Synthesis and evaluation of antioxidant activities of novel 1,3,4-oxadiazole and imine containing 1H-benzimidazoles

Article abstract of DOI:10.3906/kim-1403-44

Some novel 2-(substitutedbenzylthio)-5-((2-(4-substitutedphenyl)-1H-benzo[d]imidazol-1-yl)methyl)-1,3,4-oxadiazoles (5-12) and 2-(2-(4-chlorophenyl)-1H-benzo[d]imidazol-1-yl)-N′-(arylmethylene)acetohydrazide derivatives (13-22) were prepared and their in

Full text of DOI:10.3906/kim-1403-44

Turk J Chem
Turkish Journal of Chemistry
(2015) 39: 42 – 53
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1403-44
Research Article
Synthesis and evaluation of antioxidant activities of novel 1,3,4-oxadiazole and
imine containing 1H -benzimidazoles
1
2
Ay¸se Selen ALP1,∗, Gu¨lgu¨n KILCIGIL , El¸cin Deniz OZDAMAR ,
˙
¨
Tu¨lay C¸ OBAN2, Binay EKE2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandog˘an, Ankara, Turkey
2Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Ankara University, Tando˘gan, Ankara, Turkey
Received: 17.03.2014
•
Accepted: 21.06.2014
•
Published Online: 23.01.2015
•
Printed: 20.02.2015
Abstract: Some novel 2-(substitutedbenzylthio)-5-((2-(4-substitutedphenyl)-1H -benzo[d]imidazol-1-yl)methyl)-1,3,4-
oxadiazoles (5–12) and 2-(2-(4-chlorophenyl)-1H -benzo[d]imidazol-1-yl)-N′ -(arylmethylene)acetohydrazide derivatives
(13–22) were prepared and their in vitro antioxidant properties were investigated by determination of rat liver microsomal
NADPH-dependent inhibition of lipid peroxidation (LP) levels and microsomal ethoxyresorufin O-deethylase (EROD)
activity. Compound 18 was found to be the most active compound with 100% inhibition on LP level and 92% inhibition
on EROD. Compounds 4b, 17, and 19 showed the strongest inhibitory effect (97%) on EROD. The free radical scavenging
capacities of the compounds were also tested in vitro determining the interaction of the stable free radical 2,2,diphenyl-
1-picrylhydrazyl (DPPH), and compounds 4a and 4b exhibited good antioxidant activities.
Key words: Antioxidant, lipid peroxidation, benzimidazole, oxadiazole, imine
1. Introduction
Antioxidant defense mechanisms are required to prevent cellular damage observed in various diseases. Impair-
ment of the antioxidants and antioxidant systems could be related to increased oxidative stress.1,2 Therefore,
drugs possessing antioxidant and free radical scavenging properties are considered for preventing and/or treat-
ment of diseases that are directly involved with the lack of antioxidant capacity of organisms. It is known that
lipid peroxidation (LP) is a free radical-initiated reaction that causes the degeneration of the cell membranes3
and is involved in the evaluation of the antioxidant properties of a compound. Most products of lipid perox-
idation are known to have mutagenic and/or carcinogenic properties. Furthermore, reactive oxygen/nitrogen
species are produced by different mechanisms such as cytochrome P450 (CYP)-dependent enzymes that me-
tabolize chemicals and endogenous substances. In this system, CYP1A1/2 have an important role in NADPH-
dependent LP. Therefore, it is important to evaluate the effects of synthesized compounds on NADPH-dependent
LP and CYP systems.4 On the other hand, DPPH assay is recommended as an accurate method for measuring
the antioxidant capacity of various compounds.5
Previously, we have reported the synthesis, characterization, and antioxidant properties of some benzimi-
dazole derivatives containing thiadiazole, triazole, oxadiazole, and thiazolidinone rings at the first position.6−14
In the present study, the design and synthesis of some novel benzimidazole derivatives having an oxadiazole ring
∗Correspondence: sgurkan@pharmacy.ankara.edu.tr
42

ALP et al./Turk J Chem
Table 1. Formula of compounds 5–22.
Compound
R1
R2
Ar
5
6
Cl
Cl
H
-
-
-
-
-
-
-
-
4-Br
7
Cl
2,4-diCl
4-F
8
Cl
9
Cl
4-NO2
4-Br
10
11
12
OCH2Ph
OCH2Ph
OCH2Ph
2,4-diCl
4-NO2
13
14
Cl
Cl
-
-
15
16
Cl
Cl
-
-
17
18
19
Cl
Cl
Cl
-
-
-
20
21
Cl
Cl
-
-
22
Cl
-
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ALP et al./Turk J Chem
Table 2. In vitro effects of compounds 4a, 4b, and 5–22 on liver LP levels, EROD enzyme, and DPPH free radical
scavenging capacities*.
DPPH
EROD
LP
(nmol/mg/min)
16.57 ꢀ 0.07
Compound**
% inh.
% inh.
(% inh.) (pmol/mg/min)
4a10
4b
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
BHT
Caffeine
88
88
5
3
5
25.05 ꢀ 1.80
1.10 ꢀ 0.25
2.18 ꢀ 0.68
6.35 ꢀ 1.51
8.70 ꢀ 0.70
2.60 ꢀ 0.16
2.59 ꢀ 0.41
9.59 ꢀ 1.46
5.34 ꢀ 0.72
9.61 ꢀ 2.30
6.07 ꢀ 0.41
4.01 ꢀ 1.23
3.29 ꢀ 0.64
2.29 ꢀ 0.07
1.23 ꢀ 0.22
3.21 ꢀ 0.48
1.37 ꢀ 0.20
4.30 ꢀ 0.58
3.44 ꢀ 0.74
4.88 ꢀ 0.22
40
97
95
85
79
94
94
77
87
77
85
90
92
95
97
92
97
90
92
88
NA
78
46
49
59
58
61
54
50
54
62
51
68
NA
63
10
10
3.60 ꢀ 0.36
8.92 ꢀ 0.44
8.42 ꢀ 0.78
6.68 ꢀ 0.44
6.76 ꢀ 0.69
6.32 ꢀ 0.71
7.45 ꢀ 0.42
8.10 ꢀ 0.42
7.51 ꢀ 0.18
6.21 ꢀ 0.92
8.03 ꢀ 0.33
5.16 ꢀ 0.35
17.11 ꢀ 3.14
5.95 ꢀ 0.78
0
NA
6
33
24
76
NA
6
5
13
12
17
20
20
35
57
85
100
NA
NA
NA
43
18.77 ꢀ 4.25
18.09 ꢀ 2.27
18.32 ꢀ 2.09
9.33 ꢀ 0.98
5.68 ꢀ 022
65
6.41 ꢀ 0.36
41.53 ꢀ 0.99
85
-
Control (DMSO)
16.25 ꢀ 1.45
-
*Each value represents mean ꢀ SD of 2–4 independent experiments. **Concentration in incubation medium (10−3 M).
NA: No activity
(5–12) and arylmethyleneamino acetamide (13–22) (Table 1) were performed and their antioxidant properties
were investigated (Table 2).
2. Results and discussion
2.1. Chemistry
The desired benzimidazole derivatives were synthesized according to the Scheme. Firstly, 2-(4-chlorophenyl)-
1H -benzo[d]imidazole (1a) and 2-(4-(benzyloxy)phenyl)-1H -benzo[d]imidazole (1b) were prepared via oxida-
tive condensation of o-phenylenediamine, the corresponding aldehyde (p-chloro benzaldehyde or 4-benzyloxy
benzaldehyde, respectively), and sodium metabisulfite.10,12 Treatment of 1a (or 1b) with ethyl chloroacetate in
KOH/DMSO gave the N -alkylated products ethyl 2-(2-(4-chlorophenyl)-1H -benzo[d]imidazol-1-yl)acetate (2a)
or ethyl 2-(2-(4-(benzyloxy) phenyl)-1H -benzo[d]imidazol-1-yl)acetate (2b).10,12 Hydrazine hydrate and the es-
ter (2a or 2b) in ethanol were refluxed for 4 h to give the desired hydrazide compounds, 2-(2-(4-chlorophenyl)-
1H -benzo[d]imidazol-1-yl)acetohydrazide (3a) or 2-(2-(4-(benzyloxy) phenyl)-1H -benzo[d]imidazol-1-yl)ace-
tohydrazide (3b).12,15 Then 5-((2-(4-chloro phenyl)-1H -benzo[d]imidazol-1-yl)methyl)-1,3,4-oxadiazole-2-thiol
44

ALP et al./Turk J Chem
(4a)10 and 5-((2-(4-(benzyloxy)phenyl)-1H -benzo[d]imidazol-1-yl)methyl)-1,3,4-oxadiazole-2-thiol (4b) were
synthesized by the reaction of the hydrazide compounds 3a and 3b, respectively, with carbon disulfide/KOH in
ethanol.16 Thiol compounds 4a and 4b were alkylated with related benzylbromide in the presence of potassium
hydroxide to obtain 2-(substitutedbenzylthio)-5-((2-(4-chlorophenyl)-1H -benzo[d]imidazol-1-yl)methyl)-1,3,4-
oxadiazole derivatives 5–9 and 2-(substitutedbenzylthio)-5-((2-(4-(benzyloxy) phenyl)-1H -benzo[d]imidazol-1-
yl)methyl)-1,3,4-oxadiazole derivatives 10–12. Moreover, compounds 13–22 were obtained by condensing acyl
hydrazide 3a with the corresponding aromatic aldehyde derivatives in the presence of catalytic amounts of
ceric ammonium nitrate (CAN) in ethanol (Scheme).14 The chemical structures of the synthesized compounds
were consistent with their mass and 1 H and 13 C NMR spectra. The spectral data are summarized in the
Experimental section.
Scheme. Synthetic route to the compounds 5–22. Reagents: (a) Na2 S2 O3 Adduct of 4-chlorobenzaldehyde or
4-(benzyloxy)benzaldehyde/DMF; (b) Ethyl chloroacetate/KOH; (c) Hydrazine/EtOH; (d) CS2 /KOH; (e) Related
benzylbromide/KOH; (f) Corresponding aromatic aldehyde/CAN/EtOH.
1 H NMR and 13 C NMR spectra were measured in chloroform-d for compounds 14 and 17 and in
dimethyl sulfoxide-d6 for the other imine-containing compounds (13, 15, 16, 18–22) at ambient temperature.
N -acylhydrazones can exist as 4 isomers due to the geometric isomerism with respect to the imino group (E ,Z
isomers) and conformers about the amide linkage (syn/anti amide conformers).17 In the 1 H NMR spectra
measured in the less polar solvent chloroform-d, distinguished proton signals belonging to aromatic hydrogens
45

ALP et al./Turk J Chem
were observed. Imino hydrogen (CH=N), methylene protons (CH2 CO), and amide hydrogen (NH) displayed 1
singlet signal about the single isomer in chloroform-d. However, in polar solvent dimethyl sulfoxide-d6 , 2 singlet
signals expressing the imino hydrogen (CH=N) were observed at 8.20–8.79 and 8.01–8.62 ppm; the syn/anti
methylene protons (CH2 CO) were also observed typically as 2 singlet signals at 5.02–5.15 and 5.32–5.64 ppm,
respectively, for compounds 13–17 and 19–22 favoring 1 geometric isomer (E or Z). For compound 18, 4
singlet signals were observed at 12.01, 12.19, 14.34, and 14.68 ppm for the NH proton; at 8.61, 8.62, 8.78, and
8.79 ppm for the CH=N proton; and at 5.10, 5.32, 5.57, and 5.58 for –CH2 protons belonging to syn and anti
conformers about both of the E and Z isomers.
For establishing the solvent effect in isomerism of the N -acylhydrazones, 1 H-NMR spectra for compound
14 were determined in 2 different (less polar and polar) solvents. According to the results, methylene, CH=N,
and NH protons displayed 1 singlet signal belonging to 1 isomer at 5.34, 7.88, and 10.8 ppm in chloroform-d,
while it was observed as 2 singlet signals belonging to anti and syn conformers at 5.02 and 5.40, 8.29 and 8.48,
and 11.64 and 11.83 ppm in dimethyl sulfoxide-d6 , respectively.
The upfield lines of methylene protons have been assigned to syn amide conformers and the downfield lines
of methylene protons to anti amide conformers.17,18 The intensities of the 1 H NMR signals of methylene protons
have allowed us to make measurements of the ratio of amide syn/anti conformers. Syn amide conformers were
predominant over anti conformers when dissolved in dimethyl sulfoxide-d6 at the ratio of 6/4 for compounds
13, 14, 17, and 19; at the ratio of 7/3 for compounds 15, 16, and 20–22; and at the ratio of 6.5/3.5 for
compound 18.
2.2. In vitro antioxidant activity
The synthesized compounds were evaluated based on their antioxidant effects on the rat liver microsomal
NADPH-dependent lipid peroxidation (LP) levels by measuring the formation of 2-thiobarbituric acid reactive
substances (TBARS) (Table 2). The in vitro inhibitory effect of intermediate thiol compound 4b bearing
benzyloxyphenyl at the second position of the benzimidazole ring on LP levels was stronger (78%) than that of
the corresponding S -substituted 1,3,4-oxadiazole derivatives. On the other hand, compound 4a bearing chloro
substituent showed no activity on LP levels.10 5-Mercapto-S -substituted 1,3,4-oxadiazole derivatives (5–12)
had moderate inhibitory activity on LP levels in the range of 46%–61%, whereas imine containing compounds
(13–22) exhibited diverse levels of activity. Compounds 15 (68%) and 18 (100%) displayed the highest activity
among all of the synthesized compounds. The most active compound, 18, led to 100% inhibition on LP level,
while butylated hydroxy toluene (BHT) showed 65% inhibition at the same concentration.
The compounds were tested for their in vitro effects on liver microsomal EROD activity. The inhibitory
effects of intermediate thiol compound 4b on EROD activity were more powerful (97%) than those of the
corresponding 5-mercapto-S -substituted 1,3,4-oxadiazole derivatives 10–12, while compound 4a displayed the
lowest inhibitory activity (40%) on EROD10 among all of the compounds. All the final 1,3,4-oxadiazole
derivatives caused significant inhibition (77%–95%) of EROD activity. Typically, imine-containing compounds
(13–22) showed better EROD inhibitory effects than 1,3,4-oxadiazoles (5–12). All the compounds except 7
(79%), 10 (77%), and 12 (77%) inhibited microsomal EROD activity better than (87%–97%) standard caffeine
(85%), while compounds 6 and 13 possessed the same inhibitory effect as caffeine (85%) (Table 2).
The compounds’ interaction with the stable free radical DPPH was also examined. It was observed that
the final compounds did not show a significant inhibition in the DPPH scavenging assay, except compound
46

ALP et al./Turk J Chem
12, which exhibited the highest scavenger capacity on DPPH radical with 76%, which is close to that of BHT
(85%). Furthermore, the DPPH radical scavenger capacities of the intermediate thiol compounds 4a and 4b
were the same (88%) at 10−3 M concentration (Table 2). Compound 4b (81%) was more active than compound
4a (71%) at 10−4 M concentration, with IC50 values of 0.8 × 10−4 M and 0.65 × 10−4 M, respectively.
These findings indicate that some of the synthesized compounds possess beneficial effects on the human
antioxidant defense system and have been suggested to act as antioxidants. Therefore, they could be thought
as promising treatment candidate compounds for diseases related to excess of some reactive species.
3. Conclusion
The synthesized compounds had different effects on the systems examined. Compound 18, which bears
2-pyridinyl, showed the best inhibitory activity on liver microsomal LP levels (100%) and EROD (92%).
Intermediate thiol compound 4b and final imine compounds 17 and 19, bearing 4-pyridinyl and 5-nitrofuran-
2-yl substituents, respectively, had the most powerful inhibitory effect (97%) on EROD. Among all of the
final compounds, compound 12 displayed the highest inhibition (76%) in the DPPH scavenging assay. The
substitution of intermediate 4b with benzylic groups led to a reduction in the antioxidant activity for all
systems tested, while the substitution of intermediate 4a decreased the DPPH scavenging activity at the same
concentration.
4. Experimental section
4.1. General synthesis
All starting materials and chemical reagents used in the synthesis were high-grade commercial products pur-
chased from Aldrich or Merck (Germany). BHT and caffeine were obtained from Sigma. Analytical thin-layer
chromatography was performed with Merck precoated TLC plates, and spots were visualized with ultraviolet
light. Column chromatography was accomplished on silica gel 60 (40–63-µm particle size) (Merck, Germany).
Melting points were determined with an Electrothermal 9100 digital melting point apparatus and were uncor-
rected. The structures of all synthesized compounds were assigned on the basis of NMR and mass spectral
analyses. 1 H NMR and 13 C NMR spectra were measured with a Varian Mercury 400 MHz instrument (Varian
Inc., Palo Alto, CA, USA) using TMS internal standard and CDCl3 or DMSO-d6 ; coupling constants (J) were
reported in Hertz. All chemical shifts were reported as δ (ppm) values. ES–MS were obtained with a Waters
ZQ Micromass LC–MS spectrometer (Waters Corporation, Milford, MA, USA) with positive electrospray ion-
ization. All instrumental analyses were performed at the Central Instrumentation Laboratory of the Pharmacy
Faculty of Ankara University, Ankara, Turkey.
General procedure for the preparation of 4b
2-(2-(4-(Benzyloxy)phenyl)-1H -benzo[d]imidazol-1-yl)acetohydrazide (3b) (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 H2 O and 1 mL of ethanol. The reaction
mixture was refluxed for 3 h. The solid obtained after evaporation under reduced pressure was dissolved in
water and acidified with conc. HCl. The precipitate was filtered, washed with water, and recrystallized from
ethanol.10
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ALP et al./Turk J Chem
5-((2-(4-(Benzyloxy)phenyl)-1H -benzo[d]imidazol-1-yl)methyl)-1,3,4-oxadiazole-2-thiol (4b)
White solid (yield 77%), mp 218–220 ◦ C. MS (ESI+) M+H (%): 415 (100). 1 H NMR δ ppm (DMSO-d6):
7.72 (d, 2H, J = 8.8 Hz), 7.63–7.70 (m, 2H), 7.46 (d, 2H,J = 6.8 Hz), 7.39 (t, 2H, J = 7.6 Hz), 7.26–7.34 (m,
3H), 7.19 (d, 2H, J = 8.8 Hz), 5.67 (s, 2H, –O–CH2), 5.19 (s, 2H, –N–CH2). 13 C NMR δ ppm (DMSO-d6):
178.6, 160.6, 159.8, 153.6, 142.2, 137.4, 136.1, 131.6, 129.2, 128.7, 128.5, 123.8, 123.6, 121.8, 119.4, 115.9, 111.6,
70.2.
General procedure for the preparation of 2-(substitutedbenzylthio)-5-((2-(4-chlorophenyl)-1H -
benzo[d]imidazol-1-yl)methyl)-1,3,4-oxadiazoles (5–9) and 2-(substitutedbenzylthio)-5-((2-(4-(ben-
zyloxy)phenyl)-1H -benzo[d]imidazol-1-yl)methyl)-1,3,4-oxadiazoles (10–12)
To a mixture of the thiol compound 4a (or 4b) (0.15 mmol) in 0.1 mL of 1 N KOH and 1 mL of H2 O was
added corresponding benzylbromide (0.15 mmol). The mixture was stirred overnight at room temperature. The
end of the reaction was monitored by TLC. The solid separated was collected and dried. The crude product
was purified by column chromatography eluting with an appropriate solvent system or by recrystallization from
ethanol to give 5–12.
2-(Benzylthio)-5-((2-(4-chlorophenyl)-1H -benzo[d]imidazol-1-yl)methyl)-1,3,4-oxadiazole (5)
Crude 5 was purified by column chromatography (n-hexane/ethyl acetate (3:1)) to provide 5 (yield 40%) as a
white solid, mp 112–114 ◦ C. MS (ESI+) M+H (%): 433 (100), 435 (37). 1 H NMR δ ppm (CDCl3): 7.83–7.86
(m, 1H), 7.78 (d, 2H, J = 8.4 Hz), 7.54 (d, 2H, J = 8.8 Hz), 7.50–7.52 (m, 1H), 7.31–7.37 (m, 4H), 7.25–7.28
(m, 3H), 5.49 (s, 2H, –N–CH2), 4.42 (s, 2H, –S–CH2). 13 C NMR δ ppm (CDCl3): 165.9, 162.3, 152.5, 142.8,
136.8, 135.4, 134.9, 130.9, 129.4, 129.0, 128.8, 128.2, 127.6, 123.9, 123.5, 120.3, 110.1, 39.6, 36.9.
2-(4-Bromobenzylthio)-5-((2-(4-chlorophenyl)-1H -benzo[d]imidazol-1-yl)methyl)-1,3,4-oxadiazole
(6)
Crude 6 was recrystallized from ethanol to provide 6 (yield 44%) as a white solid, mp 159 ◦ C. MS (ESI+)
M+H (%): 511 (66), 513 (100), 515 (31). 1 H NMR δ ppm (CDCl3): 7.82–7.85 (m, 1H), 7.78 (d, 2H, J = 8.4
Hz), 7.54 (d, 2H, J = 8.8 Hz), 7.48–7.51 (m, 1H), 7.39 (d, 2H, J = 8.0 Hz), 7.34–7.37 (m, 2H), 7.21 (d, 2H,
J = 8.4 Hz), 5.49 (s, 2H, –N–CH2), 4.35 (s, 2H, –S–CH2). 13 C NMR δ ppm (CDCl3): 165.5, 162.4, 152.5,
142.9, 136.8, 135.4, 134.2, 131.9, 130.9, 130.7, 129.4, 127.6, 123.9, 123.6, 122.3, 120.3, 110.1, 39.5, 36.1.
2-((2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)methyl)-5-(2,4-dichlorobenzylthio)-1,3,4-oxa-
diazole (7)
Crude 7 was recrystallized from ethanol to provide 7 (yield 45%) as a white solid, mp 166–168 ◦ C. MS (ESI+)
M+H (%): 501 (95), 503 (100), 505 (40), 507 (10). 1 H NMR δ ppm (CDCl3): 7.82–7.85 (m, 1H), 7.78 (d, 2H,
J = 8.8 Hz), 7.54 (d, 2H, J = 8.8 Hz), 7.48–7.51 (m, 1H), 7.43 (d, 1H, J = 8.4 Hz), 7.32–7.37 (m, 3H), 7.12
(dd, 1H, J = 8.4 Hz, J = 2.0 Hz), 5.49 (s, 2H, –N–CH2), 4.47 (s, 2H, –S–CH2). 13 C NMR δ ppm (CDCl3):
165.6, 162.5, 152.5, 142.9, 136.8, 135.4, 134.9, 132.2, 131.8, 130.9, 129.6, 129.4, 127.6, 127.4, 123.9, 123.5, 120.3,
110.1, 39.5, 33.9.
48

ALP et al./Turk J Chem
2-((2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)methyl)-5-(4-fluorobenzylthio)-1,3,4-oxadiazole
(8)
Crude 8 was recrystallized from ethanol to provide 8 (yield 38%) as a white solid, mp 127–128 ◦ C. MS (ESI+)
M+H (%): 451 (100), 453 (47). 1 H NMR δ ppm (CDCl3): 7.83–7.86 (m, 1H), 7.79 (d, 2H, J = 8.8 Hz), 7.55
(d, 2H, J = 8.8 Hz), 7.50–7.52 (m, 1H), 7.30–7.39 (m, 4H), 6.96 (t, 2H, J = 8.8 Hz), 5.51 (s, 2H, –N–CH2),
4.40 (s, 2H, –S–CH2). 13 C NMR δ ppm (CDCl3): 165.7, 163.7, 162.3, 152.5, 142.9, 136.8, 135.4, 130.9, 130.8,
129.4, 127.6, 123.9, 123.5, 120.4, 115.9, 115.7, 110.1, 39.6, 36.1.
2-((2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)methyl)-5-(4-nitrobenzylthio)-1,3,4-oxadiazole
(9)
Crude 9 was recrystallized from ethanol to provide 9 (yield 40%) as a white solid, mp 154–155 ◦ C. MS (ESI+)
M+H (%): 478 (100), 480 (43). 1 H NMR δ ppm (CDCl3): 8.12 (d, 2H, J = 9.2 Hz), 7.82–7.85 (m, 1H), 7.78
(d, 2H, J = 8.4 Hz), 7.54 (d, 4H, J = 8.8 Hz), 7.47–7.50 (m, 1H), 7.33–7.37 (m, 2H), 5.51 (s, 2H, –N–CH2),
4.47 (s, 2H, –S–CH2). 13 C NMR δ ppm (CDCl3): 164.9, 162.6, 152.5, 147.6, 142.9, 142.8, 136.8, 135.4, 130.9,
129.9, 129.4, 127.6, 123.9, 123.6, 120.4, 109.9, 39.5, 35.6.
2-((2-(4-(Benzyloxy)phenyl)-1H -benzo[d]imidazol-1-yl)methyl)-5-(4-bromobenzylthio)-1,3,4-
oxadiazole (10)
Crude 10 was recrystallized from ethanol to provide 10 (yield 53%) as a white solid, mp 119–121 ◦ C. MS
(ESI+) M+H (%): 583 (100), 585 (97). 1 H NMR δ ppm (CDCl3): 7.82 (d, 1H, J = 7.2 Hz), 7.76 (d, 2H, J
= 8.0 Hz), 7.31–7.46 (m, 10H), 7.20 (d, 2H, J = 7.6 Hz), 7.14 (d, 2H, J = 8.4 Hz), 5.51 (s, 2H, –O–CH2), 5.15
(s, 2H, –N–CH2), 4.34 (s, 2H, –S–CH2). 13 C NMR δ ppm (CDCl3): 165.6, 162.9, 160.6, 153.9, 143.2, 136.6,
135.6, 134.5, 132.2, 131.3, 130.9, 128.9, 128.4, 127.7, 123.7, 123.5, 122.5, 121.8, 120.3, 115.7, 110.2, 70.4, 39.9,
36.3.
2-((2-(4-(Benzyloxy)phenyl)-1H -benzo[d]imidazol-1-yl)methyl)-5-(2,4-dichlorobenzylthio)-1,3,4-
oxadiazole (11)
Crude 11 was purified by column chromatography (n-hexane/ethyl acetate (2:1)) to provide 11 (yield 36%) as
a white solid, mp 124–125 ◦ C. MS (ESI+) M+H (%): 573 (100), 575 (91), 577 (20). 1 H NMR δ ppm (CDCl3):
7.81 (d, 1H, J = 6.8 Hz), 7.76 (d, 2H, J = 8.8 Hz), 7.28–7.48 (m, 10H), 7.14 (d, 2H, J = 9.2 Hz), 7.11 (dd,
1H, J = 8.0 Hz, J = 2.0 Hz), 5.51 (s, 2H, –O–CH2), 5.15 (s, 2H, –N–CH2), 4.46 (s, 2H, –S–CH2). 13 C NMR
δ ppm (CDCl3): 165.5, 162.8, 160.4, 153.7, 142.9, 136.4, 135.4, 134.9, 134.9, 132.2, 131.9, 131.1, 129.6, 128.7,
128.2, 127.5, 127.4, 123.4, 123.3, 121.6, 120.1, 115.4, 109.9, 70.1, 39.6, 33.9.
2-((2-(4-(Benzyloxy)phenyl)-1H -benzo[d]imidazol-1-yl)methyl)-5-(4-nitrobenzylthio)-1,3,4-oxa-
diazole (12)
Crude 12 was purified by column chromatography (n-hexane/ethyl acetate (2:1)) to provide 12 (yield 37%) as
a yellowish solid, mp 69–71 ◦ C. MS (ESI+) M+H (%): 550 (100). 1 H NMR δ ppm (CDCl3): 8.11 (d, 2H, J
= 8.4 Hz), 7.82 (d, 1H, J = 7.2 Hz), 7.76 (d, 2H, J = 8.8 Hz), 7.52 (d, 2H, J = 8.4 Hz), 7.28–7.47 (m, 8H),
7.14 (d, 2H, J = 8.8 Hz), 5.52 (s, 2H, –O–CH2), 5.15 (s, 2H, –N–CH2), 4.45 (s, 2H, –S–CH2). 13 C NMR
49

ALP et al./Turk J Chem
δ ppm (CDCl3): 164.8, 162.9, 160.4, 153.6, 147.6, 142.9, 142.8, 136.3, 135.4, 131.1, 129.9, 128.7, 128.2, 127.5,
123.9, 123.5, 123.3, 121.6, 120.1, 115.5, 109.9, 70.2, 39.6, 35.6.
General procedure for the preparation of 2-(2-(4-chlorophenyl)-1H -benzo[d]imidazol-1-yl)-N ′ -
(arylmethylene)acetohydrazide derivatives (13–22)
A mixture of acyl hydrazide 3a (0.02 mol), related aromatic aldehyde derivative (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.14
2-(2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)-N ′ -(4-methylthio)benzylidene) acetohydrazide
(13)
White solid (yield 93%), mp 247–249 ◦ C. MS (ESI+) M+H (%): 435 (100), 437 (68). 1 H NMR δ ppm (DMSO-
d6): 11.78, 11.93 (2s, 1H, NH), 8.01, 8.20 (2s, 1H, –N=CH), 7.27–7.85 (m, 12H, Ar–H), 5.06, 5.53 (2s, 2H,
–CH2), 2.50 (s, 3H, –CH3).
2-(2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)-N ′ -((3-methylthiophen-2-yl) methylene) aceto-
hydrazide (14)
White solid (yield 74%), mp 200–202 ◦ C. MS (ESI+) M+H (%): 409 (100), 411 (37). 1 H NMR δ ppm
(CDCl3): 10.8 (s, 1H, NH), 7.88 (s, 1H), 7.83 (d, 1H, J = 8.4 Hz), 7.73 (d, 2H, J = 8.0 Hz), 7.45 (d, 2H, J
= 8.0 Hz), 7.25–7.31 (m, 4H), 6.81 (d, 1H, J = 5.2 Hz), 5.34 (s, 2H, –CH2), 2.11 (s, 3H, CH3).
1 H NMR δ ppm (DMSO-d6): 11.64, 11.83 (2s, 1H, NH), 8.29, 8.48 (2s, 1H, –N=CH), 7.52–7.49 (m,
7H, Ar–H), 7.32–7.26 (m, 2H, Ar–H), 6.97 (d, 1H, Ar–H, J = 5.2 Hz), 5.02, 5.40 (2s, 2H, –CH2), 2.31 (s, 3H,
CH3).
13 C NMR δ ppm (CDCl3): 169.1, 153.3, 142.7, 141.3, 140.2, 136.3, 136.3, 131.2, 130.9, 130.7, 129.2,
128.4, 127.9, 123.4, 122.9, 119.9, 109.7, 45.6, 13.9.
2-(2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)-N ′ -(2-fluorobenzylidene) acetohydrazide (15)
White solid (yield 96%), mp 226–228 ◦ C. MS (ESI+) M+H (%): 407 (100), 409 (39). 1 H NMR δ ppm
(DMSO-d6): 11.94, 12.12 (2s, 1H, NH), 8.28, 8.48 (2s, 1H, –N=CH), 7.47–7.99 (m, 8H, Ar–H), 7.27–7.33 (m,
4H, Ar–H), 5.09, 5.57 (2s, 2H, –CH2).
N ′ -(4-Chloro-3-nitrobenzylidene)-2-(2-(4-chlorophenyl)-1H -benzo[d]imidazol-1-yl) acetohydra-
zide (16)
White solid (yield 67%), mp 217 ◦ C. (ESI+) M+H (%): 468 (100), 470 (66), 472 (12). 1 H NMR δ ppm
(DMSO-d6): 12.12, 12.29 (2s, 1H, NH), 8.33, 8.39, 8.45 (s, 2d, 1H, –N=CH), 8.12 (s, 1H, Ar–H), 8.02–8.06 (m,
1H, Ar–H), 7.59–7.86 (m, 7H, Ar–H), 7.30–7.36 (m, 2H, Ar–H), 5.15, 5.64 (2s, 2H, –CH2).
50

ALP et al./Turk J Chem
2-(2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)-N ′ -(pyridin-4-yl-methylene) acetohydrazide (17)
White solid (yield 67%), mp 76–79 ◦ C. MS (ESI+) M+H (%): 390 (100), 392 (35). 1 H NMR δ ppm (CDCl3):
10.93 (s, 1H, NH), 8.65 (br s, 2H), 7.84 (d, 1H, J = 4.8 Hz), 7.72 (s, 1H), 7.70 (d, 2H, J = 9.2 Hz), 7.46 (d,
2H, J = 8.4 Hz), 7.41 (d, 2H, J = 3.6 Hz), 7.31–7.34 (m, 3H, Ar–H), 5.38 (s, 2H, –CH2).
1 H NMR δ ppm (DMSO-d6): 12.08, 12.23 (2s, 1H, NH), 8.66 (br s, 2H), 8.03, 8.25 (2s, 1H, –N=CH),
7.59–7.83 (m, 8H, Ar–H), 7.27–7.31 (m, 2H, Ar–H), 5.10, 5.59 (2s, 2H, –CH2).
13 C NMR δ ppm (CDCl3): 169.1, 153.2, 150.4, 143.5, 142.7, 140.2, 136.5, 136.3, 130.6, 129.3, 128.2,
123.5, 123.1, 121.1, 120.1, 109.6, 45.95.
2-(2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)-N ′ -(pyridin-2-yl-methylene) acetohydrazide (18)
White solid (yield 34%), mp 201–204 ◦ C. MS (ESI+) M+H (%): 390 (100), 392 (34). 1 H NMR δ ppm (DMSO-
d6): 12.01, 12.19, 14.34, 14.68 (4s, 1H, NH), 8.61, 8.62, 8.78, 8.79 (4s, 1H, –N=CH), 7.27–8.25 (m, 12H, Ar–H),
5.10, 5.32, 5.57, 5.58 (4s, 2H, –CH2).
2-(2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)-N ′ -((5-nitrofuran-2-yl)methylene) acetohydra-
zide (19)
White solid (yield 62%), mp 242–245 ◦ C. MS (ESI+) M+H (%): 424 (100), 426 (38). 1 H NMR δ ppm
(DMSO-d6): 12.21, 12.33 (2s, 1H, NH), 8.01, 8.21 (2s, 1H, –N=CH), 7.56–7.82 (m, 7H, Ar–H), 7.26–7.34 (m,
3H, Ar–H), 5.13, 5.51 (2s, 2H, –CH2).
2-(2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)-N ′ -((1-methyl-1H -indol-2-yl) methylene) ace-
tohydrazide (20)
White solid (yield 82%), mp 268–269 ◦ C. MS (ESI+) M+H (%): 442 (100), 444 (33). 1 H NMR δ ppm
(DMSO-d6): 11.51, 11.66 (2s, 1H, NH), 8.23, 8.39 (2s, 1H, –N=CH), 8.13–8.19 (m, 1H, Ar–H), 7.49–7.92 (m,
8H, Ar–H), 7.23–7.32 (m, 3H, Ar–H), 7.12–7.16 (m, 1H, Ar–H), 5.04, 5.55 (2s, 2H, –CH2), 3.83 (s, 3H, –CH3).
2-(2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)-N ′ -((1-methyl-1H -indol-3-yl) methylene) ace-
tohydrazide (21)
White solid (yield 69%), mp 235 ◦ C. MS (ESI+) M+H (%): 442 (100), 444 (35). 1 H NMR δ ppm (DMSO-d6):
11.79, 11.97 (2s, 1H, NH), 8.19, 8.38 (2s, 1H, –N=CH), 7.50–7.88 (m, 8H, Ar–H), 7.23–7.31 (m, 3H, Ar–H), 7.08
(t, 1H, Ar–H, J = 7.6 Hz), 6.94 (d, 1H, Ar–H, J = 9.6 Hz), 5.08, 5.54 (2s, 2H, –CH2), 4.01 (s, 3H, –CH3).
2-(2-(4-Chlorophenyl)-1H -benzo[d]imidazol-1-yl)-N ′ -((2-methyl-1H -indol-3-yl) methylene) ace-
tohydrazide (22)
White solid (yield 88%), mp 299 ◦ C. MS (ESI+) M+H (%): 442 (100), 444 (48). 1 H NMR δ ppm (DMSO-d6):
11.39, 11.52, 11.60 (3s, 2H, NH, indole NH), 8.34, 8.47 (2s, 1H, –N=CH), 7.56–8.12 (m, 7H, Ar–H), 7.27–7.34
(m, 3H, Ar–H), 7.02–7.12 (m, 2H, Ar–H), 5.03, 5.54 (2s, 2H, –CH2), 2.50 (s, 3H, –CH3).
51

ALP et al./Turk J Chem
4.2. In vitro antioxidant activity
4.2.1. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay
The free radical scavenging activities of the compounds were tested based on their ability to bleach the stable
radical 2,2,diphenyl-1-picrylhydrazyl (DPPH). The stock solutions of the compounds and BHT were prepared
at 10−2 M in DMSO. A series of stock solutions in DMSO were diluted to varying concentrations in 96-well
microplates. Methanolic DPPH solution (100 µM) was then added to each well. The plate was shaken and
placed in darkness. After 30 min, the optical density of the solution was read at the wavelength 517 nm. The
methanolic solution of DPPH served as a control. Percentage inhibition was calculated using the following
formula:
Radical scavenging activity % = [(A
– A Sample)/A Control ] × 100
absorption of blank sample; A sample : absorption of tested solution)
Control
(A
control:
All tests and analyses were run in triplicate and averaged. The standard used in this assay was BHT.19
4.2.2. 7-Ethoxyresorufin O-deethylase (EROD) assay
7-Ethoxyresorufin O-deethylase (EROD) enzyme activity was determined by the spectrofluorometric method
described by Burke et al.20 A typical optimized assay mixture contained 1.0 mM ethoxyresorufin; 10−3 M test
compound; 100 mM Tris–HCl buffer pH 7.8; a 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 of liver microsomal protein in a final volume of 1.0 mL. The standard used
in this assay was caffeine. DMSO was used as a control.
4.2.3. Lipid peroxidation (LP) assay
Male albino Wistar rats (200–225 g) were used in the experiments. The animals were fed with standard
laboratory rat chow and tap water ad libitum. The animals were starved for 24 h prior to sacrifice and
were killed by decapitation under anesthesia. The livers were removed immediately and washed in ice-cold
water, and the microsomes were prepared as defined previously.21 NADPH-dependent LP was determined
using the optimum conditions as determined and described previously21 and measured spectrophotometrically
by estimation of thiobarbituric acid-reactive substances (TBARS). Amounts of TBARS were expressed in terms
of nmol malondialdehyde (MDA)/mg protein. The assay was essentially obtained from the methods of Wills22,23
as modified by Bishayee et al.24 LP was measured spectrophotometrically at 532 nm as the thiobarbituric
acid-reactive material. Compounds inhibit the production of MDA, and the color produced after addition of
thiobarbituric acid is less intensive. A typical optimized assay mixture contained 10−3 M test compound; 0.2
nM Fe++ ; 90 mM KCl; 62.5 mM potassium phosphate buffer, pH 7.4; a NADPH-generating system comprising
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 of microsomal protein in a final volume of 1.0
mL. The standard used in this assay was BHT. DMSO was used as a control.
Acknowledgment
The Central Instrumental Analysis Lab. at the Faculty of Pharmacy, Ankara University, provided the support
for acquisition of the NMR and mass data used in this work.
52

ALP et al./Turk J Chem
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