New (3-(1: H -benzo [d] imidazol-2-yl))/(3-(3 H -imidazo[4,5- b] pyridin-2-yl))-(1 H -indol-5-yl)(3,4,5-trimethoxyphenyl)methanone conjugates as tubulin polymerization inhibitors

Article abstract of DOI:10.1039/c7md00450h

A series of new (3-(1H-benzo[d]imidazol-2-yl))/(3-(3H-imidazo[4,5-b]pyridin-2-yl))-(1H-indol-5-yl)(3,4,5-trimethoxyphenyl)methanone conjugates 4-6(a-i) were synthesized and evaluated for their antiproliferative activity on selected human cancer cell lines

Full text of DOI:10.1039/c7md00450h

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DOI: 10.1039/C7MD00450H.
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New (3-(1H-benzo[d]imidazol-2-yl)/(3-(3H-imidazo[4,5-b]pyridin-
2-yl)-(1H-indol-5-yl)(3,4,5-trimethoxyphenyl)methanone
conjugates as tubulin polymerization inhibitors
Received 00th January 20xx,
Accepted 00th January 20xx
Kishore Mullagiri,a V. Lakshma Nayak, a Satish Sunkari,a Geetha Sai Mani,b Sravanthi Devi
Guggilapu,b Burri Nagaraju,a Abdullah Alarifi,c and Ahmed Kamal*a,b,c
DOI: 10.1039/x0xx00000x
www.rsc.org/
A
series of (3-(1H-benzo[d]imidazol-2-yl)/(3-(3H-imidazo[4,5-b]pyridin-2-yl)-(1H-indol-5-yl)(3,4,5-trimethoxyphenyl)
methanone conjugates 4-6(a‒i) were synthesized and evaluated for their antiproliferative activity on selected human
cancer cell lines such as prostate (DU-145), lung (A549), cervical (HeLa) and breast (MCF-7). Most of these conjugates
showed considerable cytotoxicity with IC50 values ranging from 0.54 to 31.86 µM. Among them, compounds 5g and 6f
showed significant activity against human prostate cancer cell line DU-145 with IC50 values of 0.68 µM and 0.54 µM
respectively. Tubulin polymerization assay and immunofluorescence analysis results suggest that these compounds
effectively inhibit microtubule assembly formation in DU-145. Further, the apoptosis inducing ability of these derivatives
(5g and 6f) was confirmed by Hoechst staining, measurement of mitochondrial membrane potential ROS generation and
Annexin V-FITC assays.
agents. Combretastatin A-4, isolated from the bark of the
South African tree Combretum caffrum,3 is a highly effective
natural tubulin-binding molecule affecting microtubule
Introduction
The Microtubules are dynamic in structures that together with
actin microfilaments and intermediate filaments, constitute
the cellular cytoskeleton. Besides their well-known role in cell
division, their functions involve maintenance of cell shape and
morphology, cellular motility, and trafficking of organelles and
vesicles.1 Microtubules are formed by the polymerization of
heterodimers of α and β tubulin. In the mitotic phase,
microtubules are in dynamic equilibrium with tubulin dimers
as tubulin is assembled into microtubules and which are
disassembled to tubulin. The essential role of microtubules in
mitotic spindle formation and proper chromosomal separation
makes them one of the most attractive targets for the design
and development of many small natural and synthetic
antitumor drugs.1,2 Many of them exert their effects by
inhibiting the noncovalent polymerization of tubulin into
microtubules. Therefore, there has been great interest in
identifying and developing novel antimicrotubule molecules.
Among the naturally occurring compounds, combretastatin
O
O
O
O
O
OH
O
O
O
OH
O
(2)
Phenstatin
R1
X
Combretastatin A-4 (1)
N
NH
O
O
O
O
O
H
N
S
O
NH
N
N
R
O
X = CH and N
4(a-i), 5(a-i) and 6(a-i)
3
)
Nocodazole (
Fig. 1: Tubulin polymerization inhibitors.
dynamics by binding to the colchicine site.4It shows potent
cytotoxicity against a wide variety of human cancer cell lines
and MDR cell lines.5 However, combretastatins are
characterized by poor solubility and chemical instability.
Importantly, the olefinic bond with cis configuration (Z-
geometry) plays a fundamental role in binding at the colchicine
site by positioning the rings at appropriate distance to
maximize interactions.6 Several attempts have been reported
to modify this cis-olefinic bond to prevent its isomerisation
under amenable conditions. This mostly included either
modification of the olefinic bond by the introduction of
saturation, substituents and its replacement with a three to six
membered ring system, which resulted in cis restricted
analogues of CA-4.7-11The replacement of the olefinic bridge of
CA-4 with a carbonyl group furnished a benzophenone-type
A-4 (1, Fig. 1) is one of the best characterized antimitotic
a.Medicinal Chemistry and Biotechnology, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007, India, E-mail: ahmedkamal@iict.res.in
b.Department of Medicinal Chemistry, National Institute of Pharmaceutical
Education and Research (NIPER), Hyderabad-500 037, India
c. Catalytic Chemistry Research Chair, Chemistry Department, College of Science,
King Saud University, Riyadh 11451, Saudi Arabia.
† Footnotes relaꢀng to the ꢀtle and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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CA-4 analogue named phenstatin (2, Fig. 1), which has been iodomethane (MeI) and bromoethane (EtBr) in the presence of
found to be a potent cytotoxic agent and inhibitor of tubulin sodium hydride and DMF to produce corresponding 1-
polymerization, with activities differing little from those of CA-
4.12 Benzophenone-type CA-4 analogues are attractive targets
for anti-tubulin agents as the benzophenone backbone not
only provides ease of synthesis without the need to control the
geometric selectivity (Z and E-geometry) but also increases the
pharmacological potential through increased drug stability and
water solubility.13
Agents that effect the tubulin polymerization having an
indole as their core nucleus have been recently reviewed and
in the past few years an ever increasing number of synthetic
indoles as potent tubulin polymerization inhibitors have been
reported.14 Aroylindole moiety constituted the core structure
of antimitotic compounds that have been reported as tubulin
polymerization inhibitors.15 On the other hand, benzimidazoles
are more privileged scaffold due to their biological properties
and have been reported to possess potential anticancer
activity and anti-HIV activity,16-24 apart from antibacterial,25,26
antifungal, antiviral and antioxidant activities.27-30 Nocodazole
(NSC-238189,
3, Fig. 1) is another well-known inhibitor of
tubulin polymerization that possess a benzimidazole moiety
which inhibits cell proliferation and is largely used as a
pharmacological tool and positive control.31As part of our
ongoing efforts to discover newer tubulin inhibitors, we
previously reported imidazopyridine-benzimidazole hybrids32
and phenstatin/isocombretastatin-oxindole conjugates.33
Considering the biological importance of these moieties
(phenstatin, indole and benzimidazole) attempt has been
made in the present study to synthesize indole-benzimidazole
conjugates.
Scheme 1: Reagents and conditions: (i) TBDMSCl, NaH, THF, 0 °C-rt, 3 h, 93%; (ii)
MeI, NaOH,DMSO, 0°C-rt, 3 h, 95%; (iii) EtBr, NaH, THF,0°C-rt, 3 h, 95% (iv) 3,4,5-
trimethoxy benzaldehyde, n-BuLi, THF, -78°C, 4 h, 73˗75%, (v) IBX, DMSO, 0 °C–
,
rt, 3 h, 95%; (vi) TBAF, THF, 0°C-rt, 4 h, 90% (vii) POCl3, DMF, CHCl3, Reflux, 12 h,
78-82%; (viii) Na2S2O5, EtOH:H2O, 80°C, 2 h, 68-85%.
substituted indoles (8a-c) respectively. The products 8(a-c)
were further treated with n-butyllithium (n-BuLi) and 3,4,5-
trimethoxybenzaldehyde at -78 oC to furnish the
corresponding alcohols (9a-c) with moderate yields. The
oxidized products (10a-c) were isolated with good yields from
the reaction of 9(a-c) with 2-iodoxybenzoic acid (IBX) in DMSO
and after oxidation, the deprotection of TBDMS group with
tetra-n-butylammonium fluoride (TBAF) provided 10a. Next,
Vilsmeier reaction of 10(a-c) with phosphoryl chloride (POCl3)
and DMF afforded 11(a-c) in good yields. Finally, the
derivatives 4-6(a-i) were generated by condensation of various
aldehydes 11(a-c) with substituted benzene-1,2-diamine
(OPDs) in the presence of sodium metabisulfite (Na2S2O5) as
Based on these observations, we describe the
modifications on the CA-4 scaffold which contains
a
trimethoxyphenyl moiety identical to the A-ring of CA-4.
Further congeners were generated having indole moiety at 3rd
position. Thus, the rationale of these moieties was to conserve
the privileged structures of the trimethoxyphenyl and indole
groups. Considering these modifications, an attempt has been
made in the present study to synthesize indophenstatin-
benzimidazole conjugates 4-6(a‒i) (Fig. 1). The resulting
conjugates were evaluated for antiproliferative activity and
examined their structure activity relationship (SAR) followed
by studies to elucidate the mechanism of action which
included cell cycle progression, tubulin polymerization assay
and molecular docking studies. Further to confirm the
induction of apoptotic cell death by the conjugates studies
such as Hoechst staining, mitochondrial membrane potential
and Annexin V elucidated.
shown in Scheme
1. All these new derivatives were
characterized by spectral analysis such as 1H-NMR, 13C NMR
and HRMS.
Biological studies
Cytotoxic activity
These conjugates 4-6(a-i) were evaluated for their cytotoxic
activity on a panel of deferent human cancer cell lines (All the
cell lines were purchased from The National Centre for Cell
Science (NCCS), Pune, India) such as, A549 (lung), HeLa
(cervical), MCF-7 (breast) and DU-145 (prostate) by employing
MTT assay.34 The results are summarized in Table1 and the IC50
values expressed in µM and compared with nocodazole as
control. Some of the derivatives show significant activity
against most the cell lines tested with IC50 values ranging from
Results and discussion
Chemistry
The trimethoxy aroyl indole-benzimidazole conjugates 4-6(a‒i)
were synthesized in six straight forward reactions. Initially, the
0.54 to 31.86 µM. Among them derivatives 4c-e
, 4i, 5b, 5c, 5f-
commercially available 5-bromoindole (7) was protected with
h
,
6a, 6b, 6d-f and 6h showed considerable activity with IC50
tert-butylchlorodimethylsilane (TBDMSCl), alkylated with
2 | J. Name., 2012, 00, 1-3
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Fig. 2: Flow cytometric analysis in DU-145 prostate cancer cell lines after
treatment with 5g and 6f at 0.5 and 1 µM concentrations for 48 h; A: Untreated
cells (control cells), B: 5g (0.5 µM), C: 5g (1 µM), D: 6f (0.5 µM) and E: 6f (1 µM).
values of <5 µM and majority of derivatives show good
cytotoxicity against prostate cancer cell line (DU-145).
However, derivatives 5g and 6f were found to be very active
against DU-145 cell line with IC50 values of 0.68 µM and 0.54
µM respectively. Therefore, the DU-145 cell line was chosen as
a model cell line for subsequent experiments.
Many anticancer compounds exert their cytotoxic effect either
by arresting the cell cycle at a particular checkpoint of cell
cycle or by induction of apoptosis or a combined effect of both
cycle block and apoptosis.35In this study DU-145 cells were
treated with derivatives 5g and 6f at concentrations of 0.5 and
1 µM for 48 h. The data obtained clearly indicated that these
derivatives show G2/M cell cycle arrest in comparison with the
untreated cells. These derivatives 5g and 6f showed 14.56 and
15.15% of cell accumulation in G2/M phase at 0.5 µM
concentration, whereas they exhibited 38.47 and 39.80 % of
cell accumulation at 1 µM concentration respectively (Fig. 2
and Table 2).
Table 1: aIC50 values (in µM) for derivatives 4-6(a-i) on selected human cancer cell lines.
Compound
A549b
17.53
26.79
9.13
MCF-7c
10.15
11.88
1.76
DU-145d
9.72
11.09
1.69
2.47
7.82
4.83
8.26
10.42
11.3
11.88
0.83
1.20
6.31
1.95
7.80
0.68
1.14
7.26
4.34
1.53
5.30
1.26
1.95
0.54
1.12
5.95
7.52
1.39
HeLae
10.03
12.07
2.51
4a
4b
4c
4d
16.28
19.53
27.00
28.40
31.86
15.6
3.89
3.84
4e
9.89
13.95
11.12
13.12
17.52
13.8
4f
8.97
4g
16.43
15.00
12.4
Table 2. Distribution of DU-145 cells in various phases of cell cycle.
4h
4i
Sample
A: Untreated cells
(Control)
Sub G1%
0.89
G0/G1%
87.95
S%
G2/M%
8.26
5a
18.69
15.44
3.16
14.44
1.38
14.75
5.95
0.99
5b
5c
1.73
9.80
B: 5g (0.5 µM)
C: 5g (1 µM)
D: 6f (0.5 µM)
E: 6f (1 µM)
1.61
0.65
1.77
0.48
79.15
57.53
77.75
57.05
2.23
2.07
2.32
1.83
14.56
38.47
15.15
39.80
5d
29.64
27.08
27.15
1.44
8.56
8.22
5e
2.11
6.44
5f
10.50
0.95
12.85
1.41
5g
5h
22.88
28.54
25.86
2.19
1.41
7.33
Effect of compounds on tubulin polymerization
5i
14.73
8.29
15.16
15.11
2.00
In general G2/M cell cycle arrest is strongly associated with
inhibition of tubulin polymerization and since compounds 5g
and 6f cause cell cycle arrest at G2/M phase, it was considered
of interest to investigate their microtubule inhibitory function.
Tubulin subunits are known to heterodimerize and self-
assemble to form microtubules in a time dependent manner.
The progression of tubulin polymerization36 was thus examined
by monitoring the increase in fluorescence emission at 420 nm
(excitation wavelength is 360 nm) in 384 well plate for 1 h at
37 oC with and without the conjugates in comparison with
reference compound nocodazole. The test compounds (5g and
6f) significantly inhibited tubulin polymerization by 66.65 and
70.09 % respectively, whereas the reference
6a
6b
1.65
6c
17.82
1.69
10.27
1.41
14.76
6.48
6d
6e
5.90
1.90
1.73
6f
0.94
1.05
0.91
6g
6.58
1.86
4.64
6h
6i
14.36
31.62
1.05
11.38
10.14
1.48
11.25
13.43
1.05
Nocodazole
a50% Inhibitory concentration after 48 h of drug treatment. bHuman lung cancer,
c Human breast cancer. dHuman prostate cancer. eHuman cervical cancer
.
Cell cycle analysis
Fig. 3: Effect of compounds on tubulin polymerization: tubulin polymerization
was monitored by the increase in fluorescence at 360 nm (excitation) and 420
nm (emission) for 1 h at 37°C
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Fig. 4. Hoechst staining in DU-145 prostate cancer cell line.A: Untreated cells
(control cells), B: 5g (0.5 µM) and C:6f (0.5 µM).
Table 3: Inhibition of tubulin polymerization (IC50) of compound5g and6f
Compound
IC50a ± SD (in μM)
1.53±0.02
Apoptosis is one of the major pathways that lead to the
process of cell death. Chromatin condensation and fragmented
nuclei are known as the classic characteristics of apoptosis. It
was considered of interest to investigate the apoptotic
inducing effect of the derivatives by Hoechst staining (H33258)
method in DU-145 cell line. Therefore, DU-145 cells were
treated with 5g and 6f at each 0.5 µM concentrations for 48 h.
Manual field quantification of apoptotic cells based on
cytoplasmic condensation, presence of apoptotic bodies,
nuclear fragmentation and relative fluorescence of these
derivatives (5g and 6f) revealed that there was significant
increase in the percentage of apoptotic cells (Fig. 5).
5g
6f
1.45±0.05
Nocodazole
1.60±0.03
compound (nocodazole) exhibited 65.47% inhibition (Fig. 3).
Furthermore, these conjugates (5g and 6f) were evaluated for
their in vitro tubulin polymerization assay at different
concentrations. These molecules (5g and 6f) showed potent
inhibition of tubulin polymerization with IC50 values of 1.53 and
1.45 μM respectively (Table 3) and nocodazole was employed
as a reference compound.
Immunohistochemistry studies on tubulin
To find out in vitro tubulin polymerization, we investigated
alterations in the microtubule network in DU-145 cells induced
by conjugates 5g and 6f by using fluorescence microscope of
immunohistochemistry studies, as most antimitotic agents
affect microtubules.36 Therefore, DU-145 cells were treated
with conjugates 5g and 6f at 0.5 µM concentration for 48 h.
Measurement of mitochondrial membrane potential (ΔΨm)
The maintenance of mitochondrial membrane potential (∆Ψm)
is significant for mitochondrial integrity and bioenergetic
function.37 Mitochondrial changes including loss of
mitochondrial membrane potential (∆Ψm) are key events that
take place during drug-induced apoptosis. Mitochondrial injury
by 5g and 6f was evaluated by detecting drops in
mitochondrial membrane potential (ΔΨm). In this study we
have investigated the involvement of mitochondria in the
induction of apoptosis by these derivatives. After 48 h of
The results demonstrated
a well-organized microtubular
network in control cells. However, cells treated with
conjugates 5g and 6f showed disrupted microtubule
organization as shown in Fig. 4, thus confirming the inhibition
of tubulin polymerization.
treatment with these derivatives at 0.5 and
1 µM
concentrations, it was observed that reduced mitochondrial
membrane potential (ΔΨm) of DU-145 cells which assessed by
JC-1 staining (Fig. 5).
Fig. 4. Immunohistochemistry analysis of derivativeson microtubule network.
DU-145 cells were treated with conjugates 5g and6f at 0.5 µM concentrations for
48 h followed by staining with an antitubulin antibody and FITC conjugated
secondary antibody.A: Untreated cells (control cells), B: 5g (0.5 µM) and C:6f (0.5
µM).
Hoechst staining for apoptosis
Fig. 5: Derivatives 5g and6f triggers mitochondrial injury. Drops in
mitochondrialmembrane potential (ΔΨm) was assessed by JC-1 staining of DU-
145 cells treated with derivativesand samples were then subjected to flow
cytometry analysis on a FACScan (Becton Dickinson) in the FL1, FL2 channel to
detect mitochondrialmembranepotential.A: Untreated cells (control cells), B: 5g
(0.5 µM), C: 5g (1 µM), D: 6f (0.5 µM) and E: 6f (1 µM).
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fluorescent probe, DCFDA (2',7'-dichlorofluorescindiacetate).
Experimental results revealed that these compounds enhance
the generation of ROS in DU-145 (Fig. 7).
Annexin V-FITC assay for apoptosis
The apoptotic effect of 5g and 6f was further evaluated by
Annexin V FITC/PI (AV/PI) dual staining assay38 to examine the
occurrence of phosphatidyl serine externalization and also to
understand whether it is due to physiological apoptosis or
nonspecific necrosis. In this study DU-145 cells were treated
with these derivatives for 48 h at 0.5 and 1 µM concentrations
to examine the apoptotic effect. It was observed that these
derivatives showed significant apoptosis against DU-145 cells
and results indicated that derivatives 5g and 6f showed 23.62
and 25.60% at 0.5µM concentration, while they exhibited
36.43% and 42.17% apoptosis at
respectively as shown in Fig. 6.
1 µM concentration
Fig. 7 The effect of 5g and 6f on the ROS production in DU-145 cells; A:
Untreated cells (Control cells), B: 5g (0.5 µM), C: 5g (1 µM), D: 6f (0.5 µM) and E:
6f (1 µM).
Molecular docking studies
Molecular modeling studies were performed on Glide v6.040
(Schrodinger, LLC, New York, NY) to investigate the potential
interactions between new series of synthesized compounds
and protein (PDB code: 1SA0). Compounds 5g and 6f were
docked into the colchicine binding site of β tubulin (PDB 1SA0)
for studying the binding mode of these compounds for
antitumor activity. The trimethoxyphenyl ring of compound 5g
formed hydrogen bonding interactions with Ser 140 and Gln
11. Additionally, the –NH group of benzimidazole ring and C=O
group displayed hydrogen bonding with Thr 179 and Cys 254
Fig. 6: Annexin V-FITC staining assay. Quadrants; Upper left (necrotic cells),
Lower left (live cells), Lower right (early apoptotic cells) and Upper right (late
apoptotic cells). A: Untreated cells (control cells), B: 5g (0.5 µM), C: 5g (1 µM), D:
6f (0.5 µM) and E: 6f (1 µM).
Table 4. Distribution of apoptotic cells in Annexin-V FITC experiment.
Upper
left%
Upper
right%
Lower
left%
Lower
right%
Sample
A:Untreated cells
(Control)
0.69
1.55
96.99
0.77
B: 5g (0.5 µM)
C: 5g (1 µM)
D: 6f (0.5 µM)
E: 6f (1 µM)
0.65
0.38
0.52
0.36
5.38
10.58
4.09
75.73
63.19
73.88
57.47
18.24
25.85
21.51
31.30
10.87
Fig. 7: (a) Docking of 5g (coloured by atom) with protein (1SA0) on colchicine-
binding site of β tubulin.(b) Docking of 6f (coloured by atom) with colchicine-
binding site of β tubulin. The backbone of tubulin is shown using a ribbon
representation, and the interacting amino acids are shown as stick models.
Green and Yellow dashed lines represent interaction with active site residues
within a 4.5 Å sphere, H bonding with amino acid backbone respectively.
Effect on ROS generation
Many anticancer agents have demonstrated to exert their
cytotoxic effects by the generation of reactive oxygen species
(ROS)39 which is considered as one of the key mediators of
apoptotic signalling. In this connection, DU-145 cells were
treated with these compounds (5g and 6f) at 0.5 and 1 µM
concentrations for 48 h. After 48 h of treatment, ROS
experiment was performed by using an oxidant-sensitive
respectively. The interactions with other active site residues
Gln 247, Leu 248, Leu 255, Asn 258, Met 259 and Lys 352 were
also observed (Fig. 7a). While in compound 6f; the para, meta
positions of trimethoxyphenyl ring formed hydrogen bonding
with Cys 241. Moreover, the interaction with active site
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6
7
A. B. Maya, B. del Rey, R. P. Lamamie de Clairac, E. Caballero
-
residues Gln 247, Leu 248, Ala 250, Asp 251, Leu 252, Lys 254,
Leu 255, Asn 258, Ala 316, Ala 317, Lys 352, Thr 353, Ala 354,
Val 355 and Ile 378 were also observed (Fig 7b).
and I. Barasoain, Bioorg. Med. Chem. Lett., 2000, 10, 2549
2551.
H. Chen, Y. Li, C. Sheng, Z. Lv, G. Dong, T. Wang, J. Liu, M.
Zhang, L. Li, T. Tao Zhang, D. Geng, C. Niu and K. Li, J. Med.
Chem., 2013, 56, 685-699.
M. Metzler and H. G. Neumann, Xenobiotica., 1977, 7, 117-
132.
D. Simoni, G. Grisolia, G. Giannini, M. Roberti, R. Rondanin, L.
Piccagli, R. Baruchello, M. Rossi, R. Romagnoli, F.P. Invidiata,
S. Grimaudo, M.K. Jung, E. Hamel, N. Gebbia, L. Crosta, V.
Abbadessa, A.D. Cristina, L. Dusonchet, M. Meli and M.
Conclusion
8
9
In conclusion, we have synthesized trimethoxy aroyl indole-
benzimidazole conjugates 4-6(a‒i) and evaluated for their
antiproliferative activity against human cancer cell lines.
Among them, conjugates 5g and 6f showed significant
antiproliferative activity against human prostate cancer cell
line (DU-145). The flow cytometric analysis revealed that these
conjugates arrest cell cycle at G2/M phase. However, these
derivatives effectively inhibited microtubule assembly and
disrupt microtubule network in human prostate cancer cell
line, DU-145. Further, apoptosis studies such as Hoechst
staining, mitochondrial membrane potential and Annexin V
FITC assay suggested that these derivatives induced cell death
by apoptosis in DU-145 cells. Moreover, docking studies
provided some molecular insights about the binding mode of
these conjugates 5g and 6f that bind at the colchicine-binding
site in α,β interfaces of the tubulin. Based on the above results
it is evident that conjugates of this structural class, particularly
conjugates 5g and 6f are acquiescent to further modifications
and function as a suitable template for the design of new class
of tubulin polymerization inhibitors and apoptosis inducers for
the treatment of cancer.
Tolomeo, J. Med. Chem., 2005, 48, 723
10 Q. Zhang, Y. Peng, X. I. Wang, S. M. Keenan, S. Arora and W.
J. Welsh, J. Med. Chem., 2007, 50, 749 754.
-736.
-
11 R. Romagnoli, P. G. Baraldi, M. K. Salvador, D. Preti, M. G.
Tabrizi, A. Brancale, X. H. Fu, J. Li, S. Z. E. Hamel, R.
Bortolozzi, G. Basso and G. Viola, J. Med. Chem., 2012, 55
475-488.
,
12 (a) Y. L. Liu, H. L. Chen, H. Y. Lee and J. P. Liou, Expert Opin
Ther Pat., 2014, 24, 69-88; (b) M. Kavallaris, Nature reviews-
cancer., 2010, 10, 1-12; (c) X. Wu, Q. Wang, W. Li, Anticancer
Agents Med Chem., 2016, 16, 1325-38; (d) G. R. Pettit, B.
Toki, D. L. Herald, P.V. Pinard, M. R. Boyd, E. Hamel and R. K.
Pettit, J. Med. Chem., 1998, 41, 1688-1695.
13 (a) K. Ohsumi, R. Nakagawa, Y. Fukuda, T. Hatanaka, Y.
Morinaga, Y. Nihei, K. Ohishi, Y. Suga, Y. Akiyama and T. Tsuji,
J. Med. Chem., 1998, 41, 3022-3032; (b) K. Ohsumi, T.
Hatanaka, R. Nakagawa, Y. Fukuda, Y. Morinaga, Y. Suga, Y.
Nihei, K. Ohishi, Y. Akiyama and T. Tsuji, Anticancer Drug
Des., 1999, 14, 539
14 A. Brancale and R. Silvestri, Medicinal Research Reviews.,
2007, 27, 209 238.
-548.
-
15 R. Romagnoli, P. G. Baraldi, T. Sarkar, M. D. Carrion, C. L.
Cara, O. C. Lopez, D. Preti, M. A. Tabrizi, M. Tolomeo, S.
Grimaudo, A. Di Cristina, N. Zonta, J. Balzarini, A. Brancale, H.
Conflicts of interest
P. Hsieh and Ernest Hamel, J. Med. Chem., 2008, 51, 1464
1468.
-
The authors declare no competing interest.
16 T. D. Penning, G. D. Zhu, V.B. Gandhi, J. Gong, X. Liu, Y. Shi, V.
Klinghofer, E. F. Johnson, C.K. Donawho, D. J. Frost, V.B. Diaz,
J. J. Bouska, D. J. Osterling, A. M. Olson, K. C. Marsh, Y. Luo
and V. L. Giranda, J. Med. Chem., 2009, 52, 514-523.
17 D. Hao, J. D. Rizzo, S. Stringer, R. V. Moore, J. Marty, D. L.
Dexter, G. L. Mangold, J. B. Camden, D. D. Von- Hoff and S. D.
Acknowledgements
K. S. acknowledges CSIR, New Delhi for the award of senior
research fellowship. We also acknowledge CSIR, New Delhi, for
financial support under the 12th Five Year plan project
“Affordable Cancer Therapeutics (ACT)” (CSC0301) and we
extend our appreciation to the International Scientific
Partnership Program ISPP at King Saud University for funding
this research work through ISPP#0054.
Weitman, Invest. New Drugs., 2002, 20, 261
18 H. M. Refaat, Eur. J. Med. Chem., 2010, 45, 2949
-
270.
2956.
19 S. C. Lio, J. Johnson, A. Chatterjee, J. W. Ludwig, D. Millis, H.
-
Banie, J. C. Sircar, A. Sinha and M. L. Richards, Can. Chem.
Pharmacol., 2008, 61, 1045
20 R. Abonia, E. Cortes, B. Insuasty, J. Quiroga, M. Nogueras and
J. Cobo, Eur. J. Med. Chem., 2011, 46, 4062 4070.
21 M. M. Ramla, M. A. Omar, A. M. M. El-Khamry and H. I. El-
Diwani, Bioorg. Med. Chem., 2006, 14, 7324 7332.
22 J. Styskala, L. Styskalova, J. Slouka and M. Hajduch, Eur. J.
Med. Chem., 2008, 43, 449 455.
23 S. Demirayak, A. Usama, A. C. Mohsen, K. Agri, Eur. J. Med.
Chem., 2002, 37, 255 260.
24 H. T. A. Mohsen, F. A. F. Ragab, M. M. Ramla and H. I. E.
Diwani, Eur. J. Med. Chem., 2010, 45, 2336 2344.
25 B. V. S. Kumar, S. D. Vaidya, R. V. Kumar, S. B. Bhirud and R.
B. Mane, Eur. J. Med. Chem., 2006, 41, 599 604.
26 O. O. Guven, T. Erdogan, H. Goker and S. Yildiz, Bioorg. Med.
Chem. Lett., 2007, 17, 2233 2236.
27 H. Goker, C. Kus, D. W. Boykin, S. Yildiz and N. Altanlar,
Bioorg. Med. Chem., 2002, 10, 2589 2596.
28 I. Kerimov, G. A. Kilcigil, B. C. Eke, N. Altanlar and M. Iscan, J.
Enzym. Inhib. Med. Chem., 2007, 17, 696 701.
-1058.
-
-
References
1
A. L. Risinger, F. J. Giles and S. L Mooberry, Cancer Treat.
Rev., 2009, 35, 255 261.
(a) Kingston, D. G. J. Nat. Prod. 2009, 72, 507
Dumantet and M. A. Jordan, Nat. Rev. Drug Discovery, 2010,
, 790 803; (c) C. Kanthou and G. M. Tozer, Int. J. Exp.
Pathol., 2009, 90, 284 294.
G. R. Pettit, S. B. Singh, E. Hamel, C. M. Lin, D. S. Alberts and
D. Garcia-Kendall, Experentia., 1989, 45, 209 211.
C. M. Lin, H. H. Ho, G. R. Pettit and E. Hamel, Biochemistry,
1989, 28, 6984 6991.
(a) G. R. Pettit, M. R. Rhodes, D. L. Herald, E. Hamel, J. M.
Schmidt and R. K. Pettit, J. Med. Chem., 2005, 48, 4087 4099;
(b) A. McGown and B. Fox, Cancer Chemother. Pharmacol.,
1990, 26, 79 81.
-
-
2
-
515; (b) C.
-
9
-
-
-
3
4
5
-
-
-
-
-
-
-
-
6 | J. Name., 2012, 00, 1-3
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Please do not adjust margins

Please do not adjust margins
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Page 7 of 8
View Article Online
DOI: 10.1039/C7MD00450H
Journal Name
ARTICLE
29 D. Sharma, B. Narasimhan, P. Kumar, V. Judge, R. Narang, E.
D. Clercq and J. Balzarini, J. Enzym. Inhib. Med. Chem., 2009,
24, 1161-1168.
30 G. A. Kilcgil, C. Kus, T. Coban, B. C. Eke and M. Iscan, J.
Enzym. Inhib. Med. Chem., 2004, 19, 129-135.
31 (a) C. Duanmu, L. K. Shahrik, H. H. Holly and E. Hamel, Cancer
Res., 1989, 49, 1344
Yvon, P. Wadsworth and L. Cassimeris, Mol. Biol. Cell., 1997,
, 973–985.
32 A. Kamal, A. V. S. Rao, V. L. Nayak, N. V. S. Reddy, K. Swapna,
-1348; (b) R. J. Vasquez, B. Howell, A. M.
8
G. R. Krishna and A. Mallika, Org. Bio. Chem., 2014, 12, 9864
9880.
-
33 G. B. Kumar, V. L. Nayak, I. B. Sayeed, V. S. Reddy, A. B. Shaik,
R. Mahesh, M. F. Baig, M. A. Shareef, A. R. kumar and A.
Kamal, Bioorg. Med. Chem., 2016, 24, 1729
34 M. Botta, S. Armaroli, D. Castagnolo, G. Fontana, P. Perad
and E. Bombardelli, Bioorg. Med. Chem. Lett., 2007, 17
1579 1583.
-1740.
,
-
35 (a) K. T. Chan, F. Y. Meng, Q. Li, C. Y. Ho, T. S. Lam, Y. To, W.
H. Lee, M. Li, K. H. Chu and M. Toh, Cancer Lett., 2010,
294,118
and J. Jin, Ann. Hematol., 2009, 88,743
36 (a) C. Kanthou, O. Greco, A. Stratford, I. Cook, R. Knight, O.
Benzakour and G. Tozer, Am. J. Pathol., 2004, 165, 1401
-
124; (b) J. K. Shen, H. P. Du, M. Yang, Y. G. Wang
-
752.
-
1411; (b) A. Kamal, Y. V. V. Srikanth, T. B. Shaik, M. N. A.
Khan, M. Ashraf, M. K. Reddy, K. A. Kumar and S. V.
Kalivendi, Med. Chem.Commun., 2011, 2, 819-823.
37 K. Gonda, H. Tsuchiya, T. Sakabe, Y. Akechi, R. Ikeda, R.
Nishio, K. Terabayashi, K. Ishii, Y. Matsumi, A. A. Ashla, H.
Okamoto, K. Takubo, S. Matsuoka, Y. Watanabe, Y.
Hoshikawa, A. Kurimasa and G. Shiota, Biochem. Biophys.
Res. Commun., 2008, 370, 629
38 H. Zhu, J. Zhang, N. Xue, Y. Hu, B. Yang and Q. He, Invest.
New Drugs, 2010, 28, 493 501.
-633.
-
39 (a) Y. Sanchez, G. P. Simon, E. Calvino, E. de Blas and P. Aller,
J. Pharmacol. Exp., 2010, 335, 114-123; (b) K. Liu, D. Zhang, J.
Chojnacki, Y. Du, H. Fu, S. Grant and S. Zhang, Org. Biomol.
Chem., 2013, 11, 4757-4763.
40 Glide, version 6.0, Schrödinger, LLC, New York, NY, 2013.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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DOI: 10.1039/C7MD00450H
New (3-(1H-benzo[d]imidazol-2-yl)/(3-(3H-imidazo[4,5-b]pyridin-2-yl)-
(1H-indol-5-yl)(3,4,5-trimethoxyphenyl)methanone
tubulin polymerization inhibitors
conjugates
as
Kishore Mullagiri,a V. Lakshma Nayak,a Satish Sunkari,a Geetha Sai Mani,b Sravanthi Devi
Guggilapu,b Burri Nagaraju,a Abdullah Alarifi,c and Ahmed Kamal*a,b,c
A series of (3-(1H-benzo[d]imidazol-2-yl)/(3-(3H-imidazo[4,5-b]pyridin-2-yl)-(1H-indol-5-
yl)(3,4,5-trimethoxyphenyl) methanone conjugates were synthesized and evaluated for their
antiproliferative activity on selected human cancer cell lines. Tubulin polymerization assay and
immunofluorescence analysis results suggest that these compounds effectively inhibit
microtubule assembly formation in DU-145.