0.91
2.36 € 0.54
3.29 € 0.41
9.65 € 0.30
3.41 € 0.83
3.53 € 0.04
4.22 € 0.35
1.77 € 0.53
5.68 € 0.22
84 € 0.8
70 € 2.2
88 € 1.7
nt
90 € 0.6
70 € 3.8
92 € 1.0
nt
3
4
5
The methylene protons of thiazolidinone (1t–18t) displayed two
doublets at 3.70–3.84 and 3.57–4.00 ppm because of the non-
equivalent geminal methylene protons. Thiazolidinone protons (-CH)
(1t–18t) resonated as singlets at 5.11–6.62 ppm. The methylene
protons (-CH2CO-) of compounds 1t–18t were observed at 4.81–
4.99 ppm as an AB quartet.
6
7
8
9
10
11
12
13
14
15
16
17
18
1t
72 € 3.9
nt
nt
nt
It has been recognized for some time that the two protons of a
methylene group adjacent to an asymmetrically substituted carbon
or any dissymmetric moiety are magnetically non-equivalent and
consequently split each other in the NMR spectra (38). In accord to
this expectation, methylene protons of acetamide chain (1t–5t,7t–
11t,13t, and 15t–18t) appear as an AB quartet at 4.81–
4.99 ppm and with a coupling constant 15.93–20.40 Hz. In the other
compounds (6t, 12t, and 14t), magnetically equivalent CH2 pro-
tons are observed as a sharp singlet at 4.80–4.94 ppm.
86 € 3.0
91 € 0.6
85 € 2.0
72 € 0.6
88 € 2.2
81 € 1.2
59 € 0.7
66 € 1.7
85 € 1.5
85 € 0.1
96 € 1.2
69 € 1.4
55 € 1.0
73 € 1.8
23 € 0.6
97 € 0.4
54 € 0.9
52 € 0.7
71 € 2.0
62 € 2.4
62 € 0.6
68 € 0.8
76 € 1.7
15 € 2.9
2t
3t
4t
5t
6t
The mass spectra of synthesized compounds showed a M + H ion
peak, which is conforming with the molecular formula of the com-
pounds. The structures of the synthesized compounds were consis-
tent with the 1H-, 13C-NMR, and mass spectra. Spectral data are
summarized in Tables 1, 2, and 3.
7t
8t
9t
10t
11t
12t
13t
14t
15t
16t
17t
18t
BHT
Caffeine
Controlc
Antioxidant activity in vitro
The in vitro effects of the compounds and caffeine in liver micro-
somal EROD activity are shown in Table 4. All the tested com-
pounds except compound 6t (24%) showed significant inhibition
(50–95%) of EROD activity. Compounds 1, 3, 5, 11, 16, 17, and
18 decreased liver EROD activity in the range of 87–95%, better
than that of the specific inhibitor caffeine (85%) at 10)3 M concen-
tration. In terms of inhibition on EROD activity, it can be said that
2-[2-(4-chlorophenyl) benzimidazole-1-yl]-N-(2-arylmethylene amino)
acetamide (1–18) derivatives displayed better activity than 2-[2-(4-
6.41 € 0.36
41.53 € 0.99 100
15
16.25 € 1.45
100
100
aEach value represents mean € SD of 2–4 independent experiments.
bConcentration in incubation medium (10)3 M).
cDMSO only, control for compounds.
chlorophenyl)
benzimidazole-1-yl]-N-(4-oxo-2-aryl-thiazolidine-3-yl)
nt, not tested.
Chem Biol Drug Des 2012; 79: 869–877
875

Ayhan-Kılcıgil et al.
ity. In contrast to EROD activity, in the series of thiazolidinone, all
of the compounds except compound 10t have better LP activities
than corresponding methylaminoacetamide derivatives (1–18). The
most active compound 9t (2-[2-(4-chlorophenyl) benzimidazole-1-yl]-
N-(4-oxo-2-(3-nitrophenyl-thiazolidine-3-yl) acetamide) caused 91%
inhibition (0.375 mM IC50) on LP level in rat liver microsomes at
for the control of periodontal diseases. J Med Chem;30:205–
208.
9. Soliman F.S.G., Rida S.M., Badawey E.A.M., Kappe T. (1984)
Synthesis of substituted 3-hydroxy-1H,5H-pyrido[1,2-a]ben-
zimidazol-1-ones as possible antimicrobial and antineoplastic
agents. Arch Pharm;317:951–958.
10. Podunovac-Kuzmanovic S.O., Cvetkovic D.D., Barna D.J. (2008)
The effect of lipophilicity on the antibacterial activity of some
1-benzylbenzimidazole derivatives. J Serb Chem Soc;73:967–978.
11. Islam I., Skibo E.B., Dorr R.T., Alberts D.S. (1991) Structure-activ-
ity studies of antitumor agents based on pyrrolo[1,2-a]benzimi-
dazoles: new reductive alkylating DNA cleaving agents. J Med
Chem;34:2954–2961.
12. Kruse L.I., Ladd D.L., Harrsch P.B., McCabe F.L., Mong S.M.,
Faucette L., Johnson R. (1989) Synthesis, tubulin binding, anti-
neoplastic evaluation, and structure-activity relationships of on-
codazole analogues. J Med Chem;32:409–417.
10)3
M
concentration, while BHT showed 65% inhibition
(0.408 mM IC50) at the same concentration.
We also tested the DPPH radical scavenger capacities (Table 4) by
the synthesized compounds. Almost all of the compounds' DPPH
free radical scavenger capacities were not enough, except com-
pounds 1 and 10t. Compounds 1 and 10t showed the highest
scavenger capacities among the tested compounds on DPPH radical
with 76% and 77%, respectively, which are close to BHT (85%).
Conclusion
13. Habernickel V.J. (1992) Alkyl-5-heterocyclic-benzimidazolyl-carba-
mate derivatives. Drugs Ger;35:97.
Compound 10t appeared to have similar effect for all assay sys-
tems. It is plausible to suggest that the inhibition of LP levels and
DPPH scavenger capacities could be related to decrease in produc-
tion of oxygen radicals because of the depression of CYPA1 ⁄ 2.
Moreover, the other compounds showed different effect on these
parameters. Such contradictory results have also been found in a
previous study (39). Each method relates to the generation of a dif-
ferent radical, acting through a variety of mechanism. Therefore,
the observation of different effects of the synthesized compounds
on EROD activity, LP levels, and DPPH radical scavenger capacity is
not surprising, as the mechanism of production of reactive oxygen
species using these methods is different.
14. Fukuda T., Morimoto Y., Iemura R., Kawashima T., Tsukamoto G.,
Ito K. (1984) Efffects of 1-(2- ethoxyethyl)-2-(4-methyl-1-homopip-
erazinyl)-benzimidazole difumarate (KB-2413), a new antiallergic,
on chemical mediators. Arzneim-Forsch ⁄ Drug Res;34:801–805.
15. Nakano H., Inoue T., Kawasaki N., Miyataka H., Matsumoto H.,
Taguchi T., Inagaki N., Nagai H., Satoh T. (2000) Synthesis of
benzimidazole derivatives as antiallergic agents 5-lipoxygenase
inhibiting action. Bioorg Med Chem;8:373–380.
16. Can-Eke B., Puskullu M.O., Buyukbingol E., Iscan M. (1998) Study
on the antioxidant capacities of some benzimidazoles in rat tis-
sues. Chem Biol Interact;113:65–77.
17. Siddiqui N., Arshad M.F., Khan S.A., Ahsan W. (2010) Sulfon-
amide derivatives of thiazolidin-4-ones with anticonvulsant activ-
ity against two seizure models:synthesis and pharmacological
evaluation. J Enzyme Inhib Med Chem;25:485–491.
References
18. Havrylyuk D., Mosula L., Zimenkovsky B., Vasylenko O., Gzella
A., Lesyk R. (2010) Synthesis and anticancer activity evaluation
of thiazolidinones containing benzothiazole moiety. Eur J Med
Chem;45:5012–5021.
19. Upadhyay A., Srivastava S.K., Srivastava S.D. (2010) Conven-
tional and microwave assisted synthesis of some new N-[(4-oxo-
2-substituted aryl _1, 3-thiazolidine)-acetamidyl]- 5-nitroindazoles
and its antimicrobial activity. Eur J Med Chem;45:3541–3548.
20. Cacic M., Molnar M., Sarkanj B., Has-Schon E., Rajkovic V.
(2010) Synthesis and antioxidant activity of some new coumari-
nyl-1,3-thiazolidine-4-ones. Molecules;15:6795–6809.
1. Rice-Evans C., Diplock A.T. (1991) Techniques in Free Radical
Research. Amsterdam: Elsevier, 291 p.
2. Hallivell B., Halliwell B. (1994) Free radicals and antioxidants: a
personal view. Nutr Rev;52:253–265.
3. Krishna M.C., DeGraff W., Hankovszky O.H., Sar C.P., Kalai T.,
Jeko J., Russo A., Mitchell J.B., Hideg K. (1998) Studies of
structure–activity relationship of nitroxide free radicals and their
precursors as modifiers against oxidative damage. J Med
Chem;41:3477–3492.
4. Shackelford R.E., Kaufmann W.K., Paules R.S. (2000) Oxidative
stress and cell cycle checkpoint function. Free Radical Biol
Med;28:1387–1404.
5. Cadenas E., Packer L. (2002) Handbook of Antioxidants, 2nd edn.
Revised and Expanded. New York-Basel: Marcel Dekker.
6. Sa¢nchez-Moreno C. (2002) Review: methods used to evaluate
the free radical scavenging activity in foods and biological sys-
tems. Food Sci Technol Int;8:121–137.
21. Kus C., Ayhan-Kılcıgil G., Can-Eke B., Iscan M. (2004) Synthesis
and antioxidant properties of some novel benzimidazole deriva-
tives on lipid peroxidation in the rat liver. Arch Pharm
Res;27:156–163.
22. Kus¸ C., Ayhan-Kilcigil G., Altanlar N. (2004) Antimicrobial activity
of some thiadiazolyl- and triazolylbenzimidazoles. J Fac Pharm
Ankara Univ;33:1–6.
7. Abdel-Rahman A.E., Mahmoud A.M., El-Naggar G.M., El-Sherief
H.A. (1983) Synthesis and biological activity of some new benz-
imidazolyl-azetidin-2-ones and thiazolidin-4-ones. Pharma-
zie;38:589–590.
8. Coburn R.A., Clark M.T., Evans R.T., Genco R.J. (1987) Substi-
tuted 2-(2-hydroxyphenyl) benzimidazoles as potential agents
23. Ayhan-Kılcıgil G., Kus C., Coban T., Can-Eke B., Iscan M. (2004)
Synthesis and antioxidant properties of novel benzimidazole
derivatives. J Enzyme Inhib Med Chem;19:129–135.
24. Ayhan-Kilcigil G., Kus C., Coban T., Can-Eke B., Ozbey S., Iscan
M. (2005) Synthesis, antioxidant and radical scavenging activi-
876
Chem Biol Drug Des 2012; 79: 869–877

Synthesis and Evaluation of Antioxidant Properties
ties of novel benzimidazoles. J Enzyme Inhib Med Chem;20:503–
31. Wills E.D. (1966) Mechanism of lipid peroxide formation in ani-
mal tissues. Biochem J;99:667–676.
32. Wills E.D. (1969) Lipid peroxide formation in animal tissues. Bio-
chem J;113:333–341.
33. Bishayee S., Balasubramanian A.S. (1971) Lipid peroxide forma-
tion in rat brain. J Neurochem;18:909–920.
34. Burke M.D., Thompson S., Elcombe C.R., Halpert J., Haaparanta
T., Mayer R.T. (1985) Ethoxy-, pentoxy- and benzyloxyphenoxaz-
ones and homologues: a series of substrates to distinguish
between different induced cytochromes P-450. Biochem Pharma-
col;34:3337–3345.
514.
_
25. Ayhan-Kilcigil G., Kus
¸
C., ꢀzdamar E.D., Can-Eke B., Is
¸
can M.
(2007) Synthesis and antioxidant capacities of some new benz-
imidazole derivatives. Arch Pharm Chem Life Sci;340:607–611.
26. Kerimov I., Ayhan-Kilcigil G., Can-Eke B., Altanlar N., Iscan M.
(2007) Synthesis, antifungal and antioxidant screening of some
novel benzimidazole derivatives.
Chem;22:696–701.
J
Enzyme Inhib Med
27. Kus¸ C., Ayhan-Kilcigil G., ꢀzbey S., Kaynak F.B., Kaya M., ꢁoban
T., Can-Eke B. (2008) Synthesis and antioxidant properties of
novel N-methyl-1,3,4-thiadiazol-2-amine and 4-methyl-2H-1,2,4-
triazole-3(4H)-thione derivatives of benzimidazole class. Bioorg
Med Chem;16:4294–4303.
35. Blois M.S. (1958) Antioxidant determination by the use of a sta-
ble free radical. Nature;181:1199–1200.
36. Dabiri M., Salehi P., Baghbanzadeh M., Bahramnejad M. (2006) A
facile procedure for the one-pot synthesis of unsymmetrical 2,5-
disubstituted 1,3,4-oxadiazoles. Tetrahedron Lett;47:6983–6986.
37. Karabatsos G.J., Graham J.D., Vane F.M. (1962) Syn-anti isomer
determination of 2,4-dinitrophenylhydrazones and semicarbaz-
ones by N.m.r. J Am Chem Soc;84:753–755.
38. Hill R.K., Chan T.K. (1965) Magnetic non-equivalence of methy-
lene protons in dissymmetric benzylamines: a simple method of
assignment of configuration to identically a,a¢-disubstituted het-
erocyclic bases. Tetrahedron;21:2015–2019.
28. Kus
¸ C., Ayhan-Kılcıgil G., TunÅbilek M., Altanlar N., ꢁoban T.,
_
Can-Eke B., Is
¸
can M. (2009) Synthesis, antimicrobial and antioxi-
dant activities of some benzimidazole derivatives. LDDD;6:374–
379.
29. Gurkan A.S., Karabay A., Buyukbingol Z., Adejare A., Buyukbingol
E. (2005) Syntheses of novel indole lipoic acid derivatives and
their antioxidant effects on lipid peroxidation. Arch Pharm Chem
Life Sci;338:67–73.
30. Iscan M., Arinc E., Vural N., Iscan M.Y. (1984) In vivo effects of
3-methylcholantrene, phenobarbital, pyretrum and 2,4,5-tisoocty-
lester on liver, lung and kidney microsomal mixed function oxi-
dase system of guinea-pig: a comparative study. Comp Biochem
Physiol;77C:177–190.
39. ꢀlgen S., Coban T. (2003) Antioxidant evaluations of novel N-H
and N-substituted indole esters. Biol Pharm Bull;26:736–738.
Chem Biol Drug Des 2012; 79: 869–877
877