Benzimidazole derivatives: Synthesis, leishmanicidal effectiveness, and molecular docking studies

Article abstract of DOI:10.1007/s00044-012-0375-5

Leishmanolysin GP63 is a zinc metalloprotease, expressed at the surface of Leishmania promastigotes. Studies on this protein are hindered as only a limited number of effective non-toxic inhibitors of this drug target are known. Present study describes the identification of a variety of 2-aryl- and 5-nitro-2-arylbenzimidazoles as new GP63 inhibitors. All the compounds were tested for in vitro activity against the promastigote form of Leishmania major and showed very good activity. 2-(Thiophen-2-yl)-1H-benzimidazole (19) and 2-(1H-indol-3-yl)-5-nitro-1H-benzimidazole (34) with IC₅₀ value of 0.62 μg/mL were identified as lead of this library. Molecular docking studies were performed on binding site of GP63 to study the binding mode of compounds. The results of both in vitro and in silico studies clearly indicated that benzimidazoles may serve as new drug candidates in the combat against leishmaniasis.

Full text of DOI:10.1007/s00044-012-0375-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3606–3620
DOI 10.1007/s00044-012-0375-5
ORIGINAL RESEARCH
Benzimidazole derivatives: synthesis, leishmanicidal effectiveness,
and molecular docking studies
•
Awais Shaukat Hira M. Mirza Amna H. Ansari
•
•
•
•
•
Masoom Yasinzai Sohail Z. Zaidi Sana Dilshad
Farzana L. Ansari
Received: 10 September 2012 / Accepted: 15 November 2012 / Published online: 28 November 2012
Ó Springer Science+Business Media New York 2012
Abstract Leishmanolysin GP63 is a zinc metallopro-
tease, expressed at the surface of Leishmania promastig-
otes. Studies on this protein are hindered as only a limited
number of effective non-toxic inhibitors of this drug target
are known. Present study describes the identification of a
variety of 2-aryl- and 5-nitro-2-arylbenzimidazoles as new
GP63 inhibitors. All the compounds were tested for in vitro
activity against the promastigote form of Leishmania major
and showed very good activity. 2-(Thiophen-2-yl)-1H-
benzimidazole (19) and 2-(1H-indol-3-yl)-5-nitro-1H-
benzimidazole (34) with IC50 value of 0.62 lg/mL were
identified as lead of this library. Molecular docking studies
were performed on binding site of GP63 to study the
binding mode of compounds. The results of both in vitro
and in silico studies clearly indicated that benzimidazoles
may serve as new drug candidates in the combat against
leishmaniasis.
Introduction
Leishmaniasis is a chronic infectious disease caused by
protozoan parasites belonging to the genus Leishmania
having sandfly as its vector. Leishmania is digenetic and has
several diverse clinical manifestations. It disseminates as
self-healing cutaneous leishmaniasis to the progressive
mucocutaneous and the potentially lethal visceral leish-
maniasis (Murray et al., 2005). Leishmaniasis is endemic in
more than 88 countries throughout Africa, Asia, Southern
Europe, and Latin America. It has an estimated prevalence of
12 million cases worldwide, which continues to increase,
over 1.5–2 million new cases each year (Ameen, 2010).
Conventional therapies used for the treatment of leish-
maniasis are based on pentavalent antimonials such as
sodium stibogluconate (PentostamÒ) and meglumine anti-
moniate (GlucantimeÒ). However, owing to their toxic
nature, requiring long term treatment and most importantly
due to development of resistance, the search for new drugs
has become imperative. A liposomal glycomacrolide,
amphotericin B (FungilinÒ), is a drug of choice for the
treatment of leishmaniasis, but its cost is prohibitively high
and is out of reach of poor people. More recently, mil-
tefosine (MiltexÒ), an alkylphospholipid which is known to
be effective in cancer chemotherapy too, has emerged as an
oral leishmanicidal drug that is highly effective in children,
but is plagued with the drawback of causing serious gas-
trointestinal complications and teratogenic effects (Murray
et al., 2005; Ameen, 2010; Minodier and Parola, 2007;
Reithinger et al., 2007; Singh and Sivakumar, 2004; Des-
jeux, 2004). Some other drugs, for example, pentamidine,
edelfosine (another alkyl-phospholipid) paromomycin,
tamoxifen, and simataquine are reported to give variable
cure rates (Miguel et al., 2008; Cabrera-Serra et al.,
2008; Singh and Sivakumar, 2004). Early this year,
Keywords Benzimidazoles ꢀ Antileishmanial activity ꢀ
Leishmania major ꢀ Leishmanolysin ꢀ Molecular docking ꢀ
Ro5
A. Shaukat ꢀ S. Dilshad ꢀ F. L. Ansari (&)
Department of Chemistry, Quaid-i-Azam University,
Islamabad 45320, Pakistan
e-mail: fla_qau@yahoo.com
H. M. Mirza ꢀ A. H. Ansari ꢀ M. Yasinzai
Department of Biochemistry, Quaid-i-Azam University,
Islamabad 45320, Pakistan
S. Z. Zaidi
Department of Virology, National Institute of Health,
Islamabad, Pakistan
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Med Chem Res (2013) 22:3606–3620
3607
´
Fairlamb et al. have identified fexinidazole, which is
already in clinical trials for treating sleeping sickness, as
possible new treatment for leishmaniasis (Wyllie et al.,
2012). In spite of a variety of drugs being available, the
current treatments seem to be far from ideal and innovation
of new active compounds on behalf of control, and pre-
vention of leishmaniasis is urgent necessity of the current
scenario.
Chavez et al., 1992; Navarrete-Vazquez et al., 2001). A
recent example of fexinidazole, reported to be an effective
antileishmanial drug, illustrates the significance of an
imidazole ring (Wyllie et al., 2012). Nowadays, with the
advent of fast computers and sophisticated softwares, in
silico tools like molecular modeling and docking have
emerged as acumen for the designing of novel scaffolds,
besides providing a rationale for the results achieved in wet
lab (Ivanenkov et al., 2009). Our research group has been
engaged, for a long time, in target-based drug designing
and has reported the synthesis of diverse classes of small
drug-like compounds and quite a few have been identified
as potential candidates for the inhibition of enzymes such
as urease, cholinesterase, etc. Moreover, the leads identi-
fied in different bioassays were subjected to molecular
modeling and docking studies and an attempt was made to
rationalize the results obtained in different bioassays.
Based on these in silico studies, some predictions for the
synthesis of new enzyme inhibitors with activity were also
made (Ansari et al., 2012, 2009; Nawaz et al., 2008;
Zaheer-Ul-Haq et al., 2010). In continuation to our efforts
in this drug design paradigm, a library of 2-aryl/heteroaryl-
1H-benzimidazoles and 2-aryl/heteroaryl-5-nitro-1H-benz-
imidazoles has been synthesized and evaluated for its
potential against L. major. Although GP63 is a virulent
factor for leishmaniasis (Yao et al., 2003), however,
molecular docking studies using this protein target have not
yet been reported. Hence, molecular docking studies of the
synthesized benzimidazoles were performed on leishman-
olysin (PDB code: 1LML) by means of Molecular Oper-
ating Environment (MOE) software (MOE, 2010.10 2010).
In the combat against multi-drug resistance, such in silico
studies have played a key role in the identification of new
drug targets and the designing of new scaffolds as novel
drug candidates.
Besides the generation of an adequate armory of drugs
to treat leishmaniasis, new and active drug targets are also
required to combat the disease. Enzymes present in the
parasite, but absent from their mammalian host, are con-
sidered as ideal targets for rational drug designing. One
such target is pteridine reductase (PTR1) which is exten-
sively studied, and a variety of PTR1 inhibitors are
developed (Pandey et al., 2010). Another target for leish-
maniasis is the zinc metalloprotease called leishmanolysin,
also termed as GP63, MSP, and PSP (EC 3.4.24.36). It
belongs to the metzincin class, symbolizing the major
surface protease of Leishmania sp. It is the major protein
constituent of the promastigote surface (*5 9 105 mole-
cules/cell). GP63, a glycol protein of 63 kDa, contributes
to parasite virulence and pathogenesis, participating in the
resistance of promastigotes to complement-mediated lysis
and in receptor-mediated uptake of Leishmania. Further-
more, promastigotes release active GP63 by autoproteoly-
sis of GPI-linked GP63 and/or secretion of intracellular
GP63. In fact, GP63 is a drug target and vaccine candidate
(Bianchini et al., 2006; Schlagenhauf et al., 1998; Yao
et al., 2003).
GP63 (PDB code: 1LML) consists of 478 amino acids
and contains predominantly b-sheet secondary structure. It
contains three domains: the N-terminal, the central, and C-
terminal. The N-terminal domain consists of His264 and
His268 catalytic residues within the HisGluXxxXxxHis
signature. The central domain comprises the third catalytic
His334 residue, a Met-turn (Asp342–Ala348), an a-helix C
(H12), and the 62 amino acid insertion (Phe272–Ser333)
which is unique within metzincins. The extended C-ter-
minal domain of GP63 resides mainly antiparallel b-strand
and random coil structure with only slight helical contri-
butions. In the active-site of GP63, the zinc atom is coor-
dinated to the e-nitrogen atoms of His264, His268, and
Results and discussion
Chemistry
Benzimidazoles are generally synthesized by the Philips
reaction; but, in the case of 2-aryl-benzimidazole, the yield
of the reaction is very low and condition of the reaction is
also harsh (Hein et al., 1957). Another route involves the
initial formation of sodium metabisulfite adducts by the
reaction of aryl-carboxaldehydes with sodium metabisulfite
followed by their reaction with 1,2-phenylenediamine
(Ridley et al., 1965; Goker et al., 2002). This protocol leads
to a better yield of benzimidazoles under milder conditions.
Following the strategy (Scheme 1), sodium metabisul-
fite adducts of 13 different substituted benzaldehydes and
six different heteroaryl-carboxaldehydes were synthesized
˚
His334 at a distance of 2.18, 2.18, and 2.12 A, respectively
(Schlagenhauf et al., 1998).
Currently, globally, scientists are trying to explore new
armors to fight multi-drug resistance. In this combat,
benzimidazole received a lot of attention of researchers
because they are found numerously in biologically active
compounds (Preston, 1974; Touzeau et al., 2003; Rondu
et al., 1997). Moreover, benzimidazole scaffold is found in
various synthetic pharmaceuticals revealing a broad spec-
trum of biologic activity (Cedillo-Rivera and Munoz, 1992;
123

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Med Chem Res (2013) 22:3606–3620
R1
Scheme 1 Synthesis of 2-aryl-
and 5-nitro-2-aryl-1H-
benzimidazoles
R1
NH2
NH2
HO
N
DMF
A
A
N
H
NaO3S
Cpd.
1
2
3
4
5
6
7
R1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
A
Cpd.
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
R1
A
Phenyl
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
Phenyl
4-Chlorophenyl
2-Chlorophenyl
2-Nitrophenyl
3-Nitrophenyl
4-Nitrophenyl
3-Methoxyphenyl
4-Methoxyphenyl
4-Hydroxyphenyl
4-Methylphenyl
4-Fluorophenyl
4-Butoxyphenyl
4-i-Propylphenyl
Pyridin-3-yl
4-Chlorophenyl
2-Chlorophenyl
2-Nitrophenyl
3-Nitrophenyl
4-Nitrophenyl
3-Methoxyphenyl
4-Methoxyphenyl
4-Methylphenyl
4-Fluorophenyl
4-Butoxyphenyl
4-i-propylphenyl
Pyridin-2-yl
Pyridin-3-yl
Indol-3-yl
Pyrrol-2-yl
5-Methylfuran-2-yl
Thiophen-2-yl
8
9
10
11
12
13
14
15
16
17
18
19
Pyridin-4-yl
Indol-3-yl
Pyrrol-2-yl
5-Methylfuran-2-yl
Thiophen-2-yl
in good yield ([90 %). In a subsequent step, benzimidazoles
1–19 were prepared by reacting these sodium metabisulfite
adducts with 1,2-phenylenediamine. Benzimidazoles 20–37
were synthesized by the reaction of the same adducts with
4-nitro-1,2-phenylenediamine. The reaction time recorded
was 4–7 h; however, the synthesis of nitro-substituted
counterparts required relatively longer time. The yield of all
compounds varied from good to excellent. Melting points of
literature-reported compounds were verified. The structures
inhibitory concentrations (IC50) of Leishmania growth
were calculated by GraphPad Prism software and the data
are reported as the mean SD in Table 1.
IC50 values of the tested compounds 1–37 indicated that
all the compounds exhibited significant activity (IC50
0.62–0.92 lg/mL) against L. major as compared to the
standard drug amphotericin B (IC50 0.56 lg/mL). Of the
entire series of compounds, 2-heteroarylbenzimidazoles
were found to be most active (17, 19, and 34). Furthermore,
2-arylbenzimidazoles were found to have stronger inhibi-
tory potential than their 5-nitro-substituted counterparts.
However, compounds 21, 34, and 36 were an exception to
this observation. Nitro group is well recognized as potential
candidates for chemotherapy of the hypoxic cells. The
cytotoxicity of nitro compounds against hypoxic cells may
be attributed to the reduction of the nitro group yielding
several reactive metabolites such as nitroanion, hydronitr-
oxide, etc. (Romero-Castro et al., 2011). It may, therefore,
be anticipated that these nitro benzimidazoles can be
potential candidates in cytotoxicity studies too. The general
trend in the leishmanicidal activity due to a nitro group on
the 2-aryl ring was ortho [ para [ meta (4, 5, 6, 23, 24,
and 25). Of the chloro-substituted benzimidazoles, the p-
isomers were found to be more effective than the o-isomers
(2, 3, 21, and 22).
1
of all synthesized compounds were confirmed by IR, H-,
and 13C-NMR spectroscopic data and LCMS. IR spectro-
scopic analysis indicated the formation of benzimidazole
ring by the absorption peaks corresponding to NH and C=N
group at 3,274–3,410 and 1,586–1,625 cm-1, respectively.
1H NMR supported the proposed structures by the appear-
ance of a characteristic NH signal recorded as broad singlet
at d 10.21–11.23 ppm. The presence of the nitro group at
C-5 of benzimidazole ring in compounds 20–37 was con-
firmed by a singlet d 8.39–8.63 ppm corresponding to H-4.
LCMS analysis confirmed the identity of synthesized com-
pounds by the appearance of [M?H?] peak in all the cases
besides confirming their purity.
Antileishmanial activity
In order to establish the pharmacological profile of the
synthesized benzimidazoles, these compounds were sub-
jected to in vitro study against the promastigote form of L.
major by the method reported by Zhai et al. (1999) using
amphotericin B as standard control in the assay. The 50 %
Molecular docking
As mentioned earlier, leishmanolysin (GP63) is a virulent
factor for leishmaniasis; however, to date, no molecular
123

Med Chem Res (2013) 22:3606–3620
3609
Table 1 In vitro antileishmanial activity of synthesized library against promastigote form of L. major and in silico studies on GP63
˚
Distance (A)
Compd.
IC50 (lg/mL)
London dG
score (kcal/mol)
Amino acids
Interaction
Energy (kcal/mol)
1
0.68 0.02
-7.2453
His268
Ala227
Ser273
Ser273
His268
Zn
Arene–arene
H–bonding
Arene–H
3.91
3.14
3.70
3.02
3.87
2.64
2.68
3.44
3.23
2.63
4.52
3.15
3.12
3.82
3.82
2.74
3.39
4.14
3.36
4.16
4.37
2.52
3.95
3.82
3.14
3.69
3.51
3.44
4.07
3.93
3.82
3.82
2.86
3.85
3.92
3.97
3.81
3.17
4.35
3.90
3.51
2.89
3.56
2.64
2.68
4.6
–
-2.6
-0.6
-1.4
–
2
3
4
5
0.72 0.08
0.78 0.09
0.69 0.08
0.82 0.10
-7.2570
-7.6713
-7.9387
-8.3562
H–bonding
Arene–arene
Metal contact
Metal contact
H–bonding
H–bonding
Metal contact
Arene–H
-0.6
-0.8
-1.6
-2.6
-1.2
-1.0
-0.8
-0.9
–
Zn
Ala349
Gly222
Zn
6
7
8
0.71 0.03
0.77 0.10
0.81 0.02
-7.6344
-7.7883
-7.5160
Thr229
Ser273
Thr228
His268
Ser273
Glu265
His264
Ala350
His264
Ala350
His268
Zn
H–bonding
H–bonding
Arene–arene
Arene–H
-1.1
-4.2
–
9
0.92 0.10
0.79 0.05
-8.4845
-7.2669
H–bonding
Arene–arene
Arene–H
10
-0.6
–
11
12
0.79 0.02
0.65 0.10
-7.8308
-8.7365
Arene–arene
Arene–H
-0.6
-0.8
-2.5
–
Arene–H
Metal contact
Arene–arene
Arene–arene
H–bonding
Arene–H
13
14
0.64 0.02
0.64 0.01
-7.5399
-7.8907
His268
His268
Ala227
Ser273
His264
Ala349
Ala350
His268
Pro347
His264
Pro347
His264
His264
His268
Pro347
Glu265
Val223
His268
Ala227
Gly329
Glu265
Zn
–
-2.4
-2.1
–
15
0.88 0.07
-7.6183
Arene–arene
Arene–H
-0.6
-1.2
–
Arene–H
16
17
18
19
20
21
0.87 0.05
0.63 0.04
0.84 0.10
0.62 0.05
0.80 0.07
0.64 0.09
-8.4532
-7.0279
-7.4853
-7.0881
-7.9371
-8.5248
Arene–arene
H–bonding
Arene–arene
H–bonding
Arene–arene
Arene–arene
Arene–arene
H–bonding
H–bonding
Arene–H
-1.5
–
-2.5
–
–
-1.4
-2.3
-0.7
–
Arene–arene
H–bonding
H–bonding
H–bonding
Metal contact
Metal contact
Arene–H
-0.7
-2.2
-1.7
-0.6
-2.4
-0.8
-2.4
22
23
24
0.91 0.04
0.73 0.09
0.79 0.10
-7.7101
-8.4132
-8.7291
Zn
Ala349
Thr228
25
0.75 0.01
-8.3911
H–bonding
2.84
123

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Med Chem Res (2013) 22:3606–3620
Table 1 continued
˚
Distance (A)
Compd.
IC50 (lg/mL)
London dG
score (kcal/mol)
Amino acids
Interaction
Energy (kcal/mol)
26
27
28
29
0.82 0.01
0.85 0.04
0.88 0.05
0.77 0.22
-8.5942
-8.7449
-8.2060
-8.0795
Gly329
Gly329
Gly329
His268
Gly329
His268
Ser273
Zn
H–bonding
H–bonding
H–bonding
Arene–arene
H–bonding
Arene–H
2.76
2.98
2.95
3.94
2.89
4.13
4.10
2.59
4.73
4.16
2.64
4.98
2.66
3.14
3.48
2.65
3.66
3.13
3.14
3.90
3.48
2.85
3.05
3.53
2.49
3.61
2.94
-3.0
-5.1
-4.5
–
-5.0
-1.1
-0.7
-1.6
-0.7
-1.2
-0.9
-0.6
-0.9
-2.9
-0.7
-4.7
-0.6
-3.1
-2.8
–
30
0.76 0.07
-8.8546
Arene–H
Metal contact
Arene–H
Thr229
Ser273
Zn
31
0.80 0.09
-8.3534
Arene–H
Metal contact
Arene–H
Thr229
Zn
32
33
34
0.73 0.03
0.79 0.01
0.62 0.07
-8.0065
-7.9663
-8.7914
Metal contact
H–bonding
H–bonding
H–bonding
H–bonding
H–bonding
H–bonding
Arene–arene
H–bonding
H–bonding
H–bonding
H–bonding
Metal contact
H–bonding
H–bonding
Thr229
Ala227
Gly329
Ala349
Gly222
Gly222
His268
Ala227
Gly329
Gly329
Gly329
Zn
35
36
0.91 0.08
0.64 0.06
-7.8610
-7.7326
-0.8
-2.9
-4.6
-0.6
-1.3
-0.5
-1.1
37
0.82 0.06
0.56 0.04
-8.0428
Standarda
-11.0916
Glu265
Glu220
a
Amphotericin B
docking studies on this target have been reported. Hence,
molecular docking studies were carried out by means of
MOE program of Chemical Computing Group for the
standard drug amphotericin B and benzimidazoles 1–37.
The crystal structure of GP63 was obtained from protein
data bank (PDB code: 1LML). Since this structure does not
contain any co-crystallized ligand molecular docking
studies were, therefore, conducted by means of the site
finder option given in MOE program. In order to visualize
the binding pocket, Alpha spheres were created followed
by the generation of dummy atoms on the centers of these
spheres.
Structurally, GP63 consists of three domains: N-terminal,
central, and C-terminal. The binding pocket of GP63 is built
by His264, His268, and Zn metal from N-terminal domain,
while His334, Asp342–Ala348, and Phe272–Ser333 from
the central domain (Fig. 1). The key amino acids of the
active His264, His268, and His334, are found coordinated
with Zn (Fig. 2) (Schlagenhauf et al., 1998).
Fig. 1 The ribbon form of GP63 showing three domains (Color
figure online)
After having identified the active site, the standard drug
amphotericin B was successfully docked in the binding
pocket with the rmsd value of 1.02. It was noted that the
123

Med Chem Res (2013) 22:3606–3620
3611
Fig. 2 a The ribbon form of the binding pocket of GP63. b A close-up view of binding pocket showing lipophilic (magenta) and hydrophilic
(green) surface (Color figure online)
Fig. 3 a Docking pose of amphotericin B in the binding pocket. b A 2D representation of the same showing interactions with amino acids of the
active site (Color figure online)
amino acids Glu265, Glu220, and Zn were involved in
binding with the docked standard drug through a network
of hydrogen bonding (Fig. 3). Our target compounds
(1–37) were also docked successfully in the binding
pocket. As all the compounds of the library exhibited
promising activity with a narrow range of IC50 value
(0.62–0.92 lg/mL), no significant correlation was found
with the London dG score (-7.0279 to -8.8546 kcal/mol).
All the active compounds were found to have an
interesting binding mode in the docked pocket. The
superimposition of these benzimidazoles together with
amphotericin B is shown in Fig. 4. As shown in figure, the
most active group of benzimidazoles (yellow) with IC50
values from 0.62 to 0.68 lg/mL was found fitted beauti-
fully in the center of the pocket. However, the docked
Fig. 4 Binding pocket of GP63 showing superimposition of benz-
imidazoles and amphotericin B (Color figure online)
123

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Med Chem Res (2013) 22:3606–3620
compounds (orange) with intermediate IC50 values
(0.69–0.78 lg/mL) occupied the left side, while the group
(red) with the highest values (0.79–0.92 lg/mL) docked to
the right of the gorge.
of the nitro group present on the 2-aryl ring of benzimi-
dazoles such as 4, 5, and 6 in series1 and the same pattern
was visible in 23, 24, and 25 of the other series. However,
in compounds 30 and 31, the metal ligation was due to the
involvement of 5-nitro functionality of benzimidazole ring.
It is anticipated that this metal ligation may be responsible
for the leishmanicidal effect of these nitro-benzimidazoles.
The highest activity (IC50 = 0.62 lg/mL) of the docked
compounds 19 and 34 was supported by the fitting pattern
and binding interactions with the amino acids in the gorge.
The binding interactions observed in the docked compound
19 (Fig. 5) were hydrophobic interaction with His268 at
The most important ligand-enzyme binding interactions
were arene–arene, arene–H, hydrogen bonding, and metal
ligation with Zn578. One of the common binding features
of the most potent docked compounds was that all of them
showed arene–arene interactions either with His268 (e.g.,
1, 13, 14, 19, 21,, and 36) or with His264 (12, 17, and 34).
Another general observation was the hydrogen bonding of
these ligands through NH or H-4 of benzimidazole ring
with Ala227 or, in a few cases, with Gly329 (e.g., 21 and
34). Zn metal ligation was observed with the oxygen atom
˚
distance of 3.97 A and H-bonding interaction between S of
thiophene moiety (as hydrogen bond acceptor) with Pro347
Fig. 5 a The close-up view of the binding pocket containing 2-(thiophen-2-yl)-1H-benzimidazole (19). b A 2D representation of the same
showing interaction with amino acids of the active site (Color figure online)
Fig. 6 a Docking pose of the 2-(1H-indol-3-yl)-5-nitro-1H-benzimidazole (34). b A 2D ligand interaction diagram of the same compound
showing interaction with amino acids of the active site (Color figure online)
123

Med Chem Res (2013) 22:3606–3620
3613
˚
at a distance of 3.81 A with the energy released -1.4 kcal/
Table 2 Physico-chemical properties for the synthesized library
mol. In the case of docked compound 34 (Fig. 6), the
ligand-enzyme complex was stabilized by a strong
H-bonding interaction of NH of benzimidazole ring with
Compd.
Mol. wt.
Log P
TPSA
HBD
HBA
NOR
1
194.24
228.68
228.68
239.23
239.23
239.23
224.26
224.26
210.24
208.26
212.23
266.34
236.32
195.23
195.23
233.27
183.21
198.23
200.27
239.23
273.68
273.68
284.23
284.23
284.23
269.26
269.26
253.26
257.22
311.34
281.32
240.22
240.22
278.27
228.21
243.26
245.26
924.09
3.23
3.88
3.88
3.14
3.14
3.14
3.24
3.24
2.94
3.54
3.37
4.40
4.35
2.63
2.63
3.71
2.56
3.13
3.29
3.14
3.79
3.79
3.05
3.05
3.05
3.15
3.15
3.45
3.28
4.32
4.26
2.53
2.53
3.62
2.47
3.04
3.20
2.67
28.68
28.68
28.68
74.50
74.50
74.50
37.91
37.91
48.91
28.68
28.68
37.91
28.68
41.57
41.57
44.47
44.47
41.82
28.68
74.50
74.50
74.50
120.32
120.32
120.32
83.73
83.73
74.50
74.50
83.73
74.50
87.39
87.39
90.29
90.29
87.64
74.50
445.14
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
1
1
13
1
1
1
1
1
1
2
2
2
1
1
2
1
2
2
1
1
1
1
1
1
1
1
1
1
2
2
1
1
2
1
2
2
1
1
1
1
17
1
1
1
2
2
2
2
2
1
1
1
5
2
1
1
1
1
1
1
2
2
2
3
3
3
3
3
2
2
6
3
2
2
2
2
2
2
3
˚
the oxygen of Gly329 at distance of 2.65 A with the energy
2
3
released -4.7 kcal/mol. Further, H-4 of benzimidazole
4
ring was also found interacting with carbonyl oxygen
˚
of Ala227 at distance of 3.48 A and energy released
5
6
-0.7 kcal/mol. Since the precise role of GP63 in parasite
physiology is not well understood, these molecular docking
studies (expected to be the first report) may enable the
designing of new scaffolds for GP63 inhibition.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Standarda
a
Lipinski’s rule of five
According to Lipinski’s Ro5, most drug-like molecules
have molecular weight B500, logarithm of the octanol/
water partition coefficient (log P) B5, total polar surface
2
˚
area (TPSA) \ 140 A , number of hydrogen bond donors
(HBD) B 5, and hydrogen bond acceptor (HBA) B 10
(Lipinski et al., 1997). Further modifications in the Ro5
were made by Veber et al. who suggested the number of
rotatable bond (NOR) of drug-like molecule must be fewer
or equal to 10 (Veber et al., 2002). Molecules violating
more than one of these rules may have problem with bio-
availability. These molecular descriptors were calculated
for all the synthesized compounds by the ligand property
calculation function of MOE software and all of them were
found to obey Lipinski’s Ro5 cut-off limits (Table 2),
thereby confirming that these drug-like compounds may be
potential drug candidates against leishmaniasis. Log P of
all synthesized compounds ranges from 2.47 to 4.4,
reflected no absorption risk as oral drug administration.
Furthermore, the compounds do not accumulate in deep
tissue compartments and clearance is majorly via renal (as
most compounds have MW \ 300 amu). All drugs have
HBD \ 4, and hence are not prone to phase-II metabolism.
Conclusion
Leishmaniasis has become a major health problem world-
wide because the current chemotherapy is far from being
satisfactory and urges the search of new, safe, affordable,
and effective drugs. However, besides generating an arm-
ory of drugs for treating any disease, there is also need of
new and effective drug targets to combat the disease. This
work targets GP63 protein which is a virulent factor for
leishmaniasis. The leishmanicidal effect of 37 membered
library of benzimidazole library against promastigote form
of clinical isolate of L. major was studied. All compounds
of library exhibited significant antileishmanial activity with
IC50 value of 0.62–0.92 lg/mL. These results were
Amphotericin B
corroborated by an in silico study on leishmanolysin also
known as GP63 (PDB code: 1LML). Since the precise role
of GP63 in parasite physiology is not well understood,
these molecular docking studies (expected to be the first
report), may enable the designing of new scaffolds for
GP63 inhibition. The current study is an encouraging
advance in the search of new drug candidates for
GP63 inhibition. The easy and economic synthesis of
123

3614
Med Chem Res (2013) 22:3606–3620
2-(4-Chlorophenyl)-1H-benzimidazole (2) Off-white solid;
Yield: 87 %; m.p. 287–289 °C (Patil et al., 2011). IR (KBr,
cm-1): 3,363 (NH); 3,052 (CH aromatic); 1,617 (C=N);
benzimidazoles, together with their drug-like behavior
urges further in vivo studies for their assessment as
effective and affordable drug candidates.
1
1,539 (C=C); 747 (Ar–Cl). H NMR (300 MHz, DMSO-
d6): d 7.27 (m, 2H, H-5, H-6); 7.34 (m, 2H, H-30, H-50);
7.43 (m, 2H, H-20, H-60); 7.70(m, 2H, H-4, H-7); 10.97 (bs,
1H, NH). 13C NMR (75 MHz, DMSO-d6): 115.31; 123.19;
128.59; 128.93; 129.78; 134.92; 138.87; 152.94. LCMS:
229.2 [M?H?].
Experimental
Chemistry
Melting points are uncorrected and were recorded by Open
Capillary tube method using Gallenkamp melting point
apparatus. The IR spectra were obtained on Thermo Sci-
entific Nicolet Spectrophotometer model 6700 (samples
2-(2-Chlorophenyl)-1H-benzimidazole (3) Off-white
solid; Yield: 86 %; m.p. 233–234 °C (Hein et al., 1957). IR
(KBr, cm-1): 3,369 (NH); 3,054 (CH aromatic); 1,613
1
1
taken in original form). H- and 13C NMR spectra were
(C=N); 1,542 (C=C); 743 (Ar–Cl). H NMR (300 MHz,
DMSO-d6): d; 7.16–7.34 (m, 6H, H-5, H-6, H-30, H-40,
H-50, H-60); 7.73 (m, 2H, H-4, H-7); 10.93 (bs, 1H, NH).
13C NMR (75 MHz, DMSO-d6): 115.29; 123.04; 127.21;
128.59; 129.48; 130.31; 138.26; 138.93; 152.74. LCMS:
229.1 [M?H?].
recorded on Bruker spectrometer at 300 and 75 MHz,
respectively, and chemical shifts (d) were reported in parts
per million (ppm) relative to tetramethyl silane (TMS) as
an internal standard. Mass spectra were recorded on Agi-
lent Technologies 1200 series HPLC comprising G1313
DAD and Ion Trap LCMS G2445D SL. All the chemicals
and solvents were purchased from Sigma-Aldrich or Fluka.
The library of 2-substituted-benzimidazole derivatives was
synthesized by the reported procedure (Ridley et al., 1965;
Goker et al., 2002).
2-(2-Nitrophenyl)-1H-benzimidazole (4) Brown solid;
Yield: 81 %; m.p. 240–241 °C (Sharma et al., 2009). IR
(KBr, cm-1): 3,361 (NH); 3,079 (CH aromatic); 1,615
1
(C=N); 1,553 (C=C); 1,367 (NO2). H NMR (300 MHz,
DMSO-d6): d 7.27–7.46 (m, 3H, H-5, H-6, H40); 7.70–7.74
(m, 4H, H-4, H-7, H-50, H-60); 8.25 (m, 1H, H-30); 10.89
(bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6): 115.92;
121.94; 123.56; 128.30; 132.31; 138.88; 146.74; 152.83.
LCMS: 240.1 [M?H?].
General procedure for the synthesis of 1–37
Appropriate
aryl-
and
heteroaryl-carboxaldehydes
(15 mmol) were dissolved in 50 mL ethanol followed by
the addition of 10 mL (16 %) aqueous solution of sodium
metabisulfite in portions along with vigorous stirring.
During the course of reaction, more ethanol was added.
After that, the mixture was kept in refrigerator for several
hours to yield precipitates which were filtered and dried in
oven at 50 °C (yield [ 90 %). The mixture of the obtained
sodium metabisulfite adduct (7 mmol) and appropriate 1,2-
phenylenediamine (5 mmol) in DMF (15 mL) was heated
at 110 °C under reflux condition for 4–7 h. The reaction
mixture was then cooled to room temperature and poured
into the ice cold water (150 mL); the resulting solid was
filtered and recrystallized by a mixture of ethanol and
water.
2-(3-Nitrophenyl)-1H-benzimidazole (5) Brown solid;
Yield: 80 %; m.p. [300 °C (Patil et al., 2011). IR (KBr,
cm-1): 3,361 (NH); 3,079 (CH aromatic); 1,619 (C=N);
1
1,548 (C=C); 1,358 (NO2). H NMR (300 MHz, DMSO-
d6): d 7.28 (m, 2H, H-5, H-6); 7.58 (m, 1H, H-50); 7.73 (m,
2H, H-4, H-7);7.89 (m, 1H, H-60); 8.15 (m, 1H, H-40); 8.42
(s, 1H, H-20); 11.12 (bs, 1H, NH). 13C NMR (75 MHz,
DMSO-d6): 115.39; 121.11; 122.53; 123.39; 130.36;
131.94; 134.10; 139.18; 148.93; 152.72. LCMS: 240.2
[M?H?].
2-(4-Nitrophenyl)-1H-benzimidazole (6) Brown solid;
Yield: 81 %; m.p.[300 °C (Sharma et al., 2009). IR (KBr,
cm-1): 3,359 (NH); 3,082 (CH aromatic); 1,609 (C=N);
1
2-Phenyl-1H-benzimidazole (1) White solid; Yield: 89 %;
m.p. 291–292 °C (Gogoi and Konwar, 2006). IR (KBr,
cm-1): 3,365 (NH); 3,057 (CH aromatic); 1,612 (C=N);
1,547 (C=C); 1,357 (NO2). H NMR (300 MHz, DMSO-
d6): d 7.25 (m, 2H, H-5, H-6); 7.72–7.74 (m, 4H, H-4, H-7,
H-20, H-60); 8.21 (m, 2H, H-30, H-50); 11.10 (bs, 1H, NH).
13C NMR (75 MHz, DMSO-d6): 116.13; 121.72; 123.70;
128.29; 136.63; 138.64; 148.19; 152.59. LCMS: 240.2
[M?H?].
1
1,542 (C=C). H NMR (300 MHz, DMSO-d6): d 7.22 (m,
1H, H-40); 7.26 (m, 2H, H-5, H-6); 7.31 (m, 2H, H-30,
H-50); 7.51 (m, 2H, H-20, H-60); 7.71(m, 2H, H-4, H-7);
11.01 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
115.25; 123.22; 127.57; 128.78; 129.92; 130.95; 138.75;
152.70. LCMS: 195.1 [M?H?].
2-(3-Methoxyphenyl)-1H-benzimidazole (7) White solid;
Yield: 89 %; m.p. 212–213 °C. IR (KBr, cm-1): 3,354
123

Med Chem Res (2013) 22:3606–3620
3615
(NH); 3,091 (CH aromatic); 1,621 (C=N); 1,559 (C=C);
1
3,367 (NH); 3,065 (CH aromatic); 2,909 (CH aliphatic);
1,612 (C=N); 1,560 (C=C); 1,237 (C–O–C). 1H NMR
1,209 (C–O–C). H NMR (300 MHz, DMSO-d6): d 3.78
(O–CH3); 6.73 (m, 1H, H-40), 7.01–7.04 (m, 2H, H-20,
H-60); 7.23 (m, 1H, H-50); 7.27 (m, 2H, H-5, H-6); 7.71 (m,
2H, H-4, H-7) 10.86 (bs, 1H, NH). 13C NMR (75 MHz,
DMSO-d6): 55.73; 112.31, 114.67; 115.69; 120.13; 123.10;
130.54; 131.76; 138.89; 152.71, 161.26. LCMS: 225.2
[M?H?].
(300 MHz, DMSO-d6): d 0.97 (t, 3H, CH3 ); 1.34 (q, 2H,
00
CH2-300); 1.71 (m, 2H, CH2-200); 3.95 (t, 2H, CH2–O); 6.82
(m, 2H, H-30, H-50); 7.28–7.35 (m, 4H, H-5, H-6, H-20,
H-60); 7.73 (m, 2H, H-6, H-7); 10.57 (bs, 1H, NH). 13C
NMR (75 MHz, DMSO-d6): 15.18; 19.35; 32.12; 69.78;
113.52; 115.80; 123.19; 123.63; 128.36; 138.92; 152.54;
158.47. LCMS: 267.3 [M?H?].
2-(4-Methoxyphenyl)-1H-benzimidazole
(8) Off-white
solid; Yield: 92 %; m.p. 224–225 °C (Gogoi and Konwar,
2006). IR (KBr, cm-1): 3,345 (NH); 3,103 (CH aromatic);
2,918 (CH aliphatic); 1,623 (C=N); 1,563 (C=C); 1,214
(C–O–C). 1H NMR (300 MHz, DMSO-d6): d 3.75 (O–
CH3); 6.81 (m, 2H, H-30, H-50), 7.26–7.37 (m, 4H, H-5,
H-6, H-20, H-60); 7.72 (m, 2H, H-4, H-7); 10.96 (bs, 1H,
NH). 13C NMR (75 MHz, DMSO-d6): 55.92; 114.80;
116.17; 123.61; 124.34; 129.93; 138.75; 152.90; 161.31.
LCMS: 225.2 [M?H?].
2-(4-i-Propylphenyl)-1H-benzimidazole (13) Off-white
solid; Yield: 81 %; m.p. 259–260 °C. IR (KBr, cm-1):
3,364 (NH); 3,062 (CH aromatic); 2,913 (CH aliphatic);
1
1,614 (C=N); 1,563 (C=C). H NMR (300 MHz, DMSO-
d6): d 1.29 (d, 6H, CH3); 3.14 (q, 1H, CH); 7.19–7.37 (m,
6H, H-5, H-6, H-20, H-30, H-50, H-60); 7.71 (m, 2H, H-6,
H-7); 10.64 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
24.36; 36.49; 115.44; 123.07; 126.72; 127.36;
128.31;138.84; 148.77; 152.49. LCMS: 237.2 [M?H?].
2-(4-Hydroxyphenyl)-1H-benzimidazole (9) White solid;
Yield: 78 %; m.p. 225–226 °C (Sharma et al., 2009). IR
(KBr, cm-1): 3,343 (NH); 3,479 (OH); 3,087 (CH aro-
2-(Pyridin-3-yl)-1H-benzimidazole (14) Light yellow
solid; Yield 89 %; m.p. 248–249 °C (Schiffmann et al.,
2006). IR (KBr, cm-1): 3,354 (NH); 3,060 (CH aromatic);
1
1
matic); 1,613 (C=N); 1,557 (C=C). H NMR (300 MHz,
1,601 (C=N); 1,564 (C=C). H NMR (300 MHz, DMSO-
DMSO-d6): d 6.78 (m, 2H, H-30, H-50), 7.27–7.30 (m, 4H,
H-5, H-6, H-20, H-60); 7.71 (m, 2H, H-4, H-7); 7.91 (bs, 1H,
OH) 10.96 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
115.94; 116.76; 123.23; 124.76; 128.94; 138.78; 152.80;
159.12. LCMS: 211.1 [M?H?].
d6): d 7.21–7.44 (m, 3H, H-5, H-6, H-30); 7.74–7.92 (m,
3H, H-4, H-7, H-60); 8.57 (m, 1H, H-40); 8.75 (s, 1H, H-20);
11.04 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
116.34; 123.27; 124.20; 133.35; 134.13; 138.66;148.73;
149.48; 152.72. LCMS: 196.1 [M?H?].
2-(4-Methylphenyl)-1H-benzimidazole (10) White solid;
Yield: 91 %; m.p. 275–276 °C (Gogoi and Konwar, 2006).
IR (KBr, cm-1): 3,359 (NH); 3,067 (CH aromatic); 2,917
(CH aliphatic); 1,619 (C=N); 1,561 (C=C). 1H NMR
(300 MHz, DMSO-d6): d 2.36 (s, 3H, CH3); 7.17–7.33 (m,
6H, H-5, H-6, H-20, H-30, H-50, H-60); 7.73 (m, 2H, H-4,
H-7); 10.45 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
26.33; 116.12; 123.38; 127.25; 127.91; 138.18; 138.94;
152.45. LCMS: 209.2 [M?H?].
2-(Pyridin-4-yl)-1H-benzimidazole (15) Yellow solid;
Yield: 88 %: m.p. 218–219 °C (Huang et al., 2004). IR
(KBr, cm-1): 3,331 (NH); 3,057 (CH aromatic); 1,597
1
(C=N); 1,556 (C=C). H NMR (300 MHz, DMSO-d6): d
7.25 (m, 2H, H-5, H-6); 7.63–7.74 (m, 4H, H-4, H-7, H-20,
H-60); 8.67 (m, 2H, H-30, H-50); 10.94 (bs, 1H, NH). 13C
NMR (75 MHz, DMSO-d6): 115.93; 121.59; 123.36;
134.43; 138.97; 150.14; 152.91. LCMS: 196.1 [M?H?].
2-(1H-indol-3-yl)-1H-benzimidazole
(16) Off-white
2-(4-Fluorophenyl)-1H-benzimidazole (11) White solid;
Yield: 91 %; m.p. 258–259 °C (Rashid et al., 2007). IR
(KBr, cm-1): 3,347 (NH); 3,061 (CH aromatic); 1,612
solid; Yield: 75 %; m.p. 228–229 °C. IR (KBr, cm-1):
3,335 (NH); 3,061 (CH aromatic); 1,594 (C=N); 1,543
1
(C=C). H NMR (300 MHz, DMSO-d6): d 7.01–7.52 (m,
1
(C=N); 1,560 (C=C); 1,037 (Ar–F). H NMR (300 MHz,
7H, H-5, H-6, H-20, H-40, H-50, H-60, H-70); 7.74 (m, 2H,
H-4, H-7); 10.94 (bs, 1H, NH); 11.23 (bs, 1H, NH). 13C
NMR (75 MHz, DMSO): 107.38; 112.15; 116.10; 119.13;
120.16; 122.42; 123.18; 128.67; 131.69; 135.53; 138.92;
153.27. LCMS: 234.2 [M?H?].
DMSO-d6): d 7.04 (m, 2H, H-30, H-50); 7.26 (m, 2H, H-5,
H-6); 7.48 (m, 2H, H-20,H-60); 7.71 (m, 2H, H-4, H-7);
10.62 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
115.88; 117.13; 123.10; 126.73; 129.49; 138.73; 152.91.
LCMS: 213.2 [M?H?].
2-(1H-pyrrol-2-yl)-1H-benzimidazole (17) Orange solid;
Yield: 79 %; m.p. 205–206 °C. IR (KBr, cm-1): 3,356
(NH); 3,091 (CH aromatic); 1,607 (C=N); 1,534 (C=C).
2-(4-Butoxyphenyl)-1H-benzimidazole
(12) Off-white
solid; Yield: 87 %; m.p. 223–224 °C. IR (KBr, cm-1):
123

3616
Med Chem Res (2013) 22:3606–3620
1H NMR (300 MHz, DMSO-d6): d 6.12–6.53 (m, 3H, H-30,
H-40, H-50); 7.29 (m, 2H, H-4, H-7); 7.69 (m, 2H, H-4,
H-7); 10.61 (bs, 1H, NH); 10.92 (bs, 1H, NH). 13C NMR
(75 MHz, DMSO): 106.33; 108.90; 115.16; 118.38;
123.15; 129.92; 138.68; 154.65. LCMS: 184.1 [M?H?].
H-50, H-60); 7.99–8.61 (m, 3H, H-4, H-6, H-7); 10.63 (bs,
1H, NH). 13C NMR (75 MHz, DMSO-d6): 112.38; 116.31;
118.55; 127.32; 128.66; 129.45; 130.33; 133.59; 136.30;
139.83; 143.18; 144.37; 152.71. LCMS: 274.1 [M?H?].
2-(2-Nitrophenyl)-5-nitro-1H-benzimidazole (23) Orange
solid; Yield: 75 %; m.p. 130–131 °C (Romero-Castro
et al., 2011). IR (KBr, cm-1): 3,365 (NH); 3,133 (CH
2-(5-Methylfuran-2-yl)-1H-benzimidazole (18) Grayish
pink solid; Yield: 92 %; m.p. 175–177 °C. IR (KBr,
cm-1): 3,354 (NH); 3,101 (CH aromatic); 2,925 (CH ali-
1
aromatic); 1,613 (C=N); 1,541 (C=C); 1,368 (NO2). H
1
phatic); 1,612 (C=N); 1,527 (C=C); 1,218 (C–O–C). H
NMR (300 MHz, DMSO-d6): d 7.81–7.92 (m, 3H, H-7,
H-40, H-50); 8.01–8.17 (m, 3H, H-6, H-30, H-60); 8.53 (bs,
1H, H-4); 10.81 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-
d6): 110.93; 116.28; 118.14; 122.3; 128.612; 129.84;
132.19; 135.30; 139.72; 142.86; 144.59; 147.15; 152.81.
LCMS: 285.2 [M?H?].
NMR (300 MHz, DMSO-d6): d 2.19 (s, 3H, CH3);
5.91–6.13 (m, 2H, H30, H-40); 7.24 (m, 2H, H-4, H-7); 7.73
(m, 2H, H-4, H-7); 10.90 (bs, 1H, NH). 13C NMR
(75 MHz, DMSO-d6): 14.38; 101.99; 107.67; 116.31;
123.40; 138.92; 141.74; 151.96; 153.17. LCMS: 199.2
[M?H?].
2-(3-Nitrophenyl)-5-nitro-1H-benzimidazole (24) Orange
solid; Yield: 72 %; m.p. 281–282 °C (Romero-Castro
et al., 2011). IR (KBr, cm-1): 3,359 (NH); 3,069 (CH
2-(Thiophen-2-yl)-1H-benzimidazole
(19) Off-white
solid; Yield: 90 %; m.p. [300 °C (Schiffmann et al.,
2006). IR (KBr, cm-1): 3,348 (NH); 3,122 (CH aromatic);
1,613 (C=N); 1,530 (C=C); 945 (C–S). 1H NMR
(300 MHz, DMSO-d6): d 6.98–7.14 (m, 3H, H30, H-40,
H-50); 7.27 (m, 2H, H-4, H-7); 7.70 (m, 2H, H-4, H-7);
10.87 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
116.10; 124.17; 125.43; 127.51; 127.87; 138.82; 141.58;
143.80. LCMS: 201.1 [M?H?].
1
aromatic); 1,613 (C=N); 1,553 (C=C); 1,359 (NO2). H
NMR (300 MHz, DMSO-d6): d 7.86–7.97 (m, 3H, H-7,
H-50, H-60); 8.17–8.36 (m, 3H, H-6, H-20, H-40); 8.63 (bs,
1H, H-4); 10.97 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-
d6): 111.34; 116.15; 118.30; 121.97; 123.62; 130.35;
131.75; 134.66; 139.92; 142.60; 144.31;148.21; 152.50.
LCMS: 285.2 [M?H?].
2-Phenyl-5-nitro-1H-benzimidazole (20) Light yellow
´
2-(4-Nitrophenyl)-5-nitro-1H-benzimidazole (25) Brown
solid; Yield: 76 %; m.p. 265–266 °C (Romero-Castro
et al., 2011). IR (KBr, cm-1): 3,371 (NH); 3,121 (CH
solid; Yield: 79 %; m.p. 148–149 °C (Navarrete-Vazquez
et al., 2010). IR (KBr, cm-1): 3,380 (NH); 3,126 (CH
1
1
aromatic), 1,614 (C=N); 1,545 (C=C); 1,367 (NO2). H
aromatic); 1,612 (C=N); 1,537 (C=C); 1,361 (NO2). H
NMR (300 MHz, DMSO-d6): d 7.47–7.58 (m, 5H, H-20,
H-30, H-40, H-50, H-60); 7.94–8.70 (m, 3H, H-4, H-6, H-7);
10.21 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
112.37; 115.53; 119.82; 127.58; 128.64; 129.83; 130.91;
140.65; 142.93; 143.19 152.81. LCMS: 240.1 [M?H?].
NMR (300 MHz, DMSO-d6): d 7.75–7.96 (m, 3H, H-7,
H-20, H-60); 8.21–8.25 (m, 3H, H-6, H-30, H-50); 8.63 (bs,
1H, H-4); 11.03 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-
d6): 112.63; 116.30; 118.58; 122.34; 128.90; 135.18;
139.61; 142.70; 144.32; 148.85; 152.81. LCMS: 285.2
[M?H?].
2-(4-Chlorophenyl)-5-nitro-1H-benzimidazole (21) Yel-
low solid; Yield: 77 %; m.p. 275–276 °C (Jain et al.,
2010). IR (KBr, cm-1): 3,274 (NH); 3,056 (CH aromatic);
2-(3-Methoxyphenyl)-5-nitro-1H-benzimidazole
(26) Light yellow solid; Yield: 81 %; m.p. 187–189 °C.
IR (KBr, cm-1): 3,367 (NH); 3,086 (CH aromatic); 1,608
(C=N); 1,534 (C=C); 1,337 (NO2); 1,215 (C–O–C). 1H
NMR (300 MHz, DMSO-d6): d 3.81 (s, 3H, O–CH3); 5.11
(bs, 1H, NH); 6.81–7.01 (m, 4H, H-20, H-40, H-50, H-60);
7.95–8.15 (m, 2H, H-6, H-7); 8.59 (bs, 1H, H-4); 10.71 (bs,
1H, NH). 13C NMR (75 MHz, DMSO-d6): 55.68; 112.18;
113.95; 114.92; 116.46; 118.18; 121.19; 129.30; 132.71;
139.77; 142.67; 145.14; 152.73; 161.34. LCMS: 270.2
[M?H?].
1
1,614 (C=N); 1,545 (C=C); 1,377 (NO2); 742 (Ar–Cl). H
NMR (300 MHz, DMSO-d6): d 7.24–7.57 (m, 4H, H-20,
H-30, H-50, H-60); 7.91–8.63 (m, 3H, H-4, H-6, H-7); 10.86
(bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6): 113.15;
116.21; 119.54; 127.71; 129.57; 130.32; 134.56; 140.61;
142.97; 143.19; 152.83. LCMS: 274.1 [M?H?].
2-(2-Chlorophenyl)-5-nitro-1H-benzimidazole (22) Light
yellow solid; Yield: 79 %; m.p. 223–224 °C. IR (KBr,
cm-1): 3,376 (NH); 3,090 (CH aromatic); 1,613 (C=N);
1,542 (C=C); 1,356 (NO2); 744 (Ar–Cl). 1H NMR
(300 MHz, DMSO-d6): d 7.33–7.46 (m, 4H, H-30, H-40,
2-(4-Methoxyphenyl)-5-nitro-1H-benzimidazole (27) Yellow
solid; Yield: 83 %; m.p. 237–239 °C (Estrada-Soto et al.,
123

Med Chem Res (2013) 22:3606–3620
3617
2006). IR (KBr, cm-1): 3,342 (NH); 3,139 (CH aromatic);
2,921 (CH aliphatic); 1,620 (C=N); 1,564 (C=C); 1,369
(NO2); 1,225 (C–O–C). 1H NMR (300 MHz, DMSO-d6): d
3.83 (s, 3H, O–CH3); 6.91–7.21 (m, 4H, H-20, H-30, H-50,
H-60); 7.76–8.11 (m, 2H, H-6, H-7); 8.49 (bs, 1H, H-4);
10.90 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
55.49; 110.61; 114.67; 115.94; 118.72; 122.78; 128.52;
139.93; 142.81; 144.74; 152.68; 160.73. LCMS: 270.2
[M?H?].
13C NMR (75 MHz, DMSO-d6): 23.63; 37.19; 110.64;
116.23; 118.16; 126.87; 127.50; 128.33; 139.73; 142.81;
144.79; 150.10; 152.83. LCMS: 282.2 [M?H?].
2-(Pyridin-2-yl)-5-nitro-1H-benzimidazole
(32) Pink
solid; Yield: 81 %; m.p. 212–213 °C (Schiffmann et al.,
2006). IR (KBr, cm-1): 3,328 (NH); 3,056 (CH aromatic);
1,625 (C=N); 1,564 (C=C); 1,351 (NO2). 1H NMR
(300 MHz, DMSO-d6): d 7.61–7.82 (m, 3H, H-30, H-40,
H-50); 7.94–8.13 (m, 2H, H-6, H-7); 8.45–8.56 (m, 2H,
H-4, H-60); 10.61 (bs, 1H, NH). 13C NMR (75 MHz,
DMSO-d6): 112.13; 116.15; 117.98; 123.83; 120.93;
124.11; 136.82; 139.90; 142.73; 144.61; 149.69; 155.14.
LCMS: 241.2 [M?H?].
2-(4-Methylphenyl)-5-nitro-1H-benzimidazole (28) Light
yellow solid; Yield: 84 %; m.p. 205–207 °C (Jain et al.,
2010). IR (KBr, cm-1): 3,258 (NH); 3,050 (CH aromatic);
2,916 (CH aliphatic); 1,608 (C=N); 1,563 (C=C); 1,366
1
(NO2). H NMR (300 MHz, DMSO-d6): d 2.33 (s, 3H,
CH3); 7.12–7.35 (m, 4H, H-20, H-30, H-50, H-60); 7.86-8.13
(m, 2H, H-6, H-7); 8.39 (bs, 1H, H-4); 10.44 (bs, 1H, NH).
13C NMR (75 MHz, DMSO-d6): 25.97; 112.22; 115.95;
118.73; 127.18; 127.63; 128.90; 137.72; 140.10; 142.61;
144.93; 152.65. LCMS: 254.2 [M?H?].
2-(Pyridin-3-yl)-5-nitro-1H-benzimidazole
(33) Off-
white solid; Yield: 79 %; m.p. 260–261 °C (Jain et al.,
2010). IR (KBr, cm-1): 3,329 (NH); 3,059 (CH aromatic);
1,590 (C=N); 1,562 (C=C); 1,350 (NO2). 1H NMR
(300 MHz, DMSO-d6): d 7.42 (m, 1H, H-50); 7.96–8.13
(m, 3H, H-6, H-7, H-40); 8.39–8.49 (m, 2H, H-4, H-60);
8.78 (s, 1H, H-20); 10.81 (bs, 1H, NH). 13C NMR (75 MHz,
DMSO-d6): 112.26; 116.34; 118.19; 124.05; 132.96;
134.23; 138.94; 142.95; 144.34; 147.92; 149.10; 152.68.
LCMS: 241.2 [M?H?].
2-(4-Fluorophenyl)-5-nitro-1H-benzimidazole (29) Light
yellow solid; Yield: 86 %; m.p. 264–265 °C (Goker et al.,
2002). IR (KBr, cm-1): 3,410 (NH); 3,146 (CH aromatic);
1,623 (C=N); 1,545 (C=C); 1,366 (NO2); 1,040 (Ar–F). 1H
NMR (300 MHz, DMSO-d6): d 7.10–7.42 (m, 4H, H-20,
H-30, H-50, H-60); 7.83–8.16 (m, 2H, H-6, H-7); 8.45 (bs,
1H, H-4); 10.95 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-
d6): 113.59; 115.98; 116.77; 118.13; 126.82; 129.27;
139.93; 142.66; 145.34; 152.90; 171.15. LCMS: 258.2
[M?H?].
2-(1H-indol-3-yl)-5-nitro-1H-benzimidazole (34) Light
brown solid; Yield: 68 %; m.p. 213–215 °C. IR (KBr,
cm-1): 3,334 (NH); 3,067 (CH aromatic); 1,586 (C=N);
1
1,552 (C=C); 1,343 (NO2). H NMR (300 MHz, DMSO-
d6): d 7.05–7.55 (m, 5H, H-20, H-40, H-50, H-60, H-70);
7.93–8.13 (m, 2H, H-6, H-7); 8.39 (s, 1H, H-4); 10.23 (bs,
1H, NH); 10.91 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-
d6): 106.72; 110.43; 111.19; 116.37; 118.12; 119.27;
121.23; 122.70; 128.55; 131.76; 135.29; 138.74; 142.61;
144.82; 152.98. LCMS: 279.2 [M?H?].
2-(4-Butoxyphenyl)-5-nitro-1H-benzimidazole (30) Light
yellow solid; Yield: 81 %; m.p. 191–193 °C. IR (KBr,
cm-1): 3,361 (NH); 3,143 (CH aromatic); 2,883 (CH ali-
phatic); 1,618 (C=N); 1,556 (C=C); 1,378 (NO2); 1,232
1
(C–O–C). H NMR (300 MHz, DMSO-d6): d 0.96 (t, 3H,
00
CH3 ); 1.34 (q, 2H, CH2-300); 1.70 (m, 2H, CH2-200); 3.93
2-(1H-pyrrol-2-yl)-5-nitro-1H-benzimidazole (35) Orange
solid; Yield: 71 %; m.p. 186–188 °C. IR (KBr, cm-1):
3,363 (NH); 3,108 (CH aromatic); 1,615 (C=N); 1,548
(t, 2H, CH2–O); 6.87–7.37 (m, 4H, H-20, H-30, H-50, H-60);
7.86–8.17 (m, 2H, H-6, H-7); 8.53 (bs, 1H, H-4); 11.01 (bs,
1H, NH). 13C NMR (75 MHz, DMSO-d6): 15.13; 21.28;
31.70; 69.27; 110.54; 114.45; 116.34; 118.36; 124.73;
128.30; 139.59; 142.81; 144.97; 151.90; 157.69. LCMS:
312.2 [M?H?].
1
(C=C); 1,358 (NO2). H NMR (300 MHz, DMSO-d6): d
6.12–6.59 (m, 3H, H-30, H-40, H-50); 7.96–8.19 (m, 2H,
H-6, H-7); 8.49 (s, 1H, H-4); 10.13 (bs, 1H, NH); 10.35 (bs,
1H, NH). 13C NMR (75 MHz, DMSO-d6): 106.75; 108.53;
110.98; 116.14; 118.21; 120.33; 122.94; 139.79; 142.90;
145.06; 154.86. LCMS: 229.1 [M?H?].
2-(4-i-Propylphenyl)-5-nitro-1H-benzimidazole (31) Yellow
solid; Yield: 80 %; m.p. 213–214 °C. IR (KBr, cm-1):
3,258 (NH); 3,050 (CH aliphatic); 2,926 (CH aliphatic);
1,614 (C=N); 1,563 (C=C); 1,366 (NO2). 1H NMR
(300 MHz, DMSO-d6): d 1.30 (d, 6H, CH3); 3.12 (q, 1H,
CH); 7.21–7.41 (m, 4H, H-20, H-30, H-50, H-60); 7.83–8.11
(m, 2H, H-6, H-7); 8.47 (bs, 1H, H-4); 10.83 (bs, 1H, NH).
2-(5-Methylfuran-2-yl)-5-nitro-1H-benzimidazole (36) Brown
solid; Yield: 78 %; m.p. 149–150 °C. IR (KBr, cm-1):
3,342 (NH); 3,139 (CH aromatic); 2,916 (CH aliphatic);
1,612 (C=N); 1,527 (C=C); 1,369 (NO2); 1,225 (C–O–C).
1H NMR (300 MHz, DMSO-d6): d 2.16 (s, 3H, CH3);
123

3618
Med Chem Res (2013) 22:3606–3620
6.12–6.24 (m, 2H, H-30, H-40); 7.87–8.08 (m, 2H, H-6,
H-7); 8.45 (s, 1H, H-4); 10.43 (bs, 1H, NH). 13C NMR
(75 MHz, DMSO-d6): 14.28; 102.13; 107.87; 112.34;
116.79; 118.10; 139.92; 141.31; 142.74; 144.89; 150.90;
152.14. LCMS: 244.1 [M?H?].
Canada. The program operated under ‘‘Windows Server
2003 R2’’ operating system installed on an IBM System
93400 with four Intel(R) Xeon(TM) CPU 3.00 GHz pro-
cessors and 2048 RAM.
The target compounds were built using the builder
interface of the MOE program and subjected to energy
minimization tool by means of the included MOPAC 7.0.
The produced model was subjected Systematic Confor-
mational Search where all the atoms were set as default
with RMS gradient of 0.01 kcal/mol and RMS distance of
2-(Thiophen-2-yl)-5-nitro-1H-benzimidazole (37) Light
yellow solid; Yield: 79 %; m.p. 146–147 °C. IR (KBr,
cm-1): 3,380 (NH); 3,126 (CH aromatic); 1,613 (C=N);
1,530 (C=C); 1,367 (NO2); 940 (C–S). 1H NMR
(300 MHz, DMSO-d6): d 6.97–7.25 (m, 3H, H-30, H-40,
H-50); 7.92–8.15 (m, 2H, H-6, H-7); 8.43 (s, 1H, H-4);
10.81 (bs, 1H, NH). 13C NMR (75 MHz, DMSO-d6):
110.78; 116.25; 118.01; 125.31; 127.68; 128.14; 139.73;
141.59; 142.64; 143.51; 144.89. LCMS: 246.1 [M?H?].
˚
0.1 A.
The data of high resolution crystal structure of GP63
were obtained from Protein Data Bank (PDB code: 1LML)
(Schlagenhauf et al., 1998). The crystal structure was
imported into MOE and all hydrogen atoms were added to
structure with their standard geometry followed by their
energy minimization using MOPAC 7.0. The water mole-
cules were removed using sequence editor interface of the
MOE program. The resulting model was subjected to sys-
tematic conformational search at default parameters with
RMS gradient of 0.01 kcal/mol using Alpha Site Finder for
the active site search in the enzyme structure and dummy
atoms were created from the obtained Alpha spheres. The
docking studies were performed on the prepared enzyme
by Triangle Matcher method in conjunction by means of
post-placement refinement method Forcefield. Rescoring 1
and 2 were selected London dG and Affinity dG, respec-
tively. The obtained ligand–enzyme complex model was
then used in calculating the energy parameters by means of
MMFF94x force field energy calculation and predicting the
ligand–enzyme interactions at the active site. The lowest
energy minimized pose was used for further analysis. Rule
of five parameters (Ro5) were calculated by ligand prop-
erty calculation function of MOE.
Antileishmanial activity
Antileishmanial activity of the library was assayed by
following the method reported by Zhai et al. (1999) using a
pre-established culture of clinical isolate of L. major
obtained from National Institute of Health (NIH), Islama-
bad, Pakistan. Promastigotes were cultured in medium 199
(Cassion Laboratories, Inc. USA) containing 10 % fetal
bovine serum (ICN Flow, UK), HEPES (4-(2-hydroxy-
ethyl)-1-piperazineethanesulfonic acid), sodium bicarbon-
ate (Sigma), penicillin (ICN Biochemicals, Inc. Germany),
and streptomycin (Sigma). Incubation and growth of the
parasite were carried out at 24 °C. Briefly after 6–7 days of
the culture, the promastigotes were centrifuged at
3,000 rpm for 3 min, the supernatant was discarded and the
pellet was washed three times with phosphate buffer saline
(MP-Biomedicals, Inc. France) and resuspended in medium
199 at 2 9 106 cell/mL.
Promastigotes were seeded in 96-well round bottom
microtiter plates (TPP, Switzerland) containing serial
dilution of the compound and media 199. Amphotericin B
(BioChemica, Germany) and DMSO were used as positive
and negative controls, respectively. The plates were incu-
bated for 72 h at 24 °C in shaker incubator. All the com-
pounds were assayed in triplicate and the number of alive
parasites was determined by counting in Neubauer cham-
ber. The inhibitory concentrations for the 50 % of the
inhibition IC50 were calculated by GraphPad Prism
(GraphPad Prism Software, Inc. USA) software and data
reported as the mean SD.
Acknowledgments Mr. Awais Shaukat gratefully acknowledges the
Higher Education Commission, Pakistan (HEC), for the award of
Indigenous PhD 5000 Fellowship Program Phase III scholarship. Prof.
Farzana Latif Ansari owes her thanks to HEC for financial support to
conduct this study.
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