Synthesis, biological evaluation and molecular docking studies of trans-indole-3-acrylamide derivatives, a new class of tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.04.027

In this study, we synthesized a series of trans-indole-3-acrylamide derivatives (3a-k) and investigated their activity for inhibition of cell proliferation against five human cancer cell lines (HeLa, MCF7, MDA-MB-231, Raji and HL-60) by MTT assay. Compound 3e showed significant antiproliferative activity against both the Raji and HL-60 cell lines with IC₅₀ values of 9.5 and 5.1 μM, respectively. Compound 3e also exhibited moderate inhibitory activity on tubulin polymerization (IC₅₀ = 17 μM). Flow cytometric analysis of cultured cells treated with 3e also demonstrated that the compound caused cell cycle arrest at the G2/M phase in HL-60 and HeLa cells. Moreover, 3e, the most active compound, caused an apoptotic cell death through the activation of caspase-3. Docking simulations suggested that 3e binds to the colchicine site of tubulin.

Full text of DOI:10.1016/j.bmc.2014.04.027

Bioorganic & Medicinal Chemistry 22 (2014) 3096–3104
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular docking studies
of trans-indole-3-acrylamide derivatives, a new class of tubulin
polymerization inhibitors
a
b
a
b
Sultan Nacak Baytas a, , Nazan Inceler , Akın Yılmaz , Abdurrahman Olgac , Sevda Menevse ,
⇑
Erden Banoglu a, Ernest Hamel c, Roberta Bortolozzi d, Giampietro Viola d
a Division of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, 06330 Etiler, Ankara, Turkey
b Department of Medical Biology and Genetics, Faculty of Medicine, Gazi University, 06500 Besevler, Ankara, Turkey
c Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick National Laboratory for Cancer Research,
National Cancer Institute, National Institutes of Health, Frederick, MD 21702, United States
d Department of Women’s and Children’s Health, Laboratory of Oncohematology, University of Padova, 35128 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we synthesized a series of trans-indole-3-acrylamide derivatives (3a–k) and investigated
their activity for inhibition of cell proliferation against five human cancer cell lines (HeLa, MCF7, MDA-
MB-231, Raji and HL-60) by MTT assay. Compound 3e showed significant antiproliferative activity
Received 20 January 2014
Revised 11 April 2014
Accepted 15 April 2014
Available online 20 April 2014
against both the Raji and HL-60 cell lines with IC50 values of 9.5 and 5.1
l
M, respectively. Compound
M). Flow cytometric
3e also exhibited moderate inhibitory activity on tubulin polymerization (IC50 = 17
l
analysis of cultured cells treated with 3e also demonstrated that the compound caused cell cycle arrest at
the G2/M phase in HL-60 and HeLa cells. Moreover, 3e, the most active compound, caused an apoptotic
cell death through the activation of caspase-3. Docking simulations suggested that 3e binds to the colchi-
cine site of tubulin.
Keywords:
Synthesis
Anticancer activity
Indole
Tubulin polymerization
Colchicine binding
Apoptosis
Ó 2014 Elsevier Ltd. All rights reserved.
Cell cycle arrest
Molecular docking
1. Introduction
Among them, colchicine was the first tubulin-binding agent to
have antivascular effects causing hemorrhagic necrosis in human
Cancer remains one of the leading causes of death worldwide
and requires a pressing need for the development of novel and
more effective treatments. Although the chemotherapy is the
major method for treatment for various cancer types, the narrow
dosing window of current drugs with regard to their efficacy and
safety and significant drug resistance resulting a failure of antitu-
mor drugs to exert their effects in certain cancer types limit the
use of contemporary cancer chemotherapy. Therefore, the design
and discovery of more effective and safer anticancer drug candi-
dates are of interest in contemporary medicinal chemistry.1,2
Microtubules are important in mitosis and have been recog-
nized as an important target for the development of novel antican-
cer drugs.3 Agents targeting tubulin such as the vinca alkaloids and
taxoids are potent chemotherapeutics currently used in the clinic.
tumors.4 Combretastatins which are isolated from the South
African tree Combretum caffrum are also a group of antimitotic
compounds and combretastatin A-4 (CA-4, Fig. 1) is one of the
well-known natural tubulin-binding molecule affecting microtu-
bule dynamics.5 CA-4 has provided researchers a simple structural
template for the design of related compounds with potent activity
and a large number of combretastatin analogues have been pre-
pared as potential anticancer agents including chalcones, some of
which have recently been reviewed.6–10 Chalcones (1,3-diaryl-2-
propen-1-ones) with an ionone system between two aromatic
rings (Fig. 1) serve as precursors for the preparation of various
flavonoids and exhibit interesting pharmacological activities11,12
such as anticancer13–15 and antiproliferative activities.16–21 Their
broad biological properties are reported to be due to the
urated ketone moiety.22 Chalcones in which both the 1,3-diaryl
rings are separated by ,b-unsaturated carbonyl system with
a,b-unsat-
a
three-carbon lengths are structurally similar to indolyl heterocy-
cles (Fig. 1). There are many indole-based compounds found to
⇑
Corresponding author. Tel.: +90 (312) 202 3525; fax: +90 (312) 223 5018.
E-mail address: baytas@gazi.edu.tr (S.N. Baytas).
http://dx.doi.org/10.1016/j.bmc.2014.04.027
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

S. N. Baytas et al. / Bioorg. Med. Chem. 22 (2014) 3096–3104
3097
H3CO
H3CO
OCH3
OCH3
O
HO
HO
OCH3
OCH3
OCH3
H3CO
OCH3
H3CO
O
HN
OCH3
CH3
OCH3
O
Chalcone
Combretastatin A-4
Colchicine
OCH3
OCH3
H
N
O
OCH3
n
OCH3
O
OCH3
OCH3
n
O
OCH3
N
H
N
H
N
H
OCH3
Phenylcinnamides
Indolyl chalcone
Figure 1. Chemical structures of known tubulin inhibitors and synthesized compounds.
be effective as tubulin assembly inhibitors such as the recently
reported 3-arylthioindoles, which induced significant apoptotic
cell death.23 However, indole-based chalcones remain largely
unexplored for their anticancer potential.24–26 Kumar et al. showed
that indolyl chalcones inhibited the growth of A549, PaCa-2 and
PC-3 cancer cell lines at micromolar concentrations.27 Cinnamon
(HRMS), IR and 1H- and 13C NMR spectral data. Final acryl amide
derivatives exhibited a characteristic strong absorption peaks in
the area of 1638–1733 cmꢀ1, which was attributable to the C@O
of the amide moiety. In the 1H NMR spectra of compounds 3a–k,
one of the olefinic protons (CH@CHACO) was observed as a
doublet at about 7.61–7.83 ppm, while the other (CH@CHACO)
was observed as a doublet at about 6.64–7.15 ppm, with coupling
constants of 15.6 or 16.0 Hz indicating the presence of the (E)
isomer.
amides having an
a,b-unsaturated ketone moiety (phenylcinna-
mides in Fig. 1) are shown to bind to tubulin, thereby causing an
inhibition of its polymerization and alteration in the tubulin-
microtubule equilibrium.28–31
Encouraged with these results and to discover novel anticancer
agents, we have synthesized a series of novel indolylacrylamide
derivatives and evaluated their anticancer activities. The newly
synthesized compounds structurally resemble the indolyl chalcone
structure (Fig. 1).
2.2. Biological evaluations
2.2.1. Effects of the compounds on the viability of cancer cells
trans-Indolyl-3-acrylamide derivatives 3a–k were screened
against five human cancer cell lines (HeLa, MCF7, MDA-MB-231,
Raji and HL-60) using the 3-(4,5-dimethyldiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assay. The MTT cell proliferation
assay has been widely accepted as a reliable way to measure the
cell proliferation rate when metabolic events lead to apoptosis or
necrosis.33–36 After incubation with compounds at different con-
centrations for 48 h, the cells were treated with MTT to measure
2. Results and discussion
2.1. Chemistry
We synthesized a series of amide derivatives of trans-indole-
3-acrylic acid as illustrated in Scheme 1. First, trans-indole-
3-acrylic acid 2 was generated by Knoevenagel condensation of
indole-3-carbaldehyde with malonic acid in the presence of piper-
idine as reported previously.32 Treatment of 2 with appropriate
amines in the presence of triethylamine and ethyl chloroformate,
which was used as the carboxylate activator, produced trans-
indole-3-acrylamide derivatives 3a–k in moderate to good yields
(40–69%). Compounds were purified by automated flash chroma-
tography and checked for purity with UPLC before being tested
in biological assays (purity was >97%). The structures of these com-
pounds were confirmed by high resolution mass spectrometry
their growth/viability (% of the untreated control) using
a
spectrophotometer as described previously.37,38 Experiments were
performed in quadruplicate. The IC50 values (Table 1) were calcu-
lated from concentration–response curves by means of the PRISM
5, GraphPad Software.39
In general, compounds having a phenylamidic moiety (3a–e)
displayed greater antiproliferative potency than the molecules
possessing a benzylamidic moiety (3g–k). Compounds having
methoxy substituent at positions 3 and 4 or at 3 and 5 on the phe-
nyl amidic moiety of the molecules (3c, 3d) exerted potency on the
Raji and HL-60 cell lines (IC50 values of 6.2-10.3
showed appreciable antiproliferative activity against both Raji and
HL-60 cell lines, with IC50 values of 9.5 and 5.1 M, respectively.
lM). Compound 3e
l
Breast cancer cell lines MDA-MB-231 and MCF7 were not sensitive
towards the newly synthesized compounds with the exception of
the 3,4,5-trimethoxy-substituted phenyl amide derivative 3e,
which was the most effective compound found in the phenyl
amide series.
In the series of benzylamide derivatives, compound 3h, with
2,3-dimethoxy substitutions on the amide moiety, showed weak
activity against HeLa cells. Derivatives 3g (3-methoxy-4-hydroxy-
substituted benzylamide derivative) and 3i (2,5-dimethoxy-substi-
tuted benzylamide derivative) had weak activity against Raji cells.
Scheme 1. Reagent and conditions; (a) Malonic acid, piperidine, pyridine, 5 h,
40 °C; (b) 2, ethyl chloroformate, NEt3, amine derivative, CH2Cl2, overnight, rt.

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S. N. Baytas et al. / Bioorg. Med. Chem. 22 (2014) 3096–3104
Table 1
In vitro cytotoxic activity of compounds 3a–k
H
N
O
Ar
n
N
H
a
Compound
n
IC50 (lM)
Ar
HeLa
>100
MDA-MB-231
>100
MCF7
Raji
HL-60
OCH3
OCH3
3a
0
>100
22.6 1.45
17.8 1.54
OCH3
3b
0
>100
>100
>100
22.9 1.76
10.1 0.87
H3CO
OCH3
OCH3
OCH3
3c
0
0
31.4 9.01
>100
>100
>100
>100
10.3 2.43
9.3 1.93
6.2 0.44
7.1 0.31
3d
>100
H3CO
OCH3
3e
0
23.7 2.46
>100
36.3 3.07
9.5 1.28
5.1 0.32
OCH3
OCH3
H3CO
H3CO
3f
0
1
>100
>100
>100
>100
>100
>100
>100
>100
>100
OCH3
OH
3g
33.1 1.40
OCH3
3h
3i
1
1
36.5 6.26
>100
>100
>100
>100
>100
>100
>100
>100
OCH3
H3CO
H3CO
32.9 0.94
OCH3
OCH3
3j
1
>100
>100
>100
>100
>100
>100
>100
>100
H3CO
OCH3
3k
1
>100
>100
OCH3
Each experiment was independently performed four times, and data are shown as means SD.
a
2.2.2. Inhibition of tubulin polymerization and colchicine
binding
To investigate whether the antiproliferative activity of these com-
pounds is due to an interaction with tubulin, we evaluated the
effects of 3a–k on the polymerization of purified tubulin, using
the highly potent CA-4 as reference (Table 2).40
Based on their structural resemblance to phenylcinnamides, we
considered tubulin as a potential target31 for our active compounds.

S. N. Baytas et al. / Bioorg. Med. Chem. 22 (2014) 3096–3104
3099
Only 3e inhibited tubulin polymerization with IC50 values
below 20 M. Compounds 3e with 3,4,5-trimethoxy substituents
on the amide moiety had IC50 value of 17 M. This was in agree-
ment with 3e being the compound with the greatest antiprolifera-
tive activity. Three compounds (3b, 3c, 3d) of the five best
antiproliferative agents were inactive as antitubulin agents. There-
fore, the results suggest that there may be another, more dominant
molecular mechanism of action for these three compounds for
their antiproliferative activity.
A100
G1
G2/M
S
l
l
80
60
40
20
0
All synthesized compounds were also examined for their inhib-
itory effects at 5 and/or 50
lM on the binding of 5 l
M [3H]colchi-
cine to 1
l
M tubulin (Table 2).41 Significant inhibition was only
observed with 3e at 50 lM, in accord with their effects on the
assembly reaction.
0
10
20
30
Concentration (µM)
2.2.3. Analysis of cell cycle effects
Considering the cytotoxic activity of 3e and its antitubulin
properties, we further analyzed its effects on cell cycle distribution
of cultured HL-60 and HeLa cells, as determined by flow cytometry
(Fig. 2). After treatment of cells with 3e for 24 h, we observed a
concentration dependent increase of G2/M-phase cells with a con-
comitant reduction in the proportion of cells in G1 and S phases
with both cell lines. The increase in the G2/M phase cells occurred
B 100
G1
G2/M
S
80
60
40
20
0
at a concentration as low as 10
HeLa and 60% of the HL-60 cells were blocked in G2/M at higher
concentrations (15–30 M). These results were in good agreement
lM, while more than 80% of the
l
with the inhibitory effect of 3e on tubulin polymerization and also
on the proliferation of both cell lines.
2.2.4. Compound 3e induces apoptosis
To characterize the mode of cell death by 3e, the annexin-V/
propidium iodide (PI) biparametric cytofluorimetric assay was
performed on both the HeLa and HL-60 cell lines. Since PI, which
stains DNA and only enters the dead cells, and annexin-V [conju-
gated to fluorescein isothiocyanate (FITC)], which binds to phos-
phatidylserine (PS) located only on the outer membrane of
apoptotic cells, the assay permits quantitation of live cells
(annexin-Vꢀ/PIꢀ), early apoptotic cells (annexin-V+/PIꢀ), late
apoptotic cells (annexin-V+/PI+) and necrotic cells (annexin-Vꢀ/
PI+). As shown in Figure 3, treatment of both cell lines with com-
pound 3e induced a concentration- and time-dependent increase
in annexin-V+/PIꢀ and annexin-V+/PI+ fractions, indicating activa-
tion of the apoptotic cell death machinery.
0
10
20
30
Concentration (µM)
Figure 2. Percentage of cells in each phase of the cell cycle in HL-60 (panel A) and
HeLa cells (panel B) treated with 3e at the indicated concentrations for 24 h. Cells
were fixed and labeled with PI and analyzed by flow cytometry as described in the
experimental section. Data are represented as means SEM of three independent
experiments.
(PARP) protein is cleaved into 89 and 24 kDa fragments, which
can be shown by Western blot analysis. As shown in Figure 4, 3e
induced the activation of caspase-3 with the appearance of the
cleaved fragments and the subsequent cleavage of its substrate
PARP, after 24 and 48 h treatments. This observation indicated that
the 3e-induced apoptosis was caspase-dependent.
2.2.5. Effects of 3e on caspase activation
To further evaluate the apoptotic process induced by 3e, we
performed immunoblot analysis on HL-60 protein extracts. During
apoptosis, the endogenous 113 kDa Poly-ADP-ribosyl-polymerase
2.2.6. Molecular docking studies
To determine the possible binding modes of the most active
compound 3e to tubulin, docking studies were carried out using
Table 2
Inhibition of tubulin polymerization and colchicine binding by synthesized compounds and CA-4
Compound
Inhibition of tubulin assemblyaIC50
(lM) SD
Inhibition of colchicine bindingb % inhibition SD
50
lM
5
lM
1
lM
3a
3b
3c
3d
3e
3f
3g
3h
3i
>40
>40
>40
>40
13
15
11
26
50
11
14
12
13
23
5
5
2
5
4
1
5
4
4
2
—
—
—
—
11
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
17
1
4
>40
>40
>40
>40
>40
>40
3j
3k
CA-4
13 0.2
1.3 0.07
98 0.8
88 0.3
a
Tubulin was at 10 lM.
Tubulin and colchicine were at 1 and 5 lM concentrations, respectively.
b

3100
S. N. Baytas et al. / Bioorg. Med. Chem. 22 (2014) 3096–3104
A-/PI-
A+/PI-
A+/PI+
A-/PI+
B 100
A-/PI-
A 100
A+/PI-
A+/PI+
A-/PI+
80
80
60
40
20
0
60
40
20
0
0
1
10
0
1
10
Concentration (µM)
Concentration (µM)
A-/PI-
A+/PI-
A+/PI+
A-/PI+
100
A-/PI-
A+/PI-
A+/PI+
A-/PI+
C
D 100
80
60
40
20
0
80
60
40
20
0
0
1
10
0
1
10
Concentration (µM)
Concentration (µM)
Figure 3. Flow cytometric analysis of apoptotic cells after treatment of HL-60 (panels A and B) and HeLa (panels C and D) cells with 3e at the indicated concentrations after
incubation for 24 (panels A and C) or 48 h (panels B and D). The cells were harvested and labeled with annexin-V-FITC and PI and analyzed by flow cytometry. Data are
represented as means SEM of three independent experiments.
polymerization and inhibited the binding of [3H]colchicine to tubu-
lin. Compound 3e caused HeLa and HL-60 cells to accumulate in
the G2/M phase of the cell cycle as was the case with most antitub-
ulin agents. 3e also induced apoptotic cell death by activation of
caspase-3 activity. Moreover, molecular docking studies demon-
strated a potential binding mode for compound 3e in the colchicine
site of tubulin resembling the co-crystallized DAMA-colchicine
binding mode.
4. Experimental
4.1. Chemistry
Figure 4. Western blot analysis of caspase-3 and PARP after treatment of HL-60
cells with 3e at the indicated concentrations and for the indicated times. To confirm
equal protein loading, each membrane was stripped and reprobed with anti-b-actin
antibody.
Chemicals purchased from commercial vendors were used
without purification. Thin-layer chromatography (TLC) was per-
formed on Merck 60F254 plates. Reactions were monitored by
TLC on silica gel, with detection by UV light (254 nm) or charring
Dragendorff reagent.46 Melting points were determined with an
SMP-II Digital Melting Point Apparatus and are uncorrected (Scho-
rpp Geaetetechnik, Germany). IR spectra were obtained using a
Perkin Elmer Spectrum 400 FTIR/FTNIR spectrometer equipped
with a Universal ATR Sampling Accessory. 1H NMR spectra were
recorded in CDCl3 or DMSO-d6 on a Varian Mercury 400 MHz High
Performance Digital FT-NMR spectrometer using tetramethylsilane
as the internal standard at the NMR facility of the Faculty of Phar-
macy, Ankara University. 13C NMR spectra were recorded in CDCl3
on a Varian Mercury 300 MHz FT-NMR spectrometer using tetra-
methylsilane as the internal standard at the NMR facility of FAR-
GEM (Pharmaceutical Research and Development Center) Inc. All
chemical shifts were recorded as d (ppm). High resolution mass
spectra data (HRMS) were collected using a Waters LCT Premier
XE Mass Spectrometer (high sensitivity orthogonal acceleration
time-of-flight instrument) using ESI (+) method. The instrument
was coupled to an AQUITY Ultra Performance Liquid Chromatogra-
phy system (Waters Corporation, Milford, MA, USA). Flash chroma-
tography was performed with a CombiflashÒ Rf automated flash
chromatography system with RediSep columns (Teledyne-Isco,
Lincoln, NE, USA) using CH2Cl2–methanol (0–5%) solvent gradients.
the high-resolution crystal structure of the tubulin/DAMA-colchi-
cine complex (PDB ID: 1SA0).42
By following the reported docking technique43,44 for 1SA042, a
Glide 5.845 run in single precision mode (GlideScore SP) was used
to collect the best ranked pose of each ligand as shown in Figure 5.
Ligand 3e assumed a V-shaped conformation in the same binding
region of DAMA-colchicine (X-ray ligand, Fig. 5A). The trimethoxy-
phenyl group of 3e was positioned in a similar orientation of the
corresponding ring in the co-crystallized DAMA-colchicine. In both
structures, the middle methoxy group formed a hydrogen bond
with Cys241.
3. Conclusions
We synthesized a series of indolylacrylamides as potential anti-
cancer agents and determined their cytotoxic activity against five
human cancer cell lines. Compound 3e, the most potent derivative
in this series, displayed antiproliferative activity with IC50 values in
the range of 5.1–36.3
lM against the tested cancer cell lines.
Compound 3e was also the most active inhibitor of tubulin

S. N. Baytas et al. / Bioorg. Med. Chem. 22 (2014) 3096–3104
3101
Figure 5. 3D visualization of (A) DAMA-colchicine (X-ray ligand)42 and docking results of the best ranked docking pose of 3e. (B) The main interacting residues are shown and
labeled. Hydrogen bonds are indicated by black dashed lines.
4.2. Synthesis of trans-indole-3-acrylic acid 2
4.3.1. (E)-N-(2,4-Dimethoxyphenyl)-3-(1H-indol-3-
yl)acrylamide 3a
trans-Indole-3-acrylic acid was synthesized by Knoevenagel
condensation between indole-3-carbaldehyde and malonic acid
as previously reported.32 (Yield 91%, mp 195 °C).
4.3.1.1. (CAS registry number: 953135-26-7).
Yield 40%, mp
165–167 °C; IR (FTIR/FTNIR-ATR): 1644 cmꢀ1 (C@O), 3172 cmꢀ1
(NAH). 1H NMR (DMSO-d6) d: 11.60 (1H, s), 9.15 (1H, s), 8.11
(1H, d, J = 6.8 Hz), 8.04 (1H, d, J = 9.2 Hz), 7.97 (1H, s), 7.72 (1H,
d, J = 16 Hz), 7.47 (1H, d, J = 7.2 Hz), 7.20 (2H, m), 7.07 (1H, d,
J = 15.6 Hz), 6.64 (1H, d, J = 2.8 Hz), 6.52 (1H, m), 3.87 (3H, s),
3.75 (3H, s). 13C NMR (DMSO-d6) d: 165.6, 156.7, 151.2, 138.1,
134.8, 131.6, 125.5, 123.1, 122.9, 121.9, 121.2, 121.0, 117.1,
113.1, 112.9, 104.6, 99.3, 56.4, 55.9; HRMS C19H19N2O3 [M+H]+
Calcd 323.1396, Found m/z 323.1397.
4.3. General procedure for the preparation of amide derivatives
of trans-indole-3-acrylic acid
To a solution of the acid derivative (1 mmol) in CH2Cl2 were
added triethylamine (2 mmol) and ethyl chloroformate (1 mmol),
followed by stirring at 0 °C for 30 min. After addition of the appro-
priate amine derivative (1.2 mmol), the mixture was stirred for an
additional 1 h at 0 °C. Then, the reaction mixture was warmed to
room temperature and stirred overnight. After the solvent was
evaporated under reduced pressure, acetone was added, filtered,
and evaporated. The residue was dissolved in CH2Cl2, and the
organic phase was washed with a 1% NaHCO3 solution and brine,
dried over Na2SO4, and evaporated under vacuum. The final residue
was purified by flash column chromatography (CombiflashÒ Rf)
using CH2Cl2–MeOH (0–5%) as eluents.
4.3.2. (E)-N-(2,5-Dimethoxyphenyl)-3-(1H-indol-3-
yl)acrylamide 3b
4.3.2.1. (CAS registry number: 953194-76-8).
Yield 63%, mp
158–160 °C; IR (FTIR/FTNIR-ATR): 1655 cmꢀ1 (C@O), 3403 cmꢀ1
(NAH). 1H NMR (DMSO-d6) d: 11.64 (1H, s), 9.29 (1H, s), 8.15
(1H, d, J = 7.2 Hz), 8.02 (1H, m), 7.81 (1H, s), 7.75 (1H, d,
J = 15.6 Hz), 7.47 (1H, d, J = 7.6 Hz), 7.20 (2H, m), 7.15 (1H, d,
J = 15.6 Hz), 6.97 (1H, d, J = 9.2 Hz), 6.60 (1H, m), 3.84 (3H, s),
3.70 (3H, s). 13C NMR (DMSO-d6) d: 166.0, 153.6, 143.7, 138.1,

3102
S. N. Baytas et al. / Bioorg. Med. Chem. 22 (2014) 3096–3104
135.6, 132.0, 129.6, 125.4, 123.0, 121.3, 121.0, 116.8, 113.1, 112.9,
112.1, 108.3, 107.7, 56.9, 55.9; HRMS C19H19N2O3 [M+H]+ Calcd
323.1396, Found m/z 323.1393.
8.21 (1H, t, J = 5.6 Hz), 7.91 (1H, d, J = 7.6 Hz), 7.75 (1H, s), 7.63
(1H, d, J = 15.6 Hz), 7.45 (1H, d, J = 7.6 Hz), 7.18 (2H, m), 6.88 (3H,
m), 6.71 (1H, d, J = 16 Hz), 4.40 (2H, d, J = 6 Hz), 3.80 (3H, s), 3.76
(3H, s). 13C NMR (DMSO-d6) d: 166.9, 152.9, 146.9, 138.0, 133.8,
133.5, 131.0, 125.5, 124.5, 122.8, 121.1, 120.9, 120.6, 116.8,
112.9, 112.8, 112.3, 60.8, 56.3, 37.7; HRMS C20H21N2O3 [M+H]+
Calcd 337.1552, Found m/z 337.1544.
4.3.3. (E)-N-(3,4-Dimethoxyphenyl)-3-(1H-indol-3-yl)
acrylamide 3c
4.3.3.1. (CAS registry number: 953243-47-5).
Yield 68%, mp
244–246 °C; IR (FTIR/FTNIR-ATR): 1649 cmꢀ1 (C@O), 3328 cmꢀ1
(NAH). 1H NMR (DMSO-d6) d: 11.63 (1H, s), 9.86 (1H, s), 7.94
(1H, m), 7.81 (1H, s), 7.73 (1H, d, J = 15.6 Hz), 7.48 (1H, m), 7.43
(1H, s), 7.21 (3H, m), 6.92 (1H, d, J = 8.8 Hz), 6.78 (1H, d,
J = 15.6 Hz), 3.76 (3H, s), 3.73 (3H, s). 13C NMR (DMSO-d6) d:
165.3, 149.2, 145.1, 138.1, 134.9, 134.1, 131.6, 125.5, 123.0,
121.1, 120.6, 116.8, 113.0, 112.8, 112.8, 111.2, 104.5, 56.3, 55.9;
HRMS C19H19N2O3 [M+H]+ Calcd 323.1396, Found m/z 323.1393.
4.3.9. (E)-N-(2,4-Dimethoxybenzyl)-3-(1H-indol-3-yl)
acrylamide 3i
Yield 48%, mp 164–166 °C; IR (FTIR/FTNIR-ATR): 1644 cmꢀ1
(C@O), 3241 cmꢀ1 (NAH). 1H NMR (DMSO-d6) d: 11.54 (1H, s),
8.07 (1H, t, J = 6 Hz), 7.92 (1H, d, J = 8 Hz), 7.74 (1H, s), 7.61 (1H,
d, J = 15.6 Hz), 7.45 (1H, d, J = 7.6 Hz), 7.18 (3H, m), 6.71 (1H, d,
J = 16 Hz), 6.57 (1H, s), 6.50 (1H, d, J = 8 Hz), 4.28 (2H, d,
J = 5.6 Hz), 3.81 (3H, s), 3.74 (3H, s). 13C NMR (DMSO-d6) d:
166.9, 160.4, 158.4, 138.0, 133.6, 131.0, 129.8, 125.5, 122.8,
120.9, 120.7, 119.8, 116.9, 112.9, 112.8, 104.9, 98.8, 56.0, 55.8,
4.3.4. (E)-N-(3,5-Dimethoxyphenyl)-3-(1H-indol-3-yl)
acrylamide 3d
Yield 45%, mp 218–221 °C; IR (FTIR/FTNIR-ATR): 1649 cmꢀ1
(C@O), 3206 cmꢀ1 (NAH). 1H NMR (DMSO-d6) d: 11.68 (1H, s),
9.96 (1H, s), 7.95 (1H, m), 7.84 (1H, s), 7.75 (1H, d, J = 15.6 Hz),
7.49 (1H, m), 7.23 (2H, m), 6.96 (2H, s), 6.79 (1H, d, J = 15.6 Hz),
6.21 (1H, m), 3.74 (6H, s). 13C NMR (DMSO-d6) d: 165.7, 161.2,
142.1, 138.1, 135.6, 131.9, 125.5, 123.1, 121.2, 120.7, 116.5,
113.1, 112.8, 97.8, 95.5, 55.7. HRMS C19H19N2O3 [M+H]+ Calcd
323.1396, Found m/z 323.1391.
37.7; HRMS
337.1539.
C
20H21N2O3 [M+H]+ Calcd 337.1552, Found m/z
4.3.10. (E)-N-(2,5-Dimethoxybenzyl)-3-(1H-indol-3-yl)
acrylamide 3j
Yield 57%, mp 190–193 °C; IR (FTIR/FTNIR-ATR): 1638 cmꢀ1
(C@O), 3299 cmꢀ1 (NAH). 1H NMR (DMSO-d6) d: 11.56 (1H, s),
8.18 (1H, t, J = 5.6 Hz), 7.93 (1H, d, J = 8 Hz), 7.76 (1H, s), 7.63
(1H, d, J = 15.6 Hz), 7.46 (1H, d, J = 8 Hz), 7.18 (2H, m), 6.93 (1H,
d, J = 8.8 Hz), 6.79 (2H, m), 6.73 (1H, d, J = 15.6 Hz), 4.34 (2H, d,
J = 5.6 Hz), 3.77 (3H, s), 3.68 (3H, s). 13C NMR (DMSO-d6) d:
172.2, 168.4, 168.3, 164.1, 155.2, 155.0, 136.7, 133.2, 132.7,
125.6, 121.2, 121.0, 119.8, 115.1, 115.0, 112.5, 111.6, 68.4, 24.4;
HRMS C20H21N2O3 [M+H]+ Calcd 337.1552, Found m/z 337.1548.
4.3.5. (E)-3-(1H-Indol-3-yl)-N-(3,4,5-trimethoxyphenyl)
acrylamide 3e
Yield 53%, mp 246–248 °C; IR (FTIR/FTNIR-ATR): 1733 cmꢀ1
(C@O), 3304 cmꢀ1 (NAH). 1H NMR (DMSO-d6) d: 11.68 (1H, s),
9.96 (1H, s), 7.95 (1H, m), 7.83 (1H, s), 7.74 (1H, d, J = 15.6 Hz),
7.48 (1H, m), 7.23 (2H, m), 7.10 (2H, s), 6.79 (1H, d, J = 15.6 Hz),
3.77 (6H, s), 3.63 (3H, s). 13C NMR (DMSO-d6) d: 165.5, 153.4,
138.1, 136.6, 135.2, 133.7, 131.8, 125.5, 123.0, 121.1, 120.6,
116.7, 113.1, 112.8, 97.1, 60.8, 56.3; HRMS C20H21N2O4 [M+H]+
Calcd 353.1501, Found m/z 353.1503.
4.3.11. (E)-N-(3,4-Dimethoxybenzyl)-3-(1H-indol-3-yl)
acrylamide 3k47
Yield 68%, mp 176–178 °C; IR (FTIR/FTNIR-ATR): 1657 cmꢀ1
(C@O), 3292 cmꢀ1 (NAH). 1H NMR (DMSO-d6) d: 11.52 (1H, s),
8.24 (1H, t, J = 5.6 Hz), 7.86 (1H, d, J = 7.6 Hz), 7.72 (1H, s), 7.61
(1H, d, J = 16 Hz), 7.42 (1H, d, J = 7.6 Hz), 7.13 (2H, m), 6.89 (2H,
m), 6.80 (1H, m), 6.64 (1H, d, J = 16 Hz), 4.30 (2H, d, J = 5.2 Hz),
3.71 (3H, s), 3.69 (3H, s). 13C NMR (DMSO-d6) d: 166.8, 149.2,
148.4, 138.0, 133.9, 132.7, 131.0, 125.5, 122.8, 120.9, 120.6,
120.2, 116.8, 112.9, 112.7, 112.3, 112.1, 56.2, 56.0, 42.8; HRMS
4.3.6. (E)-3-(1H-Indol-3-yl)-N-(2,4,6-trimethoxyphenyl)
acrylamide 3f
Yield 41%, mp 221–223 °C; IR (FTIR/FTNIR-ATR): 1651 cmꢀ1
(C@O), 3324 cmꢀ1 (NAH). 1H NMR (DMSO-d6) d: 11.57 (1H, s),
8.68 (1H, s), 7.96 (1H, d, J = 7.2 Hz), 7.77 (1H, s), 7.61 (1H, d,
J = 16 Hz), 7.47 (1H, d, J = 7.2 Hz), 7.20 (2H, m), 6.83 (1H, d,
J = 15.6 Hz), 6.27 (2H, s), 3.79 (3H, s), 3.73 (6H, s). 13C NMR
(DMSO-d6) d: 165.9, 159.7, 157.2, 138.0, 134.0, 131.1, 125.5,
122.8, 121.0, 120.6, 117.0, 112.9, 112.8, 108.7, 91.6, 56.3, 56.0;
HRMS C20H21N2O4 [M+H]+ Calcd 353.1501, Found m/z 353.1490.
C
20H21N2O3 [M+H]+ Calcd 337.1552, Found m/z 337.1544.
4.4. Anticancer activity
4.4.1. Cell lines and cell culture
The human cancer cell lines, cervical carcinoma (HeLa), estro-
gen receptor positive breast carcinoma (MCF7), estrogen receptor
negative breast carcinoma (MDA-MB-231), Burkitt’s lymphoma
(Raji) and human promyelocytic leukemia (HL-60), were obtained
from ATCC.
4.3.7. (E)-N-(4-Hydroxy-3-methoxybenzyl)-3-(1H-indol-3-yl)
acrylamide 3g
Yield 55%, mp 213–215 °C; IR (FTIR/FTNIR-ATR): 1702 cmꢀ1
(C@O), 3266 cmꢀ1 (NAH). 1H NMR (DMSO-d6) d: 11.55 (1H, s),
8.87 (1H, s), 8.23 (1H, t, J = 5.6 Hz), 7.89 (1H, d, J = 7.2 Hz), 7.67
(1H, m), 7.63 (1H, d, J = 15.6 Hz), 7.45 (1H, d, J = 7.6 Hz), 7.18 (2H,
m), 6.88 (1H, s), 6.72 (1H, m), 6.67 (1H, d, J = 15.6 Hz), 4.29 (2H,
d, J = 5.2 Hz), 3.75 (3H, s). The OH-signal was not observed in the
spectrum, due to solvent exchange. 13C NMR (DMSO-d6) d: 166.8,
148.1, 146.1, 138.0, 133.8, 131.0, 130.9, 125.5, 125.2, 122.8,
120.9, 120.7, 116.9, 115.9, 112.9, 112.8, 112.6, 56.2, 42.9; HRMS
4.4.2. Cytotoxicity assay
MDA-MB-231 and MCF7 cells were cultured in DMEM whereas
HeLa, HL-60 and Raji cells were grown in RPMI-1640 medium in a
humidified atmosphere containing 5% CO2 at 37 °C. Both DMEM
and RPMI-1640 medium were supplemented with 10% fetal bovine
serum (FBS), 200 mM
L-glutamine, 100 IU/mL penicillin and
C
19H19N2O3 [M+H]+ Calcd 323.1396, Found m/z 323.1397.
100 g/mL streptomycin (all from Hyclone Laboratories, Logan,
l
UT, USA). Cell viability was determined using the MTT assay (Cell
Proliferation Kit I, Roche, Germany). Briefly, cells were seeded in
a 96-well plate at 10,000 cells per well and cultured overnight in
growth medium containing 1% FBS. Then cells were treated with
4.3.8. (E)-N-(2,3-Dimethoxybenzyl)-3-(1H-indol-3-yl)
acrylamide 3h
Yield 69%, mp 211–213 °C; IR (FTIR/FTNIR-ATR): 1639 cmꢀ1
(C@O), 3206 cmꢀ1 (NAH). 1H NMR (DMSO-d6) d: 11.56 (1H, s),
test compounds at different concentrations (from 0.1 to 125 lM)

S. N. Baytas et al. / Bioorg. Med. Chem. 22 (2014) 3096–3104
3103
for 48 h. As a solvent control, cells were also treated with dimethyl
4.4.6. Annexin-V assay
sulfoxide (DMSO) at a final concentration of 0.1%. At the end of the
incubation time, MTT (final concentration, 0.5 mg/mL) was added
to each well, and the plate was incubated for an additional 4 h.
After formation of blue formazan crystals, medium containing
MTT was discarded, and DMSO was added to the wells to dissolve
the MTT crystals. The absorbance of samples was measured with a
Spectra Max M3 microplate reader (Molecular Devices, Sunnyvale,
CA, USA) at 570 nm. Average absorbance values from quadruplicate
replicates per test compound and solvent control (DMSO) were cal-
culated. Mean solvent control values were set to 100% viability,
and then the effects of test compounds on cell viability were calcu-
lated by comparing mean values obtained from compound treated
culture wells with those of the solvent controls. IC50 values were
calculated from concentration–response curves by means of PRISM
5, Graph Pad Software.39
Surface exposure of PS on apoptotic cells was measured by flow
cytometry with a Coulter Cytomics FC500 (Beckman Coulter) by
adding annexin-V-FITC to cells according to the manufacturer’s
instructions (Annexin-V Fluos, Roche Diagnostic). Simultaneously,
the cells were stained with PI. Excitation was set at 488 nm, and
the emission filters were at 525 and 585 nm, respectively, for FITC
and PI.
4.4.7. Western blot analysis
HL-60 cells were incubated in the presence of test compounds
and, after different times, were collected, centrifuged and washed
twice with ice-cold phosphate-buffered saline. The pellet was then
resuspended in lysis buffer. After the cells were lysed on ice for
30 min, lysates were centrifuged at 15,000ꢁg at 4 °C for 10 min.
The protein concentration in the supernatant was determined using
the BCA protein assay reagents (Pierce, Italy). Equal amounts of
4.4.3. Tubulin polymerization assays
protein (20 lg) were resolved using sodium dodecyl sulfate
Purified bovine brain tubulin48 was used in turbidimetric poly-
merization studies and in the colchicine binding assay. Tubulin and
the desired concentrations of compound were preincubated at
30 °C for 15 min in a 0.24 mL reaction volume. Reaction mixtures
polyacrylamide gel electrophoresis (7.5–15% acrylamide gels) and
transferred to a PVDF Hybond-p membrane (GE Healthcare). Mem-
branes were blocked with 5% bovine serum albumin for 2 h. Mem-
branes were then incubated with primary antibodies against
caspase-3 (Alexis) and PARP (Cell Signalling) overnight at 4 °C.
Membranes were next incubated with peroxidase-labeled second-
ary antibodies for 60 min. All membranes were visualized using
ECL Select (GE Healthcare) and exposed to Hyperfilm MP (GE
Healthcare). To ensure equal protein loading, each membrane was
stripped and reprobed with anti-b-actin antibody (Sigma–Aldrich).
were then placed on ice, and 10
concentrations are in terms of the final reaction volume of
0.25 mL: tubulin at 1.0 mg/mL (10 M), 0.8 M monosodium gluta-
lL of 10 mM GTP was added. All
l
mate (adjusted to pH 6.6 in a 2 M stock solution with HCl), 4% (v/v)
DMSO, and 0.4 mM GTP. The ice-cold reaction mixtures were
transferred to cuvettes held at 0 °C in recording spectrophotome-
ters (Beckman models DU7400 and DU7500) equipped with elec-
tronic temperature controllers. After baselines were established
at 350 nm, the temperature was jumped to 30 °C over about 30 s,
and the IC50 is defined as the concentration of compound that
inhibits turbidity development by 50% at 20 min. Detailed method
description has been published previously.40 The method is gener-
4.5. Docking and modeling studies
A structure based procedure was applied to discover and study
the putative binding mode of the most potent compounds in our
compound series. The PDB used (PDB ID: 1SA042) in this work
was first submitted to the Protein Preparation Wizard (only Chain
A and B) protocol of the Schrödinger Suite45 by following similar
procedures described previously.43,44 Later, ligand preparation
was applied by the LigPrep 2.545 run to correctly assign the proton-
ation states and atom types of the molecule. An additional confor-
mational search by using the Mixed torsional/Low-mode sampling
method present in MacroModel 9.945 and setting 50 conformations
ally most reliable at compound concentrations up to 20 lM. At
higher concentrations, compound precipitation and/or absorbance
often leads to interference with the turbidity readings caused by
tubulin assembly.
4.4.4. Inhibition of colchicine binding assays
The colchicine binding assay was performed in 0.1 mL reaction
volumes. Each assay tube contained 0.1 mg/mL (1.0
l
M) tubulin,
as output for each compound was needed to reproduce correctly
0
5% (v/v) DMSO, 5.0
l
M [3H]colchicine (Perkin Elmer), varying con-
the binding pose of the X-ray ligand (RMSD 0.877 ÅA on heavy
atoms) and was then applied to all the other chemical entities.
The grid generation and all docking runs were performed by leav-
ing all the variables at default values in the Glide 5.845 program
used in single precision docking mode (SP) and saving up to the
10 best poses per conformation. The best ranking pose was visual-
ized with PyMOL 1.6.45
centrations of potential inhibitors, as indicated, and additional
components, including 1.0 M glutamate, shown to stabilize the col-
chicine binding activity of tubulin41,49 for prolonged times at 37 °C.
Samples were incubated for 10 min at 37 °C, at which time the
reaction in control samples is 40-60% complete. The reactions were
stopped with water at 0 °C, and the diluted samples (total volume,
about 2 mL, with several 2 mL rinses) were filtered through a stack
of two DEAE-cellulose filters (Whatman) in a 12-place manifold
(Millipore) under a weak vacuum (filtration time about 10 min).
The filters were then rapidly washed three times (2 mL each) with
ice-cold water, using a stronger vacuum. Radiolabel bound to the
filters was measured in a liquid scintillation counter.
Acknowledgment
This research was supported by Scientific Research Grant 02/
2011-09, awarded by Gazi University BAP.
Supplementary data
4.4.5. Flow cytometric analysis of cell cycle distribution
For flow cytometric analysis of DNA content, 5 ꢁ 105 HeLa and
HL-60 cells were treated with different concentrations of the test
compounds for 24 h. After the incubation period, the cells were
collected, centrifuged and fixed with ice-cold ethanol (70%). The
cells were then treated with lysis buffer containing RNAse A and
0.1% Triton X-100 and then stained with PI. Samples were analyzed
on a Cytomic FC500 flow cytometer (Beckman Coulter). DNA histo-
grams were analyzed using MultiCycle for Windows (Phoenix Flow
Systems).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.04.027.
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