An efficient synthesis of benzimidazoles via a microwave technique and evaluation of their biological activities

Article abstract of DOI:10.1007/s00706-012-0916-0

A simple and practical protocol was developed for the synthesis of benzimidazoles. The protocol uses iminoester hydrochloride which is very useful in the reaction with 4,5-dichloro-1,2-phenylenediamine under microwave irradiation leading to the products with good yields and in short reaction times. This method can be used as a general technique for synthesizing benzimidazoles. The synthesized compounds were evaluated for their biological properties such as anti-lipase, antiviral, and antitumor activities. Five benzimidazol-1-acetic acid hydrazides showed slight antiviral activity at 25 μg/cm³ concentration despite their low toxicity. Substituted 2-benzylbenzimidazoles were active against adenocarcinoma (CT26) and melanoma (B16F10) cancer cell lines at concentrations below 10 μg/cm³. Six of the compounds showed anti-lipase activities at various concentrations; the IC₅₀ value of one compound was 0.35 μg/cm³, which is similar activity to that of orlistat (0.32 μg/cm³).

Full text of DOI:10.1007/s00706-012-0916-0

Monatsh Chem
DOI 10.1007/s00706-012-0916-0
ORIGINAL PAPER
An efficient synthesis of benzimidazoles via a microwave
technique and evaluation of their biological activities
¨
Bahittin Kahveci Emre Mentes¸e Musa Ozil
•
•
•
¨
•
Serdar Ulker Murat Ertu¨rk
Received: 4 June 2012 / Accepted: 28 December 2012
Ó Springer-Verlag Wien 2013
Abstract A simple and practical protocol was developed
for the synthesis of benzimidazoles. The protocol uses
iminoester hydrochloride which is very useful in the
reaction with 4,5-dichloro-1,2-phenylenediamine under
microwave irradiation leading to the products with good
yields and in short reaction times. This method can be used
as a general technique for synthesizing benzimidazoles.
The synthesized compounds were evaluated for their bio-
logical properties such as anti-lipase, antiviral, and
antitumor activities. Five benzimidazol-1-acetic acid
hydrazides showed slight antiviral activity at 25 lg/cm3
concentration despite their low toxicity. Substituted
2-benzylbenzimidazoles were active against adenocarci-
noma (CT26) and melanoma (B16F10) cancer cell lines at
concentrations below 10 lg/cm3. Six of the compounds
showed anti-lipase activities at various concentrations; the
IC50 value of one compound was 0.35 lg/cm3, which is
similar activity to that of orlistat (0.32 lg/cm3).
Keywords Benzimidazole drug Á Microwave Á
Iminoester hydrochloride Á Antitumor activity Á
Anti-lipase activity
Introduction
Benzimidazoles are an important group of heterocyclic
compounds in the field of medicinal chemistry because they
frequently have interesting biological and pharmacological
properties. For example, in 1944, Wayne Walley, one of the
leading exponents of antimetabolite therapy, noted the
resemblance of benzimidazole to adenine and speculated
that it might act as an adenine antimetabolite (Scheme 1).
He even demonstrated that it could inhibit the growth of
bacteria and fungi and that this could be reversed by either
adenine or guanine [1]. Obesity is widely recognized as a
major public health problem which is caused by an imbal-
ance between energy intake and expenditure. Obesity can
cause different serious diseases, including hypertension,
hyperlipidemia, arteriosclerosis, and type II diabetes [2].
Pancreatic lipase plays a key role for fat digestion. More-
over, pancreatic lipase inhibitors, such as orlistat, are used
as therapeutic agents for curing obesity [3].
B. Kahveci (&)
Department of Nutrition and Dietetics, Faculty of Health
Sciences, Karadeniz Technical University, Trabzon, Turkey
e-mail: bahittin@yahoo.com
Benzimidazole structures are classified under several
ATC groups [4]. Benzimidazole drugs (e.g., anthelmintics,
albendazole, thiabendazole, and the proton pump inhibitor
omeprazole) represent substances used in both human and
veterinary medicine (Scheme 2) [5–13].
¨
E. Mentes¸e Á M. Ozil
Department of Chemistry, Art and Science Faculty,
Recep Tayyip Erdogan University, 53100 Rize, Turkey
1-Benzyl-5,6-dichloro-1H-benzimidazole-2-amine is as
a potent inhibitor of viral RNA synthesis (Scheme 3) [14].
Human cytomegalovirus (HCMV) is an important patho-
gen in immunocompromised conditions, such as bone
marrow and organ transplant patients and individuals with
AIDS [15].
¨
S. Ulker
Department of Biology, Faculty of Arts and Sciences,
Recep Tayyip Erdogan University, 53100 Rize, Turkey
M. Ertu¨rk
Medical Microbiology, School of Medicine, Karadeniz
Technical University, 61080 Trabzon, Turkey
123

B. Kahveci et al.
l
l
C
NH2
O
N
N
X
N
l
l
C
N
N
N
N
N
NH2
NH
N
C
C
H
O
O
N
H
N
H
N
H
NH2
N
Benzimidazole
Adenine
Guanine
Scheme 1
H
H
O
O
Viral RNA synthesis inhibitor
Benzimidazole ribonucleosides
Scheme 3
In the literature, there are many synthetic routes that are
common to the preparation of benzimidazoles. However,
many of these procedures are associated with several
drawbacks such as expensive reagents, harsh reaction
conditions, extended reaction times, the occurrence of side
products, unsatisfactory yields, and complicated experi-
mental procedures. Electron deficiency of the starting
material in terms of the nature of the substituents on the
aromatic ring can affect the success of these reactions. In
general, when 4,5-disubstituted-1,2-phenylenediamine with
electron-withdrawing groups, such as chloro, are used the
yield and purity of the product are significantly worse,
reaction times are long, and catalysts are required [16–31].
Therefore, there is a need for a new synthetic route for
benzimidazoles which is short and economical.
Results and discussion
This is the first report on the synthesis of benzimidazoles
2a–2f from iminoester hydrochlorides 1a–1f [32, 33] both
in methanol under microwave irradiation and on a solid
support using microwave irradiation in the absence of
organic solvents, which make the procedure a clean, effi-
cient, and cheap method to afford various useful
heterocyclic compounds. The new heterocyclic compounds
2a–2f were also obtained by using conventional heating in
methanol [34].
In this new method we obtained products within short
reaction times and with high yields (Table 1). In addition,
the reaction was carried out catalyst-free under mild con-
ditions. Moreover, the use of solid supports under solvent-
less conditions is particularly important in terms of the
development of green technologies.
In this study we developed a novel and practical method
for the synthesis of 2-substituted benzimidazole deriva-
tives. This method can provide a convenient way to
synthesize potentially bioactive benzimidazoles. The
structures of new compounds were confirmed by FT-IR, 1H
NMR, 13C NMR spectroscopy, and mass spectrometry. All
the synthesized compounds were screened for their bio-
logical activities. The synthetic path of the target
compounds is shown in Scheme 4.
Acyl hydrazides 4a–4f were synthesized as key inter-
mediates for combinatorial benzimidazoles. Since the acyl
hydrazides could serve as both a hydrogen donor and
acceptor, they could be potentially more potent than the
parent benzimidazoles. Acyl hydrazides 4a–4f were easily
O
N
S
S
O
N
H
3C
NH
H
C
3
N
N
H
N
H
Albendazole
Thiabendazole
H
3C
O
N
H
C
3
O
N
H
H
C
S
3
N
O
H
C
3
Omerazolep
Scheme 2
123

Synthesis of benzimidazoles via a microwave technique
R
l
l
NH2
NH2
C
l
l
C
N
MW, MeOH
R
+
HN
H l
C
·
N
H
O
C
C
1a-1f
2a-2f
MW,
BrCH2COOMe
R
R
MW
H2N-NH2·H2O
,
l
l
C
l
l
C
N
N
N
N
C
C
O
O
4a-4f
3a-3f
O
NH
H2N
a
b
c
:
:
:
=
-
:
= -
R H
2
C
d
R
2
l
C
3
e
:
:
-
-
3 Cl
-
3 CH3
-
f
4
l
C
4
H
C
3
Scheme 4
Table 1 Comparison of yields and reaction time under microwave
irradiation and conventional method for compounds 2a–2f
Table 2 Residual lipase activity
Residual activity/%
% SD
Product Method i
Method ii
Method iii
T?
100
39
30
10
6
2
5
0
1
1
5
2
2
3
Time/
min
Yield/ Time/
%
Yield/ Time/
%
Yield/
%
2d (5 mg)
3a (5 mg)
3b (5 mg)
3c (3 mg)
3f (5 mg)
min
min
2a
2b
2c
2d
2e
2f
6
7
5
6
7
6
72
69
71
68
66
69
10
12
8
93
92
96
87
85
90
600
600
600
600
600
600
73
71
88
62
69
70
19
43
5
10
12
7
4b (5 mg)
Orlistat (0.5 mg)
Orlistat (0.1 mg)
13
prepared in excellent yields from 2a–2f by alkylation with
methyl a-bromoacetate in acetone followed by a nucleo-
philic displacement of the methoxy group with hydrazine.
drug, inhibited activity by 87, 95, and 99 % at the same
concentrations. IC50 values of orlistat and compound 3c
were calculated as 0.32 and 0.35 lg/cm3, respectively.
Orlistat is the only approved anti-obesity medication [3]
but it has some side effects, such as fecal incontinence,
flatulence, and steatorrhea [35, 36]. Synthesized com-
pounds such as 3b and 3c can be good alternatives to
orlistat.
Anti-lipase activity results
All compounds were evaluated with regard to pancreatic
lipase activity and 2d, 3a–3c, 3f, and 4d showed anti-lipase
activities at various concentrations (Table 2). No inhibitory
effect was detected for the other compounds. Dose-
dependent pancreatic lipase activity was observed as
shown in Fig. 1. Among the tested compounds, 3c showed
the best anti-lipase activity. The compound inhibited pan-
creatic lipase activity by 62, 92, and 96 % at concentrations
of 0.625, 1.25, and 6.25 lg/cm3, respectively. Orlistat, a
known pancreatic lipase inhibitor used as an anti-obesity
Antiviral activity results
All compounds tested were toxic at 100 and 25 lg/cm3 in
Vero and MDCK cells used to grow HSV-1 and influenza
A virus, respectively. Only compounds 4a, 4b, and 4d–4f
showed a slight antiviral activity at 100 and 25 lg/cm3
concentration despite their low toxicity. 3f and 2d were
123

B. Kahveci et al.
Table 4 Antitumor activity of the compounds
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Orlistat
3b
Compound
Tumor cell growth inhibition
(GI50, log lg/cm3)
3c
Cell line
3f
CT26 (adenocarcinoma)
B16F10 (melanoma)
2a
2b
2c
2d
2e
2f
0.7999
0.8162
0.7253
0.744
0.6427
0.6584
1.416
1.187
1.688
1.258
1.799
0.9827
1.501
1.466
1.248
1.592
1.401
1.404
0.6654
0.6667
0.6305
0.7094
0.7239
0.7474
2.171
4.348
5.645
1.296
1.867
1.506
1.574
1.697
1.506
1.593
1.554
1.549
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
Concentration/µg/cm3
3a
3b
3c
3d
3e
3f
Fig. 1 Dose-dependent inhibitory effect of the compounds 3b, 3c,
and 3f. Orlistat was used as a positive control. All compounds were
measured at final concentrations of 0.625, 1.25, and 6.25 lg/cm3.
Residual activities of compounds are expressed as the mean SD in
triplicate
4a
4b
4c
4d
4e
4f
Table 3 Anti-HSV and anti-influenza A virus activity of the compounds
CNc HSV % plaque reductiona
Anti-influenza A activity ( )
b
Concentration/lg/cm3
Concentration/lg/cm3
6.25
12.5
25
50 100 50 25 12.5 6.2 3.1 1.5 0.7
2a
2b
2c
2d
2e
2f
Td
T
T
T
T
T
T
0
T
T
T
T
T
T
0
T
–
–
–
–
–
–
–
–
–
–
–
–
–
?
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
T
T
–
–
Antitumor activity results
T
T
–
–
79 (T)
T
?
–
?
–
The tumor cell growth inhibition results in Table 4 indi-
cated that some of the compounds exhibited a dose-
dependent inhibitory effect on adenocarcinoma (CT26) and
melanoma (B16F10) cells. Six of the compounds, namely
2a–2f, were active against both cancer cell lines at con-
centrations below 10 lg/cm3. It was interesting to see that
although compounds 3a–3c at concentrations less than
100 lg/cm3 were not active against melanoma, but they
were inhibitory toward adenocarcinoma cells.
T
T
0
0
0
0
0
T
T
–
–
3a
3b
3c
3d
3e
3f
0
–
–
0
0
0
–
–
0
0
0
–
–
8 (T) 8 (T)
T
–
–
0
0
T
8
8
8
8
8
8
0
–
–
33 (T) 88 (T)
T
?
?
–
?
?
–
4a
4b
4c
4d
4e
4f
0
0
0
0
0
0
0
0
0
0
0
0
41
33
T
?
?
–
?
–
16
33
33
Conclusion
–
?
–
The present synthetic methods are rapid, inexpensive, and
efficient routes for the synthesis of 2-substituted benz-
imidazole derivatives. These new methods can be
efficiently used to synthesize many new benzimidazole
derivatives which have a broad spectrum of biological
activities. Synthesized compounds such as 3b and 3c could
be good alternatives to orlistat. The IC50 value of 3c was
0.35 lg/cm3, which is similar to that of orlistat (0.32 lg/
cm3). Compounds 2a–2f were active against adenocarci-
noma (CT26) and melanoma (B16F10) cancer cell lines at
concentrations below 10 lg/cm3.
a
Percentage of plaque reduction: [(mean number of plaques in control - mean
number of plaques in test)/(mean number of plaques in control)] 9 100
b
? and - indicate ‘no virus growth’ and ‘virus growth’, respectively, as
determined by hemagglutination assay using chicken erythrocytes
c
Compound number
d
Toxic: cell monolayer integrity is lost partially or completely
active against HSV-1 at 12.5 and 6.25 lg/cm3 concentra-
tion albeit with some degree of associated toxicity
(Table 3). No anti-influenza virus activity of the com-
pounds was detected.
123

Synthesis of benzimidazoles via a microwave technique
General procedure for the synthesis of 5,6-dichloro-2-
(substituted benzyl)-1H-benzimidazoles 2a–2f,
conventional method (method iii)
Experimental
Melting points were determined in open capillaries on a
Bu¨chi digital melting point apparatus. IR spectra were
recorded in KBr pellets on a Perkin-Elmer 100 FTIR
spectrophotometer. H NMR and 13C NMR spectra were
A
mixture
of
4,5-dichloro-1,2-phenylenediamine
1
(0.010 mol) and iminoester hydrochlorides 1a–1f
(0.013 mol) in 30 cm3 dry methanol was taken in a round-
bottom flask. The solution was stirred for 10 h at room
temperature. After the completion of the reaction (moni-
tored by TLC, ethyl acetate/hexane 3:1), the mixture was
poured into water. The precipitate was collected by filtra-
tion and recrystallized from ethanol/water (1:3) to give
pure 2a–2f.
measured on a Varian 200 spectrometer using DMSO-d6 as
solvent and TMS as internal standard. Chemical shifts are
given in parts per million, coupling constants J in Hz. Mass
spectra (MS) were determined in H-ESI mode on a Thermo
Quantum Mars. Elemental analyses were performed on a
Carlo Erba 1106 CHN analyzer, and the results agreed
favorably with calculated values. Starting materials were
obtained from Fluka or Aldrich. A monomode CEM-Dis-
cover Microwave was used in the standard configuration as
delivered, including proprietary software. All experiments
were carried out in microwave process vials (30 cm3) with
temperature control by an infrared detection temperature
sensor. The temperature was computer monitored and
maintained constant by a discrete modulation of delivered
microwave power. After completion of the reaction, the
vial was cooled to 60 °C by air jet cooling.
5,6-Dichloro-2-(2-chlorobenzyl)-1H-benzimidazole
(2a, C14H9Cl3N2)
ꢀ
M.p.: 237–238 °C; IR (KBr): m = 3,422 (NH), 3,071,
1
2,914, 1,629, 1,574, 1,287, 765, 744, 536 cm-1; H NMR
(200 MHz, DMSO-d6): d = 4.31 (s, 2H, CH2), 7.28–7.77
(m, 6H, Ph), 12.63 (s, 1H, NH ? D2O exchangeable) ppm;
13C NMR (50 MHz, DMSO-d6): d = 32.66 (CH2), 119.60,
123.80, 124.46, 127.29, 128.75, 129.24, 131.50, 133.23,
134.48 (Ar–C), 155.18 (C=N) ppm; ESI–MS (70 eV):
m/z = 311 ([M ? H]?).
General procedure for the synthesis of 5,6-dichloro-2-
(substituted benzyl)-1H-benzimidazoles 2a–2f
under microwave irradiation (method i)
5,6-Dichloro-2-(3-chlorobenzyl)-1H-benzimidazole
(2b, C14H9Cl3N2)
ꢀ
M.p.: 202–203 °C; IR (KBr):m = 3,430 (NH), 3,004,
2,932, 1,619, 1,599, 1,286, 1,097, 862, 796 cm-1 1H
;
A mixture of 4,5-dichloro-1,2-phenylenediamine (0.010 mol),
iminoester hydrochlorides 1a–1f (0.013 mol), and 9 g acidic
alumina oxide was taken in a 20-cm3 round-bottom flask and
solubilized in 10 cm3 dichloromethane. The solvent was
completely evaporated under reduced pressure. The mixture
was microwave-irradiated at 80 °C for 2 9 3 min (hold time)
at 60 W. After the completion of the reaction (monitored by
TLC, ethyl acetate/hexane3:1), themixture was extractedwith
ethanol (3 9 15 cm3) and poured into water. The precipitate
was filtered and recrystallized from ethanol/water (1:3) to give
pure 2a–2f.
NMR (200 MHz, DMSO-d6): d = 4.25 (s, 2H, CH2), 7.31–
7.82 (m, 6H, Ph), 12.68 (s, 1H, NH ? D2O exchangeable)
ppm; 13C NMR (50 MHz, DMSO-d6): d = 34.09 (CH2),
115.97, 123.93, 126.69, 127.60, 128.66, 130.33, 132.97,
138.15, 139.21 (Ar–C), 155.81 (C=N) ppm; ESI–MS
(70 eV): m/z = 311 ([M ? H]?).
5,6-Dichloro-2-(4-chlorobenzyl)-1H-benzimidazole (2c)
M.p.: 215–216 °C (Ref. [29] 214–215 °C)
5,6-Dichloro-2-(2-methylbenzyl)-1H-benzimidazole
(2d, C15H12Cl2N2)
ꢀ
M.p.: 192–196 °C; IR (KBr): m = 3,427 (NH), 3,027,
General procedure for the synthesis of 5,6-dichloro-2-
(substituted benzyl)-1H-benzimidazoles 2a–2f
under microwave irradiation (method ii)
1
2,977, 1,628, 1,528, 1,296, 1,096, 867, 750, 698 cm-1; H
NMR (200 MHz, DMSO-d6): d = 1.67 (s, 3H, CH3), 4.34
(s, 2H, CH2), 7.20–7.83 (m, 6H, Ph), 12.53 (NH, s,
1H ? D2O exchangeable) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 20.15 (CH3), 39.20 (CH2), 112.35,
119.50, 123.44, 123.92, 126.63, 127.20, 128.46, 133.82,
142.62, 142.98 (Ar–C), 160.17 (C=N) ppm; ESI–MS
(70 eV): m/z = 291 ([M ? H]?).
A mixture of 4,5-dichloro-1,2-phenylenediamine (0.010 mol)
and iminoester hydrochlorides 1a–1f (0.013 mol) in
15 cm3 dry methanol was irradiated in closed vessels
with pressure control at 65 °C for 10 min (hold time) at
300 W maximum power. After the completion of the
reaction (monitored by TLC, ethyl acetate/hexane 3:1),
the mixture was poured into water. The precipitate was
collected by filtration and recrystallized from ethanol/
water (1:3) to give pure 2a–2f.
5,6-Dichloro-2-(3-methylbenzyl)-1H-benzimidazole
(2e, C15H12Cl2N2)
ꢀ
M.p.: 197–198 °C; IR (KBr): m = 3,435 (NH), 3,067,
2,923, 1,609, 1,580, 1,283, 862, 803, 756 cm-1; H NMR
1
123

B. Kahveci et al.
(200 MHz, DMSO-d6): d = 2.10 (s, 3H, CH3), 4.11 (s, 2H,
CH2), 7.10–7.76 (m, 6H, Ph), 12.58 (s, 1H, NH ? D2O
exchangeable) ppm; 13C NMR (50 MHz, DMSO-d6):
d = 20.85 (CH3), 34.66 (CH2), 115.88, 119.42, 123.66,
125.79, 127.21, 128.34, 129.31, 133.32, 135.90, 136.78,
137.54 (Ar–C), 156.48 (C=N) ppm; ESI–MS (70 eV):
m/z = 291 ([M ? H]?).
124.18, 124.53, 126.53, 128.66, 130.07, 132.78, 135.11,
138.14, 141.43 (Ar–C), 155.90 (C=N), 167.74 (C=O) ppm;
ESI–MS (70 eV): m/z = 383 ([M ? H]?).
Methyl 5,6-dichloro-2-(4-chlorobenzyl)-1H-benzimidazol-
1-acetate (3c, C17H13Cl3N2O2)
ꢀ
M.p.: 154–155 °C; yield: 92 %; IR (KBr): m = 3,050,
1
2,956, 1,737 (C=O), 1,614, 1,227 (C–O), 761 cm-1; H
5,6-Dichloro-2-(4-methylbenzyl)-1H-benzimidazole
(2f, C15H12Cl2N2)
NMR (200 MHz, DMSO-d6): d = 3.53 (s, 3H, OCH3),
4.25 (s, 2H, CH2), 5.24 (s, 2H, NCH2), 7.24–7.92 (m, 6H,
Ph) ppm; 13C NMR (50 MHz, DMSO-d6): d = 31.90
(CH2), 44.54 (NCH2), 52.18 (OCH3), 112.10, 119.71,
124.23, 124.60, 128.23, 130.78, 134.68, 135.18, 141.45
(Ar–C), 156.15 (C=N), 167.79 (C=O) ppm; ESI–MS
(70 eV): m/z = 383 ([M ? H]?).
ꢀ
M.p.: 185–186 °C; IR (KBr): m = 3,430 (NH), 3,004,
1
2,920, 1,625, 1,550, 1,288, 864, 768, 522 cm-1; H NMR
(200 MHz, DMSO-d6): d = 2.23 (s, 3H, CH3), 4.10 (s, 2H,
CH2), 7.10–7.71 (m, 6H, Ph), 12.54 (s, 1H, NH ? D2O
exchangeable) ppm; 13C NMR (50 MHz, DMSO-d6):
d = 21.30 (CH3), 35.09 (CH2), 111.58, 119.30, 124.35,
129.39, 129.76, 134.62, 136.42, 141.90 (Ar–C), 157.45
(C=N) ppm; ESI–MS (70 eV): m/z = 291 ([M ? H]?).
Methyl 5,6-dichloro-2-(2-methylbenzyl)-1H-benzimidazol-
1-acetate (3d, C18H16Cl2N2O2)
ꢀ
M.p.: 117–118 °C; yield: 91 %; IR (KBr): m = 3,027,
2,921, 1,741 (C=O), 1,621, 1,590, 1,223 (C–O), 1,099,
General procedure for the synthesis of methyl 5,6-
dichloro-2-(substituted benzyl)-1H-benzimidazol-1-
acetates 3a–3f
1
702 cm-1; H NMR (200 MHz, DMSO-d6): d = 2.20 (s,
3H, CH3), 3.63 (s, 3H, OCH3), 4.32 (s, 2H, CH2), 5.30 (s,
2H, NCH2), 7.29–7.97 (m, 6H, Ph) ppm; 13C NMR
(50 MHz, DMSO-d6): d = 21.48 (CH3), 36.80 (CH2),
44.24 (NCH2), 51.94 (OCH3), 112.06, 119.85, 124.03,
124.48, 126.63, 127.28, 128.45, 135.33, 141.30, 141.78
(Ar–C), 159.46 (C=N), 167.34 (C=O) ppm; ESI–MS
(70 eV): m/z = 363 ([M ? H]?).
A mixture of compounds 2a–2f (0.01 mol), methyl a-bro-
moacetate (0.01 mol), and K2CO3 (0.025 mol) in 10 cm3
acetone was irradiated in closed vessels with pressure
control at 85 °C for 7 min (hold time) at 300 W maximum
power. After the completion of the reaction (monitored by
TLC, ethyl acetate/hexane 3:1), the mixture was poured
into water. The precipitate was collected by filtration and
recrystallized from acetone/water (1:3) to give pure 3a–3f.
Methyl 5,6-dichloro-2-(3-methylbenzyl)-1H-benzimidazol-
1-acetate (3e, C18H16Cl2N2O2)
M.p.: 165–166 °C; yield: 92 %; IR (KBr):
ꢀm = 3,040,
2,949, 1,738 (C=O), 1,607, 1,512, 1,227 (C–O), 1,094,
Methyl 5,6-dichloro-2-(2-chlorobenzyl)-1H-benzimidazol-
1-acetate (3a, C17H13Cl3N2O2)
1
763 cm-1; H NMR (200 MHz, DMSO-d6): d = 2.22 (s,
3H, CH3), 3.49 (s, 3H, OCH3), 4.21 (s, 2H, CH2), 5.20 (s,
2H, NCH2), 7.00–7.91 (m, 6H, Ph) ppm; 13C NMR
(50 MHz, DMSO-d6): d = 21.62 (CH3), 38.93 (CH2),
45.42 (NCH2), 52.90 (OCH3), 112.91, 120.52, 124.96,
125.30, 126.70, 128.20, 129.10, 130.17, 136.10, 136.30,
138.25, 142.35 (Ar–C), 157.30 (C=N), 168.54 (C=O) ppm;
ESI–MS (70 eV): m/z = 363 ([M ? H]?).
ꢀ
M.p.: 155–156 °C; yield: 92 %; IR (KBr): m = 3,063,
2,943, 1,738 (C=O), 1,618, 1,570, 1,250 (C–O), 1,096,
758 cm-1; H NMR (200 MHz, DMSO-d6): d = 3.55 (s,
1
3H, OCH3), 4.32 (s, 2H, CH2), 5.27 (s, 2H, NCH2), 7.29–
7.96 (m, 6H, Ph) ppm; 13C NMR (50 MHz, DMSO-d6):
d = 31.61 (CH2), 45.36 (NCH2), 53.14 (OCH3), 112.85,
120.58, 124.97, 125.32, 127.98, 129.51, 129.97, 132.41,
134.13, 134.60, 136.00, 142.30 (Ar–C), 156.37 (C=N),
168.78 (C=O) ppm; ESI–MS (70 eV): m/z = 383
([M ? H]?).
Methyl 5,6-dichloro-2-(4-methylbenzyl)-1H-benzimidazol-
1-acetate (3f, C18H16Cl2N2O2)
ꢀ
M.p.: 126-127 °C; yield: 96 %; IR (KBr): m = 3,043,
2,946, 1,734 (C=O), 1,615, 1,512, 1,223 (C–O), 1,094,
Methyl 5,6-dichloro-2-(3-chlorobenzyl)-1H-benzimidazol-
1-acetate (3b, C17H13Cl3N2O2)
1
769 cm-1; H NMR (200 MHz, DMSO-d6): d = 2.23 (s,
3H, CH3), 3.49 (s, 3H, OCH3), 4.19 (s, 2H, CH2), 5.19 (s,
2H, NCH2), 7.08–7.90 (m, 6H, Ph) ppm; 13C NMR
(50 MHz, DMSO-d6): d = 20.50 (CH3), 38.08 (CH2),
44.54 (NCH2), 52.15 (OCH3), 112.06, 119.66, 123.62,
124.10, 124.42, 128.64, 128.87, 132.48, 133.76, 135.24,
135.65, 141.49 (Ar–C), 156.63 (C=N), 167.72 (C=O) ppm;
ESI–MS (70 eV): m/z = 363 ([M ? H]?).
ꢀ
M.p.: 187–188 °C; yield: 95 %; IR (KBr): m = 3,065,
2,952, 1,731 (C=O), 1,596, 1,231 (C–O), 1,094, 702 cm-1
;
1H NMR (200 MHz, DMSO-d6): d = 3.54 (s, 3H, OCH3),
4.27 (s, 2H, CH2), 5.25 (s, 2H, N-CH2), 7.23–7.91 (m, 6H,
Ph) ppm; 13C NMR (50 MHz, DMSO-d6): d = 32.00
(CH2), 44.49 (NCH2), 52.12 (OCH3), 112.09, 119.72,
123

Synthesis of benzimidazoles via a microwave technique
General procedure for the synthesis of 5,6-dichloro-2-
(substituted benzyl)-1H-benzimidazole-1-acetic acid
hydrazides 4a–4f
5,6-Dichloro-2-(2-methylbenzyl)-1H-benzimidazol-1-acetic
acid hydrazide (4d, C17H16Cl2N4O)
ꢀ
M.p.: 204–205 °C; yield: 79 %; IR (KBr): m = 3,303
1
(NH2), 3,188 (NH), 1,671 (C=O), 1,542 (C=N) cm-1; H
A mixture of compounds 3a–3f (0.01 mol) and hydrazine
hydrate (0.01 mol) in 10 cm3 absolute ethanol was irradi-
ated in closed vessels with pressure control at 120 °C for
5 min (hold time) at 300 W maximum power. After the
completion of the reaction (monitored by TLC, ethyl ace-
tate/hexane 3:1), the mixture was cooled to room
temperature. The precipitate was washed with ethanol and
dried over CaCl2 to give pure 4a–4f.
NMR (200 MHz, DMSO-d6): d = 1.65 (s, 3H, CH3), 4.27
(s, 2H, CH2), 4.42 (s, 2H, NH2 ? D2O exchangeable), 4.87
(s, 2H, NCH2), 7.19–7.93 (m, 6H, Ph), 9.41 (s, 1H,
NH ? D2O exchangeable) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 22.14 (CH3), 37.59 (CH2), 44.90
(NCH2), 112.77, 120.63, 124.48, 124.87, 127.27, 127.85,
129.10, 135.87, 142.25, 142.04 (Ar–C), 160.49 (C=N),
165.89 (C=O) ppm; ESI–MS (70 eV): m/z = 363
([M ? H]?).
5,6-Dichloro-2-(2-chlorobenzyl)-1H-benzimidazol-1-acetic
acid hydrazide (4a, C16H13Cl3N4O)
5,6-Dichloro-2-(3-methylbenzyl)-1H-benzimidazol-1-acetic
acid hydrazide (4e, C17H16Cl2N4O)
ꢀ
M.p.: 246–247 °C; yield: 83 %; IR (KBr): m = 3,325,
3,310 (NH2), 3,212 (NH), 1,651 (C=O), 1,573 (C=N)
ꢀ
M.p.: 235–236 °C; yield: 81 %; IR (KBr): m = 3,300
1
1
cm-1; H NMR (200 MHz, DMSO-d6): d = 4.34 (s, 2H,
(NH2), 3,163 (NH), 1,653 (C=O), 1,535 (C=N) cm-1; H
CH2), 4.36 (s, 2H, NH2 ? D2O exchangeable), 4.93 (s, 2H,
NCH2), 7.27–7.87 (m, 6H, Ph), 9.52 (s, 1H, NH ? D2O
exchangeable) ppm; 13C NMR (50 MHz, DMSO-d6):
d = 31.69 (CH2), 45.44 (NCH2), 112.61, 120.52, 124.73,
125.09, 127.97, 129.45, 129.93, 132.35, 134.12, 134.93,
135.95, 142.35 (Ar–C), 156.60 (C=N), 166.20 (C=O) ppm;
ESI–MS (70 eV): m/z = 383 ([M ? H]?).
NMR (200 MHz, DMSO-d6): d = 2.49 (s, 3H, CH3), 4.19
(s, 2H, CH2), 4.32 (s, 2H, NH2 ? D2O exchangeable), 4.84
(s, 2H, NCH2), 7.07–7.84 (m, 6H, Ph), 9.48 (s, 1H,
NH ? D2O exchangeable) ppm; 13C NMR (50 MHz,
DMSO-d6): d = 21.44 (CH3), 33.33 (CH2), 45.20
(NCH2), 112.50, 120.21, 124.47, 124.76, 126.51, 127.79,
128.83, 130.00, 135.84, 136.47, 138.04, 142.22 (Ar–C),
157.50 (C=N), 165.97 (C=O) ppm; ESI–MS (70 eV):
m/z = 363 ([M ? H]?).
5,6-Dichloro-2-(3-chlorobenzyl)-1H-benzimidazol-1-acetic
acid hydrazide (4b, C16H13Cl3N4O)
ꢀ
M.p.: 215–216 °C; yield: 82 %; IR (KBr): m = 3,311
5,6-Dichloro-2-(4-methylbenzyl)-1H-benzimidazol-1-acetic
acid hydrazide (4f, C17H16Cl2N4O)
1
(NH2), 3,164 (NH), 1,655 (C=O), 1,598 (C=N) cm-1; H
ꢀ
NMR (200 MHz, DMSO-d6): d = 4.25 (s, 2H, CH2), 4.39
(s, 2H, NH2 ? D2O exchangeable), 4.89 (s, 2H, NCH2),
7.30–7.84 (m, 6H, Ph), 9.50 (s, 1H, NH ? D2O exchange-
able) ppm; 13C NMR (50 MHz, DMSO-d6): d = 32.11
(CH2), 44.58 (NCH2), 111.58, 119.66, 123.95, 124.24,
126.52, 127.77, 128.83, 130.06, 132.77, 135.07, 138.58,
141.53 (Ar–C), 156.26 (C=N), 165.36 (C=O) ppm;
ESI–MS (70 eV): m/z = 383 ([M ? H]?).
M.p.: 256–257 °C; yield: 82 %; IR (KBr): m = 3,324,
3,307 (NH2), 3,173 (NH), 1,660 (C=O), 1,513 (C=N)
cm-1; H NMR (200 MHz, DMSO-d6): d = 2.23 (s, 3H,
1
CH3), 4.15 (s, 2H, CH2), 4.48 (s, 2H, NH2 ? D2O
exchangeable), 4.83 (s, 2H, NCH2), 7.07–7.83 (m, 6H,
Ph), 9.48 (s, 1H, NH ? D2O exchangeable) ppm; 13C
NMR (50 MHz, DMSO-d6): d = 21.09 (CH3), 32.99
(CH2), 45.17 (NCH2), 112.46, 120.18, 124.45, 124.73,
129.31, 129.50, 133.49, 135.84, 136.19, 142.22 (Ar–C),
157.49 (C=N), 165.97 (C=O) ppm; ESI–MS (70 eV): m/
z = 363 ([M ? H]?).
5,6-Dichloro-2-(4-chlorobenzyl)-1H-benzimidazol-1-acetic
acid hydrazide (4c, C16H13Cl3N4O)
ꢀ
M.p.: 266–267 °C; yield: 85 %; IR (KBr): m = 3,341,
3,317 (NH2), 3,164 (NH), 1,655 (C=O), 1,560 (C=N)
Anti-lipase activity
1
cm-1; H NMR (200 MHz, DMSO-d6): d = 4.23 (s, 2H,
CH2), 4.33 (s, 2H, NH2 ? D2O exchangeable), 4.86 (s, 2H,
NCH2), 7.28–7.82 (m, 6H, Ph), 9.48 (s, 1H, NH ? D2O
exchangeable) ppm; 13C NMR (50 MHz, DMSO-d6):
d = 31.95 (CH2), 44.57 (NCH2), 104.14, 111.84, 119.62,
123.92, 124.23, 128.18,130.85, 131.21, 135.08, 135.12,
141.52 (Ar–C), 156.43 (C=N), 165.34 (C=O) ppm;
ESI–MS (70 eV): m/z = 383 ([M ? H]?).
The inhibitory effects of the compounds were evaluated
against porcine pancreatic lipase (15 lg/cm3). Lipase
activity assays were done according to Verger et al. [37].
Microtiter plates were coated with purified TAGs of tung
oil. Compounds and the lipase were incubated for 30 min
at a ratio 1:2 (v/v). The microtiter plates containing purified
tung oil, lipase solution, and assay buffer (10 mM Tris–
123

B. Kahveci et al.
HCl buffer, pH 8.0, containing 150 mM NaCl, 6 mM CaCl2,
1 mMEDTA, and3 mg/cm3)were recordedcontinuouslyfor
40 min against only the buffer alone by using a microplate
reader (SpectraMax M5, Molecular Devices) at 272 nm. The
inhibitory activity of those compounds and orlistat (as posi-
tive control) against pancreatic lipase was measured at
concentrations of 6.25, 1.25, and 0.625 lg/cm3. Residual
activities were calculated by comparing to control without
inhibitor (T?). The assays were done in triplicate. The IC50
value was determined as the concentration of compound that
give 50 % inhibition of maximal activity.
[40] using murine tumor cell lines CT26 (adenocarcinoma)
and B16F10 (melanoma). Briefly, 1 9 105 viable cells
from each cell line in RPMI-1640 growth medium sup-
plemented with 10 % FBS were seeded in a 96-well plate
and incubated for 24–48 h. When cells reached greater than
80 % confluence, the medium was decanted and cells were
incubated with twofold dilutions (100, 50, 25, 12.5, 6.2,
3.1, and 1.5 lg/cm3) of the test compounds prepared in
0.5 % dimethyl sulfoxide in triplicate. After 48 h of incu-
bation at 37 °C, the treated and untreated cells (controls)
were fixed by the addition of 1 % glutaraldehyde solution
for 15 min, washed with deionized water, and dried in air.
The cells were then stained with 0.4 % crystal violet for
30 min, then extensively washed with phosphate buffered
saline and allowed to dry overnight before dissolving the
retained dye with 75 % ethyl alcohol. The absorbance of
developing color was determined by measuring the optical
density (OD) at 570–630 nm using a multiwell spectropho-
tometer. The cell growth inhibiting (GI50) concentration of
thecompounds given as lg/cm3 in logunits was calculated by
GraphPad Prim 4 software. All determinations were per-
formed in triplicate.
Antiviral activity testing
Antiviral activity of the compounds against HSV-1 (wal
strain) and influenza A virus (A/PR/8, H1N1) was tested by
plaque reduction and hemagglutination assays using Vero
and MDCK cells, respectively, as described [38, 39]. For
the anti-HSV-1 activity test, briefly monolayers of Vero
cells grown in 24-well plates were infected with the virus
(ca. 100 pfu/well). After incubation for 1 h to allow viral
adsorption, the inoculum was aspirated and the infected
cells were overlaid with 0.8 % methylcellulose in mainte-
nance medium (minimal essential medium with 2 % fetal
bovine serum) containing various concentrations of the
compounds in duplicate. Controls included mock-infected
wells with and without compounds. After 72 h of incuba-
tion, the cell monolayers were washed with phosphate
buffer and then stained with naphthol blue black dye. The
plaques were counted and the percentage of plaque
reduction was calculated as follows: [(mean number of
plaques in control - mean number of plaques in test)/
(mean number of plaques in control)] 9 100. For the anti-
influenza activity test, briefly monolayers of MDCK cells
were grown in 96-well plates and infected with 0.1 cm3
of 9 100 TCID50 of influenza A virus (A/PR/8, H1N1)
prepared in a maintenance medium (minimal essential
medium with no serum but containing 1 lg/cm3 trypsin).
After incubation for 1 h to allow viral adsorption at 37 °C,
the inoculum was decanted and the infected cells were
overlaid with fresh maintenance medium containing vari-
ous concentrations (200, 100, 50, 25, 12.5, 6.2, 3.1, 1.5 lg/
cm3) of the compounds in triplicate. After 72 h of incu-
bation at 37 °C and 5 % CO2, 50 mm3 of culture
supernatant from each well was transferred into U-bottom
microwell plates to detect the presence of virus by the
hemagglutination assay. The result were reported as the
presence (?) or absence (-) of the virus growth.
Acknowledgments The authors gratefully acknowledge the finan-
cial support from the Scientific and Technical Research Council of
¨
Turkey (TUBITAK) through Project 108T356.
˙
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