Design, synthesis and biological evaluation of imidazopyridine/imidazopyrimidine-benzimidazole conjugates as potential anticancer agents

Article abstract of DOI:10.1039/c4md00400k

A series of imidazopyridine/imidazopyrimidine-benzimidazole conjugates (11a-t) were synthesized and evaluated for their antiproliferative activity. All of these conjugates showed moderate to improved cytotoxic activity against the human cervical (Hela), l

Full text of DOI:10.1039/c4md00400k

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Medicinal Chemistry Communications
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DOI: 10.1039/C4MD00400K
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ARTICLE TYPE
Design, synthesis and biological evaluation of
imidazopyridine/imidazopyrimidine-benzimidazole conjugates as
potential anticancer agents
Ahmed Kamal,* a,b G. Bharath Kumar,a V. Lakshma Nayaka, Vangala Santhosh Reddya, Anver Basha
5
Shaika, Rajendera, M. Kashi Reddya
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A series of imidazopyridine/imidazopyrimidineꢀbenzimidazole conjugates (11a-t) were synthesized and
evaluated for their antiproliferative activity. All these conjugates showed moderate to better cytotoxic
10 activity against the human cervical (Hela), lung (A549), prostate (DUꢀ145) and melanoma (Bꢀ16) cancer
cell lines. Among them, conjugates 11i and 11p showed significant antiproliferative activity against lung
cancer cell line A549 with IC50 values 1.48 and 1.92 µM respectively. Flow cytometric analysis revealed
that these conjugates induced cell cycle arrest at G2/M phase in A549 cell line leading to caspaseꢀ3
dependent apoptotic cell death. The tubulin polymerization assay (IC50 of 11i is 2.06 µM and 11p is 2.26
15 µM) and immuofluorescence analysis displayed that these conjugates effectively inhibit microtubule
assembly at both molecular and cellular levels in A549 cells. Further, Hoechst staining, caspase 3
activation assay, DNA fragmentation analysis and Annexin VꢀFITC assay also suggested that these
compounds induced cell death by apoptosis. Furthermore, molecular docking studies indicated that these
conjugates interact and bind efficiently with tubulin protein. Overall, the present study demonstrates that
20 the synthesis of imidazopyridine/imidazopyrimidineꢀbenzimidazole conjugates as promising tubulin
inhibitors with G2/M phase cell cycle arrest and apoptoticꢀinducing ability.
Nocodazole is another antimitotic drug that has high affinity to
bind to tubulin and inhibit microtubule.10 In addition, nocodazole
exhibits significant effect on microtubule dynamic instability in
interphase cells and purified brain tubulin.11
Introduction
According to WHO reports, cancer is an important cause of death
worldwide and accounted for 8.2 million deaths (around 13% of
25 all deaths) in 2012. Deaths due to cancer, are expected to rise to
over 13.1 million in 2030.1 The word cancer often invokes the
spectral of an relentlessly lethal process. However, cancer is
diverse and can follow different paths, not all of which develop to
metastates and deaths and include lethargic disease that causes
30 not much harm during the patient’s lifetime. 2
Microtubules are important cellular targets for anticancer therapy.
The suppressing of microtubule dynamics, which is required for
proper mitotic function, effectively blocks the cell cycle
progression and results in apoptosis.3 These microtubules have
50
55
60
65
O
O
O
NH
O
S
O
O
O
O
N
OH
O
O
NH
HO
O
N
H
(2)
HO
H
O
O
O
(1)
35 structural subunits of heterodimer as
α and βꢀtubulin, which are
N
NH2
NH
N
cytoskeletal elements that are important for intracellular transfer
as well as cell division in all eukaryotes.4 Interfering with the
energetic instability of microtubules, spindle poisons capture
dividing cells in G2/M phase of the cell cycle, causing mitotic
40 catastrophy and this lead to apoptotic cell death. The wellꢀknown
natural tubulin binding molecules affect by stabilizing (Paclitaxel,
1) (Fig 1.) or destabilizing (nocodazole, 2) microtubule
assembly.5ꢀ6 Paclitaxel and related compounds are important
antitumor drugs, currently used for the treatment that induce
45 tubulin polymerization and microtubule stabilization.7ꢀ9
HN
N
H
N
N
N
H
N
N
(4)
OH
Ph
N
O
(3)
Fig 1. Chemical structures of microtubule targeting agents. Paclitaxel (1)
Nocodazole (2), imidazopyridines (3), Hoechst 32258 (4).
Over the past few years there is considerable interest in the
70 synthesis and pharmacological studies of heteroaromatic organic
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compounds like imidazopyridines (3), possess promising
anticancer activity12 (Fig 1.). Recently we reported that
imidazopyridineꢀoxindole derivatives and conjugates of pyrazoleꢀ
inhibition of topoisomerase ability (Fig 1.).19 Some of the novel
benzimidazoleꢀ2ꢀcarbamate derivatives (BzCs) inhibit the
microtubule polymerization mechanism through selectively
oxindole exhibit excellent antiproliferative activity by inhibiting 15 binding to the βꢀtubulin subunit in which mutations have been
5
the polymerization of tubulin and inducing apoptosis.13,14
Benzimidazoles are important due to their wide range of
biological activities such as antitumor,15,16 antiꢀangiogenesis17
and anti vascular activity.18
identified that lead to drug resistance.20
The excellent biological activity exhibited by these conjugates
prompted us to explore some newer conjugates by linking two
pharmacophores such as imidazopyridine and benzimidazoles
Some
of
the
benzimidazole
pharmacophores
like 20 scaffolds to enhance the antimitotic activity. The results of our
10 bisbenzimidazole Hoechst 33258 (4) displayed broad spectrum of
investigations along this direction are presented below.
antiproliferative activity with DNA minor groove binding and
25
30
35
40
45
Scheme 1 Reagents & conditions: (a) acetone, reflux, 6ꢀ8 h; (b) 2N HCl, reflux,1ꢀ2 h, 85ꢀ92%; (c) POCl3, DMF, reflux,12 h, 80ꢀ85%; (d) Na2S2O5, EtOH,
8 h, reflux, 70ꢀ77%.
sodium metabisulphite in ethanol. as shown in Scheme 1. The
50 Results and discussion
imidazo[1,2ꢀa]pyridine/ imidazo[1,2ꢀa] pyrimidine ꢀ3ꢀaldehyde
Chemistry
(9a-d) were obtained by the Vilsmeier reaction with the
corresponding imidazo[1,2ꢀa] pyridine/ imidazo[1,2ꢀa]
60 pyrimidine (8a-d), that were obtained from compounds (7a-d).
The crucial intermediates such as compounds (7a-d) were
obtained by the reaction of appropriate 2ꢀbromoꢀ1ꢀarylethanone
The imidazopyridine
/
imidazopyrimidinebenzimidazole
conjugates (11a–t) were prepared by the oxidative cyclization
of substituted oꢀphenylenediamines (10a-e) and imidazo[1,2ꢀ
55 a]pyridine/ imidazo[1,2ꢀa]pyrimidineꢀ3ꢀaldehyde (9a-d) with
2
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(6a-c) and 2ꢀaminopyridine/pyrimidine. (5a, b)
Evaluation of biological activity
cytotoxic activity
On over view, electron donating substitution on Dꢀring like
methyl and hydrogen shows significant cytotoxity compared to
halo substitution like fluoro and chloro on Dꢀring.
To explore the structure activity relationship of
imidazopyridine/imidazopyrimidineꢀbenzimidazole conjugates
(11a-t) consisting A, B, C, Dꢀrings (Fig 2.) derived from
scheme 1. These 20 compounds prepared with respect to
different modifications made on the Cꢀring and Dꢀring were
evaluated for cytotoxic activity against a panel of four human
35
5
10 cancer cell lines such as A549 (lung), Hela (cervical), DUꢀ145
(prostate cancer) and Bꢀ16 (mouse melanoma) by employing
MTT assay.21 Nocodazole was used as reference drug. The
results are summarized in Table 1 and expressed as IC50 values.
The in vitro screening results revealed that these conjugates
15 possess considerable cytotoxic activity with IC50 values
ranging from 1.48 ꢀM to >30 ꢀM. particularly, the conjugate
11i having electron donating substituents like paraꢀmethoxy
group on Cꢀring and paraꢀmethyl group on Dꢀring possess
significant cytotoxicity against lung and cervical cancer cells
20 with IC50 value 1.48 µM and 1.92 µM respectively.
40
Fig 2. Structure activity relationship of imidazopyridine
/imidazopyrimidineꢀbenzimidazole conjugates
Effect on cell cycle arrest
45
50
55
60
Table 1: in vitro antiproliferative activity IC50 (in µM)a
imidazopyridine/imidazopyrimidineꢀbenzimidazole
conjugates (11a-11t)
IC50 values (in µM)
Compound
11a
Helab
20.38
17.58
14.45
26.36
> 30
A549c
23.15
14.57
19.37
> 30
DUꢀ145d
24.0
15.88
19.49
> 30
Bꢀ16e
> 30
14.79
16.02
> 30
11b
11c
11d
11e
> 30
> 30
> 30
11f
11g
11h
11i
9.06
10.51
15.07
10.22
1.48
13.25
13.80
19.89
7.08
16.67
19.35
26.80
3.63
16.96
13.97
2.57
11j
11k
11l
11m
11n
11o
11p
11q
11r
15.84
8.19
15.26
20.31
12.89
19.95
2.25
6.91
5.62
7.78
14.42
12.58
10.18
16.0
2.34
4.89
> 30
19.95
16.60
15.56
> 30
15.11
> 30
10.47
16.98
> 30
10.98
19.19
19.68
> 30
18.62
1.92
65 Fig 3. Flow cytometric analysis in A549 lung cancer cell line: Control
(A549), Nocodazole (2) (1 µM), 11i (1µM), 11i (2µM), 11p (1µM),
and 11p (2µM).
6.91
> 30
8.51
> 30
11s
7.58
2.19
2.34
8.91
11t
Nocodazole
9.33
2.23
2.12
1.62
3.71
0.25
10.23
0.69
The blockade of cell cycle progress by antiꢀcancer agents
70 prevents the proliferation of cancer cells, which is also
exploited for cancer therapy. In our in vitro screening results
revealed that compound 11i and 11p showed significant
activity against human lung cancer cells A549. It was of
interest to understand whether this inhibition of cell growth
75 was on account of cell cycle arrest. Hence, we studied the cell
cycle distribution by flow cytometry in A549 lung cancer cells.
In this study A549 cells were treated with compounds 11i and
11p for 48 h at concentrations 1 and 2 µM. The data obtained
clearly indicated that these compounds show G2/M cell cycle
80 arrest in comparison to the untreated control. These compounds
(11i and 11p) showed 11.70 and 12.59 % of cell accumulation
In vitro antiproliferative activity of
imidazopyridine/imidazopyrimidineꢀ benzimidazole conjugates
determined by MTT assay. a 50% Inhibitory concentration and the
values are average of three individual experiments after 48 h of drug
treatment. bCervical cancer, cLung cancer, dProstate cancer, eMouse
melanoma cancer
In contrast, substitution with fluoro at para position of Cꢀring
25 and chloro at para position of Dꢀring as in 11r proved
deleterious for its antiproliferative activity against same cell
lines like A549 cells (IC50 value is >30 µM) and Hela cells ꢀ
(IC50 value is >30 µM). However, imidazopyrmidine
conjugates (11f-t) were exhibited better cytotoxicity compared
30 to imidazopyridine conjugates (11a-e) against tested cell lines.
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in G2/M phase at 1 µM concentration, whereas they exhibited
28.97 and 33.84 % of cell accumulation at 2 µM concentration,
respectively (Fig 3.).26
In addition to in vitro tubulin polymerization studies, we
investigated alterations in the microtubule network induced by
50 conjugates 11i and 11p in A549 cell culture by
immunofluoresence microscopy of immunohistochemistry
studies, as most antimitotic agents affect microtubules.28
Therefore, A549 cells were treated with 11i and 11p at 1 µM
concentration for 48 h. Nocodazole was used as reference
55 compound. The test results, demonstrated a well organized
microtubular network in control cells. However, cells treated
with test conjugates showed disrupted microtubule organization
as seen in Fig 5, thus demonstrating the aspect of inhibition of
tubulin polymerization.
Effect on Tubulin polymerization
5
Compounds that alter cellꢀcycle parameters with preferential
G2/M blockade are known to exhibit effects on tubulin
assembly Moreover, inhibition of tubulin polymerization is
strongly associated with G2/M cellꢀcycle arrest.27 The cell
cycle analysis results revealed that these compounds (11i and
10 11p) arrested the cell cycle at G2/M phase as compared to
control. Thus it was considered of interest to investigate the
effect on tubulin polymerization. As tubulin subunits
heterodimerize and selfꢀassemble to form microtubules in a
time dependent manner, we have investigated the progression
60
23
15 of tubulin polymerization22, by monitoring the increase of
fluorescence emission at 420 nm (excitation wavelength is 360
o
nm) in 384 well plate for 1 h at 37 C with and without the
compounds at 3 µM concentration. Among the two compounds
examined, 11i and 11p compounds inhibited tubulin
20 polymerization by 61.12% and 60.49% respectively, whereas
the standard compound nocodazole inhibited 66.93% of tubulin
polymerization (Fig 4.).
65
25
70
Fig 5. IHC analysis of compounds on the microtubule network: A549
cells were treated with compounds 11i and 11p at 1 µM concentrations
for 48 h followed by staining with α−tubulin antibody. Nocodazole
was used as the reference compound.
30
75 Hoechst staining
Fig. 4 Effect of compounds on tubulin polymerization: tubulin
polymerization was monitored by the increase in fluorescence at 360
35 nm (excitation) and 420 nm (emission) for 1 h at 37 oC. All the
compounds were included at a final concentration of 3 µM.
Nocodozole was used as a positive controls. Values indicated are the
mean ± SD of two different experiments performed in triplicates.
80
85
90
95
Table 2. Inhibition of tubulin polymerization (IC50) of compound
11i and 11p.
Compound
IC50 a ± SD (in µM)
2.06±0.09
11i
11p
2.26±0.38
Nocodazole
1.82±0.06
Effect of congeners on tubulin polymerization. IC50 (µM) values for
11i and 11p were determined from the tubulin polymerization
assays. a Concentration of drug to inhibit 50% of tubulin assembly
40
Furthermore, these three potential conjugates (11i and 11p)
were evaluated for their in vitro tubulin polymerization assay at
different concentrations. These molecules showed potent
inhibition of tubulin polymerization with IC50 values 2.06 and
Fig 6. Hoechst staining in A549 lung cancer cell line. A: Control cells
(A549), B: 10x at 0.5 µM concentration and C: 10x at 1 µM
45 2.26 µM, respectively (Table 2). Nocodazole was used as
positive control.
100
concentration
Immunohistochemistry of tubulin
Disruption of microtubule formation leads to cellꢀcycle arrest
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in the G2/M phase, followed by apoptotic cell death.24
Chromatin condensation and fragmented nuclei are known as
the classic characteristics of apoptosis and apoptosis is one of
the major pathways that lead to the process of cell death. It was
considered of interest to investigate the apoptotic inducing
effect of the two potent conjugates (11p and 11i) by Hoechst
staining (H 33342) method in A549 cancer cell line. Therefore
cells were treated with 11i and 11p at 1 µM concentration for
48 h wherein nocodazole was used as the reference compound.
55
5
60
10 Apoptotic cells were observed based on cytoplasmic
condensation, presence of apoptotic bodies, nuclear
fragmentation and relative fluorescence of the test compounds
(11p and 11i) revealed that there is significant increase in the
percentage of apoptotic cells (Fig 6.).
15 Caspases-3 activation
Fig. 8 DNA fragmentation of compounds 11i and 11p in Aꢀ549 lung
65 cancer cells: Laneꢀ1: Marker (100 bp), Laneꢀ2: Untreated control DNA,
Laneꢀ3: 11p 1 µM, Laneꢀ4: 11i at 1µM.
Caspases or cysteineꢀaspartic proteases are a family of cysteine
proteases, which are crucial mediators of apoptosis. Among
them, caspaseꢀ3 is the best understood in the mammalian
caspases in terms of its specificity and role in apoptosis.
20 Caspaseꢀ3 is also required for some typical hallmarks of
apoptosis.22 A549 cells were treated with 11p and 11i at 1 and
2 µM concentrations for 48 h and were examined for the
activation of caspaseꢀ3 activity. Results indicated that there
was nearly 2 to 5ꢀfold induction in caspaseꢀ 3 levels compared
25 to control (Fig 7.).
Annexin V-FITC for apoptosis
The apoptotic effect of these compounds (11p and 11i) was
further evaluated by Annexin V FITC/PI (AV/PI) dual staining
70 assay29 to examine the occurrence of phosphatidylserine
externalization and also to understand whether it is due to
physiological apoptosis or nonspecific necrosis. In this study
A549 cells were treated with compounds 11p and 11i for 48 h
at 1 and 2 µM concentration to examine the apoptotic effect. It
75 was observed that these compounds showed significant
apoptosis against A549 cells as shown in Fig 6.
30
80
Fig. 7 Effect of compounds 11i and 11p on caspaseꢀ3 activity: A549
cells were treated with compounds 11i and 11p at 1 and 2 µM
35
concentrations for 48 h. Values indicated are the mean ± SD of two
different experiments performed in triplicates.
85
DNA fragmentation analysis
DNA laddering was carried out in order to elucidate the mode
of action of the compound especially for their ability to induce
40 oligonucleosomal DNA fragmentation (DNA ladder), which is
a characteristic feature of the programmed cell death or
apoptosis.25, 26 During apoptosis, DNA is cleaved into smaller
fragments and fragmented DNA produces a series of bands
which are described as DNA ladders. These fragments can be
45 observed by gel electrophoresis as ladders. A549 cells were
treated with 11i and 11p at 1µM concentrations for 48 h and
DNA was isolated from these cells. The DNA was run on 2%
agarose gel electrophoresis after staining with ethidium
bromide under UV illumination. It was observed that
50 compounds produced significant DNA fragmentation (Fig 8.),
which is indicative of apoptosis.
90
95
Fig. 9 Annexin VꢀFITC staining. A549 cells were treated with
compounds 11p, 11i and Nocodazole (2).
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Results indicated that compounds 11p and 11i showed 7.77 and
9.61 % of apoptosis at 1 µM concentration, whereas they
showed 11.45 and 14.79 % of apoptosis at 2 µM concentration
respectively (Fig 9.). In this study the untreated control cells
showed 0.14 % of apoptosis from this experiment it was
suggested that these compounds induced cell death by
apoptosis (Table 3.).
αVal355, βAla354, βThr353. All the conjugates interact with
both αβ interface tubulin in the colchicine binding pocket
(αSer178, αThr179, αVal181, βLys352, βThr353, αAsn101,
βGln247, βLeu248). In case of 11i methoxy substituent on
50 phenyl group induced change in the pose orientation that
caused the formation of hydrogen bond interaction with
βLys254, this in turn suggest a structural justification for
increased activity of 11i over 11p.These results support the
tubulin polymerization inhibition potential of these conjugates
55 at the molecular level.
5
Table 3. Annexin VꢀFITC staining of A549 cells in the presence
of compounds 11p, 11i and nocodazole.
Sample
UL % UR % LL % LR %
Control
0.07
0.04
0.35
0.18
0.22
0.07
0.14
1.29
2.69
1.20
1.43
1.30
99.79
86.96
91.88
88.37
90.17
85.14
0.00
11.71
5.08
10.25
8.18
13.49
Conclusion
Noc (1µM)
11p (1 µM)
11p (2 µM)
11i (1 µM)
11i (2 µM)
In summary, twenty congeners of imidazopyridine
/imidazopyrimidineꢀbenzimidazole conjugates (11a-t) have
been synthesized and evaluated for their cytotoxic activity
60 against four human cancer cell lines such as Aꢀ549 (lung), Hela
(cervical), DUꢀ145 (prostate) and Bꢀ16 (melonoma). Some of
these conjugates exhibited significant cytotoxic activity at
micromolar (ꢀM) concentration. Two of the most potent
compounds (11i and 11p) exhibited promising cytotoxic
65 activity (IC50, 1.48 and 1.92 ꢀM respectively) against Aꢀ549
cancer cell line. Flow cytometric analysis revealed that these
conjugates arrested the cell cycle at the G2/M phase. These
potent conjugates (11i and 11p) exert their cytotoxic activity by
inhibition of tubulin polymerization, with an IC50 value of 2.06
70 µM and 2.26 µM respectively. Further, Hoechst staining, DNA
fragmentation assay and activation of caspaseꢀ3 and Annexin
VꢀFITC assay suggested that these conjugates induce cell death
by apoptosis. Furthermore, molecular modeling analysis
suggests that these conjugates preferably bind to the colchicine
75 binding site of tubulin. Overall, this investigation describes the
10 Molecular modelling
The conjugates that are synthesised in the present study are
designed on the basis of nocodazole which is an
aminobenzimidazole with antimiotic and antitumoral activity
by tubulin inhibition. Nocodazole and colchicine show
15 resemblance in binding pattern at αβ ꢀinterface of the tubulin.30
To investigate the possible binding interactions for the
conjugates (11p and 11k), we performed molecular docking
studies at the colchicine binding pocket. Our docking studies
suggests that these conjugates occupy the binding site of the
20 colchicine and interacted with both αꢀ and βꢀtubuline
interface.The imidazopyrimidine ring of 11i binds to the
hydrophobic region with βGln247, βLeu248. Apart from this
the 4ꢀmethoxyphenyl group at the imidazopyrimidine ring
involved in hydrogen bond interaction with βLys254 and
25 hydrophobic interactions with αAsn101, αGly143. The
benzimidazole ring of conjugates (11i and 11p) involved in the
hydrogen binding interactions with αThr179 and these
interactions are in the range of 1.7ꢀ2.2Å.
synthesis
of
imidazopyridine/imidazopyrimidineꢀ
benzimidazoles conjugates as potential anticancer agents with
apoptosis inducing ability by targeting tubulin.
Acknowledgements
80 G.B.K, V.S.R and A.B.S, acknowledge CSIR, New Delhi for
the award of senior research fellowship. We also acknowledge
CSIR for financial support under the 12th Five Year plan
project “Affordable Cancer Therapeutics (ACT)” (CSC0301).
30
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Medicinal Chemistry Communications
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Introduction
Results and discussion
Chemistry
Evaluation biological
activity
Ahmed Kamal,a,b G. Bharath Kumar,V.
Lakshma Nayak, Vangala Santhosh Reddy,
Anver Basha Shaik, Rajender, M. Kashi
Reddy.
HN
N
Conclusion
Acknowledgements
Notes and references
Page No. 1– Page No. 7
N
O
N
Design, synthesis and biological
evaluation of
N
imidazopyridine/imidazopyrimidine-
benzimidazole conjugates as
potential anticancer agents
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Graphical Abstract
Design, Synthesis and Biological Evaluation of
Imidazopyridine/imidazopyrimidine-Benzimidazole Conjugates
as Potential Anticancer Agents
Ahmed Kamala,b,* G. Bharath Kumara, V. Lakshma Nayaka, Vangala Santhosh Reddya, Anver
Basha Shaika, Rajendera, M. Kashi Reddya