Synthesis of thiazole linked indolyl-3-glyoxylamide derivatives as tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.06.025

A series of thiazole linked 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 13d displayed cytotoxicity of IC₅₀ = 93 nM towards DU145 cancer cell line. The most active compound 13d was also tested on RWPE-1 cells and was found to be safe compared to the DU145 cells. The target compounds were also evaluated for their inhibition activity of tubulin polymerization. Further, the treatment of compound 13d on DU145 cells led to the inhibition of cell migration ability. The detailed studies such as acridine orange/ethidium Bromide (AO/EB), DAPI, annexin V-FITC/propidium iodide staining assay suggested that the compound 13d induced apoptosis in DU145 cells. The influence of the cytotoxic compound 13d on the cell cycle distribution was assessed on the DU145 cell line, exhibiting a cell cycle arrest at the G2/M phase. Moreover, the treatment with compound 13d caused collapse of mitochondrial membrane potential and elevated intracellular ROS levels in DU145 cells. The results from molecular modelling studies revealed that these compounds bind at the colchicine binding site of the tubulin. Thus, this new molecular scaffold could be a new lead for the development of anticancer agents that target tubulin.

Full text of DOI:10.1016/j.ejmech.2017.06.025

Accepted Manuscript
Synthesis of thiazole linked indolyl-3-glyoxylamide derivatives as tubulin
polymerization inhibitors
Sravanthi Devi Guggilapu, Lalita Guntuku, T. Srinivasa Reddy, Atulya Nagarsenkar,
Dilep Kumar Sigalapalli, V.G.M. Naidu, Suresh K. Bhargava, Bathini Nagendra Babu
PII:
S0223-5234(17)30471-3
10.1016/j.ejmech.2017.06.025
EJMECH 9520
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 January 2017
Revised Date: 30 May 2017
Accepted Date: 14 June 2017
Please cite this article as: S.D. Guggilapu, L. Guntuku, T.S. Reddy, A. Nagarsenkar, D.K. Sigalapalli,
V.G.M. Naidu, S.K. Bhargava, B.N. Babu, Synthesis of thiazole linked indolyl-3-glyoxylamide derivatives
as tubulin polymerization inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.06.025.
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ACCEPTED MANUSCRIPT
Synthesis of thiazole linked indolyl-3-glyoxylamide derivatives as tubulin polymerization
inhibitors
Sravanthi Devi Guggilapu,a Lalita Guntuku,b T. Srinivasa Reddy,c Atulya Nagarsenkar,a Dilep
Kumar Sigalapalli,a V. G. M. Naidu,b Suresh K. Bhargava,c 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
c Centre for Advanced Materials & Industrial Chemistry (CAMIC), School of Science, RMIT
University, GPO BOX 2476, Melbourne-3001, Australia.

ACCEPTED MANUSCRIPT
Synthesis of thiazole linked indolyl-3-glyoxylamide derivatives as tubulin polymerization
inhibitors
Sravanthi Devi Guggilapu,a Lalita Guntuku,b T. Srinivasa Reddy,c Atulya Nagarsenkar,a Dilep
Kumar Sigalapalli,a V. G. M. Naidu,b Suresh K. Bhargava,c 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
c Centre 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 thiazole linked 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
13d displayed cytotoxicity of IC50 = 93 nM towards DU145 cancer cell line. The most active
compound 13d was also tested on RWPE-1 cells and was found to be safe compared to the
DU145 cells. The target compounds were also evaluated for their inhibition activity of tubulin
polymerization. Further, the treatment of compound 13d on DU145 cells led to the inhibition of
cell migration ability. The detailed studies such as acridine orange/ethidium Bromide (AO/EB),
DAPI, annexin V-FITC/propidium iodide staining assay suggested that the compound 13d
induced apoptosis in DU145 cells. The influence of the cytotoxic compound 13d on the cell
cycle distribution was assessed on the DU145 cell line, exhibiting a cell cycle arrest at the G2/M
phase. Moreover, the treatment with compound 13d caused collapse of mitochondrial membrane
potential and elevated intracellular ROS levels in DU145 cells. The results from molecular
modelling studies revealed that these compounds bind at the colchicine binding site of the
tubulin. Thus, this new molecular scaffold could be a new lead for the development of anticancer
agents that target tubulin.
Keywords: Indolyl-3-glyoxylamide, thiazole, anticancer, apoptosis, tubulin polymerization
inhibitor.
‘

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1.0 Introduction
Microtubules are dynamic polymers of α- and β-tubulin in all eukaryotic cells controlling various
cellular functions, including the movement of organelles, intracellular transportation, and the
formation and maintenance of cell shape [1-3]. Interference with microtubule assembly, either by
inhibition of tubulin polymerization or by blocking microtubule disassembly, leads to an increase
in the number of cells in metaphase arrest. Due to the multiple functions of microtubules in the
cell cycle, tubulin has become an attractive target for the development of compounds useful in
anticancer chemotherapy [4-7]. 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
[8]. Despite the advances in treatment microtubule targeting agents have undoubtedly offered,
existing agents suffer from some significant drawbacks [9a] which often compromise clinical
efficacy and can lead to treatment failure. Drug resistance (either innate or acquired) is a serious
problem [9b], and adverse toxicities such as peripheral neuropathy (PN) can prove dose-limiting,
indicative of a narrow therapeutic window [9c]. This has encouraged medicinal chemists to
develop novel antimitotic agents for cancer therapy.
The indole nucleus is the core structure of a great number of tubulin polymerization inhibitors
including the indolyl-3-glyoxamide D-24851 and the 2-aroylindoles D-64131(Figure 1) [10]. D-
24851, known as indibulin, is a novel synthetic compound that was identified in a cell-based
screening assay to discover cytotoxic drugs. Indibulin destabilizes microtubules and blocks cell
cycle transition specifically at G2-M phase subsequently leading to cell death [11-14].
On the other hand, thiazole derivatives are one of the most revered compounds with many
applications and biological activities attributed to them. Indolylthiazole and its related
heterocyclic systems are found in many natural products. Camalexin, a thiazole-substituted

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indole derivative causes apoptosis in human cancer cell lines via increasing the reactive oxygen
species (ROS) generation and activation of caspases [15, 16]. A series of 4-substituted
methoxybenzoyl-aryl-thiazoles (SMART) compounds exert their anticancer activity through
inhibition of tubulin polymerization via the colchicine binding site. Among this series, a phenyl-
aminothiazole derivative,
(2-((4-fluorophenyl)amino)thiazol-4-yl)(3,4,5
trimethoxyphenyl)methanone was designed which displayed nanomolar range potency against
cancer cell lines via inhibition of tubulin polymerization [17a].
Molecular hybridization is an effective tool to design more active and novel chemical entities by
covalently combining two or more drug pharmacophores into a single molecule [17b]. In this
context, inspired by the structural features of indolyl-3-glyoxylamides and therapeutic
significance of the thiazole nucleus, we envisioned that the merging of these two bioactive
scaffolds in a single chemical entity may lead to the development of promising new leads with
potential anticancer activity [18]. Therefore, owing to our interest in the synthesis of biologically
active scaffolds [19], herein we incorporated thiazole moiety to the indolyl-3-glyoxylamide
structural motif connected through alkyl linker with a view to produce thiazole linked indolyl-3-
glyoxylamide. The synthesized compounds were then assayed for their anti-proliferative activity
in vitro against four human cancer cell lines: DU145 (prostate), PC-3 (prostate), A549 (lung) and
HCT-15 (colon).
<Insert Figure 1 here>
2.0 Results and discussion
2.1 Chemistry

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The synthesized target compounds [13(a-o) and 14(a-e)] comprise two core structural elements:
(i) indolyl-3-glyoxylamide, and (ii) a substituted thiazole moiety. Initially, for the preparation of
the various 4-aryl-1,3-thiazol-2-amine 2(a-c) required for the synthesis of proposed target
compounds, iodine catalyzed condensation-cyclization of thiourea with the respective
acetophenone (1a-c) was carried out at reflux for 3-4 h [20] (Scheme 1). The starting materials,
ethyl 2-amino-5-methylthiazole-4-carboxylate (4) and 5-methoxy-benzothiazol-2-ylamine (6)
were synthesized according to the reported literature [21, 22].
On the other hand, the indolyl-3-glyoxylamides were prepared by treatment of indole (7a) or 5-
bromo indole (7b) with oxalyl chloride at 0 °C in diethyl ether produced 2-(1H-indol-3-yl)-2-
oxoacetyl chloride or 2-(5-bromo-1H-indol-3-yl)-2-oxoacetyl chloride due to the high
nucleophilic character at 3-position of indole and followed by substitution reaction with suitable
amines in presence of mild base triethylamine affording compounds (8a-d) in 78-92% yields
[10]. Furthermore, the N-alkylation of indoles was carried out using ethyl 2-bromoacetate and
methyl 2-chloropropionate in acetonitrile employing potassium carbonate as base to obtain the
desired N-alkyl esters [(9a-d) and 10b]. Later, the esters were hydrolyzed using lithium
hydroxide in methanol at room temperature to generate the acids in quantitative yields [(11a-d)
and 12b]. Finally, the acids [(11a-d) and 12b] were coupled with synthesized amines [(2a-c), 4,
and 6] using HBTU/DMAP in dichloromethane to accomplish the target compounds [13(a-o)
and 14(a-e)].
All the synthesized compounds [13(a-o) and 14(a-e)] were confirmed by using FT-IR, 1H NMR,
13C NMR and HRMS (ESI) spectroscopic techniques. The amide protons in compound 13a-13o
appeared in the range of 13.04–10.71 ppm. All the aromatic protons were found in the range of
8.97-6.95 ppm and the characteristic aliphatic –CH2 protons appeared as singlet. While in

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compound 13c, -COCH2CH3 group, two protons appeared as quartet at 4.20 ppm, three protons
as triplet at 1.22 ppm and the three protons of –CH3 group appeared as singlet at 2.56 ppm. In
compound 13d, three protons of –OCH3 group gave singlet at 3.78 ppm. While compound 13e
displayed piperidine protons in upfield region and spectral data of compounds 13f-13h were in
1
similar pattern to 13e. In compounds 13i-13l, the H NMR displayed the cyclopropyl group
protons as multiplets at 2.93–2.81 ppm (–CH), 1.31–0.60 ppm (–2CH2) and in 13C NMR, for
example 13i, the aliphatic carbons of cyclopropyl group appeared at 79.1 ppm (–CH), 22.4 and
5.4 ppm (–2CH2). The close examination of 13C NMR of compounds 13a-13o revealed carbonyl
groups in range of 186.4–161.7 ppm. All the other aromatic carbon was found in the range of
159.0–104.6 ppm. In case of compound 13c, aliphatic carbon corresponding to ethoxy group
appeared at 60.4, 16.9 ppm and methyl group at 14.0 ppm respectively. The aliphatic carbon
corresponding to the methoxy group in compound 13d was found at 55.6 ppm. The spectral data
of compounds 14a, 14b, 14d, and 14e were in similar pattern like 13e-13h except in the side
chain. In the compounds 14a-14e, the –NCH2CH2 protons of side chain appeared as two triplets.
In compound 14c, the characteristic –CH3 protons appeared at 2.31 ppm. and also the 13C NMR
were in good agreement with the molecular formula.
<Insert Scheme 1 here>
2.2 Pharmacology
2.2.1 In vitro anticancer activity
All the synthesized compounds [13(a-o) and 14(a-e)] were evaluated for their in vitro anti-
proliferative activity against selected human cancer cell lines of prostate (PC-3 and DU-145),

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lung (A549) and colon (HCT-15) by using 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay [23]. The IC50 (µM) values (concentration required to inhibit 50% of
cancer cells growth) of tested compounds [13(a-o) and 14(a-e)] and reference standard
podophyllotoxin has been listed in the Table 1. From analysis of IC50 values it could be inferred
that most of the compounds were sensitive towards prostate (DU145, PC-3) cancer cell lines.
Compounds 13c, 13d, 13g, 13h, 13k, and 13l displayed significant cytotoxicity of IC50 <10µM
in both DU145, PC-3 cell lines. It was also observed that most of the compounds were inactive
towards A-549 and HCT-15 cell lines. Gratifyingly, the compound 13d was found to be most
active in prostate cancer (DU145 and PC-3) cell lines. The compound 13d showed significant
cytotoxicity with IC50 = 93 nM towards DU145 cancer cell line. Most importantly, the
compound 13d was found to be comparatively safe towards normal cell line RWPE-1.
Furthermore, C5 substitution of indolyl-3-glyoxylamide with bromo group (13m-13o) was also
explored. The compounds 13n and 13o were found to be moderately active against DU145 cell
line.
Next in the series of compounds (14a-14e) containing propanamide side chain were inactive
against all the tested cancer cell lines. Furthermore on comparison the IC50 values of compounds
13(e-h) having acetamide side chain with 14(a-e) having propanamide side chain, it could be
inferred that the increase in the alkyl group at N1 position lead to dramatic decrease in the
activity. Interestingly, the compounds containing acetamide side chain substituted with R group
such as 5-methoxybenzo[d]thiazol-2-amine and ethyl 2-amino-5-methylthiazole-4-carboxylate
were found to active (below 10 µM) against all the tested cancer cell lines. Based on the ring
substituents NR3R4, the cytotoxic activity decreases in the order of phenyl > piperidine-1-yl >
cycloprop-1-yl. Moreover, the introduction of Br group at 5th position of indole ring did not

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improve the activity as it is evident from comparison of MTT results of compounds 13a with
13m and, 13d with 13o.
2.2.2 Effect on tubulin polymerization
Based on the obtained IC50 values against the four cancer cell lines, the synthesized analogues
were evaluated for in vitro inhibitory effects on tubulin polymerization at 5 µM concentration.
Paclitaxel and podophyllotoxin were used as reference and the results expressed as a percentage
are summarized in Table 1. As shown the Figure 2, the compound 13d with the potent
cytotoxicity also displayed significant anti-tubulin activity among the tested compounds with an
inhibition percentage of 68.5% in comparison to the podophyllotoxin (78.0%). However, the
compounds 13a, 13c, 13h, 13k, 13o, 14d also exhibited moderate to low tubulin polymerization
inhibition with 23.5%, 58.4%, 38.5%, 21.3%, 23.3% and 12.2%, respectively. These results are
in correlation with the cytotoxicity of the compounds and suggest that the compounds may
inhibit the cell growth through tubulin polymerization inhibition. Based on the aforementioned
results of MTT assay and tubulin polymerization assay, it could be inferred that 4, 5-
disubstituted thiazole or fused thiazole ring such as benzthiazole scaffold are vital for significant
activity.
Since compound 13d displayed excellent anti-proliferative activity and significant tubulin
polymerization inhibitory activity in the initial screening, we selected it as the optimized
compound in further studies to determine whether the observed cell death induced by compound
13d was due to apoptosis.
<Insert Table 1 here>
<Insert Figure 2 here>

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2.2.3 Wound healing assay
Metastasis is a multistep process involving cancer cell motility and invasion which accounts for
more than 90% of cancer related deaths. Microtubule or tubulin network plays an important role
in cell motility and migration. As the compound 13d inhibits tubulin polymerization, we have
investigated their migration inhibition capacity through wound healing assay [24]. In this assay,
standardized scratches (wounds) were made in confluent monolayers of DU145 cells and
incubated with increasing concentrations of the compound 13d. The number of cells migrated in
to the wound area were captured using phase contrast microscope after 0, 12 and 24 h incubation.
As shown in the Figure 3, the compound 13d treatment resulted in significant inhibition of
migration capacity of DU145 cells and the effect was more prominent after 24 h. For instance,
compound 13d treatment at 100, 200 and 400 nM concentration led to strong inhibition of
migration in to the scratch area by 74.8%, 44.0% and 34.8% respectively in relative comparison
to control after 24 h. It can be inferred from the results that the compound 13d suppresses the
migration potential of the DU145 cells in dose dependent manner.
<Insert Figure 3 here>
2.2.4 Acridine orange–ethidium bromide (AO–EB) staining
Acridine orange/ethidium bromide (AO/EB) fluorescent staining assay was performed to
distinguish the live, apoptotic and necrotic cells [25]. 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 healthy morphology with intact nuclear architecture and appeared green in colour.
Fluorescence microscopic images of 13d treated DU145 cells have clearly demonstrated

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morphological changes which are the characteristic features of apoptotic cells such as cell
shrinkage, membrane blebbing and apoptotic body formation. This confirms that the compound
13d induced cell death in DU145 cancer cells.
<Insert Figure 4 here>
2.2.5 DAPI Nucleic Acid Staining
DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye that binds sturdily to nucleus and
detect the nuclear damage or chromatin condensation. The DAPI stains the apoptotic cells as
bright colored owing to the condensed nucleus which is a distinctive apoptotic characteristic.
Hence, it was of our interest to detect nuclear damage or chromatin condensation persuaded by
the compound 13d in DU145 cells. DAPI staining technique was performed according to earlier
reported method [26]. The results from the Figure 5 illustrated that the nuclear structure of
untreated cells was intact whereas compound 13d treated cells exhibited condensed, or
fragmented nuclei.
<Insert Figure 5 here>
2.2.6 Cell cycle analysis
As most microtubule polymerization inhibitors disrupt cell mitosis and exert cell cycle arrest
effects [27], we examined the effect of the compound 13d on cell cycle using propidium iodide
staining method [28]. DU145 cells were treated with increasing concentrations of compound 13d
for 24 h. They were stained with propidium iodide and samples were further analysed by flow
cytometry. Treatment with the compound 13d at 100, 200 and 400 nM in DU145 cells displayed
rise in G2/M population from 25.88% (control) to 30.15%, 34.07% and 50.9% respectively in a

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dose dependent manner (Figure 6). These results clearly indicate G2/M cell cycle arrest by the
test compound suggesting the induction of apoptotic death in the treated cells.
<Insert Figure 6 here>
2.2.7 Annexin V binding assay
The apoptosis inducing effect of compound 13d on DU145 cancer cells was further investigated
using annexin V-FITC/propidium iodide staining assay [29]. DU145 cells were treated with 200
and 400 nM of compound 13d for 24 h and stained with Annexin V-FITC and propidium iodide,
and samples were analysed by flow cytometry.
As depicted in Figure 7, 13d treated DU145 cells demonstrated rise in the total
percentage of apoptotic (early and late apoptotic cells- Annexin V +ve cells) and dead
cells from 14.29% (untreated) to 15.28% and 54.53% respectively in a dose dependent
manner.
<Insert Figure 7 here>
2.2.8 Analysis of Mitochondrial Membrane Potential (DΨm)
Literature reports indicate that the loss or collapse of mitochondrial membrane potential may be
an early event in the process of apoptosis [30]. Thus, the effect of compound 13d on
mitochondrial membrane potential (DΨm) was determined by staining with lipophilic cationic
JC-1 dye [31]. 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, and 400 nM of 13d for 24 h and stained by JC-1

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dye. Flow cytometric analysis of the treated cells clearly displayed increase in depolarised cell
population (P3) from control (22.28%) to 57.05 % and 64.65% respectively in concentration
dependent manner (Figure 8). Thus, the results clearly indicate loss of mitochondrial membrane
potential by the compound 13d and suggest the involvement of mitochondria dependent
apoptotic pathway in their mechanism of action.
<Insert Figure 8 here>
2.2.9 Measurement of intracellular mitochondrial superoxide levels
In order to confirm the role of ROS in compound 13d induced cell death, we further investigated
the mitochondrial superoxide levels using MitoSOX™ red, a novel fluorogenic dye that
accumulates in mitochondria and selectively detects superoxide in the mitochondria of live cells.
MitoSOX™ red is quickly oxidized by superoxide species and the oxidized product emits red
fluorescence upon binding with nucleic acids [32]. As shown in the Figure 9, the treatment with
compound 13d led to a dose-dependent increase in red fluorescence in DU145 cancer cells. The
control untreated cells displayed weak red fluorescence whereas, the cells treated with increasing
concentrations of 100, 200 and 400 nM of compound 13d showed strong increasing red
fluorescence suggesting involvement of superoxide generation in compound 13d induced cell
death.
<Insert Figure 9 here>
3.0 Molecular docking
To rationalize the experimental results obtained, molecular docking studies were performed on
the synthesized indolyl-3-glyoxylamide derivatives and co-crystallized ligand against the

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colchicine binding site of tubulin crystal structure (PDB ID: 1SA0) [33]. Molecular docking
studies were performed by using GLIDE docking module of Schrödinger suite 2014-3 [34].
From the docking study, it was observed that the top ranked conformation of all the selected
compounds were well accommodated inside the colchicine binding site of the tubulin. In case of
the most active compound 13d has shown four hydrogen bond interactions with the catalytically
active residues Ala250, Asp251, Lys254 and Lys352. Figure 10 illustrate the predicted binding
mode and the detailed protein−inhibitor interactions of compound 13d with the colchicine
binding site of tubulin. Computationally it is worthy to note that the glyoxyl group attached to
indole scaffold established three hydrogen bonds with the important active site residues Ala250,
Asp251 and Lys254. Additionally, several hydrophobic interactions were observed between the
compound 13d and other active site residues, like Ala180, Val181, Leu248, Ala250, Leu252,
Leu255, Met259, Val315, Ala316 and Val351 that stabilized the binding of the compound 13d in
the active site of tubulin. Similar interactions were found in compound 13c with active site
residues Gln11, Lys254, Lys352 and some of the hydrophobic residues such as Ala12, Ile171,
Ala180, Val181, Tyr224, Leu248, Ala250, Leu255, Met259, Val315, Ala316 and Val351.
Likewise, the compounds 13a, 13h, 13k and 13o also displayed protein-ligand interactions with
the active site residues of tubulin. On the basis of our molecular docking study, it was found that
the synthesized indolyl-3-glyoxylamide derivatives display biological activity via occupying the
colchicine binding site of the tubulin and forming favorable interactions with its key residues.
<Insert Figure 10 here>
4.0 Conclusion

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In the current study, a series of thiazole linked 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 13d was found to be the most active against DU145 (prostate
cancer) cell lines. Most importantly, the compound 13d was found to be comparatively safe
towards normal cell line RWPE-1. The treatment of DU145 cells with 13d 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 13d induced apoptosis in DU145 cells.
The cell cycle analysis confirmed G2/M phase of cell cycle arrest in a dose dependent manner
and inhibited 68.5% tubulin polymerization compared to control at 5 µM. Moreover, the
treatment with compound 13d caused collapse of mitochondrial membrane potential and
elevated intracellular ROS levels in DU145 cells. The molecular modelling studies provided
insight into the binding mode of the synthesized compounds in active site of tubulin. Overall, the
current studies demonstrated that indolyl-3-glyoxylamide derivatives could also offer potential
anti-tubulin leads.
5.0 Experimental protocols
5.1 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
plates was achieved UV light. H and 13C NMR spectra were recorded on bruker 500 MHz

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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.
5.2 General procedure for the synthesis of N- substituted 2-oxo-2-(1-(2-oxo-2-(4-arylthiazol-
2-ylamino)ethyl)-1H-indol-3-yl)-N-phenylalkylamide [(13a-13o) and (14a-14e)]
The solution of acid [11(a-d), 12b] (0.5 mmol) and O-(benzotriazol-1-yl)-N,N,N',N'-
tetramethyluronium hexafluorophosphate (0.54 mmol) in dry dichloromethane (10 ml) and
dimethyl aminopyridine (0.1 mmol) was added to substituted thiazol-2-amine [2(a-c), 4, or 6]
(0.5 mmol) at 0 °C. The reaction mixture was stirred at room temperature for 16-20 hours. Later
the reaction was quenched with water (5.0 ml), extracted with dichloromethane (10.0 ml x 2),
and evaporated under reduced pressure. The residue was subjected to silica gel chromatography
to give compound [(13a-13o) and (14a-14e)] in good yields.
5.2.1 2-oxo-2-(1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-1H-indol-3-yl)-N-phenylacetamide (13a)
Orange solid; yield: 78%; mp 283-285 o C; IR (KBr): 3352, 1698, 1580, 1511, 1468, 1385, 1283,
1168, 959, 778, 624 cm-1; 1H NMR (500 MHz, DMSO–d6): δ 12.65 (1H, bs), 10.71 (1H, s), 8.90
(1H, s), 8.32–8.29 (1H, m), 7.87–7.82 (2H, m), 7.59–7.52 (1H, m), 7.51–7.47 (1H, d, J = 3.6
Hz), 7.40–7.30 (4H, m), 7.25–7.21 (1H, m), 7.16–7.10 (1H, m), 5.40 (2H, s); 13C NMR (125
MHz, DMSO–d6): 182.4, 162.7, 143.1, 138.4, 137.5, 129.2, 126.9, 124.7, 124.3, 123.6, 121.8,

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120.7, 114.4, 111.9, 111.5, 49.2; HRMS (ESI): m/z calcd for C21H16N4O3S+ [M+H]+ 405.1016;
found: 405.1046.
5.2.2 2-oxo-2-(1-(2-oxo-2-(4-phenylthiazol-2-ylamino)ethyl)-1H-indol-3-yl)-N-
phenylacetamide (13b)
o
Orange solid; yield: 80%; mp 223-225 C; IR (KBr): 1686, 1592, 1465, 1273, 1162, 966, 778,
1
690 cm-1; H NMR (500 MHz, DMSO–d6): δ 12.84 (1H, bs), 10.71 (1H, s), 8.92 (1H, s), 8.35–
8.30 (1H, m), 7.92–7.83 (4H, m), 7.62–7.58 ( 2H, m), 7.47–7.40 (2H, m), 7.38–7.30 (5H, m),
7.14 (1H, t, J = 7.4 Hz), 5.42 (2H, s); 13C NMR (125 MHz, DMSO–d6): 181.9, 170.3, 166.0,
162.2, 157.7, 148.9, 142.6, 138.0, 137.0, 134.1, 128.7, 127.8, 126.4, 125.6, 124.2, 123.8, 123.1,
121.4, 120.2, 111.5, 111.1, 108.3, 48.9; HRMS (ESI): m/z calcd for C27H20N4O3S+ [M+H]+
481.1329; found: 481.1341.
5.2.3 Ethyl-5-methyl-2-(2-(3-(2-oxo-2-(phenylamino)acetyl)-1H-indol-1-yl)-
acetamido)thiazole-4-carboxylate (13c)
Yellow solid; yield: 75%; mp 150-152 o C; IR (KBr): 3302, 1711, 1683, 1511, 1467, 1444, 1315,
1259, 1167, 942, 866, 779 cm-1; 1H NMR (500 MHz, DMSO–d6): δ 10.71 (1H, s), 8.90 (1H, s),
8.34–8.28 (1H, m), 7.85 (2H, d, J = 8.0 Hz), 7.69–7.56 (1H, m), 7.40–7.29 (4H, m), 7.16–7.10
(1H, m), 5.43 (2H, s), 4.20 (2H, q, J = 7.1 Hz), 2.56 (3H, s), 1.22 (3H, t, J = 7.1 Hz); 13C NMR
(125 MHz, DMSO–d6): 181.8, 166.7, 162.1, 161.8, 159.0, 156.0, 142.5, 137.9, 137.0, 128.6,

ACCEPTED MANUSCRIPT
126.3, 124.2, 123.7, 123.0, 121.1, 120.1, 114.3, 111.4, 111.0, 60.4, 48.8, 16.9, 14.0; HRMS
(ESI): m/z calcd for C25H22N4O5S+ [M+H]+ 491.1384; found: 491.1389.
5.2.4 2-(1-(2-(5-methoxybenzo[d]thiazol-2-ylamino)-2-oxoethyl)-1H-indol-3-yl)-2-oxo-N-
phenylacetamide (13d)
Yellow solid; yield: 74%; mp 209-211 o C; IR (KBr): 3376, 1681, 1572, 1468, 1445, 1389, 1261,
1
831, 780 cm-1; H NMR (500 MHz, DMSO–d6): δ 12.77 (1H, bs), 10.72 (1H, s), 8.93 (1H, s),
8.34–8.29 (1H, m), 7.86 (2H, d, J = 7.9 Hz), 7.68–7.61 (2H, m), 7.58 (1H, d, J = 2.4 Hz), 7.40–
7.32 (4H, m), 7.14 (1H, t, J = 7.4 Hz), 7.03 (1H, dd, J = 8.9, 2.5 Hz), 5.45 (2H, s), 3.78 (3H, s);
13C NMR (125 MHz, DMSO–d6): 181.9, 166.4, 162.2, 156.2, 155.3, 142.6, 138.0, 137.1, 132.7,
128.7, 126.4, 124.2, 123.8, 123.1, 121.3, 120.2, 115.1, 111.5, 111.1, 104.7, 55.6, 48.9; HRMS
(ESI): m/z calcd for C26H20N4O4S+ [M+H]+ 485.1278; found: 485.1305.
5.2.5 2-(3-(2-oxo-2-(piperidin-1-yl)acetyl)-1H-indol-1-yl)-N-(thiazol-2-yl)acetamide (13e)
Yellow solid; yield: 76%; mp 240-243 o C; IR (KBr): 2931, 1693, 1571, 1437, 1351, 1191, 1065,
955, 844,754 cm1;1H NMR (500 MHz, DMSO–d6): δ 12.66 (1H, bs), 8.29 (1H, s), 8.16–8.12
(1H, m), 7.61–7.55 (1H, m), 7.51 (1H, d, J = 3.6 Hz), 7.35–7.29 (2H, m), 7.25 (1H, d, J = 3.5
Hz), 5.37 (2H, s), 3.59–3.56 (2H, m), 3.31–3.28 (2H, m), 1.61 (4H, bs), 1.46 (2H, bs); 13C NMR
(125 MHz, DMSO–d6): δ 186.4, 165.4, 140.7, 137.5, 125.0, 123.7, 122.8, 121.0, 113.88, 112.6,
111.0, 48.6, 46.4, 41.2, 25.7, 24.9, 23.8; HRMS (ESI): m/z calcd for C20H20N4O3S+ [M+H]+
397.1339; found: 397.1316.
5.2.6 2-(3-(2-oxo-2-(piperidin-1-yl)acetyl)-1H-indol-1-yl)-N-(4-phenylthiazol-2-yl)acetamide
(13f)

ACCEPTED MANUSCRIPT
Orange solid; yield: 71%; mp 245-248 o C; IR (KBr): 2938, 1703, 1614, 1464, 1387, 1280, 1176,
1072, 967, 849 cm1; 1H NMR (500 MHz, DMSO–d6): δ 12.83 (1H, bs), 8.30 (1H, s), 8.16 (1H, d,
J = 6.8 Hz), 7.91 (2H, d, J = 7.5 Hz), 7.66 (1H, s), 7.60 (1H, d, J = 7.0 Hz), 7.45 (2H, t, J = 7.6
Hz), 7.36-7.30 (3H, m), 5.40 (2H, s), 3.58 (2H, bs), 2.69 (2H, bs), 1.62 (4H, bs), 1.46 (2H, bs);
13C NMR (125 MHz, DMSO–d6): δ 186.4, 165.8, 165.4, 157.3, 148.9, 140.7, 137.5, 134.0,
128.7, 127.8, 125.6, 123.7, 122.9, 121.0, 112.6, 111.1, 108.4, 48.7, 46.4, 41.2, 38.1, 25.7, 24.9,
23.8; HRMS (ESI): m/z calcd for C26H24N4O3S+ [M+H]+ 473.1642; found: 473.1679.
5.2.7 Ethyl 5-methyl-2-(2-(3-(2-oxo-2-(piperidin-1-yl)acetyl)-1H-indol-1-
yl)acetamido)thiazole-4-carboxylate (13g)
Yellow solid; yield: 72%; mp 210-213 o C; IR (KBr): 2937, 1704, 1615, 1555, 1465, 1136, 1014,
1
749 cm1; H NMR (500 MHz, DMSO-d6): δ 13.04 (1H, s), 8.28 (1H, s), 8.17–8.13 (1H, m),
7.60–7.56 (1H, m), 7.34–7.29 (2H, m), 5.39 (2H, s), 4.21 (2H, q, J = 7.1 Hz), 3.58 (2H, s), 3.30
(2H, bs), 2.57 (3H, s), 1.61 (4H, bs), 1.46 (2H, bs), 1.24 (3H, t, J = 7.1 Hz); 13C NMR (125 MHz,
DMSO-d6) δ 13C NMR (125 MHz, DMSO–d6): δ 186.4, 165.3, 161.8, 140.6, 137.5, 125.0, 123.7,
122.9, 120.9, 114.3, 112.6, 111.1, 60.5, 48.8, 46.4, 41.2, 25.7, 24.9, 23.8, 16.9, 14.0; HRMS
(ESI): m/z calcd for C24H26N4O5S+ [M+H]+ 483.1697; found: 483.1725.
5.2.8 N-(5-methoxybenzo[d]thiazol-2-yl)-2-(3-(2-oxo-2-(piperidin-1-yl)acetyl)-1H-indol-1-
yl)acetamide (13h):
Yellow solid; yield: 68%; mp 219-222 o C; IR (KBr): 2920, 1713, 1604, 1526, 1364, 1235, 1227,
1175, 977, 846, 7492 cm1;1H NMR (500 MHz, DMSO–d6): δ 12.77 (1H, bs), 8.31 (1H, s), 8.17–
8.14 (1H, m), 7.68 (1H, d, J = 8.9 Hz), 7.63–7.60 (1H, m), 7.57 (1H, d, J = 2.6 Hz ), 7.35–7.31
(2H, m), 7.04-7.02 (1H, m, J = 8.9, 2.6 Hz), 5.42 (2H, s), 3.79 (3H, s), 3.59 (2H, m), 3.33 (2H,
m), 1.61 (4H, bs), 1.46 (2H, bs); 13C NMR (125 MHz, DMSO–d6): δ 186.4, 165.4, 156.2, 140.6,

ACCEPTED MANUSCRIPT
137.5, 125.0, 123.7, 122.8, 120.9, 115.0, 112.6, 111.1, 104.6, 55.5, 48.7, 46.4, 41.2, 25.7, 24.9,
23.7; HRMS (ESI): m/z calcd for C25H24N4O4S+ [M+H]+ 477.1591; found: 477.1589.
5.2.9 N-cyclopropyl-2-oxo-2-(1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-1H-indoly-3-yl)acetamide
(13i)
Orange solid; yield: 67%; mp 205-207 o C; IR (KBr): 3334, 2901, 1697, 1579, 1495, 1354, 1282,
1
1164, 839, 749 cm-1; H NMR (500 MHz, DMSO–d6): δ 12.67 (1H, bs), 8.87 (1H, s), 8.80 (1H,
d, J= 4.9 Hz ), 8.28–8.22 (1H, m), 7.58–7.54 (1H, m), 7.51 (1H, t, J= 4.4 Hz), 7.34–7.28 (2H,
m), 7.25 (1H, d, J= 3.5 Hz), 5.39 (2H, s), 2.90–2.81 (1H, m), 0.72–0.67 (2H, m), 0.66–0.60 (2H,
m); 13C NMR (125 MHz, DMSO–d6): 182.2, 165.6, 164.8, 157.5, 142.3, 136.9, 126.4, 123.6,
122.9, 121.3, 113.9, 111.5, 110.9, 79.1, 48.7, 22.4, 5.4; HRMS m/z calcd for C18H16N4O3S+
[M+H]+: 369.1016; found: 369.1023.
5.2.10
N-cyclopropyl-2-oxo-2-(1-(2-oxo-2-(4-phenylthiazol-2-ylamino)ethyl)-1H-indoly-3-
yl)acetamide (13j)
White solid; yield: 68%; mp 213-215 o C; IR (KBr): 3432, 2922, 1677, 1543, 1463, 1385, 1282,
1
1072, 904, 736 cm-1; H NMR (500 MHz, DMSO–d6): δ 12.84 (1H, bs), 8.88 (1H, s), 8.81 (1H,
d, J= 4.8 Hz ), 8.28–8.22 (1H, m), 7.92 (2H, d, J= 7.3 Hz ), 7.66 (1H, s), 7.61–7.56 (1H, m), 7.45
(2H, t, J= 7.7 Hz), 7.38–7.28 (3H, m), 5.42 (2H, s), 2.91–2.82 (1H, m), 0.74–0.68 (2H, m), 0.67–
13
0.61 (2H, m); C NMR (125 MHz, DMSO–d6): 182.2, 165.9, 164.8, 157.3, 148.9, 142.3, 136.9,
134.0, 128.7, 127.8, 126.4, 125.6, 123.6, 122.9, 121.3, 111.5, 110.9, 108.4, 79.1, 48.7, 22.4, 5.4;
HRMS (ESI): m/z calcd for C24H20N4O3S+ [M+H]+ 445.1329; found: 445.1343.
5.2.11
Ethyl
2-(2-(3-(2-(cyclopropylamino)-2-oxoacetyl)-1H-indol-1-yl)acetamido)-5-
methylthiazole-4-carboxylate (13k)

ACCEPTED MANUSCRIPT
o
Creamish white solid; yield: 69%; mp 162-164 C; IR (KBr): 3413, 2940, 1715, 1684, 1536,
1469, 1385, 1362, 1169, 1015, 907, 746 cm-1; 1H NMR (500 MHz, DMSO–d6): δ 13.04 (1H, bs),
8.87 (1H, s), 8.80 (1H, d, J= 5.0 Hz ), 8.28–8.22 (1H, m), 7.98 (1H, d, J= 8.5 Hz ), 7.46–7.38
(1H, m), 7.35–7.27 (1H, m), 5.42 (2H, s), 4.21 (2H, q, J= 7.1 Hz), 2.93–2.81 (1H, m), 2.57 (3H,
s), 1.24 (3H, t, J=7.1 Hz), 0.75–0.67 (2H, m), 0.66–0.61 (2H, m); 13C NMR (125 MHz, DMSO–
d6): 182.2, 164.8, 161.9, 142.3, 136.9, 127.7, 127.2, 126.3, 124.4, 123.6, 122.9, 121.3, 111.6,
110.9, 79.1, 60.5, 48.8, 22.4, 16.9, 14.0, 5.4; HRMS (ESI): m/z calcd for C22H22N4O5S+ [M+H]+
455.1384; found: 455.1393.
5.2.12 N-cyclopropyl-2-(1-(2-(5-methoxybenzo[d]thiazol-2-ylamino)-2-oxoethyl)-1H-indol-
3-yl)-2-oxoacetamide (13l)
Orange solid; yield: 72%; mp 135-137 o C; IR (KBr): 3353, 2920, 1671, 1573, 1224, 1180, 972,
1
831, 751 cm-1; H NMR (500 MHz, DMSO–d6): δ 12.78 (1H, bs), 8.89 (1H, s), 8.81 (1H, d, J=
4.8 Hz ), 8.33–8.18 (1H, m), 7.98 (1H, d, J= 8.4 Hz ), 7.74–7.65 (1H, m), 7.64–7.52 (1H, m),
7.41 (1H, t, J= 7.3 Hz ), 7.34–7.28 (1H, m), 7.07–6.95 (1H, m), 5.44 (2H, s), 3.79 (3H, s), 2.90–
2.82 (1H, m), 1.31–1.15 (2H, m), 0.73–0.62 (2H, m); 13C NMR (125 MHz, DMSO–d6): 182.2,
164.8, 156.2, 142.3, 136.9, 127.2, 126.3, 124.4, 123.6, 122.9, 121.3, 115.0, 111.6, 110.9, 109.5,
104.6, 79.1, 55.5, 48.8, 38.1, 22.4, 5.4; HRMS (ESI): m/z calcd for C23H20N4O4S+ [M+H]+
449.1278; found: 449.1269.
5.2.13
2-(5-bromo-1-(2-oxo-2-(thiazol-2-ylamino)ethyl)-1H-indoly-3-yl)-2-oxo-N-
phenylacetamide (13m)
Yellow solid; yield: 73%; mp 174-176 o C; IR (KBr): 3641, 2961, 1690, 1591, 1512, 1385, 1283,
1
1163, 1068, 828, 651 cm-1; H NMR (500 MHz, DMSO–d6): δ 12.66 (1H, bs), 10.72 (1H, s),

ACCEPTED MANUSCRIPT
8.95 (1H, s), 8.42 (1H, s), 7.85 (2H, d, J= 7.7 Hz), 7.62 (1H, d, J= 8.8 Hz), 7.49 (2H, dd, J= 7.1,
2.8 Hz), 7.37 (2H, t, J= 7.9 Hz), 7.24 (1H, d, J= 3.6 Hz), 7.14 (1H, t, J= 7.4 Hz), 5.42 (2H, s);
13C NMR (125 MHz, DMSO–d6): 182.3, 163.2, 144.0, 138.4, 136.5, 129.2, 128.6, 126.9, 124.8,
123.9, 120.7, 116.4, 114.4, 114.0, 111.4, 49.5; HRMS (ESI): m/z calcd for C21H15BrN4O3S+
[M+2H]+ 485.0106; found: 485.0104.
5.2.14
2-(5-bromo-1-(2-oxo-2-(4-p-tolylthiazol-2-ylamino)ethyl)-1H-indoly-3-yl)-2-oxo-N-
phenylacetamide (13n)
Yellow solid; yield: 75%; mp 116-118 o C; IR (KBr): 3441, 2920, 1683, 1592, 1443, 1387, 1280,
1168, 966, 780 cm-1; 1H NMR (500 MHz, DMSO–d6): δ 12.81 (1H, bs), 10.74 (1H, s), 8.97 (1H,
s), 8.44 (1H, d, J= 1.4 Hz ), 7.86 (2H, d, J= 8.1 Hz), 7.80 (2H, d, J= 8.1 Hz), 7.58 (1H, s), 7.51
(1H, dd, J= 8.7, 1.9 Hz), 7.38 (2H, t, J= 7.9 Hz), 7.15 (1H, t, J= 7.4 Hz), 7.05 (1H, dd, J= 8.8,
2.5 Hz), 5.45 (2H, s), 2.33 (3H, s); 13C NMR (125 MHz, DMSO–d6): 181.7, 165.6, 161.7, 157.1,
148.9, 143.4, 137.8, 137.1, 136.0, 131.3, 129.2, 128.6, 128.1, 126.3, 125.5, 124.3, 123.3, 120.2,
115.9, 113.5, 110.8, 107.4, 48.9, 20.7; HRMS (ESI): m/z calcd for C28H21BrN4O3S+ [M+H]+
573.0591; found: 573.0590.
5.2.15 2-(5-bromo-1-(2-(5-methoxybenzo[d]thiazol-2-ylamino)-2-oxoethyl)-1H-indol-3-yl)-2-
oxo-N-phenylacetamide (13o)
Yellow solid; yield: 65%; mp 225-227 o C; IR (KBr): 3355, 2920, 1698, 1637, 1574, 1469, 1395,
1262, 1166, 979, 868, 754 cm-1; 1H NMR (500 MHz, DMSO–d6): δ 12.81 (1H, bs), 10.74 (1H,
s), 8.97 (1H, s), 8.44 (1H, d, J= 1.9 Hz ), 7.85 (2H, d, J= 8.0 Hz), 7.80 (2H, d, J= 8.0 Hz), 7.67
(1H, t, J= 7.9 Hz), 7.57 (1H, d, J= 2.4 Hz), 7.51 (1H, dd, J= 8.7, 1.8 Hz), 7.38 (2H, t, J= 7.9 Hz),
7.15 (2H, t, J= 7.4 Hz), 7.04 (1H, dd, J= 8.9, 2.5 Hz), 5.47 (2H, s), 3.79 (3H, s); 13C NMR (125

ACCEPTED MANUSCRIPT
MHz, DMSO–d6): 181.7, 166.2, 161.7, 156.1, 143.4, 137.8, 136.0, 128.6, 128.1, 126.3, 124.3,
123.4, 120.2, 115.9, 115.0, 113.4, 110.8, 104.6, 55.5, 38.1; HRMS (ESI): m/z calcd for
C26H19BrN4O4S+ [M+2H]+ 565.0368; found: 565.0362.
5.2.16
(14a)
3-(3-(2-oxo-2-(piperidin-1-yl)acetyl)-1H-indol-1-yl)-N-(thiazol-2-yl)propanamide
Yellow solid; yield: 71%; mp 195-198 o C; IR (KBr): 3210, 2925, 1673,1581, 1530, 1387, 1277,
1
1169, 1067, 840,740 cm1; H NMR (500 MHz, DMSO–d6): δ 12.19 (1H, bs), 8.12 (1H, d, J =
7.7 Hz), 8.09 (1H, s), 7.73 (1H, d, J = 8.1 Hz), 7.44 (1H, d, J = 3.5 Hz), 7.35 (1H, t, J = 7.6 Hz),
7.30 (1H, t, J = 7.4 Hz), 7.22 (1H, d, J = 3.4 Hz), 4.65 (2H, t, J = 6.5 Hz), 3.54 (2H, bs), 3.18
(2H, bs), 3.03 (2H, t, J = 6.6 Hz), 1.56 (4H, bs), 1.33 (2H, bs); 13C NMR (125 MHz, DMSO–d6):
δ 186.3, 168.6, 165.4, 157.6, 139.1, 137.6, 136.6, 125.2, 123.7, 123.0, 121.1, 113.5, 112.2, 111.3,
46.3, 42.2, 41.1, 35.1, 25.7, 25.0, 23.8; HRMS (ESI): m/z calcd for C21H22N4O3S+ [M+H]+
411.1485; found: 411.1487.
5.2.17
3-(3-(2-oxo-2-(piperidin-1-yl)acetyl)-1H-indol-1-yl)-N-(4-phenylthiazol-2-
yl)propanamide (14b)
Creamish white solid; yield: 74%; mp 222-225 o C; IR (KBr): 3640, 2941, 1624, 1524, 1445,
1389, 1276, 1173, 1013, 991, 836, 743 cm1; 1H NMR (500 MHz, DMSO–d6): δ 12.37 (1H, bs),
8.13 (1H, d, J = 7.6 Hz), 8.09 (1H, s), 7.87–7.84 (2H, m), 7.76–7.73 (1H, m), 7.64 (1H, s), 7.43–
7.39 (2H, m), 7.37–7.34 (1H, m), 7.33–7.28 (2H, m), 4.68 (2H, t, J = 6.6 Hz), 3.53 (2H, bs), 3.17
(2H, bs), 3.06 (2H, t, J = 6.7 Hz), 1.53 (4H, bs), 1.30 (2H, bs); 13C NMR (125 MHz, DMSO–d6):
δ 186.2, 168.9, 165.4, 157.4, 148.7, 139.0, 136.6, 134.1, 128.6, 127.7, 125.5, 125.2, 123.7, 122.9,
121.1, 112.2, 111.3, 108.0, 46.2, 42.2, 41.0, 35.1, 25.7, 24.9, 23.7; HRMS (ESI): m/z calcd for
C27H26N4O3S+ [M+H]+ 487.1798; found: 487.1798.

ACCEPTED MANUSCRIPT
5.2.18 3-(3-(2-oxo-2-(piperidin-1-yl)acetyl)-1H-indol-1-yl)-N-(4-p-tolylthiazol-2-
yl)propanamide (14c)
Yellowish white solid; yield: 70%; mp 190-193 oC; IR (KBr): 3565, 2938, 1614, 1552, 1463,
1
1325, 1274, 1172, 841, 742, 557 cm1; H NMR (500 MHz, DMSO–d6): δ 12.34 (1H, bs), 8.13
(1H, d, J = 7.6 Hz), 8.09 (1H, s), 7.75 (2H, d, J = 8.1 Hz), 7.56 (1H, s), 7.39–7.28 (3H, m), 7.21
(2H, d, J = 8.2 Hz), 4.67 (2H, t, J = 6.6 Hz), 3.53 (2H, bs), 3.16 (2H, bs), 3.05 (2H, t, J = 6.6
Hz), 2.31 (3H, s), 1.53 (4H, bs), 1.29 (2H, bs). 13C NMR (125 MHz, DMSO–d6): δ 186.2, 168.8,
165.4, 157.3, 148.8, 139.0, 137.0, 136.6, 131.4, 129.2, 125.4,125.2, 123.6, 122.9, 121.1, 112.2,
111.2, 107.1, 46.2, 42.2, 41.0, 38.1, 35.1, 25.7, 24.9, 23.7, 20.7; HRMS (ESI): m/z calcd for
C28H28N4O3S+ [M+H]+ 501.1955; found: 501.1960.
5.2.19 Ethyl 5-methyl-2-(3-(3-(2-oxo-2-(piperidin-1-yl)acetyl)-1H-indol-1-
yl)propanamido)thiazole-4-carboxylate (14d)
o
Creamish white solid; yield: 68%; mp 178-181 C; IR (KBr): 3164, 2932, 1703, 1622, 1643,
1526, 1388, 1318, 1197, 1089, 957, 856, 790 cm1; 1H NMR (500 MHz, DMSO–d6): δ 12.62 (1H,
bs), 8.12 (1H, d, J = 7.7 Hz), 8.08 (1H, s), 7.73 (1H, d, J = 8.2 Hz), 7.38–7.33 (1H, m), 7.32–
7.28 (1H, m), 4.66 (2H, t, J = 6.6 Hz), 4.24 (2H, q, J = 7.1 Hz), 3.54 (2H, bs), 3.18 (2H, bs), 3.05
(2H, t, J = 6.6 Hz), 2.51 (3H, s), 1.57 (4H, bs), 1.30 (2H, bs), 1.27 (3H, t, J = 7.1 Hz); 13C NMR
(125 MHz, DMSO–d6): δ 186.2, 169.6, 165.4, 161.9, 159.0, 156.0, 139.3, 136.5, 125.2, 123.6,
122.9, 121.1, 114.0, 112.2, 111.2, 60.4, 46.2, 42.0, 41.0, 35.1, 25.7, 24.9, 23.7, 16.8, 14.0;
HRMS (ESI): m/z calcd for C25H28N4O5S+ [M+H]+ 497.1853; found: 497.1848.
5.2.20 N-(5-methoxybenzo[d]thiazol-2-yl)-3-(3-(2-oxo-2-(piperidin-1-yl)acetyl)-1H-indol-1-
yl)propanamide (14e)

ACCEPTED MANUSCRIPT
Yellow solid; yield: 62%; mp 122-125 oC; IR (KBr): 3433, 2922, 1690, 1552, 1466, 1387, 1262,
1224, 1135, 1065, 950, 825,772; 1H NMR (500 MHz, DMSO-d6): δ 12.33 (1H, bs), 8.13 (1H, d,
J = 7.8 Hz), 8.10 (1H, s), 7.81–7.70 (1H, m), 7.65–7.58 (1H, m), 7.57 (1H, d, J = 2.6 Hz), 7.39–
7.27 (2H, m), 7.02 (1H, dd, J = 8.8, 2.6 Hz), 4.68 (2H, t, J = 6.7 Hz), 3.80 (3H, s), 3.52 (2H, bs),
3.14 (2H, bs), 3.08 (2H, t, J = 6.6 Hz,), 1.53 (2H, bs), 1.45 (2H bs), 1.28 (2H, bs) 13C NMR (125
MHz, DMSO–d6): δ 186.2, 169.4, 165.4, 156.1, 155.4, 142.4, 139.0, 136.6, 132.6, 125.2, 123.6,
122.9, 121.1, 114.8, 112.3, 111.3, 104.6, 55.5, 46.2, 42.2, 41.0, 35.3, 25.7, 24.9, 23.7; HRMS
(ESI): m/z calcd for C26H26N4O4S+ [M+H]+ 491.1748; found: 491.1753.
5.3 Pharmacology
5.3.1 Cell culture
Cells were obtained from National Centre for Cell Science (NCCS) Pune, India and stocks were
maintained in the laboratory. Breast (BT549 and MDA-MB-231), prostate (PC-3 and DU145),
lung (A549) and cervical (HeLa) cancer cells were grown in tissue culture flasks in DMEM
(Dulbecco modified Eagle medium, Sigma) or MEM (Minimum Essential Medium, Sigma)
supplemented with 10% fetal bovine serum with 1X stabilized antibiotic-antimycotic solution
(Sigma) in a CO2 incubator at 37 oC with 5% CO2 and 90% relative humidity.
5.3.2 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

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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.
5.3.3 Tubulin polymerization assay
Tubulin polymerization kit was purchased from Cytoskeleton, Inc. (BK011). To evaluate the
effect of compounds 13a, 13c, 13d, 13h, 13k, 13o and 14d on tubulin assembly, a fluorescence
based in vitro tubulin polymerization 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 MgCl2, 0.5 mM EGTA,1.0 mM GTP and glycerol in the presence and absence of
test compounds (final concentration of 5 µM) 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 proceeds.
Fluorescence emission at 420 nm (excitation wavelength is 360 nm) was measured by using a
Spectramax M4 Multi mode Micro plate Detection System. The IC50 value was calculated from
the drug concentration required for inhibiting 50% of tubulin assembly compared to control.
5.3.4 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 13d were added to the petridishes. Cells which migrated across the

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wound area were photographed under the phase contrast microscope (Nikon) after 0, 12 and 24 h
treatment.
5.3.5 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 13d. 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 (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.
5.3.6 DAPI Nucleic Acid Staining
Nuclear morphological changes were observed through DAPI staining. After treatment with 13d
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.
5.3.7 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
13d at varied concentration of 100, 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 aldrich) in the presence of RNase A (20 µg/mL) containing 0.1% Triton X-

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o
100 for 30 min at 37 C in dark, and about 10000 events were analyzed by flow cytometer (BD
FACSVerseTM, USA).
5.3.8 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 13d at 200 and 400 nM
concentrations. 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 Apoptosis Detection Kit, BD PharmingenTM)
according to the manufacturer’s instruction. Further, flow cytometric analysis was performed
using a flow cytometer (BD FACSVerseTM, USA).
5.3.9 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 and 400 nM concentrations of the compound 13d 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).
5.4 Molecular docking
All the molecules were built on Maestro Molecule Builder of Schrödinger. The built molecules
were optimized using OPLS-2005 force field in LigPrep module of Schrödinger software.
Docking procedure was followed using the standard protocol implemented in Maestro, version

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9.9 and the molecules were docked against tubulin. Each complex was analyzed for interactions
and the 3D pose of most active compound was taken using PyMOL v0.99.
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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