Synthesis, anticancer activity and docking studies of N-phenyl-2-(2-((4-phenyl piperazin-1-yl) methyl)-1H-benzo [d] imidazol-1-yl) acetamides

Article abstract of DOI:10.1016/j.molstruc.2018.04.057

A series of novel N-phenyl-2-(2-((4-phenyl piperazin-1-yl) methyl)-1H-benzo [d] imidazol-1-yl) acetamides (7a-o) have been synthesized in multiple steps with suitable reaction procedures and well characterized by various analytical techniques. All the synthesized compounds were evaluated for their in vitro anticancer activity against three human cancer cell lines includes human cervical carcinoma (HeLa), human breast carcinoma (MCF-7) and human embryonic kidney (HEK 293) cell lines at various concentrations. The results were shown in terms of percentage cell viability reduction and IC₅₀values were compared against standard anti cancer drug doxorubicin. Among all the synthesized compounds, compound 7k has shown highest activity against HeLa and MCF-7. The compounds 7b, 7l, 7m, 7n and 7o also showed significant activity over HeLa and MCF-7. Furthermore, the structure and anticancer activity relationship was supported by molecular docking study of the active compounds against quinone reductase-2 (PDB ID 4ZVM) protein.

Full text of DOI:10.1016/j.molstruc.2018.04.057

Accepted Manuscript
Synthesis, anticancer activity and docking studies of N-phenyl-2-(2-((4-phenyl
piperazin-1-yl) methyl)-1H-benzo [d] imidazol-1-yl) acetamides
Lingaiah Boddu, Ashok Kumar Pagudala, Durgaiah Gandamalla, Saikrishna
Balabadra, Vijjulatha Manga, Yellu Narsimha Reddy, N.J.P. Subhashini
PII:
S0022-2860(18)30499-X
10.1016/j.molstruc.2018.04.057
MOLSTR 25130
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 26 January 2018
Revised Date: 14 April 2018
Accepted Date: 15 April 2018
Please cite this article as: L. Boddu, A.K. Pagudala, D. Gandamalla, S. Balabadra, V. Manga, Y.N.
Reddy, N.J.P. Subhashini, Synthesis, anticancer activity and docking studies of N-phenyl-2-(2-((4-phenyl
piperazin-1-yl) methyl)-1H-benzo [d] imidazol-1-yl) acetamides, Journal of Molecular Structure (2018),
doi: 10.1016/j.molstruc.2018.04.057.
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ACCEPTED MANUSCRIPT
Synthesis, anticancer activity and docking studies of N-phenyl-2-(2-((4-
phenyl piperazin-1-yl) methyl)-1H-benzo [d] imidazol-1-yl) acetamides
Lingaiah Boddua, Ashok Kumar Pagudalaa, Durgaiah Gandamallab, Saikrishna
Balabadrac, Vijjulatha Mangac Yellu Narsimha Reddyb and N.J.P. Subhashinia*
a Department of Chemistry, University College of Science, Osmania University, Hyderabad,
India.
b Department of Pharmacology and Toxicology, University College of Pharmaceutical
Sciences (UCPSc), Kakatiya University, Warangal, India.
c Molecular Modelling and Medicinal Chemistry Group, Department of Chemistry, Osmania
University, Hyderabad, india.
*Corresponding author: njsubhashini@yahoo.co.in
Abstract
A series of novel N-phenyl-2-(2-((4-phenyl piperazin-1-yl) methyl)-1H-benzo [d]
imidazol-1-yl) acetamides (7a-o) have been synthesized in multiple steps with suitable
reaction procedures and well characterized by various analytical techniques. All the
synthesized compounds were evaluated for their in vitro anticancer activity against three
human cancer cell lines includes human cervical carcinoma (HeLa), human breast carcinoma
(MCF-7) and human embryonic kidney (HEK 293) cell lines at various concentrations. The
results were shown in terms of percentage cell viability reduction and IC50values were
compared against standard anti cancer drug doxorubicin. Among all the synthesized
compounds, compound 7k has shown highest activity against HeLa and MCF-7. The
compounds 7b, 7l, 7m, 7n and 7o also showed significant activity over HeLa and MCF-7.
Furthermore, the structure and anticancer activity relationship was supported by molecular
docking study of the active compounds against quinone reductase-2 (PDB ID 4ZVM) protein.

ACCEPTED MANUSCRIPT
Synthesis, anticancer activity and docking studies of N-phenyl-2-(2-((4-
phenyl piperazin-1-yl) methyl)-1H-benzo [d] imidazol-1-yl) acetamides
Lingaiah Boddua, Ashok Pagudalaa, Durgaiah Gandamallab, Saikrishna Balabadrac,
Vijjulatha Mangac Yellu Narsimha Reddyb and N.J.P. Subhashinia*
aDepartment of Chemistry, University College of Science, Osmania University, Hyderabad,
India.
bDepartment of Pharmacology and Toxicology, University College of Pharmaceutical
Sciences (UCPSc), Kakatiya University, Warangal, India.
cMolecular Modelling and Medicinal Chemistry Group, Department of Chemistry, Osmania
University, Hyderabad, India.
*Corresponding author: njsubhashini@yahoo.co.in
Abstract
A series of novel N-phenyl-2-(2-((4-phenyl piperazin-1-yl) methyl)-1H-benzo [d]
imidazol-1-yl) acetamides (7a-o) have been synthesized in multiple steps with suitable
reaction procedures and well characterized by various analytical techniques. All the
synthesized compounds were evaluated for their in vitro anticancer activity against three
human cancer cell lines includes human cervical carcinoma (HeLa), human breast carcinoma
(MCF-7) and human embryonic kidney (HEK 293) cell lines at various concentrations. The
results were shown in terms of percentage cell viability reduction and IC50 values were
compared against standard anti cancer drug doxorubicin. Among all the synthesized
compounds, compound 7k has shown highest activity against HeLa and MCF-7. The
compounds 7b, 7l, 7m, 7n and 7o also showed significant activity over HeLa and MCF-7.
Furthermore, the structure and anticancer activity relationship was supported by molecular
docking study of the active compounds against quinone reductase-2 (PDB ID 4ZVM) protein.
Key words: o-phenylene diamine, phenyl piperazines, acid-amide coupling, anticancer
activity, docking.
Introduction
Cancer is a generic term used for the collection of related diseases involving
uncontrolled cell growth beyond their limit, invade and spread to distinct parts of the body.
There by healthy cells are destroyed and displaced by cancer cell tissues and can have severe
health consequences and leading to cause death. In order to reduce the unhealthy cell growth,
so many chemical agents available for various cancer types includes lung, prostate,
colorectal, stomach, liver, breast and uterine cervix cancer common for human. It is one of
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the leading causes of morbidity and mortality worldwide, with approximately 14 million new
cases in 2012 and the number expected raise by 20301. Globally, one in six deaths due to
cancer and it was responsible for 8.8 million deaths in 2015. Cancer causing infections
hepatitis and human papilloma virus (HPV) are responsible up to 25% in low and middle
income countries2. The most important risk factor for cancer is tobacco products3 and other
risk factors also include overweight or obesity, physical inactivity and consuming unhealthy
diet. The primary goal is cure cancer or considerably prolongs life. In order to cure the
cancer, diagnosis and identification of the stage of cancer is crucial. At initial stage, it can be
cured and final stage it may not be cured but prolongs life using certain drugs. Even though
many chemo therapeutic agents are exists, they fail to stop the proliferation resistance
developed in them by the existing drugs. So, some other new chemo therapeutic agents are
required to prevent the cell growth or destroy the cancer cell.
Benzimidazole structure is a privileged structure of heterocyclic compounds and it is
essential moiety in medicinal chemistry, which shows broad spectrum of biological activity
including antimicrobial4, antiprotozoal5, antihypertensive6, antiulcer7, anti diabetics8 and
anticancer9-12. Most of anti hypertension and antiulcer drugs were incorporated with
benzimidazole moiety as a substructure and marketed under various trade names, viz.
nocodazole (anticancer), candesartan, telmisartan (antihypertension), omeprazole,
pantoprazole, lansoprazole, dexlansoprazole and rabeprazole (antiulcer).
In present work, we describe the synthesis of N-Phenyl-2-(2-((4-phenylpiperazin-1-yl)
methyl)-1H-benzo[d]imidazol-1-yl) acetamides derivatives (7a-o) with widely utilized
procedures, but benzimidazole framework was appeared in most of the drugs for numerous
diseases. So, we planned to synthesize new molecules containing additional rings or side
chain with functional group on benzimidazole moiety. In addition to this, all these
compounds were evaluated for their in vitro anticancer activity against 3 cell lines, viz.
human cervical carcinoma (HeLa), human breast carcinoma (MCF-7) and human embryonic
kidney (HEK 293) cell lines was performed at various concentrations. The results of cell
viability in terms of IC50 values were compared with the standard anticancer drug
doxorubicin. Furthermore, all these compounds were explored for molecular docking studies.
Results and discussion
Chemistry
The synthetic route for the desired N-phenyl-2-(2-((4-phenylpiperazin-1-yl) methyl)-
1H-benzo [d] imidazol-1-yl) acetamides (7a-o) were summarised in scheme 1.
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Benzimidazole core was constructed by commercially available o- phenylene diamine and
chloro acetic acid in presence of 4N HCl at harsh dehydrating conditions and it forms 2-
(chloromethyl)-1H-benzo[d]imidazole (2). N- alkylation of phenyl pipirazine (3) with 2-
(chloromethyl)-1H-benzo[d]imidazole produced 2-((4-phenylpiperazin-1-yl)methyl)-1H-
benzo[d] imidazole (4) in presence of sodium hydride, which on further alkylation with
bromo ethyl acetate produce (2-((4-phenylpiperazin-1-yl) methyl)-1H-benzo[d]imidazol-1-yl)
acetate derivatives (5a-c), upon hydrolysis produce 2-(2-((4-phenylpiperazin-1-yl)methyl)-
1H-benzo[d]imidazol-1-yl)acetic acid (6a-c) in presence of LiOH.H2O. The titled amide
compounds
(7a-o)
obtained
with
2-(2-((4-phenylpiperazin-1-yl)
methyl)-1H-
benzo[d]imidazol-1-yl) acetic acid (6) and corresponding amines in presence of EDC.HCl in
good yields.
Scheme 1: Conditions. (i) 4N HCl, Reflux, 3-4h. (ii) Sodium hydride, 0ºC to r.t., DMF, 2h.
(iii) Sodium hydride, 0ºC to r.t., Bromo ethyl acetate, 2h. (iv) LiOH.H2O, THF: H2O (4:1),
r.t., 2h. (v) EDC.HCl, HoBt, TEA, r.t., 5-6h.
All the synthesized compounds were confirmed by analytical and spectroscopic data.
The compound 7b was confirmed by representative peaks in 1H NMR, δ 3.44 due to methoxy
protons and 13C NMR, δ 164.7 due to amide carbonyl, which is not present in acid
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compound (13C NMR of acid, δ 170.4), and it was further confirmed by IR spectrum, which
shows that, peaks at 3306 cm-1(amide I) and 1667 cm-1(amide II) were not coincidence with
acid peaks at 3388 cm-1(acid OH) and 1647 cm-1(acid carbonyl). Finally it also confirmed by
mass spectrum of the compound have molecular mass 455 (m/z. 456)
Anticancer activity
Synthesized novel N-Phenyl-2-(2-((4-phenylpiperazin-1-yl) methyl)-1H-
benzo[d]imidazol-1-yl) acetamide derivatives (7a-o) were evaluated for their in vitro
anticancer activity against a panel of human cancer cell lines namely HeLa, MCF-7 and
HEK 293 by MTT colorimetric assay as per ATCC protocol and the results were performed
in Table 1 as percentage of inhibition and in terms of IC50 values and also presented in
graphical form (Fig.1). Cell viability of all the three human cell lines was also presented
separately as shown in fig.2, 3 and 4. Fig.2 demonstrates cell viability on HeLa cell lines;
Fig.3 reveals the cell viability on MCF 7 and Fig.4 showed the cell viability on HEK 293
cells at various concentrations. The whole thing was presented in Fig.1. From the results, the
amide compound 7k shows highest activity against HeLa and MCF-7 among all the tested
compounds. By close examination of data reveals that 7b, 7l, 7m, 7n and 7o shown superior
activity and the rest of the compounds shown moderate activity against HeLa and MCF-7.
Few products shown medium activity and remaining products shows less activity towards
HEK 293. So, it is indicating that these products were selectively works towards particular
type of cell lines only. By analysing the results, compounds embedded nitro (NO2)
substitution shown highest activity, where as other substitutions containing compoundss
shown moderate activity. Either of the groups (OCH3, Cl) slightly reduces the activity of the
unsubstituted amide compound (7k) compare with amides 7l, 7m, 7n and 7o.
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Table 1: Anticancer activity of synthesized compounds (7a-o) with cell viability
(in terms of IC50 values)
IC50 Values (µM)*
S.No
Compound
HeLaA
MCF 7B
HEK 293C
1
2
3
57.57 ± 2.04 65.48 ±3.20 57.34 ± 3.19
40.43 ± 2.39 43.36 ±3.49 80.82 ± 3.30
68.03 ± 3.20 64.88 ± 3.26 61.0 ± 3.28
59.65 ± 3.19 63.97 ± 3.60 73.34 ± 3.42
55.92 ± 3.50 58.86 ± 3.62 72.63 ± 3.15
59.97 ± 3.73 56.82 ± 3.92 62.47 ± 3.05
64.03 ± 3.28 68.38 ± 3.83 71.74± 3.29
64.27 ± 3.10 56.77 ± 3.20 93.14 ± 2.70
87.11 ± 3.27 95.04 ± 4.28 84.77 ± 2.14
54.80 ± 3.20 71.02 ± 3.60 70.14 ± 2.59
14.05 ± 3.40 17.64 ± 3.29 90.89 ± 3.82
26.40 ± 3.21 22.86 ± 3.25 46.38 ± 3.71
22.39 ± 2.37 25.02 ± 2.14 64.0 ± 3.20
25.35 ± 2.05 24.71 ± 2.30 52.51 ± 3.60
28.95 ± 2.40 26.59 ± 3.19 50.78 ± 3.91
7a
7b
7c
4
7d
5
7e
6
7f
7
7g
8
9
7h
7i
10
11
12
13
14
15
7j
7k
7l
7m
7n
7o
Doxorubicin
3.56 ± 3.17
4.94 ± 3.28 65.61 ± 2.30
* Values are expresses as mean±SD (n=4).
1. HeLa: Human cervical cancer cells
2. MCF-7: Human Breast cancer cells
3. HEK 293: Human embryonic kidney cells
HeLa
MCF-7
100
80
60
40
20
0
HEK 293
Fig. 1: Percentage Cell viability reduction, IC50 Values (µM) of compounds (7a-o) on three human
cell lines
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100
80
60
40
20
0
7a
7m
7n
7o
7e
7i
7j
7k
7l
7b
7c
7d
7f
7g
7h
STD
0
20
40
60
80
100
Con (µM)
Fig. 2: Anticancer activity of compounds (7a-o) measured by MTT assay on Human
Cervical Cancer (HeLa) with concentrations (1 - 100 µM) for 48 h. Data as mean ± standard
deviation (S.D.) n = 4. Stastical analysis was performed using one-way ANOVA followed by
$
#
a
Bonferroni post-hoc test. *p<0.05, p <0.01, p<0.001versus Standard; p<0.001versus
Control.
100
80
7m
7a
7b
7f
7i
7e
7g
7n
7o
7j
7k
7l
7c
7d
STD
7h
60
40
20
0
0
20
40
60
80
100
Con (µM)
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Fig. 3: Anticancer activity of compounds (7a-o) measured by MTT assay on Human breast
cancer cells with concentrations (1 - 100 µM) for 48 h. Data as mean ± standard deviation
(S.D.) n = 4. Stastical analysis was performed using one-way ANOVA followed by
$
#
a
Bonferroni post-hoc test. *p<0.05, p <0.01, p<0.001versus Standard; p<0.001 versus
Control
100
7i
7j
7e
7f
7g
7m
7n
7o
7a
7b
7c
7d
80
60
40
20
0
7k
7l
7h
STD
0
20
40
60
80
100
Con (µM)
Fig. 4: Anticancer activity of compounds (7a-o) measured by MTT assay on Human kidney
cells (HEK 293) with concentrations (1 - 100 µM) for 48 h. Data as mean ± standard
deviation (S.D.) n = 4. Stastical analysis was performed using one-way ANOVA followed by
$
#
a
Bonferroni post-hoc test. *p<0.05, p <0.01, p<0.001versus Standard; p<0.001 versus
Control.
Docking Studies
GLIDE 5.613 was used for molecular docking studies. Protein quinone reductase-2
(PDB ID 4ZVM) 14-15 was prepared using protein preparation wizard in Maestro 9.0 applying
default parameters; a grid was generated around the active site by selecting the co-
crystallized ligand. Receptor van der Waals scaling for non polar atoms was kept at 0.9.
Molecules were built using Maestro build panel and prepared by LigPrep 2.0 application.
Low energy conformation of the ligands were selected and docked into the grid generated for
the protein using an extra precision (XP) docking mode. Dock pose of each ligand was
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analyzed for interactions with the receptor. From the docking studies, it was observed that the
standard Doxorubicin showed hydrogen bond interactions with Leu 103, Gly 149, Gly 150,
Tyr 155 and Glu 193 and one hydrophobic/π-π stacking interaction with Tyr 104 residues.
The best active compounds 7k showed hydrogen bond interactions with Leu 103 (1.635 Å
bond length), Gly 149 (2.364 Å) and Asn 161(1.92 Å) and 7m showed hydrogen bond
interactions with Leu 103 (1.732 Å), Gly 149 (2.121 Å), Tyr 155 (2.461 Å) and Asn 161
(2.429 Å) residues. Figure A, B, C represents the docked poses with ligand interaction
diagrams of 7k, 7m and Doxorubicin molecules in the target 4ZVM active site. The dock
scores, Emodels and Glide energies were given in Table 2.
A
Fig. A: Ligand interaction diagram of 7k with active site of PDB ID 4ZVM (Protein Quinone
reductase-2)
B
Fig. B: Ligand interaction diagram of 7m with active site of PDB ID 4ZVM (Protein
Quinone reductase-2)
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C
Fig. C: Ligand interaction diagram of Doxorubicin with active site of PDB ID 4ZVM
(Protein Quinone reductase-2)
From the docking results we have observed that the most potent compounds 7k and
7m showed common hydrogen bond interactions (Leu 103, Gly 149, Tyr 155) as compared to
Doxorubicin which is in agreement with experimental anticancer activities. The other
synthesized compounds also showed good and comparable binding interactions with target
protein.
Table 2. Glide scores, Glide energies and Glide emodels of synthesized compounds
Compound
GScore (XP)
-3.67
-3.71
-3.67
-3.82
-1.92
-3.6
glide energy
-36.1
glide emodel
-46.78
-50.1
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
-37.45
-33.44
-37.49
-37.45
-33.06
-35.33
-36.05
-33.11
-34.12
-40.68
-38.6
-48.8
-53.45
-49.02
-48.36
-49.97
-47.6
-44.15
-40.08
-52.68
-53
-57.28
-40.99
-50.82
-64.1
-3.76
-3.53
-4.15
-3.98
-4.08
-3.82
-4.31
-3.62
-3.53
-7.94
-41.41
-35.56
-38.62
-45.28
Doxorubicin
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ADME Calculations
ADME (Drug like) properties of all synthesized ligands were checked using
QikProp4.0. ADME explains about Absorption, Distribution, Metabolism and Excretion of
drug candidates. These are considered as drug like properties need to fulfil by the drug
molecule to pass through phase-I clinical trials. QikProp helps in analyzing pharmacokinetics
and pharmacodynamics of the ligand by accessing drug like properties. It predicts both
physically significant descriptors and pharmaceutically relevant properties. It also evaluates
an acceptability of the analogues based on the Lipinski's rule of 5 (number of violations of
Lipinski's rule of five) that is essential for rational drug design. Poor absorption or
permeation are more likely when a ligand molecule violates Lipinski's rule of five i.e., more
than 5 hydrogen bond donors, the molecular weight over 750, the log P over 5 and the sum of
N's and O's over 10. The calculated values for the synthesized molecules ranged within the
acceptable limits, indicating that the compounds may be considered for further development
(Table 3).
Table 3. ADME properties of synthesized compounds
Comp M.Wt QPlogPo/wa QPlogSb QPPCacoc QPlogBBd QPPMDCKe %Human
ound
Oral
Absorptionf
100
425.5
455.6
460
4.93
4.83
5.13
4.95
5.33
5.01
4.95
5.17
5.02
5.53
3.83
4.15
4.41
4.29
4.73
-5.78
-5.43
-5.78
-5.83
-6.41
-5.98
-5.69
-6
900.5
816.7
917.6
899.4
829.4
900.4
828.2
866.5
878.4
1189
0.15
0.06
0.27
0.08
0.28
0.08
-0.01
0.18
0
488.7
439.7
814.1
488.1
1104
7a
7b
7c
7d
7e
7f
100
100
455.6
460
100
100
455.6
485.6
490
488.6
446.4
783.1
475.7
1629
96.179
100
7g
7h
7i
96.859
96.085
100
485.6
490
-6.01
-6.62
-4.36
-5.61
-5.9
0.39
-0.55
-1.09
-0.86
-1.13
-0.73
7j
470.5
500.6
505
187.8
99.15
109.9
107.3
142.5
89.78
45.01
82.16
49.04
164.5
90.052
74.007
76.365
75.443
80.24
7k
7l
7m
7n
7o
500.6
505
-6.11
-6.54
a
b
c
Predicted octanol/water partition coefficient log P (Acceptable range_2.0 to 6.5).
Predicted aqueous solubility's in mol/L (Acceptable range_6.5 to 0.5).
Predicted BBB permeability (Acceptable range_3 to 1.2).
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d
e
Predicted Caco cell permeability in nm/s (Acceptable range: < 25 is poor and >500 is great).
Predicted apparent MDCK cell permeability in nm/s (Acceptable range in nm/s (Acceptable
range: <25 is poor and>500 is great).
f
Percentage of human oral absorption (Acceptable range: <25 is poor and >80% is high).
Experimental
All the reagents and solvents purchased from commercially available sources.
Analytical TLC was performed on Merck Silica Gel GF254 plates and visualization by I2
vapours / UV light. Melting points were determined in open capillary tubes on SISCO
electrical melting point apparatus and are uncorrected. IR spectra were recorded on a Perkin–
Elmer spectrophotometer using potassium bromide optics. 1H and 13C NMR spectra recorded
1
13
on a Bruker Avance 400 spectrometer (400 MHz for H NMR and 100 MHz for C) in
CDCl3/DMSO using TMS as internal standard. ESI Mass Spectra recorded on Quatro LC
Micromass (Waters Manchester, UK) mass Spectrometer.
The o- phenylene diamine (1, 18.5 mmol, 2g) reacts with chloroacetic acid (27.75
mmol, 2.6g) in presence of 4N HCl at reflux condition for 3-4 h yields 2-(chloromethyl)-1H-
benzo[d]imidazole (2, 9.03 mmol, 1.5g), which is subsequently treated with substituted
phenyl piperazine (3a, 9.03 mmol, 1.46g) in presence of sodium hydride gives sustituted 2-
((4-phenylpiperazin-1-yl)methyl)-1H-benzo[d]imidazole (4a). Further, Substituted 2-((4-
phenylpiperazin-1-yl) methyl)-1H-benzo[d]imidazole (4a, 6.84 mmol, 2g) treated with bromo
ethylacetate (8.21 mmol, 1.36g) in presence of sodium hydride to obtain corresponding (2-
((4-phenylpiperazin-1-yl) methyl)-1H-benzo[d]imidazol-1-yl) acetate derivatives (5a). Then,
hydrolysis of (2-((4-phenylpiperazin-1-yl) methyl)-1H-benzo[d]imidazol-1-yl) acetate
derivative (5a, 3.96 mmol,1.56g) with LiOH.H2O (5.95 mmol, 244mg) in THF: H2O (4:1)
produces substituted 2-(2-((4-phenylpiperazin-1-yl) methyl)-1H-benzo[d]imidazol-1-yl)
acetic acid (6a). The amide coupling reactions of substituted 2-(2-((4-phenylpiperazin-1-yl)
methyl)-1H-benzo[d]imidazol-1-yl) acetic acid (6a, 0.571 mmol, 200mg) with o- anisidine
(7a, 0.571 mmol, 69 mg) in presence of EDC.HCl and HoBt in TEA, DMF gives the titled
compounds in good yields (73-86%).
Anticancer activity
MTT assay Method
In vitro anti cancer activity of the compounds was tested using MTT colorimetric
assay as per ATCC protocol. Cell lines used for testing in-vitro cytotoxicity are HeLa, MCF-
7 and HEK 293 being received with job number 1769 from NCCS, Pune. Cell lines were
0
maintained at 37 C in a humidified 5% CO2 incubator (Thermo scientific) using suitable
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media prescribed in NCCS Protocol. Decontaminated flasks were incubated for subculture.
Cells were passed by 14 numbers, After getting 70% confluence, from culture flasks take
100ꢀL cell suspension and make a cell count using haemocytometer and found 8,000 –
10,000 per well in a 96-well plate. The cell suspension was mixed thoroughly by pipetting
several times to get a uniform single cell suspension. Different dilutions of solutions 3, 10,
30, 100 ꢀg/ mL were made in media DMEM, 0.2% DMSO and ultrapure water H20. 100ꢀl of
cell suspension was transferred aseptically to each well of a 96 well plate and to it 100ꢀl of
nanoparticle solution (in quadruplicate) in media was added. The plate was then incubated at
370C for 72 h in CO2 incubator. After 48 h of incubation, 20ꢀl of MTT was added to each
well aseptically. The plate was again incubated for 2 h. 80ꢀl of lyses buffer was added to
each well the plate was wrapped in aluminium foil to prevent the oxidation of the dye and
the plate was placed on a shaker for 30 minutes. The absorbance was recorded on the ELISA
reader (Biotech EL x 800) at 570 nm wavelength. We have calculated the % inhibition by
following formula % inhibition = Control ODs – Test ODs ÷ Control ODs × 100 and finally
IC50 values to asses anticancer activity. Doxorubicin was used as the standard drug in the
assay.
Principle
The cytotoxicity assay was performed using MTT reagent. In this colorimetric assay
MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) a water soluble
yellow coloured tetrazolium salt, was converted to an insoluble purple formazan by cleavage
of the tetrazolium ring by succinate dehydrogenase present within the mitochondria. The
formazan product being impermeable to the cell membranes it accumulates in healthy cells.
Since reduction of MTT could only occur in metabolically active cells, the intensity of purple
colour was used as a measure of the viability of the cells. The intensity was measured by
spectrophotometer after dissolving formazan crystal in DMSO.
Conclusion
A series of novel N-phenyl-2-(2-((4-phenyl piperazin-1-yl) methyl)-1H-benzo [d]
imidazol-1-yl) acetamides (7a-o) were synthesized and well characterized by using various
spectral techniques. All the synthesized compounds were examined for their anticancer
activity against three human cell lines, viz. Hela, MCF-7 and HEK-293 at various
concentrations and the results were performed by means cell viability with reference to
standard anticancer drug doxorubicin. Among all the compounds, compound 7k showed
highest activity against HeLa and MCF-7. The compounds 7b, 7l, 7m, 7n and 7o also showed
good activity over HeLa and MCF-7. Based on the anticancer activity, docking and ADME
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studies, the synthesized molecules can be considered as promising lead molecules for the
development of new anticancer agents.
Acknowledgements
The authors are grateful to Head, Department of chemistry, Osmania University for
providing laboratory facilities and also thankful to CFRD, Osmania University for analytical
facilities. One of the authors BL, thankful to UGC for providing fellowship.
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Date: 14.04.2018
To
The Editor,
Journal of Molecular Structure
Dear Sir/Madam,
Kindly find enclosed our revised manuscript entitled “Synthesis, anticancer activity and
docking studies of N-phenyl-2-(2-((4-phenyl piperazin-1-yl) methyl)-1H-benzo [d] imidazol-1-
yl) acetamides” for publication in your esteemed journal, the “Journal of Molecular Structure”.
This manuscript has not been published elsewhere or submitted to another journal and all authors
are familiar with and agree with the contents of the manuscript.
Highlights of the manuscript are
•
•
A series of novel N-phenyl-2-(2-((4-phenyl piperazin-1-yl) methyl)-1H-benzo [d]
imidazol-1-yl) acetamides were synthesize in conventional methods.
The newly synthesized compounds were screened for anticancer activity against 3 cell
lines. Viz, HeLa, MCF-7 and HEK 293. Compound 7k had shown excellent activity
against HeLa and MCF-7 and compounds 7b, 7l, 7m, 7n and 7o also showed significant
activity over HeLa and MCF-7.
•
In-silico molecular docking studies were performed using Schrodinger software. (quinone
reductase-2 (PDB ID 4ZVM) protein.
Thank you.
With best reguards
Yours Sincerely,
Dr. N.J.P. Subhashini
Department of chemistry,
University College of science,
Osmania University,
Hyderabad, Telangana, 500007, India
Tel.: +91 9849941559;
E-mail: njsubhashini@yahoo.co.in