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

Article abstract of DOI:10.1016/j.bmc.2008.02.077

Some novel 1-methyl-4-(2-(2-substitutedphenyl-1H-benzimidazol-1-yl)acetyl)thiosemicarbazides (16a-20a), 5-[(2-(substitutedphenyl)-1H-benzimidazol-1-yl)methyl]-N-methyl-1,3,4-thiadiazol-2-amines (17b-20b), and 5-[(2-(substitutedphenyl)-1H-benzimidazol-1-yl)methyl-4-methyl-2H-1,2,4-triazole-3(4H)-thiones (16c-20c) were synthesized and tested for antioxidant properties by using various in vitro systems. Compounds 16a-20a were found to be a good scavenger of DPPH radical (IC₅₀, 26 μM; IC₅₀, 30 μM; IC₅₀, 43 μM; IC₅₀, 55 μM; IC₅₀, 74 μM, respectively) when compared to BHT (IC₅₀, 54 μM). Noteworthy results could not be found on superoxide radical. Compound 19b, which is the most active derivative inhibited slightly lipid peroxidation (28%) at 10^-3 M concentration. Compound 17c inhibited the microsomal ethoxyresorufin O-deethylase (EROD) activity with an IC₅₀ = 4.5 × 10^-4 M which is similarly better than the specific inhibitor caffeine IC₅₀ = 5.2 × 10^-4 M.

Full text of DOI:10.1016/j.bmc.2008.02.077

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4294–4303
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
a
b
b
Canan Kus,a, Gulgun Ayhan-Kılcıgil, Suheyla Ozbey, F. Betul Kaynak,
*
¨
ß
¨ ¨
¨
¨
Melek Kaya,c Tulay C¸ obanc and Benay Can-Ekec
¨
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Ankara, Turkey
bDepartment of Engineering Physics, Faculty of Engineering, Hacettepe University, 06800 Ankara, Turkey
cDepartment of Toxicology, Faculty of Pharmacy, Ankara University, 06100 Ankara, Turkey
Received 27 August 2007; revised 20 February 2008; accepted 26 February 2008
Available online 29 February 2008
Abstract—Some novel 1-methyl-4-(2-(2-substitutedphenyl-1H-benzimidazol-1-yl)acetyl)thiosemicarbazides (16a–20a), 5-[(2-(substi-
tutedphenyl)-1H-benzimidazol-1-yl)methyl]-N-methyl-1,3,4-thiadiazol-2-amines (17b–20b), and 5-[(2-(substitutedphenyl)-1H-ben-
zimidazol-1-yl)methyl-4-methyl-2H-1,2,4-triazole-3(4H)-thiones (16c–20c) were synthesized and tested for antioxidant properties
by using various in vitro systems. Compounds 16a–20a were found to be a good scavenger of DPPH radical (IC50, 26 lM; IC50,
30 lM; IC50, 43 lM; IC50, 55 lM; IC50, 74 lM, respectively) when compared to BHT (IC50, 54 lM). Noteworthy results could
not be found on superoxide radical. Compound 19b, which is the most active derivative inhibited slightly lipid peroxidation
(28%) at 10ꢀ3 M concentration. Compound 17c inhibited the microsomal ethoxyresorufin O-deethylase (EROD) activity with an
IC50 = 4.5 · 10ꢀ4 M which is similarly better than the specific inhibitor caffeine IC50 = 5.2 · 10ꢀ4 M.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
pounds, thiosemicarbazide derivatives (Fig. 1) usually
had stronger inhibitory effects on LP levels and more
considerable scavenger effects on DPPH radical than
those of their cyclic counterparts namely, 1,3,4-thiadiaz-
oles and 2H-1,2,4-triazole-3(4H)-thiones.
Nowadays antioxidants arouse researchers’ interest in
both medical plants and synthetic compounds. In our
previous research,1–3 we reported some novel benzimid-
azole derivatives in which substituted aminothiocarba-
moylhydrazinecarbonyl, substituted 1,3,4-thiadiazole,
and substituted 4H-1,2,4-triazole groups were attached
to the 1st position of benzimidazole ring via a methyl
chain. At the 2nd position of these derivatives, there
are phenyl, p-chlorophenyl, p-methoxyphenyl, and
pyridinyl rings. Antioxidant properties of these com-
pounds were investigated by employing various
in vitro systems interaction of 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH), and scavenging of superoxide
radical, microsomal NADPH-dependent inhibition of
lipid peroxidation (LP), microsomal ethoxyresorufin
O-deethylase (EROD) activity. Among these com-
In this study, we synthesized some novel benzimidazole
derivatives (Fig. 2) bearing alkyl (methyl) group at the
4th position of triazole ring and at the 5th position of
thiadiazole ring instead of aryl group. In addition
1
to their IR, H and 13C NMR, Mass, and Elemental
H
O
H
N
N
N
H
S
N
R
X
Y
N
X = C, N
Y = -H, -Cl, -OCH3
Keywords: N-Methylthiosemicarbazides; N-Methyltriazolylbenzimi-
dazoles; N-Methylthiadiazolyl-benzimidazoles; Antioxidant; X-ray
structure analysis.
R = -H, -Cl, di-Cl, -F, -Br, -CH3, -OCH3,
*
Figure 1. Structures of previously synthesized thiosemicarbazido-
benzimidazoles.
Corresponding author. Tel.: +90 312 203 30 75; fax: +90 312 213 10
81; e-mail: kus@pharmacy.ankara.edu.tr
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.077

C. Kußs et al. / Bioorg. Med. Chem. 16 (2008) 4294–4303
4295
R
N
X
Y
N
X = -H, 5,6-dichloro
Y = -H, 4-chloro, 4-methoxy, 3,4-dimethoxy, 4-benzyloxy, 4-hydroxy
NHCH3
S
H3C
S
N
,
-CONHNHCSNHCH3
R =
N H
N
,
N H
N
Figure 2. Structures of the synthesized compounds in this study.
analysis, the structure of 5-[(2-phenyl-1H-benzimidazol-
1-yl)methyl-4-methyl-2H-1,2,4-triazole-3(4H)-thione (16c)
was determined by X-ray analysis as well. All the synthe-
sized compounds were evaluated by their scavenger effects
on DPPH radical, superoxide radical, and rat liver micro-
somal NADPH-dependent lipid peroxidation levels by
measuring the formation of 2-thiobarbituric acid reactive
substances and also examined the inhibition capacity of
their microsomal EROD activity.
(1–5) were prepared via the oxidative condensation of
related o-phenylenediamines with sodium metabisulfite
adduct of appropriate benzaldehydes such as p-chloro,
p-methoxy, 3,4-dimethoxy, and 4-benzyloxy in DMF.4
Treatment of these compounds (1–5) with ethyl chloro-
acetate in KOH/DMSO yielded N-alkylated products
(6–10).5 Hydrazine hydrate and esters (6–10) in ethanol
were refluxed for 4 h to give the desired hydrazide com-
pounds (11–15).6
Thiosemicarbazides (16a–20a) were obtained upon the
reaction of acid hydrazide with methylisothiocyanate
in absolute ethanol. The cyclization of 17a–20a with
sulfuric acid resulted in the formation of thiadiazoles
(17b–20b) and the cyclization of 16a–20a with sodium
hydroxide resulted in the formation of triazoles
(16c–20c). Unexpectedly, 20b was obtained from the
2. Results and discussion
2.1. Synthesis
For the synthesis of the target compounds, the reaction
sequences are outlined in Scheme 1. 1H-Benzimidazoles
CH 2COOEt
N
H
N
NH 2
HOC
a
b
+
X
X
NH 2
X
N
Y
Y
N
Y
6-10
1-5
c
Y
X
-H
-H
-H
11
12
-H
1
2
6
CH 2CONHNHCSNHCH3
N
CH 2CONHNH 2
N
4-Cl
7
8
4-OCH3
d
3
4
5
13
X
X
5,6-dichloro
-H
14
15
3,4-dimethoxy
4-benzyloxy
N
9
Y
N
Y
10
16a-20a
11-15
e
f
S
H3C
NHCH3
N
NH
S
N
N
N
2'
3'
N
2'
3'
5'
4'
Y
N
N
X
N
4'
Y
6'
5'
X
16c-20c
6'
17b-20b
Scheme 1. Synthetic route for the preparation of compounds 1–15, 16a–20a, 17b–20b, and 16c–20c. Reagents: (a) Na2S2O5; (b) ClCH2COOEt/KOH–
DMSO; (c) NH2NH2ÆH2O/EtOH; (d) methyl isothiocyanate; (e) H2SO4; (f) NaOH.

Table 1. Physical and spectral data of 16a–20a, 17b–20b, and 16c–20c
Compound
Formulas
17H17N5OS
% Yield
78
Mp (ꢁC)
1H NMR (400 MHz, DMSO-d6); (d ppm)
ESI-MS (M+H)
340 (100%)
16a
C
210–212
2.91 (d, 3H, NHCH3, J = 4), 4.97 (s, 2H, CH2), 7.25–7.76 (m, 9H, Ar-
H), 8.11, 9.43, 10.31 (3H, CONHNHCSNH)
17a
18a
C17H16ClN5OSÆ0.3H2O
62
59
237–239
257
2.90 (d, 3H, NHCH3, J = 4), 4.94 (s, 2H, CH2), 7.25–7.31 (m, 2H, H-
5,6), 7.52–7.79 (m, 6H, other Ar-H), 8.08, 9.39, 10.30 (3H,
CONHNHCSNH)
374 (100%), 376 (33%)
370 (100%)
C18H19N5O2S
2.90 (d, 3H, NHCH3, J = 4), 3.84 (s, 3H, OCH3), 4.93 (s, 2H, CH2),
7.10 (d, 2H, Jo = 8.99, H-20,60), 7.22–7.28 (m, 2H, H-5,6), 7.48–7.69
(m, 4H, H-4,7,30,50), 8.11, 9.42, 10.31 (3H, CONHNHCSNH) 13C
NMR (DMSO-d6) d: 31.6, 46.5, 56.0, 111.4, 114.9, 119.5, 122.7, 122.9,
131.4, 137.1, 143.1, 154.1, 161.1, 167.8, 171.2, 182.9
19a
C
C
19H19Cl2N5O3SÆ1.1H2O
63
229
2.89 (d, 3H, NHCH3, J = 4), 3.78 (s, 3H, OCH3), 3.82 (s, 3H, OCH3),
4.97 (s, 2H, CH2), 7.08–7.28 (m, 3H, H-20,50,60), 7.82 (s, 1H, H-4), 7.95
(s, 1H, H-7), 8.09, 9.42, 10.29 (3H, CONHNHCSNH)
468 (100%), 470 (68%),
472 (12%)
20a
17b
24H23N5O2S
71
61
206
275
2.90 (d, 3H, N-CH3, J = 4), 4.93 (s, 2H, OCH2), 5.20 (s,2H, CH2),
7.16–7.69 (m,13H, Ar-H), 8.09, 9.41, 10.29 (s, 3H, CONHNHCSNH)
446 (100%)
C17H14N5ClSÆ0.9H2OÆ0.6 C3H8O
2.76 (d, 3H, NHCH3, J = 4), 5.73 (s, 2H, CH2), 7.26–7.29 (m, 2H, H-
5,6), 7.63–7.64 (m, 4H, H-4,20,60, NH), 7.68 (d, 1H, H-7, Jo = 7.76 Hz),
7.85 (d, 2H, H-30,50, Jo = 8.20 Hz). 13C NMR (DMSO-d6) d: 31.9, 43.9,
111.8, 120.1, 123.3, 123.8, 129.4, 129.7, 131.8, 135.6, 136.3, 143.2,
152.5, 153.3, 170.7
356 (100%), 358 (34%)
18b
19b
20b
C18H17N5OS
52
49
37
187–188
194–196
262
2.78 (d, 3H, NHCH3, J = 4), 3.84 (s, 3H, O-CH3), 5.70 (s, 2H, CH2),
7.12 (d, 2H, Jo = 8.10 Hz, H-20,60), 7.22–7.28 (m, 2H, H-5,6), 7.59–7.68
(m, 3H, H-4,7, NH), 7.78 (d, 2H, Jo = 8.11 Hz, H-30,50); 13C NMR
(DMSO-d6) d: 31.8, 43.9, 56.0, 111.5, 114.9, 119.7, 122.6, 123.1, 123.3,
131.4, 136.2, 143.3, 153.6, 161.2, 170.7
352 (100%)
450 (100%)
338 (100%)
C19H17Cl2N5O2SÆ0.4 H2O
2.78 (d, 3H, NHCH3, J = 4.8), 3.78 (s, 3H, O-CH3), 3.82 (s, 3H, O-
CH3), 5.76 (s, 2H, CH2), 7.11 (d, 1H, Jo = 8.98 Hz, H-50), 7.31–7.42
(m, 2H, H-20,60), 7.66 (q, 1H, NH), 7.96 (s, 1H, H-4), 8.04 (s, 1H, H-7);
13C NMR (DMSO-d6) d: 31.9, 44.2, 56.3, 112.5, 113.2, 113.4, 120.9,
121.8, 122.9, 125.6, 125.7, 136.0, 142.8, 149.4, 151.3, 153.0, 156.1, 170.8
C17H15N5OSÆ0.7H2O
2.78 (d, 3H, NHCH3, J = 4.8), 5.69 (s, 2H, CH2), 6.92 (d, 2H, Jo = 8.6,
H-30, 50), 7.23–7.25 (m, 2H, H-5,6), 7.57–7.59 (m, 1H, H-4), 7.64-7.67
(m, 4H, H-7,20,60,NH), 10.01 (s, 1H, OH); 13C NMR (DMSO-d6) d:
31.8, 43.9, 111.4, 116.3, 119.6, 120.9, 122.9, 123.0, 131.4, 136.1, 143.3,
153.6, 153.9, 159.7, 170.6
16c
17c
C17H15N5S
64
51
258–261
279–280
3.39 (s, 3H, N-CH3), 5.70 (s, 2H, CH2), 7.25–7.29 (m, 2H, H-5,6), 7.55–
7.75 (m, 7H, H-4,7,20,30,4’,50,60), 13.64 (s, 1H, NH)
322 (100%)
C17H14ClN5SÆ0.75H2O
3.41 (s, 3H, N-CH3), 7.27–7.30 (m, 2H, H-5,6), 7.58–7.74 (m, 6H, H-
4,7,20,30,50,60), 13.62 (s, 1H, NH)
356 (100%), 358 (32%)

C. Kußs et al. / Bioorg. Med. Chem. 16 (2008) 4294–4303
4297
cyclization reaction of 20a in the cold H2SO4 by missing
the benzyl group.
All physical and spectral data of 16a–20a, 17b–20b, and
16c–20c were seen in Table 1. In the IR spectra of
compounds 16c–20c no absorption bands were detected
about 1671–1696 cmꢀ1 indicating the absence of CO
group of acylthiosemicarbazides (16a–20a) which is an
evidence for the conversion of thiosemicarbazides to
triazoles. Although two types of tautomers (thione or
thiole) could be expected from the cyclization of
compounds 16a–20a under alkaline conditions, only
the thione type compounds (16c–20c) were observed.
This cyclization and formation of thione tautomer also
demonstrated both by the presence of two absorption
maxima at 1310–1322 and 1265–1290 cmꢀ1 belonging
to the C@S group and X-ray data of compound 16c.
X-ray analysis results show that compound 16c
exists in the thione tautomeric form in the solid state.
The location of the H-atom on the atom N2 rather
than the atom S, and the C1–S1, N2–C1, and C1–N1
bond lengths support this idea. The IR spectra of novel
esters and hydrazides revealed sharp bands at around
1731–1752 and 1660–1698 cmꢀ1
,
respectively. As
expected, in the mass spectra of 14, 17a–17c, 19a, and
19c both mono and two chlorine isotopes of this
atom were seen. The structures of the synthesized com-
pounds were consistent with the 1H and 13C NMR spec-
tra. In the 1H NMR spectra, NH–CH3 coupling
constants (J) are seen as 4 Hz (16a–20a, 17b, 18b) and
4.8 Hz (19b, 20b). In the 13C NMR spectra of the triazol-
ethione derivatives (18c, 19c), CH2– peak was observed
at about 41.0–41.5 ppm which was close to the DMSO-
d6 peaks. In the 13C NMR spectra of thiadiazole deriv-
atives (17b, 18b, and 19b), these peaks were seen at
43.85, 43.96, and 44.22 ppm, respectively. The crystal
data, intensity data collection parameters, and final
refinement results for compound 16c are summarized
in Table 2.
˚
Hydrogen bonding geometry (A, ꢁ) for 16c is shown in
Table 3. In all essential details, the geometry of the mol-
ecule in terms of bond lengths and angles is in good
agreement within experimental errors, with those ob-
served in other benzimidazole derivatives. As expected,
the benzimidazole moiety of the molecule is planar due
to the wide electronic delocalization effect [maximum
˚
deviations 0.032(2) A for N8]. The dihedral angle be-
tween the imidazole and benzene ring planes is 1.9(1)ꢁ.
Selected bond lengths and angles are listed in Table 4.
The molecular structure with atom numbering scheme
and the packing arrangement of the molecules are pre-
sented in Figures 3 and 4.
The phenyl and triazole groups at C16 and N8 are essen-
tially planar, and twisted out of the plane of the benz-
imidazole ring with torsion angles of ꢀ54.6(3)ꢁ and
ꢀ85.4(2)(3)ꢁ for N15–C16–C17–C18 and C16–N8–C7–
C1, respectively. The dihedral angle between the 2,4-
dihydro-1,2,4-triazole ring and the benzimidazole nu-
cleus is 76.9(1)ꢁ. The methyl and thione groups attached
to triazole ring almost lie linearly in the triazole ring

4298
C. Kußs et al. / Bioorg. Med. Chem. 16 (2008) 4294–4303
Table 2. Crystal data and details of the structure determination of 16c
Crystal formula
Formula weight
Crystal dimensions [mm]
Temperature [K]
Crystal system
C
321.4
17H15N5S
Z; Dcalc [g cmꢀ3
hmax [ꢁ]
]
8; 1.303
74.27
1.8
0.33 · 0.48 · 0.52
295
l (MoKa) [cmꢀ1
]
Reflections collected
Reflections used in refinement
No. of refined parameters
R/Rw values
3323
3225
Monoclinic
C2/c
Space group
˚
213
a [A]
˚
17.3529(14)
9.6909(13)
19.6297(9)
97.036(5)
0.0388/0.1124
1.049
0.000
b [A]
GOF
Final shift
˚
c [A]
ꢀ3
˚
b [ꢁ]
(Dq)min, (Dq)max [e A
]
ꢀ0.227, 0.232
˚
Table 3. Hydrogen bonding geometry (A, ꢁ) for 16c
D–H ꢁ ꢁ ꢁ A
D–H
H ꢁ ꢁ ꢁ A D ꢁ ꢁ ꢁ A
2.77 3.253
D–H ꢁ ꢁ ꢁ A
112
C6–H6A ꢁ ꢁ ꢁ S1
N3–H3 ꢁ ꢁ ꢁ N15i
C18–H18 ꢁ ꢁ ꢁ N2ii
0.96
0.91(3) 1.96(3)
2.831(2) 159
0.93
2.56
2.55
3.389
3.423
148
156
C22–H22 ꢁ ꢁ ꢁ N15iii 0.93
Symmetry code: (i) 1/2 ꢀ x, ꢀ1/2 + y, 1/2 ꢀ z, (ii) 1/2 ꢀ x, 1/2 + y,
1/2 ꢀ z, (iii) 1 ꢀ x,y, 1/2 ꢀ z.
˚
Table 4. Selected bond distances and bond angles (A, ꢁ) for 16c
C1–N2
C1–N5
C1–C7
C4–N3
C4–N5
C4–S1
1.295(2)
1.376(2)
1.497(3)
1.341(2)
1.372(2)
1.6751(19)
1.459(2)
1.462(2)
1.387(2)
1.391(3)
1.393(3)
1.378(3)
1.393(4)
C12–C13
C13–C14
C14–N15
C16–N15
C16–N8
C16–C17
C17–C18
C17–C22
C18–C19
C19–C20
C20–C21
C21–C22
N2–N3
1.373(3)
1.397(3)
1.395(2)
1.321(2)
1.371(2)
1.474(2)
1.381(3)
1.399(3)
1.384(3)
1.376(3)
1.375(3)
1.381(3)
1.372(2)
C6–N5
C7–N8
C9–N8
C9–C14
C9–C10
C10–C11
C11–C12
Figure 3. Molecular structure of 16c, showing the atom-numbering
scheme. Displacement ellipsoids are drawn at the 50% probability
level.
(data are not shown), NADPH-dependent lipid peroxi-
dation levels, and EROD enzyme activity (Table 5).
C4–N5–C1
C4–N5–C6
C1–N5–C6
N2–C1–N5
N2–C1–C7
N5–C1–C7
N3–C4–N5
N3–C4–S1
107.74(15)
124.74(16)
127.52(16)
111.19(16)
122.56(17)
126.24(16)
103.54(16)
127.76(15)
N5–C4–S1
N15–C16–N8
C16–N8–C7
C16–N8–C9
C9–N8–C7
N8–C7–C1
C1–N2–N3
C4–N3–N2
128.65(14)
112.18(15)
127.15(15)
107.09(14)
124.49(16)
110.89(14)
104.29(15)
113.20(16)
Each method relates to the generation of a different rad-
ical, acting through a variety of mechanisms.
The inhibitory effects of different concentrations of syn-
thesized compounds on DPPH radical are presented in
Table 5. It appears that compounds 16a–20a were found
to be a good scavenger of DPPH radical (IC50, 26 lM;
IC50, 30 lM; IC50, 43 lM; IC50, 55 lM; IC50, 74 lM,
respectively) when compared to BHT (IC50, 54 lM).
Compounds 18c, 16c, 19c, 17c, and 20c exhibited mod-
erate effect on DPPH radical with IC50 values of 140,
271, 271, and 462 lM, respectively. All thiadiazole series
of compounds (17b–20b) have the weakest effect on
DPPH radical with IC50 values of >1000 lM, when
compared to BHT.
plane, with the greatest deviation being ꢀ0.100(1) and
˚
0.051(2) A for S1 and C6 atoms, respectively.
The phenyl group makes a dihedral angle of 56.2(1)ꢁ
and 48.3(1)ꢁ with the benzimidazole and triazole rings,
respectively. Two types of intermolecular hydrogen
bonds, N–H. . .N and C–H. . .N, and one intramolecular
interaction, C–H. . .S, are observed in the structure
(Fig. 4 and Table 3). Screw related molecules are
interconnected by hydrogen bonds between triazole
and benzimidazole groups and phenyl C18 and C22
hydrogen forming infinite chains along a direction
(Fig. 4).
The superoxide radical scavenging activities of the com-
pounds were also investigated by using the xanthine/
xanthine oxidase system, and there was no significant
activity pattern obtained from this experiment (data
are not shown). Only compounds 17a, 19b, 19a, 16a,
and 17b showed moderate inhibitory effect on superox-
ide radical at 0.1 mM concentration and inhibition rates
were 38%, 31%, 26%, 22%, and 21%, respectively. The
scavenging rates of the rest of the compounds were in
The antioxidant properties of the targeted compounds
(16a–20a, 17b–20b, and 16c–20c) were evaluated by the
interaction of DPPH, scavenging of superoxide radical

C. Kußs et al. / Bioorg. Med. Chem. 16 (2008) 4294–4303
4299
Figure 4. Crystal packing of 16c projected onto ac plane. The dashed lines indicated the intermolecular hydrogen bonds.
Table 5. Effects of the compounds on liver LP levels, on EROD and on DPPH free radical scavenging activities in vitroa
NHCH3
S
NH
H3C
S
N
N
CH2 CONHNHCSNHCH3
N
N
2'
N
3'
5'
2'
N
3'
N
X
4'
Y
N
Y
4'
Y
X
X
N
N
6'
16a-20a
5'
6'
17b-20b
16c-20c
Compoundb
X
Y
DPPH
IC50 (lM)
LP
(nmol/mg per min)
% of
control
EROD
(pmol/mg per min)
% of
control
16a
17a
–H
–H
–H
4-Cl
26 4.2
30 1.4
43 2.8
55 3.5
74 4.2
—
19.19 0.27
13.08 0.14
16.12 0.98
14.81 0.09
96.56 0.38
16.12 0.15
14.39 0.11
11.78 0.27
129.22 0.74
20.95 1.27
26.60 1.50
23.10 1.76
27.48 0.71
69.58 0.56
5.68 0.22
—
118
80
50.98 2.10
28.26 0.31
34.55 0.45
12.31 0.11
28.58 0.27
2.46 0.05
34.29 0.47
12.20 0.18
36.08 0.56
13.13 0.91
7.89 0.26
15.85 0.16
18.32 0.27
17.96 0.19
—
128
68
83
30
69
60
83
34
87
32
19
38
44
43
—
20
100
18a
19a
–H
5,6-Dichloro
4-OCH3
99
91
3,4-Dimethoxy
4-Benzyloxy
4-Cl
20a
17b
–H
–H
594
99
18b
19b
–H
5,6-Dichloro
4-OCH3
3,4-Dimethoxy
4-Hydroxy
–H
>1000
>1000
>1000
89
72
20b
16c
–H
–H
795
129
164
142
169
428
35
271 14
462 21
140 7.0
271 28
605 35
54 4.9
17c
18c
–H
–H
4-Cl
4-OCH3
3,4-Dimethoxy
4-Benzyloxy
—
19c
20c
5,6-Dichloro
–H
—
—
—
BHT
Caffeine
Controlc
—
—
—
100
8.31 0.42
41.53 0.99
16.25 1.45
a Each value represents mean SD of 2–4 independent experiments.
b Concentration in incubation medium (10ꢀ3 M).
c DMSO only, control for compounds.

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C. Kußs et al. / Bioorg. Med. Chem. 16 (2008) 4294–4303
the range of 5–10%. Compounds 17c, 18a, and 19c had
no effect on superoxide radical.
As we reported before, It is well known that there exists
two mechanisms for an antioxidant to scavenge DPPH.
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).7
The effect of compounds (16a–20a, 17b–20b, and 16c–
20c) were evaluated on lipid peroxidation levels. The
NADPH-dependent lipid peroxidation inhibition pro-
duced by all new compounds in rat liver microsomes
was examined by measuring the formation of 2-thiobar-
bituric acid reactive substance for their antioxidant
capacity. As can be seen from Table 5, the BHT de-
creased the LP level by about 65% at 10ꢀ3 M concentra-
tion. However, the synthesized compounds have no
significant effect on lipid peroxidation level in rat liver
microsomes at the same concentration.
DPPHÅ þ RXH ! DPPHH þ RXHÅ
DPPHÅ þ RXH ! DPPHꢀ þ RXHÅþ
! DPPHH þ RXÅ
ð1Þ
ð2Þ
DPPH-scavenging mechanism of our compounds could
not clarified yet but our effort on this way is going on.8
Compound 19b which is the most active derivative
slightly inhibited lipid peroxidation (28%) at 10ꢀ3 M
concentration. Bearing N-phenyl and N-substitutedphe-
nyl derivatives (triazoles, thiadiazoles and especially thi-
osemicarbazides) it had stronger inhibitory effects on LP
levels than those of N-methyl analogues.2,3
Compounds 17a (20%) and 18b (11%) showed rather
limited lipid peroxidation at 10ꢀ3 M concentration but
compounds 16a, 16c, 17c, 18c, 19c, 20a, 20b, and 20c en-
hanced LP levels. The most active compound in this ser-
ies is 19b, which caused only 28% inhibition.
Compound 17c inhibited the EROD activity with an
IC50 = 4.5 · 10ꢀ4 M which is similarly better than the
specific inhibitor caffeine IC50 = 5.2 · 10ꢀ4 M.
The in vitro effects of compounds and caffeine on liver
microsomal EROD activity are also shown in Table 5.
The concentrations required for 50% inhibition (IC50)
of liver microsomal EROD activity were 8.4 · 10ꢀ4
,
7.4 · 10ꢀ4, and 4.5 · 10ꢀ4 lg/ml for 19a, 16c, and 17c,
respectively. Compound 17c inhibited the microsomal
EROD activity (81%) which was similarly better than
the specific inhibitor caffeine (80%) at 10ꢀ3 M concen-
tration. Significant inhibitory activities were observed
also by compounds 19a (70%), 19b (66%), and 16c
(68%). In this series of compounds, particularly the tri-
azole derivatives (16c–20c) were the most active accord-
ing to the method.
Since the mechanisms of these methods are different, it is
quite difficult to explain the observed different effects of
synthesized compounds. Different effects of compounds
in these systems have been noticed previously.3,9,10
Therefore, the observation of distinct effects of synthetic
compounds on DPPH radical, superoxide radical, LP
levels, and EROD is not surprising since the mecha-
nisms of production of oxidative stress using these meth-
ods are different.11–13 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.14 Therefore, it is extremely difficult to compare
the results from different assays.
3. Conclusion
In this study, a series of new benzimidazole deriva-
tives 16a–20a, 17b–20b, and 16c–20c having [N-methyl-
thiosemicarbazide], [2-methylamino-1,3,4-thiadiazole],
and [4-methyl-2H-1,2,4-triazole-3(4H)-thione] moieties,
respectively, at 1st position was synthesized, and their
antioxidant activities were evaluated. It was observed
that the effects of the compounds on DPPH radical,
superoxide radical, lipid peroxidation, and EROD
activity levels were variable. The strong scavenging ef-
fects of compounds 16a–20a were noted on the level of
DPPH radical but not superoxide radical. These and
previous published results2 clearly indicate that both
N-aryl and N-methyl thiadiazole derivatives were found
to have no interaction with DPPH. If we compare with
N-methyl and N-aryl series reported before,2,3 in all the
series, triazole derivatives have moderate activity
and thiosemicarbazides have the best effect on DPPH.
IC50 values of N-methylthiosemicarbazides 16a–20a
(26–74 lM) are similar to N-aryl analogues 1-[(phen-
ylthiocarbamoylhydrazinecarbonyl)methyl]-2-(4-pyrid-
inyl)-1H-benzimidazole (13 lM) and 1-[(p-meth-
oxyphenylthiocarbamoylhydrazinecarbonyl)-methyl]-2-
(4-pyridinyl)-1H-benzimidazole (12 lM) so it can be
said that there is no noteworthy difference between their
scavenger effects on DPPH radical.
As a result, data obtained from all our researches asso-
ciated with this field guide us for the development of no-
vel antioxidant compounds.
4. Experimental
4.1. Synthesis
Melting points were determined with an Electrothermal
and a Buchi SMP-20 melting point apparatus and are
¨
uncorrected. IR spectra were recorded on a Jasco FT/
IR 420 spectrometer as potassium bromide discs. 1H
and 13C NMR spectra were measured with a Varian
Mercury 400, 400 MHz instrument using TMS internal
standard and DMSO-d6, coupling constants (J) are re-
ported 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 Electro-
spray Ionization method. Elemental analyses (C, H, N,
and S) were determined on a Leco CHNS 932 instru-
ment, and were within 0.4% of the theoretical values.
All instrumental analyses were performed at Ankara

C. Kußs et al. / Bioorg. Med. Chem. 16 (2008) 4294–4303
4301
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 was performed
with Merck precoated TLC plates and spots were visu-
alized with ultraviolet light.
1731 (C@O); 1H NMR (DMSO-d6) d: 1.13 (t, 3H, H2C–
CH3), 3.80 (s, 3H, O-CH3), 3.83 (s, 3H, O-CH3), 4.09–
4.15 (q, 2H, H2C–CH3), 5.35 (s, 2H, N-CH2), 7.16 (d,
1H, Jo = 8.2), 7.28 (d, 1H, Jo = 8.2), 7.3 (s, 1H), 7.99 (s,
1H), 8.24 (s, 1H).
4.1.2.3. 2-(4-Benzyloxyphenyl-1H-benzimidazol-1-yl)-
acetic acid ethyl ester (10). Mp 127 ꢁC; ES (+) (M+H):
387 (100%); IR (KBr cmꢀ1) 1749 (C@O); 1H NMR
(DMSO-d6) d: 1.13 (t, 3H, H2C–CH3), 4.12 (q, 2H,
H2C–CH3), 5.2 (s, 4H, N-CH2, O-CH2), 7.15–7.68 (m,
13H, Ar-H).
2-Phenyl-1H-benzimidazole (1), 2-(4-chlorophenyl)-1H-
benzimidazole (2), 2-(4-methoxy-phenyl)-1H-benzimid-
azole (3), (2-phenyl-1H-benzimidazol-1-yl)-acetic acid
ethyl ester (6), (2-(4-chlorophenyl)-1H-benzimidazol-1-
yl)-acetic acid ethyl ester (7), (2-phenyl-1H-ben-
zimidazol-1-yl)-acetic acid hydrazide (11), and 2-(4-chlo-
rophenyl-1H-benzimidazol-1-yl)-acetic acid hydrazide
(12) were previously prepared in our laboratory.1–3
4.1.3. General procedure for the preparation of [2-
(substitutedphenyl)-1H-benzimidazol-1-yl]-acetic acid hydra-
zides (11–15). Hydrazine hydrate (4 ml) and related 1H-
benzimidazole acetic acid ethyl esters (6–10) (1.5 mmol)
in ethanol (5 ml) were refluxed for 4 h. The reaction mix-
ture was cooled and poured into water. The crude product
was filtered off and recrystallized from ethanol to give the
desired hydrazide compounds (11–15).
4.1.1. General procedure for the preparation of 2-substi-
tuted benzimidazoles (1–5). For the synthesis of
compounds 1–5, the mixture of appropriate o-phenylen-
diamine (1 mmol) and sodium metabisulfite adduct of
substituted benzaldehyde (1.25 mmol) in DMF was
heated at 110 ꢁC for 5 h.4 Water was added to the reac-
tion medium and the solid product was collected by fil-
tration and washed with water. The crude product was
crystallized from EtOH.
4.1.3.1. [2-(4-Methoxyphenyl)-1H-benzimidazol-1-yl]-
acetic acid hydrazide (13). Yield: 73%; mp 197–198 ꢁC;
ES (+) (M+H): 297 (100%); IR (KBr cmꢀ1) 1694
1
(C@O); H NMR (DMSO-d6) d: 3.8 (s, 3H, O-CH3),
4.1.1.1. 5,6-Dichloro-2-(3,4-dimethoxyphenyl)-1H-benz-
imidazole (4). Yield: 87%; mp 269 ꢁC; ES (+) (M+H): 323
4.43 (s, 2H, –NH2–), 4.79 (s, 2H, –CH2–), 7.07–7.77
(m, 8H, Ar-H), 9.59 (s, 1H, NH).
1
(100%); H NMR (DMSO-d6) d: 3.85 (s, 3H, O-CH3),
3.88 (s, 3H, O-CH3), 7.16 (s, 1H), 7.75 (3H), 7.89 (s, 1H),
13.01 (s, 1H, NH).
4.1.3.2. [5,6-Dichloro-2-(3,4-dimethoxyphenyl)-1H-ben-
zimidazol-1-yl]-acetic acid hydrazide (14). Yield: 68%;
mp 265–266 ꢁC; ES (+) (M+H): 395 (100%), 397 (67%),
399 (11%); IR (KBr cmꢀ1) 1698 (C@O); 1H NMR
(DMSO-d6) d: 3.81 (s, 3H, O-CH3), 3.85 (s, 3H, O-
CH3), 4.40 (br s, 2H, –NH2), 4.90 (s, 2H, CH2–), 7.10
(d, 1H, Jo = 8.0), 7.31–7.35 (2H), 7.83 (s, 1H), 7.93 (s,
1H), 9.54 (s, 1H).
4.1.1.2. 2-(4-Benzyloxyphenyl)-1H-benzimidazole (5).
1
Yield: 79%; mp 276 ꢁC; ES (+) (M+H): 301 (100%); H
NMR (DMSO-d6) d: 5.20 (s, 2H, O-CH2), 7.17–7.20 (m,
4H), 7.33–7.61 (7H), 8.13 (d, 2H, J = 8), 12.70 (br s, 1H,
NH).
4.1.2. General procedure for the preparation of [2-
(substitutedphenyl)-1H-benzimidazolyl]-acetic acid ethyl
esters (6–10). Dimethylsulfoxide (15 ml) was added to
potassium hydroxide (27 mmol) (crushed pellets) and
the mixture was stirred 15 min and related benzimid-
azole derivative (6.7 mmol) was added and then the mix-
ture was stirred for 2 h. Ethylchloroacetate (27 mmol)
was added and the mixture was cooled briefly and stir-
red 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 ethylchloroace-
tate was removed under reduced pressure. The residue
was recrystallized from ethanol and gave the desired es-
ter compounds (6–10).
4.1.3.3. [2-(4-Benzyloxyphenyl)-1H-benzimidazol-1-yl]-
acetic acid hydrazide (15). Yield: 61%; mp 227–229 ꢁC; ES
(+) (M+H): 373 (100%); IR (KBr cmꢀ1) 1660 (C@O); 1H
NMR (DMSO-d6) d: 4.39 (s, 2H, NH2), 4.80 (s, 2H,
–CH2–), 5.20 (s, 2H, –CH2–), 7.17–7.79 (m, 13H, Ar-H),
9.59 (s, 1H, NH).
4.1.4. General procedure for the preparation of thiosemic-
arbazides [(1-(methylaminothio carbamoylhydrazine-
carbonyl)-methyl-2-substitutedphenyl-1H-benzimidazoles)]
(16a–20a). Appropriate acid hydrazides (11–15) 0.54 g
(2.03 mmol) in absolute ethanol (20 ml) and methyl isothi-
ocyanate (3.05 mmol) were heated under reflux for 30 min.
Precipitate formed was cooled, filtered, and recrystallized
from ethanol.
4.1.2.1. [2-(4-Methoxyphenyl)-1H-benzimidazol-1-yl]-
acetic acid ethyl ester (8). Yield: 68%; mp 96–97 ꢁC; ES
(+) (M+H): 311 (100%); IR (KBr cmꢀ1) 1752 (C@O);
1H NMR (DMSO-d6) d: 1.17 (t, 3H, H2C–CH3), 3.85
(s, 3H, O-CH3), 4.15 (q, 2H, H2C–CH3), 5.28 (s, 2H,
N-CH2), 7.11–7.69 (m, 8H, Ar-H).
4.1.5. General procedure for the preparation of 5-[(2-
(4-substitutedphenyl)-1H-benzimidazol-1-yl)methylN-
methyl-1,3,4-thiadiazol-2-amines (17b–20b). Appropriate
thiosemicarbazides 17a–20a (3.4 mmol) in 10 ml ice-cold
concentrated sulfuric acid were stirred for 10 min, and
then left for another 10 min at room temperature. The
resulting solution was poured slowly into ice-cold water,
made alkaline to pH 8 with aqueous ammonia and the
precipitated product was filtered, washed with water,
4.1.2.2. [5,6-Dichloro-2-(3,4-dimethoxyphenyl)-1H-ben-
zimidazol-1-yl]-acetic acid ethyl ester (9). Yield: 76%; mp
207–208 ꢁC; ES (+) (M+H): 409 (100%); IR (KBr cmꢀ1
)

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C. Kußs et al. / Bioorg. Med. Chem. 16 (2008) 4294–4303
and crystallized from ethanol or ethanol/isopropanol
(10:1).
in a final volume of 1 ml, 0.05 M phosphate buffer, pH
7.8, 0.32 U xanthine oxidase, 50 lM xanthine, 60 mM
cytochrome c, and different concentration of synthesized
compounds at 100 ll. The absorbance was measured
spectrophotometrically at 550 nm for cytochrome c
reduction.
4.1.6. General procedure for the preparation of the 5-[(2-
(substitutedphenyl)-1H-benzimidazol-1-yl)methyl]-4-methyl-
2H-1,2,4-triazole-3(4H)-thiones (16c–20c). Appropriate
thiosemicarbazides (3.4 mmol) 16a–20a in 10 ml 1 N
sodium hydroxide were refluxed for 1 h. The reaction mix-
ture was cooled and then acidified to pH 6 with 1 N hydro-
chloric acid. The precipitate was filtered, washed with water,
and recrystallized from ethanol.
4.3.3. Assay of lipid peroxidation. Male albino Wistar
rats (200–225 g) were used in the experiments. 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 pre-
pared as described previously.21
4.2. Single crystal X-ray structure determinations
Single crystals suitable for X-ray diffraction analysis
were obtained by crystallization from ethanol. Measure-
ments were performed on a Enraf-Nonius CAD4 four
circle diffractometer equipped with graphite monochro-
mator MoKa radiation.15 Intensity of peaks was cor-
rected for standard reflections automatically. Structure
was solved by direct methods using SHELXS16 and,
refinement was performed with SHELXL.17 Non-hydro-
gen atom parameters were refined anisotropically. All
hydrogen atoms, except for N3, were placed in idealized
positions and refined using a riding model with
Ueq(H) = 1.3Ueq(C), and fixed distances of C–H =
NADPH-dependent LP was determined using the opti-
mum conditions determined and described previously.21
NADPH-dependent was measured spectrophotometri-
cally by the estimation of thiobarbituric acid reactant
substances (TBARS). Amounts of TBARS were ex-
pressed in terms of nanomole malondialdehyde
(MDA)/mg protein. The assay was essentially derived
from the methods of Wills22,23 as modified by Bish-
ayee.24 A typical optimized assay mixture contained
0.2 nM Fe++, 90 mM KCl, 62.5 mM potassium-phos-
phate buffer, pH 7.4, NADPH generating system con-
sisting of 0.25 mM NADP+, 2.5 mM MgCl2, 2.5 mM
glucose-6-phosphate, 1.0 U glucose-6-phosphate dehy-
drogenase, and 14.2 mM potassium phosphate buffer
pH 7.8 and 0.2 mg microsomal protein in a final volume
of 1.0 ml.
˚
˚
0.93 A (aromatic), C–H = 0.96 A (methyl), and C–H =
˚
0.97 A (ethyl). Hydrogen atom of N3 was found from
a difference Fourier map and refined isotropically. The
geometric calculations were performed using the pro-
gram Platon.18
4.3. Antioxidant properties of novel benzimidazole
derivatives
4.3.4. Assay of EROD. EROD activity was measured by
the spectrofluorometric method of Burke et al.25
A
4.3.1. 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,di-
phenyl-1-picrylhydrazyl (DPPH) as described by Blois.19
This assay has often been used to estimate the antiradi-
cal activity of antioxidants. Because of its odd electra,
DPPH gives a strong absorption bound at 517 nm in vis-
ible spectroscopy. DPPH was dissolved in methanol to
give a 100 lM solution. To 1.0 ml of the methanolic
solution of DPPH was added 0.1 ml of the test com-
pounds and BHT dissolved in dimethylsulfoxide
(DMSO). Absorbance at 517 nm was determined after
30 min at room temperature and the scavenging activity
was calculated as a percentage of the 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 ex-
pressed as IC50 which was determined from a calibration
curve for each compound.
typical optimized assay mixture contained 1.0 mM eth-
oxyresorufin, 100 mM Tris–HCl buffer, pH 7.8,
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 liver microsomal protein in a final volume of
1.0 ml. EROD activity of compounds was expressed as
IC50 which determined from a calibration curve.
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