Design, synthesis and biological evaluation of imidazopyridine-propenone conjugates as potent tubulin inhibitors

Article abstract of DOI:10.1039/c7md00043j

A library of imidazopyridine-propenone conjugates (8a-8u) were synthesized and evaluated for their antitumor activity against four human cancer cell lines, namely, prostate (DU-145), lung (A549), cervical (Hela) and breast (MCF-7) cancer cell lines. These conjugates showed good to moderate activity against the tested cell lines. Among them, two conjugates (8m and 8q) showed significant antiproliferative activity against the human lung cancer cell line (A549) with IC₅₀ values of 0.86 μM and 0.93 μM, respectively. Flow cytometry analysis revealed that these compounds arrested the cell cycle at the G₂/M phase in the human lung cancer cell line (A549), inhibiting tubulin polymerization leading to apoptosis. Further, Hoechst staining, decrease in mitochondrial membrane potential and Annexin V-FITC assay suggested that the cell death was due to apoptosis induction. Overall, the present investigation demonstrated that the synthesized imidazopyridine-propenone conjugates are promising tubulin inhibitors and apoptotic inducers.

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ARTICLE
Design, synthesis and biological evaluation of imidazopyridine-
†
propenone conjugates as potent tubulin inhibitors
Received 00th January 20xx,
Accepted 00th January 20xx
a,b
a
a
a
Ibrahim Bin Sayeed, V. Lakshma Nayak, Mohd Adil Shareef, Neeraj Kumar Chouhan and
*a
DOI: 10.1039/x0xx00000x
Ahmed Kamal.
A library of imidazopyridine-propenone conjugates (8a-8u) were synthesized and evaluated for their antitumor activity
against four human cancer cell lines namely prostate (DU-145), lung (A549), cervical (Hela) and breast (MCF-7) cancer.
These conjugates showed good to moderate activity against the tested cell lines. Among them two conjugates (8m and 8q)
showed significant antiproliferative activity against human lung cancer cell line (A549) with an IC50 values of 0.86 µM and
www.rsc.org/
0
2
.93 µM respectively. Flow cytometry analysis revealed that these compounds arrested the cell cycle at G /M phase in
human lung cancer cell line (A549) inhibiting tubulin polymerization leading to apoptotis. Further, Hoechst staining,
decrease in mitochondrial membrane potential and Annexin V-FITC assay suggested the cell death was due to apoptosis
induction. Overall, the present investigation demonstrated that the synthesized imidazopyridine-propenone conjugates
are promising tubulin inhibitors and apoptotic inducers.
6
effective approach to treat cancer. Many literature reports
-7
Introduction
are available for inhibition of tubulin polymerization with
In eukaryotic cells, microtubules are key components and
essential in a variety of elemental cellular processes such as
cell formation, cell division, maintenance of cell shape,
regulation of motility, secretion and cytoplasmic transport
making microtubules an important target for anticancer
8
subsequent arrest of cells during mitosis leading to apoptosis.
Microtubule polymerization inhibitors are effective in the
treatment of breast, lung, ovarian and other cancers.
Colchicines (
I) and nocodazole (II) are the prominent examples
1
-3
of compounds (Figure 1) that inhibit assembly of microtubule
drugs. In addition, microtubules are also involved in cell
signalling pathways which are accountable for cellular
apoptosis. The microtubule dynamics are regulated by
9
-10
by binding to tubulin.
nocodazole shows
Among the said compounds,
a
preferential interference in the
4
destabilization of microtubule polymerization.
different important proteins such as dynein and kinesin.
Various reports have recognized that microtubule dependence
force is essential for chromosomal translocation and spindle
Imidazopyridine is a fused bicylic heterocycle that represents
1
1
an important class of privileged scaffold. This scaffold
displays a broad spectrum of biological activity such as
inflammation, tumour suppression, viral, apoptosis, fungal and
5
formation. The irregular microtubule dynamics result in the
obstruction of cell division at metaphase; as a result, various
efforts aimed to block mitosis, like inhibition of tubulin
polymerization by tubulin targeting agents has emerged as an
1
2
bacterial. This heterocyclic core can also be found in drugs
like olprinone, nicopidem, saripidem, zolpidem and
1
zolimidine. Investigations demonstrated that this scaffold can
3
a
Medicinal Chemistry and Pharmacology Division, CSIR - Indian Institute of Chemical be extensively used for various therapeutic benefits. Many
Technology, Hyderabad 500007, India
attempts to discover new drugs through creative discovery
b
Academy of Scientific and Innovative Research (AcSIR), CSIR - Indian Institute of
technologies have fallen short of producing the expected
Chemical Technology, Hyderabad 500007, India
results. Henceforth, privileged structure guided scaffold re-
†The authors declare no compeꢀng interests.
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Journal Name
evolution is a primary strategy to identify structurally novel Colchicine (I), Nocodazole (II), Imidazopyridine guanylhydrazones
chemo types by modifying either the central core of the (III) Imidazopyridine-benzimidazoles (IV), aryl propenones (V) and
scaffold or by modification of the side-chain of existing active Imidazopyridine propenone conjugates 8(a-u).
1
compounds. In this regard, our group previously reported
4
1
5
imidazopyridine-benzimidazoles
(
IV) as apoptosis inducers Results and Discussions
that inhibit tubulin polymerization by binding at the colchicine Chemistry
binding site on tubulin. Thus imidazopyridine motifs provide Synthesis of the imidazopyridine-propenones 8(a-u) is shown
immense scope to exploit undescribed bioactivities by making in Scheme 1 (also given in supplementary information). To
use of readily derivatized motifs with well established obtain 2-arylimidazopyridine 4(a-c), equimolar mixtures of
synthetic protocols of imidazopyridine.
substituted 2-bromoacetophenones and 2-aminopyridine were
refluxed for 4-5h followed by 2N HCl under reflux conditions.
The intermediates, imidazopyridine aldehydes 5(a–c) were
prepared by means of Vilsmeier-Hack reaction on the
corresponding 2-arylimidazopyridine 4(a–c). These aldehydes
16
Aryl-propenones are new class of compounds reported
recently as potent tubulin binders which block mitotic cell
division leading to apoptotic cell death. This, combined with
our previous work on aryl-propenone containing molecules as
5
(a-c) were further treated with ethynylmagnesium bromide in
1
7
tubulin polymerization inhibitors prompted us to combine an
aryl-propenone chain to the imidazopyridine scaffold that may
exhibit interesting cytotoxicity profile. Our continued efforts to
discover effective anticancer agents through the combination
of the said two scaffolds led us to design and synthesize a new
series of twenty one imidazopyridine-propenone conjugates
that constitutes a hybrid molecule with three rings. In addition,
a comprehensive structure activity relationship has been
established by varying the substitutions on the rings. These
synthesized conjugates were tested for their antiproliferative
effect in human lung cancer cell line (A549). The two most
active molecules in the series (conjugates 8m and 8q) were
further investigated for their ability to inhibit tubulin assembly
and inducing apoptosis. The results of our investigations along
this direction are presented in this work.
THF to obtain the intermediates 6(a-c) followed by oxidation
with IBX in DMSO providing the corresponding precursors 7(a-
c)
. Subsequently, the desired compounds 8(a-u) were
prepared by reaction of corresponding precursors 7(a-c) with
arylamines in ethanol. The list of substitutions along with their
corresponding yields and respective melting points are listed in
Table 1
.
Scheme 1: Synthesis of imidazopyridine-propenones
NH
2
OMe
O
MeO
OMe
H
N
NH
N
Table 1: Structures of compounds 8(a−u) and their yields
MeO
O
S
NH
NH
O
Yield
%
Melting
N
O
N
Compound
8a
R
2
R
1
0
point ( C)
N
HN
I
II
3
,5-
III
O
OCH3
74%
152-154
R
2
dimethoxyphenyl
4-methoxyphenyl
4-bromophenyl
4-flourophenyl
5-indolyl
R
2
8b
8c
OCH3
75%
71%
74%
71%
66%
62%
64%
78%
130-132
174-176
118-120
218-220
198-200
210-212
232-234
151-153
HN
Y
OCH3
HN
N
O
O
HN
8
d
OCH
3
N
R
1
X
N
R1
8e
OCH3
N
N
8
f
6-indolyl
OCH
OCH
OCH
H
3
3
3
IV
V
8(a-u)
8
8
g
3-quinolyl
h
6-quinolyl
Figure 1: Chemical structures of microtubule targeting agents
8
i
3,5-
2
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1
8
dimethoxyphenyl
4-methoxyphenyl
4-bromophenyl
3-quinolyl
employing MTT assay using Nocodazole as reference drug. The
structure activity relationship was analysed based on the
cytotoxicity results given in Table 2. For ease of understanding, the
rings in the molecule are labelled A, B and C as shown in Figure 2.
8
j
H
H
H
H
82%
75%
65%
69%
172-174
160-162
188-190
204-206
8k
8
l
8
m
6-quinolyl
th
3
,4,5-
trimethoxyphenyl
,5-
Structural modifications were performed 4 position of rings B and
8
n
o
Cl
Cl
72%
73%
194-196
120-122
C respectively whereas ring A was left as such. The IC50 values of
these conjugates showed considerable cytotoxic activity ranging
from 0.86 µM – 22.78 µM. The synthesized conjugates fall into
three categories depending on the substitutents on ring B as
follows: compounds 8(a-h) with 4-methoxy substitution, conjugates
3
8
dimethoxyphenyl
4-methoxyphenyl
4-bromophenyl
5-indolyl
8
8
p
q
Cl
Cl
Cl
Cl
Cl
Cl
67%
79%
61%
64%
69%
71%
96-98
208-210
251-253
219-221
229-231
214-216
8
r
8s
6-indolyl
8
(i-m) with no substitution and compounds 8(n-u) with chlorine
8
t
3-quinolyl
th
substituted at the 4 position. Among the conjugates 8(a-h), it can
be observed from the data that conjugate 8g (IC50 – 1.05 µM) and
conjugate 8h (IC50 – 1.21 µM) have displayed improved cytotoxicity,
on human lung cancer cells, than nocodazole (IC50 – 1.39 µM).
Conjugate 8c (IC50 – 1.34 µM) is equally active as nocodazole when
tested on lung cancer cells. The presence of three methoxy groups
in conjugate 8a has shown moderate cytotoxicity on the four tested
cancer cell lines. Among conjugates 8(i-m), particularly, conjugate
8
u
6-quinolyl
Biological evaluation
a
Table 2: IC50 (µM) values for compounds 8(a-u) on selected human
cancer cell lines
b
c
d
e
Compound
DU-145
6.261
11.13
1.452
6.130
11.33
10.69
5.280
4.720
2.924
2.190
1.820
2.443
1.076
3.250
11.97
10.79
1.500
1.288
2.630
4.168
13.630
1.259
A549
Hela
MCF-7
8
a
b
4.467
12.65
1.346
3.251
10.97
8.800
1.052
1.211
2.239
1.570
1.349
1.750
0.861
1.138
9.897
6.458
0.933
1.014
2.291
2.884
13.040
1.393
4.571
19.95
4.545
10.95
13.60
15.32
9.440
4.852
3.090
14.90
2.042
3.076
1.219
3.020
14.56
11.85
1.479
3.243
10.93
3.715
16.350
1.611
14.86
16.50
9.833
13.00
18.94
19.35
14.95
11.63
10.22
7.609
8.667
5.167
1.807
3.388
13.58
16.33
2.692
7.125
9.600
18.860
22.780
1.086
8
8
c
8
m with quinolyl substituted on ring C possessed significant activity
8
d
e
8
against human lung cancer and prostate cancer cells with IC50
8
f
values of 0.86 µM and 1.07 µM respectively. Also, conjugate 8m
8
8
g
(
IC50 – 1.21 µM) was found to exert potent cytotoxicity on Hela cells
h
in comparison with the rest of conjugates. Conjugate 8k (IC50 – 1.34
8
8
i
j
th
µM) with bromine at 4 position on ring C exerted cytotoxicity close
8
k
to that of nocodazole. An overview on cytotoxicity results of
8
l
conjugates 8(n-u) reveals that conjugate 8q, having chlorine atom
8m
th
th
at 4 position of ring B and bromine atom at 4 position on ring C,
displayed IC50 value 0.93 µM on human lung cancer cell line.
Conjugate 8r (IC50 – 1.28 & 1.01 µM) with 5-indolyl substitution on
ring C was found to display potent cytotoxicity against human
prostate cancer cells and human lung cancer cells respectively. An
outlook on the substituents on ring B shows that the potency is
maximum when the ring is unsubstituted followed by good profile
with electron withdrawing halogen (chlorine) and less with electron
donating substituent (methoxy). Similarly on ring C heterocyclic
substituents (quinolyl, indolyl) were found to be highly active
followed by withdrawing substituents (bromine, fluorine) compared
to donating substituent (methoxy) on ring C.
8
n
o
p
q
8
8
8
8
r
8
s
8
t
8
u
Nocodazole
a
5
b
0% Inhibitory concentration after 48 h of drug treatment;
c
d
Human prostate cancer; Human lung cancer; Human cervical
e
cancer; Human breast cancer.
Cytotoxic activity
The synthesized conjugates were evaluated for their cytotoxic
potential against four human cancer cell lines namely prostate (DU-
145), lung (A549), cervical (Hela) and breast (MCF-7) cancer cells by
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Journal Name
R
2
C
HN
O
A
N
R
1
B
N
R
1
= 4-OCH
=3,4,5-(OCH
-F, 4-Br, indolyl, quinolyl
3
, H, 4-Cl
R
2
3
)
3
, 3,5-(OCH ) , 4-OCH ,
3 2 3
4
Figure 2: Structure activity relationship of imidazopyridine-
propenone conjugates
Effect on cell cycle arrest
Many anticancer compounds exert their growth inhibitory
effect either by arresting the cell cycle at a particular
checkpoint or by induction of apoptosis or a combination of
1
9-20
both.
The screening results revealed that both the
compounds, 8m and 8q, showed improved antiproliferative
activity against lung cancer cell line (A549). To understand that Figure 3: Flow cytometric analysis in A549 lung cancer cell lines
the cell growth inhibition was due to cell cycle arrest, we after treatment with compounds, 8m and 8q at 0.5 and 1 µM
performed a cell cycle analysis using human lung cancer (A549) concentrations for 48 h. A: Control cells (A549); B: Nocodazole (1
cells. In this study, A549 cells were treated with compounds µM); C: 8m (0.5 µM); D: 8m (1 µM); E: 8q (0.5 µM) and F: 8q (1 µM).
8
m
and 8q at concentrations of 0.5 and 1 µM for 48 h. The
data obtained clearly indicated that these compounds
exhibited G /M cell cycle arrest in comparison with the
untreated cells. The compounds, 8m and 8q, showed 15.69%
and 14.40% of cell accumulation in G
Table 3: Distribution of A549 cells in various phases of cell cycle
Sample
Sub G
1
%
G
0
/G
1
%
S%
G
2
/M%
2
A: Control (A549)
B: Nocodazole (1 µM)
1.11
0.79
0.50
0.91
0.66
0.86
90.52
66.92
82.37
66.32
83.28
65.94
1.92
4.55
2.77
4.28
2.68
5.62
7.40
29.68
15.69
30.50
14.40
29.84
/M phase at 0.5 µM C: 8m (0.5 µM)
2
D: 8m (1 µM)
E: 8q (0.5 µM)
F: 8q (1 µM)
concentration, whereas they exhibited 30.50 and 29.84% of
cell accumulation at 1 µM concentration, respectively (Figure 3
and Table 3).
Effect of compounds on tubulin polymerization
In general, G /M cell cycle arrest is strongly associated with
2
2
1
inhibition of tubulin polymerization and since compounds 8m and
8
q caused cell cycle arrest at G /M phase, it was considered of
2
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
2
2-23
tubulin polymerization
was thus examined by monitoring the
increase in fluorescence emission at 420 nm (excitation wavelength
0
is 360 nm) in 384 well plate for 1 h at 37 C with and without the
conjugates in comparison with reference compound nocodazole.
The test compounds, 8m and 8q, inhibited tubulin polymerization
by 68.21% and 66.53% respectively, whereas the reference
4
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compound (nocodazole) exhibited 66.83% of tubulin polymerization
Figure 4). This was followed by the evaluation of IC50 values for
(
these conjugates and the results are shown in Table 4. It was
observed that these conjugates (8m and 8q) showed tubulin
assembly inhibition with IC50 values of 1.82 and 1.93 µM
respectively.
80.00
60.00
40.00
20.00
0
.00
Control
8m
8q
Nocodazole
Figure 5: Immunohistochemistry analysis on microtubule network
A549 cells were treated with conjugates 8m and 8q at 0.5 µM
concentrations for 48 h followed by staining with an antitubulin
antibody and FITC conjugated secondary antibody. A: Control
Figure 4: Effect of conjugates on tubulin polymerization: tubulin
polymerization was monitored by the increasing fluorescence at
0
3
60 nm (excitation) and 420 nm (emission) for 1 h at 37 C.
(
A549); B: Nocodazole (0.5 µM); C: 8m (0.5 µM) and D: 8q (0.5 µM).
Table 4: Inhibition of tubulin polymerization (IC50) of compounds
Hoechst staining for apoptosis
8
m and 8q
Apoptosis, with its classic characteristics of chromatin condensation
and fragmented nuclei, is one of the major pathways that lead to
the process of cell death. The study was performed on A549 cell line
by Hoechst staining (H33258) method. In the study, A549 cells were
treated with the test compounds at 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 the test compounds (8m and 8q)
revealed that the compounds exerted apoptotic activity as evident
from the increase in the percentage of apoptotic cells (Figure 6).
a
Compound IC50 ± SD (in µM)
8
8
m
q
1.82±0.06
1.93±0.03
Nocodazole 1.98±0.04
a
Note
:
Concentration of drug to inhibit 50% of tubulin
assembly
Immunohistochemistry studies on tubulin
As most antimitotic agents affect microtubules, the alterations in
the microtubule network in A549 cells induced by 8m and 8q by
using fluorescence microscope were investigated. The cells were
treated with 8m and 8q at 0.5 µM concentration for 48 h. The
results demonstrated a well-organized microtubular network in
control cells, however cells treated with these compounds and
nocodozole resulted in disrupted microtubule organization (Figure
5
) thus confirming the inhibition of tubulin polymerization.
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Figure 6: Hoechst staining in A549 lung cancer cell line. A: control
cells (A549); B: nocodazole (0.5 µM); C: 8m (0.5 µM) and D: 8q (0.5
µM). The arrows indicate apoptotic cells.
Measurement of mitochondrial membrane potential (ΔΨm)
The maintenance of mitochondrial membrane potential (∆Ψm) is
considered vital for mitochondrial integrity and bioenergetic
2
4
function. Loss of mitochondrial membrane potential (∆Ψm) is a
key event that takes place during drug-induced apoptosis.
Mitochondrial injury by compounds 8m and 8q was evaluated by
detecting drop in mitochondrial membrane potential (ΔΨm). In this
study we investigated the involvement of mitochondria in the
induction of apoptosis by these compounds. After 48 h of drug
treatment with these compounds at 0.5 and 1 µM concentrations, it
Figure 7: Compounds 8m and 8q triggers mitochondrial injury.
Drops in membrane potential (ΔΨm) was assessed by JC-1 staining
of A549 cells treated with test compounds and samples were then
subjected to flow cytometry analysis on a FAC Scan (Becton
Dickinson) in the FL1, FL2 channel to detect mitochondrial potential.
A: Control cells (A549); B: Nocodazole (1 µM); C: 8m (0.5 µM); D:
was observed that there was
a considerable reduction in
mitochondrial membrane potential (ΔΨm) of A549 cells, assessed
8
m (1 µM); E: 8q (0.5 µM) and F: 8q (1 µM).
by JC-1 staining (Figure 7).
Annexin V-FITC for apoptosis
The apoptotic effect of 8m and 8q was further evaluated by
2
5
Annexin V FITC/PI dual staining assay to examine the occurrence
of phosphatidyl-serine externalization and also to understand
whether it was due to physiological apoptosis or nonspecific
necrosis. In this study A549 cells were treated with these
compounds for 48 h at 0.5 and 1 µM concentrations to examine the
apoptotic effect. It was observed that compounds 8m and 8q
showed good apoptotic effect against A549 cells as shown in Figure
8
. Results indicated that compounds 8m and 8q showed 34.27%
and 31.85% apoptosis at 0.5 µM concentration, whereas they
6
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27
exhibited 53.51% and 52.03% respectively at 1 µM concentration docking program . Binding mode was predicted and analysed
(
Figure 8 and Table 5).
using Glide docking which showed that these compounds bind in
the colchicine binding pocket at the interface of α- and β-chain. For
8
m, the N-atom in imidazopyridine ring has shown H-bonding with
Asnα101 (1.96Å) residue of α-chain. Further, in case of 8m the
protein ligand complex was stabilized by hydrophobic interactions
with other residues, like Alaα180, Valα181, Tyrα224, Metβ259,
Leuβ255, Cysβ241, Alaβ250, Ileβ378, Ileβ318, Alaβ354, Alaβ316,
Tyrβ202, Valβ238. The N-atom in imidazopyridine ring and O-atom
of carbonyl group of 8q has shown H-bondings with Asnα101
(
1.97Å) and Serα178 (2.62 Å) residue respectively. Other residues
like Valα181, Alaα180, Tyrα224, Alaβ250, Cysβ241, Leuβ255,
Alaβ354, Alaβ316, Ileβ318, Ileβ378 and Metβ259 were found to be
involved in hydrophobic interactions with 8q. The binding models
for compounds 8m and 8q are shown in Figure 9.
Figure 8: 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: control cells (A549);
B: nocodazole (1 µM); C: 8m (0.5 µM); D: 8m (1 µM); E: 8q (0.5 µM)
and F: 8q (1 µM).
Table 5: Distribution of apoptotic cells in Annexin-V FITC
experiment
Sample
Upper
left %
Upper
right %
3.83
Lower
left %
93.97
Lower
right %
1.03
Figure 9A
A: Control(A549) 1.17
B: Nocodazole
1 µM)
1.11
16.23
51.48
31.19
(
C: 8m (0.5 µM)
D: 8m (1 µM)
E: 8q (0.5 µM)
F: 8q (1 µM)
0.75
1.07
1.64
1.38
11.35
18.52
9.94
64.98
45.42
66.51
46.59
22.92
34.99
21.91
33.97
18.06
Molecular modelling study
Molecular docking studies were performed on the selected two
compounds 8m and 8q to authenticate the obtained experimental
results. These compounds were succesfully docked in the colchicine
2
6
binding site of tubulin (PDB code: 3UT5) using the SchrÖdinger
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Acknowledgements
I.B.S acknowledges CSIR, New Delhi for the award of senior research
fellowship. We also acknowledge CSIR, New Delhi, for financial
th
support under the 12 Five Year plan project “Affordable Cancer
Therapeutics (ACT)” (CSC0301).
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Design, synthesis and biological evaluation of imidazopyridine-propenone
conjugates as potent tubulin inhibitors
a,b
a
a
a
*a
Ibrahim Bin Sayeed, V. Lakshma Nayak, Mohd Adil Shareef, Neeraj Kumar Chouhan and Ahmed Kamal.
Graphical abstract