Synthesis and biological evaluation of curcumin inspired indole analogues as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.12.043

In our endeavour towards the development of potent cytotoxic agents, a series of some new curcumin inspired indole analogues, in which indole and phenyl moieties are linked on either sides of 1,5-diaryl-1,4-pentadien-3-one system have been synthesized and characterized by spectral data. All the newly synthesized analogues were tested for their cytotoxic potential against a panel of eight cancer cell lines namely, lung (A549), breast (MDA-MB-231, BT549 and 4T1), prostate (PC-3, DU145), gastric (HGC-27) and cervical (HeLa). Notably, among all the compounds tested, compounds 11c, 11d and 11f showed potent growth inhibition on PC-3 and BT549 with IC₅₀values in the range of 3.12–6.34?μM and 4.69–8.72?μM respectively. The most active compound (11c) was also tested on RWPE-1 (normal prostate) cells and was found to be safe compared to the PC-3?cells. In tubulin polymerization assay, compounds 11c and 11f effectively inhibited microtubule assembly with IC₅₀values of 10.21?±?0.10 and 8.83?±?0.06?μM respectively. The results from molecular modelling studies revealed that these compounds bind at the colchicine binding site of the tubulin. Moreover, DAPI and acridine orange/ethidium bromide staining studies indicated that compounds 11c and 11f can induce apoptosis in PC-3?cells. Further flow-cytometry analysis revealed that compound 11c arrests PC-3?cells in G2/M phase of the cell cycle while compound 11f treatment resulted in moderate increase in the G2/M population. Additionally, the treatment by these compounds led to the impairment of mitochondrial membrane potential (DΨm) in PC-3?cells.

Full text of DOI:10.1016/j.ejmech.2016.12.043

European Journal of Medicinal Chemistry 127 (2017) 100e114
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of curcumin inspired indole
analogues as tubulin polymerization inhibitors
a
a
b
a
P.V. Sri Ramya , Srinivas Angapelly , Lalita Guntuku , Chander Singh Digwal ,
a
b,
**
, Ahmed Kamal a
, *
Bathini Nagendra Babu , V.G.M. Naidu
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500037, India
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500037, India
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 November 2016
Received in revised form
In our endeavour towards the development of potent cytotoxic agents, a series of some new curcumin
inspired indole analogues, in which indole and phenyl moieties are linked on either sides of 1,5-diaryl-
1,4-pentadien-3-one system have been synthesized and characterized by spectral data. All the newly
19 December 2016
synthesized analogues were tested for their cytotoxic potential against a panel of eight cancer cell lines
namely, lung (A549), breast (MDA-MB-231, BT549 and 4T1), prostate (PC-3, DU145), gastric (HGC-27)
and cervical (HeLa). Notably, among all the compounds tested, compounds 11c, 11d and 11f showed
Accepted 21 December 2016
Available online 23 December 2016
potent growth inhibition on PC-3 and BT549 with IC50 values in the range of 3.12e6.34
e8.72 M respectively. The most active compound (11c) was also tested on RWPE-1 (normal prostate)
cells and was found to be safe compared to the PC-3 cells. In tubulin polymerization assay, compounds
11c and 11f effectively inhibited microtubule assembly with IC50 values of 10.21 0.10 and
8.83 ± 0.06 M respectively. The results from molecular modelling studies revealed that these com-
mM and 4.69
Keywords:
Curcumin mimics
Indole
Cytotoxic
Apoptosis
m
±
m
Cell cycle
Claisen-Schmidt condensation
pounds bind at the colchicine binding site of the tubulin. Moreover, DAPI and acridine orange/ethidium
bromide staining studies indicated that compounds 11c and 11f can induce apoptosis in PC-3 cells.
Further flow-cytometry analysis revealed that compound 11c arrests PC-3 cells in G2/M phase of the cell
cycle while compound 11f treatment resulted in moderate increase in the G2/M population. Additionally,
the treatment by these compounds led to the impairment of mitochondrial membrane potential (D
in PC-3 cells.
Jm)
©
2016 Published by Elsevier Masson SAS.
1
. Introduction
metastasis through the down-regulation of androgen receptor and
epidermal growth factor receptor, but also through the induction of
cell cycle arrest [11,12]. Although, curcumin is a potent anticancer
agent with remarkable safety profile, it is highly metabolically
unstable and has poor systemic bioavailability that prevents itself
from becoming a clinical chemotherapeutic [13]. This has triggered
extensive research in search for curcumin analogues/mimics with
improved potency and pharmacokinetic profiles for the potential
clinical treatment of cancers [14e25].
Attempts have been made by researchers to chemically modify
curcumin in order to improve its activity against cancer. In this
process, a number of curcumin analogues have been synthesized
and studied for their chemopreventive effects [14e16]. Curcumin
mimic 3 (Fig. 1) differs from curcumin in having a five-carbon
Curcumin (1, Fig. 1), the active ingredient in the traditional
herbal remedy and dietary Indian spice turmeric, is originally iso-
lated from the rhizomes of Curcuma longa [1]. Curcumin, the
principal curcuminoid of turmeric and its derivatives are charac-
terized by a wide range of medicinal properties such as antimi-
crobial [2,3], anticancer [4], antioxidant [5], anti-inflammatory [6,7]
and anti-HIV [8] activities. Moreover, curcumin has been reported
to influence many cell-signaling pathways such as NF-kB, STAT,
TNF-
9,10]. Also, curcumin exhibits therapeutic promise for prostate
cancer by interfering with cell proliferation, development of
a and regulates the expression of p53 tumor suppressing gene
[
dienone linear linker instead of a seven-carbon b-diketone linker,
showed 5e5.5 fold increase in the cytotoxic potency relative to
*
Corresponding author.
*
* Corresponding author.
E-mail address: ahmedkamal@iict.res.in (A. Kamal).
curcumin towards PC-3 cell line [17]. Further, curcumin analogues
http://dx.doi.org/10.1016/j.ejmech.2016.12.043
223-5234/© 2016 Published by Elsevier Masson SAS.
0

P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
101
Fig. 1. Structure of curcumin and its analogues/mimics.
possessing imidazole [18], quinazoline [19], resveratrol [20],
piperidone [21], thiazole [22] and chromone [23] moieties have
been well documented in the literature for their enhanced potency
than curcumin. From these observations, it can be noticed that 1,5-
diheteroaryl/arylheteroaryl penta-1,4-dien-3-one, represented by
these compounds (Fig. 1, curcumin analogues 3e4 and 6e9), is an
optimal scaffold for developing curcumin mimics as potential
antitumor agents due to their superior in vitro potency and better
pharmacokinetic profiles compared to curcumin. It is therefore
very meaningful to launch in-depth investigation of this class of
curcumin mimics as potential drug candidates for the treatment of
cancer.
antimicrobial [25], anticancer [26], antiHIV [27], antioxidant [28]
and antitubercular [29]. Therapeutically interesting drug candi-
dates; for example (Fig. 2), vincristine [30], vinblastine [31], pan-
obinostat [32] (anti-tumor), vincamine (antihypertensive) [33],
pindolol (antidepressant) [34], ateviridine (antiHIV) [35] and sir-
amesine (antidepressant) [36] are known to contain indole nucleus.
In this context, we desire to explore the pharmacophoric po-
tential of curcumin inspired indole analogues (Fig. 3) and to the
best of our knowledge, these analogues have not been described in
the literature yet. Herein, we report the synthesis of a series of
novel curcumin inspired indole analogues, possessing a five-carbon
dienone linear linker attached to indole/indole derivative and
phenyl ring on either sides of it to give 1,5-arylheteroarylpenta-1,4-
dien-3-ones and evaluated for their cytotoxic activity on selected
human cancer cell lines.
On the other hand, the chemistry of indole/its derivtaives has
received substantial attention due to their synthetic and effective
biological importance. Compounds bearing this ring display a va-
riety of biological activities such as anti-inflammatory [24],
Fig. 2. Structure of some representative bioactive compounds possessing indole moiety.

102
P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
Fig. 3. Design of novel curcumin inspired indole analogues as anticancer agents.
1
13
2
. Results and discussion
similar patterns were observed in H and C NMR spectra of rest of
ꢀ
1
the compounds (11a-11f). A sharp band at ~1658.5 cm in the FT-
IR spectrum confirmed the presence of ketone functionality in
compound 9b whereas a broad band at 3123.5 cm and a sharp
band at ~1647 cm confirmed the presence of N-H and ketone
functionalities respectively in compound 11b. The HRMS (ESI) of all
the compounds showed an [MþH] peak equivalent to their mo-
2.1. Chemistry
ꢀ
1
ꢀ
1
Two core structural elements (i) an aldehyde, and (ii) a
substituted chalcone are required for achieving the target com-
pounds (1E,4E)-1-(1-benzyl-1H-indol-3-yl)-5-phenylpenta-1,4-
dien-3-one (9a-s) and (1E,4E)-1-(1H-indol-3-yl)-5-phenylpenta-
,4-dien-3-one (11a-f). Initially, for the preparation of aldehydes
þ
lecular formulae.
1
(
[
5), the appropriately substituted benzaldehydes (1) were reduced
37] to produce corresponding alcohols (2), which were then
2
2
.2. Biological evaluation
treated with phosphorous tribromide [38] to furnish the benzyl
bromide derivatives (3). Then N-benzylation of 1H-indole-3-
carboxaldehyde (4) with benzyl bromides (3) in the presence of
NaH [39] produced the target aldehydes (5). Chalcones 8a-f were
prepared by NaOH-catalysed Claisen-Schmidt condensation of
benzaldehyde (6) with acetone [40]. Similarly 1H-indole-3-
carboxaldehyde (4) was treated with acetone in the presence of
.2.1. Evaluation of antiproliferative activity
All the synthesized compounds 9a-s and 11a-f were evaluated
for their antiproliferative activity against a panel of tumor cell lines
such as lung (A549), breast (MDA-MB-231, BT549 and 4T1), pros-
tate (PC-3, DU145), gastric (HGC-27) and cervical (HeLa) using 3-
(
(
4,5-dimethylthiazol-2-yl)-2,5-diphenyl
MTT) assay [41]. Concentration response course analysis was
tetrazolium
bromide
1
5% NaOH solution to give intermediate 8g. Finally, NaOH-catalysed
performed to determine the drug concentration required to inhibit
the growth of cancer cells by 50% (IC50) after incubation for 48 h.
Curcumin and vincristine were used as reference standards and the
assay results have been tabulated in Table 1.
In order to get the insight in to the structure-activity relation-
ship (SAR), we varied the substitutions on both the heteroaryl and
aryl rings (indole and phenyl) of these conjugates. The cytotoxic
activities of compounds (9a-s and 11a-f) revealed that generally N-
benzylated indole analogues (9a-s) exhibited lesser activity
compared to N-unsubstituted indole analogues (11a-f). Among N-
benzylated indole analogues (9a-s), some of the synthesized
compounds showed different levels of anticancer activities. Ana-
logues without substitution (9a-b, 9g, 9k and 9n) or 4-chloro (9c,
Claisen-Schmidt condensation of chalcone 8 and aldehyde (5 or 10)
furnished the target compounds 9a-s and 11a-f in good yields.(see
Scheme 1)
All the synthesized compounds were purified by column chro-
matography and well characterized by spectroscopic techniques
such as IR, NMR, HRMS which were in full accordance with the
1
depicted structures. The H NMR spectrum of (1E,4E)-1-(1-benzyl-
1
H-indol-3-yl)-5-(4-methoxyphenyl)penta-1,4-dien-3-one,
9b
showed a sharp singlet of benzylic protons at
methoxy protons at
aromatic region. In the C NMR spectrum of 9b, the carbonyl car-
bon appeared at 188.9 and the aromatic carbons appeared in the
range of 141.7e110.6. Methoxy carbon and benzylic carbon
appeared at 55.4 and 50.5 respectively. Almost similar patterns
d
5.35, a singlet of
d
3.86 and rest all protons appeared in the
13
d
d
9
o) or 3-fluoro substitution on benzyl ring (9d, 9p) were found to
d
1
13
be inactive towards all the tested cell lines. Also, analogues con-
taining 4-methoxy (9e, 9f), 2,5-dimethoxy (9h, 9i), 2,4,6-
trimethoxy (9q, 9r) substitutions on the phenyl ring were inac-
tive on all the cell lines studied. The target compound 9j with 4-
fluoro substitution on benzyl ring and 3,4-dimethoxy substitution
on phenyl ring was moderately active selectively against BT549 cell
line. Further, the compound 9l having 4-fluoro substitution on
benzyl ring and 2,4,5-trimethoxy substitution on phenyl ring was
found to be active on BT549, 4T1 cell lines and moderately active on
were observed in H and C NMR spectra of all the synthesized
compounds (9a-9s) of this series. The H NMR spectrum of (1E,4E)-
1
1
-(1H-indol-3-yl)-5-(4-methoxyphenyl)penta-1,4-dien-3-one, 11b
showed a broad singlet at 10.37, which is characteristic of indole
N-H proton and all aromatic protons appeared in the range of
d
.11e6.55. In the 1 C NMR spectrum of 11b, the carbonyl carbon
appeared at 189.0, the aromatic carbons appeared in the range of
141.5e112.3 and the methoxy carbon appeared at 55.3. Almost
3
8
d
d
d

P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
103
º
º
Scheme 1. Synthesis of curcumin inspired indole analogues 9a-s and 11a-f; Reagents and conditions: (i) NaBH
NaH, dry DMF, 0 C-rt, 2e3 h, 75e90%; (iv) 15% NaOH, ethanol 0 C - rt, 2e3 h, 70e85%.
4
, methanol, 0 C-rt, 1 h, 94%; (ii) PBr
3
, ether 0 C-rt,1e2 h, 91%; (iii)
º
ꢁ
HeLa cell line. Moreover, the compound 9m containing 4-
trifluoromethyl substitution on benzyl ring and 2,4,5-trimethoxy
substitution on phenyl ring was active on both BT549 and 4T1
cell lines. Additionally, the analogue 9s with 4-trifluoromethoxy
substitution on benzyl ring was found to be inactive on all the
cell lines examined. Among N-unsubstituted indole (N-H) ana-
logues (11a-f), analogue 11a with 4-chloro substitution on phenyl
ring was selectively active on HeLa cell line and moderately active
on BT549 cell line. Analogues (11b-f) having electron donating
substitution such as methoxy showed remarkable activity against
some of the cell lines studied. Particularly, compounds 11c, 11d, 11e
and 11f displayed potent cytotoxicity on PC-3 cell lines (IC50 values
analogues (11a-f) were found to be more active than the other N-
substituted analogues (9a-s) (Fig. 4). The impact of substitution on
phenyl ring is interesting in the light of the results of the SAR study,
which demonstrates that the presence of electron donating groups
such as methoxy (11b-f) is favourable for activity, in contrast
electron withdrawing group such as 4-chloro (11a) resulted in
significant loss of activity. Moreover, analogues [11c (2,5-
dimethoxy), 11d (3,4-dimethoxy), 11e (2,4,6-trimethoxy) and 11f
(3,4,5-trimethoxy)] with increasing number of methoxy sub-
stitutions (more than one) on phenyl ring showed enhanced cyto-
toxicity. On the other hand, the presence of 4-methoxy (11b) and
2,4,6-trimethoxy substitution (11e) demonstrated increase in
selectivity towards PC-3 cell line. Based on the promising cytotoxic
activity, the most active compounds 11c-f from this series were
taken-up for further detailed studies.
Compounds 11c-f were appraised as more potent by comparing
their IC50 values with that of curcumin (Table 2). They are 2.4e5.3
times, 1.9 to 3.9 times, and 2.1 to 3.7 times, respectively, more
potent than curcumin on human breast cancer cell line (BT549) and
two human prostate cancer cell lines (PC-3 and DU145). This vali-
dates the promising scaffold containing indole and phenyl ring on
each sides of a central linear dienone linker as novel curcumin
of 6.34, 5.52, 3.12 and 3.15
standard, curcumin displayed IC50 of 12.22
compounds 11c, 11d and 11f were significantly active on HGC-27
IC50 of 5.21, 7.45 and 8.65 M respectively), HeLa (IC50 of 6.59,
M respectively) and BT549 cell lines (IC50 of 8.34,
M respectively). The compounds (11c and 11f) with
mM respectively), whereas the reference
mM. Interestingly,
(
m
6
4
.44 and 3.31
.69 and 8.72
m
m
promising cytotoxicity were also tested on normal prostate (RWPE-
1
) cells and were found to be safe compared to the PC-3 cells.
The structure-activity relationship (SAR) studies of these ana-
logues can be summed up by stating that N-unsubstituted indole

104
P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
Table 1
IC50 valuesa
(mM) of analogues 9a-s and 11a-f against selected human cancer cell lines.
Compound
A549b
MDA-MB-231c BT549c
4T1d
PC-3e
DU145e
HGC-27f
HeLag
RWPE-1h
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
j
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50 ± 0.56
>50
>50
>50
>50
50.87 ± 0.64
>50
e
40.12 ± 0.41 50.19 ± 0.46 >50
50.44 ± 0.22 >50
40.36 ± 0.52 >50
>50
>50
>50
50.12 ± 0.58 >50
>50 >50
30.12 ± 0.91 40 ± 0.87
40.65 ± 0.81 40.84 ± 0.97 >50
25.12 ± 0.75 25.17 ± 0.82 >50
25.16 ± 0.84 25.92 ± 0.72 >50
>50
>50
>50
>50
>50
50.13 ± 0.75 >50
30.12 ± 0.93 40.24 ± 0.56 >50
31.4 ± 0.29
8.34 ± 0.12
4.69 ± 0.29
10.45 ± 0.31 13.94 ± 3.35 3.12 ± 0.12
8.72 ± 3.32
24.93 ± 0.9
e
>50
>50
>50
>50
>50
>50
>50
>50
e
e
>50
>50
>50
e
e
e
e
e
50.32 ± 0.84 >50
>50
e
k
l
50.12 ± 0.94
e
50.12 ± 0.92 >50
>50
>50
>50
>50
>50
>50
>50
>50
45.12 ± 2.1
18.96 ± 0.42 6.02 ± 0.09
30.22 ± 0.97 15.72 ± 0.46
NA
40.19 ± 0.65 30.66 ± 0.64
40.12 ± 0.92 50.56 ± 0.81
>50
>50
>50
50.78 ± 0.47 >50
>50
>50
e
m
n
o
p
q
>50
>50
>50
>50
>50
>50
>50
50.24 ± 0.76
32.01 ± 2.00
e
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50.10 ± 0.35
40.91 ± 0.65
40.15 ± 0.57
e
e
e
e
r
s
>50
50.54 ± 0.27
e
e
1a
1b
1c
1d
1e
1f
50.01 ± 0.75 25.16 ± 0.65
13.62 ± 1.19 24.01 ± 0.45 NA
e
21.55 ± 1.26 3.51 ± 0.1
16.07 ± 0.21 6.34 ± 0.25
21.93 ± 0.45 5.52 ± 0.55
e
7.71 ± 0.41
13.32 ± 0.18 7.45 ± 0.49
NA
8.92 ± 0.31
5.21 ± 0.18
6.59 ± 1.03
6.44 ± 0.40
NA
3.31 ± 0.04
25.41 ± 0.27
ND
18.61 ± 0.16
e
46.44 ± 0.91
11.44 ± 0.96
15.81 ± 0.49
NA
8.65 ± 0.29
e
8.01 ± 1.65
ND
9.79 ± 0.5
ND
3.15 ± 1.91
12.22 ± 0.11 28.41 ± 0.24 ND
27.6 ± 2.4 72.3 ± 6.6 ND
6.42 ± 0.09
e
Curcumin
Vincristine sulphate (nM) 171.5 ± 20.1 116.6 ± 9.45
54.72 ± 1.18 ND
e
ND e Not determined.
a
5
0% inhibitory concentration after 48 h of drug treatment and mean ± SD of three individual experiments performed in triplicate.
b
c
Human lung cancer cell line.
Human breast cancer cell line.
Mouse breast cancer cell line.
Human prostate cancer cell line.
Human gastric cancer cell line.
Human cervical cancer cell line.
Normal human prostate epithelial cells.
d
e
f
g
h
Fig. 4. SAR analysis of curcumin inspired indole analogues.
mimics with promising cytotoxic effects against cancer cells.
.2.2. Effect of compounds 11c, 11d and 11f on tubulin
investigate whether the synthesized compounds are interacting
with microtubule system in order to elicit antiproliferative activity,
the selected compounds 11c, 11d and 11f were evaluated for their
tubulin polymerization inhibitory activity in a cell-free in vitro
assay. Initially, all the compounds were screened at 10 mM and %
inhibition by compounds 11c, 11d and 11f at this concentration was
found to be 51.7%, 39.7% and 57.3% respectively (Fig. 5).
Nocodazole and paclitaxel, were used as reference standards.
The IC50 value was calculated from the drug concentration required
for inhibiting 50% of tubulin assembly compared to control. Among
2
polymerization
Tubulin is a key component of the cytoskeletal network and is
important in a wide range of cellular functions including mitosis.
Tubulin is the target of several small molecule anti-proliferative
ligands including chalconoids, 4,5-Diaryl-3H-1,2-dithiole-3-
thiones and macrocyclic bisbibenzyl analogues which have been
reported recently as tubulin polymerization inhibitors [42e44]. To

P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
105
Table 2
deduce whether it is due to apoptosis or non-specific necrosis [45].
Acridine orange permeates through the intact cell membrane and
stains the cell in green, while ethidium bromide is taken up by a cell
only when the membrane integrity is impaired and tinge the nu-
cleus red. The compound 11c and 11f treated as well as untreated
PC-3 cells were stained with acridine orange/ethidium bromide
and observed under fluorescence microscope. It can be interpreted
from Fig. 7 that the control cells showed the normal healthy
morphology with intact nuclear architecture and appeared green in
colour whereas treated PC-3 cells have clearly demonstrated
morphological changes which are the characteristic features of
apoptotic cells such as condensed chromatin, cell membrane
blebbing, non-uniform distribution of chromatin which marginated
into a horse-shoe shaped structure and destructive fragmentation
Relative potency of curcumin mimics.
IC50 of curcumin/IC50 of compounda
MDA-MB-231c
BT549c
PC-3e
DU145e
Compound
Curcumin
1
1
1
1
1
1
1
1
1c
1d
1e
1f
2.6
1.0
0.3
1.4
2.9
5.3
2.4
2.8
1.9
2.2
3.9
3.9
3.7
2.1
NA
3.2
a
The relative potency of curcumin mimics obtained by dividing the IC50 value of
curcumin by that of each curcumin mimic.
c
Human breast cancer cell line.
Human prostate cancer cell line.
e
of the nuclei at 2.5
confirms that both of these compounds induced cell death in PC-
mM, 5 mM of compounds 11c and 11f. This
4
4
3
3
2
2
1
1
500
000
500
000
500
000
500
000
3
cells.
Ctrl
Paclitaxel
Nocodazole
2.2.3.2. DAPI staining. To clearly examine nuclear morphological
changes induced by compounds 11c and 11f on PC-3 cells, DAPI
nuclear staining was performed. Blue-fluorescent DAPI is a nuclear
stain, which can cross intact membrane of live cells and stains the
nucleus of the live cells as light blue, wherein the apoptotic cell
nuclei appears as bright blue due to chromatin condensation. Thus,
it was of our interest to examine nuclear fragmentation or chro-
matin condensation induced by compounds 11c and 11f. For this,
PC-3 cells were stained with DAPI following the treatment with
both the compound 11c, 11f and cells were observed under fluo-
rescence microscope for nuclear morphological changes. The re-
sults from Fig. 8 indicate that the nuclear structure of untreated
cells was intact while 11c and 11f treated cells showed nuclear
shrinkage, fragmented, pyknotic and horse shoe shaped nuclei
1
1c
11d
1f
5
00
0
1
-
500
0
5
10 15 20 25 30 35 40 45 50 55 60
Time (min)
Fig. 5. % Inhibition of tubulin polymerization by compound 11c, 11d and 11f at 10 mM.
(white arrow) and chromatin condensation, these are the remark-
able features of apoptosis. These findings demonstrate that these
compounds could induce apoptosis in PC-3 cells.
2
.2.3.3. Effect of compounds 11c and 11d on mitochondrial mem-
brane potential (D m). The maintenance of mitochondrial mem-
brane potential (D m) is essential for mitochondrial integrity and
J
J
cellular bioenergetic functions. During drug-induced apoptosis, a
mitochondrial change such as loss of mitochondrial membrane
potential (DJm) is the key event that usually takes place. It is re-
ported that collapse of mitochondrial membrane potential can be
correlated to the early events that appear during the apoptotic
process. Mitochondrial injury by compounds 11c and 11f was
assessed based on the loss of mitochondrial membrane potential
Fig. 6. % Dose response inhibition of tubulin polymerization by compound 11f.
(DJm) as indicated by green to red fluorescence ratio [46]. It was
determined by using spectrofluorometer with JC-1, a cell perme-
able cationic, tracer dye that emits green and red fluorescence
based on the membrane potential of mitochondria. In cells with
healthy mitochondria, JC-1 accumulates in mitochondria and forms
JC-1 oligomers that exhibit red fluorescence. However, JC-1 remains
as monomers which emit green fluorescence in the depolarized
mitochondria containing cells. Spectrofluorimetric analysis of PC-
the compounds studied, compounds 11c and 11f were found to be
more potent inhibitors of tubulin polymerization with IC50 value of
10.23 ± 0.10 and 8.81 ± 0.06 mM (Fig. 6).
2.2.3. Apoptosis induction studies
Apoptosis is a process of programmed cell death that occurs in
cells after suffering severe irrepairable damage to their key bio-
molecules and organelles. Many cytotoxic agents act by inducing
the apoptosis as their common mechanism. Therefore, it was
interesting to examine the apoptosis inducing effect of these
compounds on PC-3 cells by the DAPI staining and acridine orange/
ethidium bromide (AO/EB) staining methods.
3 cells after treatment with 5 mM of 11c and 11f disclosed that both
the compounds significantly increased green/red fluorescence ratio
from 0.103 (control) to 0.135 (11c), 0.132 (11f) (Fig. 9). These results
prove the collapse of mitochondrial membrane potential by the
compounds 11c and 11f and suggest the involvement of mito-
chondria dependent pathway in their mechanism of action.
2.2.3.1. Acridine orange and ethidium bromide staining (AO/EB).
2.2.4. Flow-cytometry analysis
The morphological changes induced by compounds 11c and 11f in
prostate cancer PC-3 cells were further studied by employing ac-
ridine orange/ethidium bromide (AO/EB) staining technique to
Cytotoxic agents usually alter the regulation of cell cycle
resulting in the arrest of cell division in various phases, thereby
prevents the growth and proliferation of cancer cells. The screening

106
P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
Fig. 7. Morphological changes in PC-3 cells treated with and without compound 11c and 11f for 48 h. Green live cells show normal morphology of control and early signs of
apoptosis such as extremely condensed chromatin, horse-shoe shaped nuclei and destructive fragmentation of the nuclei were clearly observed in 11c and 11f treated cells at
indicated concentrations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 8. Compounds 11c and 11f induced nuclear morphological changes in PC-3 cells after treatment for 48h.
results revealed that 11c could exhibit significant antiproliferative
activity on PC-3 cells. To understand whether this inhibition of
growth of PC-3 cells is on account of cell cycle arrest, cell cycle
distribution of PC-3 cells was determined by flow-cytometry
Dickinson, US). The results from Fig. 10 demonstrate that the con-
trol cells showed 12.2% cells in G2/M phase, while the treatment
with 11c resulted in the increase of G2/M population to 57.5% (at
2.5 mM) in 24 h. Treatment with 11f at 2.5, 5 and 10 mM displayed
analysis [47]. PC-3 cells were treated with 11c at 2.5, 5
1f at 2.5, 5, 10 M and stained with propidium iodide, which was
analyzed further by using flow cytometer (FACS verse, Becton
m
M and
rise in G2/M population from 24.74% (control) to 27.04%, 29.87% and
31.42% respectively. These findings clearly indicate that the treat-
ment of PC-3 cells with 11c results in G2/M cell cycle arrest.
1
m

P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
107
similar position with respect to 2-methoxycyclohepta-2,4,6-trien-
-one ring of colchicine and the carbonyl group in 11c superim-
poses with the amide carbonyl of the colchicine as seen in Fig. 12.
1
3. Conclusion
In summary, a series of curcumin inspired indole analogues
were synthesized, and evaluated for their anticancer potential. An
initial screening was performed against lung (A549), breast (MDA-
MB-231, BT549 and 4T1), prostate (PC-3, DU145), gastric (HGC-27)
and cervical (HeLa) cancer cell lines by using MTT assay. Interest-
ingly, compounds 11c (1E, 4E)-1-(2,5-dimethoxyphenyl)-5-(1H-
indol-3-yl)penta-1,4-dien-3-one and 11f (1E, 4E)-1-(3,4,5-
trimethoxyphenyl)-5-(1H-indol-3-yl)penta-1,4-dien-3-one
Fig. 9. Effect of compounds 11c and 11f on mitochondrial membrane potential (DJm).
The compound treated PC-3 cells were stained with JC-1 and the intensity of red and
green fluorescence in each sample was analyzed by spectroflourometer. Data were
mean ± SEM of three independent experiments.*P < 0.05 when compared to control
showed the most potent antiproliferative activity on PC-3 cell lines
(IC50 of 6.34 ± 0.25 and 3.15 ± 1.91 mM respectively). Most impor-
(
One way ANNOVA followed by Dunnet's multiple comparison test).
tantly, these compounds were found to be safe towards normal
prostate epithelial cells (RWPE-1) compared to PC-3 cell line. Both
1
1c and 11f effectively inhibited the polymerization of tubulin with
However, the % inhibition of cell cycle at G2/M phase is less for the
compound 11f when compared to 11c.
IC50 values 10.21 ± 0.10 and 8.83 ± 0.06 M respectively. Molecular
m
modelling studies suggest that 11c and 11f bind at the colchicine
binding site of the tubulin. The detailed studies like AO/EB staining
and DAPI nuclear staining suggest that the cell proliferation is
inhibited through induction of apoptosis. In addition, the treatment
with 11c and 11f results in the collapse of mitochondrial membrane
potential in PC-3 cells. The flow-cytometry analysis confirmed the
arrest of PC-3 cells in the G2/M phase of cell cycle by 11c whereas
11f showed modertae increase in the G2/M population. Overall, the
current studies demonstrate that curcumin inspired indole ana-
logues like 11c and 11f have the potential to be developed as leads
and their further structural modifications may produce promising
new cancer therapeutics.
2
.3. Molecular modelling studies
Molecular modelling studies were performed extensively on
tubulin (PDB code: 3E22) [48] to gain better understanding on the
potency of the studied compounds (11c and 11f) and to support the
discussion of the biological results obtained in the previous sec-
tions. As the active site, at which colchicine binds, is located at the
interface of a,b-tubulin subunits, both the a and b chains have been
considered for molecular modelling studies [49]. Fig. 11 shows the
binding of 11c and 11f at the interface (colchicine binding site of
tubulin) of a,b-tubulin subunits. The significant hydrogen bonding
interactions are shown as yellow dotted lines and the amino acid
residues that interact with ligands are highlighted as shown in
Fig. 11.
4. Experimental section
4.1. General
The promising compounds (11c and 11f) were docked in a
similar fashion at the active site and sandwiched between the
All solvents were purified and dried using standard methods
prior to use. Commercially available reagents were used without
further purification. The reactions were monitored by thin layer
chromatography (TLC), using MERCK pre-coated silica gel 60-F254
aluminum plates. Visualization of spots on TLC plates was done by
UV light. Column chromatography with 60e120 mesh silica gel was
used as separation and purification method. Ethyl acetate and
hexane were used as eluent. Melting points were obtained on
Stuart digital melting-point apparatus/SMP 30 and were uncor-
rected. All IR spectra were recorded on a Perkin Elmer, FT-IR
amino acid residues like
a
Asn101, Ala180,
Tyr224 of -chain and Leu255,
Val315, Thr353 and Ala354 of b-chain of
a
Ser178,
a
Thr179,
a
a
b
Val181,
Asn258,
a
b
Arg221,
Met259,
a
b
a
b
Lys254, b
b
b
tubulin. Compound 11c showed significant hydrogen bond inter-
action between one of the hydrogens of CONH group of Asn101
2
a
and carbonyl oxygen of penta-1,4-dien-3-one linear linker (H ….O)
at a distance of 2.38 Å. In addition, one more hydrogen bond was
observed between carbonyl oxygen of bThr353 and NH hydrogen of
indole ring (O ….H) at a distance of 2.13 Å. Moreover, some of the
1
hydrophobic interactions were also observed between the ligand
spectrometer using KBr discs. H NMR spectra were recorded on
13
and
a
Val181,
bLeu255, and
b
Lys254. Compound 11f was also
an Avance NMR instrument operated at 500 MHz. C NMR spectra
were recorded on an Avance NMR instrument operated at 125 MHz.
Chemical shift values were reported in ppm with TMS as an internal
reference and J values were given in Hertz. The following abbre-
docked in a similar fashion at the interface of two chains and
showed a strong hydrogen bond interaction between carbonyl
oxygen of COOH group of
a
Glu71 and NH hydrogen of indole ring (O
1
….H) at a distance of 1.92 Å while the other surrounded amino acid
viations were used for H NMR spectra to indicate the signal mul-
residues such as
b
Leu248,
b
Asn258,
b
Leu255 and
b
Ala250 experi-
tiplicity: s (singlet), d (doublet), t (triplet), q (quartet) and m
(multiplet). HRMS were determined with Agilent QTOF mass
spectrometer 6540 series instrument and were performed in the
ESI techniques at 70 eV.
enced hydrophobic contacts. However the indole ring and carbonyl
oxygen of linear linker played a significant role in showing the
hydrogen bond interactions with the amino acid residues, whereas
the phenyl rings of both the conjugates were burried at the hy-
drophobic region. The linear linker also played a bridging role be-
tween the phenyl and indole rings, that leads to a successful
docking at the target binding site (Fig. 12) suggesting that 11f and
4.2. Synthetic procedures and spectral data
4.2.1. General synthetic procedure for the synthesis of 2
11c are potent inhibitors of tubulin. To gain further insight of our
To a magnetically stirred solution of an appropriately
findings, we demonstrated the superimposition of colchicine with
the ligands and the corresponding superimposition poses suggest
that methoxy phenyl ring of ligands under investigation occupied
substituted benzaldehyde 1 (1 mmol) in methanol (5 mL) was
added sodium borohydride (2 mmol) slowly over 10 min at 0 C.
The resulting solution was stirred at room temperature for the
ꢁ

108
P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
Fig. 10. Effect of compounds 11c and 11f on cell cycle progression of PC-3 cells. Cells were treated with compounds 11c and 11f for 24 h followed by analysis of cell cycle distribution
using propidium iodide cell staining method. All assays were done in triplicate.

P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
109
Fig. 11. Poses represent the binding of compounds 11c and 11f at the interface of a,b-tubulin subunits (colchicine binding site of tubulin).
Fig. 12. (a), (b) Docking poses of ligands 11c, 11f respectively and the interacting amino acids at the colchicine binding domain of tubulin. (c), (d) Poses represent the superim-
position of potential ligands (11c and 11f) and colchicine at the colchicine binding domain: The potential ligands were shown as ball and stick model, while the interacting amino
acids were denoted as thin tubes and the colchicine was shown as green ball and stick model in the black background. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
required time. On completion, the reaction mixture was quenched
with 3% aqueous HCl solution, and evaporated to dryness. The
residue was extracted with CH Cl , dried over Na SO and
2 2 2 4
the reaction mixture was stirred for 0.5 h. After completion of the
reaction (TLC monitoring), the reaction mixture was carefully
poured over ice, and the aqueous phase was extracted with ether.
concentrated under vaccum to obtain corresponding alcohol 2
which was used without further purification.
The combined organic layers were washed with NaHCO
3
and water,
followed by brine. Drying over Na SO and evaporation of the
2
4
solvents under reduced pressure affords the desired benzyl bro-
mide 3 which was used without further purification.
4.2.2. General synthetic procedure for the synthesis of 3
To a stirred solution of alcohol 2 (1 mmol) in dry ether (10 mL)
ꢁ
was added phosphorus tribromide (0.5 mL) dropwise at 0 C and

110
P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
4
.2.3. General procedure for the synthesis of 5a-f
123.4,121.8,121.2,120.9,113.3,110.7, 50.6; HRMS (ESI): m/z calcd for
þ
To a suspension of NaH 60% in oil (2.25 mmol) in dry DMF
C
26
H21ClNO 398.1306, found 398.1309 [MþH] .
ꢁ
(
0.8 mL/mmol) was added, at 0 C and under nitrogen atmosphere,
a solution of indole-3-carboxaldehyde 4 (1 mmol) in dry DMF
2.5 mL/mmol). After stirring for 30 min at room temperature,
benzyl bromide 3 (1.2 mmol) was slowly added. On completion, the
reaction was quenched by addition of water and the product was
extracted with diethyl ether. The organic layer was dried over
Na SO , filtered off and concentrated under vacuum. The crude
2 4
residue was purified by column chromatography (Silica gel, 60e120
mesh, 9:1 hexane/ethyl acetate) to obtain the desired aldehyde 5a-
f.
4.2.8.2. (1E,4E)-1-(1-benzyl-1H-indol-3-yl)-5-(4-methoxyphenyl)
(
penta-1,4-dien-3-one (9b). Yellow solid, yield 78.4%; mp:
ꢁ
ꢀ1
140e142 C; IR (KBr, cm ): 3109.2, 2988.5, 1660.5, 1612.4, 1390.0,
1
3
980.3; H NMR (500 MHz, CDCl ): d 8.09e7.95 (m, 2H), 7.71 (d,
J ¼ 15.8 Hz, 1H), 7.59 (d, J ¼ 8.7 Hz, 2H), 7.51 (s, 1H), 7.41e7.27 (m,
6H), 7.16 (d, J ¼ 6.8 Hz, 2H), 7.09 (d, J ¼ 15.8 Hz, 1H), 6.99 (d,
1
3
J ¼ 15.8 Hz, 1H), 6.94 (d, J ¼ 8.7 Hz, 2H), 5.35 (s, 2H), 3.86 (s, 3H);
C
NMR (125 MHz, CDCl
3
): d 188.9, 161.3, 141.7, 137.8, 136.6, 136.0,
133.3, 129.9, 129.0, 128.1, 127.9, 127.0, 126.4, 124.0, 123.2, 121.6,
1
C
21.5, 120.9, 114.3, 113.4, 110.6, 55.4, 50.5; HRMS (ESI): m/z calcd for
þ
4.2.4. General procedure for the synthesis of 8a-f
27
H
24NO
2
394.1802, found 394.1770 [MþH] .
To a stirred solution of benzaldehyde 6 (1 mmol) in ethanol
(
3 mL) was added 0.5 mL of acetone and 15% aqueous NaOH (1 mL)
4.2.8.3. (1E,4E)-1-(1-(4-chlorobenzyl)-1H-indol-3-yl)-5-(4-
ꢁ
solution at 0 C. The reaction was allowed to stir at room temper-
ature till it was completed. The reaction mixture was evaporated to
dryness, extracted twice with ethyl acetate, the combined organic
methoxyphenyl)penta-1,4-dien-3-one (9c). Yellow solid, yield 74.8%;
ꢁ
ꢀ1
mp: 134e136 C; IR (KBr, cm ): 3097.4, 2959.4, 1656.3, 1621.1,
1
1395.0, 1170.2, 974.9, 836.1; H NMR (500 MHz, CDCl
3
): d 8.15e7.93
2 4
layers were dried over anhydrous Na SO and concentrated under
(m, 2H), 7.74 (d, J ¼ 15.7 Hz, 1H), 7.66e7.56 (m, 2H), 7.51 (s, 1H),
13
reduced pressure. The crude product was purified by column
chromatography (Silica gel, 60e120 mesh, 9:1 hexane/ethyl ace-
tate) to obtain the desired chalcone 8a-f.
7.38e7.28 (m, 4H), 7.20e6.87 (m, 7H), 5.33 (s, 2H), 3.87 (s, 3H);
NMR (125 MHz, CDCl ): 188.9, 161.3, 141.9, 137.6, 136.4, 134.6,
134.0, 133.1, 133.0, 129.2, 128.2, 127.9, 126.4, 124.0, 123.4, 121.8,
C
3
d
1
C
21.7, 120.9, 114.3, 113.6, 110.5, 55.4, 49.9; HRMS (ESI): m/z calcd for
þ
4.2.5. Procedure for the synthesis of 8g
27
H
23ClNO
2
428.1412, found 428.1413 [MþH] .
Similarly, to a stirred solution of indole-3-carboxaldehyde 4
(
1 mmol) in ethanol (5 mL) was added 0.5 mL of acetone and 15%
4.2.8.4. (1E,4E)-1-(1-(3-fluorobenzyl)-1H-indol-3-yl)-5-(4-
methoxyphenyl)penta-1,4-dien-3-one (9d). Yellow solid, yield
78.1%; mp: 164e166 C; IR (KBr, cm ): 3093.6, 3002.3, 1657.3,
ꢁ
aqueous NaOH (3 mL) solution at 0 C. The reaction was allowed to
stir at room temperature till it was completed. The reaction mixture
was evaporated to dryness and similar work up procedure as
described for chalcones 8a-f was followed to extract the compound
from reaction mixture. Further, it was purified by column chro-
matography (Silica gel, 60e120 mesh, 9:1 hexane/ethyl acetate) to
obtain pure chalcone 8g.
ꢁ
ꢀ1
1
1620.9, 1394.4, 1253.5, 1107.5, 966.2; H NMR (500 MHz, CDCl
8.12e7.99 (m, 2H), 7.74 (d, J ¼ 15.8, 1H), 7.61 (d, J ¼ 8.5 Hz, 2H),
7.53 (s, 1H), 7.37e7.29 (m, 4H), 7.13 (d, J ¼ 15.8 Hz, 1H), 7.07e6.92
3
):
d
13
(m, 5H), 6.86 (d, J ¼ 9.3 Hz, 1H), 5.35 (s, 2H), 3.87 (s, 3H); C NMR
(125 MHz, CDCl ): 188.8, 164.1, 162.2, 161.2, 141.9, 138.7, 137.6,
36.4, 133.1, 130.6, 130.0, 127.9, 126.4, 124.0, 123.4, 122.4, 121.8,
3
d
1
4
.2.6. General procedure for the synthesis of 9a-s
To a stirred solution of chalcone 8a-f (0.5 mmol) in ethanol
121.7, 120.9, 115.1, 114.3, 113.8, 110.4, 55.4, 50.0; HRMS (ESI): m/z
þ
calcd for C27
H
23FNO
2
412.1707, found 412.1710 [MþH] .
(
3 mL) was added 15% aqueous NaOH (1 mL) solution and aldehyde
ꢁ
5
(0.5 mmol) at 0 C. The resulting solution was stirred at room
4.2.8.5. (1E,4E)-1-(1-(4-fluorobenzyl)-1H-indol-3-yl)-5-(4-
methoxyphenyl)penta-1,4-dien-3-one (9e). Yellow solid, yield
temperature till the completion of the reaction. The reaction
mixture was evaporated to dryness, extracted twice with ethyl
acetate, the combined organic layers were dried over anhydrous
ꢁ
ꢀ1
1
76.3%; mp: 153e154 C; IR (KBr, cm ): 3096.5, 3003.0, 1656.6,
1617.9, 1381.2, 1169.8, 1102.3, 971.9; H NMR (500 MHz, CDCl
8.07e7.95 (m, 2H), 7.72 (d, J ¼ 15.8 Hz, 1H), 7.59 (d, J ¼ 8.6 Hz, 2H),
7.49 (s, 1H), 7.35e7.27 (m, 3H), 7.17e6.90 (m, 8H), 5.31 (s, 2H), 3.86
3
):
Na
2
SO
4
and concentrated under reduced pressure. The crude mass
d
was purified by column chromatography (Silica gel, 60e120 mesh,
8
13
:2 hexane/ethyl acetate) to give the pure product 9a-s.
(s, 3H); C NMR (125 MHz, CDCl
3
): d 184.1, 158.7, 156.6, 137.1, 132.9,
131.7, 128.3, 127.0, 125.2, 124.0, 123.1, 121.7, 119.2, 118.6, 116.9, 116.2,
4
.2.7. General procedure for the synthesis of 11a-f
To a stirred solution of chalcone 8g (0.5 mmol) in ethanol (5 mL)
111.3, 111.1, 109.6, 108.8, 106.7, 50.0, 45.1; HRMS (ESI): m/z calcd for
þ
C
27
H
23FNO
2
412.1707, found 412.1709 [MþH] .
was added 15% aqueous NaOH (2 mL) solution and aldehyde 10
ꢁ
(
0.5 mmol) at 0 C. The resulting solution was stirred at room
4.2.8.6. (1E,4E)-1-(4-methoxyphenyl)-5-(1-(4-(trifluoromethyl)
benzyl)-1H-indol-3-yl)penta-1,4-dien-3-one (9f). Yellow solid, yield
temperature till the completion of the reaction. The reaction
mixture was evaporated to dryness, extracted twice with ethyl
acetate, the combined organic layers were dried over anhydrous
ꢁ
ꢀ1
1
74.2%; mp: 148e150 C; IR (KBr, cm ): 3097.1, 2969.8, 1644.9,
3
1597.9, 1323.6, 1168.9, 1109.2, 976.4; H NMR (500 MHz, CDCl ):
Na
2
SO
4
and concentrated under reduced pressure. The crude mass
d
8.07e7.97 (m, 2H), 7.72 (d, 1H, J ¼ 15.8), 7.62e7.55 (m, 4H), 7.52 (s,
was purified by column chromatography (Silica gel, 60e120 mesh,
1H), 7.34e7.26 (m, 3H), 7.25e7.21 (m, 2H), 7.12 (d, J ¼ 15.8 Hz, 1H),
6.99 (d, J ¼ 15.8 Hz, 1H), 6.94 (d, J ¼ 8.6 Hz, 2H), 5.41 (s, 2H), 3.85 (s,
3
:2 hexane/ethyl acetate) to give the pure product 11a-f.
13
3
3
H); C NMR (125 MHz, CDCl ): d 188.9, 161.4, 142.0, 140.2, 137.6,
4
4
.2.8. Spectral data
.2.8.1. (1E,4E)-1-(1-benzyl-1H-indol-3-yl)-5-(4-chlorophenyl)
136.3, 133.0, 130.0, 127.8, 127.0, 126.5, 126.0, 124.0, 123.5, 122.0,
121.8, 121.0, 114.4, 113.8, 110.3, 55.4, 50.0; HRMS (ESI): m/z calcd for
þ
penta-1,4-dien-3-one (9a). Yellow solid, yield 75.3%; mp:
C
28
H F
23 3
NO
2
462.1675, found 462.1673 [MþH] .
ꢁ
ꢀ1
136e138 C; IR (KBr, cm ): 3092.9, 2922.0, 1658.8, 1619.4, 1385.0,
1
974.8; H NMR (500 MHz, CDCl
3
):
d
8.11e7.97 (m,1H), 7.76e7.66 (m,
4 . 2 . 8 . 7 . ( 1 E , 4 E ) - 1 - ( 1 - b e n z yl - 1 H - i n d o l - 3 - yl ) - 5 - ( 2 , 5 -
1
H), 7.59e7.50 (m, 2H), 7.45e7.29 (m, 6H), 7.22e6.78 (m, 6H), 5.36
dimethoxyphenyl)penta-1,4-dien-3-one (9g). Yellow solid, yield
13
ꢁ
ꢀ1
81.2%; mp: 154e156 C; IR (KBr, cm ): 3094.2, 2988.7, 1662.0,
(
s, 2H); C NMR (125 MHz, CDCl
3
):
d
188.6,142.1,140.4,137.8, 136.5,
1
135.9, 133.8, 133.2, 129.5, 129.3, 129.1, 128.2, 127.0, 126.5, 125.7,
3
1612.1, 1383.0, 1100.2, 984.2; H NMR (500 MHz, CDCl ): d 8.12e7.98

P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
111
(
6
m, 3H), 7.54 (s, 1H), 7.38e7.29 (m, 6H), 7.20e7.11 (m, 5H),
4.2.8.13. (1E,4E)-1-(1-(4-(trifluoromethyl)benzyl)-1H-indol-3-yl)-5-
(2,4,5-trimethoxyphenyl)penta-1,4-dien-3-one (9m). Yellow solid,
yield 79.5%; mp: 214e216 C IR (KBr, cm ): 3109.2, 2988.5, 1660.5,
13
.97e6.92 (m, 2H), 5.37 (s, 2H), 3.90 (s, 3H), 3.85 (s, 3H); C NMR
): 189.3, 153.5, 153.0, 137.7, 137.0, 136.7, 136.0,
33.2,128.9,128.1,127.1,126.9,126.4,124.7,123.2,121.5,121.3,120.8,
ꢁ
ꢀ1
(
125 MHz, CDCl
3
d
1
1
1
1604.5, 1325.8, 1285.5, 1087.0, 980.3; H NMR (500 MHz, CDCl
8.11e7.98 (m, 3H), 7.63e7.50 (m, 3H), 7.36e7.27 (m, 4H), 7.24e7.12
(m, 3H), 7.00 (d, J ¼ 16 Hz, 1H), 6.53 (s, 1H), 5.42 (s, 2H), 3.95 (s, 3H),
3
):
16.9, 113.3, 113.0, 112.4, 110.5, 56.1, 55.8, 50.5; HRMS (ESI): m/z
d
þ
calcd for C28
H26NO
3
424.1907, found 424.1928 [MþH] .
13
3
.92 (s, 3H), 3.91 (s, 3H); C NMR (125 MHz, CDCl
52.1,143.3,140.2,137.5,137.1,135.8,132.7, 130.4,130.2,126.9,126.5,
25.9, 124.6, 123.4, 121.7, 121.0, 115.6, 113.8, 110.7, 110.3, 96.8, 56.4,
3
): d 189.2, 154.2,
1
1
4
.2.8.8. (1E,4E)-1-(2,5-dimethoxyphenyl)-5-(1-(4-fluorobenzyl)-1H-
indol-3-yl)penta-1,4-dien-3-one (9h). Yellow solid, yield 78.6%; mp:
1
ꢁ
ꢀ1
56.0, 49.9; HRMS (ESI): m/z calcd for C30H27F NO 522.1887, found
52e154 C; IR (KBr, cm ): 3095.8, 2956.8, 1662.6, 1644.6, 1347.8,
3
4
þ
1
522.1906 [MþH] .
1
3
218.0, 1107.7, 984.4; H NMR (500 MHz, CDCl
H), 7.49 (s, 1H), 7.33e7.30 (m, 3H), 7.19e7.08 (m, 5H), 7.05e6.99
m, 2H), 6.94e6.85 (m, 2H), 5.31 (s, 2H), 3.88 (s, 3H), 3.82 (s, 3H);
3
): d 8.07e7.96 (m,
4
.2.8.14. (1E,4E)-1-(1-benzyl-1H-indol-3-yl)-5-(2,4,6-
(
1
3
trimethoxyphenyl)penta-1,4-dien-3-one (9n). Yellow solid, yield
3
C NMR (125 MHz, CDCl ): d 189.3, 163.4, 161.4, 153.5, 153.0, 137.6,
ꢁ
ꢀ1
7
1
9.3%; mp: 151e153 C; IR (KBr, cm ): 3091.3, 2934.6, 2836.6,
1
1
37.1, 136.5, 132.9, 131.8, 128.6, 127.1, 126.5, 124.7, 123.3, 121.5, 120.9,
1
651.2, 1595.1, 1340.7, 1285.1, 1098.7, 972.5; H NMR (500 MHz,
17.0, 115.9, 113.5, 113.0, 112.4, 110.4, 56.1, 55.8, 49.8; HRMS (ESI): m/
þ
CDCl ):
d
8.19 (d, J ¼ 16.1 Hz, 1H), 8.07e8.01 (m, 1H), 7.98 (d,
z calcd for C28
H25FNO
3
442.1813, found 442.1804 [MþH] .
3
J ¼ 11.7 Hz, 1H), 7.48 (s, 1H), 7.42 (d, J ¼ 16.1 Hz, 1H), 7.37e7.26 (m,
6
3
H), 7.20e7.09 (m, 3H), 6.14 (s, 2H), 5.33 (s, 2H), 3.91 (s, 6H), 3.86 (s,
H); C NMR (125 MHz, CDCl ): d 190.7, 162.7, 161.4, 137.6, 136.2,
3
4
.2.8.9. (1E,4E)-1-(2,5-dimethoxyphenyl)-5-(1-(4-(trifluoromethyl)
13
benzyl)-1H-indol-3-yl)penta-1,4-dien-3-one (9i). Yellow solid, yield
7
1
d
ꢁ
ꢀ1
1
135.3, 133.4, 132.6, 128.9, 128.0, 126.9, 126.6, 126.5, 123.0, 122.2,
5.4%; mp: 181e183 C; IR (KBr, cm ): 3097.1, 2988.6, 1662.9,
121.3, 120.8,113.5,110.4,106.6, 90.5, 55.7, 55.3, 50.4; HRMS (ESI): m/
610.1, 1325.0, 1177.2, 1106.6, 984.3; H NMR (500 MHz, CDCl
3
):
þ
z calcd for C29
H
28NO
4
454.2013, found 454.2009 [MþH] .
8.132e7.95 (m, 3H), 7.58 (d, J ¼ 7.9 Hz, 2H), 7.52 (s, 1H), 7.48e7.27
(
m, 3H), 7.25e7.08 (m, 5H), 6.96e6.85 (m, 2H), 5.41 (s, 2H), 3.88 (s,
H), 3.82 (s, 3H); 1 C NMR (125 MHz, CDCl
40.2,137.5, 137.2,136.3,132.9,130.4,127.1,127.0,126.5,126.0,124.7,
23.5, 121.7, 121.0, 117.0, 113.8, 113.1, 112.5, 110.4, 56.1, 55.8, 50.0;
3
4.2.8.15. (1E,4E)-1-(1-(4-chlorobenzyl)-1H-indol-3-yl)-5-(2,4,6-
3
1
1
3
): d 189.3, 153.5, 153.1,
trimethoxyphenyl)penta-1,4-dien-3-one (9o). Yellow solid, yield
ꢁ
ꢀ1
3.4%; mp: 143e145 C; IR (KBr, cm ): 3096.0, 2971.2, 1651.8,
8
1
1596.3, 1324.1, 1213.0, 1157.7, 991.4; H NMR (500 MHz, CDCl
3
):
HRMS (ESI): m/z calcd for C29
H
25
F NO
3 3
492.1781, found 492.1815
þ
d
8.35e7.88 (m, 3H), 7.62e7.28 (m, 6H), 7.25e6.88 (m, 4H), 6.16 (s,
[MþH] .
13
2
CDCl
1
5
H), 5.32 (s, 2H), 3.93 (s, 6H), 3.88 (s, 3H); C NMR (125 MHz,
): 190.7, 162.8, 161.5, 137.5, 135.1, 134.8, 133.9, 133.6, 132.4,
29.1, 128.2, 126.6, 123.2, 122.5, 121.4, 120.9, 113.8, 110.3, 106.5, 90.5,
3
d
4
1
.2.8.10. (1E,4E)-1-(3,4-dimethoxyphenyl)-5-(1-(4-fluorobenzyl)-
H-indol-3-yl)penta-1,4-dien-3-one (9j). Yellow solid, yield 73.5%;
5.8, 55.4, 49.8; HRMS (ESI): m/z calcd for C29
H
27ClNO
4
488.1623,
ꢁ
ꢀ1
mp: 177e178 C; IR (KBr, cm ): 3095.2, 2989.1, 1657.6, 1616.9,
1
d
þ
found 488.1629 [MþH] .
1
380.0, 1253.0, 1133.7, 972.1;
8.09e7.96 (m, 2H), 7.71 (d, J ¼ 15.8 Hz, 1H), 7.50 (s, 1H), 7.36e7.28
m, 3H), 7.25e6.93 (m, 8H), 6.91 (d, J ¼ 8.3 Hz, 1H), 5.32 (s, 2H), 3.97
s, 3H), 3.94 (s, 3H); 13C NMR (125 MHz, CDCl
): 188.7, 163.4, 161.4,
51.0, 149.2, 142.0, 137.6, 136.5, 133.1, 131.7, 128.6, 128.1, 126.4, 124.3,
23.3, 122.9, 121.5, 120.9, 115.9, 113.5, 111.0, 110.4, 109.8, 55.9, 49.8;
3
H NMR (500 MHz, CDCl ):
4
.2.8.16. (1E,4E)-1-(1-(3-fluorobenzyl)-1H-indol-3-yl)-5-(2,4,6-
(
(
1
trimethoxyphenyl)penta-1,4-dien-3-one (9p). Yellow solid, yield
3
d
ꢁ
ꢀ1
1
8
1
1.0%; mp: 131e132 C; IR (KBr, cm ): 3093.8, 2988.4, 1649.6,
601.4, 1341.6, 1260.5, 1176.6, 975.0; H NMR (500 MHz, CDCl
3
):
1
d
8.43e8.13 (m, 3H), 7.66e7.27 (m, 6H), 7.21e6.78 (m, 4H), 6.16 (s,
H), 5.34 (s, 2H), 3.93 (s, 6H), 3.87 (s, 3H); C NMR (125 MHz,
HRMS (ESI): m/z calcd for C28
H25FNO
3
442.1813, found 442.1804
13
2
þ
[MþH] .
3
CDCl ): d 190.7, 164.1, 162.8, 162.1, 161.5, 138.9, 137.5, 135.6, 135.2,
1
33.6, 132.9, 132.5, 130.6, 126.5, 123.3, 123.2, 122.5, 122.3, 121.6,
4
.2.8.11. (1E,4E)-1-(1-benzyl-1H-indol-3-yl)-5-(2,4,5-
121.4, 120.9, 115.0, 113.9, 110.3, 106.5, 90.5, 55.8, 55.4, 49.8; HRMS
trimethoxyphenyl)penta-1,4-dien-3-one (9k). Yellow solid, yield
8
þ
(ESI): m/z calcd for C29
H27FNO
4
472.1919, found 472.1919 [MþH] .
ꢁ
ꢀ1
2.1%; mp: 174e176 C; IR (KBr, cm ): 3096.9, 2962.8, 1661.5,
1
1602.3, 1389.1, 1099.4, 985.2;
3
H NMR (500 MHz, CDCl ):
4
.2.8.17. (1E,4E)-1-(1-(4-fluorobenzyl)-1H-indol-3-yl)-5-(2,4,6-
d
8.11e7.94 (m, 3H), 7.50 (s, 1H), 7.38e7.26 (m, 6H), 7.20e7.11 (m,
H), 6.91 (d, J ¼ 16 Hz, 1H), 6.53 (s, 1H), 5.34 (s, 2H), 3.94 (s, 3H),
trimethoxyphenyl)penta-1,4-dien-3-one (9q). Yellow solid, yield
4
3
ꢁ
ꢀ1
82.1%; mp: 152e154 C; IR (KBr, cm ): 2972.2, 2901.7, 1659.9,
.91 (s, 6H); 13C NMR (125 MHz, CDCl
):
d
189.3, 154.2, 152.1, 143.3,
1
3
1594.0, 1391.9, 1336.9, 977.9;
3
H NMR (500 MHz, CDCl ):
137.7,136.9,136.2,136.1,133.1,128.9,128.0,127.0,126.5,124.7,123.2,
d
8.38e7.81 (m, 3H), 7.72e7.27 (m, 5H), 7.25e6.75 (m, 5H), 6.14 (s,
121.4, 120.8, 115.7, 113.4, 110.8, 110.5, 96.9, 56.4, 56.0, 50.5; HRMS
13
2H), 5.30 (s, 2H), 3.91 (s, 6H), 3.86 (s, 3H); C NMR (125 MHz,
þ
(
ESI): m/z calcd for C29
H28NO
4
454.2013, found 454.2057 [MþH] .
CDCl
28.6, 126.5, 123.1, 122.4, 121.3, 120.9, 115.8, 113.6, 110.3, 106.5, 90.5,
3
): d 190.7, 163.3, 162.7, 161.4, 137.5, 135.2, 133.5, 132.4, 131.9,
1
4
.2.8.12. (1E,4E)-1-(1-(4-fluorobenzyl)-1H-indol-3-yl)-5-(2,4,5-
55.7, 55.3, 49.7; HRMS (ESI): m/z calcd for C29
found 472.1898 [MþH] .
H
27FNO
4
472.1919,
þ
trimethoxyphenyl)penta-1,4-dien-3-one (9l). Yellow solid, yield
8
ꢁ
ꢀ1
1
4.3%; mp: 170e172 C; IR (KBr, cm ): 3097.1, 2988.6, 1658.8,
1600.5, 1387.7, 1209.3, 1092.3, 982.4; H NMR (500 MHz, CDCl
3
):
4.2.8.18. (1E,4E)-1-(1-(4-(trifluoromethyl)benzyl)-1H-indol-3-yl)-5-
(2,4,6-trimethoxyphenyl)penta-1,4-dien-3-one (9r). Yellow solid,
d
8.13e7.91 (m, 3H), 7.48 (s, 1H), 7.32e7.24 (m, 3H), 7.20e6.90 (m,
13
ꢁ
ꢀ1
yield 78.6%; mp: 203e205 C; IR (KBr, cm ): 3105.0, 2970.7,
7
H), 6.52 (s, 1H), 5.30 (s, 2H), 3.94 (s, 3H), 3.90 (s, 6H); C NMR
): 189.2, 163.3, 161.4, 154.1, 152.1, 143.3, 137.5,
37.0, 136.0, 132.8, 131.8, 128.6, 126.4, 124.6, 123.2, 121.5, 120.9,
1
(
125 MHz, CDCl
3
d
3
1633.8, 1601.9, 1321.6, 1292.2, 992.7; H NMR (500 MHz, CDCl ):
1
d
8.19 (d, J ¼ 16.1 Hz, 1H), 8.05 (d, J ¼ 7.4 Hz, 1H), 7.97 (d, J ¼ 15.8 Hz,
116.0, 115.8, 115.6, 113.5, 110.7, 110.4, 96.9, 56.5, 56.4, 56.0, 49.7;
1H), 7.58 (d, J ¼ 7.8 Hz, 2H), 7.50 (s, 1H), 7.42 (d, J ¼ 16.1 Hz, 1H),
7.35e7.27 (m, 2H), 7.25e7.19 (m, 3H), 7.16 (d, J ¼ 15.8 Hz, 1H), 6.14
HRMS (ESI): m/z calcd for C29
H27FNO
4
472.1919, found 472.1957
þ
13
[MþH] .
(s, 2H), 5.40 (s, 2H), 3.91 (s, 6H), 3.86 (s, 3H); C NMR (125 MHz,

112
P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
CDCl
3
):
d
190.6, 162.8, 161.5, 140.4, 137.4, 135.0, 133.6, 132.3, 126.9,
55.1, 54.6; HRMS (ESI): m/z calcd for C22
364.1539 [MþH] .
H
22NO
4
364.1543, found
þ
126.5, 125.9, 123.3, 122.7, 121.5, 121.0, 114.0, 110.2, 106.5, 90.5, 55.7,
5
5
5.3, 49.7; HRMS (ESI): m/z calcd for C30
22.1870 [MþH] .
H F NO
27 3 4
522.1887, found
þ
4.2.8.25. (1E,4E)-1-(1H-indol-3-yl)-5-(3,4,5-trimethoxyphenyl)
penta-1,4-dien-3-one (11f). Yellow solid, yield 70.2%; mp:
ꢁ
ꢀ1
4
5
.2.8.19. (1E,4E)-1-(1-(4-(trifluoromethoxy)benzyl)-1H-indol-3-yl)-
-(3,4-dimethoxyphenyl)penta-1,4-dien-3-one (9s). Yellow solid,
166e168 C; IR (KBr, cm ): 3240.1, 2988.5, 2901.7, 1638.8, 1608.6,
1
1332.6, 1100.4, 981.1; H NMR (500 MHz, CDCl
3
þDMSO-d
6
): d 11.47
ꢁ
ꢀ1
yield 75.8%; mp: 132e134 C; IR (KBr, cm ): 3100.7, 2922.9, 1712.8,
(s, 1H), 8.11e7.92 (m, 2H), 7.77e7.66 (m, 3H), 7.63 (d, J ¼ 15.7 Hz,
1
1
d
669.0, 1635.9, 1396.7, 1249.2, 985.4; H NMR (500 MHz, CDCl
8.13e7.93 (m, 2H), 7.71 (d, J ¼ 15.8 Hz, 1H), 7.51 (s, 1H), 7.38e7.27
m, 3H), 7.23e7.10 (m, 6H), 6.97 (d, J ¼ 15.8 Hz, 1H), 6.90 (d,
3
):
1H), 7.48 (s, 1H), 7.25 (s, 1H), 7.10 (d, J ¼ 15.7 Hz, 2H), 6.94 (s, 1H),
13
3.93 (s, 6H), 3.85 (s, 3H); C NMR (125 MHz, CDCl
3
þ DMSO-d
6
):
(
d 187.1, 152.1, 140.3, 138.5, 136.7, 130.7, 129.5, 124.6, 124.1, 121.6,
120.0, 119.2, 111.9, 111.3, 104.3, 59.4, 54.9; HRMS (ESI): m/z calcd for
13
J ¼ 8.3 Hz, 1H), 5.35 (s, 2H), 3.96 (s, 3H), 3.93 (s, 3H); C NMR
125 MHz, CDCl ): 188.8, 151.1, 149.2, 148.9, 142.2, 137.6, 136.5,
34.8,133.1,128.2,128.1,126.4,124.4,123.5,122.9,121.8,121.6,121.5,
þ
(
1
3
d
C
22
H22NO
4
364.1543, found 364.1547 [MþH] .
1
21.0, 113.7, 111.1, 110.4, 109.8, 56.0, 55.9, 49.7; HRMS (ESI): m/z
4.3. Biological evaluation
þ
calcd for C29
H
25
F
3
NO
4
508.1730, found 508.1769 [MþH] .
4.3.1. Cell cultures
4
.2.8.20. (1E,4E)-1-(4-chlorophenyl)-5-(1H-indol-3-yl)penta-1,4-
The lung (A549), breast (MDA-MB-231, BT549 and 4T1), prostate
ꢀ1
dien-3-one (11a). Yellow solid, yield 76.4%; mp: ºC; IR (KBr, cm ):
(PC-3, DU145), gastric (HGC-27) and cervical (HeLa) tumor cell lines
were obtained from National center for Cell science (NCCS), Pune,
India. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium
bromide], Trypsin-EDTA were purchased from Sigma Chemicals Co
(St. Louis, MO). DMEM, RPMI 1640 medium, Fetal bovine serum
were purchased from GIBCO-Invitrogen. The 12, 24 and 96 well flat
bottom tissue culture plates were purchased from Corning.
1
3
(
7
149.1, 3049.9, 2988.4, 1644.7, 1610.7, 1338.5, 961.9; H NMR
500 MHz, CDCl ): 8.58 (s, 1H), 8.11e8.00 (m, 1H),
þ DMSO-d
.75e7.28 (m, 10H), 7.19e7.01 (m, 2H); C NMR (125 MHz, CDCl
DMSO-d ): 188.2, 139.8, 138.5, 137.9, 135.3, 134.0, 132.4, 129.8,
3
6
d
13
3
þ
6
d
129.1, 126.9, 125.4, 122.9, 121.3, 120.6, 120.4, 113.1, 112.7; HRMS
þ
(
ESI): m/z calcd for C19
H
15ClNO 308.0837, found 308.0835 [MþH] .
4
.2.8.21. (1E,4E)-1-(1H-indol-3-yl)-5-(4-methoxyphenyl)penta-1,4-
4.3.2. MTT assay
ꢁ
dien-3-one (11b). Yellow solid, yield 72.1%; mp: 163e165 C; IR
The cytotoxic activity of the compounds (9a-s, 11a-f) was
ꢀ
1
3
(
KBr, cm ): 3123.5, 2987.9, 2929.7, 1647.5, 1585.8, 1332.3, 1100.6,
determined by using MTT assay. 5 ꢂ 10 cells per well were seeded
1
9
8
83.2; H NMR (500 MHz, CDCl
.14e7.30 (m, 7H), 7.25e6.68 (m, 6H), 3.83 (s, 3H); C NMR
þ DMSO-d ): 189.0, 161.2, 141.5, 137.7, 137.5,
30.9, 129.9, 127.8, 125.4, 124.0, 122.9, 121.2, 121.0, 120.4, 114.3,
13.6, 112.3, 55.3; HRMS (ESI): m/z calcd for C20 304.1332,
3
þDMSO-d
6
):
d
10.37 (s, 1H),
in 100
mL DMEM or RPMI, supplemented with 10% FBS in each well
13
ꢁ
of 96-well microculture plates and incubated for 24 h at 37 C in a
(
125 MHz, CDCl
3
6
d
CO
culture medium, were added to the wells with respective vehicle
control. After 48 h of incubation, medium was removed, 100 L of
MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bro-
2
incubator. Compounds, diluted to the desired concentrations in
1
1
H18NO
2
m
þ
found 304.1327 [MþH] .
ꢀ1
mide) (0.5 mg mL ) containing medium was added to each well
and the plates were further incubated for 4 h. Then, the supernatant
from each well was carefully removed, formazon crystals were
dissolved in 200 mL of DMSO and absorbance was recorded at
570 nm wavelength with multimode plate reader (Spectramax M4,
Molecular devices, USA).
4
.2.8.22. (1E,4E)-1-(2,5-dimethoxyphenyl)-5-(1H-indol-3-yl)penta-
ꢁ
1,4-dien-3-one (11c). Yellow solid, yield 70%; mp: 98e100 C; IR
ꢀ
1
(
KBr, cm ): 3123.7, 2987.9, 2930.1, 1647.5, 1585.7, 1332.3, 1100.4,
1
9
8
83.1; H NMR (500 MHz, CDCl
3
þ DMSO-d
6
):
d
10.05 (s, 1H),
.06e7.96 (m, 2H), 7.61e7.57 (m, 1H), 7.35e7.28 (m, 3H), 7.21e6.82
13
(
m, 6H), 3.87 (s, 3H), 3.82 (s, 3H); C NMR (125 MHz, CDCl
DMSO-d ): 189.8, 153.5, 153.1, 137.5, 137.4, 130.2, 127.0, 124.3,
23.4, 122.9, 121.4, 121.6, 120.6, 117.1, 114.2, 113.0, 112.5, 56.1, 55.8;
3
þ
6
d
4.3.3. Evaluation of tubulin polymerization inhibitory activity
Tubulin polymerization kit was purchased from Cytoskeleton,
Inc. (BK011). To evaluate the effect of compound 11c, 11d and 11f on
tubulin assembly, a fluorescence based in vitro tubulin polymeri-
zation assay was conducted following the manufacturer's protocol.
The reaction mixture having porcine brain tissue (2 mg/mL) in
80 mM PIPES at pH 6.9, 2.0 mM MgCl
and glycerol in the presence and absence of test compounds (final
concentration of 10 M) was prepared and added to each well of
96-well plate. Tubulin polymerization was followed by a time
dependent increase in fluorescence due to the incorporation of a
fluorescence reporter into microtubules as polymerization pro-
ceeds. Fluorescence emission at 440 nm (excitation wavelength is
360 nm) was measured by using a Spectramax M4 Multi mode
Micro plate Detection System. Nocodazole was used as positive
control in the assay. The IC50 value was calculated from the drug
concentration required for inhibiting 50% of tubulin assembly
compared to control.
1
HRMS (ESI): m/z calcd for C21
H20NO
3
334.1438, found 334.1442
þ
[MþH] .
4
.2.8.23. (1E,4E)-1-(3,4-dimethoxyphenyl)-5-(1H-indol-3-yl)penta-
ꢁ
1,4-dien-3-one (11d). Yellow solid, yield 72.3%; mp: 136e138 C; IR
2
, 0.5 mM EGTA,1.0 mM GTP
ꢀ
1
(
KBr, cm ): 3179.2, 3111.2, 2970.2, 1634.9, 1610.8, 1358.9, 1262.1,
1
9
8
3
82.0; H NMR (500 MHz, CDCl
.04e7.85 (m, 2H), 7.68e7.30 (m, 3H), 7.25e6.73 (m, 7H), 3.88 (s,
H), 3.85 (s, 3H); 13C NMR (125 MHz, CDCl
þ DMSO-d ): 188.7,
50.9, 149.1, 141.7, 137.7, 131.2, 128.0, 125.3, 124.3, 122.8, 121.1, 120.6,
3
þ DMSO-d
6
):
d
10.70 (s, 1H),
m
3
6
d
1
120.3, 113.4, 112.3, 111.1, 109.7, 55.9; HRMS (ESI): m/z calcd for
þ
C
21
H20NO
3
334.1438, found 334.1435 [MþH] .
4
.2.8.24. (1E,4E)-1-(1H-indol-3-yl)-5-(2,4,6-trimethoxyphenyl)
penta-1,4-dien-3-one (11e). Yellow solid, yield 74.1%; mp:
2
ꢁ
ꢀ1
26e228 C; IR (KBr, cm ): 3123.7, 2987.9, 2929.9, 1647.5, 1585.5,
1
1332.2, 1100.3, 983.1; H NMR (500 MHz, CDCl
3
þDMSO-d
6
): d 8.51
(
5
s, 1H), 8.20 (d, J ¼ 16.1 Hz, 1H), 8.06e7.94 (m, 2H), 7.61e7.27 (m,
4.3.3.1. Acridine orange/ethidium bromide (AO/EB) staining.
The morphological changes of treated and control cells were
examined by Acridine Orange/Ethidium Bromide (AO/EB) staining.
PC-3 cells were grown in 24 well plates (25,000 cells/well) for 24 h
13
H), 7.18 (d, J ¼ 15.8 Hz, 1H), 6.15 (s, 2H), 3.96e3.82 (m, 9H);
þDMSO-d ): 189.6,162.1,160.6,136.9,132.3,
30.0, 125.5, 124.7, 121.9, 120.8, 120.2, 119.5, 112.6, 111.6, 105.4, 89.8,
C
NMR (125 MHz, CDCl
1
3
6
d

P.V. Sri Ramya et al. / European Journal of Medicinal Chemistry 127 (2017) 100e114
113
and were treated with 2.5 and 5
1c and 11f for 48 h. After the incubation, medium was removed,
washed with PBS and treated with dyes 10 g/ml acridine orange
and 10 g/mL ethidium bromide. Morphological features were
m
M concentration of compound
(Extra Precision) flexible program. The best model was picked
based on the best stabilization energy as shown in Fig. 12.
1
m
m
Acknowledgements
observed and photographs were taken immediately under fluo-
rescence inverted microscope (Model: Nikon, Japan) with excita-
tion (488 nm) and emission (550 nm) at 200ꢂ maginification.
P.V.S. and A.S. are thankful to DoP, Ministry of Chemicals &
Fertilizers, Govt. of India, New Delhi, for the award of NIPER
fellowship.
4.3.3.2. DAPI staining. Human prostate cancer PC-3 cells were
seeded on 24 well plates at the density of 25,000 cells/well and
allowed to adhere overnight. Then cells were treated with 2.5 and
Appendix A. Supplementary data
5
m
M concentration of compound 11c and 11f for 48 h, washed with
PBS and fixed with 4% neutral buffered formalin solution for
0 min. Then, cells were washed twice with PBS and permeabilized
with 0.2% triton-x for 5min and stained with DAPI (1 g/mL) for
0 min at room temperature. Cells were examined for morpho-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.12.043.
2
m
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