Design and synthesis of substituted (1-(benzyl)-1: H -1,2,3-triazol-4-yl)(piperazin-1-yl)methanone conjugates: Study on their apoptosis inducing ability and tubulin polymerization inhibition

Article abstract of DOI:10.1039/d0md00162g

A library of substituted (1-(benzyl)-1H-1,2,3-triazol-4-yl)(piperazin-1-yl)methanone derivatives were designed, synthesized and screened for their in vitro cytotoxic activity against BT-474, HeLa, MCF-7, NCI-H460 and HaCaT cells by employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Among all the synthesized analogues, compound 10ec displayed the highest cytotoxicity with the IC50 value of 0.99 ± 0.01 μM towards BT-474 cancer cell line. The target compound (10ec) was also evaluated for its tubulin polymerization inhibition study. Detailed biological studies such as acridine orange/ethidium bromide (AO/EB), DAPI and annexin V-FITC/propidium iodide staining assay suggested that compound 10ec induced the apoptosis of BT-474 cells. The clonogenic assay revealed that the inhibition of colony formation in BT-474 cells by 10ec in concentration-dependent manner. Moreover, the flow cytometric analysis revealed that 10ec induced apoptosis via cell cycle arrest at the sub-G1 and G2/M phase. In silico studies of sulfonyl piperazine-integrated triazole conjugates unveil that they possess drug-like properties. According to the molecular modelling studies, compound 10ec binds to the colchicine binding site of the tubulin.

Full text of DOI:10.1039/d0md00162g

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RESEARCH ARTICLE
Design and synthesis of substituted (1-(benzyl)-
1H-1,2,3-triazol-4-yl)(piperazin-1-yl)methanone
conjugates: study on their apoptosis inducing
ability and tubulin polymerization inhibition†
Cite this: DOI: 10.1039/d0md00162g
Kesari Lakshmi Manasa,ab Sowjanya Thatikonda,c Dilep Kumar Sigalapalli,ab
Sowmya Vuppaladadium,d Ganthala Parimala Devi,a Chandraiah Godugu,c
a
Mallika Alvala,b Narayana Nagesh
d and Bathini Nagendra Babu
*
*
A
library of substituted (1-(benzyl)-1H-1,2,3-triazol-4-yl)(piperazin-1-yl)methanone derivatives were
designed, synthesized and screened for their in vitro cytotoxic activity against BT-474, HeLa, MCF-7, NCI-
H460 and HaCaT cells by employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Among all the synthesized analogues, compound 10ec displayed the highest cytotoxicity with the IC50
value of 0.99 0.01 μM towards BT-474 cancer cell line. The target compound (10ec) was also evaluated
for its tubulin polymerization inhibition study. Detailed biological studies such as acridine orange/ethidium
bromide (AO/EB), DAPI and annexin V-FITC/propidium iodide staining assay suggested that compound 10ec
induced the apoptosis of BT-474 cells. The clonogenic assay revealed that the inhibition of colony formation
in BT-474 cells by 10ec in concentration-dependent manner. Moreover, the flow cytometric analysis
revealed that 10ec induced apoptosis via cell cycle arrest at the sub-G1 and G2/M phase. In silico studies of
sulfonyl piperazine-integrated triazole conjugates unveil that they possess drug-like properties. According to
the molecular modelling studies, compound 10ec binds to the colchicine binding site of the tubulin.
Received 18th May 2020,
Accepted 29th July 2020
DOI: 10.1039/d0md00162g
rsc.li/medchem
stability under in vivo conditions, are responsible for their
Introduction
enhanced biological properties. Fig.
1
demostrated a
Over the past few decades, 1,2,3-triazole and its derivatives
have played a prominent role in the field of medicinal
chemistry. The formation of 1,2,3-triazoles using click
chemistry has become an efficient method for the preparation
of diverse building blocks for chemical synthesis,1 material
and surface science,2 medicinal chemistry3 and combinatorial
chemistry.4 Moreover, 1,2,3-triazoles play a noteworthy role
among the pharmaceutically important nitrogen-containing
heterocyclic scaffolds due to their broad spectrum of
biological activities such as anticancer,5 antitubercular,
anticonvulsant,6 antiallergic,7 antifungal,8 antibacterial,9
antiviral10 and anti-inflammatory activities.11 The desirable
properties of the 1,2,3-triazole ring, such as hydrogen
bonding capability,12 high dipolar character,13 rigidity and
biologically important natural products14 containing the
triazole moiety.
It is evident from the literature that sulphonamide-based
scaffolds are known to exhibit excellent antibacterial,
anticancer,
antithyroid,
antidiabetic,
antiviral
and
antihypertensive activities. Moreover, piperazine and its
derivatives are also important scaffolds. Besides having
a Department of Fluoro-Agrochemicals, CSIR-Indian Institute of Chemical
Technology, Hyderabad-500007, India. E-mail: bathini@iict.res.in
b Department of Medicinal Chemistry, National Institute of Pharmaceutical
Education and Research, Hyderabad-500037, India
c Department of Regulatory Toxicology, National Institute of Pharmaceutical
Education and Research (NIPER), Hyderabad-500037, India
d CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007, India
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00162g
Fig.
1 Chemical structures of a few biologically active sulfonyl
piperazine derivatives (I–V); triazole derivatives (VI and VII) and
designed congeners (10aa–ef).
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antibacterial activity, they also possess potent anticancer
properties. AK301 was able to possess a potent ability to
hinder the mitosis and induce cell apoptosis.15 Chopra
et al.16 reported piperazine-based colchicine binding site
inhibitors, such as XRP44X (Fig. 1). CB694, a piperazine
containing compound binds to the colchicine binding site
and inhibits the tubulin with an IC50 value of 2.3 μM.17
Plinabulin18 showed the effective inhibition of tubulin
polymerization at the nanomolar scale. Herein, the
sulphonamide scaffold containing the piperazine group was
introduced into the triazole nucleus (Fig. 2) and these
heterocyclic scaffolds may display a synergistic effect on
antitumor activity.
In the recent scenario of drug discovery, more efficient
antitumor compounds can be designed by mimicking two or
more structural domains into a single framework.19 Ample
amount of literature survey reveals that to date enough
efforts have not been made to combine 1,2,3-triazole and
sulphonamide moieties into a single molecular scaffold. As a
part of the continued research for the development of new
antitumor agents, we have designed bifunctional heterocyclic
systems containing sulfonyl piperazine-linked 1,2,3-triazole
conjugates. In this study, we report the synthesis of
Scheme
1
Synthetic pathway for the preparation of sulfonyl
piperazine-linked triazole conjugates (10aa–ef).
propiolate (4) and 3a–e by using sodium ascorbate,
CuSO4·5H2O, t-butanol : H2O (2 : 1) for 8 h. These were further
hydrolyzed in the presence of 4 N NaOH solution to get 1,2,3-
triazole acids (6a–e) in good yields. Next, a variety of
substituted phenyl sulfonyl piperazine intermediates (9a–f)
were synthesized according to the previous synthetic
procedures20 by the reaction of different substituted
benzenesulfonyl chlorides (7a–f) with piperazine (8) and Et3N
in DCM at 0 °C for 30 min.
substituted
(1-(benzyl)-1H-1,2,3-triazol-4-yl)(piperazin-1-yl)
methanone derivatives (10aa–ef), the evaluation of their
in vitro cytotoxic profile on selected human cancer cell lines
and further studies on their mechanistic aspects.
Results and discussion
Chemistry
Finally, the key intermediate benzyl-1H-1,2,3-triazole-4-
carboxylic acids (6a–e) with different substitution were
coupled with 9a–f using HBTU, HOBt in DMF to afford the
desired conjugates 10aa–ef, and the yields were obtained in
the range of 75–90%. All the structures of these conjugates
The synthetic plan was executed as planned and outlined in
Scheme 1. The two intermediates substituted benzyl-1,2,3-
triazole-4-carboxylic acids (6a–e) and substituted sulfonyl
piperazine derivatives (9a–f) were prepared via the
conventional methods for the synthesis of desired products.
Substituted benzyl-1,2,3-triazole-4-carboxylic acids (6a–e) were
synthesized from commercially available starting materials.
The reaction was employed with different substituted benzyl
alcohols (1a–e) treated with PBr3 to give the corresponding
benzyl bromides (2a–e). We envisioned that the treatment of
2a–e with sodium azide would result in corresponding benzyl
azides (3a–e). The key intermediate ethyl 1-benzyl-1H-1,2,3-
triazole-4-carboxylates (5a–e) with different substitutions were
synthesized by a standard copper(I) catalyzed azide–alkyne
cycloaddition protocol yielding 1,4-adduct from ethyl
were characterized via H and 13C NMR spectroscopy and by
HRMS data.
1
The structure of compound 10aa could be easily identified
by the appearance of a benzylic proton at δ 5.47 ppm and the
signals at δ 3.78 and 2.42 ppm, correspond to the methoxy
and methyl groups. All the aromatic protons of 10aa were in
the range of δ 7.94–6.79 ppm. Compound 10aa showed 4
signals at δ 4.42, 3.82, 3.11 and 3.07 ppm, which account for
the methylene protons of piperazine. Assessment of 13C NMR
of compound 10aa revealed that the characteristic peak of
carbonyl compound is at 158.86 ppm. All the aromatic and
aliphatic carbons of the synthesized compounds were in the
range of 158.86–20.47 ppm. The HRMS spectra of the
designed hybrids showed characteristic [M + H]+ peaks to
their corresponding molecular formula.
Evaluation of biological activity
In vitro cytotoxic activity. All the as-synthesized
compounds (10aa–ef) were evaluated for their in vitro anti-
proliferative activity against the selected human cancer cell
Fig.
2 Design of substituted (1-(benzyl)-1H-1,2,3-triazol-4-yl)
(piperazin-1-yl)methanone conjugates (10aa–ef).
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lines of the breast (BT-474 and MCF-7 were procured from
NCCS, Pune, India), cervical (HeLa were procured from
NCCS, Pune, India), non-small cell lung (NCI-H460 were
obtained from ATCC, USA) and human epidermal
keratinocyte (HaCaT was a kind gift from Dr. Munia Ganguly,
IGIB, New Delhi, India) by employing the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Here, nocodazole was used as a control standard for
comparing the cytotoxicity of the new chemical entities
(NCEs). The IC50 (μM) values (concentration required to
inhibit 50% of cancer cell growth) of tested compounds
(10aa–ef) and reference (nocodazole) have been calculated in
Microsoft Excel. The drug concentrations against the percent
inhibition are plotted. The estimate of IC50 is by plotting x–y
and fit the data with a straight line (linear regression). Using
the linear (y = mx + c) equation on this graph, the IC50 value
was determined and summarized in Table 1. Compound 10ec
was the most potent against BT-474 and HeLa with IC50
values of 0.99 0.01 μM and 1.02 0.18 μM, respectively.
Moreover, 10ec also showed to be active on MCF-7, NCI-H460
and HaCaT with IC50 values of 2.34 0.05 μM, 4.88 0.02
μM and 3.68 0.19 μM, respectively. Among the series tested,
compounds 10dc, 10de and 10ee showed considerable
cytotoxicity in all cancer cell lines.
It is evident from the IC50 values (Table 1) that the
substitution of the R-group with m-phenoxy and R1 is
substituted with 4-bromo showed better cytotoxic activity
among all derivatives. Furthermore, the removal of the
substituent or introducing an electron-donating group
(p-tolyl) at R1; without changing the substitution pattern at R
(m-phenoxy), progressively decreases the cytotoxicity (Fig. 3).
Moreover, R1 substituted with electron-withdrawing groups
(4-Cl) showed considerable cytotoxicity. Replacing the
m-phenoxy with 4-iodo at position R, and R1 substituted with
electron-withdrawing substituents (4-Cl and 4-Br) showed a
detrimental effect on cytotoxicity. Considering the steric
effect of the substituents such as Cl, Br, CH3, OCH3 and
t-butyl, it can be seen that moderately bulkier Br, shows the
highest potency among all of the other substitutions with less
A-value or very high A-value, when R1 = I or phenoxy (bulky
groups). Therefore, further biological studies were carried out
on one of the most potent compounds (10ec) from the series.
Tubulin polymerization assay. Tubulin is an important
component of the cytoskeletal network and it is essential for
the cellular functions mainly during mitosis. Tubulin is the
target of several small molecules. Recently, several anti-
proliferative compounds have been shown to function as
tubulin polymerization inhibitors.21,22 To investigate the
Table 1 Cytotoxicity of target compounds (10aa–ef) against selected human cancer cell lines (IC50 values in μM)a
Compound
bBT-474
cHeLa
dMCF-7
eNCI-H460
fHaCaT
10aa
10ab
10ac
10ad
10ae
10af
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
10ba
10bb
10bc
10bd
10be
10bf
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
10ca
10cb
10cc
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
10cd
10ce
10cf
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
10da
10db
10dc
10dd
10de
10df
>30
>30
>30
>30
>30
4.96 0.13
2.33 0.11
>30
1.21 0.17
>30
5.74 0.02
6.52 0.01
>30
3.58 0.05
>30
1.34 0.04
2.73 0.02
>30
3.06 0.18
>30
2.25 0.11
3.55 0.18
>30
1.42 0.03
>30
2.22 0.71
2.48 0.09
>30
2.06 0.05
>30
10ea
10eb
10ec
10ed
10ee
10ef
>30
>30
>30
>30
>30
1.96 0.14
0.99 0.01
>30
3.89 0.16
5.13 0.15
0.87 0.01
8.61 0.24
1.02 0.18
>30
3.70 0.02
3.72 0.04
1.11 0.08
4.41 0.24
2.34 0.05
>30
3.28 0.11
2.20 0.19
1.76 0.14
5.68 0.15
4.88 0.02
>30
5.05 0.18
8.20 0.01
2.29 0.05
2.96 0.05
3.68 0.19
>30
4.34 0.04
2.59 0.17
1.19 0.18
Nocodazoleg
a
b
c
d
e
50% inhibitory concentration after 48 h of compounds treatment. Breast cancer cells. Cervical cancer cells. Breast cancer cells. Non-
f
g
small cell lung cancer cells. Human epidermal keratinocyte. Reference compound. All the values are expressed as mean SEM in which
each treatment was performed in triplicates.
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Fig. 3 SAR summary of sulfonyl piperazine integrated 1,2,3-triazole
conjugates.
synthesized compound (compound 10ec) used in the present
study, which showed maximum cytotoxicity in the MTT assay
and inhibited the cell cycle at the G2/M phase, was evaluated
for its tubulin polymerization inhibitory activity in a cell-free
in vitro assay. The assay was performed where nocodazole
and paclitaxel were considered as standards and tubulin
inhibition in the absence of test compounds was considered
as control. The data obtained with standards and control was
compared with the tubulin inhibition activity of the test
compound. It was found that tubulin targeting ability of the
test compound was moderate. Since it was moderate, some
other additional targets may also be involved in controlling
the cellular activity besides the tubulin polymerization
inhibition. The details were shown in Fig. 4.
Fig. 5 BT-474 cells were treated with both CA-4 (control) and 10ec
for 48 h. A. Cells in the absence of 10ec (untreated cells); B. Cells after
the treatment of 10ec; C. Cells in the absence of CA-4 (untreated
cells); D. Cells after the treatment of CA-4.
in
a
concentration-dependent
manner.
However,
morphological features were not present in control cells.
After incubation of 48 h with compound 10ec, the images
were captured under a phase-contrast microscope.
Acridine orange/ethidium bromide (AO/EB) staining. To
differentiate live, apoptotic and necrotic cells, the AO/EB
staining assay was performed.
AO stains the nuclei in green that impregnate the intact cell
membrane, whereas EB stains the nuclei in red that have lost
membrane integrity. BT-474 cells were stained with AO/EB.
Normal morphology was observed in the control cells. Fig. 7
clearly stated that morphological changes, such as non-
uniform distribution of chromatin, chromatin condensation
and membrane blebbing, were observed under a fluorescence
microscope in dose-dependent manner, thus it indicates that
compound had induced apoptosis in breast cancer cells.
DAPI staining. DAPI (4′,6-diamidino-2-phenylindole) is a
blue fluorescent dye that binds firmly to the nucleus and
distinguishes chromatin condensation or nuclear damage.
DAPI stains the apoptotic nuclei as bright blue colour due to
Immunohistochemistry assay to find the extent of tubulin
polymerization inhibition. Results from the cell cycle
experiments indicated that 10ec is efficient in blocking the
cell cycle at the G2/M phase. To check the possible
mechanism of action of these molecules in exhibiting
cytotoxicity
among
BT-474
cancer
cells,
immunohistochemistry assay was performed.23
BT-474 cells were treated with 10ec at 2 μM concentration
for 48 h. Combretastatin A-4 (CA-4), a known tubulin
polymerization inhibitor used as a positive control. Confocal
microscopic results suggested that untreated cancer cells
displayed the normal distribution of microtubules, whereas
cells treated with 10ec and CA-4 compounds showed the
disrupted microtubule organization as shown in Fig. 5.
Morphological
observations
using
phase-contrast
microscope. Morphological changes were observed using a
phase-contrast microscope. As depicted in Fig. 6, the
characteristic morphological changes such as cell wall
deformation, shrinkage of cells, loss of cell–cell adhesion and
reduction in the number of viable cells have been observed
Fig. 6 BT-474 cells were treated with and without compound 10ec at
concentrations 0.5, 1 and 2.5 μM. Images were captured at 200×
magnification. The scale bar in the image indicates 50 μm.
Fig. 4 Effect of compound 10ec on tubulin polymerization.
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Fig. 7 AO/EB staining of compound 10ec. Cells were treated with
10ec in the concentration of 0.5, 1 and 2.5 μM and compared with
control. Images were captured at 200× magnification. The red scale
bar in the image indicates 50 μm.
Fig. 9 Clonogenic assay was performed with compound 10ec. BT-474
cells were treated with 10ec in the concentration of 0.5, 1, 2.5 μM and
compared with control (untreated cells).
the condensed nucleus and stains the live-cell nucleus as
light blue. Hence, to observe the chromatin condensation or
nuclear damage, DAPI staining was performed. It can be
observed in Fig. 8 that the control cells were intact with
normal morphology, while compound 10ec treated cells
showed horse shoe shaped nuclei, nuclear shrinkage,
fragmented, pyknotic and chromatin condensation, these are
the remarkable features of apoptosis.
Clonogenic growth inhibition assay. Disruption of the
colony-forming ability in BT-474 cells by 10ec was evaluated by
the clonogenic assay, as shown in Fig. 9. We observed the
formation of colonies in control, whereas after treatment with
compound 10ec, apparent reduction in the number of colonies
was observed in a concentration-dependent manner (0.5, 1 and
2.5 μM). Hence, these results specify the importance of
compound 10ec in inhibiting colony formation in BT-474 cells.
The total numbers of colonies were counted by a molecular
imaging system (Vilber Fusion Fx software), and the values
were represented as a percent colony-forming ability.
Cell cycle analysis. To understand the effect of 10ec on cell
cycle progression, the cell cycle analysis was performed using
the propidium iodide staining method and the cells were
treated with increasing concentrations (0.5, 1 and 2.5 μM) of
10ec. It can be seen in Fig. 10 that a significant increase in the
population of cells at sub G1 (from 4.06% to 11.30%) and G2/
M phase (from 19.35% to 63.41%) happen as compared to the
control cells. However, the moderate accumulation of cells in
the S phase (from 2.22% to 5.57%) and extreme decrement of
cells in the G0/G1 phase (60.53% to 9.09%) was also observed.
Collectively, these results indicate that compound 10ec induces
blockade at the sub-G1 and G2/M phase.
Effect on mitochondrial membrane potential (ΔΨm). The
effect of compound 10ec on the mitochondrial membrane
potential (ΔΨm) was determined using the JC-1 staining
method. JC-1 is a lipophilic cationic dye. Normal polarised
Fig. 8 DAPI staining of compound 10ec. BT-474 cells were treated
with 10ec in the concentration of 0.5, 1, 2.5 μM and compared with
control (untreated cells). Images were captured at 200× magnification.
Red scale bar in the image indicates 50 μm.
Fig. 10 Effect of 10ec on the cell cycle progression of BT-474 cells.
Cells were treated with 10ec (0.5, 1 and 2.5 μM) and cell cycle analysis
was performed by the flow cytometric analysis using PI staining.
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mitochondria stain red due to the formation of JC-1
oligomers, whereas depolarised mitochondria stain green
due to the formation of J-monomers. Cells were treated with
increasing concentrations (0.5, 1 and 2.5 μM) of compound
10ec for 48 h and mitochondrial membrane potential was
observed with respect to the control. The flow cytometric
analysis clearly demonstrates that increase in the depolarized
cell population (P2) from control (0.48%) to 50.48% (0.5 μM),
61.44% (1 μM) and 89.84% (2.5 μM), respectively in a
concentration-dependent manner (Fig. 11). Thus, compound
10ec triggered the depolarisation of ΔΨm which indicates that
cytotoxic effect associated with mitochondria.
Annexin V-Alexa fluor 488/PI assay. To quantify the
apoptotic cell death induced by compound 10ec, the Annexin
V-Alexa fluor 488/propidium iodide staining assay was used.
Cells were treated with 0.5, 1 and 2.5 μM of 10ec for 48 h
were stained with Annexin V-Alexa fluor 488/propidium
iodide, and samples were analysed using flow cytometry.
Annexin assay expedites the detection of necrotic cells (Q1-
UL; AV−/PI+), live cells (Q2-LL; AV−/PI−), early apoptotic cells
(Q3-LR; AV+/PI), and late apoptotic cells (Q1-UR; AV+/PI+).
Fig. 12 Effect of compound 10ec on the apoptotic cell death in BT-
474 cells. The compound 10ec treated cells were stained with Annexin
V-Alexa fluor 488/propidium iodide and analysed for apoptosis using a
flow cytometer. The 10 000 cells from each sample were analysed via
flow cytometry.
10ec and 10ef) have one or zero violations except 10ee.
Moreover, compounds that violated Lipinski's rules either have
a molecular weight larger than 500 or showed logP > 5
(octanol–water partition coefficients). The prediction of
physicochemical parameters for good bioavailability can be
useful for the design of safe and effective compounds at the
advanced stages of the drug discovery process.
Molecular docking studies. To understand the binding
view and the nature of contacts with tubulin protein (PDB ID:
3E22),26 we have performed molecular docking studies on the
most active compound 10ec by employing the GLIDE module
of Schrödinger 2017-1 (ref. 27) software. The molecular
docking results for compound 10ec are depicted in Table S3
in the ESI† file. Compound 10ec has made three hydrogen
Therefore, compound 10ec induces apoptosis in
a
concentration-dependent manner in comparison to the control,
as shown in Fig. 12.
In silico computational studies. Lipinski parameters (TPSA,
NRotB, logP, nOH–NH, nO–N, molecular weight and number
of violations) can be assessed using molinspiration online
property calculation tool kit.24 The solubility and drug score
values were predicted by using molsoft software25 and the
results are depicted in Table S2 in the ESI† file. Molecules likely
to be having TPSA ≤ 140 Å2, NRotB ≤ 10, milogP value must
be in the range of −0.4 to 5, clogS < −4, molecular weight ≤
500, hydrogen bond donor sites (nOHNH) ≤ 5 and hydrogen
bond acceptor sites (nON) ≤ 10. On this basis, all the
synthesized analogues were found to be within the acceptable
range and are anticipated to have good oral absorption. It is
noteworthy that analyzed compounds (10db, 10dc, 10de, 10eb,
Fig. 13 a) Molecular docking view of the compound 10ec (maroon
color ball and stick) and b) its ligand–protein interactions in the active site
of α/β-tubulin (PDB ID: 3E22). The red dashed line represents a hydrogen
bond. The atom colors for all amino acids are as follows: for all amino
acids residues [nitrogen (dark blue), oxygen (red) and hydrogen (white)],
Ser178 [carbon (yellow)], Ala250 [carbon (cyan)], Lys254 [carbon (light
blue)], Cys241 and Met325 [carbon (green), sulphur (pale yellow)], for all
other amino acids residues [carbon (green)].
Fig. 11 Effect on compound 10ec on mitochondrial membrane
potential. BT-474 cells were treated 10ec in the concentration of 0.5,
1, 2.5 μM and compared with control (untreated cells).
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bond contacts with the binding site residues Ser178, Ala250 References
and Lys254. The sulfonyl group of compound 10ec has
established strong hydrogen bond interaction with Ser178 (d
= 2.7 Å). The triazole moiety has formed two hydrogen bond
interactions with binding site residues Ala250 and Lys254
with a distance of 3.7 Å and 3.4 Å respectively. In addition,
compound 10ec showed hydrophobic interactions that
stabilize the compound in the binding pocket of α/β-tubulin.
As shown in Fig. 13, compound 10ec has shared hydrophobic
contacts with Val177, Ala180, Tyr224, Cys241, Leu248, Ala250,
Leu252, Leu255, Ala316, Ala317, Val318, Met325, Ala354,
Val355 and Ile378 of α/β-tubulin. Moreover, compound 10ec
was well lodged in the binding site interphase of α/β-tubulin
and established a number of interactions with its key
residues. From the rational justification of docking studies,
we understood that compound 10ec has the good tubulin
polymerization inhibitory activity and obtained many key
instructions for future design and synthesis of potential
tubulin polymerization inhibitors.
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Conclusion
In the current study a series of substituted (1-(benzyl)-1H-
1,2,3-triazol-4-yl)(piperazin-1-yl)methanone derivatives were
synthesized and evaluated for their in vitro cytotoxic potential
against a selected panel of human cancer cell lines such as
BT-474 (breast), HeLa (cervical), MCF-7 (breast), NCI-H460
(non-small cell lung cancer) and HaCaT (human epidermal
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value of 0.99
0.01 μM. The flow cytometric analysis
indicated that compound 10ec inhibits the cell cycle at sub-
G1 and G2/M phase. Compound 10ec could also effectively
inhibit tubulin polymerization with an increase in the
concentration. Moreover, biological studies such as phase
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10ec induced morphological changes and cell proliferation is
inhibited through the induction of apoptosis in BT-474 cell
line. Molecular modelling studies revealed that compound
10ec interacted with the tubulin via colchicine binding site.
Overall, the current studies suggest that compound 10ec
could be a potential lead for the development of a library of
compounds, and further modifications in the structure may
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Conflicts of interest
There is no conflict of interest to declare.
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Acknowledgements
The authors thank and acknowledge the Department of
Pharmaceuticals, Ministry of Chemicals & Fertilizers and
CSIR-Indian Institute of Chemical Technology, Hyderabad,
India for providing funds, Scientific and Instrumental support
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