Synthesis, crystal studies, anti-tuberculosis and cytotoxic studies of 1-[(2E)-3-phenylprop-2-enoyl]-1H-benzimidazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.04.017

Series of 1-[(2E)-3-phenylprop-2-enoyl]-1H-benzimidazole derivatives were synthesized and characterized by spectral methods. Among 21 derivatives, single crystals of 3a and 3l were grown and their structural parameters were evaluated. Newly synthesized compounds were screened for anti-tubercular activity and the MIC was determined against Mycobacterium tuberculosis H37Rv by Microplate Alamar Blue Assay (MABA) method. Majority of the compounds exhibited a promising inhibition of M. tuberculosis and the molecules functionalized with electron-donating groups at C-2 carbon of benzimidazole moiety were found to be more active in inhibiting M. tuberculosis. Further, more promising compounds viz., 3b, 3i and 3l were tested for their cytotoxic activity. Compound 3l was found to display excellent activity (IC₅₀ < 10 μg mL ^-1) with 100% cell lysis at 30 μg mL^-1 concentration against A549 (Human lung carcinoma) and 8E5 (Human; Acute Lymphoblastic Leukemia) cell lines.

Full text of DOI:10.1016/j.ejmech.2014.04.017

European Journal of Medicinal Chemistry 79 (2014) 194e202
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, crystal studies, anti-tuberculosis and cytotoxic studies of
1-[(2E)-3-phenylprop-2-enoyl]-1H-benzimidazole derivatives
,
a
Veerendra Kumar A. Kalalbandi a, J. Seetharamappa a , Umesha Katrahalli ,
*
Kishore G. Bhat b
a Department of Chemistry, Karnatak University, Dharwad 580 003, India
b Department of Microbiology, Maratha Mandal’s NGH Institute of Dental Sciences and Research Centre, Belgaum 590 010, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of 1-[(2E)-3-phenylprop-2-enoyl]-1H-benzimidazole derivatives were synthesized and charac-
terized by spectral methods. Among 21 derivatives, single crystals of 3a and 3l were grown and their
structural parameters were evaluated. Newly synthesized compounds were screened for anti-tubercular
activity and the MIC was determined against Mycobacterium tuberculosis H37Rv by Microplate Alamar
Received 15 July 2013
Received in revised form
1 April 2014
Accepted 4 April 2014
Available online 5 April 2014
Blue Assay (MABA) method. Majority of the compounds exhibited
a promising inhibition of
M. tuberculosis and the molecules functionalized with electron-donating groups at C-2 carbon of benz-
imidazole moiety were found to be more active in inhibiting M. tuberculosis. Further, more promising
compounds viz., 3b, 3i and 3l were tested for their cytotoxic activity. Compound 3l was found to display
Keywords:
1H-benzimidazoles
Anti-tuberculosis
Cytotoxic activity
Crystal structure
excellent activity (IC50 < 10
(Human lung carcinoma) and 8E5 (Human; Acute Lymphoblastic Leukemia) cell lines.
Ó 2014 Elsevier Masson SAS. All rights reserved.
m m
g mLꢀ1) with 100% cell lysis at 30 g mLꢀ1 concentration against A549
1. Introduction
the direction of benzimidazole derivatives, as these compounds
exhibit a wide spectrum of biological activities including antitu-
berculous activity [10]. Specifically, this nucleus is a constituent of
vitamin-B12 and many currently existing medications [11]. Almost
all benzimidazoles with different heterocyclic substituents led to
essential modification in their physico-chemical, metabolic and
pharmacokinetic properties [12].
Tuberculosis (TB) is an aerial contagious disease caused by
species found in Mycobacterium tuberculosis complex that includes
M. tuberculosis (Mtb). The number of cases of MDR-TB have been
increased in 27 high burden countries in 2011 and 20% of previously
treated cases were estimated to have MDR-TB [1]. About 9 million
new cases are estimated each year with almost two million death
tolls [2,3]. Thus, tuberculosis becomes a significant threat to global
health. So, the novel therapeutics are necessary to treat both drug-
susceptible TB and progressively common drug resistant strains
since, no new chemical entities are emerged in the past 4 decades
for the treatment of TB [4e8].
It is known that most of the currently existing tubercular
medications are constituted by the group of nitrogen heterocyclic
compounds such as isoniazid, pyrazinamide etc. Further, most of
them are derived from pyridine and pyrazines [9]. In an attempt to
look for better bioactive heterocyclic compounds containing ni-
trogen hetero atom (since most of antituberculosis compounds are
based on either pyridine or pyrazines), our consideration curved in
Only a few reports are available in the literature on antituber-
culous activity of benzimidazoles [13,14]. Further, literature survey
revealed that most of the first-line anti-tuberculous drugs were
constituted by amide linker (shown in Fig. 1.). However, remarkable
antitumor/antiproliferative/anticancer [15e18] activity of 1-
substituted benzimidazole derivatives prompted us to carry out
the cytotoxic activity. Taking into account of the functional group
similarity of the amide linkage [19,20], the structural similarity of
the pyridopyrazine moiety, albendazole and thiabendazole [21,22]
and with the aim of obtaining pharmacologically active com-
pounds, we have envisioned that the benzimidazole scaffold could
be a good starting point for the development of good Mtb in-
hibitors. In the present work, we report the synthesis of a new class
of 1H-benzimidazole derivatives. Single crystals of some of the
synthesized compounds were developed to elucidate the structural
properties. Finally, the synthesized compounds were tested for
their antitubercular activity against Mtb H37Rv strains. Further,
the cytotoxicity of potential 1-[(2E)-3-phenylprop-2-enoyl]-1H-
* Corresponding author.
E-mail addresses: jseetharam@yahoo.com, j_seetharamappa@rediffmail.com
(J. Seetharamappa).
http://dx.doi.org/10.1016/j.ejmech.2014.04.017
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

V.K.A. Kalalbandi et al. / European Journal of Medicinal Chemistry 79 (2014) 194e202
195
confirmed that, the condensation of two moieties occurred at 1
position of benzimidazole. The 1H NMR spectra of all compounds
showed two doublets each around
d 6.12e7.49 ppm and d 7.57e
8.09 ppm due to vinylic protons. The coupling constants of vinylic
protons (3JHH) at ca 16 Hz proved the E configuration of the double
bond in all compounds.
The 13C NMR of all the compounds exhibited signals around
d
128.9e133.4 ppm and
atoms. The carbonyl carbon appeared around
and the peaks appeared between 111.1e160.2 ppm were assigned
d
145.5e149.2 ppm due to vinylic carbon
d
164.4e168.1 ppm
d
to aromatic carbon atoms. The mass spectral analyses of all the
compounds have shown the m/z values which corresponds to their
respective molecular mass. Fragmentation peaks suggests that all
the molecules get fragmented as respective substituted benzimid-
azole and cinnamoyl group with m/z 131 in all the compounds.
HPLC was run on gradient mode to determine the purity of the
compounds. The compounds were found to be 97e99.8% pure and
eluted with retention time (tR) ranging from 13 to 19 min. The
purity of the compounds is reflected in NMR and IR spectra with
good and sharp peaks.
Fig. 1. Currently available anti-tubercular drugs containing amide linkage.
benzimidazole derivatives against two cell lines namely, A549
(Human lung carcinoma) and 8E5 (Human; Acute Lymphoblastic
Leukemia) was also investigated.
2.2. X-ray diffraction analysis
2. Results and discussion
The single crystals of compounds of 3a and 3l were developed
by slow evaporation of chloroform and alcohol respectively. Crystal
data were refined [25a,b] and their properties were studied. Com-
pounds, 3a and 3l were crystallized under monoclinic system with
space groups P21/n and P121/c1 respectively. The unit cell parame-
2.1. Chemistry
The key starting compounds, 2-aryl substituted benzimidazoles
were prepared by the condensation of aryl aldehyde with o-phe-
nylenediamine in the presence of p-TsOH [23]. Then, the 1-[(2E)-3-
phenylprop-2-enoyl]-1H-benzimidazole derivatives, portrayed in
Scheme 1, were prepared by slow addition of cinnamoyl chloride to
substituted benzimidazoles in dry THF in the presence of sodium
hydride at 0e5 ꢁC.
ꢀ
ters of the compound, 3a were as follows: a ¼ 13.466 (4) A,
b ¼ 4.9191 (13) A, c ¼ 19.168 (7) A,
¼ 90,
b
¼ 96.38(4),
g
¼ 90ꢁ,
ꢀ
ꢀ
a
Z ¼ 4 while the unit cell parameters of compound 3l were found to
ꢀ
ꢀ
ꢀ
a
be: a ¼ 10.9768 (5) A, b ¼ 13.3148 (6) (A), c ¼ 12.5164 (7) A,
¼ 90,
b
¼ 96.591 (4)ꢁ,
g
¼ 90ꢁ, Z ¼ 4.
The molecules of 3a were stabilized by weak intermolecular Ce
Nemoto et al. have [24] reported the synthesis of (E)-1-(1H-
benzo[d]imidazol-1-yl)-3-phenylprop-2-en-1-one (3a) by the re-
action of cinnamic acid and benzimidazole in the presence of
equimolar ratio of 1-ethyl-3-(dimethylaminopropyl)carbodimide
hydrochloride for 12 h at room temperature. However, this re-
ported method [24] suffered from disadvantages like high reaction
time, use of hazardous reagents and tedious product isolation
method. In view of this, we have developed a new practicable route
for the synthesis of (E)-1-(1H-benzo[d]imidazol-1-yl)-3-
phenylprop-2-en-1-one and its derivatives using sodium hydride
as a base at 0e5 ꢁC. In addition, the present route results in good
yield in about only 2 h. The optimized experimental conditions are
given in Table 1 for the synthesis of 1-[(2E)-3-phenylprop-2-enoyl]-
1H-benzimidazole derivatives.
The newly synthesized compounds were characterized by FTIR,
1H NMR, 13C NMR, Mass and elemental analysis data. The physical
and elemental analyses of all compounds are given in experimental
section. The spectral data of newly synthesized compounds are in
good agreement with proposed structures and are given in exper-
imental section.
H . N interactions in the solid state, while the molecules of 3l were
stabilized by weak intermolecular CeH . H. The crystal data and
structure refinement data of both 3a and 3l are given in
supplementary material. Fig. 2 represented the ORTEP diagrams of
3a and 3l.
The compound 3a was almost planar and the cinnamoyl group
did not make any significant dihedral angle with benzimidazole
moiety. But, when compared to crystal structure of 3a, compound
3l has a dihedral angle of 17.8 (3)ꢁ with cinnamoyl group due to
steric hindrance of bulkier 4-CH3-phenyl ring C16eC17 at C2 po-
sition of benzimidazole. Further, 4-CH3-phenyl ring C16eC17
formed a dihedral angle of 115.7 (2)ꢁ with the mean plane of the
nine membered benzimidazole ring system. Bond lengths and an-
gles of both 3a and 3l were observed to be in normal ranges. Crystal
information files (CIF) have been deposited at Cambridge Crystal-
lographic Data Centre. The CCDC numbers for 3a and 3l are CCDC
946546 and CCDC 946547, respectively.
2.3. Pharmacological evaluation
IR spectra of newly synthesized compounds showed sharp
bands w1650e1697 cmꢀ1 and 1619e1649 cmꢀ1 are due to the
amide carbonyl and alkenyl C]C stretching frequency, respectively.
The bands at w1340e1344 cmꢀ1 and 1511e1515 cmꢀ1 indicated the
presence of NO2 group in nitro substituted compounds (3d, 3e and
3n). Further, alkyl CeH stretching frequencies were observed at
2885e2928 cmꢀ1 in the cases of methyl substituents. The OeH
stretching frequencies were observed at 3292 and 3368 cmꢀ1 in
phenol derivatives (3k, 3m).
2.3.1. Anti-tubercular activity assay
In vitro antituberculosis activity of all the newly synthesized
compounds was investigated against Mycobacterium tuberculosis
H37Rv strain by microplate Alamar Blue assay (MABA) method and
the corresponding results are shown in Table 2 and Fig. 3. As
evident from the table, all the newly synthesized compounds
exhibited anti-tubercular activity, ranging from moderate to
excellent values, with a minimum inhibitory concentration (MIC)
range of 1.6e100.0
concentration (
g mLꢀ1) of the compound required to inhibit the
bacterial growth, completely. Compounds 3b, 3h, 3i and 3l showed
m
g mLꢀ1. The MIC is defined as the lowest
In 1H NMR spectrum of 3a, the presence of a singlet at
m
d
8.56 ppm and absence of amine proton of the title compound

196
V.K.A. Kalalbandi et al. / European Journal of Medicinal Chemistry 79 (2014) 194e202
R1
H
R2
H
R3
H
a
b
c
d
e
f
CH3
H
H
H
CH3
H
CH3
NO2
NO2
H
CH3
H
H
CH2Cl
H
g
h
i
H
H
CH3
H
C6H5
H
4-OCH3-C6H4
4-Cl-C6H4
2-OH-C6H4
4-CH3-C6H4
4-OH-C6H4
2-NO2-C6H4
4-NO2-C6H4
4-F-C6H4
3-Br-C6H4
3-Cl-C6H4
3-NO2-C6H4
2-Cl-C6H4
3,4-di-Cl-C6H4
H
H
j
H
H
k
l
H
H
H
H
m
n
o
p
q
r
H
H
H
H
H
H
H
H
H
H
H
H
s
H
H
t
H
H
u
H
H
Scheme 1. Synthetic route for the substituted 1-[(2E)-3-phenylprop-2-enoyl]-1H-benzimidazole.
excellent in vitro activity against H37Rv strain as compared to
pyrazinamide and streptomycin (MIC ¼ 3.12 and 6.25
g mLꢀ1
respectively), although compound 3j revealed the poorest activity
(MIC > 100
g mLꢀ1). From Table 2, no clear relation was observed
results in better antitubercular activity. Thus, the importance of the
amide and cinnamic double bond part was evident from these
biological results. However, the mechanism of action was still un-
known, but the presence of enoyl-acyl backbone to the benzimid-
azole moiety contributes more to become a potent antitubercular
agent along with the selective alkyl groups [30]. Further, extensive
SAR experiments on both the enzymatic target would be required
to establish unique modes of action. Furthermore, the compounds
viz., 3b, 3i, and 3l, exhibited potent cytotoxic effect against lung
carcinoma (A549 cells) and Leukemic cells (8E5 lymphocyte T-
human).
m
,
m
between MIC and the lipophilicity (calculated log P). However, the
physico-chemical properties seem to be one of the factors influ-
encing the antimycobacterial activity [26]. Further, the most active
compounds carry alkyl and alkoxy groups at the benzimidazole 2-
position or in the para-position of phenyl substituent. The 4-
methoxy derivative was observed to be less active compared to
that of 4-methyl derivative indicating that the volume of the sub-
stituent might play a significant role. Further, it was evident that
the methyl substituted compounds showed better activity
compared to that of the 2-chloromethyl and chloro-substituted
compounds. Thus, electron-donating substituents reinforced the
antitubercular activity of 1-substituted benzimidazole derivatives
[27,28]. De et al. [29a] have observed that the replacement of
double bond of the enoyl-acyl backbone by other group such as
isosteric cyclopropyl resulted in decreased activity. Lee et al. [29b]
have shown from SAR studies that the presence of amide linker
2.3.2. MTT assay for cell proliferation
The potent antitubercular compounds viz., 3b, 3i and 3l were
examined against two cancer cell lines (A549 and 8E5) for their cell
proliferation. The compounds showed IC50 ranging between <10
and >30 m
g mLꢀ1. It was evident from Table 3 that the 1-[(2E)-3-
phenylprop-2-enoyl]-1H-benzimidazole derivatives exhibited
good to moderate activity towards both cell lines. Among the tested
compounds, compound 3l displayed excellent cytotoxicity

V.K.A. Kalalbandi et al. / European Journal of Medicinal Chemistry 79 (2014) 194e202
197
Table 1
inhibit thioredoxin reductase through nucleophilic addition of
glutathione cystine-SH residues and can potentially be exploited
for use in different concentrations in chemotherapeutic and che-
mopreventive strategies. From a chemical point of view, the com-
pounds having an electron-donating group in the para-position of
the phenyl ring at C2 position of the benzimidazole can enhance
the activity. These results illustrated that the presence of amide and
olefin to the benzimidazole moiety exhibited better activity against
both cancerous cell lines.
Optimized conditions for production of 1-[(2E)-3-phenylprop-2-enoyl]-1H-
benzimidazole.
Entry
Base (equivalents)
Solvent
Time
Yield (%)
3a
3a
3a
3a
3a
3a
NaOH (1.2)
KOH (1.2)
NaOCH3 (1.2)
Piperidine (1.2)
Pyridine (1.2)
NaH (1.2)
Ethanol
Ethanol
Methanol
Methanol
Methanol
THF
8 h (Reflux)
8 h (Reflux)
8 h (Reflux)
8 h (Reflux)
8 h (Reflux)
2 h (RT)
10
15
18
24
21
82
Fig. 2. ORTEP diagrams of compounds 3a and 3l.
(IC50 < 10
m
g mLꢀ1) with 100% cell lysis at 30
m
g mLꢀ1 concentra-
-unsatu-
3. Conclusions
tion. E. H. Chew et al. [31] have recently showed that a, b
rated carbonyl moiety, also known as the Michael acceptor can
An efficient, simple and viable method for the synthesis of 1-
[(2E)-3-phenylprop-2-enoyl]-1H-benzimidazole derivatives with
yields ranging 65e85% was reported. All the derivatives were
characterized by FT-IR, 1H NMR, 13C NMR and Mass spectroscopic
data. Single crystals of molecules 3a and 3l were developed and
their crystal parameters were evaluated. All the newly synthesized
compounds were screened for their anti-tubercular activity. Cyto-
toxicity activity of the more promising tuberculosis inhibitor was
investigated against 2 cancerous cell lines, A549 and 8E5.
Table 2
Anti-tubercular activities and log
P measurements of 1-[(2E)-3-phenylprop-2-
enoyl]-1H-benzimidazole derivatives.
Compound
R1
R2
R3
MIC
m
g mLꢀ1
log Pa
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
H
CH3
H
CH3
H
CH2Cl
H
C6H5
4-OCH3-C6H4
4-Cl-C6H4
2-OH-C6H4
4-CH3-C6H4
4-OH-C6H4
2-NO2-C6H4
4-NO2-C6H4
4-F-C6H4
3-Br-C6H4
3-Cl-C6H4
3-NO2-C6H4
2-Cl-C6H4
3,4-di-Cl-C6H4
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
50
3.12
50
25
100
25
3.73
3.81
4.53
2.92
2.83
4.41
4.15
5.84
5.90
6.52
5.58
6.29
5.36
4.92
5.80
6.01
6.63
6.50
5.78
6.47
7.13
L0.71
L5.35
2.62
CH3
NO2
NO2
H
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
4. Experimental protocol
4.1. Chemistry
50
6.25
3.12
>100
25
1.6
12.5
12.5
50
25
50
12.5
50
50
All chemicals and solvents were purchased from Spectrochem,
sd-fine Chemicals and HiMedia (India) and used without further
purification. Melting points were determined in open capillary
tubes on a melting point apparatus of Concord Instruments (P) Ltd.,
Bangalore, and are reported as uncorrected values. Carbon,
hydrogen and nitrogen were estimated by using CHN analyzer. 1H
NMR and 13C NMR spectra were recorded on a Bruker DPX-
500 MHz/Bruker Avance DPX-300 MHz/Bruker Avance FT 400 MHz
3q
3r
3s
3t
NMR spectrometer in DMSO-d6/CDCl3. Chemical shifts (d) are given
in ppm relative to TMS and coupling constants (J) are expressed in
Hz. Mass spectra were recorded on a Shimadzu spectrometer model
QP2010 S and IR spectra were recorded on a Bruker Alpha-T FT-IR
spectrophotometer using KBr optics. High resolution X-ray
diffraction data were collected on Xcalibur, Eos, Gemini diffrac-
tometer and Bruker SMART APEX II diffractometer. The purity of the
compounds was determined on Agilent 1200 Series HPLC
3u
H
25
3.12
6.25
0.12
Pyrazinamide
Sreptomycin
Rifampicin
Results shown in bold letters indicate more antituberculosis activity of compounds
compared to others.
a
Calculated by http://www.molinspiration.com/.

198
V.K.A. Kalalbandi et al. / European Journal of Medicinal Chemistry 79 (2014) 194e202
Fig. 3. Antitubercular activity of synthesized compounds 3aeu against Mtb H37Rv strains.
instrument. The anti-tubercular and cytotoxicity activity evaluation
was carried out at the Department of Microbiology and Immu-
nology, NGH College of Dental sciences, Belagavi, Karnataka, India.
solution was prepared by dissolving the sample (mg mLꢀ1) in
methanol; MeCN (1:1 v/v) diluent. The injection volume was 5 mL
and the column temperature was maintained at 25 ꢁC. The flow rate
of 1.0 mL minꢀ1 was maintained throughout the run. The run time
was typically 30 min. The UV detector was set at a wavelength
range of 280e320 nm.
4.1.1. General procedure for the synthesis of 2-aryl substituted
benzimidazoles (2heu)
The series of these derivatives was synthesized using the pro-
cedure reported in the literature [23] and characterized by 1H NMR.
The melting points of these compounds were also compared with
the reported values [32].
4.1.3.1. (E)-1-(1H-benzo[d]imidazol-1-yl)-3-phenylprop-2-en-1-one
(3a). Yield: 86%; m.p: 232e234 ꢁC; IR (KBr): 1650 (C]O),1620 (C]
C) cmꢀ1; 1H NMR (CDCl3, 400 MHz,
d
ppm): 7.22 (d, 1H, J ¼ 15.2 Hz,
H-a
), 7.41 (t, 2H, J ¼ 7.2 Hz, C5, C6eH), 7.65 (t, 1H, J ¼ 7.4 Hz, C40-H),
7.81 (t, 2H, J ¼ 7.4 Hz, C30,C50-H), 7.83e7.94 (m, 4H, C4, C7,C20C60-H),
4.1.2. General method for the synthesis of 1-[(2E)-3-phenylprop-2-
enoyl]-1H-benzimidazole derivatives (3aeu)
8.09 (d, 1H, J ¼ 15.2 Hz, H-
b
), 8.56 (s, 1H, C2-H); 13C NMR (CDCl3,
d ppm): 114.12, 121.06, 123.86, 128.22, 128.94, 129.15,
Substituted benzimidazole (1 eq.) was added slowly at reduced
temperature to a solution of sodium hydride (1.1 eq.) in anhydrous
THF and the contents were stirred for 15e20 min. To this, a solution
of freshly prepared cinnamoyl chloride (1 eq.) in THF was added
dropwise at 0e5 ꢁC for about 30 min. The reaction was carried out
in an inert atmosphere. Then, the reaction mixture was allowed to
attain ambient temperature and the stirring was continued for 60e
90 min (monitored by TLC). After the completion of the reaction,
the volume of the THF was reduced to half in vacuo and poured to
crushed ice and the pH was adjusted to neutral. The separated solid
was filtered, washed with water, dried and finally purification was
achieved on silica gel (60e120 mesh) column chromatography
using ethyl acetate/hexane as the eluent.
100 MHz,
130.63, 130.94, 134.13, 140.21, 144.22, 147.94, 165.16; MS (m/z): 249
[(M þ H)þ, a], 248 (Mþ), 145, 131, 118, 103, 90, 77; Anal. Calcd. for
C16H12N2O (248.09): C, 77.40%; H, 4.87%; N, 11.28%; Found: C,
77.35%; H, 4.85%; N, 11.30%; HPLC purity: 97.05%, tR-13.7 min.
4.1.3.2. (E)-1-(2-methyl-1H-benzo[d]imidazol-1-yl)-3-phenylprop-2-
en-1-one (3b). Yield: 80%; m.p: 96e98 ꢁC; IR (KBr): 2928 (CH3),
1677 (C]O), 1632 (C]C) cmꢀ1 1H NMR (DMSO-d6, 300 MHz,
;
d
ppm): 2.64 (s, 3H, C2-CH3), 6.50 (d, 1H, J ¼ 15.9 Hz, H-
a), 6.99 (t,
2H, J ¼ 7.2 Hz C5, C6eH), 7.29 (t, 1H, J ¼ 7.4 Hz, C40-H), 7.38 (t, 2H,
J ¼ 7.4 Hz, C30,C50-H), 7.41e7.49 (m, 4H, C4, C7, C20, C60-H), 7.57 (d,
1H, J ¼ 15.9 Hz, H-
b d ppm): 14.94,
); 13C NMR (DMSO-d6, 75 MHz,
114.69, 118.93, 124.50, 127.07, 128.63, 129.18, 130.70, 131.79, 134.69,
139.15, 144.39, 147.55, 168.14; MS (m/z): 263 [(M þ H)þ, a], 262
(Mþ), 147, 131, 117, 103, 90, 77; Anal. Calcd. for C17H14N2O (262.11):
C, 77.84%; H, 5.38%; N, 10.68%; Found: C, 77.79%; H, 5.35%; N,
10.62%; HPLC purity: 98.67%, tR-14.2 min.
4.1.3. General method for the determination of purity of the
compounds by HPLC
The HPLC method was employed to determine the purity of
compounds on Eclipse XDB-C18 column (50 ꢂ 4.6 mm, 5
mM) using
the binary mobile phase consisting of 20 mM monopotassium
phosphate at pH 4.5 (eluent A) and MeCN (eluent B). The gradient
elution program was: 70% A and 30% B (0e15 min), 30% A and 70% B
(15e25 min), and 70% A and 30% B (25e30 min). The sample
4.1.3.3. (E)-1-(5,6-dimethyl-1H-benzo[d]imidazol-1-yl)-3-
phenylprop-2-en-1-one (3c). Yield: 85%; m.p: 218e220 ꢁC; IR (KBr):
2911 and 2885 (CH3), 1655 (C]O), 1623 (C]C) cmꢀ1 1H NMR
;
(DMSO-d6, 300 MHz,
d ppm): 2.48 (s, 6H, C5, C6-CH3), 6.90 (d, 1H,
J ¼ 15 Hz, H-
a
), 7.39e7.47 (m, 3H, C4, C7,C40-H), 7.53 (t, 2H,
Table 3
J ¼ 7.8 Hz, C30,C50-H), 7.59 (t, 2H, J ¼ 7.8 Hz, C20, C60-H), 7.79 (d, 1H,
IC50 values with reference to control and compound concentration.
J ¼ 15 Hz, H-
b
), 8.07 (s, 1H, C2-H); 13C NMR (DMSO-d6, 75 MHz,
ppm): 19.59, 116.29, 128.24, 128.60, 129.48, 129.64, 130.22, 130.46,
Entry
IC50 (mg/mL)
d
134.63, 135.26, 142.97, 147. 61, 164.39; MS (m/z): 277 [(M þ H)þ, a],
276 (Mþ), 146, 131, 116, 103, 77; Anal. Calcd. for C18H16N2O (276.33):
C, 78.24%; H, 5.84%; N,10.14%; Found: C, 77.85%; H, 5.85%; N,10.25%;
HPLC purity: 99.26%, tR-14.8 min.
A549
8E5
3b
3i
>30
30
>30
20
3l
<10
<10

V.K.A. Kalalbandi et al. / European Journal of Medicinal Chemistry 79 (2014) 194e202
199
4.1.3.4. (E)-1-(2-methyl-6-nitro-1H-benzo[d]imidazol-1-yl)-3-
phenylprop-2-en-1-one (3d). Yield: 59%; m.p: 204e206 ꢁC; IR
(KBr): 2920 (CH3), 1696 (C]O), 1608 (C]C), 1514 and 1344 (NO2)
4.1.3.9. (E)-1-(2-(4-methoxyphenyl)-1H-benzo[d]imidazol-1-yl)-3-
phenylprop-2-en-1-one (3i). Yield: 78%; m.p: 98e100 ꢁC; IR (KBr):
2918 (CH3), 1681 (C]O), 1621 (C]C) cmꢀ1 1H NMR (CDCl3,
;
cmꢀ1; 1H NMR (DMSO-d6, 500 MHz,
d
ppm): 2.86 (s, 3H, C2-CH3),
400 MHz,
H-
d
ppm): 3.60 (s, 3H, O-CH3 of Ph), 6.12 (d, 1H, J ¼ 15.2 Hz,
7.49 (d, 1H, J ¼ 16 Hz, H-
a
), 7.69 (t, 1H, J ¼ 7.4 Hz, C40-H), 7.76 (t, 2H,
a
), 6.83 (d, 2H, J ¼ 8.8 Hz, C3,C5-H of Ph), 7.09 (t, 2H, J ¼ 7.4 Hz,
J ¼ 7.4 Hz, C30,C50-H), 7.82e7.92 (m, 3H, C4, C20, C60-H) 8.01 (d, 1H,
C5, C6-H of Benzimidazole), 7.16 (t,1H, J ¼ 7.2 Hz, C40-H), 7.43 (t, 2H,
J ¼ 16 Hz, H-
b
), 8.16 (d, 1H, J ¼ 7.6 Hz, C5-H), 8.23 (s, 1H, C7-H); 13
C
J ¼ 7.2 Hz, C30,C50-H), 7.44e7.52 (m, 4H, AreH), 7.75 (d, 1H,
NMR (DMSO-d6, 125 MHz,
d
ppm): 14.80, 112.02, 116.82, 119.08,
J ¼ 15.2 Hz, H-
b
), 8.52 (d, 2H J ¼ 8.0 Hz, C2,C6-H of Ph); 13C NMR
128.53, 129.15, 129.32, 131.12, 132.56, 135.86, 142.16, 144.36, 145.67,
147.14, 166.06; MS (m/z): 307 (Mþ), 147, 131, 120, 103, 91, 77; Anal.
Calcd. for C17H13N3O3 (307.30): C, 66.44%; H, 4.26%; N, 13.67%;
Found: C, 66.36%; H, 4.25%; N, 13.67%; HPLC purity: 98.97%, tR-
14.1 min.
(CDCl3, 100 MHz,
d ppm): 54.86, 114.11, 114.92, 115.31, 122.96,
123.12,128.23,128.64,128.88,130.46,130.52,130.86,135.34,138.93,
141.87, 147.63, 160.82, 165.63; MS (m/z): 357 (Mþ3), 227, 147, 131,
121, 103, 91, 78; Anal. Calcd. for C23H18N2O2 (354.40): C, 77.95%; H,
5.12%; N, 7.90%; Found: C, 77.89%; H, 5.06%; N, 7.88%; HPLC purity:
98.76%, tR-18.8 min.
4.1.3.5. (E)-1-(6-nitro-1H-benzo[d]imidazol-1-yl)-3-phenylprop-2-
en-1-one (3e). Yield: 65%; m.p: 110e112 ꢁC; IR (KBr): 1682 (C]O),
1619 (C]C), 1515 and 1342 (NO2) cmꢀ1; 1H NMR (CDCl3, 400 MHz,
4.1.3.10. (E)-1-(2-(4-chlorophenyl)-1H-benzo[d]imidazol-1-yl)-3-
phenylprop-2-en-1-one (3j). Yield: 84%; m.p: 116e118 ꢁC; IR (KBr):
1686 (C]O), 1614 (C]C) cmꢀ1; 1H NMR (CDCl3, 400 MHz,
d ppm):
d
ppm): 7.38 (d, 1H, J ¼ 15.2 Hz, H-
a
), 7.46 (t, 1H, J ¼ 7.4 Hz, C40-H),
7.52 (t, 2H, J ¼ 7.4 Hz, C30,C50-H), 7.66e7.78 (m, 3H, C4, C20, C60-H),
6.41 (d, 1H, J ¼ 16 Hz, H- ), 7.16 (t, 2H, J ¼ 7.4 Hz, C5, C6-H of
a
Benzimidazole), 7.37 (t, 1H, J ¼ 7.2 Hz, C40H), 7.44 (t, 2H, J ¼ 7.2 Hz,
7.98 (d, 1H, J ¼ 15.2 Hz, H- ), 8.62 (s, 1H, 2-H), 8.71 (d, 1H, J ¼ 7.2 Hz,
b
C30,C50-H), 7.60e7.70 (m, 6H, AreH), 7.83 (d,1H, J ¼ 16 Hz, H-
b
), 8.11
C5-H), 8.79 (s, 1H, C7-H); 13C NMR (CDCl3, 100 MHz,
d ppm): 111.34,
(d, 2H, J ¼ 8.1 Hz, C2,C6-H of Ph); 13C NMR (CDCl3, 100 MHz,
d ppm):
116.82, 119.24, 127.46, 128.84, 130.64, 131.33, 135.21, 141.88, 144.13,
147.56, 149.49, 166.02; MS (m/z): 294 [(M þ H)þ, a], 165, 147, 131,
125, 103, 91, 77, 63; Anal. Calcd. for C16H11N3O3 (293.28): C, 65.53%;
H, 3.78%; N, 14.33%; Found: C, 65.46%; H, 3.77%; N, 14.33%; HPLC
purity: 98.47% tR-14.5 min.
114.53, 116.75, 125.02, 128.43, 128.60, 129.09, 129.12, 129.81, 130.99,
133.73, 134.29, 136.86, 142.68, 147.11, 165.32; MS (m/z): 358/360/
359 (Mþ, a), 228, 147, 131, 103, 90, 77; Anal. Calcd. for C22H15ClN2O
(358.82): C, 73.64%; H, 4.21%; N, 7.81%; Found: C, 73.55%; H, 4.20%;
N, 7.77%; HPLC purity: 99.41%, tR-19.2 min.
4.1.3.6. (E)-1-(2-(chloromethyl)-1H-benzo[d]imidazol-1-yl)-3-
4.1.3.11. (E)-1-(2-(2-hydroxyphenyl)-1H-benzo[d]imidazol-1-yl)-3-
phenylprop-2-en-1-one (3f). Yield: 63%; m.p: 150e152 ꢁC; IR (KBr):
phenylprop-2-en-1-one (3k). Yield: 56%; m.p: 198e200 ꢁC; IR
1678 (C]O), 1623 (C]C) cmꢀ1; 1H NMR (CDCl3, 400 MHz,
d
ppm):
(KBr): 3315 (OH), 1681 (C]O), 1633 (C]C) cmꢀ1 1H NMR (CDCl3,
;
3.72 (s, 2H, C2-CH2), 7.16 (d, 1H, J ¼ 15 Hz, H-
a), 7.24 (t, 2H,
400 MHz,
d
ppm): 6.73 (d, 1H, J ¼ 16 Hz, H- ), 6.91 (d, 1H, J ¼ 6.8 Hz,
a
J ¼ 7.2 Hz, C5, C6-H), 7.45 (t, 1H, J ¼ 7.4 Hz, C40-H), 7.56 (t, 2H,
J ¼ 7.4 Hz, C30,C50-H), 7.69e8.78 (m, 4H, C4, C7, C20, C60-H), 7.86 (d,
C3-H of Ph), 7.04 (t, 1H, J ¼ 7.0 Hz, C5-H of Ph), 7.31 (m, 3H, C4-H of
Ph, C5, C6-H of Benzimidazole), 7.38 (t, 1H, J ¼ 7.4 Hz, C40-H) 7.48 (t,
2H, J ¼ 7.4 Hz, C30,C50-H), 7.61e7.69 (m, 5H, AreH), 7.78 (d, 1H,
1H, J ¼ 15 Hz, H-
b d ppm): 35.80,113.97,
); 13C NMR (CDCl3,100 MHz,
115.06, 123.15, 127.29, 128.34, 128.47, 130.54, 130.89, 135.94, 139.23,
141.92, 147.63, 165.82; MS (m/z): 296/298 (Mþ, a), 165, 147, 131, 120,
103, 91, 77; Anal. Calcd. for C17H13ClN2O (296.75): C, 68.81%; H,
4.42%; N, 9.44%; Found: C, 68.71%; H, 4.38%; N, 9.41%; HPLC purity:
97.98%, tR-15.2 min.
J ¼ 16 Hz, H-
b
), 8.03 (s, 1H, eOH D2O exchangeable); 13C NMR
(CDCl3,100 MHz,
d
ppm): 13.50, 46.92,114.51, 115.36,123.10,128.06,
128.61, 128.83, 128.92, 130.43, 130.58, 130.76, 135.36, 139.13, 141.93,
147.33, 165.88; MS (m/z): 340/341 (Mþ, a), 209, 131, 103, 90, 77;
Anal. Calcd. for C22H16N2O2 (340.37): C, 77.63%; H, 4.74%; N, 8.23%;
Found: C, 77.56%; H, 4.76%; N, 8.21%; HPLC purity: 98.74%, tR-
17.7 min.
4.1.3.7. (E)-1-(6-methyl-1H-benzo[d]imidazol-1-yl)-3-phenylprop-2-
en-1-one (3g). Yield: 82%; m.p: 218e220 ꢁC (Dec); IR (KBr): 2920
(CH3), 1695 (C]O), 1622 (C]C) cmꢀ1 1H NMR (CDCl3, 500 MHz,
;
4.1.3.12. (E)-3-phenyl-1-(2-(p-tolyl)-1H-benzo[d]imidazol-1-yl)
prop-2-en-1-one (3l). Yield: 82%; m.p: 103e105 ꢁC; IR (KBr): 2921
d
ppm): 2.50 (s, 3H, C6-CH3), 7.20 (d,1H, J ¼ 15.5 Hz, H-
(m, 2H, C4,C40-H), 7.46e7.52 (m, 3H,C7,C30,C50-H), 8.05 (d, 1H,
J ¼ 15.5 Hz, H-
), 8.14e8.18 (m, 3H, C5, C20C60-H), 8.50 (s, 1H, C2-H);
13C NMR (CDCl3, 125 MHz,
ppm): 21.56, 115.82, 115.94, 126.49,
a), 7.25e7.32
(CH3), 1683 (C]O), 1621 (C]C) cmꢀ1
;
1H NMR (DMSO-d6,
b
500 MHz,
d
ppm): 2.37 (s, 3H, C2-CH3 of Ph), 6.54 (d, 1H, J ¼ 16 Hz,
H-
a
), 7.26e7.28 (m, 4H, AreH), 7.36 (t, 1H, J ¼ 7.6 Hz, C40-H), 7.42 (t,
d
2H, J ¼ 7.6 Hz, C30,C50-H), 7.64e7.69 (m, 4H, AreH), 7.76 (d, 1H,
128.60, 129.15, 129.87, 131.35, 133.90, 134.95, 140.01, 140.54, 148.51,
162.52; MS (m/z): 262/263 (Mþ, a), 146, 131, 103, 77; Anal. Calcd. for
J ¼ 16 Hz, H-
b
), 8.04 (d, 2H, J ¼ 7.2 Hz, C2,C6-H of Ph); 13C NMR
C
17H14N2O (262.31): C, 77.84%; H, 5.38%; N, 10.68%; Found: C,
(DMSO-d6, 125 MHz,
d ppm): 20.88, 114.08, 119.63, 121.03, 124.57,
77.80%; H, 5.31%; N, 10.65%; HPLC purity: 98.17% tR-15.5 min.
124.87, 128.19, 128.35, 128.91, 129.19, 129.59, 131.15, 133.75, 133.83,
140.30, 142.30, 145.48, 165.49; MS (m/z): 338/339 (Mþ, a), 207, 147,
131, 103, 77; Anal. Calcd. for C23H18N2O (338.40): C, 81.63%; H,
5.36%; N, 8.28%; Found: C, 81.55%; H, 5.32%; N, 8.25%; HPLC purity:
99.87%, tR-18.9 min.
4.1.3.8. (E)-3-phenyl-1-(2-phenyl-1H-benzo[d]imidazol-1-yl)prop-2-
en-1-one (3h). Yield: 72%; m.p: 130e132 ꢁC; IR (KBr): 1697 (C]O),
1630 (C]C) cmꢀ1 1H NMR (CDCl3, 500 MHz,
; d ppm): 6.85 (d, 1H,
J ¼ 16 Hz, H- ), 7.28 (t, 2H, J ¼ 7.4 Hz, C5, C6-H of Benzimidazole),
a
7.39 (t, 1H, J ¼ 7.2 Hz, C40-H)7.46e7.51 (m, 3H, C30,C50,C4 of Ph),
4.1.3.13. (E)-1-(2-(4-hydroxyphenyl)-1H-benzo[d]imidazol-1-yl)-3-
7.56e7.72 (m, 6H, AreH), 7.93 (d, 1H, J ¼ 16 Hz, H-
b
), 8.36 (d, 2H,
phenylprop-2-en-1-one (3m). Yield: 71%; m.p: 120e122 ꢁC; IR
J ¼ 8.4 Hz, C2,C6-H of Ph); 13C NMR (CDCl3, 125 MHz,
d
ppm):
(KBr): 3280 (OH), 1680 (C]O), 1649 (C]C) cmꢀ1 1H NMR (CDCl3,
;
114.23, 115.46, 124.41, 127.68, 127.98, 128.63, 128.82, 129.45, 130.43,
130.82, 130.96, 131.29, 135.43, 139.43, 142.01, 147.85, 167.94; MS (m/
z): 324/325 (Mþ, a), 193, 147, 131, 103, 77; Anal. Calcd. for C22H16N2O
(324.38): C, 81.46%; H, 4.97%; N, 8.64%; Found: C, 81.41%; H, 4.99%;
N, 8.61%; HPLC purity: 98.88%, tR-18.2 min.
400 MHz,
d
ppm): 6.62 (d, 1H, J ¼ 16 Hz, H- ), 6.84 (d, 2H, J ¼ 8.4 Hz,
a
C3,C5-H of Ph), 7.26 (t, 2H, J ¼ 7.4 Hz, C5, C6-H of Benzimidazole),
7.39 (t, 1H, J ¼ 7.6 Hz, C40-H), 7.51 (t, 2H, J ¼ 7.6, C30,C50-H), 7.60e
7.66 (m, 4H, AreH), 7.86 (d, 1H, J ¼ 16 Hz, H-
b), 7.92 (d, 2H,
J ¼ 7.8 Hz, C2,C6-H of Ph), 8.12 (s, 1H, eOH D2O exchangeable); 13
C

200
V.K.A. Kalalbandi et al. / European Journal of Medicinal Chemistry 79 (2014) 194e202
NMR (CDCl3,100 MHz,
d
ppm): 114.15,115.82,116.93,123.12,123.23,
1687 (C]O), 1619 (C]C) cmꢀ1; 1H NMR (CDCl3, 400 MHz,
d ppm):
128.03, 128.42, 128.56, 130.61, 130.83, 135.23, 138.93, 142.62, 147.45,
158.82, 165.33; MS (m/z): 340/341 (Mþ, a), 209, 131, 103, 90, 77;
Anal. Calcd. for C22H16N2O2 (340.37): C, 77.63%; H, 4.74%; N, 8.23%;
Found: C, 77.55%; H, 4.72%; N, 8.21%; HPLC purity: 98.55%, tR-
18.3 min.
6.73 (d, 1H, J ¼ 16 Hz, H- ), 7.28 (t, 2H, J ¼ 7.2 Hz, C5, C6-H of
a
Benzimidazole), 7.39 (t,1H, J ¼ 7.6 Hz, C40-H), 7.48e7.54 (m, 4H, Are
H), 7.62e7.71 (m, 4H, AreH), 7.88 (d, 1H, J ¼ 16 Hz, H-
b), 8.06 (s, 1H,
C2-H of Ph), 8.15 (d, 1H, J ¼ 6.8 Hz, C6-H of Ph); 13C NMR (CDCl3,
100 MHz,
d ppm): 114.15, 115.34, 123.55, 124.86, 126.48, 127.69,
128.59, 128.86, 129.08, 129.65, 130.65, 131.27, 132.67, 135.28, 136.12,
139.19,141.58,148.00,165.31; MS (m/z): 358 (Mþ), 228,147,131,103,
63; Anal. Calcd. for C22H15ClN2O (358.82): C, 73.64%; H, 4.21%; N,
7.81%; Found: C, 73.60%; H, 4.10%; N, 7.72%; HPLC purity: 98.89%, tR-
19.5 min.
4.1.3.14. (E)-1-(2-(2-nitrophenyl)-1H-benzo[d]imidazol-1-yl)-3-
phenylprop-2-en-1-one (3n). Yield: 59%; m.p: 168e170 ꢁC; IR (KBr):
1680 (C]O), 1625 (C]C), 1515 and 1340 (NO2) cmꢀ1
;
1H NMR
(CDCl3, 400 MHz,
d
ppm): 6.72 (d, 1H, J ¼ 16 Hz, H-
a), 7.14 (t, 2H,
J ¼ 7.3 Hz, C5, C6-H of Benzimidazole), 7.28 (t, 1H, J ¼ 7.6 Hz, C40-H),
7.40 (t, 2H, J ¼ 7.6, C30,C50-H), 7.59e7.67 (m, 5H, AreH), 7.94 (d, 1H,
4.1.3.19. (E)-1-(2-(3-nitrophenyl)-1H-benzo[d]imidazol-1-yl)-3-
J ¼ 16 Hz, H-
b
), 8.01 (t, 1H, J ¼ 6.4 Hz, C5-H of Ph), 8.12 (d, 1H,
phenylprop-2-en-1-one (3s). Yield: 91%; m.p: 100e102 ꢁC; IR (KBr):
J ¼ 6.7 Hz, C3-H of Ph), 8.20 (d, 1H, J ¼ 6.4 Hz, C6-H of Ph); 13C NMR
1671 (C]O), 1629 (C]C), 1511 and 1337 (NO2) cmꢀ1
;
1H NMR
(CDCl3, 100 MHz,
d
ppm): 114.23, 115.63, 123.08, 124.61, 126.43,
(CDCl3, 400 MHz,
d
ppm): 6.62 (d, 1H, J ¼ 16 Hz, H-
a), 7.35 (t, 2H,
128.03, 128.46, 128.73, 129.74, 130.43, 130.86, 133.45,135.88, 139.14,
142.63, 147.27, 150.23, 165.85; MS (m/z): 369/370 (Mþ, a), 238, 131,
121, 103, 91, 77, 63; Anal. Calcd. for C22H15N3O3 (369.37): C, 71.54%;
H, 4.09%; N, 11.38%; Found: C, 71.45%; H, 4.01%; N, 11.37%; HPLC
purity: 98.92%, tR-19.2 min.
J ¼ 7.2 Hz, C5, C6-H of Benzimidazole), 7.39 (t, 1H, J ¼ 7.4 Hz, C40-H)
7.46 (t, 2H, J ¼ 7.4, C30,C50-H), 7.52e7.66 (m, 5H, AreH), 7.71 (d, 1H,
J ¼ 16 Hz, H- ), 8.31 (d, 1H, J ¼ 6.8 Hz, C4-H of Ph), 8.69 (s, 1H, C2-H
b
of Ph), 8.73 (d,1H, J ¼ 6.9 Hz, C6-H of Ph); 13C NMR (CDCl3,100 MHz,
d
ppm):114.18, 115.23, 122.78, 123.23, 124.12, 127.95, 128.65, 128.76,
130.21, 130.87, 131.04, 131.69, 133.88, 135.20, 138.86, 141.55, 147.20,
148.45, 165.12; MS (m/z): 369/370 (Mþ, a), 238, 131, 121, 103, 90, 79,
62; Anal. Calcd. for C22H15N3O3 (369.37): C, 71.54%; H, 4.09%; N,
11.38%; Found: C, 71.42%; H, 4.01%; N, 11.41%; HPLC purity: 98.47%,
tR-19.8 min.
4.1.3.15. (E)-1-(2-(4-nitrophenyl)-1H-benzo[d]imidazol-1-yl)-3-
phenylprop-2-en-1-one (3o). Yield: 85%; m.p: 215e216 ꢁC; IR (KBr):
1684 (C]O), 1628 (C]C), 1522 and 1334 (NO2) cmꢀ1
;
1H NMR
(CDCl3, 400 MHz,
d
ppm): 6.63 (d, 1H, J ¼ 16 Hz, H-
a), 7.31 (t, 2H,
J ¼ 7.2 Hz, C5, C6-H of Benzimidazole), 7.38 (t, 1H, J ¼ 7.6 Hz, C40-H),
7.48 (t, 2H, 7.6 Hz, C30,C50-H), 7.49e7.51 (m, 4H, AreH), 7.96 (d, 1H,
4.1.3.20. (E)-1-(2-(2-chlorophenyl)-1H-benzo[d]imidazol-1-yl)-3-
J ¼ 16 Hz, H-
b
), 8.03 (d, 2H, J ¼ 6.8 Hz, C2,C6-H of Ph), 8.36 (d, 2H,
phenylprop-2-en-1-one (3t). Yield: 76%; m.p: 186e188 ꢁC; IR (KBr):
J ¼ 8.8 Hz, C3,C5-H of Ph); 13C NMR (CDCl3, 100 MHz,
d
ppm):
1680 (C]O), 1625 (C]C) cmꢀ1; 1H NMR (CDCl3, 400 MHz,
d
ppm):
114.09, 119.69, 120.82, 123.82, 125.22, 126.07, 128.57, 129.28, 130.63,
131.91, 133.41, 134.23, 137.31, 142.88, 148.60, 150.54, 165.06; MS (m/
z): 369/370 (Mþ, a), 238, 131, 121, 103, 90, 79, 62; Anal. Calcd. for
6.68 (d, 1H, J ¼ 16 Hz, H-
a
), 7.25 (t, 2H, J ¼ 7.2 Hz, C5, C6-H of
Benzimidazole), 7.36e7.40 (m, 3H, AreH), 7.46 (t, 2H, J ¼ 7.5 Hz,
C30,C50-H), 7.60e7.74 (m, 6H, AreH), 7.82 (d, 1H, J ¼ 16 Hz, H-
b C
); 13
C
22H15N3O3 (369.37): C, 71.54%; H, 4.09%; N, 11.38%; Found: C,
NMR (CDCl3, 100 MHz, d ppm):114.16, 115.33, 123.34, 127.32, 128.01,
71.46%; H, 4.03%; N, 11.35%; HPLC purity: 98.12%, tR-17.0 min.
128.54, 128.72, 129.04, 129.54, 130.19, 130.66, 130.85, 131.94, 135.45,
137.81, 138.61, 142.00, 147.50, 165.22; MS (m/z): 358 (Mþ), 228, 147,
131, 103, 63; Anal. Calcd. for C22H15ClN2O (358.82): C, 73.64%; H,
4.21%; N, 7.81%; Found: C, 73.67%; H, 4.20%; N, 7.78%; HPLC purity:
99.14%, tR-19.5 min.
4.1.3.16. (E)-1-(2-(4-fluorophenyl)-1H-benzo[d]imidazol-1-yl)-3-
phenylprop-2-en-1-one (3p). Yield: 85%; m.p: 215e216 ꢁC; IR
(KBr):1682 (C]O), 1619 (C]C) cmꢀ1 1H NMR (CDCl3, 400 MHz,
;
d
ppm): 6.73 (d, 1H, J ¼ 16 Hz, H- ), 7.28 (t, 2H, J ¼ 7.3 Hz, C5, C6-H
a
of Benzimidazole), 7.33e7.38 (m, 3H, AreH), 7.46 (t, 2H, J ¼ 7.4 Hz,
4.1.3.21. (E)-1-(2-(3,4-dichlorophenyl)-1H-benzo[d]imidazol-1-yl)-
C30,C50-H), 7.62e7.68 (m, 4H, AreH) 7.82 (d, 1H, J ¼ 16 Hz, H-
b
), 7.98
3-phenylprop-2-en-1-one (3u). Yield: 82%; m.p: 166e168 ꢁC; IR
(d, 2H, J ¼ 8.2 Hz, C2,C6-H of Ph); 13C NMR (CDCl3,100 MHz,
d
ppm):
(KBr): 1675 (C]O), 1613 (C]C) cmꢀ1 1H NMR (CDCl3, 400 MHz,
;
114.19, 115.69, 117.82, 123.65, 125.22, 128.11, 128.94, 129.35, 130.63,
131.91, 133.41, 136.23, 139.31, 142.88, 147.60, 163.54, 165.43; MS (m/
z): 342 (Mþ), 212, 131, 121, 103, 90, 77, 63; Anal. Calcd. for
d
ppm): 6.58 (d,1H, J ¼ 16 Hz, H- ), 7.18 (t, 2H, J ¼ 7.2 Hz, C5, C6-H of
a
Benzimidazole), 7.29 (t,1H, J ¼ 7.6 Hz, C40-H), 7.38e7.46 (m, 3H, Are
H), 7.57e7.63 (m, 4H, AreH), 7.68 (d, 1H, J ¼ 16 Hz, H-
b), 8.01 (s, 1H,
C
22H15FN2O (342.37): C, 77.18%; H, 4.42%; N, 8.18%; Found: C,
C2-H of Ph), 8.12 (d, 1H, J ¼ 6.8 Hz, C6-H of Ph); 13C NMR (CDCl3,
77.22%; H, 4.38%; N, 8.16%; HPLC purity: 99.26%, tR-17.6 min.
100 MHz, d ppm):114.26, 115.22, 123.14, 127.32, 127.96, 128.46,
128.68, 129.02, 130.14, 130.68 139.90, 133.01, 133.80 135.25, 139.15,
141.50, 147.46, 165.08; MS (m/z): 392 (Mꢀ1), 394 [(M þ H)þ, a], 261,
131, 103, 63; Anal. Calcd. for C22H14Cl2N2O (393.27): C, 67.19%; H,
3.59%; N, 7.12%; Found: C, 67.24%; H, 3.63%; N, 7.08%; HPLC purity:
99.52%, tR-19.2 min.
4.1.3.17. (E)-1-(2-(3-bromophenyl)-1H-benzo[d]imidazol-1-yl)-3-
phenylprop-2-en-1-one (3q). Yield: 82%; m.p: 210e212 ꢁC; IR (KBr):
1676 (C]O), 1612 (C]C) cmꢀ1; 1H NMR (CDCl3, 400 MHz,
d ppm):
6.69 (d, 1H, J ¼ 16 Hz, H- ), 7.18 (t, 2H, J ¼ 7.2 Hz, C5, C6-H of
a
Benzimidazole), 7.29 (t,1H, J ¼ 7.4 Hz, C40-H), 7.42e7.50 (m, 4H, Are
H), 7.62e7.68 (m, 5H, AreH), 7.76 (d, 1H, J ¼ 16 Hz, H-
b
), 8.35 (d, 1H,
4.2. Methodology for the evaluation of anti-tubercular activity
assay
J ¼ 6.8 Hz, C6-H of Ph); 13C NMR (CDCl3, 100 MHz,
d ppm): 114.09,
115.29, 122.32, 123.11, 126.67, 127.87, 128.06, 128.63, 129.04,130.45,
130.78, 131.42, 131.68, 132.93, 135.34, 139.02, 141.50, 147.20, 165.00;
MS (m/z): 404 [(M þ H)þ, a], 272, 147, 131, 120, 103, 77, 63; Anal.
Calcd. for C22H15BrN2O (403.27): C, 65.52%; H, 3.75%; N, 6.95%;
Found: C, 65.58%; H, 3.78%; N, 6.91%; HPLC purity: 99.31%, tR-
18.4 min.
All the synthesized molecules were screened for their potential
to inhibit the growth of M. tuberculosis strain H37Rv using the
microplate Alamar Blue assay (MABA) [33] method. The experi-
ment was carried out in triplicate using a 96-welled plate under the
standard conditions. Initially, 200
added to each well of the plate to minimize evaporation of medium
in the test wells during incubation. Subsequently 100 L of Mid-
dlebrook 7H9 broth was added to each well. Further, the test
mL sterile deionized water was
4.1.3.18. (E)-1-(2-(3-chlorophenyl)-1H-benzo[d]imidazol-1-yl)-3-
phenylprop-2-en-1-one (3r). Yield: 88%; m.p: 133e134 ꢁC; IR (KBr):
m

V.K.A. Kalalbandi et al. / European Journal of Medicinal Chemistry 79 (2014) 194e202
201
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method. Consequently, the concentrations of tested compounds
were in the range of 100e0.2
sealed with parafilm and incubated at 37 ꢁC for 5 days. Further,
25 L of freshly prepared 1:1 mixture of Almar Blue reagent and
mg/mL. The plates were covered and
m
10% tween-80 was added to the plate and incubated for 24 h. The
blue color in the well indicated the inhibition of bacterial growth
while the pink color was scored as growth. Further, the minimum
concentration of the compound required to inhibit the bacterial
growth was determined.
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cells were incubated for additional 2e3 h. After careful removal of
incubation medium from the incubator, 100
mL of dimethyl
sulphoxide was added and the plates were gently shaken to re-
suspend formed formazan and waited for few mins to form a
homogenized color. The suspension was placed on a microvibrator
for 5 min, and absorbances of wells containing cells and blanks
were recorded at 490 nm. The experiment was executed in trip-
licate. The mean of the absorbance of wells was calculated with
the same treatment after subtracting of blank absorbance. The
results were normalized by considering control wells as 100%
(maximum absorbance obtained), expressing then the results as
percentage of controls.
Acknowledgments
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The authors acknowledge the University Scientific Instrumen-
tation Centre (USIC), Karnatak University, Dharwad for IR, NMR,
Mass, SC-XRD and analytical data. We are also grateful to NMR
Research Centre, Indian Institute of Science (IISc), Bengaluru, India
for carrying out the spectral analyses.
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THF: tetrahydrofuran
FTIR: Fourier transform infrared spectroscopy
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13C NMR: carbon-13 nuclear magnetic resonance
MIC: minimum inhibitory concentration
IC50: half maximal inhibitory concentration
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MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
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SC-XRD: single crystal X-ray diffraction.
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