Nitric oxide inhibition, antioxidant, and antitumour activities of novel copper(ii) bis-benzimidazole diamide nanocoordination complexes

Article abstract of DOI:10.1039/c4nj02147a

Novel copper(ii) nanocoordination complexes [LCuCl]Cl C1, [LCu(SCN)]SCN C2, and [LCu(NO₃)]NO₃C3 capped by bis-benzimidazole diamide ligand N¹,N⁵-bis{(1H-benzo[d]imidazole-2-yl)methyl}glutarimide L were synthesized and characterized by spectroscopic methods viz. transmission electron microscopy (5-100 nm), dynamic light scattering, elemental analysis, EPR, and infrared spectroscopy. These complexes were also tested for their ability to inhibit nitric oxide release during culture. Lipopolysaccharide-induced superoxide production decreased most significantly for complex C2. Nanocoordination complex C2 also showed the most significant in vitro cytotoxicity against HeLa human cervical cancer cells, MCF-7 breast cancer cells, and U-87 glioblastoma cells, ranging from 50 μg ml^-1 to 500 μg ml^-1. In vivo study revealed an increase in body weight (11.5 g) for the tumour control groups, whereas in the case of the mouse groups treated with test sample C3, the difference in body weight as compared to the control was approximately 4.7 g. In further studies, it decreased to 2.2 g and 0.6 g in the case of mouse groups treated with nanocoordination complexes C1 and C2, respectively. The mean mouse survival time was 21 days, which increased to 100, 120, and 170 days for C3, C1, and C2, respectively.

Full text of DOI:10.1039/c4nj02147a

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Nitric oxide inhibition, antioxidant, and antitumour
activities of novel copper(II) bis-benzimidazole
diamide nanocoordination complexes†
Cite this:DOI: 10.1039/c4nj02147a
Manisha Singla,a Rajeev Ranjan,b Kuldeep Mahiya,c Subash C. Mohapatra*d and
Sharif Ahmade
Novel copper(II) nanocoordination complexes [LCuCl]Cl C1, [LCu(SCN)]SCN C2, and [LCu(NO3)]NO3 C3
capped by bis-benzimidazole diamide ligand N1,N5-bis{(1H-benzo[d]imidazole-2-yl)methyl}glutarimide L
were synthesized and characterized by spectroscopic methods viz. transmission electron microscopy
(5–100 nm), dynamic light scattering, elemental analysis, EPR, and infrared spectroscopy. These complexes
were also tested for their ability to inhibit nitric oxide release during culture. Lipopolysaccharide-induced
superoxide production decreased most significantly for complex C2. Nanocoordination complex C2 also
showed the most significant in vitro cytotoxicity against HeLa human cervical cancer cells, MCF-7 breast
cancer cells, and U-87 glioblastoma cells, ranging from 50 mg mlꢀ1 to 500 mg mlꢀ1. In vivo study revealed
an increase in body weight (11.5 g) for the tumour control groups, whereas in the case of the mouse
groups treated with test sample C3, the difference in body weight as compared to the control was
approximately 4.7 g. In further studies, it decreased to 2.2 g and 0.6 g in the case of mouse groups treated
with nanocoordination complexes C1 and C2, respectively. The mean mouse survival time was 21 days,
which increased to 100, 120, and 170 days for C3, C1, and C2, respectively.
Received (in Montpellier, France)
28th November 2014,
Accepted 16th March 2015
DOI: 10.1039/c4nj02147a
www.rsc.org/njc
peroxynitrite (ONOOꢀ), and nitrogen dioxide (NO2) also play an
important role in inflammatory processes6,7 and carcinogenesis.8
1. Introduction
The development of modern technologies allows us to solve Thus, it is very important to keep ROS and RNS at normal levels,
many therapeutic, medical, and vetting problems. In the develop- otherwise they can play a major role in mutations, which cause
ment of new materials for effective drug delivery systems, diag- DNA damage.9–11
nostics, and control over patient treatment, nanocoordination
Metal complexes have been extensively used as potential
complexes are used for new medicines, biological applications, antitumour agents viz. the use of cis-platin in cancer chemo-
and preparations. Many natural biological sites, such as the therapy,12 while mixed ligand complexes play a key role in a
active centres of several metalloenzymes, function with the variety of biological systems.13 These inventions lead us to
assistance of transition metal ions; thus, synthetic mononuclear the specific structural shapes of drug-like entities13,14 that have
and binuclear receptors can be used to mimic the active been implicated in the storage and transport of active substances
centres,1–4 which might be helpful for healthy organisms. In through membranes. Among these ligands, benzimidazole-
healthy organisms, the production of reactive oxygen species (ROS) containing metal complexes and their derivatives are progres-
and reactive nitrogen species (RNS) is approximately balanced by sively used in bioinorganic systems,15 while monomer copper(II)
antioxidant defence systems and is related to oxidative stress.5 benzimidazole complexes exhibit a broad spectrum of biological
Recent studies have shown that RNSs such as nitric oxide (NO), activities.16,17 The benzimidazole moiety is a structural isostere of
naturally occurring nucleotides, and it has been extensively
utilized as a drug scaffold. Compounds containing the benzimi-
dazole nucleus have a wide spectrum of biological activity such as
antifungal,18,19 antibacterial,20,21 antimicrobial,22,23 antiamoebic,24,25
antiparasitic,26 and antitumour.27,28
a Department of Chemistry, Keshav Mahavidhyalya, University of Delhi,
New Delhi, India
b Department of Zoology, University of Delhi, New Delhi, India
c Department of Chemistry, Ch. Devi Lal University, Sirsa, Haryana, India
d Department of Chemistry, Atma Ram Sanatan Dharma College,
University of Delhi, New Delhi, India. E-mail: subashcm@gmail.com;
Fax: +91 11 24111390; Tel: +91 11 24113436, ext. 3268
e Department of Chemistry, Jamia Millia Islamia, New Delhi, India
Scientists have recently reported that benzimidazole analo-
gues can be suitably modified by the introduction of different
heterocyclic moieties that exhibit a broad spectrum of biological
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nj02147a activities such as potent anticancer and antifungal activity.29,30
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Benzimidazole derivatives serve as good ligands for transition surfactant that has changed the morphology and size of the
metal ions due to the large conjugated p-systems and the imine nanocoordination complex, resulting in more positive and effec-
nitrogen, which can positively affect the structures of the tive properties of the complex. Due to a change in the surfactant
complexes.31 The literature on different metal-containing nano- from neutral to cationic, the morphology of compound C3 has
coordination complexes shows that their toxicity is 7–50 times shifted from rod to sphere.35a Dynamic light scattering experi-
less than normal, due to their multifunctional and durable ments (Fig. 2) provide different information regarding the size
action, easy penetration into all organs and tissues, and stimu- of the nanocoordination complexes than that given by the TEM
lation of metabolic processes when used in biotic doses.32–34 studies.
Herein, we are reporting the synthesis and characterization of
The number of determinations/cumulants: 3 and standard
novel benzimidazole-containing copper(II) nanocoordination deviations of results ꢁ1–3 nm (Table 1). However, the hydration
complexes with significant in vivo and in vitro antioxidant as sphere was found to be smaller for the particles of SCNꢀ
well as antitumour activity. These complexes also significantly containing nanocoordination complex than those of the other
inhibit NO release in culture.
nanocoordination complexes.
2.2 UV-visible spectroscopy
2. Results
2.1 Transmission electron microscopy (TEM) and dynamic
The electronic spectra of the nanocoordination complexes were
recorded in methanol and show two strong bands in the UV
region, i.e., in the range of 273–284 nm assigned to the p–p*
transition in the benzimidazole group with extinction coeffi-
cients (log e) ranging from 4.09–4.30. A broad and much less
intense d–d band is observed in the region from 650–750 nm,
indicating the presence of coordinated copper(II) ion with
ligand L, while a shoulder at 300 nm is observed for the
complexes due to ligand to metal charge transfer (LMCT).36a,37
light scattering (DLS)
TEM images show a nearly spherical distribution of the nano-
coordination complexes (Fig. 1).
The average size of the nanocoordination complexes were
5–10 nm for C1 (with Clꢀ as the co-anion) and C2 (wiꢀth SCNꢀ
as the co-anion), and 80–100 nm for C3 (with NO3 as the
co-anion) (Table 1).
In a previous study, we reported the synthesis, performance,
and application of the C3 complex using Triton X-100, whereas
in this work we report the synthesis of C3 in the presence of
cetyl-trimethyl ammonium bromide (CTAB), which is a cationic
2.3 Infrared spectroscopy
Infrared spectra of diamide ligand L: 3198 (nNH amide), 3034
(nNH benzimidazole), 1643 (nCQO amide-I), 1569 (nC–N amide-II),
1441 (nCQN–CQC benzimidazole), 742 (nCQC benzene). 1643,
1569, and 1441 cmꢀ1 are assigned to amide-I (mainly nCQO
amide stretch), amide-II (mainly nC–N amide stretch), and
benzimidazole nCQN–CQC stretching frequencies, respectively.
The benzene ring vibrations appear at 742 cmꢀ1 (due to the
benzimidazole ring). The green coloured nanocoordination
complexes of copper(II) show an IR spectrum wherein the
amide-I shifts at approximately 1620 cmꢀ1, while the amide-II
and benzimidazole (nCQN–CQC) bands shift at approximately
1556 and 1450 cmꢀ1, respectively.
2.4 Electron paramagnetic resonance (EPR) spectroscopy
Fig. S1–S3 (ESI†) shows the EPR spectra of nanocoordination
complexes C1–C3. The value of gJ ranges from 2.27–2.32 and
g> from 2.03–2.07, which is greater than 2.0023 (Table 2).
2.5 Energy-optimized structure
Fig. 1 TEM micrograph of C2 (A), C1 (B), and C3 (C) nanocoordination The energy optimization of C1 was found to be 4745.5517 kcal molꢀ1
complexes.
(Fig. 3). The H-bond of this operation was found to be 299.576 with
Table 1 Size of the nanocoordination complexes obtained by TEM and DLS
Cu(II) nanocoordination
complex
DLS (nm) ꢁ 1–3 nma
Width of hydrational
sphere (nm)
TEM (nm)
number of determinations/cumulants: 3
[LCuCl]Cl (C1)
[LCuSCN]SCN (C2)
[LCu(NO3)]NO3 (C3)
8
8
90
37
27
107
15
10
15
a
Standard deviation in DLS measurement.
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Fig. 2 Size distribution plot for C2 (A), C1 (B), and C3 (C) nanocoordination complexes.
Table 2 X-band EPR data for copper(II) nanocoordination complexes at
120 K
Complex
gJ
g>
AJ (G) a2
(gJ/AJ)/10ꢀ4
[Cu(GBGA)Cl]Cl (C1)
[Cu(GBGA)(NO3)]NO3 (C3) 2.32 2.07 135
[Cu(GBGA)(SCN)]SCN (C2) 2.31 2.03 170
2.27 2.05 156
0.56 145
0.68 171
0.68 135
Fig. 4 The effect of pretreatment with 100 mg mlꢀ1 of three different nano-
coordination complexes on nitric oxide production by LPS-stimulated murine
macrophage cell line RAW 264.7. After 24 hours of treatment, the cell-free
supernatant from each well was taken, and nitrite levels were measured. Values
are expressed from six independent experiments performed in triplicate.
ligand L was found to be 5130.3028 kcal molꢀ1 (Fig. 3) with
stretch = 865.318, angle = 1358.690, stretch bend = ꢀ0.094,
dihedral = ꢀ0.014, improp torsion = 0.000, torsion stretch =
ꢀ6.612, bend bend = 15.385, van der Waals = 2142.139,
electrostatics = 12.940, hydrogen bond = 357.
2.6 Effect of copper(II) nanocoordination complexes on nitric
oxide synthesis
Lipopolysaccharide (LPS) (1 mg mlꢀ1) stimulated RAW 264.7
cells to release substantial amounts of NO during 24 h incuba-
tion (0.3 mM). The inhibitory effect on LPS-induced NO produc-
tion by the compounds is shown in Fig. 4. The test compounds
Fig. 3 Energy-optimized structures of monocationic copper(II) nano-
coordination complexes C1 (above left), C2 (above right), C3 (below left),
and ligand L (below right).
stretch
= 1236.581, angle = 1233.654, stretch bend =
ꢀ6.917, dihedral = 231.136, improp torsion = 63.858, torsion inhibited NO release when added to the culture. C1, C2, and C3
stretch = ꢀ10.790, bend bend = ꢀ5.960, van der Waals = reduced NO levels to 0.24, 2.1, and 2.5, mM respectively.
2187.081, electrostatics = ꢀ97.915. The energy optimization of
C2 was found to be 805.8313 kcal molꢀ1 (Fig. 3) with stretch =
186.692, angle = 177.754, stretch bend = ꢀ0.404, dihedral =
2.7 Effect of copper(II) nanocoordination complexes on
superoxide production
174.896, improp torsion = 35.482, torsion stretch = ꢀ1.839, A significant decrease in LPS-induced superoxide levels was
bend bend = ꢀ2.056, van der Waals = 165.035, electrostatics = observed 24 hours after administration of the test compounds
32.596, hydrogen bond = 405.861. The energy optimization of to RAW-264.7 cells (Fig. 5). All of the data points were plotted with
C3 was found to be 6758.2864 kcal molꢀ1 (Fig. 3) with stretch = respect to the LPS-induced superoxide level. The concentration of
1333.362, angle = 1050.934, stretch bend = ꢀ18.474, dihedral = the produced superoxide in LPS-treated cells was 0.25 mM. When
247.415, improp torsion = 29.944, torsion stretch = ꢀ12.656, test compounds were administered with LPS to the cells, super-
bend bend = 9.063, van der Waals = 4065.143, electrostatics = oxide levels decreased in terms of mM concentration. C1 and C3
ꢀ352.305, hydrogen bond = 37.676. The energy optimization of reduced the LPS-induced superoxide production from 0.235 and
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Fig. 7 The effect of copper(II) nanocoordination complexes on the inhibition
of cancer cell lines. The in vitro anti-cancer effect against HeLa cells was
determined by MTT assay. Cells were seeded in 96-well tissue culture
plates and treated with two concentrations (50 mg mlꢀ1 and 100 mg mlꢀ1
of three different nanocomplexes of copper(II) for different time periods
(24 h, 48 h, and 72 h). Values are expressed from six independent experiments
performed in triplicate.
)
Fig. 5 Effect of copper(II) nanocoordination complexes on superoxide
production in murine macrophage cell line RAW 264.7. Cells were pre-
treated with LPS (1 mg mlꢀ1) to induce a sufficient amount of superoxide
in the culture medium and were then treated with three different nano-
complexes of copper(II). After 24 hours of treatment, superoxide levels
were measured. Values are expressed from six independent experiments
performed in triplicate.
0.232 mM to 0.215 and 0.200 mM, respectively, while C2 also
significantly reduced superoxide from 0.244 mM to 0.152 mM.
2.8 Effect of copper(II) nanocoordination complexes on
cytotoxicity
The cytotoxicity of the test compounds was tested in vitro
against normal cell lines (murine peritoneal macrophage cells)
and human cancer cell lines consisting of HeLa human cervical
cancer cells, MCF-7 breast cancer cells, and U-87 glioblastoma
cells. As shown in Fig. 6 and 7, C2 exhibited significant
cytotoxicity towards HeLa cells at a concentration of 100 mg mlꢀ1
as compared to C1 and C3 under similar conditions.
Further cytotoxicity analysis of C2 showed that there was a
significant enhancement in toxicity in both a dose- and time-
dependent manner (Fig. 8–10).
Fig. 8 The inhibitory effect of copper(II) nanocoordination complex C2 on
cancer cell lines. The in vitro anti-cancer effect against HeLa cells was
determined by MTT assay. Cells were seeded in 96-well tissue culture plates
and treated with four different concentrations (50 mg mlꢀ1, 100 mg mlꢀ1
,
200 mg mlꢀ1, and 500 mg mlꢀ1) of C2 for different time periods (24 h, 48 h,
and 72 h). Values are expressed from six independent experiments performed
in triplicate.
HeLa, MCF-7, and U-87 cells exposed to different concen-
trations of C2 ranging from 50 mg mlꢀ1 to 500 mg mlꢀ1 showed
Fig. 9 The effect of copper(II) nanocoordination complex C2 on cancer cell
line inhibition. The in vitro anti-cancer effect against breast cancer cell line
MCF-7 was determined by MTT assay. Cells were seeded in 96-well tissue
culture plates and treated with four different concentrations (50 mg mlꢀ1
,
100 mg mlꢀ1, 200 mg mlꢀ1, and 500 mg mlꢀ1) of C2 for different time periods
(24 h, 48 h, and 72 h). Values are expressed from six independent experiments
performed in triplicate.
Fig. 6 Effect of copper(II) nanocoordination complexes on the toxicity of
normal cells. The in vitro cytotoxic effect against murine peritoneal
macrophage cells was determined by MTT assay. Cells were seeded in
96-well tissue culture plates and then treated with two concentrations
(50 mg mlꢀ1 and 100 mg mlꢀ1) of three different nanocomplexes of
copper(II) for different time periods (24 h, 48 h, and 72 h). Values are
expressed from six independent experiments performed in triplicate.
maximum toxicity at 500 mg mlꢀ1 concentrations at the 72 h
interval. In contrast, there was no toxicity against normal cell
lines (murine peritoneal macrophage cells).
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case of mouse groups treated with C1 and C2, respectively.
Fig. 11a shows a pictographic representation of the treated
mice whereas Fig. 11b is the graphical representation of the
body weight of the treated mice.
2.10 Effect of test compounds on survival of tumour-
transplanted mice
In the tumour control group, the mean survival time was
21 days, which increased to 100 days for mice treated with test
compound C3, while it was found to extend even up to 120 days
for mice treated with C1. In the case of C2 treatment, animals
survived even after 170 days with the gradual regression of the
tumour load (Fig. 12).
Fig. 10 The effect of copper(II) nanocoordination complex C2 on cancer
cell line inhibition. The in vitro anti-cancer effect against the U-87
glioblastoma cell line was determined by MTT assay. Cells were seeded
in 96-well tissue culture plates and treated with four different concentra-
tions (50 mg mlꢀ1, 100 mg mlꢀ1, 200 mg mlꢀ1, and 500 mg mlꢀ1) of C2 for
different time periods (24 h, 48 h, and 72 h). The values are expressed from
six independent experiments performed in triplicate.
3. Discussion
2.9 Effect of the test compounds on body weight of tumour-
transplanted mice
TEM images reveal that the nanocoordination complexes are
spherical, and each particle follows a symmetrical pattern. The
above results slightly differ in agreement with dynamic light
scattering experiments, giving a different size range in the
liquid state. However, the hydration sphere of the particles
was found to be larger in comparison to that of TEM values.
This may be due to solvent interaction with water. Some of our
In the case of the tumour control groups, the difference in body
weight increased to approximately 11.5 g, whereas in the case of
the mouse groups treated with test sample C3, the difference in
body weight as compared to the control was approximately
4.7 g. In further studies, it decreased to 2.2 g and 0.6 g in the
Fig. 11 (a) (above) Pictographic representations of Balb/c mice showing the effect of copper(II) nanocoordination complexes C1, C2, and C3 on body
weight of mice transplanted with tumours 7 days prior. (b) (below) The effect of copper(II) nanocoordination complexes C1, C2, and C3 on the body
weight of mice transplanted with tumours 7 days prior. Seven days after tumour transplantation, treatment was given to all the treated groups. C1 control,
C2 control, and C3 control groups received only test compounds C1, C2, and C3, respectively, whereas C1 treated, C2 treated, and C3 treated groups
were tumour transplanted 7 days prior to treatment with test compounds C1, C2, and C3, respectively. The above graph was plotted on the basis of body
weight differences of the treated groups with respect to tumour control groups after 30 days of treatment.
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Fig. 12 The effect of copper(II) nanocoordination complexes C1, C2, and C3 on the survival of mice that were tumor-transplanted 7 days prior.
Treatment was given after 7 days of tumour transplantation in all the treated groups. The C1 control, C2 control, and C3 control groups received only test
compounds C1, C2, and C3, respectively, whereas the C1 treated, C2 treated, and C3 treated groups were tumour-transplanted 7 days prior to treatment
with test compounds C1, C2, and C3, respectively.
complexes show extensive H-bonding interactions involving the amide carbonyl is shifted by approximately 15–20 cmꢀ1
coordinated, lattice, and solvent water molecules, the nitrogen with respect to the free ligand. This suggests that in the nano-
of the benzimidazole moiety, amide groups, and co-anions,35b coordination complexes, the ligand is bound to copper(II)
which could be the main reason to enhance the size of particles through the oxygen of amide carbonyl. This band shifting is
in the liquid state. However, the hydration sphere was found indicative of copper(II) bound to the ligand in the nanocoordi-
to be smaller for the SCNꢀ particles than those of other nation complexes.36a,37a The presence of nitrate and the thio-
complexes. The electronic spectra revealed two peaks in the cyanate anions in the respective complexes is justified by the
range of 272–284 nm, due to a pꢀp* transition in the –CQN– presence of strong IR bands at 1384 cmꢀ1 (nO–N–O sym str, ionic
CQC– group of benzimidazole (these bands are slightly blue- nitrate). Nitrate ion is confirmed by a strong band in the region
shifted with a lowered extinction coefficient as compared to of 1340 to 1385 cmꢀ1 (bound monodentate nitrate) for the
the ligand).36 The d–d band was observed in the region 650– compounds and 2074 cmꢀ1 (nC–N str.), which resembles
750 nm, while a shoulder at 300 nm was observed for the N-coordinated nC–N stretching, 846 cmꢀ1 (nC–S str.). Thus, the
copper(II) nanocoordination complexes due to LMCT.37 In general, IR data clearly reveal that the copper ions are bound to the bis-
these bands are assigned to the overlapping transition: dyz,zx
-
benzimidazole ligand in the nanocoordination complexes. The
dx2ꢀy2 and dxy - dx2ꢀy2. The low energy of the d–d transition X-band EPR data of copper(II) nanocoordination complexes
suggests a severely distorted tetragonal copper(II) nanocoordina- typically indicate a dx2ꢀy2 ground state (gJ 4 g> 4 2.0023).
tion complex in the solution state. CuCl2 salts in methanol show The solution state spectra of the nanocoordination complexes
d–d bands (lmax) at 900 nm. CuCl2 also gives a d–d band (lmax) at a result in less than four lines and the broadening of the g> line
greater wavelength than C1, which indicates that free copper(II) in some cases, thus indicating a severe distorted tetragonal
ions are different from coordinated Cu(II) nanocoordination geometry. The broadening of g> reveals a decreased symmetry
complexes. On the basis of the d–d spectra, we can say that there that manifests itself through rhombic splitting.
is no possibility of free copper(II) ion production from our nano-
The AJ and gJ values for the complexes do not lie on the
coordination complexes in the solution phase. Henceforth, Peisach Blumberg plots37a of AJ vs. gJ for two nitrogen and
copper(II) metal capped by the bis-benzimidazole group could two oxygen atoms in the structural plane, which suggests a
provide suitable anchoring sites on the nanosurface.
distortion of the equatorial plane. The factor gJ/AJ also
The free ligand shows characteristic single IR bands at 1643, indicates the distortion of coordination geometry. The higher
1569, and 1441 cmꢀ1. These are assigned to amide-I (mainly the value of this factor, the greater will be the distortion. This is
nCQO amide stretching), amide-II (mainly nC–N amide stretching), because when the geometry is no longer planar, the decreasing
and benzimidazole nCQN–CQC stretching frequencies, respec- interaction of the ground state dx2ꢀy2 orbital with ligand
tively. Benzene ring vibrations appear at 742 cmꢀ1 due to the orbitals results in decreased energy, and thus repulsion
benzimidazole ring. The ligand has a band at 3194 cmꢀ1, which between unpaired electrons in the dx2ꢀy2 orbital and the ligand
is assigned to the stretching mode of nN–H benzimidazole, which electron decreases. As a result, this unpaired electron is now
has previously been reported.35 The green coloured nanocoordi- more delocalised and couples with the copper nucleus to a
nation complexes of copper(II) show an IR spectrum wherein the lesser extent. Here, no nitrogen super hyperfine splitting could
amide-I shifted at 1620 cmꢀ1, while the amide-II and benzimi- be observed, implying non-planarity of the complexes. It was
dazole (nCQN–CQC) bands shifted to 1556 and 1450 cmꢀ1, respec- observed that gJ/AJ follows the order SCNꢀ o Clꢀ o NO3
,
ꢀ
tively, for the compounds containing the co-anions nitrate and indicating that the least amount of distortion in the coordination
chloride, respectively. In all cases, the stretching frequency for geometry exists for the thiocyanate nanocoordination complex.
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The thiocyanate nanocoordination complex might be one of cell lines and mediated cellular oxidative stress, promoted
the reasons for the significant inhibitory effect on NO syntheses membrane lipid peroxidation, and killed the cancer cells. The
and superoxide production, and it possesses more power to kill bis-2-benzimidazoles have also received attention in the past
cancer cells due to an increased planer and less distorted structure few years due to their anti-cancer activities. Recent develop-
as compared to the other two nanocoordination complexes.
ments for 2-substituted benzimidazoles have revealed varied
The covalency factor a2 has been calculated (Table 2) by heterocycles at the 2-position to yield potent anticancer agents
using the relationship37 gJ = 2.0023 ꢀ 8la2/XY. The ‘a2’ depends for various carcinoma cell lines. These include pyrimidine
on the nature of the copper–ligand bond and decreases with derivatives,41 pyrazoline derivatives,42 and thiazole derivatives.43
increasing covalency of the Cu–L bond. The minimum theore- Bis-benzimidazoles44a are another class of compounds exploited
tical value of a2 is 0.5, and the maximum is 1.0. The a2 of these as anticancer agents. Hoechst 3334244b,45 and Hoechst 3325846,47
nanocoordination complexes ranges from 0.56–0.68, revealing are the openers of this series that exhibit in vitro antitumour
a very good amount of covalent character in the Cu–L bond. It as well as DNA topoisomerase I inhibitory activities.
is very convincing to note that the chloro nanocoordination
complex in this series is relatively more covalent, while the nitrate were found to be the most potent anticancer synthetic precursor
and thiocyanate nanocoordination complexes are more ionic. of bis-benzimidazoles against human A-549, BFTC-905, RD,
Huang et al. have synthesized benzimidazole isosteres that
The increasing order of covalency reflects the decreasing MES-SA, and HeLa carcinoma cell lines.48 Benzimidazolyl-1,2,4-
order of visible band energy, i.e., the greater covalency results in triazino[4,5-a]benzimidazol-1-one is another bis-benzimidazole
a lowering of energy of the orbital, and henceforth, the energy analogue having significant activity against multidrug-resistant
of the dxz,yz/dxy - dx2ꢀy2 transition is lowered. This is proven in P-glycoprotein-expressing cell lines.49 Complexes of benzimid-
the lmax of the d–d band in the visible spectral data of these azoles acting as ligands with transition metal ions possess
nanocoordination complexes.
antitumour activity. The Cu(II) complex of benzimidazolylmethyl-
The present study reports the inhibitory effect of nanocoor- 1,3-diaminopropane has the ability to intercalate into the double
dination complexes on the release of nitric oxide production, helix of DNA.50 A Cu(II) complex of 2-pyridinylbenzimidazole-5-
which acts as an antioxidant and exhibits in vitro cytotoxicity carboxylic acid has been found to exert potent topoisomerase II
and anti-tumour activity. C2 exhibited strong dose-dependent inhibitory activity.51a Thus, interest in the study of amide
cytotoxic activity against the different cell lines due to a poten- complexes is derived from their ability to model active sites
tially quantum dot range (5–10 nm) for co-anion thiocyanate. present in some metalloproteins and enzymes. The polypeptide
Thiocyanate has back-bonding36b ability and donates electrons chain surrounding the active site provides a suitable hydro-
from ligand to metal and vice versa. Another structural aspect philic environment. The imine nitrogen of benzimidazole and
of these nanocoordination complexes is that they attain more histidine has similar coordinating abilities (pKa 5.5 and 6.0,
surface area, which subsequently enhances the surface and bulk respectively) that favor the pH control of metal ion coordination
properties. The other two nanocoordination complexes C1 and to the imidazole nitrogen atom. Hence, benzimidazole has
C3 also exhibited a significant inhibitory effect on NO syntheses been considered to be a good mimic of histidine. The inducing
and superoxide production, but they were found to exert a agent LPS (1 mg mlꢀ1) was used to produce reactive oxygen,
decreased inhibitory effect as compared to C2 due to a larger which is also inhibited by protons of the amide and benzimidazole
nanosize and the effect of co-anions. The shape and size of a of the ligand moiety.
nanocomplex is important for how easily it can penetrate into a
The energy-optimized structural analysis of nanocoordination
tumour.37b The bis-benzimidazole diamide ligand, which is complexes and ligand L reveals a significant interconnection
coordinated by metallic salts, also plays a vital role by acting between the stability of nanocoordination complexes and inhibi-
as an antioxidant and an antitumour agent. The presence of tory capability. The energy and number of hydrogen bonds of the
imine nitrogen and carbonyl groups might be functionalized by optimized structure of C2 was found to be 805.8313 kcal molꢀ1
a H-bonding network that would further inhibit NO release. The and 405, respectively, which is quite different from ligand L. C2
presence of the proton of the NH amide, NH benzimidazole, and has lower optimized energy in comparison to compounds C1
CO amide may be attributed to a greater extent of H-bonding, and C3, reemphasizing the increased stability and additional
which also contributes to inhibiting the production of NO and hydrogen-bonding network that might tend to act as an anti-
superoxide.
tumour agent. The energy and number of hydrogen bonds
Recently, several reports have appeared in the literature for optimized structures of C1, C3, and ligand L are found to
describing the anti-cancer activity of copper(II) derivatives of be 4745.5517 kcal molꢀ1, 299; 6758.2864 kcal molꢀ1, 37; and
several classes of nitrogen donors. Their mechanism of action 5130.3028 kcal molꢀ1, 357, respectively. These data are attri-
appears to be different from that of cis-platin. There are many buted to the inter- and intra-connection of their stabilities,
copper complexes that cause induced apoptosis in cultured particle size, and their reactivity.
mammalian cells39,40 and are known to mediate significant
Various anticancer agents (also referred to as antitumour,
cellular oxidative stress, promote membrane lipid peroxidation, antiproliferative, and antineoplastics) reported for the treatment
and interfere with mitochondrial respiratory activity in fungal of various types of cancers act through different mechanisms.
cells. Similarly C1, C2, and C3 reported in the present work However, the major side effect associated with these agents is
induced apoptosis in HeLa, MCF-7, and U-87 cultured cancer cytotoxicity towards normal cells (murine macrophage cells) due
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to lack of selectivity for the abnormal cells, which is why the nanocoordination complexes could be used as a potent anti-cancer
search for an anticancer agent has been conducted for many inhibitor or drug in the future.
years. Benzimidazole is an isostere of purine-based nucleic acids
and is an important scaffold in various biologically active mole-
cules; it has been widely explored as an anticancer agent.51b
In the in vivo study, the initial mean survival time for the
tumour control group was 21 days. For the mice treated with
5. Experimental section
5.1 Materials and methods
test compound C3, C1, and C2, the survival time extended to Roswell Park Memorial Institute media (RPMI 1640) was pur-
100 days, 120 days, and 170 days, respectively, with the gradual chased from Gibco TM, Griess reagent, aprotinin, leupeptin,
regression of tumour load. The in vivo results also show the phenylmethylsulfonylfluoride (PMSF), [N-(2-hydroxyethyl)-
impact of size and the nature of the co-anion, which were piperazine-N0-(2-ethanesulfonic acid)] (HEPES), dimethyl sulfoxide,
attributed to the function of the benzimidazole moiety, the NaHCO3, Escherichia coli LPS, and all other required chemicals
size of the nanocoordination complexes, and the role of the were purchased from Sigma (St. Louis, MO, USA). Fetal bovine
co-anion. These results confirmed that the shape and size of serum (FBS) and trypsin–EDTA were purchased from Gibco-BRL
nanocoordination complexes could influence their in vivo (Gaithersburg, MD, USA). Ligand L was synthesized as previously
toxicity.37b
All compounds exhibited an inhibitory effect upon LPS-
reported.35a,37a
In vitro studies were performed in order to evaluate the nitric
induced NO production. Pre-incubation of macrophage cells oxide inhibition, antioxidant, and anti-cancer activity of the
with different solids effectively inhibited NO production in a test compounds. This study used an in vitro model of mouse
concentration-dependent manner. This inhibition was not due macrophage cell line RAW 264.7 cells activated with LPS or
to the cytotoxicity of the compounds, as indicated by cell PMA to induce nitric oxide and superoxide production. The
viability values. The role of the free radical (NO) in inflamma- inhibitory mechanism of nitric oxide production by the extract
tory processes is well known.52 Free radicals liberated from was limited to the measurement of nitrite concentration in
phagocyte cells are important in inflammatory processes micromolar concentrations. Inhibition of superoxide was assessed
because they are implicated in the activation of nuclear factor by the NBT reduction assay. Swiss albino mice were used as a
kB (NF-kB), which induces the transcription of inflammatory source of macrophages for in vitro studies. An inbred strain of
cytokines and COX-2. Furthermore, antioxidants have been shown pathogen-free mice of either sex was used for various studies. The
to be able to effectively block the activation of NF-kB through the mice were maintained at the animal facility of the Institute of
stabilization of the NF-kB/IkB-a complex.53 The results of this Genomics and Integrative Biology, New Delhi, India. They were
study showed that these compounds contain constituents that housed in polycarbonate cages and were provided with tap water
can inhibit the production of NO and superoxide.
and an animal diet ad libitum. Animals were weighed, individually
identified by tail marks, and assigned to homogeneous weight
groups. The animal room was maintained between 18–26 1C with
light/dark cycles at 12 h intervals.
4. Conclusions
In vitro studies were performed in order to evaluate the
A novel class of copper(II) nanocoordination complexes (C1, C2, immunomodulatory and anti-cancer activity of the test com-
and C3) functionalized by bis-benzimidazole diamide ligand L pounds. This study used an in vitro model of mouse macrophage
have been synthesized and characterized by various spectroscopic cell line RAW 264.7 cells activated with LPS or phorbol-12-
methods. These complexes inhibited nitric oxide release and LPS- myristate-13-acetate (PMA) to induce nitric oxide and super-
induced superoxide production, and also exhibited significant oxide production. The inhibitory mechanism of nitric oxide
in vitro cytotoxicity against the human cervical cancer cell line production by the extract was limited to the measurement of
HeLa, breast cancer cell line MCF-7, and glioblastoma cell line nitrite concentration in mM. Inhibition of superoxide was
U-87 at different concentrations, whereas they were non-toxic assessed by NBT reduction assay. The cells (RAW 264.7, MCF-7,
against normal cells (murine peritoneal macrophage cells). and glioblastoma cell line U-87) were cultured and maintained in
In vivo study also revealed the same pattern of toxicity against RPMI 1640 medium containing 2 mM glutamine, 10 mM HEPES,
tumour-bearing mice. The body weight of tumour-bearing mice 1 mM sodium pyruvate adjusted to contain 1.5 g lꢀ1 sodium
significantly decreased by varying degrees for all the compounds bicarbonate, 10% heat-inactivated fetal bovine serum, 100 U mlꢀ1
tested. C2 produced the maximum reduction in body weight, penicillin, 100 mg mlꢀ1 streptomycin, and 50 U mlꢀ1 gentamicin.
whereas C3 produced the least, which is totally interconnected Cells were grown at 37 1C with 5% CO2 in humidified air. Dalton’s
with the function of size and the nature of co-anions of nano- lymphoma (DL) cells were maintained in vivo in Swiss albino
coordination complexes. Furthermore, the survival of the mice, by intraperitoneal transplantation of 1 ꢂ 106 cells per
treated mice potentiated the pattern of action of the nano- mouse after every 7 days. The animal ethics committee of
coordination compounds, as the tumour-bearing mice treated Department of Zoology, University of Delhi approved the pro-
with C2 survived approximately 150 days more as compared to the posal, and animals were handled according to the guidelines
tumour control group. It decreased to 100 and 80 days in the case of the Committee for Purpose of Control and Supervision of
of the C3- and C1-treated groups, respectively. In conclusion, these Experiments on Animals (CPCSEA), India.
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5.2 Physical measurement
by adding a nonpolar solvent and surfactant with subsequent
centrifugation followed by washing with HPLC-grade chloro-
form. The resultant compound was then dried in vacuum and
used without any further purification. Yield: 60% (155 mg).
UV-vis: lmax (nm, log e) in methanol: 272 (4.13), 284 (4.09), 695
(1.62). IR (cmꢀ1): 3241 (nNH amide), 3162 (nNH benzimidazole),
1621 (nCQO amide-I), 1552 (nC–N amide-II), 1455 (nCQN–CQC
benzimidazole), 736 (nCQC benzene). Anal. calcd for
Cu(C21H22N6O2)Cl2: Cu, 11.52; C, 51.33; H, 3.87; N, 15.23; found:
Cu, 11.39; C, 51.66; H, 3.72; N, 15.43.
Before the synthesis of C2, we synthesized Cu(SCN)2 reagent
by dissolving CuCl2ꢃ2H2O in dry methanol (2 ml), followed by
the dropwise addition of potassium thiocyanate solution in dry
methanol. The KCl precipitate were filtered off, and the filtrate
was used for the synthesis of C2 as described in the procedure
for C1. The resultant green precipitate was separated by a
nonpolar solvent and surfactant with subsequent centrifuga-
tion followed by washing with HPLC-grade chloroform.
The resultant compound was then dried in vacuum and used
without any further purification. Yield: 55% (100 mg). UV-vis:
lmax (nm, log e) in methanol: 272 (4.35), 282 (4.30), 300 (3.80),
680 (1.68). IR (cmꢀ1): 3275 (nNH amide), 3141 (nNH benzimidazole),
1620 (nCQO amide-I), 1556 (nC–N amide-II), 1450 (nCQN–CQC
benzimidazole), 739 (nCQC benzene), 2074 (nC–N str.), 846 (nC–S
str.). Anal. calcd for Cu(C21H22N6O2)(SCN)2: Cu, 11.01; C, 48.27;
H, 4.13; N, 19.48; O, 5.88; S, 11.02; found: Cu, 11.23; C, 48.05; H,
4.31; N, 19.26; O, 6.07; S, 11.29.
TEM studies were carried out using a Philips Morgagni 268
electron microscope operating at 80 kV at the All India Institute
of Medical Sciences (AIIMS), New Delhi, India. The TEM speci-
mens were prepared by dispersing the powdered compounds in
double distilled water and sonicating them. A drop of the
sonicated solution was placed onto a porous carbon copper
grid and then air dried. A PerkinElmer FTIR-2000 spectro-
photometer was used for comparable study of IR bands of the
nanocoordination complexes and the free ligand L by using KBr
pellets at the Department of Chemistry, University of Delhi,
Delhi, India. The light scattering experiments were performed
on a Photocor FC, and all the measurements were done at a
scattering angle of 901 at a temperature of 20 1C. Dilute
solutions of the samples were prepared by dispersing the
powder in double distilled water followed by ultrasonic treatment
for 15 min. Electronic spectra were obtained on a Shimadzu
UV-vis 1601 spectrometer. Elemental analysis was obtained from
the University Science Instrumentation Centre (USIC), University
of Delhi, Delhi, India. 1H NMR spectra were recorded on a
300 MHz Bruker-spin instrument at the Indian Institute of
Technology (IIT), Delhi, India. The X-Band EPR spectra of the
copper(II) nanocoordination complexes were recorded on a
BRUKER-Spectrospin with a variable temperature liquid nitrogen
cryostat at 120 K at IIT Kanpur, India; the samples were in a
solution of methanol as the solvent that was frozen at the
temperature of liquid nitrogen.
5.4 Measurement of nitrite
Griess reagent54 was used for the detection of nitrite concen-
5.3 Synthesis of nanocoordination complexes C3, C1, and C2
This synthesis method has been previously reported.35a,c Micro- tration with minor modifications. Briefly, RAW 264.7 cells were
emulsion I is composed of a mixture of CTAB as the surfactant, plated at a density of 2 ꢂ 105 cells per well in a 96-well plate.
n-butanol as the co-surfactant, iso-octane as the hydrocarbon The cells were incubated for 2 h, followed by the addition of
phase, conductive water, and 0.227 mmol of ligand L. Similarly, compounds. The final concentrations of the compounds in the
microemulsion II was prepared containing the same constitu- cultures were 100 mg mlꢀ1. After 30 min incubation, RAW 264.7
ents as microemulsion I except that instead of the diamide cells were stimulated with 1 mg mlꢀ1 LPS. The activated cells
ligand L, it contained 0.227 mmol of Cu(NO3)2ꢃ3H2O. The were further incubated for 24 h. Then, 100 mL supernatants
weight fractions of the various constituents in these micro- were collected to determine the nitrite concentration. The cell-
emulsions were 16.66% of CTAB, 17.70% of n-butanol, 57.4% of free supernatant from each well was transferred to another
iso-octane, and 8.1% of the aqueous phase. The two micro- 96-well flat-bottom plate, and Greiss reagent (100 mL 1%
emulsions were very slowly mixed together and stirred over- sulphanilamide in 30% glacial acetic acid and 0.1% naphthyl-
night on a magnetic stirrer, resulting in the formation of green ethylenediamine dihydrochloride in 60% glacial acetic acid)
precipitates that were separated using a nonpolar solvent and was added. The absorbance of the samples was measured at
surfactant with subsequent centrifugation followed by washing 540 nM, and the nitrite in the samples was quantified by
with HPLC-grade chloroform. The resultant compounds were comparison with a standard curve. A nitrite multipoint linear
then dried in vacuum and used without any further purifica- standard curve was determined using known concentrations of
tion. Yield: 54% (160 mg). UV-vis: lmax (nm, log e) in methanol: NaNO2, ranging from 1.56 mM to 100 mM. Negative control cells
276 (4.16), 283 (4.11), 710 (1.69). IR (KBr pellet, cmꢀ1): 3276 (cells only) were grown under identical conditions but were not
(nNH amide), 3061 (nNH benzimidazole), 1621 (nCQO amide-I), exposed to the compound or LPS.
1557 (nC–N amide-II), 1452 (nCQN–CQC benzimidazole), 738
(nCQC benzene), 1384 (nO–N–O sym stretch). Anal. calcd for
5.5 Nitroblue tetrazolium (NBT) reduction assay
Cu(C21H22N6O2)(NO3)2: Cu, 10.91; C, 43.60; H, 3.87; N, 19.48; The NBT reduction assay was carried out according to a previously
O, 21.09; found: Cu, 10.80; C, 43.80; H, 3.71; N, 19.59; O, 21.22. described method55 with minor modifications. Briefly, 100 mL
The same procedure as described above was used for the (stock solution 100 mg mlꢀ1) of the test compounds was added
preparation of C1 except that CuCl2ꢃ2H2O was used in place of to 2 ꢂ 105 RAW-264.7 cells. Inducing agent LPS (1 mg mlꢀ1) was
Cu(NO3)2ꢃ3H2O. The green coloured precipitates were separated used to produce reactive oxygen. The final volume was adjusted to
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200 mL with RPMI-1640 medium in a flat-bottom 96-well plate nanocoordination complexes C1, C2, and C3, respectively. On day
(Tarsons, India). The final concentrations of the compounds were one, the body weight and girth size of each mouse of all groups
100 mg mlꢀ1 per well. After incubation for 24 h at 37 1C in a were measured. Dalton’s lymphoma cells were collected from the
humidified 5% CO2 incubator, the medium was removed, and donor mouse and were suspended in sterile isotonic saline. The
the cells were washed once with warm PBS (37 1C). To each well, viable DL cells were counted (trypan blue indicator) under the
100 mL of fresh medium was added that contained 5 mL of PMA microscope and were adjusted at 1 ꢂ 106 cells per ml. Then, 0.1 ml
(1 mg mlꢀ1) and 20 mL of NBT (1 mg mlꢀ1), except for the control. of Dalton’s lymphoma cells per 10 g body weight of the animals
The plates were then returned to the incubator, and after 2 h of was injected (i.p.) into group III, IV, V, and VI mice. After seven
incubation, the medium was removed and the cells were washed days of tumour transplantation, the mice of groups IV, V, and VI
once with warm PBS (37 1C). The cells were fixed with 100% were treated with test compounds C1, C2, and C3 at the concen-
methanol for 1 min, and after removing the methanol, 10 mL of tration of 3 mg per mice per day for 30 days.
0.1% Triton X-100 was added along with 120 mL of 2 M KOH and
140 mL of 100% DMSO. Cells were incubated for 5 min at room
5.9 Body weight
temperature on an orbital shaker, and the absorbance was The body weights of the experimental mice were recorded
measured using an ELISA microplate reader at a reference both in the treated and control groups at the beginning of
wavelength of 620 nm.
the experiment (day 1) and then sequentially on every 4th day
during the treatment period.
5.6 Cytotoxicity assay
5.10 Mouse survival
The cytotoxicity of compounds was investigated using cancer
cell lines of different origin (HeLa, MCF-7, and U-87 cells). The The effect of test compounds C1, C2, and C3 on tumour-
cell lines were grown in RPMI-1640 medium supplemented transplanted mice was monitored by recording the mortality.
with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM
of glutamine, 100 U mlꢀ1 of penicillin, and 100 mg mlꢀ1 of
5.11 Murine peritoneal macrophage isolation
streptomycin in a highly humidified atmosphere of 95% air Peritoneal macrophage cells were cultured using a previously
with 5% CO2 at 37 1C. Cell viability was assessed by the established method.38 Balb/c mice were euthanized by cervical
microculture tetrazolium [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- dislocation, sterilized with alcohol, and placed on a dissecting
tetrazolium bromide] (MTT) assay.56 In brief, cells were seeded tray that was earlier sterilized with alcohol and flamed. The
into a 96-well culture plate at 2 ꢂ 105 cells per well in 100 mL mouse was stretched and pinned on the tray, and then the
culture medium. After incubation for 24 h, test compounds at two outer layer of its skin was excised to expose the peritoneum.
different concentrations were added to each well, respectively. The Then, 5 ml of ice-cold incomplete medium (RPMI-1640) was
cells were incubated for another 24 h, 48 h, and 72 h followed by injected into the animal with a flame-sterilized needle (22 G1).
the addition of 20 mL MTT solution (5 mg mlꢀ1) to each well and Gentle finger pressure was applied to the point of injection for
further cultivation for 4 h. The media with MTT were removed, and 10 seconds. The medium in the mouse body was checked for an
100 mL of DMSO was added to dissolve the formazan crystals orange or pinkish colour; contamination with body medium
at room temperature for 30 min. The absorbance of each cell was indicated by a whitish color, and such contaminated
was measured with a UV-vis spectrophotometer at 570 nm. The animals were not used. The peritoneal cavity was gently tapped
percentage of viable cells was calculated relative to untreated cells. by hand so that macrophages properly diffused into the suspen-
sion. The injected medium was then retrieved from the perito-
5.7 Viable/non-viable tumour cell count
neal cavity with a needle (18 G); 3–4 ml was usually retrieved
The ascitic fluid was added to a centrifuge tube and then from each animal. The cell suspension was centrifuged at 25 1C
diluted 100 times. Next, a drop of the diluted cell suspension for 20 min. The erythrocytes in the cell pellets were lysed with a
was placed on a Neubauer counting chamber, and the number hypotonic solution (0.2% NaCl), and isotonicity was restored
of cells in the 64 small squares was counted. The cells were with 1.6% NaCl solution. The cell suspension was centrifuged,
then stained with trypan blue (0.4% in normal saline) dye. The and the cells were washed twice and re-suspended in complete
cells that did not take up the dye were viable, and those that RPMI 1640 medium. The cell number was adjusted to 1 ꢂ 106
took the stain were nonviable. These viable and nonviable cells cells per ml, which was determined by counting in a hemo-
were counted.
cytometer, and then cell viability was tested using the trypan
blue dye exclusion technique.
5.8 In vivo study
5.12 Energy-optimized structure of copper(II)
nanocoordination complexes
Swiss albino mice were divided into 6 groups, with each group
consisting of nine mice. Group I served as the control in which
mice were given no treatment. Group II was a DMSO control To obtain computational energy-optimized structures of copper(II)
group in which mice were given an intraperitoneal injection nanocoordination complexes and ligand L, Scigress 2.2.0 (Fujitsu)
containing 0.3% DMSO. The animals of group III were tumour docking software was used. The minimized structures of these
controls into which Dalton’s lymphoma (1 ꢂ 106 cells per mouse) nanocomplexes were calculated using Mechanics Compute
was injected. Groups IV, V, and VI were treated with or without test Engine 2.9.0.a1 software. Molecular mechanics and reading
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17 M. Devereux, D. O’Shea, M. O’Connor, H. Grehan, G. Connor,
M. McCann, G. Rosair, F. Lyng, A. Kellett, M. Walsh, D. Egan
and B. Thati, Polyhedron, 2007, 26, 4073–4084.
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19 H. Kucukbay, R. Durmaz, E. Orhan and S. Gunal, II Farmaco,
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20 V. Klimesova, J. Koci, K. Waisser and J. Kaustova, II Farmaco,
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parameters were obtained for all molecules by performing
molecular mechanics calculations. The MM3 force field and
energy terms for the following interactions included bond
stretch, bond angle, dihedral angle, improper torsion, torsion
stretch, bend bend, van der Waals, electrostatics, and hydrogen
bond. A conjugate gradient was used to locate the energy
minimum, and all atoms were moved at once during minimiza-
tion. The dielectric value was set to 1.50. van der Waals
interactions between atoms separated by greater than 9.00 Å
were excluded, and the van der Waals interactions list was
updated every 50 interactions. Optimization continued until
the energy change was less than 0.00100000 kcal molꢀ1, or until
the molecule was updated 300 times.
23 N. S. Pawar, D. S. Dalal, S. R. Shimpi and P. P. Mahulikar,
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Acknowledgements
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One author (Dr S. C. Mohapatra) is thankful for the Dr D. S.
Kothari UGC post-doctoral fellowship (No. F.4-2/2006 (BSR)/
13-215/2008 (BSR)) New Delhi for financial support. Authors are
thankful to Dr Gyantosh Jha, Principal, ARSD College, Delhi
University.
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