Microwave-assisted synthesis of new benzimidazole derivatives with lipase inhibition activity

Article abstract of DOI:10.3109/14756366.2012.753880

A practical protocol has been used for the synthesis of benzimidazoles. The reaction of iminoester hydrochlorides of phenylacetic with 4,5-dichloro-1,2- phenylenediamine under microwave irradiation leads to the benzimidazole derivatives with good yields and in short reaction times. After the synthesis of benzimidazoles, we synthesized ester and hydrazide derivatives under microwave irradiation with good yields. All compounds were evaluated with regard to pancreatic lipase activity and 3b, 3c, 5a and 6a showed lipase inhibition at various concentrations.

Full text of DOI:10.3109/14756366.2012.753880

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(1): 64–68
2014 Informa UK Ltd. DOI: 10.3109/14756366.2012.753880
!
Microwave-assisted synthesis of new benzimidazole derivatives with
lipase inhibition activity
3
, Serdar Ulker , Olcay Bekircan , and Bahittin Kahveci
4
5
e1, Hakan Bektas2
ꢀ
¨
Emre Mentes
ꢀ
2
1Department of Chemistry, Faculty of Arts and Sciences, Recep Tayyip Erdogan University, Rize, Turkey, Department of Chemistry, Faculty of Arts
3
and Sciences, Giresun University, Giresun, Turkey, Department of Biology, Faculty of Arts and Sciences, Recep Tayyip Erdogan University, Rize,
Turkey, 4Department of Chemistry, Faculty of Science, and 5Department of Nutrition and Dietetics, Faculty of Health Sciences, Karadeniz Technical
University, Trabzon, Turkey
Abstract
Keywords
A practical protocol has been used for the synthesis of benzimidazoles. The reaction of iminoester
hydrochlorides of phenylacetic with 4,5-dichloro-1,2-phenylenediamine under microwave
irradiation leads to the benzimidazole derivatives with good yields and in short reaction times.
After the synthesis of benzimidazoles, we synthesized ester and hydrazide derivatives under
microwave irradiation with good yields. All compounds were evaluated with regard to pancreatic
lipase activity and 3b, 3c, 5a and 6a showed lipase inhibition at various concentrations.
Benzimidazole, iminoester hydrochloride,
lipase inhibition, microwave
History
Received 13 July 2012
Revised 10 September 2012
Accepted 30 September 2012
Published online 18 January 2013
Introduction
yields and complicated experimental procedures. Also, microwave
technology has been used to synthesize benzimidazole derivatives,
and important changes have been seen on yield and reaction
time1,21,22. In this study, synthesis of benzimidazoles has shown
that iminoester hydrochlorides could be a useful intermediate in the
reactionwith 4,5-dichloro-1,2-phenylenediamine under microwave
irradiation in methanol and solvent-free conditions. This method
can provide a convenient way of synthesizing potential bioactive
benzimidazoles. Furthermore, a practical method has been
developed for the synthesis of bis-benzimidazoles. Iminoester
hydrochlorides of phenylacetic acids were used as intermediates in
the reaction with 3,30-diaminobenzidine under microwave irradia-
tion, leading to the products with good yields and short reaction
times. This method can be a general technique for the synthesis of
bis-benzimidazoles. The synthetic path of the target compounds is
shown in Scheme 1.
Benzimidazoles are an important group of heterocyclic com-
pounds in the field of medicinal chemistry1. Some benzimidazole
derivatives with different biological activity such as anticancer2,
antihelmintic3, antimicrobial4, antifungal5, antitubercular6, anti-
allergic7, antioxidant8, antihistaminic9, antitumor10, anti-inflam-
matory and analgesic activities11 have been revealed in the
literature. Benzimidazole derivatives exhibit significant activity
against several viruses such as HIV12, herpes (HSV-1)13 and
human cytomegalovirus12. In addition, the treatment potency of
benzimidazoles in diseases such as ischemia–reperfusion injury14,
hypertension15, obesity16 has been recently reported. Because of
these reasons, benzimidazole derivatives have been studied by
numerous scientists. Obesity is widely recognized as a major
public health problem which is caused by an imbalance between
energy intake and expenditure. Obesity can be a cause to different
serious diseases, including hypertension, hyperlipidemia, arterio-
sclerosis and type II diabetes17. Pancreatic lipase plays a key role
for fat digestion. Moreover, pancreatic lipase inhibitors, such as
Orlistat, are used as therapeutic agent for curing obesity18.
In the literature, there are many synthetic routes that are
common to the preparation of benzimidazoles. Generally, the
condensation of ortho-phenylenediamines and carboxylic acids (or
their derivatives such as nitriles, imidates and orthoesters) had been
widely used for the synthesis of benzimidazole19,20. 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
Results and discussion
Iminoester hydrochloride could be a useful intermediate in the
reaction with o-phenylenediamine under microwave irradiation in
methanol for the synthesis of benzimidazole derivatives. In this
method, we obtained products within short reaction times and
with high yields. In addition, the reaction was carried out catalyst-
free under mild conditions. Compounds (2a–c) were also obtained
using conventional heating in methanol23. We also synthesized
bis-benzimidazole from iminoester hydrochloride reaction of the
3,30-diaminobenzidine under microwave irradiation, leading to the
product with good yield. After the synthesis of benzimidazoles,
we synthesized esters (3a–c, 6a) and hydrazides (4a–c, 7a) under
microwave irradiation with good yields. Simple alkylation
reaction was applied to 2a–c, 6a with methyl a-bromoacetate in
acetone and then, the MeO group was displayed with
NH2NH2ꢀH2O in EtOH. The structures of new compounds were
Address for correspondence: Emre Mentesꢀ e, Department of Chemistry,
Faculty of Arts and Sciences, Recep Tayyip Erdogan University, 53100
Rize, Turkey. Tel: þ90 46 42236126. Fax: þ90 46 42234019. E-mail:
emre.mentese@rize.edu.tr

DOI: 10.3109/14756366.2012.753880
Synthesis of new benzimidazole derivatives 65
Scheme 1. Synthetic path of the target
compounds.
confirmed by FT-IR (infrared), 1H-NMR, 13C-NMR spectroscopy effects, such as fecal incontinence, flatulence and steatorrhea24,25
and mass spectrometry. All the synthesized compounds were The synthesized compounds such as 3b and 6a can be a good
.
screened for their anti-lipase activities.
alternative to Orlistat.
Anti-lipase activity results
Experimental
All the compounds were evaluated with regard for pancreatic Melting points were determined in open capillaries on Bu¨chi
lipase activity and different concentrations of 3b, 3c, 5a and 6a (Essen, Germany) digital melting point apparatus and were
showed anti-lipase activities at various concentrations (Table 1). uncorrected. IR spectra were recorded in KBr pellets on a Perkin-
1
No inhibitory effect was observed in other compounds. Dose- Elmer 100 FT-IR spectrophotometer (California). H- and 13C-
dependent pancreatic lipase activity is shown in Figure 1. Among NMR spectra were measured on a Varian 400 spectrometer
the compounds tested, 3b showed the best anti-lipase activity. The (Varian, Darmstadt, Germany) using DMSO-d6 as solvent and
compound inhibited pancreatic lipase activity by 84%, 97% and TMS as internal standard. Chemical shifts are given in parts per
98% at concentrations of 0.625, 1.25 and 6.25 mg/mL, respec- million (ppm), coupling constants J in hertz (Hz). Mass spectra
tively. Orlistat (Xenical; Hoffmann-La Roche, Segrate, Italy), (MS) were taken in H-ESI mode on Thermo Quantum Mars
known pancreatic lipase inhibitor used as anti-obesity drug, (Thermo-scientific, Florida). A monomode CEM-Discover micro-
showed inhibitory effect by 98%, 99% and 100% at the same wave (Linfort, Germany) was used in the standard configuration
concentrations. IC50 values of Orlistat and compound 3b were as delivered, including proprietary software. All experiments were
calculated as 0.04 and 0.17 mg/mL, respectively. Orlistat is the carried out in microwave process vials (30 mL) with control of the
only approved anti-obesity medication18 but it has some side temperature by IR detection temperature sensor. The temperature

66 E. Mentes¸e et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 64–68
Table 1. Residual lipase activity of chemical compounds.
General procedure for the synthesis of methyl [5,6-dichloro-2-
(substitutedbenzyl)-1H-benzimidazole-1-yl] acetate (3a–c)
Residual activity (%)
SD (%)
A mixture of compounds 2a–c (0.01 mol), methyl-a-bromoacetate
(0.01 mol) and K2CO3 (0.025 mol) in acetone (10 mL) was
irradiated in closed vessels with the pressure control at 85 ꢁC for
7 min (hold time) at 300 W maximum power. After the completion
of the reaction, (monitored by TLC, ethylacetate:hexane, 3:1), the
mixture was poured onto water. The precipitate formed was filtered
and recrystallized from acetone:water (1:3) to give pure 3a–c.
Methyl [5,6-dichloro-2-(2-bromobenzyl)-1H-benzimidazole-1-
yl] acetate (3a). Yield (90%); m.p. 162–163 ꢁC; IR (ꢀmax, cmꢂ1):
1738 (C¼O), 1618 (C¼N) and 1250 (C–O); 1H-NMR (DMSO-d6,
200 MHz) ꢁ (ppm): 3.48 (s, 3H, OCH3), 4.31 (s, 2H, CH2), 5.30
(s, 2H, N–CH2) and 7.19–7.89 (m, 6H, Ar-H); 13C-NMR (DMSO-
d6, 50 MHz) ꢁ (ppm): 30.75 (CH2), 44.48 (N–CH2), 52.27 (O–
CH3), 112.84, 119.68, 124.10, 124.41, 127.11, 128.56, 129.03,
131.39, 133.19, 133.25, 135.02, 141.42 (Ar–C), 155.44 (C¼N)
and 167.67 (C¼O). Anal. Calcd for C17H13Cl2BrN2O2: C, 47.69;
H, 3.06 and N, 6.54; found: C, 47.65; H, 3.11 and N, 6.53. ESI-
MS m/z calculated for C17H13Cl2BrN2O2 [M]þ 428.10 and
430.10; found: 428.80 and 430.88.
Methyl [5,6-dichloro-2-(3-bromobenzyl)-1H-benzimidazole-1-
yl] acetate (3b). Yield (93%); m.p. 193–194 ꢁC; IR (ꢀmax, cmꢂ1):
1731 (C¼O), 1594 (C¼N) and 1227 (C–O); 1H-NMR (DMSO-d6,
200 MHz) ꢁ (ppm): 3.55 (s, 3H, OCH3), 4.28 (s, 2H, CH2), 5.29
(s, 2H, N–CH2) and 7.24–7.95 (m, 6H, Ar–H); 13C-NMR
(DMSO-d6, 50 MHz) ꢁ (ppm): 32.57 (CH2), 45.12 (N–CH2),
52.79 (O–CH3), 112.75, 120.37, 122.06, 124.83, 128.67, 130.06,
131.03, 132.17, 135.24, 135.75, 139.06, 142.06 (Ar–C), 156.56
(C¼N) and 168.39 (C¼O). Anal. Calcd for C17H13Cl2BrN2O2: C,
47.69; H, 3.06 and N, 6.54; found: C, 47.65; H, 3.11 and N, 6.55.
ESI-MS m/z calculated for C17H13Cl2BrN2O2 [M]þ 428.10 and
430.10; found: 428.80 and 430.91.
Tþ
100
16
26
58
63
5
ꢃ4
3b (0.125 mg)
3c (0.125 mg)
5a (0.125 mg)
6a (0.125 mg)
Orlistat (0.125 mg)
ꢃ0.3
ꢃ1
ꢃ1
ꢃ1
ꢃ2.3
Tþ: porcine pancreatic lipase (PPL) without inhibitor (control).
was computer monitored and maintained constant by a discrete
modulation of the delivered microwave power. After completion
of the reaction, the vial was cooled to 60 ꢁC by air jet cooling.
Synthesis of hydrochloride of substitute benzeneimidic acid ethyl
ester (1a–c)
To an ice-cooled solution of the substitute benzonitrile (1 mol) in
dry EtOH (1.1 mol), dry hydrogen chloride was added until 1.1 mol
had been absorbed. The resulting solutionwas then allowed to stand
at 0 ꢁC in the refrigerator for 12 h, after which cold Et2O was added.
The precipitated crystals were filtered off immediately, washed
with cold Et2O and dried in a dessicator.
General procedure for the synthesis of 5,6-dichloro-2-(substitu-
tedbenzyl)-1H-benzimidazole (2a–c) under microwave irradiation
A mixture of 4,5-dichloro-1,2-phenylenediamine (0.010 mol) and
iminoester hydrochlorides (0.013 mol; 1a–c) in dry methanol
(15 mL) was irradiated in closed vessels with the pressure control
at 65 ꢁC for 10 min (hold time) at 300 W maximum power. After
the completion of the reaction, (monitored by TLC, ethylaceta-
te:hexane, 3:1), the mixture was poured onto water. The
precipitate formed was filtered and recrystallized from
ethanol–water (1:3) to give pure 2a–c.
Methyl [5,6-dichloro-2-(4-bromobenzyl)-1H-benzimidazole-1-
yl] acetate (3c). Yield (97%); m.p. 152–153 ꢁC; IR (ꢂmax, cmꢂ1):
1738 (C¼O), 1591 (C¼N) and 1226 (C–O); 1H-NMR (DMSO-d6,
200 MHz) ꢁ (ppm): 3.64 (s, 3H, OCH3), 4.23 (s, 2H, CH2), 4.66
(s, 2H, N–CH2) and 7.09–7.85 (m, 6H, Ar–H); 13C-NMR
(DMSO-d6, 50 MHz) ꢁ (ppm): 29.11 (CH2), 40.28 (N–CH2),
48.25 (O–CH3), 105.73, 116.31, 116.65, 121.93, 122.41, 125.57
(2C), 127.34 (2C), 129.10, 130.09, 137.02 (Ar–C), 149.80 (C¼N)
and 162.07 (C¼O). Anal. Calcd for C17H13Cl2BrN2O2: C, 47.69;
H, 3.06 and N, 6.54; found: C, 47.64; H, 3.10 and N, 6.57. ESI-
MS m/z calculated for C17H13Cl2BrN2O2 [M]þ 428.10 and
430.10; found: 428.81 and 430.84.
5,6-Dichloro-2-(2-bromobenzyl)-1H-benzimidazole
Yield (90%); m.p. 228–229 ꢁC; IR (ꢀmax, cmꢂ1): 3422 (NH),
(2a).
1
1629 (C¼N); H-NMR (DMSO-d6, 200 MHz) ꢁ (ppm): 4.31 (s,
2H, CH2), 7.28–7.77 (m, 6H, Ar–H) and 12.63 (s, 1H, NH);
13C-NMR (DMSO-d6, 50 MHz) ꢁ (ppm): 32.65 (CH2), 119.62,
123.79, 124.26, 127.21, 128.77, 129.10, 131.50, 133.23, 134.58
(Ar–C) and 155.24 (C¼N). Anal. Calcd for C14H9Cl2BrN2: C,
47.23; H, 2.55 and N, 7.87; found: C, 47.20; H, 2.56 and N, 7.89.
ESI-MS m/z calculated for C14H9Cl2BrN2 [M]þ 356.10 and
358.10; found: 356.70 and 358.65.
5,6-Dichloro-2-(3-bromobenzyl)-1H-benzimidazole
(2b).
Yield (91%); m.p. 208–209 ꢁC; IR (ꢀmax, cmꢂ1): 3200 (NH),
1627 (C¼N); 1H-NMR (DMSO-d6, 200 MHz) ꢁ (ppm): 4.21 (s, 2H,
CH2), 7.10–7.95 (m, 6H, Ar–H) and 12.64 (s, 1H, NH); 13C-NMR
(DMSO-d6, 50 MHz) ꢁ (ppm): 34.89 (CH2), 113.47, 120.32, 122.39
(2C), 124.48 (2C), 128.76, 130.30, 131.37, 132.32, 132.83, 140.39
(Ar–C) and 156.61 (C¼N). Anal. Calcd for C14H9Cl2BrN2: C,
47.23; H, 2.55 and N, 7.87; found: C, 47.21; H, 2.59 and N, 7.85.
ESI-MS m/z calculated for C14H9Cl2BrN2 [MþH]þ 356.10 and
358.10; found: 356.78 and 358.67.
General procedure for the synthesis of 2-[5,6-dichloro-2-(sub-
stitutedbenzyl)-1H-benzimidazole-1-yl] acetohydrazide (4a–c)
A mixture of compounds 3a–c (0.01 mol) and hydrazine hydrate
(0.025 mol) in absolute ethanol (10 mL) was irradiated in closed
vessels with the pressure control at 120 ꢁC for 5 min (hold time) at
300 W maximum power. After the completion of the reaction,
(monitored by TLC, ethylacetate:hexane, 3:1), the mixture was
cooled to room temperature. The precipitate formed was
filtered, washed with excess ethanol and dried over CaCl2 to
give pure 4a–c.
5,6-Dichloro-2-(4-bromobenzyl)-1H-benzimidazole
(2c).
Yield (95%); m.p. 234–235 ꢁC; IR (ꢀmax, cmꢂ1): 3208 (NH),
1
1579 (C¼N); H-NMR (DMSO-d6, 200 MHz) ꢁ (ppm): 4.10 (s,
2-[5,6-Dichloro-2-(2-bromobenzyl)-1H-benzimidazole-1-yl]
acetohydrazide (4a). Yield (80%); m.p. 248–249 ꢁC; IR (ꢀmax
,
2H, CH2), 7.10–7.71 (m, 6H, Ar–H) and 12.64 (s, 1H, NH);
13C-NMR (DMSO-d6, 50 MHz) ꢁ (ppm): 34.77 (CH2), 113.25,
120.20, 120.61, 124.58, 131.84 (2C), 132.07 (2C), 134.69,
137.09 (2C), 143.84 (Ar–C) and 156.74 (C ¼ N). Anal. Calcd
for C14H9Cl2BrN2: C, 47.23; H, 2.55 and N, 7.87; found: C,
47.22; H, 2.58 and N, 7.87. ESI-MS m/z calculated for
C14H9Cl2BrN2 [MþH]þ 357.10 and 359.10; found: 357.78
and 359.10.
cmꢂ1): 3331, 3302 (NH2), 3173 (NH), 1674 (C¼O) and 1588
(C¼N); 1H-NMR (DMSO-d6, 200 MHz) ꢁ (ppm): 4.33 (s, 2H,
CH2), 4.46 (s, 2H, NH2), 4.83 (s, 2H, N–CH2), 7.17–7.82 (m, 6H,
Ar–H) and 9.51 (s, 1H, –NH); 13C-NMR (DMSO-d6, 50 MHz) ꢁ
(ppm): 31.63 (CH2), 45.34 (N–CH2), 112.56, 120.42, 124.49,
125.01, 127.87, 129.34, 129.83, 132.31, 134.10, 134.87, 135.91,
142.32 (Ar–C), 156.58 (C¼N) and 166.19 (C¼O). Anal. Calcd for

DOI: 10.3109/14756366.2012.753880
Synthesis of new benzimidazole derivatives 67
Figure 1. Dose-dependent inhibitory effect of
the compounds 3b, 3c, 5a and 6a. Orlistat
was used positive control. All compounds
and Orlistat were measured at final concen-
trations of 0.625, 1.25 and 6.25 mg/mL.
Residual activities of compounds are
5a
3b
3c
6a
100
90
80
70
60
50
40
30
20
10
0
Orlistat
expressed as the mean ꢃ SD in triplicate.
0
1
2
3
4
5
6
µg/mL
C16H13Cl2BrN4O1: C, 44.89; H, 3.06 and N, 13.09; found: C, AcOEt:hexane, 3:1), the mixture was poured onto H2O. The
44.86; H, 3.07 and N, 13.10. ESI-MS m/z calculated for precipitate formed was filtered and recrystallized from EtOH:H2O
C16H13Cl2BrN4O1 [MþH]þ 429.10 and 431.10; found: 429.89 (1:3) to give pure compounds, 5a.
and 431.92.
2,20-Bis(4-bromobenzyl)-1H,10H-5,50-bibenzimidazole (5a).
2-[5,6-Dichloro-2-(3-bromobenzyl)-1H-benzimidazole-1-yl] Yield (90%); m.p. 312–313 ꢁC; IR (ꢀmax, cmꢂ1): 3335 (NH),
1
acetohydrazide (4b). Yield (83%); m.p. 231–232 ꢁC; IR (ꢀmax
,
1569 (C¼N); H-NMR (DMSO-d6, 200 MHz) ꢁ (ppm): 4.30 (s,
cmꢂ1): 3304, 3150 (NHNH2), 1658 (C¼O) and 1550 (C¼N); 1H- 4H, 2CH2), 7.19–7.56 (m, 14H, Ar–H) and 12.33 (s, 2H, 2NH);
NMR (DMSO-d6, 200 MHz) ꢁ (ppm): 4.27 (s, 2H, CH2), 4.35 (s, 13C-NMR (DMSO-d6, 50 MHz) ꢁ (ppm): 34.69 (2CH2), 120.44,
2H, NH2), 4.91 (s, 2H, N–CH2), 7.26–7.86 (m, 6H, Arom-H) and 121.91, 130.98, 131.15, 131.79, 132.06, 132.15, 135.99, 137.68
9.53 (s, 1H, NH); 13C-NMR (DMSO-d6, 50 MHz) ꢁ (ppm): 32.72 (Ar–C) and 154.34 (2C¼N). Anal. Calcd for C28H20Br2N4:
(CH2), 45.23 (N–CH2), 112.50, 120.14, 120.29, 122.07, 124.61, C, 58.76; H, 3.52 and N, 9.79; found: C, 58.75; H, 3.54 and N,
124.90, 128.79, 130.04, 130.98, 132.30, 135.69, 139.49, 139.60, 9.80. ESI-MS m/z calculated C28H20Br2N4 [M]þ 572.29 and
142.16 (Ar–C), 156.89 (C¼N) and 166.00 (C¼O). Anal. Calcd for 574.26; found: 572.33, 574.16 and symmetric division
C16H13Cl2BrN4O1: C, 44.89; H, 3.06 and N, 13.09; found: C, signal 286.14.
44.86; H, 3.08 and N, 13.11. ESI-MS m/z calculated for
C16H13Cl2BrN4O1 [MþH]þ 429.10 and 431.10; found: 429.02
General procedure for the synthesis of dimethyl 2,20-[2,20-
bis(4-bromobenzyl)-1H,10H-5,50-bibenzimidazole-1,10-diyl]diace-
and 430.98.
2-[5,6-Dichloro-2-(4-bromobenzyl)-1H-benzimidazole-1-yl]
tate (6a)
acetohydrazide (4c). Yield (85%); m.p. 264-265 ꢁC; IR (ꢀmax
,
cmꢂ1): 3310, 3190 (NHNH2), 1678 (C¼O), and 1596 (C¼N);
1H-NMR (DMSO-d6, 200 MHz) ꢁ (ppm): 4.23 (s, 2H, CH2), 4.35
(s, 2H, NH2), 4.88 (s, 2H, N–CH2), 7.26 (d, 2H, Ar–H,
J ¼ 8.4 Hz), 7.51 (d, 2H, Ar–H, J ¼ 8.4 Hz), 7.84 (s, 2H, Ar–H)
and 9.50 (s, 1H, NH); 13C-NMR (DMSO-d6, 50 MHz) ꢁ (ppm):
32.64 (CH2), 45.19 (N–CH2), 112.48, 120.24, 120.34, 124.55,
124.85, 131.74 (2C), 131.86, 135.74, 136.13, 142.14 (Ar–C),
157.00 (C¼N) and 165.97 (C¼O). Anal. Calcd for
C16H13Cl2BrN4O1: C, 44.89; H, 3.06 and N, 13.09; found: C,
44.85; H, 3.04 and N, 13.12. ESI-MS m/z calculated for
C16H13Cl2BrN4O1 [M]þ 428.11 and 430.11; found: 428.81
and 430.91.
A mixture of compounds 5a (0.01 mol), methyl-a-bromoacetate
(0.02 mol) and K2CO3 (0.05 mol) in acetone (15 mL) was
irradiated in closed vessels with the pressure control at 85 ꢁC
for 20 min (hold time) at 300 W maximum power. After the
completion of the reaction, (monitored by TLC, ethylacetate:hex-
ane, 3:1), the mixture was poured onto water. The precipitate
formed was filtered and recrystallized from acetone:water (1:3) to
give pure 6a.
Yield (85%); m.p. 104–105 ꢁC; IR (ꢀmax, cmꢂ1): 1740
(C¼O), 1622 (C¼N), 1215, and 1180 (C–O); 1H-NMR
(DMSO-d6, 200 MHz) ꢁ (ppm): 3.34 (s, 6H, 2OCH3), 4.22 (s,
4H, 2CH2), 5.25 (s, 4H, 2N–CH2) and 7.11–7.95 (m, 14H, Ar–
H); 13C-NMR (DMSO-d6, 50 MHz) ꢁ (ppm): 34.96 (2CH2),
47.34 (2N–CH2), 55.11 (2OCH3), 113.29, 119.77, 121.61,
122.63, 124.46, 124.72, 134.13, 138.25, 138.78, 139.21,
144.11, 145.63 (Ar–C), 156.91 (2C¼N) and 171.23 (2C¼O).
General procedure for the synthesis of 2,20-bis(4-bromobenzyl)-
1H,10H-5,50-bibenzimidazole under microwave irradiation (5a)
A
mixture of 3,30-diaminobenzidine (0.010 mol) and 1c Anal. Calcd for C34H28Br2N4O4: C, 57.00; H, 3.94 and N, 7.82;
(0.026 mol) in dry MeOH (15 mL) was irradiated with found: C, 57.05; H, 3.90 and N, 7.81. ESI-MS m/z calculated
microwave at 60 ꢁC for 10 min at 300 W maximum power. After C34H28Br2N4O4 [M]þ 716.42 and 718.41; found: 716.56, 718.71
the completion of the reaction, (monitored by TLC, and symmetric division signal 358.14.

68 E. Mentes¸e et al.
J Enzyme Inhib Med Chem, 2014; 29(1): 64–68
General procedure for the synthesis of 2,20-[2,20-bis(4-
bromobenzyl)-1H,10H-5,50-bibenzimidazole-1,10-diyl]diacetohy-
drazide (7a)
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A mixture of compounds 6a (0.01 mol) and hydrazine hydrate
(0.05 mol) in absolute ethanol (10 mL) was irradiated in closed
vessels with the pressure control at 125 ꢁC for 13 min (hold time)
at 300 W maximum power. After the completion of the reaction,
(monitored by TLC, ethylacetate:hexane, 3:1), the mixture was
cooled to room temperature. The precipitate formed was filtered,
washed with excess ethanol and dried over CaCl2 to give pure 7a.
Yield (80%); m.p. 185–186 ꢁC; IR (ꢀmax, cmꢂ1): 3292, 3200
(NHNH2), 1658 (C¼O), 15 621 (C¼N); 1H-NMR (DMSO-d6,
200 MHz) ꢁ (ppm): 4.25 (s, 4H, 2CH2), 4.45 (bs, 4H, 2NH2), 4.85
(s, 4H, 2N–CH2), 7.16–7.85 (m, 14H, Ar–H) and 9.60 (s, 2H,
2NH); 13C-NMR (DMSO-d6, 50 MHz) ꢁ (ppm): 36.20 (2CH2),
45.33 (2N–CH2), 109.27, 110.57, 121.25, 122.33, 124.16, 132.13,
132.28, 136.62, 138.10, 139.21, 141.96, 144.03 (Ar–C), 155.34
(2C¼N) and 166.33 (2C ¼ O). Anal. Calcd for C32H28Br2N8O2:
C, 53.65; H, 3.94 and N, 15.64; found: C, 53.63; H, 3.95 and N,
15.65. ESI-MS m/z calculated C32H28Br2N8O2 [M]þ 716.43 and
718.43; found: 716.60, 718.53 and symmetric division signal
358.23.
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Anti-lipase activity
The inhibitory effects of those compounds were evaluated against
PPL (15 ng/mL). Lipase activity assay were done according to
Verger and Chahinian26. Microtiter plates were coated with
purified tung oil TAGs. Compounds were mixed with PPL 1:2 (v/
v) and incubated for 30 min. The microtiter plates containing
purified tung oil, lipase solution and assay buffer (10 mM Tris-
HCl buffer, pH 8.0, containing 150 mM NaCl, 6 mM CaCl2, 1 mM
EDTA and 3 mg/mL b-cyclodextrin) were recorded continuously
for 40 min against the buffer alone using microplate reader
(SpectraMax M5, Molecular Devices) at 272 nm. The inhibitory
activities of those compounds and Orlistat, a positive control
against pancreatic lipase, were measured at concentrations of
6.25, 1.25 and 0.625 mg/mL. Residual activities were calculated
by comparing to control without inhibitor (Tþ). The assays were
done in triplicate. The IC50 value was determined as the
concentration of compound that give 50% inhibition of maximal
activity.
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Conclusion
In conclusion, we have developed a novel and practical method
for the synthesis of benzimidazole derivatives under microwave
irradiation. This method can provide a convenient way of
synthesizing potential bioactive benzimidazoles. This is the first
example of benzimidazoles with their inhibitory properties toward
pancreatic lipase. The results could be an inspiration for further
investigation of potential lipase inhibitors within heterocycles.
_
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their benzoderivatives. Compr Heterocyc Chem 1984;5:345–72.
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benzimidazoles in high-temperature water. Green Chem
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substituted benzimidazoles using PPA as a catalyst under solvent-
free conditions. Synthetic Commun 2002;32:3703–9.
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conventional and microwave synthesis of some benzimidazole,
benzothiazole and indole derivatives and testing on inhibition of
hyaluronidase. Molecules 2008;13:736–48.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
23. Hunger A, Kebrle J, Rossi A, Hoffmann K. Benzimidazol-derivate
und verwandte heterocyclen VI. Synthese von phenyl-[1-ami-
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