Synthesis and biological evaluation of cinnamido linked benzophenone hybrids as tubulin polymerization inhibitors and apoptosis inducing agents

Article abstract of DOI:10.1016/j.bmcl.2014.03.076

A new class of hybrid molecules containing cinnamide subunit linked to benzophenone as inhibitors of tubulin polymerization were synthesized and evaluated for their anticancer potential. These hybrids exhibit anticancer activity with IC₅₀ values ranging from 0.06 to 16.3 μM. Compounds 4f and 4g possessing fluoro and trifluoromethyl on the cinnamido subunit showed significant cytotoxic activity with IC₅₀ values 0.06 and 0.09 μM against HeLa cell line, respectively. These compounds showed cell cycle arrest at G2/M phase of the cell cycle and inhibited tubulin polymerization followed by activation of caspase-3 activity and apoptotic cell death. Further in vitro tubulin polymerization assay showed that the level of tubulin inhibition was comparable to that of 2a for the compounds 4f and 4g. Moreover, Hoechst 33258 staining and DNA fragmentation assay suggested that these compounds induce cell death by apoptosis. Overall, the current study demonstrates that the synthesis of benzophenone linked cinnamide subunit conjugates as promising anticancer agents with G2/M arrest and apoptotic-inducing ability via targeting tubulin.

Full text of DOI:10.1016/j.bmcl.2014.03.076

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2309–2314
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of cinnamido linked
benzophenone hybrids as tubulin polymerization inhibitors
and apoptosis inducing agents
a
a
a
a
Ahmed Kamal a, , Ch. Ratna Reddy , M. V. P. S. Vishnuvardhan , R. Mahesh , V. Lakshma Nayak ,
⇑
S. Prabhakar a, C. Suresh Reddy b
a Medicinal Chemistry and Pharmacology, CSIR—Indian Institute of Chemical Technology, Hyderabad 500007, India
b Department of Chemistry, Sri Venkateswara University, Tirupati 517502, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of hybrid molecules containing cinnamide subunit linked to benzophenone as inhibitors of
tubulin polymerization were synthesized and evaluated for their anticancer potential. These hybrids
Received 12 November 2013
Revised 13 March 2014
Accepted 25 March 2014
Available online 3 April 2014
exhibit anticancer activity with IC50 values ranging from 0.06 to 16.3
ing fluoro and trifluoromethyl on the cinnamido subunit showed significant cytotoxic activity with IC50
values 0.06 and 0.09 M against HeLa cell line, respectively. These compounds showed cell cycle arrest at
lM. Compounds 4f and 4g possess-
l
G2/M phase of the cell cycle and inhibited tubulin polymerization followed by activation of caspase-3
activity and apoptotic cell death. Further in vitro tubulin polymerization assay showed that the level
of tubulin inhibition was comparable to that of 2a for the compounds 4f and 4g. Moreover, Hoechst
33258 staining and DNA fragmentation assay suggested that these compounds induce cell death by apop-
tosis. Overall, the current study demonstrates that the synthesis of benzophenone linked cinnamide sub-
unit conjugates as promising anticancer agents with G2/M arrest and apoptotic-inducing ability via
targeting tubulin.
Keywords:
Tubulin polymerization
Apoptosis
Caspase-3
Hoechst staining
FACS analysis
Ó 2014 Elsevier Ltd. All rights reserved.
Cancer,
a
disease of uncontrolled mitosis that represents
resulting in mitotic arrest.5 Well known examples include paclit-
axel, vinblastine, epothilone, dolastatin and colchicine. In addition
some of these ligands target tumor endothelial cells which results
in a rapid occlusion of tumor vasculature, leading to vascular
shutdown.
Belonging to this class of molecules, phenstatin (1a, Fig. 1), a ben-
zophenone motif reported by Pettit and co-workers, showed potent
anticancer and antimitotic activities.6,7 A phosphate prodrug of
phenstatin (1b) was also reported. Later, Hsieh and co-workers,
introduced an amino group at C-3 position of the B-ring resulted
in 2a, that exerts potent tubulin polymerization inhibition and
shows promising cytotoxic activity against selected human cancer
cell lines.8 Very recently Lee et al. also demonstrated a new class
of benzophenone series with insertion of small heterocyclic groups
in the B-ring.9 In addition several others reported analogues of
phenstatin10 and aryl–heteroaryl ketones (indole, quinoline, carba-
zole, thiophenes) as potential tubulin inhibitors.11–14
approximately 200 different diseases, is now one of the most fre-
quent causes of death and is the second leading cause of death. It
is estimated that new cancer diagnoses will double by 2020 and
nearly triple by 2030.1 Despite the fact, that many anticancer drugs
are in clinical use through different mechanism of action, their ac-
tion is not specific. Moreover the drugs used in chemotherapy are
limited by drug resistance and thereby there is an urgency to de-
velop molecules that could selectively deliver their action at the
tumor site.
Among the current cellular targets in eukaryotes, alongside
DNA, tubulin-microtubule dynamics are critical for mitotic spindle
formation during cell division and are a promising target for anti-
cancer agents.2–4 These microtubules, comprising of
a and b-tubu-
lin heterodimers, are always in a state of equilibrium with the
latter with a certain amount of composition. Ligands perturb these
dynamics by binding to either tubulin or microtubule, leading to
the inhibition of microtubule polymerization or depolymerization
towards forming abnormal mitotic spindle and ultimately
At the same time we came across several antitumor molecules
possessing cinnamido scaffold that exhibit anticancer activity by
different mechanism of action.15 Hergenrother and coworkers in
2010 reported simple phenyl cinnamides acting as antimitotic
agents by disruption of microtubule dynamics that leads to cell
⇑
Corresponding author. Tel.: +91 40 27193157; fax: +91 40 27193189.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
http://dx.doi.org/10.1016/j.bmcl.2014.03.076
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2310
A. Kamal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2309–2314
Table 1
substituted benzophenone derivatives 14 and 15. Reduction of ni-
tro group of 14 and 15 was achieved using Fe-AcOH/EtOH under re-
flux conditions to provide the benzophenone amine precursors 2a
and 16.8
Structural representation of the synthesized compounds and their codes
Compd R1
R2
R3
Compd R1
R2
R3
H
4a
4b
4c
4d
OMe
OMe
H
OMe OMe 4l
H
NO2
OMe OMe
OMe
OMe
OMe
OMe
H
H
H
H
5a
5b
5c
OMe
OMe
H
H
H
OMe
4e
4f
H
F
H
H
5d
5e
OMe
NH2
H
H
H
O
Table 2
Cytotoxicity (IC50
H
OMe
l
M)a data of compounds 4a–l and 5a–i
4g
H
CF3
H
5f
H
H
O
Compd HeLab ME-180b DU-145c PC-3c COLO205d HT-29d B-16e
4h
4i
4j
OMe
OCF3
F
F
H
F
NH2
H
H
H
H
5g
5h
5i
H
H
F
F
CF3
F
H
H
F
4a
4b
4c
4d
4e
4f
4g
4h
4i
1.5
7.8
10.7
3.1
2.1
8.9
11.8
4.3
15.3
0.3
0.8
15.5
9.2
8.9
16.0
9.3
3.9
6.7
12.5
8.6
13.9
8.4
1.0
3.6
7.1
12.2
0.08
17.3
6.3
10.2
12.8
6.2
15.8
3.9
3.9
7.5
11.1
4.7
13.1
3.2
3.5
14.1
7.7
8.3
15.9
10.4
4.8
5.4
6.3
13.3
11.1
10.1
4.1
4.2
7.4
7.4
1.1
5.2
8.2
10.9
5.1
13.4
3.4
2.6
11.8
7.9
6.02
10.3
11.4
5.3
5.7
4.8
10.7
10.1
11.2
3.1
2.5
4.3
4.4
0.2
3.1
2.5
5.01
1.9
7.4
1.8
1.6
7.7
5.2
4.2
5.6
5.1
3.1
2.3
3.8
8.7
7.7
8.1
1.8
1.9
3.3
3.8
0.05
2.4
1.2
6.3
8.7
3.1
12.2
0.4
4k
H
14.0
0.06
0.09
13.6
7.9
8.1
14.7
8.5
2.5
2.7
11.2
6.9
15.8
7.3
0.9
cycle arrest in G2/M phase; among which 3 displayed promising
activity.16 In continuation of our efforts towards the design of
new anticancer agents,17–19 we considered it worthwhile to pursue
further modifications on the benzophenone part by appending cin-
namide subunit to the amine functionality situated at 30-position
on the B-ring of the benzophenone, that may allow better interac-
tions at the receptor site and could affect the biological activity.
This report describes the synthesis and evaluation of their antican-
cer activity apart from their effect on tubulin polymerization, cell
cycle and apoptosis inducing ability of these hybrids.
4.3
0.1
12.1
10.2
10.2
13.5
15.6
6.6
16.3
6.3
6.5
7.9
6.4
2.5
1.3
9.4
6.7
12.7
5.1
0.9
3.3
5.2
12.5
0.03
8.5
4j
4k
4l
5a
5b
5c
5d
5e
5f
5g
5h
5i
7.8
10.4
12.5
12.7
13.5
6.7
5.1
7.9
10.0
2.1
3.2
Results and discussions
3.0
5.0
13.5
0.06
12.3
16
2a
3
Benzophenone–cinnamides 4a–l and 5a–i listed in Tables 1
were synthesized as depicted in Schemes 1 and 2. The required
aldehydes 8 and 10 were prepared by nitration of 6 and 7 using
70% HNO3 according to the reported literature procedures.20 As
presented in Scheme 1, benzhydroxy compounds 12 and 13
obtained from Grignard reaction between 11 and 8 or 10, were
oxidized using pyridinium dichromate to afford the nitro
2.1
4.2
a
b
c
Compound concentration required to inhibit cell proliferation by 50%.
Cervical cancer cell lines.
Prostate cancer cell lines.
Colon carcinoma.
Mouse melanoma carcinoma.
d
e
CHO
CHO
i
R
R
8
6
R = 3-NO2-4-OMe
R = 4-OMe
7 R = 3-OMe-4-OH
9 R = 3-OMe-4-OH-5-NO2
10
ii
R = 3,4-di-OMe-5-NO2
CHO
MgBr
O
OH
MeO
MeO
MeO
iii
OMe
iv
R
R
+
R
MeO
R = 3-NO2-4-OMe
10 R = 3,4-di-OMe-5-NO2
MeO
OMe
11
OMe
R = 3-NO2-4-OMe
8
OMe
12
14
R = 3-NO2-4-OMe
v
13 R = 3,4-di-OMe-5-NO2
15 R = 3,4-di-OMe-5-NO2
2a
R = 3-NH2-4-OMe
16 R = 3-NH2-4,5-di-OMe
Scheme 1. Reagents and conditions: (i) 70% HNO3, 8 h, rt for 6; HNO3, AcOH, 10 h, rt for 7; (ii) Bu4NBr, NaOH, Me2SO4, CH2Cl2/H2O, 24 h, rt for 9; (iii) THF, 0 °C for 10 min–rt,
1 h; (iv) PDC, dry CH2Cl2, 8 h; (v) Fe, AcOH–EtOH, 1–2 drops of HCl, reflux, 1 h.
R1
R2
R3
O
O
O
H
N
MeO
MeO
NH2
MeO
MeO
X
ii
+
R
O
R
OMe
19
X = OH
X = Cl
OMe
2a
R
4a l
OMe
i
R = 4-OMe
16 R = 4,5-di-OMe
- R = H
5a-i R = OMe
4l, 5j
iii
4k, 5e
Scheme 2. Reagents and conditions: (i) (COCl)2, dry CH2Cl2, 0 °C, DMF (cat), 4 h; (ii) cinnamoyl chloride, Et3N, THF, 0 °C 6 h; (iii) Zn, HCO2NH4, MeOH, rt, 6 h.

A. Kamal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2309–2314
2311
O
PC-3, HT-29 and B-16 cells; while activity was reduced for com-
pound 4b and 4c (bearing 3,4-di-OMe and 4-OMe, respectively)
in the cell lines tested. Interestingly 4d (3-OMe substitution)
exhibited cytotoxicity almost similar to 4a with IC50 value of
O
R
MeO
MeO
R
MeO
MeO
B
A
B
A
OMe
OMe
OMe
OMe
1.96.2 lM range in all the tested cell lines. Whereas 4e, replacing
2a
R = NH2
1a
R = OH Phenstatin
1b R = OP(O)(ONa)2
the methoxy group on 4d with an O-propargyl substituent, re-
sulted in reduced cytotoxicity. In this series 4f and 4g, having 4-F
and 4-CF3 substitution on the cinnamide phenyl ring, showed
R1
R2
R3
OMe
O
promising activity with IC50 of 0.06 and 0.09
0.3 and 0.8 M against ME-180 and 0.4 and 0.1
cell lines, respectively, while with an IC50 range of 1.6–4.3
l
l
M against HeLa;
M against B-16
M on
H
N
OMe
OMe
MeO
MeO
O
l
O
l
N
H
OMe
the other cell lines (DU-145, PC-3 and COLO-205). 4f and 4g dis-
played same activity against HeLa as the corresponding parent core
amine 2a (IC50 of 0.06 lM) which is a positive control and showed
far better activity than the cinnamide 3. Introduction of methoxy
or fluoro group at 30-position to 4f resulted in compounds 4h
and 4j that showed decreased cytotoxicity and 4i, possessing
OMe
R
4a-l
5a-i
R = H
R = OMe
OMe
3
R1,R2,R3 = OMe,F, NO2, NH2, OCF3,
3-O-propargyl, CF3
Figure 1. Tubulin polymerization inhibitors.
3-OCF3, exhibited moderate activity (IC50 of 5.2–10.2
Whereas 4l with 4-NO2 substitution exhibited good activity (IC50
of 5.1–10.4 M range) compared to 4k (5.6–16.0 M) that has a
4-NH2 substituent.
lM).
l
l
Finally the targeted hybrids 4 and 5 were obtained by the cou-
pling of the substituted cinnamoyl chlorides to the amine interme-
diates 2a or 16 in dry THF at 0 °C using triethyl amine (Et3N) as
base (Scheme 2). Cinnamoyl chlorides were obtained from the cor-
responding acids 19 using oxalyl chloride and catalytic amount of
DMF. Some of the cinnamic acids were prepared according to the
standard literature procedures; propargylation, Wittig reaction fol-
lowed by saponification. Compound 4k and 5e were obtained by
reduction of the corresponding nitro intermediates using zinc-
ammoniumformate.
We then evaluated the cytotoxicity of compounds 5a–i, that has
3,4-di-OMe substitution on the B-ring of the benzophenone sub-
unit apart from other substitutions in the cinnamido subunit. Com-
pounds 5a and 5b, having tri-OMe and di-OMe substitution on the
cinnamido ring, respectively, were found to display almost similar
range of activity with IC50 1.3–7.8 lM in the corresponding tested
cell lines, unlike in case of 4a and 4b of the previous series. Com-
pound 5c with 4-OMe substitution showed reduced activity com-
pared to 5b (3,4-di-OMe) against HeLa, ME-180 and B-16 cell
The synthesized compounds 4a–l and 5a–i were initially
screened for their cytotoxic activity in selected human cancer cell
lines of cervical, prostate, colorectal and mouse melanoma origin
by using MTT assay.21 All the compounds exhibit cytotoxicity with
lines with IC50 of 11.2, 12.5 and 9.4
same range of activity on the other cell lines. Whereas 5d (3-
OMe) showed increased cytotoxicity (IC50 of 6.9 and 8.6 M)
lM, while maintaining the
l
against HeLa and ME-180 compared to 5c with reduced activity
on PC-3, COLO-205 and HT-29 cell lines. Moreover 5e (4-OMe-
3-NH2) also showed loss of activity on all the cell lines when
compared to 5b. Amongst the compounds of this series, 5g bearing
4-F substitution showed significant IC50 value of 0.9, 1.0 and
IC50 values ranging from 0.06 to 16.0
trated in Table 1.
Firstly the effect of the cinnamide moiety in compounds 4a–l,
possessing 4-OMe substitution on B-ring is examined. As shown
in the Table 2, of the hybrids bearing methoxy substitution
(4a–c); 4a, possessing tri-OMe unit, exhibited good cytotoxicity
lM and the results are illus-
0.9
lM against HeLa, ME-180 and B-16 cell lines. However 5h,
bearing 4-CF3 substitution, showed reduced cytotoxic activity with
with IC50 of 1.5, 2.1, 3.9, 3.1 and 1.2 lM against HeLa, ME-180,
Figure 2. Effects of compounds A (control), B (4f), C (4g), and D (2a) on DNA content/cell following the treatment of HeLa cells at 0.5 lM for 24 h. Cell cycle distribution was
analyzed by the standard propidium iodide procedure as described in Experimental section.

2312
A. Kamal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2309–2314
Table 3
Cell cycle distribution of HeLa cells (%) for 4f, 4g and 2a
Compd
SUB-G1
G1 phase
S phase
G2/M
Control
4f
4g
3.07
2.96
5.66
4.56
71.21
53.02
49.59
53.00
4.62
7.40
5.92
5.07
21.10
36.62
38.82
37.36
2a
Figure 5. Effect on tubulin polymerization: tubulin polymerization assay was
carried out in a reaction mixture that contained PEM buffer and GTP (1 mM) in the
presence or absence of test compounds (4f, 4g, and 2a) at 5 lM concentration. The
reaction was initiated by the addition of GTP to all the wells. Tubulin polymeri-
zation was monitored by the increase in fluorescence at 420 nm (excitation
wavelength is 360 nm) was measured for 1 h at 1 min interval in a multimode plate
reader (Tecan) at 37 °C.
Figure 3. Effect of compounds 4f, 4g and 2a on caspase-3 activity: HeLa cells were
treated for 48 h with 2 M concentrations of compounds 4f and 4g with 2a as a
positive control. Values indicate the mean SD of two different experiments
performed in triplicates.
activity showed by compounds 4f and 4g prompted us to evaluate
their cell viability in the HeLa cells, with a view to study their de-
tailed biological effects in this cell line.
l
In order to understand the role of these compounds (4f, 4g, 2a)
in cell cycle, FACS analysis22 was conducted in Cervical cancer cells
(HeLa). The cells were treated for 24 h with compounds 4f, 4g, 2a
an IC50 value of 3.0 and 3.6
while maintaining the same range of activity (1.9–5.1
DU-145, PC-3, COLO-205, HT-29 and B-16. Compound 5i, with
3,4,5-tri-F substitution, showed IC50 range of 3.3–7.9 M. Overall
majority of the compounds in this series displayed better activity
compared to its corresponding parent amine 16 against HeLa and
ME-180 cell lines.
It is observed from these results that 4f and 4g with fluoro and
trifluoromethyl groups located at 4-position on the cinnamide aro-
matic ring exhibited enhanced activity. The promising anticancer
l
M on the HeLa and ME-180 cell lines,
at 0.5 lM concentration. It was observed that HeLa cells showed
lM) against
36.6%, 38.8%, 37.3% G2/M cell cycle arrest, respectively, whereas
control (untreated cells) exhibited 21% as shown in Figure 2 and
Table 3.
l
It is well known23 that the cell cycle arrest at G2/M phase is
shown to induce cellular apoptosis, hence it was considered of
interest to examine whether the cytotoxicity of 4f, 4g, and 2a is
by virtue of apoptotic cell death. Cysteine aspartase group, namely,
caspases play a crucial role in the induction of apoptosis and
Figure 4. IHC analyses of compounds on the microtubule network: HeLa cells were treated with compounds 4f, 4g, and 2a at 1
staining with -tubulin antibody. Microtubule organization was clearly observed by green color tubulin network like structures in control cells and was found to be disrupted
in cells treated with compounds 4f and 4g with 2a as positive control.
lM concentration for 48 h followed by
a

A. Kamal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2309–2314
2313
Table 4
amongst them caspase-3 happens to be one of the effector caspase.
This prompted to treat HeLa cells with compounds, 4f, 4g and 2a to
examine the activation of caspase-3, The results indicate that there
is nearly 5 to 8-fold induction in caspase-3 activity in cells treated
a
Inhibition of tubulin polymerization (IC50
) of compounds 4f, 4g and 2a
Compd
Tubulin polymerization inhibition IC50 SDb
(lM)
4f
4g
2a
0.6 0.9
0.7 0.5
0.6 0.3
with 2 lM concentration by these compounds as compared to con-
trol, thus suggesting the activation of caspase-3 by 4f, 4g, 2a indi-
cate that they have the ability to induce apoptosis in HeLa cells
(Fig. 3).
a
Drug concentration required inhibiting 50% of tubulin assembly.
Standard deviation.
b
In order to substantiate the observed effects of these com-
pounds on the inhibition of tubulin polymerization to functional
microtubules immunohistochemistry studies have been carried
out to examine the in situ effects of compounds 4f and 4g on cel-
lular microtubules in HeLa cancer cells. Therefore, HeLa cells were
treated with 4f, 4g, and 2a at 1 l
M concentration for 48 h.24 In this
study, untreated human cervical cancer cells displayed the normal
distribution of microtubules (Fig. 4). However, cells treated with
compounds 4f and 4g showed disrupted microtubule organization
as seen in Figure 4, thus demonstrating the inhibition of tubulin
polymerization. This immunofluorescence study showed that the
level of tubulin polymerization inhibition was comparable to that
of 2a for the compounds 4f and 4g.
One of the possibilities that these compounds exhibit antican-
cer activity as well as G2/M cell cycle arrest is by the inhibition
of tubulin polymerization25 as this has been observed in many
antimitotic agents such as combretastatins. Hence it was consid-
ered of interest to investigate the tubulin polymerization aspect.
As tubulin subunits heterodimerize and self-assemble to form
microtubules in a time dependent manner, we have investigated
the progression of tubulin polymerization by monitoring the in-
crease in fluorescence emission at 420 nm (excitation wavelength
is 360 nm) in 384 well plate for 1 h at 37 °C with and without
the compounds at 5 lM concentration. The compounds 4f, and
4g inhibited tubulin polymerization by 60.5%, and 59.4%, respec-
tively, compared to control, (Fig. 5). Tubulin polymerization inhibi-
tion was also observed in case of standards like, 2a (58.3%).
Furthermore, these two potential compounds (4f and 4g) were
evaluated for their in vitro tubulin polymerization assay at differ-
ent concentrations. These molecules showed potent inhibition of
Figure 6. DNA fragmentations of compounds 4f and 4g in HeLa (Cervical cancer
cells. Lane-1: (untreated control DNA (1 l), lane-2: 1(4f-1 M), lane-3: 2(4f-2 M),
lane-4: 3(4g-1 M), lane-5; 4(4g-2 M), lane-6: (5) marker, 50 bpair (loaded DNA
for 4f and 4g at 1 L).
l
l
l
tubulin polymerization with IC50 values 0.6 and 0.7
tively, compared to the control 2a having IC50 of 0.6
l
M, respec-
l
l
l
l
M (Table 4).
Figure 7. Hoechst staining in HeLa cervical cancer cell line, A: HeLa control cells, 4f, 4g, 2a treated with 1 lM.

2314
A. Kamal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2309–2314
The ability of 4f and 4g for the induction of intranucleosomal
References and notes
DNA fragments at 48 h of exposure to HeLa cells have been demon-
strated by agarose gel electrophoresis technique.26 DNA laddering
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is significantly observed in the HeLa cells exposed to 1 and 2 lM of
4f and 4g. The results show that 4f and 4g harbor DNA fragmenta-
tion in HeLa cells, evident by Figure 6, which is associated with the
last events of drug induced apoptosis.
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 these
compounds (4f and 4g) by Hoechst staining23a (H33258) method
in HeLa cancer cell line. Therefore cells were treated with 4f, 4g,
and 2a at 1 lM 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 (4f, 4g, and 2a) revealed that there was sig-
nificant increase in the percentage of apoptotic cells (Fig. 7).
A new series of benzophenone hybrids were synthesized and
evaluated for their anticancer potential against panel of cancer cell
lines. All these compounds exhibited cytotoxicity with IC50 values
ranging from 0.06 to 16.3 lM and compounds 4f and 4g, bearing
4-F and 4-CF3 on the cinnamido subunit, showed significant anti-
cancer activity in HeLa cell line. Further the most active hybrids
4f and 4g induce apoptotic cell death by inhibition of tubulin
polymerization leading to cell cycle arrest at G2/M phase of the cell
cycle followed by caspase-3 activity. Hoechst 33258 staining and
DNA fragmentation assay also suggests that 4f and 4g induces cell
death by apoptosis. The in vitro tubulin polymerization assay
showed that these compounds showed that the level of tubulin
inhibition was comparable to that of 2a. Based on these results it
is evident that these new benzophenone–cinnamide hybrids, partic-
ularly 4f and 4g, have the potential to be developed as a new class of
anticancer agents by further structural modifications. Overall, the
current study demonstrates that the synthesis of cinnamido linked
benzophenone conjugates as promising anticancer agents with
G2/M arrest and apoptotic-inducing activities via targeting tubulin
deserving further research and development, for exploiting new
tubulin binding agents leading to apoptotic cell death.
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Acknowledgments
The authors (R.R., M.V.P.S.V. and R.M.) are thankful to CSIR, New
Delhi for award of fellowships and also thankful for financial sup-
port under the 12th Five Year Plan project ‘Affordable Cancer Ther-
apeutics’ (CSC0301).
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Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
03.076.