Synthesis of Combretastatin-A4 Carboxamidest that Mimic Sulfonyl Piperazines by a Molecular Hybridization Approach: in vitro Cytotoxicity Evaluation and Inhibition of Tubulin Polymerization

Article abstract of DOI:10.1002/cmdc.201900541

Molecular hybridization approach is a promising structural modification tool to design new chemical entities (NCEs) by mimicking two different pharmacophoric units into one scaffold to enhance the biological properties. With this aim, combretastatin-A4 ac

Full text of DOI:10.1002/cmdc.201900541

Accepted Article
Title: Synthesis of CA4 carboxamides mimicking with sulfonyl
piperazines by a molecular hybridization approach: In vitro
cytotoxicity evaluation and tubulin polymerization inhibition.
Authors: Ahmed Kamal, Chetna Jadala, Manda Sathish, Anchi
Pratibha, Ramya Tokala, Uppu Jaya Lakshmi, Velma Ganga
Reddy, Nagula Shankaraiah, and Chandaraiah Godugu
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Synthesis of combretastatin-A4 carboxamides mimicking with
sulfonyl piperazines by a molecular hybridization approach: In
vitro cytotoxicity evaluation and tubulin polymerization inhibition
Chetna Jadala[a], Manda Sathish[b], Pratibha Anchi[c], Ramya Tokala[a], Uppu Jaya Lakshmi[a], Velma
Ganga Reddy[b], Nagula Shankaraiah[a]*, Chandraiah Godugu[c]* and Ahmed Kamal[b,d]*
Abstract: Molecular hybridization approach is a promising structural modification tool to design new chemical entities (NCEs) by mimicking
two different pharmacophoric units into one scaffold to enhance the biological properties. With this aim, combretastatin A4 acids were
integrated with sulfonyl piperazine scaffolds as a one molecular platform and evaluated for their in vitro antiproliferative activity against a
panel of human cancer lines cell lines namely, lung (A549), mouse melanoma (B16F10), breast (MDA MB-231, MCF-7) and colon (HCT-15)
by MTT assay. Amongst which the compound (E)-3-(4-Chlorophenyl)-1-(4-((4-chlorophenyl)sulfonyl)piperazin-1-yl)-2-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (5ab) displayed significant IC50 values in the range of 0.36 to 7.08 µM against the selected cancer cell lines.
Moreover, 5ab was found to be the most potent member of this series with IC50 0.36±0.02 µM. Further investigations revealed that the
compound 5ab displayed significant inhibition of tubulin assembly with IC50 5.24±0.06 µM and molecular docking studies also disclosed the
binding of 5ab effectively at the colchicine binding site. The flow cytometric analysis demonstrated that the compound 5ab caused cell cycle
arrest at G2/M phase in A549 cells. Compound 5ab induced apoptosis in A549 cells which was further evaluated by different staining assays
such as DAPI and AO which undoubtedly speculated, the induction of apoptosis. To study the anti-migration with 5ab, cell migration/scratch
wound assay was performed and the extent of apoptosis was studied by Annexin-V, including mitochondrial potential by JC-1 staining.
armacodynamic profile with lower toxicity is the main spotlight
Introduction
for many researchers.[11] Tubulin has three binding sites, the
taxane binding site, vinca binding site and the colchicine binding
site [12, 13]. Molecules that act as microtubule stabilizing agents
Microtubules are vital components of cytoskeleton composed of
α, β-tubulin heterodimer assembly, majorly engaged in cell
division which is responsible for spindle formation and
separating duplicated chromosomes prior to cell division; this
makes them as an imperative target for anticancer drugs[1]. In
this breakthrough, both natural and synthetic derivatives are
capable of targeting microtubules assembly or disassembly that
[14,
prevent depolymerisation by targeting the taxane binding site
15]. While the agents which inhibit α, β-tubulin assembly towards
[16]
polymerization, target the vinca and colchicine binding sites
.
The ligands interacting at colchicine binding site were
extensively studied and many other interesting compounds such
as podophyllotoxin, colchicine and CA4 were identified. To that
has attracted much attention [2]. The typical paradigms that are
[17]
end, CA4 was isolated from Combretum caffrum
has
acting as antimitotic agents are epothilone A [3], paclitaxel
,
[4]
imperative pharmacological profile, structural simplicity and
potent antitumor properties. The cis-configuration of the double
bond between the two aryl rings and the 3, 4, 5-trimethoxy
system on the A ring are essential requirements for its biological
activity [18-20], however it suffers from lower water solubility and
[5]
vinblastine
and combretastatin A4 (Figure 1) [6], which are
mainly categorized in the management of ovarian, prostate and
breast cancers [7, 8]. Besides the efficacy and medical usefulness
of presently available drugs, the main drawback is systemic
toxicity and poor biopharmaceutical properties [9, 10]. As a result,
the search for novel antimicrotubule scaffolds with improved ph-
readily isomerization of double bond to inactive trans form [21, 22]
.
It is evident from the previous reports that diverse ligands
mimicking CA4 were designed and explored their structure
activity relationships and chemical modifications and were
evaluated for their anti-proliferative activities. C. Borrel et al.
have prepared two series of compounds bearing substituent
either a carboxamide or a carbamate group, on C1 position of
the olefin bridge adjacent to the A-ring of these compounds,
[a]
[b]
[c]
[d]
C. Jadala, R. Tokala, U.J. Lakshmi, Dr. N. Shankaraiah
Department of Medicinal Chemistry
National Institute of Pharmaceutical Education and Research (NIPER),
Hyderabad, 500037, India.
E-mail: shankar@niperhyd.ac.in
M. Sathish, V.G. Reddy, Dr. Ahmed Kamal
Medicinal Chemistry and Pharmacology,
CSIR-Indian Institute of Chemical Technology,
Hyderabad, 500007, India.
Email: ahmedkamal@iict.res.in
P. Anchi, C. Godugu
Department of Regulatory Toxicology,
National Institute of Pharmaceutical Education and Research (NIPER),
Hyderabad, 500037, India.
Email: chandra.niperhyd@gov.in
Dr. Ahmed Kamal
carboxamide substitution was less cytotoxic but displayed
[23, 24]
increased antitubulin activity (Figure 1, CA4 carboxamide)
.
Therefore, the above findings inspired us to design CA4 ligands
having amide linkage by different motifs with the goal of
including a new profile or a novel set of molecules with improved
cytotoxicity and lower toxicity profile.
School of Pharmaceutical Education and Research (SPER) Jamia
Hamdard,
New Delhi, 110062, India.
Email: ahmedkamal@iict.res.in
Supporting information for this article is given via a link at the end of the
document.
On the other hand, a theme has been gaining importance in
cancer chemotherapy by the use of combinations of two or more
agents [25]. With respect to molecular hybridization approach, two
important fragments are merged which could be beneficial for
the treatment of cancer, considering the fact that the
1
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sulfonamide and piperazine derivatives represent a class of
pharmacologically proficient scaffolds having diverse biological
activities. Sulfonamides were extensively used owing to their
benzenesulfonyl chlorides 1 in CH2Cl2 as solvent. Next, phenyl
acetic acid (3) was treated with substituted aldehydes to afford
the (E)-3-phenyl-2-(2,3,4-trimethoxyphenyl)acrylic acids 4a–f.
Then the desired combretastatin CA-4-linked sulfonyl piperazine
hybrids 5a–ad were successfully synthesized by acid amine
coupling between the intermediates 2a–e and 4a–f using EDCI
and HOBt in dry DMF. The corresponding crude compounds
were purified via column chromatography and obtained with
good to excellent yields (85-90%). All the final compounds 5a–
ad were unambiguously characterized by spectroscopic studies
[26]
[27, 28]
clinical usage as anti-inflammatory
antimicrobial
,
anticancer
,
.
[29,
[31]
30], antiviral and HIV protease inhibitors
Earlier reports have shown significant insights and used in
clinical studies about the effective antitumor activity of novel
sulfonamide derivatives and several colchicine binding site
[32]
agents bearing the sulfonamide group, such as J30
T138067
and
[33]
(Figure 1). Furthermore, in recent years the
1
piperazine derivatives were also reported to be diverse structural
motifs and procured much attention due to their versatile
properties [34-36]. It has been found that N-substituted piperazine
compounds possess a wide range of pharmaceutical activities,
(1H NMR, 13C NMR and HRMS). The H NMR spectrum of 5a
showed a significant broad peak of 4 protons of piperazine from
δ 2.7-3.0 ppm and another four protons were observed at δ 3.68
ppm and a prominent cis proton appeared at δ 6.62 ppm and
rest of the aromatic protons appeared in the range of δ 7.9‒6.0
ppm.
[38]
[39]
such as anticancer [37], antifungal
and antimicrobial
activities. Piperazine and its derivatives are one of the important
scaffolds, besides having potent antibacterial activity such as
[40]
piperazinyl-linked ciprofloxacin dimers
,
also possess
anticancer activity. Plinabulin which is a piperazine derivative
with colchicine-like tubulin depolymerization activity displayed
IC50 values at nanomolar scale [41]. Inspired by the biological
profile and synthetic accessibility of combretastatin A-4 and
sulfonyl piperazine and our continued interest in developing
[42]
novel anticancer agents led to molecular hybridization
of
these molecules shown in Figure 1. In this perspective, a series
of novel compounds were synthesized and evaluated for their in
vitro anticancer activity and the most promising compound was
also further evaluated for its in vitro cytotoxicity.
Figure 1. Chemical structures of novel tubulin inhibitors and molecular
hybridization approach to combretastatin A-4 inspired sulfonyl piperazine
scaffolds 5a–ad.
Results and Discussion
Chemistry
The synthetic route for the preparation of desired combretastatin
CA-4-linked sulfonyl piperazine hybrids 5a–ad was outlined in
Scheme 1. We began with the synthesis of different substituted
sulfonamides 2a–e by reacting piperazine with different
Scheme 1. Synthesis of combretastatin A-4 linked sulfonyl piperazine hybrids
5a–ad.
2
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In the 13C NMR spectrum of 5a, the carbonyl and methyl
carbons appeared at δ 170.0 and 15.4 ppm, respectively.
methoxy carbons obtained at 56.0 and 60.0 ppm and remaining
aromatic carbons appeared in the range of δ 156.5‒105.8 ppm.
From the close analysis of Table 1, the structure activity
relationship (SARs) for these compounds has been constructed
by considering the sulfonamide ring was selected, which is
diversified with 5 substituents of which 4-chloro substitution on
C4 of sulfonamide phenyl ring has shown potent activity as in
case of 5z, 5aa and 5ab of which the compound 5ab was most
potent. The other substituents on sulfonamide ring such as H,
methyl, methoxy and tertiary butyl were comparatively less
active than 4-chloro substitution. From this, we could speculate
that 4-chloro substitution on C4 of sulfonamide ring was
responsible for the compound to show cytotoxicity. It was also
noteworthy to consider the varaiable groups on CA4 ring for eg.,
the compounds 5z and 5aa having 4-chloro substitution at C4 of
CA4 B-ring were moderately active towards the tumor cell lines
investigated but the other compounds such as 5p and 5v with
chloro substitution on C4 of CA4 ring were comparatively less
active. Furthermore, the compounds such as 5a, 5g, 5m, 5s and
5y with ethoxy substitution on C4 of CA4 B-ring were less potent
on all the cell lines investigated. Finally, keeping in view of
above speculations, we have arranged the compounds in order
of their cytotoxicity based on substitution on sulfonamide and
CA4 ring as Cl-Cl>Cl-Me>Cl-OMe. To find out the selectivity
towards cancer cells, the most active compound 5ab was tested
on normal human keratinocyte cell line (HaCaT). Interestingly,
compound 5ab was found to be almost 3 times more selective
on A549 cells compared to normal HaCaT cells. The promising
cytotoxicity of compound 5ab on A549 cells provoked us to
explore for its effect at cellular level.
1
Similarly, all the remaining compounds H and 13C NMR spectra
were noticed in the similar pattern. The HRMS (ESI) of 5a‒ad
showed characteristic [M + H]+. peaks equivalent to their
molecular formulae.
Biology
Antiproliferative activity
The newly synthesized hybrids 5a–ad were evaluated for their
invitro antiproliferative potency against selected human cancer
cell lines such as lung (A549), mouse melanoma (B16F10)
breast (MDA-MB-231, MCF-7) and colon (HCT-15) by the aid of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) assay [43]. The results of respective cytotoxicity data were
displayed in the form of IC50 (concentration required to inhibit
50% of the cancer cells), along with combretastatin A4 IC50
which was included as the standard (Table 1). From the results,
it can be inferred that compounds 5d, 5f, 5h, 5j, 5t, 5u, 5ab, 5ac
and 5ad exhibited significant cytotoxicity in almost all the cancer
cell lines with IC50 values ranging from 0.43±0.06 to 29.72±4.53
µM. Amongst all the compounds tested, 5ab exhibited
remarkable cytotoxicity in all the cancerous cell lines displaying
IC50 ranging from 0.36±0.02 µM to 7.05±3.36 µM.
Cell Migration/Scratch Wound assay
Cancerous cells multiply and migrate throughout the body, with
the help of extracellular matrix by intravasing into blood
circulation, subsequently attaching to a distant site, and finally
extravasing by making its own identity. This invasion is currently
considered as a main reason for death in cancer patients termed
as metastatic progression [44]. Hence, to evaluate this property
by the potent hybrid 5ab on A549 cancer cells, the assay was
performed by cell culture wound closure assay. It can be
observed from Figure 3 that after 24 h the wound area is filled
with 17.40±4.03% of untreated control cells, while the treatment
with compound 5ab in 0.1 µM and 0.25 µM concentration
depicted significant inhibition of cell migration with 10.48±6.43%,
5.55±1.83% cells respectively in the wounded area. While in 0.5
µM and CA4 there was no migration observed. From the above
findings, it can be scrutinized that the compound inhibited cell
migration in a dose dependent manner.
Apoptosis detection studies
The human cancerous cell have the capability to escape the
apoptosis or programmed cell death for cell survival and
contribute for the tumor progression, invasion and drug
resistance; therefore most of the anticancer drugs strive for
regenerating apoptosis in the cancer cells which is now
considered as an attractive strategy in cancer therapy.
Therefore, we considered to evaluate the apoptosis inducing
effect of compound 5ab on A549 cell lines using CA4 as a
positive control by using staining studies such as AO, DAPI,
Annexin-V propidium and JC-1 assay.
Figure 2. Cytospecificity of compound 5ab towards different cell lines. IC50
values were calculated after 48 h treatment with compound 5ab Values are
represented by mean ± SEM of three independent experiments.
Moreover, the compound 5ab is active as much as CA4 showing
IC50 value as 0.36±0.02 µM whereas CA4 exhibited IC50 of
0.43±0.16 µM on A549 cell line and it was noteworthy that the
compound 5ab was comparitively less potent for the other
studied cell line showing IC50 7.05±3.36 µM on HCT cell line.
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Table 1. IC50 (µM In vitro cytotoxic activity (IC50 in µM)[a] of combretastatin A4 linked sulfonyl piperazine hybrids 5a–ad
Compound
5a
A549[b]
>30
B16F10[c]
>30
MDA MB-231[d]
>30
HCT-15[e]
>30
MCF-7[f]
>30
HaCaT[g]
-
5b
>30
>30
>30
>30
>30
-
5c
>30
>30
>30
>30
18.20±1.37
29.72±4.53
>30
-
5d
15.46±4.17
>30
7.50±0.85
>30
17.43±1.25
>30
>30
-
5e
>30
-
5f
24.71±0.51
>30
11.108±0.37
>30
5.49±0.65
>30
>30
3.70±0.81
>30
-
5g
>30
-
5h
6.38±2.77
>30
4.11±0.96
>30
1.83±0.47
>30
>30
>30
-
5i
>30
>30
-
5j
15.93±2.81
>30
7.36±1.51
>30
6.46±1.17
>30
3.27±2.13
>30
24.68±5.16
>30
-
5k
-
5l
6.38±2.77
>30
3.96±1.29
>30
14.33±2.45
>30
>30
2.23±0.23
>30
-
5m
5n
>30
-
>30
>30
>30
>30
18.94±1.822
>30
-
5o
>30
>30
6.36±0.78
>30
3.25±0.80
>30
-
5p
>30
>30
>30
-
5q
>30
>30
>30
>30
>30
-
5r
>30
>30
>30
>30
>30
-
5s
>30
>30
>30
>30
>30
-
5t
8.44±0.80
18.16±1.04
>30
1.01±0.07
>30
4.20±1.08
4.50±0.27
>30
17.07±1.32
4.10±0.95
>30
18.87±0.85
>30
-
5u
-
5v
>30
>30
-
5w
5x
>30
>30
>30
>30
>30
-
>30
>30
>30
>30
>30
-
5y
>30
>30
>30
>30
>30
-
5z
2.66±0.26
0.72±0.20
0.36±0.02
>30
5.42±3.83
2.14±0.16
1.75±0.44
>30
2.23±0.14
2.52±1.19
2.16±0.83
>30
>30
0.78±0.06
>30
-
5aa
5ab
5ac
5ad
CA-4 [h]
6.20±2.19
7.05±3.36
>30
-
4.08±1.10
>30
0.92±0.01
-
>30
>30
>30
>30
>30
-
0.43±0.16
0.03±0.00
0.79±0.21
1.92±0.92
2.12±0.21
0.63±0.17
[a] 50% Inhibitory concentration after 48 h of drug treatment and the values are average of three individual experiments. [b] Human lung cancer cells, [c] Mouse
Skin cancer cells, [d] Human Breast cancer cells [e] Human colon cancer cells, [f] Human Breast cancer cells, [g] Human keratinocyte, [h] Combretastatin A-4 =
CA-4. All the values are expressed as Mean ± SEM in which each treatment was performed in triplicate wells.
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Figure 3. (A) Images of scratch wound assay (at magnification 100X). After reaching near confluency, monolayer was scratched and treated with the compound
5ab and CA4 for 24 h. Respective images were taken at 0 and 24 h. (B) Represents % wound closure. Data presented were mean ±SEM of similar three
independent experiments ****p<0.0001 versus control at 24 h
Phase contrast imaging
irregular distribution of chromatin in analogous with CA4 as
indicated in Figure 5.
To examine whether the treatment with the compound 5ab could
lead to loss of cell viability and induce apoptosis, after 48 h of
treatment with compound at variable concentrations (0.1, 0.25,
0.5 and CA4 (1 µM), the cells were observed for the
morphological changes under phase contrast microscope.[45]
From the Figure 4, it is evident that increasing the compound
concentration has led to significant cell death in comparison to
the positive control (CA4). Apart from viability, compounds
induced apoptosis related morphological changes like cell
blebbing and condensation of nucleus. This signifies that the
compound 5ab induced typical morphological changes in A549
lung cancer cells.
Figure 5. Representative images of AO staining in A549 cells. Cells were
treated with compound 5ab at concentrations of 0.1, 0.25, 0.5 µM and CA4 (1
µM) for 48 h. The images were captured with a fluorescence microscope at
200X magnification
DAPI staining assay
DAPI diffuses easily into disrupted cell membrane rather than
intact cell membrane [47]. Therefore, stained cells showed less
intensity than normal cells. From the results, it is clear that
compound 5ab in comparison to standard (CA4) induced
massive nuclear damage with increasing concentration of
treatment. It can be observed that a bright condensed and
fragmented nucleus is identified by the DAPI staining and is a
clear indication of apoptosis. Furthermore, margination of
nucleus due to which horseshoe shaped structure was also
observed as shown in Figure 6.
Figure 4. Phase contrast imaging of A549 cells after treatment with compound
5ab.and CA-4. Images were taken after 48 h treatment and observed under
phase contrast microscope at magnification of 200X.
Acridine orange staining
Acridine orange is a stain technique which enables to identify
the apoptotic cells [46]. This assay demonstrates on the principle
that acridine orange can penetrate the live cells by which they
appear healthy with dark green color fluorescence. Herein, upon
treatment of compound 5ab and CA4, various apoptotic phase
cells were identified by this staining. From the Figure 5, it depicts
that at the lowest concentration of 0.1 µM, the cells have least
apoptotic activity and with increasing the concentration we
observed membrane blebbing, destructive fragmentation and
Annexin V/ PI dual binding assay
Various events are reported in cell changes during the apoptosis,
which includes fragmentation of the nucleus, chromosomal DNA
and chromatin condensation. Along with this externalization of
phosphatidyl serine is also considered as one of the
characteristic feature of cell undergoing apoptosis. Therefore to
evaluate this, Annexin V-PI staining technique was performed on
A549 cells with compound 5ab at different concentrations (0.1,
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0.25 and 0.5 µM), CA4 and compared with untreated cells for
about 48 h. As shown in Figure 7, the compound 5ab and CA4
caused increase in the percentage of apoptotic cells with
11.67%, 16.77%, 35.55%, and 16.88% respectively. This
suggests that compound 5ab inhibited cell proliferation by
inducing apoptosis in A549 cells in a dose dependent manner.
(generally occurs in ill stated cells/apoptotic cells) and emits
fluorescence at 590±17.5 nm. However, normal healthy cells
contain more J aggregates and less monomers due to which
JC-1 dye couldn’t bind and emits fluorescence at 530±15 nm.
Flow cytometry analysis of A549 cells after treatment with
compound 5ab revealed that the compound has disrupted the
mΨΔ in contrast to control cells. The mΨΔ is represented in
terms of P1 (normal mΨΔ) and P2 (altered mΨΔ) populations in
Figure 8. When total of 10,000 cells were analyzed for this assay,
compound 5ab increased the P2 (J monomers) population from
36.62 to 69.88% in comparison to control. Followed by this, on
comparison with a positive control, the compound 5ab at 0.25
µM, was found to be on par with the standard CA-4.
Figure 6. Representative images of DAPI staining in A549 cells. Cells were
treated with compound 5ab at concentrations of 0.1, 0.25, 0.5 µM and CA4 for
48 h. The images were captured with a fluorescence microscope at 200X
magnification
Figure 8. Image representing ratio of J aggregates (P1-red colour) to J
monomers (P2-purple colour). Fluorescence of JC-1 was analyzed using Flow
cytometer (BD FACSVerseTM, USA). Here 10,000 events were calculated for
the analysis.
Cell cycle analysis
To further study whether the compound 5ab could arrest the
process of mitosis, cell cycle distribution of A549 cells was
assessed using well studied PI staining procedure. The cells
were treated with different concentrations (0.5 and 1 µM) of
compound 5ab for 24 h, after which cell cycle distribution was
analyzed through flow cytometry [49]. The results revealed that
the treatment with compound 5ab resulted with significant
accumulation of A549 cells in G2/M phase from 25.44% to
65.44% when compared with control in a dose dependent
manner. From the Figure 9, it can be inferred that the compound
5ab arrested the lung cancer cells in G2/M phase.
Figure 7. Cells were treated with 0.1, 0.25 and 0.5 µM concentration of
compound 5ab and CA4 for 48 h and stained with Annexin V and PI. Cells
were then washed with PBS and dispersed in PBS until analysis by Flow
cytometer (BD FACSVerseTM, USA). 10,000 events were analyzed by the
instruments.
Measurement of Mitochondrial Membrane Potential
Health of mitochondria is crucial for the normal physiology of
cells by which it preserves the bioenergetics functions. This can
be represented by means of mitochondrial membrane potential
(mΨΔ). Alteration in this potential is an indication of early events
that occur during the apoptotic process. To evaluate the health
status of cancerous cells in terms of apoptosis upon treatment
with compound 5ab, tetraethylbenzimidazolylcarbocyanine
iodide (JC-1) mΨΔ assay was performed [48]. This assay works
on the principle that JC-1 a cationic dye binds dominantly to the
Figure 9. Effect of compound 5ab on the progression of cell cycle in A549
cells treated at concentrations of 0.5 and 1 µM and compared with control.
After 24 h of treatment time, the cells cycle profile was performed by using
propidium iodide staining method and analyzed by Flow cytometry analysis
(BD FACSVerseTM, USA analysis). The percentages of G1, S and G2/M phase
cells were calculated from the DNA content histograms.
J
monomers formed during hyperpolarisation in the cells
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Tubulin polymerization inhibition
Molecular docking study
The antimitotic effect of combretastatin A4 is due to inhibition of
the function of microtubules. CA-4 binds to tubulin at the
colchicine binding site. It binds to tubulin heterodimer leads to
disruption and consequently causing inhibition of its
polymerization into microtubules. This further causes formation
To further reveal the mode of binding with the tubulin, the potent
compound 5ab was docked into the combretastatin-binding site
of tubulin protein (PDB ID: 5LYJ) [52] located at the interface of α,
β-subunits. Molecular docking studies were performed by using
GLIDE docking module of Schrödinger suite 2017-1[53]. The best
of irregular mitotic spindles and metaphase arrest of mitotic cells. docked pose of ligand 5ab was well accommodated in the
Colchicine is known to be toxic and has few therapeutic uses but
the binding of CA-4 to the colchicine site of tubulin has shown
potent cytotoxicity against cancer cells [50]. Furthermore, the
newly synthesized scaffolds mimics the CA-4 structurally, in this
context, to investigate the correlation between cytotoxicity of
these compounds and tubulin, compound 5ab was examined for
combretastatin binding pocket between α,β-subunits and has
been represented in Figure 11 (A). The most active compound
5ab exhibited two strong hydrogen-bonding interactions with the
active residues. The 4-methoxy phenyl of 5ab displayed
hydrogen bond interactions with the hydroxyl group of Tyr202
(C-O…H-O) and the carbonyl oxygen and piperazine entity
displays hydrogen-bonding with amine of Asp251 (C-O…H-N)
as represented in Figure 11 (B). The piperazine moiety acts as
linker that holds the sulfonyl phenyl moiety towards α-chain of
the active pocket. Further, several hydrophobic interactions were
observed for the piperazine and sulfonyl phenyl of ligand 5ab
with amino acid residues of α-chain Ala 180, Val 181 and β-
chain residues such as Leu248, Ala250, Leu252, Leu255, and
Met259. The combretastatin moiety of the compound 5ab
extend into the active pocket on β-chain and is surrounded by
Tyr202, Phe169, Val238, Cys241, Leu242, Phe268, Val315,
Ala316, Ala317, Ile318, Phe377 and Ile378 amino acid residues.
The superimposition of the co-crystallized ligand and active
ligand 5ab was further established for the correlation of docking
study with the in vitro tubulin assay and has been represented in
Figure 11 (C). From the results, it was observed that the hydroxy
phenyl group of co-crystal was superimposed with the sulfonyl
phenyl of the ligand 5ab and the cis oriented double bond of co-
crystal gets superimposed with the amide bond of piperazine
linker. However, the ligand 5ab was well accommodated in the
active pocket with good hydrogen bonds and numerous
hydrophobic interactions with different amino acid residues, yet
there lies a slight deviation by lacking the hydrogen bond
interaction with Thr179 residue, which was actually observed in
the co-crystal, which therefore, accounts for the weak inhibition
of tubulin polymerization than CA4.
its effect on tubulin polymerization in a cell-free in vitro assay [51]
,
Paclitaxel and CA-4 were chosen as positive control where
Paclitaxel being the tubulin enhancer and CA-4 as tubulin
inhibitor. Tubulin polymerization was monitored by the increase
in fluorescence at 360 nm (excitation) and 440 nm (emission) for
1 h at 37 oC. The compounds were included at a final
concentration of 1.25, 2.5, 5.0 and 10.0 µM respectively. The
experiment was performed in duplicates. The % inhibition of
Compound 5ab screened at 10 µM was found to be 50%. As
illustrated in Figure 10, 5ab efficiently inhibited tubulin
polymerization in a dose-dependent manner with IC50 value of
5.24±0.06 µM.
Figure 11. A) Compound 5ab docked into the interface between α,β-subunits
of tubulin protein (PDB ID: 5LYJ). Green color: α-subunit, Cyan color: β-
subunit, Ligand is represented in magenta with ball and stick model. B)
Hydrogen bond interactions of the ligand 5ab with amino acid residues Tyr202
and Asp251; Ligand is represented in magenta and hydrogen bond
interactions are denoted in yellow color. C) Superimposition of co-crystallized
ligand (green) and best docked pose of ligand 5ab (magenta) in the
combretastatin binding site of α/β-tubulin interface (PDB ID: 5LYJ).
Figure 10. Effect of compound 5ab on tubulin polymerization inhibition. Data
presented were mean ±SEM of three similar independent experiments.
7
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10.1002/cmdc.201900541
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FULL PAPER
Conclusion
[9] P. M. Checchi, J. H. Nettles, J. Zhou, J. P. Snyder, H. C. Joshi,
Trends Pharmacol.Sci. 2003, 24, 361–365.
In conclusion, we have synthesized
a
series of new
[10] M. Sridhare, M. J. Macapinlac, S. Goel, D. Verdier-Pinard, T. Fojo,
M. Rothenberg, D. Colevas, Anti-Cancer Drugs 2004, 15, 553–
555.
combretastatin A-4 linked sulfonyl piperazine hybrids 5a–ad and
further evaluated for their in vitro antiproliferative activity on a
panel of human cancer cell lines. This in vitro screening
outcome signifies that the most active compound 5ab displayed
extensive range of activity on all the tested cancer cell lines with
IC50 value of 0.36±0.02 µM on A549 cells. The flow cytometric
analysis indicated the cell cycle arrest in A549 cells at G2/M
phase. In addition, the compound 5ab efficiently inhibited tubulin
polymerization with IC50 value of 5.24±0.06 µM and in silico
studies also declared that the compound 5ab binds at the
colchicine binding site of the tubulin. Furthermore, the
compound 5ab showed apoptotic changes by decreasing the
viable cells as in case of AO and DAPI staining assays. The
other studies such as JC-1, Annexin-V, cell migration/scratch
wound assay and phase contrast imaging, the compound 5ab
revealed the dose dependant inhibition by inducing apoptosis
and mitochondrial damage that gave insights about cell
migration and cell viability. Finally, the new series of compounds
are potent to be proceeded as microtubule targeting cytotoxic
agents in treating cancer.
[11] M. N. Islam, Y. Song, M. N. Iskander, J. Mol. Graphics Modell.
2003, 21, 263–272.
[12] E. Nogales, M. Whittaker, R. A. Milligan and K. H. Downing, Cell
1999, 96, 79–88.
[13] R. B. Ravelli, B. Gigant, P. A. Curmi, I. Jourdain, S. Lachkar, A.
Sobel, M. Knossow, Nature, 2004, 428, 198–202.
[14] S. B. Horwitz, Trends Pharmacol. Sci. 1992, 13, 134–136.
[15] J. J. Manfredi and S. B. Horwitz, Pharmacol. Ther. 1984, 25, 83–
125.
[16] M. Jordan, Curr. Med. Chem. Anti-Cancer Agents, 2002, 2, 1–17.
[17] G. R. Pettit, S. B. Singh, E. Hamel, C. M. Lin, D. S. Alberts, D.
Garcia-Kendal, Experientia, 1989, 45, 209–211.
[18] a) A. Brancale, R. Silvestri, Med. Res. Rev. 2007, 27, 209-238; b)
A. Chaudhary, S. N. Pandeya, P. Kumar, P. P. Sharma, S. Gupta,
N. Soni, K. K. Verma, G. Bhardwaj, Mini-Rev. Med. Chem. 2007, 7,
1186-1205.
[19] D. J. Chaplin, G. R. Pettit, S. A. Hill, Anticancer Res. 1999, 19,
189–195.
[20] J. W. Lippert III, Bioorg. Med. Chem. 2007, 15, 605–615.
[21] G. J. Rustin, S. M. Galbraith, H. Anderson, M. Stratford, L. K.
Folkes, L. Sena, L. Gumbrell, P. M. Price, J. Clin. Oncol. 2003, 21,
2815–2822
Supporting information
Synthetic procedures, experimental details, spectral information
(1H and 13C spectra) and biological evaluation are found in the
supporting information of this article.
[22] G. C. Tron, F. Pagliai, G. E. Del, A. A. Genazzani, G. Sorba, J.
Med. Chem. 2005, 48, 3260-3268.
[23] K. Ohsumi, R. Nakagawa, Y. Fukuda, T. Hatanaka, Y. Morinaga,
Y. Nihei, K. Ohishi, Y. Suga, Y. Akiyama, T. Tsuji, J. Med. Chem.
1998, 41, 3022-3032
Acknowledgments
[24] a) E. T. Haar, H. S. Rosenkranz, E. Hamel, B. W. Day, Bioorg.
Med.Chem. 1996, 4, 1659-1671; b) C. Borrel, S. Thoret, X.
Cachet, D. Guenard, F. Tillequin, et al, Bioorg. Med. Chem. 2005,
13, 3853–3864.
C. J., and R. T., are thankful to DoP, Ministry of Chemicals &
Fertilizers, Govt. of India, New Delhi, for the award of NIPER
fellowship. Dr. N. Shankaraiah is thankful to SERB, DST, Govt.
of India for the start-up grant (YSS-2015-001709).
[25] V. T. DeVita, E. Chu, Cancer Res. 2008, 68 (21), 8643-8653.
[26] R. F. Borne, R. L. Peden, I. W. Waters, M. Weiner, R. Jordan, et
al., J Pharm Sci. 1974, 63, 615-617.
Conflict of interest
[27] J. Mun, A. A. Jabbar, N. S. Devi, S. Yin, Y. Wang, et al., J. Med.
Chem. 2012, 55, 6738-6750.
The authors declare no conflict of interest.
[28] N. S. El-Sayed, E. R. El-Bendary, S. M. El-Ashry, M. M. El-
Kerdawy, Eur. J. Med. Chem. 2011, 46, 3714-3720.
[29] Y. Genc, R. Ozkanca, Y. Bekdemir, Ann Clin Microbiol Antimicrob
2008, 7, 17-22.
Keywords Combretastatin A-4 • sulfonyl piperazine • tubulin
assembly • cell migration/scratch wound assay • cytotoxicity.
[30] N. Ozbek, H. Katircioaylu, N. Karacan, T. Baykal, Bioorg. Med.
Chem. 2007, 15, 5105-5109.
References
[31] E. D Clercq, Curr. Med. Chem. 2001, 8, 1543-1572.
[32] P. K. Naik, B. P. Chatterji, S. N. Vangapandu, R. Aneja, R.
Chandra, S. Kanteveri, J. Comput. Aided Mol. Des. 2011, 25, 443–
454.
[1] a) A. Jordan, L. Wilson, Nat. Rev. Cancer 2004, 4, 253–265; b) R.
Stephens and K. Edds, Phys. Rev. 1976, 56, 709–777.
[2] B. A. Teicher, Clin. Cancer Res. 2008, 14, 1610–1617.
[3] M. Trivedi, I. Budihardjo, K. Loureiro, T. R. Reid, J. D. Ma, Future
Oncol. 2008, 4, 483–500.
[33] S. M. Kupchan, R. W. Britton, M. F. Ziegler, C. J. Gilmore, R. J.
Restivo, R. F. Bryan, J. Am. Chem. Soc. 1973, 95, 1335–1336.
[34] G. D. Hatnapure, A. P. Keche, A. H. Rodge, S.S. Birajdar, R. H.
Tale, V. M. Kamble, Bioorg. Med. Chem. Lett. 2012, 22, 6385-
6390.
[4] C. M. Spencer, D. Faulds, Drugs 1994, 48, 794–847.
[5] H. M. Robinson, Biochem. Cell Biol. 1991, 69, 581–582.
[6] J. Griggs, J. C. Metcalfe, R. Hesketh, Lancet Oncol. 2001, 2, 82–
87.
[35] a) N. K. Beyeh, A. Valkonen, K. Rissanen, Org. Lett. 2010, 12,
1392-1395; b) A. Kamal, P. S. M. M. Reddy, D. R. Reddy, E.
Laxman, Bioorg. Med. Chem. 2006, 14, 385-394; c) C. S. A.
Kumar, S. B. B. Prasad, K. Vinaya, S. Chandrappa, N. R.
Thimmegowda, Y. C. S. Kumar, S. Swarup, K. S. Rangappa, Eur.
[7] E. K. Rowinsky, R. C. Donehower, Pharmacol. Ther. 1991, 52, 35–
84.
[8] G. Attard, A. Greystoke, S. Kaye, J. De Bono, Pathol. Biol. 2006,
54, 72–84.
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900541
ChemMedChem
FULL PAPER
J. Med. Chem. 2009, 44, 1223-1229; d) C. S. A. Kumar, S. N.
Swamy, N. R. Thimmegowda, S. B. B. Prasad, G. W. Yip, K. S.
Rangappa, Med. Chem. Res. 2007, 16, 179-187.
[36] J. Z. Long, X. Jin, A. Adibekian, W. W. Li, B. F. Cravatt, J. Med.
Chem. 2010, 53, 1830-1842.
[37] D. H. Jiang, M. Huang, Chem. Reag. 2012, 34, 797-799.
[38] S. Liang, C. M. Liu, Y. S. Jin, Q. Q. He, J. Med. Chem. 2004, 14,
71-75.
[39] Q. Wu, Z. C. Wang, X. Wei, W. Xue, J. Synth. Chem. 2014, 22,
429-434.
[40] R. J. Kerns, M. J. Rybak, G. W. Kaatz, F. Vaka, R. Cha, R. G.
Grucz, V. U. Diwadkar, Bioorg. Med. Chem. Lett. 2003, 13, 2109–
2112.
[41] C. Avijeet, A. Amy, G. Charles, J. Biol. Chem. 2014, 289, 2978–
2991.
[42] a) R. Maia, C. Do, C. A. M. Fraga, Curr. Enzyme Inhib. 2010,
6,171−182; b) C. Lazar, A. Kluczyk, T. Kiyota, Y. Konishi, J. Med.
Chem. 2004, 47, 6973−82.
[43] a) T. Mosmann, J. Immunol. Meth. 1983, 65, 55–63; b) P. Sharma,
K. R. Senwar, M. K. Jeengar, T. S. Reddy, V. G. M. Naidu, A.
Kamal, N. Shankaraiah, Eur. J. Med. Chem. 2015, 104, 11–24.
[44] a) P. Sharma, T. S. Reddy, D. Thummuri, K. R. Senwar, N.
P. Kumar, V. Naidu, S. K. Bhargava, N. Shankaraiah, Eur. J. Med.
Chem. 2016, 124, 608-621; b) R. Tokala, S. Thatikonda, S. Sana,
P. Regur, C. Godugu, N. Shankaraiah, New J. Chem. 2018, 42,
16226–16236
[45] M. Yao, J. Yang, L. Cao, L. Zhang, S. Qu, H. Gao, Mol. Med. Rep.
2014, 10, 365-372.
[46] P. Sharma, D. Thummuri, T. S. Reddy, K. R. Senwar, V. G. M.
Naidu, G. Srinivasulu, S. K. Bharghava, N. Shankaraiah, Eur. J.
Med. Chem. 2016, 122, 584–600; b) K. R. Senwar, P. Sharma, T.
S. Reddy, M. K. Jeengar, V. L. Nayak, V. G. M. Naidu, A. Kamal,
N. Shankaraiah, Eur. J. Med. Chem. 2015, 102, 413–424.
[47] a) B. I. Tarnowski, F. G. Spinale, J. H. Nicholson, Biotech
Histochem, 1991, 66, 297–302; b) R. Tokala, S. Thatikonda, U. S.
Vanteddu, S. Sana, C. Godugu, N. Shankaraiah, ChemMedChem.
2018, 13, 1909–1922.
[48] a) N. Kulabas, E. Tatar, O. B. Ozakpinar, D. Ozsavci, C.
Pannecouque, E. D. Clercq, I. Kucukguzel, Eur. J. Med. Chem.
2016, 121, 58–70; b) R. Tokala, S. Bale, I. P. Janrao, A. Vennela,
N. P. Kumar, K. R. Senwar, C. Godugu, N. Shankaraiah, Bioorg.
Med. Chem. Lett. 2018, 28, 1919–1924.
[49] U. V. Mallavadhani, C. V. Prasad, S. Shrivastava, V. Naidu, Eur. J.
Med. Chem. 2014, 83, 84‒91.
[50] a) V. Chaudhary, J. B Venghateri, H. P. S Dhaked, A. S Bhoyar, S.
K Guchhait, D. Panda, J. Med. Chem. 2016, 59, 3439–3451; b) N.
P. Kumar, S. Thatikonda, R. Tokala, S. S. Kumari, U. J. Lakshmi,
C. Godugu, N. Shankaraiah, Bioorg. Med. Chem. 2018, 26, 1996–
2008.
[51] N. Shankaraiah, S. Nekkanti, U. R. Brahma, N. P. Kumar, N.
Deshpande, D. Prasanna, K. R. Senwar, U. J. Lakshmi, Bioorg.
Med. Chem. 2017, 25, 4805–4816.
[52] R. Gaspari, A. E. Prota, K. Bargsten, A. Cavalli, M. O. Steinmetz,
Chem, 2017, 2, 102–113.
[53] Schrödinger Suite 2017-1, Schrödinger, LLC, New York, 2018.
9
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Insert graphic for Table of Contents here. ((Please ensure your graphic is in one of following formats))
A series of CA4 linked sulfonyl piperazine derivatives via molecular hybridization were synthesized. The compound 5ab was found as
the active member by its in vitro cytotoxicity and exhibited potent tubulin polymerization inhibition.
10
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