Synthesis of C5-tethered indolyl-3-glyoxylamide derivatives as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.01.026

A series of C₅-tethered Indolyl-3-glyoxylamide derivatives were synthesized and evaluated for their in?vitro cytotoxic activity against DU145 (prostate), PC-3 (prostate), A549 (lung) and HCT-15 (colon) cancer cell lines by employing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Among all the synthesized compounds, compound 7f displayed cytotoxicity of IC₅₀?=?140?nM towards DU145 cancer cell line. The treatment of DU145?cells with 7f led to inhibition of cell migration ability. Futher, the detailed studies such as acridine orange/ethidium Bromide (AO/EB), DAPI, annexin V-FITC/propidium iodide staining assay suggested that the compound 7f induced apoptosis in DU145?cells. The influence of the cytotoxic compound 7f on the cell cycle distribution was assessed on the DU145?cell line, exhibiting a cell cycle arrest at the G2/M phase (hallmark of tubulin polymerization) and next inhibited tubulin polymerization with IC₅₀0.40?μM. Furthermore, the treatment with compound 7f caused collapse of mitochondrial membrane potential and elevated intracellular superoxide ROS levels in DU145?cells. Western blotting was performed to examine the levels of apoptotic proteins (Bcl-2 and Bax); the study confirmed that compound 7f induced apoptosis through apoptosis-related protein expression. Thus, these studies provided a new molecular scaffold for the further development of anticancer agents that target tubulin.

Full text of DOI:10.1016/j.ejmech.2017.01.026

Accepted Manuscript
Synthesis of C -tethered indolyl-3-glyoxylamide derivatives as tubulin polymerization
5
inhibitors
Sravanthi Devi Guggilapu, Guntuku Lalita, T. Srinivasa Reddy, Santosh Kumar
Prajapti, Atulya Nagarsenkar, Shymala Ramu, Uma Rani Brahma, Uppa Jaya
Lakshmi, Ganga Modi Naidu Vegi, Suresh K. Bhargava, Bathini Nagendra Babu
PII:
S0223-5234(17)30035-1
10.1016/j.ejmech.2017.01.026
EJMECH 9177
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 November 2016
Revised Date: 19 January 2017
Accepted Date: 19 January 2017
Please cite this article as: S.D. Guggilapu, G. Lalita, T.S. Reddy, S.K. Prajapti, A. Nagarsenkar, S.
Ramu, U.R. Brahma, U.J. Lakshmi, G.M.N. Vegi, S.K. Bhargava, B.N. Babu, Synthesis of C -tethered
5
indolyl-3-glyoxylamide derivatives as tubulin polymerization inhibitors, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.01.026.
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ACCEPTED MANUSCRIPT
Synthesis of C5-tethered Indolyl-3-glyoxylamide derivatives as Tubulin Polymerization
Inhibitors
Sravanthi Devi Guggilapu,a Guntuku Lalita,b T. Srinivasa Reddy,c Santosh Kumar Prajapti,a
Atulya Nagarsenkar,a Shymala Ramu,a Uma Rani Brahma,b Uppa Jaya Lakshmi,b Ganga Modi
Naidu Vegi, b Suresh K. Bhargava,c and Bathini Nagendra Babua*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
bPharmacology and Toxicology Division, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
cCentre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne-3001, Australia.

ACCEPTED MANUSCRIPT
Synthesis of C5-tethered Indolyl-3-glyoxylamide derivatives as Tubulin Polymerization
Inhibitors
Sravanthi Devi Guggilapu,a Guntuku Lalita,b T. Srinivasa Reddy,c Santosh Kumar Prajapti,a
Atulya Nagarsenkar,a Shymala Ramu,a Uma Rani Brahma,b Uppa Jaya Lakshmi,b Ganga
Modi Naidu Vegi, b Suresh K. Bhargava,c and Bathini Nagendra Babua*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
bPharmacology and Toxicology Division, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
cCentre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne-3001, Australia.
*Corresponding author:
Dr. Bathini Nagendra Babu
Department of Medicinal Chemistry
National Institute of Pharmaceutical Education and Research
Hyderabad, 500037, India
E-mail: bathini@niperhyd.ac.in; bnniper@gmail.com
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Abstract
A series of C5-tethered Indolyl-3-glyoxylamide derivatives were synthesized and
evaluated for their in vitro cytotoxic activity against DU145 (prostate), PC-3
(prostate), A549 (lung) and HCT-15 (colon) cancer cell lines by employing the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Among all the
synthesized compounds, compound 7f displayed cytotoxicity of IC50 = 140 nM
towards DU145 cancer cell line. The treatment of DU145 cells with 7f led to
inhibition of cell migration ability. Futher, the detailed studies such as acridine
orange/ethidium Bromide (AO/EB), DAPI, annexin V-FITC/propidium iodide staining
assay suggested that the compound 7f induced apoptosis in DU145 cells. The
influence of the cytotoxic compound 7f on the cell cycle distribution was assessed on
the DU145 cell line, exhibiting a cell cycle arrest at the G2/M phase (hallmark of
tubulin polymerization) and next inhibited tubulin polymerization with IC50 0.40 µM.
Furthermore, the treatment with compound 7f caused collapse of mitochondrial
membrane potential and elevated intracellular superoxide ROS levels in DU145 cells.
Western blotting was performed to examine the levels of apoptotic proteins (Bcl-2 and
Bax); the study confirmed that compound 7f induced apoptosis through apoptosis-
related protein expression. Thus, these studies provided a new molecular scaffold for
the further development of anticancer agents that target tubulin.
Keywords: indolyl-3-glyoxylamide, anticancer, apoptosis, tubulin polymerization inhibitor.
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1.0 Introduction
Cancer is one of the foremost global health burden and severe clinical problems in the
world with increasing frequency every year. Hormones, immune conditions, inherited
genes, viruses, chemicals and radiation are some of the responsible factors for the
development of various cancers [1]. The uncontrolled growth and spreading of
abnormal cells results in the disruption of tissues that leads to death in cancer patients
[2, 3]. Many of the currently available antitumor drugs are unable to differentiate
between normal and neoplastic cells or to overcome primary or secondary resistance
mechanisms evolved in the tumor cells. The high toxicity and poor tolerance of the
existing anticancer drugs, stress on the need to identify novel molecules with potent
antitumor activity, low toxicity and minimum side effects.
Recent drug discovery studies have focused on the design and synthesis of small
molecules that have an indole nucleus as the core structure and that act as tubulin
inhibitors [4]. Tubulin polymerization inhibitors interfere with the dynamic instability
of microtubules, causing disruption of microtubules, and thereby inducing cell cycle
arrest in the M-phase, resulting in the apoptotic cell death [5]. Anti-mitotic compounds
such as taxanes and vinca alkaloids have been widely used in the clinical treatment of
different human cancers over the past decade [6]. Unfortunately, clinical use of these
compounds are restricted by toxicity, drug resistance, and limited bioavailability [7,
8]. This has encouraged medicinal chemists to design and synthesize novel anti-mitotic
agents for cancer therapy.
The indole ring system is an essential part of several tubulin inhibitors identified in the
recent years including the indolyl-3-glyoxamide, Indibulin and the 2-aroylindoles, D-
64131 (Figure 1).
N-(pyridin-4-yl)-[1-(4-chlorbenzyl)-indol-3-yl]-glyoxyl-amide
(Indibulin) is a novel synthetic compound that was identified in a cell-based screening
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assay to discover cytotoxic drugs. Indibulin destabilizes microtubules via a direct
interaction with tubulin at a binding site, distinct from those of the known
destabilizing tubulin agents vincristine or colchicines [9]. Indibulin blocks cell cycle
transition specifically at G2/M-phase and leads to subsequent cell death [10].
On the other hand, aminopyrimidines, an important pharmacophore present in many
anticancer drugs, are also found as tubulin polymerization inhibitors.
Aminopyrimidines (Figure 1) have been identified to possess potent antitumour and
tubulin depolymerization activity [11, 12]. More interestingly, Hu et al. documented
that compound (
displayed threefold improvement in inhibiting tubulin polymerization compared to
compound ( ) [13]. Moreover, some of the naturally occurring the indolyl-pyrimidines
e), generated by introduction of indole moiety into pyrimidine (d)
d
[14, 15] such as meridianin, hyrtinadine, and aplicyanin are also reported for their
anticancer potentials (Figure 1) [16, 17].
<Insert Figure 1 here>
Therefore, owing to our interest in the synthesis of biologically active scaffolds [18],
we envisioned to synthesize indolyl-3-glyoxylamide derivatives with an array of
substitutions, which may lead us towards an enhancement in cytotoxic activity. In
order to develop pertinent leads for prospective tubulin inhibitors, we have introduced
appropriate substitutions at C–5 positions of indolyl-3-glyoxylamide derivatives by
performing Ullmann type C–N bond coupling reaction or constituting C–C bond via
Suzuki coupling reaction.
<Insert Figure 2 here>
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2.0 Results and discussion
2.1 Chemistry
The synthetic protocol to achieve target molecule involved five steps (Scheme 1),
which incites from synthesis of different substituted aromatic and heteroaromatic
enaminones 2(a–i) by employing respective acetophenones 1(a–i) and refluxing it with
DMF–DMA to obtain desired products in quantitative yields [19]. During the course
of product formation, methanol was formed as a by-product which was distilled off at
regular intervals, as it can intervene with the proceedings of reaction. The synthesized
enaminone intermediates 2(a–i) were further subjected to reflux with guanidine nitrate
in
high yields [20]. On the other hand, the indolyl-3-glyoxylamides were prepared by
treatment of 5-bromo indole ( ) with oxalyl chloride at 0 °C in diethyl ether produced
the 2-(5-bromo-1 -indol-3-yl)-2-oxoacetyl chloride due to the high nucleophilic
character at 3-position of indole and followed by substitution reaction with piperidine
in presence of mild base triethylamine afforded compound ( ) in 87% yield [21]. This
was succeeded by -methylation to acquire 1-(5-bromo-1 -indol-3-yl)-2-(piperidin-1-
yl)ethane-1,2-dione ( ), in high yields.
n-butanol under basic condition to afford respective aminopyrimidines 3(a–i) in
4
H
5
N
H
6
Finally, the two privileged moieties 3(a–i) and 6 were subsequently linked through Ullmann
type C–N bond coupling reaction to yield compounds 7(a–i) [22]. In this reaction, CuI was
utilized to carry out the coupling under strict anhydrous atmosphere, as the presence of
moisture impeded the progress of reaction. As aminopyrimidines are very weak nucleophiles,
a strong electron donating agent such as DMEDA was added under the basic condition of
Cs2CO3, to carry out the reaction. Additionally, the substrate (6) was subjected to Suzuki
coupling reaction with the relevant boronic acids and palladium catalyst
tetrakis(triphenylphosphine) palladium (0), to afford the compounds 8(a-f) [23]. All the
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1
synthesized compounds 7a–7i and 8a-8f were confirmed by using FT-IR, H NMR, 13C
NMR and HRMS (ESI) spectroscopic techniques.
<Insert Scheme 1 here>
2.2 Pharmacology
2.2.1. In vitro anticancer activity
All the newly synthesized compounds were evaluated for their in vitro cytotoxic
activity against DU145, PC-3, A549 and HCT-15 cancer cell lines by employing the
3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay [24].
Indibulin and podophyllotoxin were used as reference for this study. The concentration
response course analysis was performed to determine drug concentrations required to
inhibit the growth of cancer cells by 50% (IC50) after incubation for 48 h. The results
from the in vitro anticancer studies exposed that the synthesized target compounds
exhibited different levels of anticancer properties (Table 1). From the close
examination of the IC50 values, it was observed that, pyrimidine linked indolyl-3-
glyoxylamide with phenyl substitution (7a) was inactive against all tested cancer cell
lines while the phenyl substituted with electron donating groups such as –Me (7b) and
–OMe (7c) at para position did not offer much improvement in activity, except that
the compound 7c was specifically active against HCT-15. When the phenyl ring is
introduced with free hydroxyl group at para position (7d), the compound was inactive
against all the tested cancer cell lines. Interestingly, introduction of electron
withdrawing groups at para position of phenyl ring such as –F (7e), –Cl (7f) displayed
enhanced inhibitory activity against all the tested cancer cell lines. The compound 7f
displayed excellent inhibitory activity. Notably, the cytotoxicity decreased while
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substituting the phenyl ring with groups such as -CF3 (7g). Moreover, the
p
replacement of phenyl group with the heterocyclic substituents such as pyridine (7h
)
and thiophene (7i), displayed moderate activity however; the compound 7h was
specifically active against A-549.
On the other hand, the series of compounds 8a-8f bearing aromatic ring at 5th position
displayed no significant activity although the compound 8b, having –Me group at para
position of phenyl ring evidenced moderate activity towards HCT-15.
Analysis of the MTT assay results suggests that DU145 cells are more sensitive
towards the synthesized target compounds. The impact of modification of the “R”
group in series (7a-7i) is interesting in the light of the results of the SAR study, which
suggests that presence of electron withdrawing groups such as –F (7e) and –Cl (7f) is
optimal for anticancer activity. On the other hand simple substitutions at 5th position
such as in the series (8a-8f) demonstrated decrease in the activity. This represents that
bulky substitution such as 4-aryl pyrimidine moieties (7a-7i) should be present at 5th
position for improvement in anticancer potential. Among all compounds synthesized,
compound 7f showed the most significant cytotoxicity towards all the tested cancer
cell lines. These primary results persuade further investigation on the synthesized
compounds aiming to the development of novel potential anticancer agents to target
tubulin.
<Insert Table 1 here>
2.2.2. Wound healing assay
Wound healing assay was carried out to investigate the effect of compound 7f on migration
potential of DU145 cells [25]. The number of cells migrated in to the wound area were
captured using phase contrast microscope after 0, 12 and 24 h incubation. The results from
the Figure 3 clearly showed that wound gap in control has decreased considerably by
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migration of cells in control after 24 h, whereas the treatment with 100, 200 and 400 nM of
compound 7f significantly inhibited the migration of cells in to the scratch area by 61.6%,
52.1% and 47.2% respectively. It can be inferred from the results that the compound 7f
suppresses the migration potential of the DU145 cells in dose dependent manner.
<Insert Figure 3 here>
2.2.3. Acridine orange–ethidium bromide (AO–EB) staining
Acridine orange/ethidium bromide (AO/EB) fluorescent staining assay was performed
to differentiate the live, apoptotic and necrotic cells [26]. AO permeates the intact cell
membrane and stains the nuclei green, while EB can stain the cells that have lost their
membrane integrity and tinge the nucleus red. It can be interpreted from Figure 4 that
the control cells showed the normal morphology and appeared green in colour.
Fluorescence microscopic images of 7f treated DU145 cells have clearly demonstrated
morphological changes which are the characteristic features of apoptotic cells such as
cell membrane blebbing, chromatin condensation, destructive fragmentation of
chromatin, cell shrinkage and apoptotic body formation. This confirms that the
compound 7f induced cell death in DU145 cancer cells.
<Insert Figure 4 here>
2.2.4 DAPI Nucleic Acid Staining
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye that binds firmly to nucleus
and detect the nuclear damage or chromatin condensation. The DAPI stains the
apoptotic cells by forming bright colored fluorescent complexes with chromatin,
which is a unique apoptotic feature. Therefore, it was of our interest to detect nuclear
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damage or chromatin condensation induced by the compound 7f in DU145 cells.
DAPI staining technique was performed according to an earlier reported method [27].
The results from the Figure 5 revealed that the nuclear structure of control cells was
intact whereas compound 7f treated cells exhibited horse shaped or fragmented nuclei.
<Insert Figure 5 here>
2.2.5 Cell cycle analysis
Many of the cytotoxic compounds exert their growth inhibitory effect by arresting the cell
cycle at a specific phase of a cell cycle. In vitro screening results revealed that the compound
7f showed significant activity against DU145 cells. Thus, we herein examined the effect of
the compound 7f on cell cycle using propidium iodide staining method [28]. DU145 cells
were treated with increasing concentrations of compound 7f for 24 h. They were stained with
propidium iodide and samples were further analysed by flow cytometry. Treatment with the
compound 7f at 200 and 400 nM in DU145 cells displayed rise in G2/M population from
24.71% (control) to 28.65% and 34.06% respectively in a dose dependent manner (Figure 6).
In case of podophyllotoxin (standard), the ratio of cells in G2/M phase increased from
24.71% in control to 61.48% at 400 nM. These results indicate G2/M cell cycle arrest by the
test compound suggesting the induction of apoptotic death in the treated cells.
<Insert Figure 6 here>
2.2.6 Annexin V binding assay
To quantify the percentage of apoptosis induced by compound 7f, the annexin V-
FITC/propidium iodide staining assay was carried out using prostate DU145 cancer
cells [28]. DU145 cells were treated with 200 nM and 400 nM of compound 7f for 24
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h and stained with Annexin V-FITC and propidium iodide, and samples were analysed
by flow cytometry.
As depicted in Figure 7, 7f treated DU145 cells demonstrated rise in the total
percentage of apoptotic (early and late apoptotic cells- Annexin V +ve cells) and dead
cells from 4.11% (control) to 14.22% (200 nM), 61.45% (400 nM) respectively in a
dose dependent manner while the standard indibulin displayed 49.68% of apoptotic
(early and late apoptotic cells- Annexin V +ve cells) and dead cells at 400 nM.
<Insert Figure 7 here>
2.2.7 Analysis of Mitochondrial Membrane Potential (DΨm)
Mitochondria play a key role in energy metabolism, as they build their membrane
potential from respiratory substrates derived from the electron transport chain.
Literature reports indicate that the loss or collapse of mitochondrial membrane
potential may be an early event in the process of apoptosis [29]. Thus, the effect of
compound 7f on mitochondrial membrane potential (DΨm) was determined by
staining with lipophilic cationic JC-1 dye [30]. Healthy polarised mitochondria stains
red due to potential dependent formation of J-aggregates, while depolarised
mitochondria in apoptotic cells stains green because of J-monomers. DU145 cells were
treated with 200 nM and 400 nM of 7f for 24 h and stained by JC-1 dye. Flow
cytometric analysis of the treated cells clearly displayed increase in depolarised cell
population (P3) from control (19.06%) to 32.55% and 35.92% respectively and with
400 nM of podophyllotoxin the depolarised cell population (P3) increased from
control (19.06%) to 36.35% (Figure 8). Thus, the results clearly indicate loss of
mitochondrial membrane potential by the compound 7f and suggest the involvement
of mitochondria dependent apoptotic pathway in their mechanism of action.
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<Insert Figure 8 here>
2.2.8 Measurement of superoxide levels (MitoSOX™ red assay)
The production of ROS has been implicated in apoptotic induction by triggering
oxidative damage to the mitochondrial membrane potential and permeability [31]. We
were intrigued by the fact that the apoptosis induction could be due to the ROS
generation. Henceforth, we tested this possibility using MitoSOX™ red; a chemical
probe which reacts with mitochondrial generated superoxide and accumulates in
mitochondria [32]. MitoSOX™ Red is a novel fluorogenic dye which selectively
detects superoxide in the mitochondria of live cells. MitoSOX™ red is quickly
oxidized by superoxide species but not by other reactive oxygen species (ROS) and
reactive nitrogen species (RNS). The oxidized product is highly fluorescent upon
binding to nucleic acid and produces red fluorescence. Fluorescent microscopic
images of DU145 cancer cells stained with MitoSOX™ Red indicator were taken after
48 h treatment with different concentrations of the compound 7f. It can be easily
inferred from the Figure 9 that the treatment of compound 7f caused a dose-dependent
increase in MitoSOX™ Red fluorescence in DU145 cancer cells. DMSO treated
control DU145 cancer cells exhibited weak and diffuse MitoSOX™ Red fluorescence,
however the cells treated with 100 nM, 200 nM and 400 nM of compound 7f were
brightly stained suggesting superoxide generation.
<Insert Figure 9 here>
2.2.9 Effect on tubulin polymerization
The compound 7f displayed G2/M cell cycle arrest which is a hallmark of tubulin
polymerization [33], we investigated the antiproliferative activity of 7f on ability to
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inhibit tubulin polymerization by monitoring the increase in fluorescence emission at
420 nm (excitation wavelength is 360 nm) for 1 h at 37 °C (Figure 10). In the
assembly assay [34], 7f and indibulin inhibited 61.4%, 60.5% tubulin polymerization
compared to control at 0.5 µM respectively. While the podophyllotoxin inhibited
77.0% tubulin polymerization compared to control at 5 µM. The IC50 values of the
compound 7f, indibulin and podophyllotoxin were displayed in Table 2
.
<Insert Figure 10 here>
<Insert Table 2 here>
2.2.10 Western blotting analysis
Many studies have reported that tubulin polymerization inhibitors induce apoptosis by
phosphorylating Bcl-2, reducing the amount of Bcl-2 available to make heterodimers
with Bax [35]. Moreover, Bcl-2 family proteins are crucial components of
mitochondrial stress-induced cellular apoptosis [36]. Therefore, the expression of
apoptosis-related proteins was also determined. Herein, to investigate the molecular
mechanisms of compound 7f on apoptosis, we have checked the expression of Bcl-2
and Bax by using western blot method. Results from the Figure 11 indicated that
compound 7f down-regulated the expression of anti-apoptotic Bcl-2 and up-regulated
the expression of pro-apoptotic Bax proteins in a dose dependent manner, which is a
hallmark feature of apoptosis. These protein changes were compared with standard
indibulin. Increase in Bax level with compound 7f was more pronounced than with
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standard indibulin treatment. Collectively, these results illustrate that compound 7f
induced apoptosis through apoptosis-related protein expression.
<Insert Figure 11 here>
3.0 Conclusion
In the current study, a series of C5-tethered Indolyl-3-glyoxylamide derivatives were
synthesized, and were further evaluated for their in vitro anticancer potentials. An
initial screening was performed against human cancer cell lines such as DU145, PC-3,
A549 and HCT-15. In MTT assay, the compound 7f was found to be the most active
against DU145 (prostate cancer) cell lines. The treatment of DU145 cells with 7f led
to inhibition of cell migration ability. Further, the detailed studies AO/EB staining,
DAPI nuclear staining and Annexin V binding assay suggested that the compound 7f
induced apoptosis in DU145 cells. The cell cycle analysis confirmed G2/M phase of
cell cycle arrest in a dose dependent manner and inhibited tubulin polymerization with
IC50 0.40 µM. Furthermore, depolarization of mitochondrial membrane potential
(MMP) was also observed, which indicated that the mitochondrial pathway was also
involved in the apoptosis signaling pathway. Moreover, the treatment of 7f resulted in
elevated superoxide ROS levels in DU145 cells. Investigation of expression levels of
apoptotic proteins (Bcl-2 and Bax) confirmed that compound 7f induced apoptosis
through apoptosis-related protein expression. The cytotoxicity profile of 7f in terms of
in vitro cytotoxicity assay, inhibition of tubulin polymerization and Bcl-2, Bax
activation are comparable to the standard indubulin. Thus, it can be concluded that the
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C5-substitution on indolyl-3-glyoxylamide derivatives could also offer potential anti-
tubulin leads.
4.0 Experimental protocols
4.1 Chemistry
All reagents and solvents were obtained from commercial suppliers and were used without
further purification. Analytical thin layer chromatography (TLC) was performed on MERCK
precoated silica gel 60-F-254 (0.5 mm) aluminium plates. Visualization of the spots on TLC
1
13
plates was achieved UV light. H and C NMR spectra were recorded on bruker 500 MHz
spectrometers using tetramethyl silane (TMS) as the internal standard. Chemical shifts for 1H
and 13C are reported in parts per million (ppm) downfield from tetramethyl silane. Spin
multiplicities are described as s (singlet), bs (broad singlet), d (doublet), dd (double douplet),
t (triplet), q (quartet), and m (multiplet). Coupling constant (J) values are reported in hertz
(Hz). IR spectra were recorded on a Perkin Elmer, FT-IR spectrometer using KBr discs.
HRMS were determined with Agilent QTOF mass spectrometer 6540 series instrument.
Column chromatography was performed using silica gel 60-120.
4.2 General experimental procedure for the synthesis of compounds 7(a-i):
The pyrimidine amine 3a-i (1.1 mmol), CuI (1.0 mmol) and anhydrous Cs2CO3 (2.0 mmol)
were added to a round bottom flask along with magnetic stir bar and closed well with a
septum. The flask was evacuated and back filled with nitrogen gas three times. Dioxane (15
mL), 1-(5-bromo-1H-indol-3-yl)-2-(piperidin-1-yl)ethane-1,2-dione (6) (1.0 mmol) and
DMEDA (1.0 mmol) were added by syringe at room temperature. The reaction mixture was
stirred at 80°C for 20 h under nitrogen atmosphere and then cooled to room temperature.
Concentrated ammonia (4 mL) was added, and the mixture was extracted with ethyl acetate
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(3X20 mL). The combined organic layer was concentrated in vacuo, and the residue was
purified by column chromatography on silica gel.
4.3 General experimental procedure for the synthesis of compounds 8(a-f):
1-(5-bromo-1H-indol-3-yl)-2-(piperidin-1-yl)ethane-1,2-dione
combined with powdered potassium carbonate
(6)
(0.48 mmol)
was
(200 mg,
1.44 mmol),
tetrakis(triphenylphosphine)palladium (0) (3 mg, 0.04 mmol) and the appropriate arylboronic
acid (0.52 mmol) in a 25 mL round bottom flask containing toluene (7 mL) and water (3 mL)
under nitrogen. The reaction mixture was heated at 80 °C for 6 h with stirring before being
cooled, evaporated to dryness under reduced pressure, 10 mL of water was added. Extracted
with ethyl acetate (3X10 mL), the ethyl acetate was dried using anhydrous sodium 15ignalli
and evaporated under reduced pressure. The residue was purified by column chromatography
on silica gel to obtain the desired compounds 8(a-f).
4.3.1 1-(1-methyl-5-((4-phenylpyrimidin-2-yl)amino)-1H-indol-3-yl)-2-(piperidin-1-
yl)ethane-1,2-dione (7a): White solid; yield: 67%; mp 200-203 oC; IR (KBr): 3292, 2935,
2857, 1615, 1566, 1435, 1411, 1192, 1080, 800, 746 cm1; 1H NMR (500 MHz, DMSO-d6) δ
9.73 (1H, s), 9.04 (1H, bs), 8.56 (1H, d, J = 5.2 Hz), 8.39 – 8.32 (2H, m), 8.11 (1H, s), 7.60
(1H, dd, J = 8.9, 1.8 Hz), 7.57 – 7.51 (4H, m), 7.43 (1H, d, J = 5.2 Hz), 3.89 (3H, s), 3.60
(2H, bs), 3.34 (2H, bs), 1.63 (4H, bs), 1.47 (2H, bs); 13C NMR (125 MHz, DMSO-d6) δ
185.9, 165.7, 163.2, 160.2, 159.0, 139.9, 136.5, 136.4, 133.1, 130.7, 128.7, 127.0, 125.5,
116.8, 111.8, 110.7, 110.7, 107.2, 46.3, 41.1, 33.2, 25.8, 24.9, 23.8; HRMS: m/z calcd for
C26H25N5O2 [M+H]+: 440.2081; found: [M+H]+ 440.2089.
4.3.2 1-(1-methyl-5-((4-(p-tolyl)pyrimidin-2-yl)amino)-1H-indol-3-yl)-2-(piperidin-1-
yl)ethane-1,2-dione (7b): White solid; yield: 64%; mp 271-274 oC; IR (KBr): 3340, 2943,
1584, 1581, 1529, 1614, 1445, 1371, 1205, 993, 735 cm1; 1H NMR (500 MHz, DMSO-d6)
δ 9.69 (1H, s), 9.04 (1H, bs), 8.52 (1H, d, J = 5.2 Hz), 8.26 (2H, d, J = 7.9 Hz), 8.10 (1H,
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s), 7.59 (1H, dd, J = 8.8, 1.6 Hz), 7.52 (1H, d, J = 8.8 Hz), 7.38 (1H, d, J = 5.2 Hz), 7.35
(2H, d, J = 8.0 Hz), 3.89 (3H, s), 3.59 (2H, bs), 3.34 (2H, bs), 2.39 (3H, s), 1.63 (4H, bs),
13
1.48 (2H, s); C NMR (125 MHz, DMSO-d6) δ 185.8, 165.7, 163.2, 160.1, 158.8, 140.6,
139.9, 136.5, 133.7, 133.0, 129.3, 127.0, 125.5, 116.7, 111.8, 110.7, 110.6, 106.9, 46.3,
41.1, 33.3, 25.8, 24.9, 23.8, 20.9; HRMS: m/z calcd for C27H27N5O2 [M+H]+: 454.2238;
found: [M+H]+ 454.2234.
4.3.3 1-(5-((4-(4-methoxyphenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indol-3-yl)-2-
(piperidin-1-yl)ethane-1,2-dione (7c): White solid; yield: 61%; mp 239-241 oC; IR (KBr):
1
3421, 2951, 1625, 1561, 1449, 1409, 1255, 1171, 1032, 798, 736, 583 cm1; H NMR (500
MHz, DMSO-d6) δ 9.64 (1H, s), 9.01 (1H, bs), 8.48 (1H, d, J = 5.2 Hz), 8.32 (2H, d, J = 8.7
Hz), 8.10 (1H, s), 7.60 (1H, dd, J = 8.8, 1.8 Hz), 7.52 (1H, d, J = 8.8 Hz), 7.35 (1H, d, J =
5.2 Hz), 7.08 (2H, d, J = 8.8 Hz), 3.89 (3H, s), 3.85 (3H, s), 3.60 (2H, bs), 3.34 (2H, bs),
1.62 (4H, bs), 1.48 (2H, bs); 13C NMR (125 MHz, DMSO-d6) δ 185.8, 165.7, 162.9, 161.4,
160.1, 158.6, 139.9, 136.5, 133.0, 128.8, 128.7, 125.5, 116.7, 114.0, 111.8, 110.7, 110.6,
106.5, 55.2, 46.4, 41.1, 33.3, 25.9, 24.9, 23.8; HRMS: m/z calcd for C27H27N5O3 [M+H]+:
470.2187; found: [M+H]+ 470.2182.
4.3.4 1-(5-((4-(4-hydroxyphenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indol-3-yl)-2-
(piperidin-1-yl)ethane-1,2-dione (7d): White solid; yield: 59%; mp 241-244 oC; IR (KBr):
1
3340, 2937, 1624, 1427, 1212, 1085, 757 cm1; H NMR (500 MHz, DMSO-d6) δ 13.05
(1H, bs), 10.00 (1H, s), 8.58 (1H, d, J = 5.3 Hz), 8.41 (1H, s), 8.15 (1H s), 8.04 (1H, d, J =
7.9 Hz), 7.67 (1H, d, J = 7.3 Hz), 7.58 (1H, d, J = 8.8 Hz), 7.50 (1H, d, J = 5.4 Hz), 7.37
(1H, t, J = 7.6 Hz), 6.97 – 6.88 (2H, m), 3.90 (3H, s), 3.57 (2H, bs), 3.31 (2H, bs), 1.61
(4H, bs), 1.45 (2H, bs); 13C NMR (125 MHz, DMSO-d6) δ 185.8, 165.6, 164.1, 159.7,
159.2, 158.3, 140.1, 135.2, 133.9, 132.8, 127.9, 125.5, 118.9, 117.9, 117.3, 111.7, 111.0,
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106.2, 46.3, 41.1, 33.3, 25.8, 24.9, 23.8; HRMS: m/z calcd for C26H25N5O3 [M+H]+:
456.2030; found: [M+H]+ 456.2038.
4.3.5 1-(5-((4-(4-fluorophenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indol-3-yl)-2-
o
(piperidin-1-yl)ethane-1,2-dione (7e): Yellow solid; yield: 56%; mp 215-218 C; IR
1
(KBr): 3293, 2937, 1621, 1559, 1416, 1223, 1201, 815, 776 cm1; H NMR (500 MHz,
DMSO-d6) δ 9.75 (1H, s), 9.05 (1H, bs), 8.55 (1H, d, J = 5.1 Hz), 8.48 – 8.39 (2H, m), 8.11
(1H, s), 7.58 (1H, d, J = 8.8 Hz), 7.53 (1H, d, J = 8.8 Hz), 7.42 (1H, d, J = 5.2 Hz), 7.35
(2H, t, J = 8.7 Hz), 3.89 (3H, s), 3.59 (2H, bs), 3.33 (2H, bs), 1.62 (4H, bs), 1.47 (2H, bs);
13C NMR (125 MHz, DMSO-d6) δ 185.9, 165.6, 164.7, 162.7, 160.1, 159.1, 139.9, 136.4,
133.1, 133.0, 129.5, 129.4, 125.5, 116.7, 115.6, 115.5, 111.8, 110.8, 110.6, 107.0, 46.3,
41.1, 33.3, 25.8, 24.9, 23.8 ; HRMS: m/z calcd for C26H24FN5O2 [M+H]+: 458.1987; found:
[M+H]+ 458.1988.
4.3.6 1-(5-((4-(4-chlorophenyl)pyrimidin-2-yl)amino)-1-methyl-1H-indol-3-yl)-2-
o
(piperidin-1-yl)ethane-1,2-dione (7f): Yellow solid; yield: 62%; mp 211-214 C; IR
(KBr): 3306, 2930, 2854, 1616, 1540, 1442, 1251, 1086, 795, 806, 554 cm1; 1H NMR (500
MHz, DMSO-d6) δ 9.78 (1H, s), 9.07 (1H, bs), 8.58 (1H, d, J = 5.2 Hz), 8.40 (2H, d, J = 8.5
Hz), 8.12 (1H, s), 7.62 – 7.51 (4H, m), 7.44 (1H, d, J = 5.2 Hz), 3.88 (3H, s), 3.60 (2H, bs),
3.34 (2H, bs), 1.63 (4H, bs), 1.48 (2H, bs); 13C NMR (125 MHz, DMSO-d6) δ 185.9, 165.7,
161.9, 160.1, 159.3, 140.0, 136.3, 135.5, 135.3, 133.1, 128.9, 128.7, 125.5, 116.7, 111.8,
110.8, 110.6, 107.2, 46.3, 41.1, 33.3, 25.8, 24.9, 23.8; HRMS: m/z calcd for C26H24ClN5O2
[M+H]+: 474.1691; found: [M+H]+ 474.1681.
4.3.7 1-(1-methyl-5-((4-(4-(trifluoromethyl)phenyl)pyrimidin-2-yl)amino)-1H-indol-3-
yl)-2-(piperidin-1-yl)ethane-1,2-dione (7g): Yellow solid; yield: 65%; mp 231-234 oC; IR
1
(KBr): 3302, 2944, 2861, 1620, 1560, 1446, 1325, 1117, 1166, 814, 773 cm1; H NMR
(500 MHz, DMSO-d6) δ 9.86 (1H, s), 9.10 (1H, bs), 8.63 (1H, d, J = 5.1 Hz), 8.58 (2H, d, J
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= 7.9 Hz), 8.13 (1H, s), 7.89 (2H, d, J = 8.3 Hz), 7.57 (1H, d, J = 8.9 Hz), 7.55 – 7.50 (2H,
13
m), 3.89 (3H, s), 3.59 (2H, bs), 3.34 (2H, bs), 1.62 (4H, bs), 1.48 (2H, bs); C NMR (125
MHz, DMSO-d6) δ 185.9, 165.6, 161.6, 160.2, 159.7, 159.5, 140.4, 140.0, 136.2, 133.1,
127.9, 127.8, 125.5, 116.7, 111.8, 110.8, 110.7, 107.7, 46.4, 41.2, 33.3, 25.8, 25.0, 23.8;
HRMS: m/z calcd for C27H24 F3N5O2 [M+H]+: 508.1955; found: [M+H]+ 508.1946.
4.3.8 1-(1-methyl-5-((4-(18ignalli-3-yl)pyrimidin-2-yl)amino)-1H-indol-3-yl)-2-
(piperidin-1-yl)ethane-1,2-dione (7h): White solid; yield: 54%; mp 186-189 oC; IR (KBr):
3251, 2927, 1643, 1625, 1553, 1412, 1389, 1196, 1076, 790, 710 cm1; 1H NMR (500 MHz,
DMSO-d6) δ 9.82 (1H, s), 9.41 (1H, d, J = 1.2 Hz), 9.23 (1H, d, J = 1.5 Hz), 8.96 (1H, bs),
8.68 (1H, dd, J = 4.7, 1.3 Hz), 8.61 (1H, d, J = 5.1 Hz), 8.39 (1H, d, J = 8.0 Hz), 8.36 (1H,
d, J = 5.1 Hz), 8.12 (1H, s), 7.61 (1H, dd, J = 8.8, 1.6 Hz), 7.21 (1H, d, J = 5.1 Hz), 3.89
(3H, s), 3.59 (2H, bs), 3.31 (2H, bs), 1.62 (4H, bs), 1.47 (2H, bs); 13C NMR (125 MHz,
DMSO-d6) δ 185.9, 165.6, 161.2, 160.2, 159.3, 151.4, 148.1, 139.9, 136.2, 134.5, 133.2,
132.1, 125.5, 123.8, 116.9, 111.8, 110.9, 110.8, 107.5, 46.4, 41.1, 33.3, 25.8, 24.9, 23.8;
HRMS: m/z calcd for C25H24N6O2 [M+H]+:441.2034; found: [M+H]+ 441.2078.
4.3.9 1-(1-methyl-5-((4-(thiophen-2-yl)pyrimidin-2-yl)amino)-1H-indol-3-yl)-2-
o
(piperidin-1-yl)ethane-1,2-dione (7i): Yellow solid; yield: 52%; mp 214-217 C; IR
1
(KBr): 3320, 2939, 1618, 1524, 1445, 1207, 1085, 796 cm1; H NMR (500 MHz, DMSO-
d6) δ 9.68 (1H, s), 8.69 (1H, bs), 8.47 (1H, d, J = 5.1 Hz), 8.10 (1H, s), 8.05 (1H, d, J = 3.3
Hz), 7.80 (1H, d, J = 4.9 Hz), 7.74 (1H, dd, J = 8.9, 1.6 Hz), 7.52 (1H, d, J = 8.9 Hz), 7.29
(1H, d, J = 5.2 Hz), 7.26 – 7.21 (1H, m), 3.89 (3H, s), 3.58 (2H, bs), 3.31 (2H, bs), 1.62
13
(4H, bs), 1.46 (2H, bs); C NMR (125 MHz, DMSO-d6) δ 185.7, 165.7, 159.9, 158.6,
142.5, 139.8, 136.2, 133.3, 130.3, 128.5, 128.1, 125.5, 117.0, 111.8, 111.1, 110.6, 105.9,
46.3, 41.1, 33.3, 25.8, 24.9, 23.8; HRMS: m/z calcd for C24H23N5O2S [M+H]+: 446.1645;
found: [M+H]+ 446.1642.
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4.3.10 1-(1-methyl-5-phenyl-1H-indol-3-yl)-2-(piperidin-1-yl)ethane-1,2-dione
(8a):
White solid; yield: 74%; mp 154-157 oC; IR (KBr): 2986, 1624, 1525, 1386, 1208, 1008,
949, 759 cm1; 1H NMR (500 MHz, CDCl3) δ 8.60 (1H, s), 7.85 (1H, s), 7.69 (2H, d, J = 7.6
Hz), 7.60 (1H, dd, J = 8.5, 1.5 Hz), 7.48-7.39 (3H, m), 7.33 (1H, t, J = 7.4 Hz), 3.87 (3H,
s), 3.69 (2H, bs), 3.47 – 3.44 (2H, m), 1.68 (4H, s), 1.57 – 1.56 (2H, m); 13C NMR (125
MHz, CDCl3) δ 185.7, 166.1, 141.5, 139.4, 137.1, 136.8, 128.6, 127.5, 126.8, 123.6, 120.8,
113.7, 111.3, 110.1, 47.2, 42.3, 33.8, 26.4, 25.5, 24.5; HRMS: m/z calcd for C22H22N2O2
[M+H]+: 347.1754; found: [M+H]+ 347.1772.
4.3.11 1-(1-methyl-5-(p-tolyl)-1H-indol-3-yl)-2-(piperidin-1-yl)ethane-1,2-dione
(8b):
White solid; yield: 72%; mp 159-162 oC; IR (KBr): 3113, 2936, 1731, 1622, 1524, 1264,
1089, 949, 807, 774 cm1; 1H NMR (500 MHz, DMSO-d6) δ 8.34 (1H, s), 8.22 (1H, s), 7.67
(1H, d, J = 8.5 Hz), 7.62 (1H, dd, J = 8.6, 1.7 Hz), 7.57 (2H, d, J = 8.1 Hz), 7.29 (2H, d, J =
7.9 Hz), 3.93 (3H, s), 3.59 (2H, m), 3.33 – 3.28 (2H, m), 2.35 (3H, s), 1.61 (4H, s), 1.44
(2H, s); 13C NMR (125 MHz, DMSO) δ 186.1, 165.5, 140.5, 137.9, 136.9, 136.1, 135.4,
129.5, 126.6, 125.9, 122.6, 118.6, 112.1, 111.5, 46.4, 41.2, 33.4, 25.8, 25.0, 23.8, 20.5;
HRMS: m/z calcd for C23H25N2O2 [M+H]+: 361.1911; found: [M+H]+ 361.1926.
4.3.12 1-(5-((4-ethylphenyl)amino)-1-methyl-1H-indol-3-yl)-2-(piperidin-1-yl)ethane-
1,2-dione (8c): White solid; yield: 65%; mp 161-164 oC; IR (KBr): 3035, 2954, 1618, 1523,
1
1363, 1208, 1081, 738 cm1; H NMR (500 MHz, CDCl3) δ 8.59 (1H, s), 7.84 (1H, d, J =
6.3 Hz), 7.65 – 7.58 (3H, m), 7.42 (1H, dd, J = 13.1, 5.2 Hz), 7.29 (2H, d, J = 8.0 Hz), 3.87
(3H, s), 3.71 (2H, m), 3.45 (2H, m), 2.71 (2H, q, J = 7.6 Hz), 1.69 (4H, m), 1.59 (2H, m),
1.29 (3H, t, J = 7.6 Hz); 13C NMR (125 MHz, CDCl3) δ 185.7, 166.1, 142.9, 139.3, 138.8,
136.9, 136.7, 128.1, 127.4, 126.7, 123.5, 120.5, 113.6, 110.8, 47.2, 42.3, 33.8, 28.4, 26.4,
25.5, 24.5, 15.6; HRMS: m/z calcd for C24H26N2O2 [M+H]+: 375.2067; found: [M+H]+
375.2081.
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4.3.13 1-(5-(4-chlorophenyl)-1-methyl-1H-indol-3-yl)-2-(piperidin-1-yl)ethane-1,2-dione
(8d): Orange solid; yield: 62%; mp 149-152 oC; IR (KBr): 3133, 2927, 1617, 1528, 1444,
1208, 1087, 948, 848, 764 cm1; 1H NMR (500 MHz, DMSO-d6) δ 8.45 (2H, d, J = 1.5 Hz),
8.29 (1H, s), 8.25 – 8.17 (2H, m), 7.82 – 7.76 (3H, m), 3.96 (3H, s), 3.60 (2H, bs), 3.35 –
3.29 (2H, m), 1.63 (4H, s), 1.46 (2H, s) 13C NMR (125 MHz, DMSO-d6) δ 186.1, 165.4,
148.3, 142.4, 141.0, 137.6, 133.4, 132.9, 130.5, 125.9, 122.8, 121.6, 121.0, 119.3, 112.2,
112.0, 46.4, 41.2, 33.4, 25.9, 24.9, 23.8; HRMS: m/z calcd for C22H21ClN2O2 [M+H]+:
381.1364; found: [M+H] +: 381.1371.
4.3.14 1-(5-(2,4-dichlorophenyl)-1-methyl-1H-indol-3-yl)-2-(piperidin-1-yl)ethane-1,2-
dione (8e): Yellow solid; yield: 54%; mp 162-165 oC; IR (KBr): 3125, 2991, 1626, 1526,
1444, 1364, 1236, 1085, 949, 775 cm1; 1H NMR (500 MHz, DMSO-d6) δ 8.25 (1H, s), 8.24
(1H, s), 7.63 (3H, m), 7.55 (1H, m), 7.52 – 7.44 (1H, m), 3.90 (3H, s), 3.58 (2H, bs), 3.30 –
3.25 (2H, m), 1.61 (4H, bs), 1.43 (2H, bs); 13C NMR (125 MHz, DMSO-d6) δ 186.1, 165.4,
148.3, 142.4, 137.6, 133.4, 132.9, 130.5, 128.8, 125.8, 122.8, 121.6, 121.0, 119.3, 112.1,
112.0, 46.4, 41.2, 33.4, 25.8, 24.9, 23.8 HRMS: m/z calcd for C22H20Cl2N2O2 [M+2H]+:
416.1047; found: [M+2H] +: 416.1052.
4.3.15 1-(1-methyl-5-(naphthalen-2-yl)-1H-indol-3-yl)-2-(piperidin-1-yl)ethane-1,2-
dione (8f): Orange solid; yield: 52%; mp 159-162 oC; IR (KBr): 3053, 2856, 1622, 1444,
1363, 1207, 10008, 852, 788 cm1; 1H NMR (500 MHz, CDCl3) δ 8.56 – 8.47 (1H, m), 7.92-
7.88 (1H, m), 7.87-7.84 (1H, m), 7.82(1H, s), 7.69-7.64 (2H, m), 7.58 – 7.38 (5H, m), 7.22
(1H, d, J = 8.7 Hz), 3.92 (3H, s), 3.68 (2H, bs), 3.47 – 3.41 (2H, m), 1.72 – 1.64 (4H, m),
1.58 (2H, m);13C NMR (125 MHz, CDCl3) δ 185.4, 165.7, 139.5, 136.2, 132.1, 132.0,
128.5, 128.4, 128.1, 127.4, 126.9, 126.3, 126.1, 125.8, 125.5, 125.2, 124.9, 116.9, 111.3,
47.2, 42.4, 33.8, 26.4, 25.5, 24.4; HRMS: m/z calcd for C26H24N2O2 [M+H]+: 397.1911;
found: [M+H]+ 397.1918.
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4.4 Pharmacology
4.4.1 MTT assay
The anticancer activity of all newly synthesized compounds was determined using MTT
assay. 3-5x103 cells per well were seeded in 100 µL DMEM or RPMI, supplemented with
10% FBS in each well of 96-well microculture plates and incubated for 48 h at 37 °C in a
CO2 incubator. Compounds, diluted to the desired concentrations in culture medium, were
added to the wells with respective vehicle control. After 48 h of incubation, drugs containing
media was removed and 100 µL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) (0.5mg/mL) 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 µL of DMSO and absorbance was recorded at 570 nm wavelength.
4.4.2 Wound healing assay
DU-145 cells (5 x 105 cells/well) were grown in petridishes for 24 h. Scratches were made in
confluent monolayers using 200 µL pipette tip. Then, wounds were washed twice with PBS
to remove non-adherent cell debris. The media containing different concentrations (100, 200
and 400 nM) of the compound 7f were added to the petridishes. Cells which migrated across
the wound area were photographed under the phase contrast microscope (Nikon) after 0, 12
and 24 h treatment. The number of cells migrated in to the wound area was counted
manually.
4.4.3 Acridine orange–ethidium bromide (AO–EB) staining
DU145 cells were plated at a concentration of 1×106 cell/ml and treated with different
concentration of compound 7f. Plates were incubated in an atmosphere of 5% CO2 at 37 °C
for 48 h. 10 µL of fluorescent dyes containing Acridine Orange (AO) and Ethidium Bromide
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(EB) added into each well in equal volumes (10 ꢀg/mL) respectively and within 10 min the
cells were visualized under fluorescence microscope (Nikon, Inc. Japan) with excitation (488
nm) and emission (550 nm) at 200x magnification.
4.4.4 DAPI Nucleic Acid Staining
Nuclear morphological changes were observed through DAPI staining. After treatment with
7f for 48 h in DU145 cells, cells were washed with PBS and permeabilized with 0.1 % Tween
20 for 10 min followed by staining with 1µM DAPI. Control and Treated cells were observed
with fluorescence microscope (Model: Nikon, Japan) with excitation at 359 nm and emission
at 461 nm using DAPI filter at 200X magnification.
4.4.5 Cell cycle analysis
Flow cytometric analysis (FACS) was performed to calculate the distribution of the cell
population through the cell cycle phases. DU145 cancer cells were incubated with compound
7f at varied concentration of 200 and 400 nM for 24 h. Untreated and treated cells were
harvested, washed with PBS, fixed in ice-cold 70% ethanol and stained with propidium
iodide (50 µg/mL, sigma 22ignall) in the presence of Rnase A (20 µg/mL) containing 0.1%
o
Triton X-100 for 30 min at 37 C in dark, and about 10000 events were analyzed by flow
cytometer (BD FACSVerseTM, USA).
4.4.6 Annexin V binding assay
DU145 cells (1x106) were seeded in six-well plates and allowed to grow overnight. The
medium was then replaced with complete medium containing compound 7f at 200, 400 nM
concentrations and 400 nM concentration of indibulin. After 24 h of treatment, cells from the
supernatant and adherent monolayer cells were harvested by trypsinization, washed with PBS
at 3000 rpm. Then the cells were processed with Annexin V-assay kit (FITC Annexin V
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Apoptosis Detection Kit, BD PharmingenTM) according to the manufacturer’s instruction.
Further, flow cytometric analysis was performed using a flow cytometer (BD FACSVerseTM,
USA).
4.4.7 Measurement of Mitochondrial Membrane Potential
DU145 cells (1×106 cells/mL) were seeded in 6 well plates and allowed to adhere for
overnight. The cells were incubated with 200, 400 nM concentrations of the compound 7f and
400 nM concentration of podophyllotoxin for 24 h. Cells were collected, washed with PBS
and resuspended in solution of JC-1 (2.5 µg/mL) and incubated for 45 min in incubator at 37
°C . The cells were washed twice with PBS and cells were trypsinized, centrifuged and
analysed by flow cytometer (BD FACSVerseTM, USA).
4.4.8 Measurement of Superoxide levels
Mitochondrial superoxide (O2*-) generation was determined by using MitoSOX™ Red dye.
In this assay, DU-145 cells were seeded at a density of 5x105 cells/mL in a 24-well plate and
allowed to adhere overnight. Cells were treated with increasing concentrations of 7f (100,
200 and 400 nM) for 48 h. After the treatment, cells were incubated with a solution of
MitoSOX™ Red mitochondrial superoxide indicator (5 µM) in HBSS (Hank’s balanced salt
solution) for 15 min at 37 °C. Cells were then washed three times with PBS to remove the
excess dye. Images were captured using the fluorescent microscope (BIORAD).
4.4.9 In vitro tubulin polymerization
A fluorescence based in vitro tubulin polymerization assay was performed according to the
manufacturer’s protocol (BK011, Cytoskeleton, Inc.). Briefly, the reaction mixture in a total
volume of 10 µL contained PEM buffer, GTP (1 µM) in the presence or absence of test
compounds 7f (0.5 µM), indibulin (0.5 µM), podophyllotoxin (5 µM). Tubulin
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polymerization was followed by a time dependent increase in fluorescence due to the
incorporation of a fluorescence reporter into microtubules as polymerization proceeds.
Fluorescence emission at 420 nm (excitation wavelength is 360 nm) was measured by using a
Varioscan multimode plate reader (Thermo scientific Inc.). The IC50 value was defined as the
drug concentration required to inhibit 50% of tubulin assembly compared to control. The
reaction mixture for these experiments include: tubulin (3 mg/mL) in PEM buffer, GTP (1
mM), in the presence or absence of test compounds at various concentrations. Polymerization
was monitored by increase in the fluorescence as mentioned above at 37 °C.
4.4.10 Western blotting analysis
Primary antibodies against Bax, Bcl-2, PARP, Cyt c, β-actin and HRP conjugated antirabbit
secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
Equal amounts of protein sample (25 µg/lane) from each experimental group and were
separated by SDS–polyacrylamide gel electrophoresis. Then the protein bands were
transferred to polyvinylidene fluoride membrane (Pierce Biotechnology, Rockford, IL, USA)
for western blotting. Membranes were blocked with 3% bovine serum albumin in TBS buffer
(pH-7.4) and incubated with the indicated antibodies overnight at 4 °C. β-actin (1:1000) was
used for equal loading. After washing the membranes in TBST buffer, membranes were
exposed to the HRP-conjugated secondary antibodies (1:5000) for 2 h at room temperature.
The reactive bands were visualized with chemiluminescent detection reagents (Supersignal
West Pico, Pierce Biotechnology, Rockford, IL, USA). The densitometry analysis of the blots
was performed by using Image J software, NIH, USA.
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Acknowledgements
Authors are thankful to DoP, Ministry of Chemicals & Fertilizers, Govt. Of India, New
Delhi, for the award of NIPER fellowship.
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