Deciphering the anthelmintic activity of benzimidazolium salts by experimental and in-silico studies

Article abstract of DOI:10.1016/j.molliq.2018.07.029

Inspired from the facts that majority of the drug administrated in the form of salts and “poison is in the dose”; herein, we have synthesized and characterized 1-methyl-3-alkylbenzimidazolium and 1-methyl-3-alkylimidazolium derived ionic salts with varying N-alkyl chains and different anions. These ionic salts were evaluated for their vermicidal activity (VA) and cell viability test against the Pheretima posthuma and A549 cell lines (human alveolar basal epithelial cells), respectively. The morphological changes in the test organism were visualized by the Scanning Electron Spectroscopy (SEM) to get the mechanistic insight. Furthermore, results were compared with VA of 1-methyl-3-alkylimidazolium derivatives to establish their structure-activity-relationship (SAR) of the fused benzene ring in 1-methyl-3-alkylimidazolium. The current findings suggested that the activity of these salts depends on the nature of N-alkyl side-chain, anionic moieties, varying partial charge on quaternary nitrogen (due to different anions) lipophilicity and types of cationic core fused with imidazolium ring. These findings were complemented by the quantitative-structure-activity-relationship (QSAR), molecular docking approaches to unfold the features accountable for their activities and binding patterns of the ligand-receptor complex respectively. ADME/T assessment of the ionic salts shows that twelve compounds qualified the ADMET profiling test & encompasses the drug-likeness features.

Full text of DOI:10.1016/j.molliq.2018.07.029

Accepted Manuscript
Deciphering the anthelmintic activity of benzimidazolium salts by
experimental and in-silico studies
Prabodh Ranjan, Mohd Athar, Kari Vijayakrishna, L.K. Meena, R.
Vasita, Prakash C. Jha
PII:
S0167-7322(18)31195-4
doi:10.1016/j.molliq.2018.07.029
MOLLIQ 9350
DOI:
Reference:
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
7 March 2018
21 June 2018
8 July 2018
Please cite this article as: Prabodh Ranjan, Mohd Athar, Kari Vijayakrishna, L.K. Meena,
R. Vasita, Prakash C. Jha , Deciphering the anthelmintic activity of benzimidazolium salts
by experimental and in-silico studies. Molliq (2018), doi:10.1016/j.molliq.2018.07.029
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ACCEPTED MANUSCRIPT
Deciphering the Anthelmintic Activity of Benzimidazolium salts by
Experimental and In-silico Studies
1
1
4
3
3
2‡
Prabodh Ranjan , Mohd Athar , Kari Vijayakrishna , L.K. Meena , R. Vasita , Prakash C. Jha
1CCG@CUG, School of Chemical Sciences, Central University of Gujarat, Sector-30,
Gandhinagar-382030, Gujarat, India.
2CCG@CUG, Centre for Applied Chemistry, Central University of Gujarat, Sector-30,
Gandhinagar-382030, Gujarat, India.
3School of Life Sciences, Central University of Gujarat, Sector-30, Gandhinagar-382030, Gujarat,
India.
4Department of Chemistry, School of Advanced Sciences, VIT University, Vellore-632014, Tamil
Nadu, India
To whom correspondence should be addressed: Dr. Prakash Chandra Jha, Centre for Applied
Chemistry, Central University of Gujarat, Sector-30, Gandhinagar-382030, Tel.: +91 8866823510;
E-mail: prakash.jha@cug.ac.in
ORCID: 0000-0002-1709-511X
1

ACCEPTED MANUSCRIPT
Abstract:
Inspired from the facts that majority of the drug administrated in the form of salts and “poison is
in the dose“; herein, we have synthesized and characterized 1-methyl-3-alkylbenzimidazolium and
1
-methyl-3-alkylimidazolium derived ionic salts with varying N-alkyl chains and different anions.
These ionic salts were evaluated for their vermicidal activity (VA) and cell viability test against
the Pheretima posthuma and A549 cell lines (human alveolar basal epithelial cells), respectively.
The morphological changes in the test organism were visualized by the Scanning Electron
Spectroscopy (SEM) to get the mechanistic insight. Furthermore, results were compared with VA
of 1-methyl-3-alkylimidazolium derivatives to establish their structure-activity-relationship (SAR)
of the fused benzene ring in 1-methyl-3-alkylimidazolium. The current findings suggested that the
activity of these salts depend on the nature of N-alkyl side-chain, anionic moieties, varying charge
on quaternary nitrogen (due to different anions) lipophilicity and types of cationic core fused with
imidazolium ring. These findings were complemented by the quantitative-structure-activity-
relationship, molecular docking approaches to unfold the features accountable for their activities
and binding patterns of the ligand-receptor complex respectively. ADME/T assessment of the ionic
salts shows that all fifteen compounds qualified the ADMET profiling test (that means they
exhibits drug-likeness features).
Keywords: Ionic Liquids, 1-methylbenzimidazolium, Vermicidal, Pheretima posthuma and
Albendazole sulfoxide.
Abbreviations
ILs
Ionic Liquids
ABZSO
Albendazole sulfoxide
SIFts
QSAR
MLR
SEM
Structural Interaction Fingerprints
Quantitative structure activity relationship
Multi Linear Regression
Scanning Electron Microscopy
2

ACCEPTED MANUSCRIPT
1
.
Introduction
Helminthic infections are neglected tropical diseases (NTDs), caused by helmintic worms (e.g.,
tape worm, and hook worm etc.). These infections have affected a wide continuum of population
in developing countries due to poor sanitations and lack of social awareness [1-8]. Measures have
been taken to control and eradicates such infections by developing several drugs (i.e.,
Benzimidazole carbamates (BZCs) derivatives, Levomesol, Paraziquintel, and Pyrental etc.) and
by means of awareness programs [9-17]. Their poor bioavailability and excessive uses have led
to drug resistance owing to evolutionary adaptations of helminths [10, 18, 19]. Notably, BZCs
derivatives (albendazole (ABZ), fenbendazole (FBZ), mebendazole (MBZ) etc.) are the most
widely used anthelmintics due to their selective binding with colchicine binding domain of β-
tubulin [20-28]. Several efforts have been made to explore the role of different functionalities (1-
alkyl, aryl, arylalkyl acyl benzimidazoles and hydrogen atom) at N-1/N-3 and C-2 position of the
benzimidazole ring (Figure S1) [1, 27, 28]. Structure-activity-relationship (SAR) studies of such
compounds indicates that certain types of functional group (i.e., methoxy carbonyl amino and
heteroaryl group attached at C-2 position of benzimidazole derivatives through a CO, CHON,
CONH, S, SO) [10] improves anthelmintic activities. In spite of these efforts, their bioavailability
and resistance remains the main stay problem due to negligence by scientific community,
availability of < 1% fund of total pharmaceutical global funds and scarcity of molecular level
insight [10].
Owing to the fact that majority of the drugs administrated in the form of salts [17, 20-22, 24-28];
ionic liquids (ILs) (a class of salts/molten salts) can be worthy choice to appraise as an active
pharmaceutical ingredients (APIs) [29-39]. Their (ILs) adjustable physicochemical and biological
profile makes them pertinent for the uses in various domain such as biotechnology, catalysis, and
electrochemistry etc [40-59]. However, clinical applications of ILs as active-pharmaceutical
ingredients (APIs) are at premature stage due to their multiple debatable reasons such as toxicity,
biochemical and biopharmaceutical properties [40, 41]. At recent times, reasonably, efforts have
been made to provide the chemical space for their applications as APIs via abating their toxicity,
biochemical and biopharmaceutical properties [40, 41]. This can be made by tailoring their
solubility, counter anions, N-alkyl chain length and functional group. Possibly the tactic, would
reflect their therapeutic application in a greater perspective [40, 41]. It is worth to mention that the
3

ACCEPTED MANUSCRIPT
maxim “the poison is in the dose’, has encouraged to develop ILs as useful drugs for biomedical
applications by minimizing their toxicity [28, 31, 60].
Considering the above mentioned facts and the encouraging outcomes from our previous study
(that reveals the vermicidal activity of ILs rely on the nature of N-alkyl side chain and counter
anion) [61] we examined to evaluate the vermicidal and cytotoxic activity of the 1-methyl-3-
alkylbenzimidazolium and 1-methyl-3-alkylimidazolium derived ILs. Further, the SAR features
of fused benzene ring in imidazolium ring of the ILs were also established. Moreover, quantitative-
structure-activity-relationship (QSAR) and molecular docking were performed to explicate several
bred-in-the-bones factors accountable for their activity and binding patterns of ILs, respectively.
2
2
.
Experimental
.1. Material and methods
Benzimidazole, 1-methylimidazole, n-ethyl bromide, n-butyl bromide, n-hexyl bromide, n-octyl
bromide, and n-decyl bromide, NaBF and NaOH were purchased from Sigma-Aldrich and used
4
as received. Albendazole sulfoxide (ABZSO) was purchased from Hi-media, India. All the organic
solvents were purchased from Avara Synthesis Pvt. Ltd. and R & D Chemicals and distilled prior
1
13
their usages. H and C NMR spectra were recorded using Bruker 400 and 500 MHz spectrometer
in appropriate deuterated solvents (CDCl and d -DMSO) and TMS as an internal standard. Mass
3
6
spectral analysis was accomplished with Agilent Technologies G6520B LCMS (Q-TOF) mass
spectrometry with +ESI ionization method.
2
2
.2. Synthesis of ionic liquids (ILs)
.2.1. N-alkylation of Benzimidazole with Methyl bromide
Mixture of Benzimidazole (0.1 mole), 30 mL of 50% of NaOH (w/v) and methyl bromide (CH -
3
Br) (0.11 mole) were stirred at room temperature for 3 minutes (Scheme-1). Afterwards, the
reaction mixture was stirred at temperature 40 °C until biphasic layer (one was organic layer
and second was aqueous NaOH) appears in round bottom flask. Further, organic layer was
extracted from the reaction mixture using CHCl (3 × 25 mL). The extract was washed twice
3
with distilled water and sodium sulfate (anhydrous agent), later the organic solvent was
evaporated under reduced pressure. Finally, the yellowish color solution was distilled in order
to purify and to ensure the synthesis of ILs.
4

ACCEPTED MANUSCRIPT
H
N
N
30 ml. of 50% solution of NaOH
.11 mole of CH Br
0
3
N
N
CH3
0
.1 mole
Scheme 1: N-alkylation reaction of Benzimidazole with Methyl bromide.
2
.2.2. Solvent free quaternization of 1-Methylbenzimidzole
In a dry flask 1-methylbenzimidazole or 1-methylimidazole (1.0 equivalent) and 1.2 equivalents
of alkyl bromides (i.e., n-ethyl bromide) was added and sealed, heated in an oil bath maintained at
1
40 °C for 20 minutes, and cooled down to room temperature. Further, the reaction mixture was
heated at same temperature (140 °C) for 10 minutes, then cooled down to room temperature (rt).
The obtained IL was washed with ethyl acetate ( (5 × 25 mL) to remove the unreacted starting
materials), vacuum dried and characterized. The same procedure was applied for the quaternization
of the 1-methylbenzimidazole and 1-methylimidazole ring with varying N-alkyl side chain i.e., n-
butyl, n-hexyl, n-octyl, and n-decyl bromide. A representative reaction scheme has been given
bellow in (Scheme 2, Step-1) [62].
Derivatives of ILs with hydroxide as counter anion [RBZMIM]OH ([EBZMIM]OH,
[
[
BBZMIM]OH, [HBZMIM]OH, [OBZMIM]OH, [DBZMIM]OH) and [RMIM]OH ([EMIM]OH,
BMIM]OH, [HMIM]OH, [OMIM]OH, [DMIM]OH) were synthesized by altering the different
N-alkyl side chains (i.e., n-C
2
H
5
, n-C
4
H
9
, n-C6H13, n-C H17 and n-C10H21) using anion exchange
8
reaction as per reported procedure [61, 63]. Amberlite resin bearing hydroxide anion was used for
above anion transformation reactions. This Amberlite resin bearing hydroxide anion (hydroxide
resin) was prepared by stirring Amberlite® IRA-400 chloride resin with 1M NaOH in a round
bottom flask for 12 hours. Later, resin with hydroxide anions was obtained by neutralizing their
pH by washing with double distilled water, filtered and dried. Moreover, a demonstrative example
for the synthesis of [RBZMIM]OH (i.e., [EBZMIM]OH, [BBZMIM]OH, [HBZMIM]OH,
[
[
OBZMIM]OH, [DBZMIM]OH) and [RMIM]OH (i.e., [EMIM]OH, [BMIM]OH, [HMIM]OH,
OMIM]OH, [DMIM]OH) derivatives are as follows: 5 g of [RBZMIM]Br and [RMIM]Br IL was
dissolved separately in methanol and passed through a column packed with hydroxide resin
independently. Subsequently, substrate was concentrated under vacuum followed by vacuum
drying leading to 1-methyl-3-alkylimidazolium hydroxide and 1-methyl-3-alkylbenzimidazolium
hydroxide derivatives (Scheme 2, Step-2).
5

ACCEPTED MANUSCRIPT
Another anion exchange reaction was carried out to achieve IL with boron tetrafluorides as counter
anion (IL-BF
). A demonstrative example for the synthesis of [RMIM]BF
HMIM]BF [OMIM]BF [DMIM]BF and [RBZMIM]BF
BBZMIM]BF , [HBZMIM]BF , [OBZMIM]BF , [DBZMIM]BF
4
) by metathesis reaction as per reported literature with minor modifications (Scheme
2
4
(i.e., [EMIM]BF
4
, [BMIM]BF
4
,
,
[
[
4
,
4
,
4
)
4
(i.e., [EBZMIM]BF
4
4
4
4
4
) in Scheme 2 (step-3)
derivatives are as follows: 1:2 ratio of the 1-methyl-3-alkylimidazolium bromide or 1-methyl-3-
alkylbenzimidazolium bromide ([RBZMIM]Br) and NaBF were added distinctly in to the round
4
bottom flask containing 25mL of methanol and stirred at room temperature for 24 hrs. Afterwards,
the salt was removed by filtering the reaction mixture using Whatman filter paper. Further, the
filtrate was centrifuge for 10 minutes at 3000rpm to isolate the residual salts. Solvent was
1
13
evaporated under reduced pressure, vacuum dried and characterized (using H and C-NMR and
mass spectroscopy). The spectral details of the synthesized ILs are provided in the supplementary
information Figure S1 to S30.
2
.3. Evaluation of Vermicidal Activity
Pheretima posthuma (Indian earthworm) has been chosen as the model organism for evaluation of
vermicidal activity as a model organism due to their physiological and anatomical resemblance
with intestinal human parasitic worms. Indian adult earthworm (Pheretima posthuma) were
collected from wastewater treatment, Management Plant of Golden Temple, Vellore, India and
employed for the screening of vermicidal screening. Albendazole sulfoxide (ABZSO) and
sterilized distilled water were used as positive control and negative control. The assay was
performed according to the literature procedure [61, 64] at four different concentrations (2mM,
4
mM, 8mM and 16mM) in a time dependent manner (15, 30, 45, 60, 75, 90, 120 minutes). Their
activities were recorded in terms of percentage paralysis and mortality in triplicate at various time
interval. Their average value was considered for comparative analysis.
2
.4. Scanning Electron Microscope (SEM) Analysis
Sample Preparation: ILs treated and untreated earthworms were stored in 2:9:30:59 mixture of
acetic acid, formaldehyde, ethanol (95%) and distilled water [65]. Later, morphological study of
their skin was performed. Initially, earthworms were taken out from the stored solution and their
body parts such as anterior, posterior, dorsal/ ventral part and clitellum were dissected and dried
overnight. Afterwards, each part was processed for SEM analysis by following the protocol
6

ACCEPTED MANUSCRIPT
reported by Dhvani Jhala et al., [66] Briefly, each body part was fixed with 2% glutaraldehyde in
0
.1M sodium cacodylate buffer (pH 7.2) for 1 hr. and later transferred to cacodylate buffer. Next,
gradient dehydration was performed by washing samples serially with 30%, 70%, 80%, 90%, 95%
and 100% ethanol for 5 to 10 min each. The samples were air-dried, sputter coated with gold-
palladium and recorded under SEM (EVO 18, Zeiss Germany) at the distance of 10 mm.
2
.5. Cytotoxicity Study
Aalamar blue assay was employed to expose the A549 cells against the most active ILs (i.e.,
DBZMIM]Br, [DBZMIM]OH and [DBZMIM]BF ) identified during VA to evaluate their level
[
4
of cytotoxicity. Briefly, the A549 cells were grown in RPMI media supplemented with 10% FBS.
At around 70-80% confluency, cells were trypsinized and seeded in 96 well plates as 10000 cells
per well. After 24 hours, 4 mM dose of [DBZMIM]Br, [DBZMIM]OH and [DBZMIM]BF in
4
media were treated for another 24 hours. As the stocks of compounds were prepared in water,
equal volume of water was added to media and incubated in control wells. Again after 24 hours,
media was replaced with fresh one containing 10% v/v of the alamar blue solution and incubated
for one hour in the dark. Afterwards, absorbance was recorded at 570 nm to evaluate the cell
viability using multi-plate reader (SynergyTM H1, BioTek®) [66]. All the experiments were
performed in triplicates.
2
.6. Computational Details
Geometry optimization of 1-methyl-3-alkylbenallynzimidazolium derivatives were carried out at
B3LYP/6-311G(d) level of theory level with Conductor-like Polarizable Continuum Solvation
Model (CPCM) using Gaussian 09 [67]. Dissimilar guess geometry were obtained by manual
positioning of anions with respect to the cationic core of the ILs to estimate the real geometry
with minimized total energy [68]. The frequency analysis was also performed at the same level of
approximation to ensure the structures were at global minima. The details of the optimized guess
geometries have been given in electronic supplementary information (ESI) Table S1.
2
.7. Generation of QSAR model
The fifteen 1-methyl-3-alkylbenzimidazolium derive ILs with varying N-alkyl chains (n-C
2
5
H to
−
−
4
−
n-C10
H
21) and three different anions (Br , BF and OH ) were considered for the QSAR using
7

ACCEPTED MANUSCRIPT
Discovery Studio v4.0 [62]. Computationally, it is very challenging to mimic ILs real geometrical
+
−
+
features due to the presence of characteristic interaction between the [C] [X] (푋 = cations, and
−
푋 = anions) [68, 69]. For this reason, density functional theory (DFT) based descriptors of the
cations and anions were separately computed and used in QSAR model development (refer
TableS3) [68]. Notably, the critical electronic and solvation effects were considered during the
generation of independent variables (descriptors). Also, the average percentage mortality (at 4mM
concentration) were converted to log10 [% mortality] and considered as the dependent variable
[
70][67].
Multiple Linear Regression (MLR) technique was applied for the selection of optimal combination
of descriptors (Table S2) [71]. The following criteria was considered for the MLR based modelling
viz number of components 20, number of nearest neighbors 20, smoothness parameter 0.5,
decorrelation method was Pearson, max. correlation 0.99, model development finger print
FCFP_2, dynamic smoothing factor 0.5, fingerprint distance function Tanimato, Max OPS
fingerprint Bits 1000 and total number of variables were thirty.
ꢀ
The quality of MLR model was measured based on 푟 and RMSE, as governed by the following
equations.
∑
(푌표푏푠 − 푌푐푎푙)ꢂ
(푌표푏푠 − 푌̅ 표푏푠)ꢂ
푟ꢀ = 1 ꢁ ∑
(1)
Whereas, ꢃꢄꢅꢆ stand for the observed activity, ꢃꢇꢈꢉ is the model-derived calculated response and
̅
ꢃ
is the average of the observed response value. RMSE (Root Mean Square Error (RMSE) is a
ꢄꢅꢆ
measure of how close the data is around the line of best fit) was also examined.
ꢊ
ꢀ
푅푀푆퐸 = √ × ∑(푦 ꢁ 푦 )
(2)
ꢄꢅꢆ
ꢇꢈꢉ
푁
Where, N is number of components.
2
.8. Molecular docking study
Molecular docking study was performed using the DFT optimized ligand structures of cationic
+
+
+
+
+
part ([EBZMIM] , [BBZMIM] , [HBZMIM] , [OBZMIM] , [DBZMIM] ) and a 3D-homology
receptor model of H. contortus β-tubulin bound to Albendazole sulfoxide [72] (PDB entry: 1OJ0)
[
73]. Initially, protein preparation and binding site were defined by selecting the grid box size of
Å using receptor preparation wizard. Discovery Studio (DS) v4.0 program was employed to
8
dock cationic head groups in highly dynamic tubulin environment using flexible docking method.
8

ACCEPTED MANUSCRIPT
It uses the ChiFlex and CDOCKER algorithm [74] to generate the side chains (active site)
conformations and to dock the low energy conformations of cationic group in to the flexible
binding pockets respectively [75]. Refinement of the active side residues was made by ChiRotor
algorithm [74]. The simulated annealing (SA) approach was applied to minimize the poses in
various restrained steps using the parameters such as heating temperature (500 K), cooling
temperature (300 K), conformer generation method (BEST), grid extension (8.0 Å) and maximum
conformation (10). Also, SIFt binding pattern [76-78] of β-tubulin inhibitors distribution of
colchicine binding domain [79] and the interactions patterns of the dock poses have been
exemplified in 2D format using ligand-receptor interaction map. The present in-silico procedure
is an advancement over the current methods [77] for computational assessment of ILs.
2
.9. ADME/T assessment
The pharmacological properties of the 1-methyl-3-alkylbenzimidazolium derived ILs were
evaluated by the in-silico predictions to measure the biological regimen. Absorption, Distribution,
Metabolism, Excretion and Toxicity (or ADME/T) of the ILs were assessed by considering the
subsequent features: Aqueous Solubility, Blood Brain Barrier (BBB), Hepatotoxicity, Intestinal
Absorption, Plasma Protein Binding (PPB) properties (refer Table S3) [80-86].
3
. Results and discussions
Our previous findings indicates that vermicidal activities of the ILs depends on the nature of
−
−
varying alkyl chains and counter anions (Br and OH ) [61]. We further pursuit research in this
line and evaluate the vermicidal activities of 1-methyl-3-alkylimidazolium and 1-methyl-3-
alkylbenzimidazolium derived ILs, with diverse alkyl chains (i.e., n-C
2
H5, n-C
4
H
9
, n-C H13 n-
6
C
8
H17 and n-C10H21, substituted at N-3 position of imidazolium ring) and different cationic and
−
−
−
4
anionic (Br , OH , BF ) combinations.
4
4
R1
R1
H
N
3
3
3
5
6
5
N
N
4
5
2
H
H
X-
H
2
N
H
6
N 2
R2
N
X-
1
1
1
7
7
R2
Imidazolium Salts
Where, R = -CH , R = -C H , -C H , -C H , -C H , -C H and X = Br/ or OH/ BF
4
Benzimidazolium Salt
Benzimidazole
1
3
2
2
5
4
9
6
13
8
17
10 21
Figure 1: List of imidazolium and benzimidazolium salts.
9

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3
.1. Vermicidal Activities of ionic liquids (ILs)
The synthesized compounds (as mentioned in Scheme 2) were tested for the vermicidal activities
as a function of percentage paralysis and mortality. Percentage paralysis and mortality of the 1-
methyl-3-alkylbenzmidazolium bromide derivatives (i.e., [EBZMIM]Br, [BBZMIM]Br,
[
HBZMIM]Br, [OBZMIM]Br and [DBZMIM]Br), 1-methyl-3-alkylbenzmidazolium boron
tetrafluoride derivatives (i.e., [EBZMIM]BF , [BBZMIM]BF , [HBZMIM]BF , [OBZMIM]BF
and [DBZMIM]BF ), and 1-methyl-3-alkylbenzmidazolium hydroxide derivatives (i.e.,
EBZMIM]OH, [BBZMIM]OH, [HBZMIM]OH, [OBZMIM]OH and [DBZMIM]OH), 1-methyl-
4
4
4
4
4
[
3
-alkylimidazolium bromide (i.e., [EMIM]Br, [BMIM]Br, [HMIM]Br, [OMIM]Br and
[
[
DMIM]Br), 1-methyl-3-alkylmidazolium boron tetrafluoride derivatives (i.e., [EMIM]BF
4
,
BMIM]BF , [HMIM]BF , [OMIM]BF and [DMIM]BF ), and 1-methyl-3-alkylimidazolium
4
4
4
4
hydroxide derivatives ([EMIM]OH, [BMIM]OH, [HMIM]OH, [OMIM]OH and [DMIM]OH) and
Albendazole sulfoxides (ABZSO) were evaluated at four different concentrations in a time
dependent manner. The detailed information of the vermicidal activities (VAs) is given in
supplementary information (Figure S31 to S45). Based on the results and arguments mentioned in
ESI, it have been comprehended that ILs VA depends on doses and nature of N-alkyl side chain
(or hydrophobicity). Average percentage VA (as function of percentage paralysis and mortality)
at 4mM concentration of 1-methyl-3-alkylimidazolium and 1-methyl-3-alkylbenzimidaolium
derivatives have been considered and compared to elucidate the effects of different anions
−
−
−
4
(
Br , OH and BF ), cationic core and structure activity relationship (SAR) of the ILs.
1
0

ACCEPTED MANUSCRIPT
R2
Amberlite
Resin
N
Y
R1
RT Step-2
N
CH3
R2
Step-1
Y = OH-
N
X
N
N
Quaternization
Metathesis
R1
R1
R-X
Heat, 140°C
R2
N
N
CH3
CH3
X = Br-
(
a)
Z
Step-3
NaBF4
R1
Methanol, RT
N
CH3
[EMIM] BF4
[
[
[
EMIM] Br
BMIM] Br
HMIM] Br
Where, R = H, C H
[EMIM] OH
[BMIM] OH
[HMIM] OH
OMIM] OH
DMIM] OH
Y = OH-
1
4
4
9
[
[
[
[
BMIM] BF4
HMIM] BF4
OMIM] BF4
DMIM] BF4
Z = BF4-
R = n-C H , n-C H
2
2
5
4
n-C H , n-C H
6
13
21
8
17
[OMIM] Br
[DMIM] Br
[
[
n-C H
1
0
X = Br-
Scheme 2: General scheme for the synthesis of 1-methyl-3-alkylimidazolium and 1-methyl-3-
alkylbenzimidazolium derived ILs.
Structure activity relationship (SAR) of the tested ILs with vermicidal activity (VA)
Herein, effects of different anions and fused benzene ring at C-4 and C-5 position of the
imidazolium ring of IL were perceived by considering the VAs of tested ILs (at 4mM conc.) as
given in
Figure 2. ILs (1-methyl-3-alkylimidazolium or [RMIM] and 1-methyl-3-
alkylbenzimidaolium [RBZMIM] derivatives) showed very competitive percentage paralysis
(Figure 2a) with respect to the same N-alkyl side chain containing two different cationic core
+
(
[RMIM] and [RBZMIM]+) except [EMIM]Br. Conversely, a significant variation in percentage
−
−
−
4
mortality of the ILs can be observed with alteration of the different anions (Br , OH and BF )
and N-alkyl chains with fixed cationic core (Figure 2b). From, Figure 2b the difference in VAs of
1
-methyl-3-alkylimidazolium and 1-methyl-3-alkylbenzimidazolium derivatives having the same
−
−
−
4
N-alkyl side-chain and different anions (Br , OH and BF ) can be clearly noticed. Hence, it can
be comprehend that ILs-OH showed slightly better mortality than IL-BF , IL-Br and ABZSO (refer
4
Figure 2b). However, it can be conclude that IL-OH exhibits significant activities then IL-BF4,
IL-Br and ABZSO, due to the high hydrophobic and lipophilic ratio. Furthermore, Figure 2
deciphers that ILs [RBZMIM] OH/ Br/ BF
alkylimidazolium derive ILs and standard drug (ABZSO). Likewise, it can be noticed that ABZSO
exhibits better and competitive percentage paralysis than ILs i.e., [EMIM] and [BMIM] Br/ BF
OH (Figure 2a). On the other hand, ILs [EMIM] and [BMIM] Br/orBF /or OH and [HBMIM]BF
4
derivatives shows better activities than 1-methyl-3-
4
/
4
4
1
1

ACCEPTED MANUSCRIPT
shows very competitive mortality with ABZSO (Figure 2b). It may be possible due to the lesser
hydrophobic factors in comparison to ABSO. Also, these findings supports the fact that “majority
of the drugs exist in salt form” [72, 87-90]. Besides, fusion of benzene ring in imidazolium ring
of the IL affects the VA in greater extents compared to 1-methyl-3-alkylimidazolium derivatives.
A possible explanations for this trend can be provided from the earlier literature reports [72, 87-
9
0]. BZCs derivatives can efficiently binds to colchicine domain of β-tubulin monomer and
thereby block the microtubule formation. Also, they suppress the β-tubulin polymerase enzyme,
as a consequence lead to decrease in glucose level (due to the exhaustion of glycogen
levels/storage) and finally results in the death of the parasite [91-97]. Further, in quest to provide
better insight to the experimental findings QSAR and molecular docking approach have been
employed. For that, the most active series of compounds ([RBZMIM]Br/or OH/or BF
considered.
4
) have been
(a)
(b)
Figure 2: Comparison of %paralysis (a) and %mortality (b) of 1-methyl-3-alkylimidazolium and
-methyl-3-alkylbenzimidazolium derivatives (IL-Br, IL-BF and IL-OH) containing three
different anions and ABZSO (as standard drug).
1
4
3
.2. SEM Analysis:
The images of tegumental surfaces of untreated (as positive control) earthworm have been given
in Figure 3 mouth (a), anus (b) dorsal (c-e) and ventral (f-h) side surfaces appeared undamaged.
Mouth part (Figure b) and anus part (Figures d) recorded at high magnifications for better
resolution that showed the normal and undamaged prostomium (it covers the mouth of earthworm,
helps in dwell the soil and function as sensor). Further, dorsal part (c-e) and ventral part (f-h) was
recorded at 200, 300X and 1,000KX (Figure 3), clearly indicates the presence of grooves with
1
2

ACCEPTED MANUSCRIPT
setae (helps in locomotion of the earthworm) marked by red arrow (in Figure 3 e and h) were found
as in normal and healthy earthworm morphology.
Figure 3: Scanning electron microscopy of the different parts of the untreated or uninfected Indian
earthworm (P. posthuma) to study the morphological changes. (a) showing normal and regular
arrangements of mouth part recorded at 200X (b) showing no morphological change in anus part
recorded at 200 X (c, d) displaying normal and proper arrangements of grooves and spines (shown
by red arrow) present on the dorsal tegumental surface recorded at 200X, 300 X and 1,000 KX and
(e, f) ventral healthy tegumental surface with presence of clear grooves and spines (shown by red
arrow), recorded at 200X, 300 X and 1,000 K. The alternation of the teguments (helps in define
the shape of earthworms body, moisten the body wall, respiration as well as muscle contraction
and relaxations) on the earthworm skin morphology treated with ILs were analyzed by scanning
electron microscopy (SEM) Figure 4 (g-h).
1
3

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Figure 4: Scanning electron microscopy of the different parts of the ILs treated Indian earthworm
(P. posthuma) to study the morphological changes. (a-c) showing damaged and ruptured skin
around the mouth part recorded at 200X, 300X and 1,000X, (d-f) damaged, ruptured and swelled
skins around anus part recorded at 200X, 300X and 1,000X, (g-i) ruptured skins (marked with red
arrows) and no absence of grooves and spines on the dorsal tegumental surface recorded at 200X,
3
00 X and 1,000 KX, and (j-l) evidently showing ruptured skins (marked with red arrows) and no
absence of grooves and spines (blunted spine) on the ventral tegumental surface recorded at 200X,
00 X and 1,000 KX.
3
Figure 4 (a-f) illustrates the damaged and swelled tegumental surfaces on their mouth and anus
part. Similar observations can be perceived from Figure 4 of (g-i) and (j-l) recorded for dorsal and
ventral section respectively showing damaged tegumental surfaces, and the folded skin (damaged
and ruptured) marked with red arrows shown in Figure 4 g and h. Moreover, image (Figure 4 h)
1
4

ACCEPTED MANUSCRIPT
circled in red color is showing flashes of dorsal section of earthworms, and the image encircled in
red color display a small pore in the earthworm flashes. Also, images recorded for their ventral
section (h, i) are showing absence of setae’s (that help in locomotion of earthworm, made up of
chitin) or annular row. Moreover, images recorded for ventral section showing blunt spines marked
with red arrow (in Figure 4 j and k). The area encircled in red color (shown in Figure 4 k) display
the flashes with blunted setae. Their damaged tegumental surface and setae leads to stop all vital
functions of earthworm (such as respiration, absorption, secretion, protection as well as their
movement as well) which leads to death of the test organism.
These findings can be supported by the originally proposed biochemical mode of action for the
clinically available anthelmintic drugs [87, 91-93, 97]. They suppress the β-tubulin polymerase
enzyme and triggers the consumption of glucose that results in lowering of glucose level [87, 91-
9
3, 97]. Also, increasing hydrophobicity index with increase in N-alkyl side chain and polar
surface area (IL-OH > IL-BF > IL-Br) damages the cell wall and cutaneous layer of the worm
4
results in leakage of body fluids that leads to death of worms. This encouraged to evaluate the
binding mode of ILs against the β-tubulin structure using the molecular docking approach.
3
.3. Cytotoxicity evaluation of Ionic Liquids (ILs)
Cytotoxicity results presented in Figure 5 describes the different cytotoxic levels of the test
compounds (most active ILs). [DBZMIM]Br and [DBZMIM]BF showed lowest levels of
4
cytotoxicity, with 58% of cell survival and [DBZMIM]OH showed highest level of cytotoxicity
with, 2% of cell survival. Furthermore, these results are self-explanatory that cytotoxicity of these
ILs also depends on the nature of the anions with fixed cations. Also, this finding suggest that ILs
toxicity can be controlled by tailoring the different anionic and cationic combinations, it provides
balance between their toxicity and drug-likeness properties to develop as APIs.
1
5

ACCEPTED MANUSCRIPT
Figure 5: Cytotoxicity assay of [DBZMIM]Br, [DBZMIM]OH and [DBZMIM]BF on A549 cells
4
after 24 hours. The data shows decrease in the % cell growth as a function of cytotoxic level of the
compounds.
3
.4. Geometry Optimization
The geometries of 15 ILs were optimized at B3LYP/6-311G(d) level of theory using Gaussian 09
program [67]. Out of the various delineated structure of each IL, the lowest energy geometry
without imaginary frequency was selected (Table S1). Results of the optimized structures revealed
that the distance between the anionic moieties and C-2 positioned hydrogen atom of the
−
−
−
4
benzimidazolium rings varies by altering the different anion (Br , OH and BF ). With increase
in bulkiness of the anion with fixed cation, the distance between the anionic moieties and C-2
positioned hydrogen atom varied. They can be arranged in ascending order of their average
distance considered between the C-2 hydrogen and anionic moieties, which are as follows: OH−
−
−
(
0.98ꢋ3 Å) > BF ꢌ2.3652 Åꢍ > Br (2.5362 Å).
4
Quantitative structure activity relationship (QSAR) Modelling
QSAR based studies were performed via utilizing quantum mechanical based descriptors to
identify the role of cation and anion for their vermicidal activity of the synthesized ILs. Based on
the RMSE and the regression statistics, we selected the model equation with a_CP (Chemical
potential of anion), a_LUMO (Lowest unoccupied molecular orbital of anion) and c_FPSA
(Fractional polar surface area of anion) descriptors. Multiple Linear Regression model equation is
as follows:
푳풐품ퟏퟎ(풂풄풕풊풗풊풕풚) = ퟐ. ퟑퟓퟓ ꢎ ퟎ. ퟎퟎퟖퟎퟖퟐ ∗ [풂_푪푷] ꢁ ퟎ. ퟕퟒퟏퟒ ∗ [풂_푳푼푴푶] ꢁ ퟏퟎ. ퟖퟖ ∗ [풄_푭푷푺푨]
1
6

ACCEPTED MANUSCRIPT
The closeness of the respective variables (a_LUMO, a_CP, c_FPSA) with the observed values in
terms of their numeric differences can be perceived from Table 1. It can be said that in case of the
minimum value (Min.) and standard deviation (SD) of a_LUMO (Min. 0.107 and SD. 0.088) and
c_FPSA (Min. 0.026 and SD. 0.007) have showed closeness with the experimental number (Min.
1
.574 and SD. 0.103), while a_CP (Min. -1.770 and SD. 1.167) displayed large deviation from the
experimental number (Table 1). Therefore, these descriptors can be represented in order of
influencing the observed activity i.e., a_LUMO > c_FPSA > a_CP. It was evident to note that
these descriptors contributed to the %mortality and thus supported our experimental findings that
account the effects of anion exchange on vermicidal activities of ionic liquids and fused benzene
ring at imidazolium derive ionic liquids. The list of descriptors have been given in the Table 1.
Table 1: List of quantum mechanical descriptors used in MLR model
b
Xa
Descriptors
*Min.
-1.770
0.107
0.026
1.574
*Max.
1.076
0.319
0.046
1.954
Mean
-0.267
0.201
0.034
* SD
1.167
0.088
0.007
0.103
a_CP
0.008
a_LUMO
c_FPSA
activity
-0.741
-10.884
1.828
b
a
*
Min. (Minimum), Max. (Maximum), SD (Standard Deviation), X (Regression coefficient of descriptors)
Detailed statistics of the developed quantum mechanical based MLR model have been given in
2
Table 2, as the square root of correlation coefficient (r ) and predicted correlation coefficient
r2adjusted). The obtained MLR statistics with significant regression coefficient (r ) 0.88, and RMSE
2
(
0
.034, signify the model with good fitness ability. Additionally, the r2adjusted is a better measure of
2
the ratio than r that avoids the over-parameter value for the MLR was 0.857. It is clearly
indicating that the variation and correlation in the whole data is in good agreement.
The correlation graph plotted between predicted activities (on the Y-axis) Vs observed % mortality
2
(
on the X-axis) in Figure 6 shows significant correlation with r = 88.33% for the MLR model.
Thus, the predicted QSAR model can be used to predict new lead molecule against helminths
incorporating these factors. It can be noticed that the predicted values are in good agreement with
the experimental values. The observed % mortality and predicted values for the whole data set of
the MLR models are listed in Table S3 (Supplementary data).
1
7

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Table 2: Details of the statistics of the developed quantum mechanical based GFA model
Sl No.
Statistic
Value
15
0.942
0.887
0.759
1
2
3
4
N
r
2
r
ꢀ
푟
푝ꢏ푒푑푖ꢇ푡푒푑
ꢀ
5
푟
0.857
ꢈ푑푗푢ꢆ푡푒푑
6
7
RMSE residual error
0.034
0.005
= Square root of adjusted
RMS residual error (cross-validation)
2
ퟐ
N = No. of components; r = Regression coefficient; 풓
풂풅풋풖풔풕풆풅
r2= correlation coefficient; 풓ퟐ
RMSE = Root Mean Square Error; q = Cross-validation, r = correlation coefficient
= square root of predicted correlation coefficient,
풑풓풆풅풊풄풕풆풅
2
2
y = 0.8873x + 0.2062, R² = 0.8873
1
1
1
1
1
.95
.9
.85
.8
.75
.7
.65
.6
.55
.5
1
1
1
1
1
1
.5 1.55 1.6 1.65 1.7 1.75 1.8 1.85 1.9 1.95
2
X (%log_observed activity)
Figure 6: Correlation plot of observed (X-axis) versus predicted activities (Y-axis) by MLR.
3
.5. Molecular Docking study
In continuation to our previous studies based on the evaluation of anthelmintic activities of the ILs
and alternate binding sites (zone 1, 2 and 3) [61, 77], we have attempted to identify binding site
of the synthesized compounds. For that, molecular docking of 1-methyl-3-alkylbenzimidazolium
+
+
+
+
derived cationic head groups viz. [EBZMIM] , [BBZMIM] , [HBZMIM] , [OBZMIM] and
+
[
DBZMIM] was performed to investigate the binding interactions [76, 79]. The resulting 3D Dock
poses of the ligand-receptor complexes have been given in the Figure 7. The cationic part of the
ligands highlighted in red color (in ball and CPK form) with interacting amino acids (Figure 7).
1
8

ACCEPTED MANUSCRIPT
(b)
(a)
GLU198
THR136
THR136
GLU198
SER165
PHE167
LEU250
HIS6
GLN134
PHE200
HIS6
THR237
PHE200
GLN134
TYR50
PHE167
VAL236
MET257
THR237
MET233
TYR50
LEU253
LEU240
PHE20
PHE20
THR238
VAL236
SER234
ILE24
ILE24
SER165
THR238
THR136
(
c)
(d)
SER165
GLN134
SER165
GLN134
HIS6
THR136
PHE200
LEU250
TYR50
ILE16
CYS201
PHE167
PHE167
GLY17
VAL236
THR237
THR237
VAL236
PHE20
ILE24
MET2333
THR238
MET233
THR238
VAL229
VAL229
LYS19
SER230
ASN226
HIS137
(
e)
(f)
GLY13
VAL169
PHE200
CYS12
SER165
THR136
HIS6
PHE167
TYR50
GLY17
THR237
PHE20
ILE202
VAL229
MET233
THR238
SER230
+
+
+
+
Figure 7: 3D poses view of (a) [EBZMIM] (b) [BBZMIM] (c) [HBZMIM] (d) [OBZMIM] (e)
[
+
+
DBZMIM] and (f) [RBZMIM] of β-tubulin inhibitors on the H. contortus β-tubulin model.
Moreover, the details of the interacting amino acids have been listed in Table 3. It was found that
amino acids viz. HIS6, MET233, GLN134, PHE200, PHE167, and SER165 were consistent in
1
9

ACCEPTED MANUSCRIPT
each ligand and receptor complex (Table 3). In general, ligand-receptor complex possess four
major types of interactions van der Waal, ᴫ-ᴫ, hydrophobic and electrostatic interactions.
Table 3: Docking score and interacting amino acids of [RBZMIM] derive ILs
ILs
Docking Score
Interacting Amino acids
(
Kcal/mol)
[
[
EBZMIM]+
-20.568
ILE24, PHE20, SER234, PHE167, SER165, THR238, PHE200,
LEU250, GLU198, TYR50, MET233, HIS6, THR136, THR237,
GLN134, VAL236
ILE24, SER234, PHE20, THR238, PHE167, HIS6, THR237, VAL236,
PHE200, THR136, LEU250, SER166, SER165, LEU240, ALA254,
GLU198, LEU253, MET257, TYR50, MET233, GLN134
LEU250, VAL236, THR237, THR238, ILE24, SER165, PHE200,
ILE16, PHE167, ILE16, THR136, VAL229, SER234, PHE20,
SER230, GLN134, MET233, TYR50
VAL236, THR237, SER165, HIS6, ILE24, THR238, PHE167,
THR136, VAL229, SER230, SER234, PHE200, ILE16, GLY17,
PHE20, ASN226, LYS19, TYR50, MET233, GLN134
HIS6, CYS12, GLY13, PHE20, THR136, GLY17, GLN134, SER165,
VAL169, LEU250, VAL229, SER230, ILE202, PHE200, THR237,
TYR50, VAL236, MET233, MET236, PHE167
BBZMIM]+
-23.7665
-23.9864
-25.6676
-28.5137
[
[
HBZMIM]+
OBZMIM]+
[
DBZMIM]+
*
Green color residues shows van der Wall interactions, Light blue color shows ᴫ-ᴫ interaction, amino acid residues in pink color shows
interaction with positively charged nitrogen of imidazolium ring and residues in orange color shows non-classical H-bond interaction
Figure 8: (a) The amino acids participating in the interaction zones viz.1 to 3 (colchicine binding
domain) are represented in the line view (orange -zone-1, blue -zone-2, light green -zone-3),
+
identified according to SIFt patterns, (b). Superimposed structures of [RBZMIM] in red color
with reference ligands benzimidazole sulfoxide (blue color) in colchicine binding domain.
2
0

ACCEPTED MANUSCRIPT
The residue HIS6 interact with the positively charged nitrogen of imidazolium ring. Also, PHE167,
MET233 and PHE200 were engaged in ᴫ-ᴫ interactions, whereas, GLN134 form non-classical H-
bond interaction. Moreover, SER165 interact with hydrogen atom present at C-4 position in the
imidazolium ring (Table S47). It was further observed (Figure 8) that all five ligands were
considerably superimposed over the reference ligand (ABZSO). Interpretation of interactions
zones in colchicine binding domain by SIFt pattern reveals that all the docked ligands overlap in
zone-1 to 3 as highlighted in Figure 8 (with three different colors: orange -zone-1, blue -zone-2,
light green -zone-3). Notably, major part of the ligands binds in zone-2 (highlighted in blue color)
in align to our previous finding [77].
Results have shown that, anthelmintic activity increases with increase in alkyl chain and magnitude
of positive charge on the quaternary nitrogen of the imidazolium ring. It can be articulated from
the graph (Figure 9 plotted for comparing the experimentally observed percentage mortality with
docking score) that with increase in alkyl length (ranging from n-ethyl to n-decyl), docking score
were also increases (in negative magnitude). These findings are complementary to the observed
trends of the percentage paralysis and mortality. This study indicates that hydrophobic region,
positively charged quaternary nitrogen of the 1-methyl-3-alkylbenzimidazolium rings are the key
factors in influencing the anthelmintic activities.
Figure 9: The correlation plots of the average percent mortality of earthworms (in different time
interval at 4mM conc. in red color) and the docking score (in dotted black color) of the RBZMIM-
Br /or BF /or OH.
4
2
1

ACCEPTED MANUSCRIPT
3
.6. ADME/T assessment
It provides a qualitative idea about the absorption, distribution, metabolism, excretion and toxicity
in the pharmacokinetics of the employed ILs [98, 99]. The assessment was carried out by
considering the various ADME/T descriptors, given in Table S3. All fifteen (15) molecules
qualified the ADMET profiling test (pharmacokinetics, pharmacodynamics and toxicity study)
with default standards as implemented in DS. Figure 10 (For 95% and 99% confidence ellipse
biplot), clearly shows that majority of the compounds follows the bioavailability criteria and are
probably show drug-likeness features [100]. However, molecules outside of the absorption (95%)
confidence ellipse biplot ([DBZMIM] Br/OH/BF
4
), does not possess drug-likeness properties
Table S3) and considered as unreliable. That may possible due their higher hydrophobic factors
presence of n-decyl alkyl chain at N3 positions of benzimidazolium ring.
(
(
Figure 10: ADMET 2D description plot of the polar surface area (X-axis) vs LogP for 1-methyl-
-alkylbenzimidazolium derived ILs, showing 95% and 99% confidence limit ellipses
corresponding to the Blood Brain Barrier and Intestinal Absorption models.
3
2
2

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Conclusion:
Herein, we report that VA and cell viability of ionic salts depends on the nature of N-alkyl side
chain, anionic moieties, varying charge on quaternary nitrogen, lipophilicity and types of cationic
−
core fused with imidazolium ring. ILs having hydroxide (OH ) as counter anion shows significant
−
4
−
activities over BF , Br and ABZSO. Similarly, cell viability study proved that ionic salts can be
tuned to provide chemical space to develop them as novel APIs. SEM analysis advocates that
increasing hydrophobicity index and polar surface area (IL-OH > IL-BF > IL-Br) damages the
4
cell wall and cutaneous layer of the test organism. It leads to leakage of body fluids, thereby
allowed them to cross the cell membrane and interrupt the vital activities of the worms that causes
to death of the test organism. Also, this can be supported form the proposed biochemical mode of
action for the clinically available anthelmintic drugs [87, 91-93, 97]. Further, structure-activity-
relationship (SAR) study showed that 1-methyl-3-alkylbenzimidazolium derived salts exhibits
better activity than 1-methyl-3-alkylimidazolium derived salts. It is possible, due to high binding
affinity of the 1-methyl-3-alkylbenzimidazolium derived salts towards the colchicine binding
domain of the β-tubulin protein.
These experimental observations supported by QSAR study described that descriptors a_CP,
a_LUMO of the anions and c_FPSA of the cations are responsible factors for the activities of 1-
−
methyl-3-alkylbenzimidazolium derivatives. From this, it can be conclude that OH (as counter
−
4
−
anion) increases the polar surface area (PSA) of the cationic head, subsequently BF and Br
anion (as have been given in above para). Additionally, ligand-receptor interaction studies of the
cationic heads of the 1-methyl-3-alkylbenzimdiazolium derivatives suggests that they majorly
binds in zone-2 of the colchicine binding domain of the β-tubulin protein; hydrophobic region and
positively charged quaternary nitrogen of the 1-methyl-3-alkylbenzimidazolium rings are the key
factors in influencing the anthelmintic activities. Further, in-silico ADME/T assessment study
confirms that all fifteen compounds passes the ADME and toxicity profiling test. However, these
current findings open a new landscape for these ionic salts advancement as a novel potential
anthelmintic leads and for other biomedical applications.
2
3

ACCEPTED MANUSCRIPT
Acknowledgements: Prabodh Ranjan thanks financial support from University Grants
Commission (UGC), Govt. of India. Mr. Mohd Athar acknowledges generous support from the
Department of Science and Technology (DST), Govt. of India as Doctoral INSPIRE Fellowship.
Dr. Prakash Chandra Jha also would like to thanks Science and Engineering Research Board
(SERB) for providing start-up grants and Central University of Gujarat for providing basic
computational facilities.
Author contributions: Prabodh Ranjan, Mohd Athar, Kari Vijayakkrishna and Prakash C. Jha
designed the research problem. Prabodh Ranjan and Mohd Athar performed the calculations and
interpret the results.
Conflict of Interest: Authors have no personal, financial or non-financial conflicts of interest.
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