Discovery of certain benzyl/phenethyl thiazolidinone-indole hybrids as potential anti-proliferative agents: Synthesis, molecular modeling and tubulin polymerization inhibition study

Article abstract of DOI:10.1016/j.bioorg.2019.103188

A series of certain benzyl/phenethyl thiazolidinone-indole hybrids were synthesized for the study of anti-proliferative activity against A549, NCI-H460 (lung cancer), MDA-MB-231 (breast cancer), HCT-29 and HCT-15 (colon cancer) cell lines by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). We found that compound G37 displayed highest cytotoxicity with IC₅₀ value of 0.92 ± 0.12 μM towards HCT-15 cancer cell line among all the synthesized compounds. Moreover, compound G37 was also tested on normal human lung epithelial cells (L132) and was found to be safe in contrast to HCT-15 cells. The lead compound G37 showed significant G2/M phase arrest in HCT-15 cells. Additionally, compound G37 significantly inhibited tubulin polymerization with IC₅₀ value of 2.92 ± 0.23 μM. Mechanistic studies such as acridine orange/ethidium bromide (AO/EB) dual staining, DAPI nuclear staining, annexinV/propidium iodide dual staining, clonogenic growth inhibition assays inferred that compound G37 induced apoptotic cell death in HCT-15 cells. Moreover, loss of mitochondrial membrane potential with elevated intracellular ROS levels was observed by compound G37. These compounds bind at the active pocket of the α/β-tubulin with higher number of stable hydrogen bonds, hydrophobic and arene-cation interactions confirmed by molecular modeling studies.

Full text of DOI:10.1016/j.bioorg.2019.103188

Journal Pre-Proof
Discovery of certain benzyl/phenethyl thiazolidinone-indole hybrids as poten-
tial anti-proliferative agents: Synthesis, molecular modeling and tubulin poly-
merization inhibition study
Dilep Kumar Sigalapalli, Venkatesh Pooladanda, Priti Singh, Manasa
Kadagathur, Sravanthi Devi Guggilapu, Jaya Lakshmi Uppu, Neelima D.
Tangellamudi, Pavan Kumar Gangireddy, Chandraiah Godugu, Bathini
Nagendra Babu
PII:
S0045-2068(19)30657-1
DOI:
https://doi.org/10.1016/j.bioorg.2019.103188
YBIOO 103188
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 April 2019
22 June 2019
6 August 2019
Please cite this article as: D. Kumar Sigalapalli, V. Pooladanda, P. Singh, M. Kadagathur, S. Devi Guggilapu, J.
Lakshmi Uppu, N.D. Tangellamudi, P. Kumar Gangireddy, C. Godugu, B. Nagendra Babu, Discovery of certain
benzyl/phenethyl thiazolidinone-indole hybrids as potential anti-proliferative agents: Synthesis, molecular modeling
and tubulin polymerization inhibition study, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103188
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JOURNAL PRE-PROOF
Discovery of certain benzyl/phenethyl thiazolidinone-indole hybrids as
potential anti-proliferative agents: Synthesis, molecular modeling and
tubulin polymerization inhibition study
Dilep Kumar Sigalapalli,[a],[b] Venkatesh Pooladanda,[c] Priti Singh,[b] Manasa Kadagathur,[b] Sravanthi
Devi Guggilapu,[b,†] Jaya Lakshmi Uppu,[c] Neelima D. Tangellamudi,[b] Pavan Kumar Gangireddy[b],
Chandraiah Godugu*[c], Bathini Nagendra Babu*[a]
[a] Fluoro-Agrochemicals, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
[b] Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad
500037, India
[c] Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research (NIPER),
Hyderabad 500037, India
†
present address: Invasive Insect Biocontrol and Behavior Laboratory, USDA-ARS, NEA, 10300 Baltimore Avenue,
Beltsville, MD 20705, USA.
aCorresponding author. Tel.: +91-40-27191898; E-mail: bathini@iict.res.in; chandragodugu@gmail.com
KEY WORDS: benzyl/phenethyl thiazolidinone-indole hybrids, cytotoxicity, tubulin polymerization,
apoptosis, molecular modeling.
Abstract:
A series of certain benzyl/phenethyl thiazolidinone-indole hybrids were synthesized for the study of
anti-proliferative activity against A549, NCI-H460 (lung cancer), MDA-MB-231 (breast cancer), HCT-
29 and HCT-15 (colon cancer) cell lines by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT). We found that compound G37 displayed highest cytotoxicity with IC50 value of 0.92 ±
0.12 µM towards HCT-15 cancer cell line among all the synthesized compounds. Moreover, compound
G37 was also tested on normal human lung epithelial cells (L132) and was found to be safe in contrast
to HCT-15 cells. The lead compound G37 showed significant G2/M phase arrest in HCT-15 cells.
Additionally, compound G37 significantly inhibited tubulin polymerization with IC50 value of 2.92 ±
0.23 µM. Mechanistic studies such as acridine orange/ethidium bromide (AO/EB) dual staining, DAPI
nuclear staining, annexinV/propidium iodide dual staining, clonogenic growth inhibition assays inferred
that compound G37 induced apoptotic cell death in HCT-15 cells. Moreover, loss of mitochondrial
membrane potential with elevated intracellular ROS levels was observed by compound G37. These
compounds bind at the active pocket of the α/β-tubulin with higher number of stable hydrogen bonds,
hydrophobic and arene-cation interactions confirmed by molecular modeling studies.
1. INTRODUCTION
Microtubule modulators have played an essential role in cancer chemotherapy over the past decades,
where interference with microtubule assembly, either by down regulation of tubulin polymerization or
by modulation of microtubule disassembly, that leads to an increase in the number of cells in metaphase
arrest [1,2]. Tubulin protein is found to be α, β-heterodimer and forms heart of the microtubule network
in cell. Moreover, cancer cells undergo mitosis rapidly as compared to normal cells, which means that
they are more prone to tubulin-targeting agents [3]. Anti-mitotic compounds such as taxoids (paclitaxel
and docetaxel) and vinca alkaloids (vinblastine and vincristine) have been widely using in the treatment
of various human cancers since many years; however, these compounds have shown either one or
multiple inabilities such as adverse side effects, drug resistance, synthetic complexity and imperfect
bioavailability [4,5]. To prevail the above limitations, medicinal chemists are encouraged to develop
novel chemotherapeutic agents as modulators of microtubules/tubulin system with better safety profile.

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4-Thiazolidinones are proven class of pharmacophores, which display a broad spectrum of biological
activities. In view of tumor therapeutic potential, recent reports suggest that 4-thiazolidinones could
express their antitumor property through various mechanisms featuring affinity towards tumor necrosis
factor-α (TNF-α) [6], JNK stimulating phosphatase-1 (JSP-1) [7], BH3 domain and Bcl-XL [8], SHP-2
[9], integrin αvβ3 receptor [10], histone deacetylase [11] and microtubules/tubulin assembly [12] etc.
O
O
O
OH
O
S
N
N
NH
NH
S
N
HN
S
Br
N
H
O
HO
S
O
N
S
N
Isatin-thiazolidinone hybrid
Thioxothiazolidinone-indole hybrid
Benzo[d]isothiazol-thiazolidinone hybrid
O
O
O
HN
HN
O
HO
OH
HN
S
N
S
N
Cl
S
N
CDBT
HBPT
O
MHPT
N
Cl
O
O
O
N
O
N
N
O
H
N
H
H
N
O
O
D-64131
2-Phenyl Indole
D-24851
Figure 1. Representative structures of bioactive thiazolidinones and indoles as anti-proliferative agents.
Molecular hybridization approach is an efficient and often used direction for investigations to find novel
and highly active compounds in the current medicinal chemistry research [13-15]. Although several
researchers explored for novel hybrid therapeutics, some of the research groups discovered that the
thiazolidinone containing hybrids exhibit anti-cancer activity against different cancer cell lines [16,17].
These findings support that modifications of thiazolidinone ring at C-2 and C-5 positions have led to
many new chemical entities with increased antitumor activity. On the other hand, indoles represent one
of the most significant structural classes in drug discovery process. A number of indole derivatives used
as tubulin polymerization inhibitors to induce apoptosis in cancer chemotherapy [18]. Some of the
representatives are depicted in Fig. 1 such as 2-phenylindole, 2-aroylindoles (D-64131) and indolyl-3-
glyoxamide (D-24851) [19,20].
Figure 2. Design of certain benzyl/phenethyl thiazolidinone-indole hybrids as anticancer agents.
In search of target-specific therapeutics with the inspiration of molecular hybridization, we envision that
the hybridization of 4-thiazolidinone moiety with indole structural motif into a single chemical entity, as
depicted in Fig. 2, may produce synergistic anticancer activity. With our continued interest in the

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discovery of tubulin inhibitors [21], we synthesized the designed hybrid molecules and scrutinized for
in vitro anti-proliferative activity against five human cancer cell lines: A549, NCI-H460 (lung cancer),
MDA-MB-231 (breast cancer), HCT-29 and HCT-15 (colon cancer).
2. MATERIAL AND METHODS
2.1. Chemistry
All the solvents and reagents were procured from commercial suppliers. Analytical thin layer
chromatography (TLC) was performed on MERCK pre-coated silica gel 60-F-254 (0.5 mm) aluminium
plates. Visualization of the spots on TLC plates was achieved using UV light. 1H and 13C NMR spectra
were recorded on Bruker 500 MHz and 125 MHz spectrometers, respectively, using tetramethyl silane
1
13
(TMS) as the internal standard. Chemical shifts for H and C are reported in parts per million (ppm)
downfield from tetramethyl silane. Spin multiplicities are described as s (singlet), bs (broad singlet), d
(doublet), dd (doublet of doublets), t (triplet), q (quartet), and m (multiplet). Coupling constant (J)
values are reported in Hertz (Hz). Melting point was determined with the help of Stuart advanced
melting point apparatus. HRMS were determined with Agilent QTOF mass spectrometer 6540 series
instrument. Column chromatography was performed using silica gel 60-120.
2.2. Biology
Cell culture
Cell lines such as lung (A549 and NCI-H640), breast (MDA-MB-231), colon (HCT-15 and HCT-29)
and normal lung epithelial cells (L132) were procured from National Centre for Cell Science (NCCS),
Pune, India. All the cells were grown in appropriate DMEM and RPMI-1640 medium (Sigma-Aldrich,
USA). Cells were supplemented with 10% fetal bovine serum stabilized with 1% antibiotic-anti mycotic
o
solution (Sigma-Aldrich, USA) in a CO2 incubator at 37 C. When the cells reached up to 80-90% of
confluency, they were sub-cultured using 0.25% trypsin/1 mM EDTA solution for further passage.
MTT assay
Cytotoxicity of synthesized compounds was evaluated by MTT assay. Briefly, cells were seeded in 96-
well plates at a density of 4000 cells per well in 100 µL of complete medium and allowed to grow
overnight for attachment onto the wells. Then the cells were treated with various concentrations of the
compounds for a period of 72 h. After the treatment, 100 µL of MTT (0.5 mg/mL) was added and
o
incubated at 37 C for 4 h. Then MTT reagent was aspirated and the formazan crystals formed were
o
dissolved by the addition of 200 µL of DMSO for 20 mins at 37 C. The quantity of formazan product
was measured by using a spectrophotometric microtiter plate reader (Spectra max, M4 molecular
devices, USA) at 570 nm wavelength. Initially, cytotoxicity effects of the synthetic derivatives were
screened by MTT assay at 30 µM concentration. Among these, the compound which showed IC50 value
<30 µM was used for the dose dependent studies at various concentrations ranging from 0.78 µM to 30
µM in serial dilutions and the percentage of cytotoxicity was calculated.
2.3. Molecular modeling studies
Molecular modeling studies of the corresponding benzyl/phenethyl thiazolidinone-indole hybrid
compounds were performed by using Maestro, version 10.4 of Schrödinger suite 2015-4. The 3D
structures of the compounds were made by using Maestro Molecule Builder and optimized by means of
LigPrep 3.6 version of Schrödinger suite 2015-4. Thus obtained 3D structures of the compounds were
docked at the active site of tubulin protein (PDB ID: 1SA0).

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3. RESULTS AND DISCUSSION
3.1. Chemistry
We began our study with the synthesis of designed target molecules G1-G44 using the multi-step
reaction protocol as delineated in Scheme 1. The designed target compounds G1-G44 comprise of two
core structural elements: (i) benzyl/phenethyl thiazolidinone, and (ii) a substituted indole moiety.
Initially, substituted benzyl/phenethyl thiazolidinone (C) moiety was prepared from commercially
available benzyl/phenethylamine (A) using two simple synthetic steps. Firstly, benzyl/phenethylamine
(A) was treated with chloroacetyl chloride in DMF to afford the corresponding amide (B), which
undergoes cyclization in the presence of ammonium thiocyanate at reflux temperature in ethanol to
afford the desired fragment-thiazolidinone (C) in good to excellent yields [22, 23].
ii
i
R1
R1
R1
H
H
(a)
N
N
n
NH2
n
N
Cl
O
n
O
S
B
A
C
n=CH2; C2H4
R1=H; 4-F; 4-OCH3; 2-Cl; 3-Cl; 3,4-di-Cl; 3,5-tri-CF3; 4-CF3; 4-C(CH3)3; 3,4-di-OCH3; 3,4,5-tri-OCH3; 4-OH
O
O
R2
R2
R2
iv
iii
(b)
N
R3
N
N
H
H
D
E
F
R2=H; OCH3; I; Br
R3=H; CH3; 4-chloro-benzyl; phenacyl; 4-methoxyphenacyl; 4-methylphenacyl; 4-bromophenacyl
O
O
HN
v
R1
H
R2
R1
(c)
N
n
N
+
S
N
O
n
N
R3
S
N
R3
R2
C
F
G1-G44
G1: n=1, R1=H, R2=H, R3=H
G2: n=1, R1=H, R2=H, R3=CH3
G3: n=1, R1=H, R2=H, R3=4-chlorobenzyl
G4: n=1, R1=H, R2=H, R3=phenacyl
G23: n=1, R1=4-trifluoromethyl, R2=methoxy, R3=H
G24: n=1, R1=4-tert-butyl, R2=H, R3=H
G25: n=1, R1=4-tert-butyl, R2=methoxy, R3=H
G26: n=1, R1=4-tert-butyl, R2=bromo, R3=H
G27: n=1, R1=3,4,5-trimethoxy, R2=H, R3=H
G28: n=1, R1=3,4,5-trimethoxy, R2=methoxy, R3=H
G29: n=1, R1=4-hydroxy, R2=H, R3=H
G30: n=1, R1=4-hydroxy, R2=methoxy, R3=H
G31: n=2, R1=H, R2=H, R3=H
G32: n=2, R1=H, R2=H, R3=4-chlorobenzyl
G33: n=2, R1=4-methoxy, R2=H, R3=H
G34: n=2, R1=4-methoxy, R2=H, R3=4-chlorobenzyl
G5: n=1, R1=H, R2=H, R3=4-methoxyphenacyl
G6: n=1, R1=H, R2=H, R3=4-methylphenacyl
G7: n=1, R1=H, R2=H, R3=4-bromophenacyl
G8: n=1, R1=4-fluoro, R2=H, R3=H
G9: n=1, R1=4-fluoro, R2=H, R3=CH3
G10: n=1, R1=4-fluoro, R2=H, R3=4-chlorobenzyl
G11: n=1, R1=4-methoxy, R2=H, R3=H
G12: n=1, R1=4-methoxy, R2=H, R3=CH3
G13: n=1, R1=4-methoxy, R2=methoxy, R3=H
G14: n=1, R1=4-methoxy, R2=iodo, R3=H
G15: n=1, R1=2-chloro, R2=H, R3=H
G35: n=2, R1=4-methoxy, R2=H, R3=4-methoxyphenacyl
G36: n=2, R1=4-methoxy, R2=methoxy, R3=H
G37: n=2, R1=4-methoxy, R2=bromo, R3=H
G38: n=2, R1=4-methoxy, R2=iodo, R3=H
G16: n=1, R1=2-chloro, R2=methoxy, R3=H
G17: n=1, R1=3-chloro, R2=H, R3=H
G18: n=1, R1=3-chloro, R2=methoxy, R3=H
G19: n=1, R1=3,4-dichloro, R2=H, R3=H
G20: n=1, R1=3,4-dichloro, R2=H, R3=CH3
G21: n=1, R1=3,5-trifluoromethyl, R2=H, R3=H
G22: n=1, R1=4-trifluoromethyl, R2=H, R3=H
G39: n=2, R1=3,4-dimethoxy, R2=H, R3=H
G40: n=2, R1=3,4-dimethoxy, R2=H, R3=4-chlorobenzyl
G41: n=2, R1=3,4-dimethoxy, R2=H, R3=4-methoxyphenacyl
G42: n=2, R1=3,4-dimethoxy, R2=methoxy, R3=H
G43: n=2, R1=4-fluoro, R2=H, R3=H
G44: n=2, R1=4-fluoro, R2=methoxy, R3=H
Reagents and conditions: i) DMF/ClCOCH2Cl/0 °C - r.t. 4 h, (ii) NH4SCN/ ethyl alcohol/reflux 6 h, iii) POCl3/DMF/0 °C -
60°C. 6 h, iv) NaH/dry DMF/0 °C - r.t. 24 h, v) ethyl alcohol/ piperidine/ reflux 8 h.
Scheme 1. Synthesis of benzyl/phenethyl thiazolidinone-indole hybrids (compound G1-G44).

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On the other hand, the 5-substituted-1H-indole-3-carboxaldehyde (E) was prepared according to
Vilsmeier-Haack reaction by the treatment of 5-substituted-1H-indole (D) with phosphorus oxychloride
in DMF at 0-60 °C [24], and then N-alkylation/arylation was carried out using appropriate alkyl/aryl
halide and sodium hydride in DMF to obtain the desired N-substituted indole-3-caboxaldehyde (F) [25-
27]. Finally, the thiazolidinone (C) was coupled with the synthesized indole-3-caboxaldehyde derivative
(F) using piperidine in ethanol at 60°C to furnish the target compound (G1-G44) in moderate to very
good yields (scheme 1) as per our previous protocol [28]. All the synthesized compounds (G1-G44)
1
13
were well characterized by using HRMS, H and C NMR spectroscopy and all the data are produced
in supporting information. The theoretical existence of tautomeric forms of thiazoldinone (C) was
represent in Scheme 2. The 1H NMR spectrum of G1 showed a broad singlet of indole N-H proton at δ
12.02, which disappeared upon methylation, as seen in G2. A singlet was observed at δ 9.71, which is
characteristic of amide N-H proton. Based on this observation as well as reported literature [29]
supporting our prediction of the existence of thiazolidinone as phenylimino rather than phenylamino
form.
O
O
N
HN
Ar
S
Ar
S
N
N
H
amino form
imino form
Scheme 2. Tautomeric forms of 2-substituted thiazolidinone.
3.2. In vitro anti-proliferative activity
Anti-proliferative activity of all the synthesized compounds (G1-G44) were performed on various
human cancer cell lines such as lung (A549 and NCI-H460), colon (HCT-29 and HCT-15), and breast
cancer (MDA-MB-231) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Table S1 displays the IC50 (μM) values (concentration required to inhibit 50% of cell growth) of
tested compounds (G1-G44), as well as reference standard-podophyllotoxin. The screening of
preliminary results suggests that few of the synthesized compounds displayed moderate to potent
cytotoxicity against the tested cancer cells with IC50 values in the range of 0.92 ± 0.12 to 12.28 ± 0.30
μM. Close examination of IC50 values of the synthesized compounds showed that some of the
compounds are sensitive towards colon (HCT-29 and HCT-15) and lung (A549 and NCI-H460) cancer
cell lines and most of the compounds were inactive against MDA-MB-231 cancer cells. From these
experimental results we observed that, compounds G21, G26, G27, G33, G37, and G38 were the most
active in the series and the remaining compounds were roughly moderate to inactive against the tested
cancer cells. Interestingly, the compound G37 was found to be most active in colon cancer (HCT-29
and HCT-15) cell lines and showed marked cytotoxicity with IC50 of 0.92 ± 0.12 μM towards HCT-15
cells. To check the selectivity of our synthesized compounds towards cancerous cells, their cytotoxicity
was evaluated against normal human lung epithelial cells (L132). To our delight, the compound G37
showed IC50 of 10.84 ± 0.94 μM on non-cancerous cells, thus, indicating 10 fold selectivity towards
HCT-15 cells. Some of the common pharmacophoric features observed in active compounds G33, G37,
and G38 are the presence of the 4-methoxyphenyl group in the side chain connected to the
thiazolidinone nucleus with an ethylene bridge and absence of the substitution of the nitrogen in indole.
Furthermore, we noticed from the comparison of IC50 values that the phenethylthiazolidinone-

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indolehybrids (G31-G44) have more potent anti-proliferative action than benzyl thiazolidinone-indole
hybrids (G1-G30). In addition, it was also observed that N-alkyl/aryl substituted indole hybrids are
relatively less active than unsubstituted indole hybrids. The electron donating group (methoxy) on the
phenyl ring at the side chain of hybrid molecules has shown better anti-proliferative profile compared to
those with halogen substitutions. Finally, due to the encouraging anti-proliferative activity, one of the
most active compound G37 was taken-up for further mechanistic investigation of cancer cell growth
inhibition.
3.3. Structure-Activity Relationship (SAR)
The impact of substitution at benzyl/phenethyl, thiazolidinone and indole is highly significant. The
structure-activity relationship (SAR) analysis of these molecules are: 1) The presence of electron
donating (4-methoxy) group at the phenethyl group attached to the side chain of thiazolidinone (G33,
G37 and G38) showed significant cytotoxicity; 2) Compounds without any substitution at the benzyl/
phenethyl group attached to the side chain of thiazolidinone (G1-7 and G31-32) were nearly inactive at
30μM; 3) Compounds with halogen (fluoro and chloro) substitutions at the benzyl/phenethyl group
attached to the thiazolidinone (G8, G9, G15-20, G43 and G44) are less active; 4) From the SAR
studies, we also observed that the presence of free N-H group in thiazolidinone and indole rings is
essential for imparting cytotoxic activity (as depicted in Fig. 3.). Moreover, the compounds containing
phenethyl side chain on thiazolidinone-indole core showed enhanced cytotoxicity than benzyl side
chain.
Figure 3. SAR analysis of benzyl/phenethyl thiazolidinone-indole hybrids.
3.4. Cell cycle analysis
Most of the anticancer agents inhibit the cell proliferation of cancer cells by arresting the cell cycle at
particular check point. In our study, we found that compound G37 showed significant cytotoxicity
against HCT-15 cells. To know the underlying mechanism of its cytotoxicty, we performed cell cycle
analysis. Herein, cells were treated with compound G37 (0.5, 1, 2.5 and 5 µM) for 24 h and stained with
propidium iodide (PI) and further analyzed by flow cytometer. Fig. 4 infers that the control cells
exposed to DMSO showed 2.49% cells in sub G1 phase, whereas compound G37 treatment resulted in
increased sub G1 population to 10.88% with 5 μM concentration. The percentage of cells in G2/M
phases was 53.57 and 68.53% with 2.5 and 5 µM of the compound, respectively, in comparison to the
control cells where 36.49% was observed. Collectively, these results indicated that treatment of HCT-15
cells with compound G37 arrested in sub G1 and G2/M phase.

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Figure 4. Effect of G37 compound on cell cycle progression of HCT-15. Cells were treated with compound G37
(0.5, 1, 2.5 and 5 µM) and cell cycle analysis was performed by flow cytometric analysis using PI staining after
24 h of incubation.
3.5. Effect on tubulin polymerization
Microtubules are cytoskeletal filaments consisting of α and β tubulin subunits and are involved in a
wide range of cellular functions including cell division [30]. Further, tubulin is a well-established target
for a variety of successful anticancer drugs. As evident from the literature, 4-thiazolidinones and indoles
show anti-proliferative activity via inhibition of tubulin polymerization, thereby, disturbing the
microtubule formation. In addition, as these hybrids significantly induced G2/M cell cycle arrest, their
microtubule inhibitory functional aspects were also explored. The active compound G37 was evaluated
for tubulin polymerization inhibitory activity, where podophyllotoxin was taken as the positive control.
Initially, compound G37 was screened at 5 µM concentration and the percentage of inhibition was
found to be 81.46%. Fig. 5 infers that tubulin polymerization was significantly inhibited by compound
G37 in a concentration dependent manner with IC50 value of 2.92 ± 0.23 µM. Collectively, these results
showed that tubulin might be the molecular target of 4-thiazolidinone-indole hybrids. Compound G37
(0.31-5 µM) concentration-dependently inhibited the tubulin polymerization compared to
podophyllotoxin. Data represented as mean ± SEM (n=3). p<0.001 is significantly different from
podophyllotoxin.
Figure 5. Effect of compound G37 on tubulin polymerization.

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3.6. Apoptosis induction studies
As most of the anticancer agents show the anticancer activity by inducing apoptosis, we have
investigated the apoptotic inducing activity of compound G37 on HCT-15 cells. We performed various
assays including acridine orange/ethidium bromide (AO/EB), 4',6-diamidino-2-phenylindole (DAPI),
DCFDA, JC-1 staining and Annexin V binding assays and results are summarized. Initially, HCT-15
cells were treated with compound G37 and were observed under phase contrast microscopy to examine
the morphological changes of cells. Compound G37 (0.5, 1 and 2.5 µM) showed characteristic
apoptotic morphological features such as cell shrinkage and decreased number of viable cells compared
to the control group (Fig. S1 Supporting Information).
AO/EB staining is used to differentiate live, apoptotic and necrotic cells. Here, AO is a dye, which
permeates the intact cell membrane and stains the nuclei with green colour, whereas EB stains the
nucleus as red in colour. Fig. 6 demonstrated that control cells appeared green in colour without any
morphological changes. However, cells treated with 2.5 µM of compound G37 clearly exhibited
morphological changes such as cell shrinkage, nuclear condensation and apoptotic blebs formation.
Collectively, these results indicate that compound G37 induces apoptosis in HCT-15 cells in
concentration dependent manner.
Figure 6. AO/EB staining of compound G37. Cells were treated with G37 in the concentration of 0.5, 1 and 2.5
µM and compared with control (DMSO treatment). Images were captured at 200X magnification. Arrows
indicate the apoptotic changes such as nuclear condensation and membrane blebbing.
DAPI stain selectively binds to A-T rich sequences of DNA and helpful to visualize the nuclear changes
in cells. It clearly differentiates live cells from apoptotic cells by bright condensed nuclei. Nuclear
morphology of cancer cells after DAPI staining (Fig. S2 Supporting Information) demonstrated that the
compound G37 induced the formation of horse-shoe shaped or fragmented nuclei in cells in contrast to
the control group. Loss of mitochondrial membrane potential (MMP) is an earliest stage of apoptotic
signaling activation. We therefore used JC1 staining to assess the apoptotic activity of compound G37
on MMP. Treatment with compound G37 on HCT-15 cells for 48 h, resulted in increased loss of
mitochondrial membrane potential in concentration dependent manner (Fig. S3 Supporting
Information). Thus, mitochondrial membrane depolarization was significantly increased after treatment
with compound G37 in colon cancer cells.

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Quantitatively, the percentage of apoptosis induced by compound G37 was determined by Annexin
V/PI dual staining. This staining facilitates for the quantification of live (LC; AV-/ PI-), early apoptotic
(EA; AV+/PI-), late apoptotic (LA; AV+/PI+) and necrotic (NC; AV-/PI+) cell population. The
percentage of total apoptotic cells (both early and late apoptotic cells, respectively) was increased to
24.83% after treatment with 2.5 µM concentration of compound G37 in comparison to the control
(1.66%) cells (Fig. 7). Collectively, the percentage of early apoptotic cells was significantly increased
by compound G37 in colon cancer cells.
Figure 7. Effect of compound G37 on apoptotic cell death in HCT-15 cells after 48 h. The compound G37
treated cells were stained with Annexin V/PI and analysed for apoptosis using flow cytometer. The 10,000 cells
from each sample were analysed by flow cytometry.
We next determined the effect of compound G37 on cellular ROS levels in HCT-15 cancer cells using
cell permeable fluorogenic dye-DCFDA. The compound G37 (0.5, 1 and 2.5 µM) increased the ROS
levels in colon cells and increased the green fluorescence. The percentage of DCF intensity increased in
concentration-dependent manner (Fig. S4 Supporting Information). Anti-proliferative activity of the
most potent compound G37 was further tested by colony formation assay which infers the ability of
forming clone from single cells, which mimics the human tumor metastasis. The compound G37
significantly inhibited the clonogenic growth of HCT-15 cells at 0.5 µM, which was the lowest tested
concentration. However, at 2.5 µM concentration of compound G37, the colony formation was
completely inhibited as compared to the control group (Fig. S5A Supporting Information). The number
of colonies was counted by molecular imaging system Vilber Fusion Fx software and values were
represented as total number of colonies versus concentration (Fig. S5B Supporting Information).
3.7. Molecular modeling studies
Docking simulation study
Molecular docking studies were performed for the representative compounds (G21, G26, G27, G33,
G37, and G38) of synthesized benzyl/phenethyl thiazolidinone-indole hybrids and bound ligand against
the active site of X-ray crystal structure of tubulin protein and the detailed interaction pattern of most
active compound G37 with tubulin protein is shown in Fig. 8a and 8b. The docking model of the active
compound G37 revealed that, compound G37 has shown four hydrogen bonding contacts with the key
amino acid residues Thr145, Ala250, Asp251 and Lys254, and the results showing hydrogen bond,
hydrophobic and π-cation interactions of synthesized compounds with tubulin protein are depicted in

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Table S2 (supporting information). The nitrogen atom of thiazolidinone moiety has shown H-bond
interaction with side chain amino group of Lys254 with distance of 2.4 Å and oxygen atom of keto
functional group of thiazolidinone ring has established two H-bond interactions with the back bone
amino group of Ala250 (d = 1.9 Å) and Asp251 (d = 3.3 Å), respectively. Similarly, p-methoxy group
of phenethyl side chain had shown hydrogen bond contact with Thr145 (d = 3.3 Å). Furthermore, the
benzene ring of this compound established one important π-cation interaction with Lys254.
Additionally, compound G37 has shown several hydrophobic interactions with the key amino acid
residues of α/β tubulin. Moreover, docked compound G37 and cocrystallized ligand suggested that G37
also lodged at the active site in a comparable manner as that of cocrystallized ligand which
demonstrates the superimposition of best docked pose and bound ligand at the active site of α/β tubulin
(Fig. S6 Supporting Information). This docking analysis gave us a discerning avocation to astounding
tubulin polymerization inhibitory action of compound G37 and some significant information for future
structural modifications.
Figure 8. a) Predicted binding pose of G37 (ball and stick); b) pattern of interactions in the active pocket of α/β
tubulin. The red colour lines represent hydrogen bond interactions and black line indicates cation-arene
interaction with Lys254.
Prime MM/GBSA binding energy calculations
We observed that the synthesized compounds have shown comparable binding energies to that of bound
ligand using MM/GBSA binding energy calculations (Table 1). The aim of MM-GBSA and docking
studies in the present work is to examine the ligand-protein complexation energy and ligand affinity to
α/β tubulin, thereby giving more perceptive results of ligands pose and pattern of interaction at active
pocket of human α/β tubulin. Some of the recognized ligands confirmed great binding energy similar to
the bound ligand, signifying stable ligand-protein complex formation, leading to the stronger binding.
Table 1. Binding energies (ΔGbind) obtained for some of the synthesized benzyl/phenethyl thiazolidinone-indole
hybrids and co-crystallized ligand.
S.No
Ligand ID
Binding Energy
(Kcal/mol)
1
2
G21
G26
-53.195
-70.353

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3
4
5
6
7
G27
G33
G37
G38
Cocrystallized
ligand
-52.844
-63.735
-64.325
-39.718
-71.189
In silico ADME/T studies
In order to examine drug-likeliness, physicochemically important descriptors and pharmacokinetically
key properties of synthesized molecules were assessed through QikProp program of Schrödinger
software. Some of the computed ADME/T parameters (Table 2) are within their recommended range
and are mentioned in supporting information. From this in silico study, we can compare the ADME/T
properties of the synthesized molecules with that of known drugs. ADME/T prediction studies depicts
that the synthesized hybrids conform to the good PSA and that they have appropriate logP values and
show no violation in the recommended ranges of physico-chemical descriptors.
Table 2. ADME/T Profile of some of the synthesized benzyl/phenethyl thiazolidinone-indole hybrids and other
known tubulin inhibitors.
Parameters
Ligand
ID
PSA
QPlog
Khsa
Predicte Predict Predic
Predicted
apparent
Predict Predicte Ioniza Percent
d
ed
ted
ed
d skin
tion
Human
Oral
octanol/
water
polariz
ability
CNS
Caco-2 cell brain/b permea Potent
Activit permeabili
lood
partitio
n
bility
ial
Absorpt
ion
partition
coefficie
nt
y
ty in
nm/sec.
coeffici
ent
G21
G26
G27
G33
G37
G38
C
68.428
68.439
87.819
77.665
77.716
77.521
92.246
97.519
174.957
0.944
1.099
0.506
0.506
0.584
0.707
-0.050
-0.125
1.435
5.706
5.551
4.141
3.932
4.245
4.702
2.573
2.361
5.226
43.656
45.855
43.140
38.916
39.634
42.177
40.449
36.743
80.172
0
649.268
656.605
649.812
536.906
531.574
549.938
542.309
1745.470
15.509
-0.499
-0.941
-1.234
-1.106
-0.970
-1.057
-0.780
-0.314
-0.918
-2.280
-2.190
-2.012
-2.167
-2.324
-2.183
-2.798
-2.242
-7.316
8.573
8.600
9.005
8.467
8.627
8.563
8.571
8.915
8.142
100.000
96.911
100.000
100.000
100.000
90.565
90.950
100.000
39.976
-1
-2
-2
-1
-2
-1
-1
0
P
V
C # Colchicine; P # Podophyllotoxin; V # Vincristine
4. CONCLUSION
In summary, we have synthesized a series of benzyl/phenethyl thiazolidinone-indole hybrid compounds
1
13
(G1–G44) and characterized them by various analytical techniques such as HRMS, H and C NMR.
Anti-proliferative activity of these compounds was evaluated in A549, NCI-H460 (lung cancer), MDA-
MB-231 (breast cancer), HCT-29 and HCT-15 (colon cancer) cell lines using MTT assay. Amongst all
the synthesized hybrids, compound G37 showed excellent anti-proliferative activity on all the tested
cell lines, especially in colon cancer cell lines HCT-15, HCT-29 (IC50 of 0.92 ± 0.12 and 2.802 ±
0.02μM, respectively). Additionally, compound G37 significantly down-regulated the tubulin

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polymerization with IC50 value of 2.92 ± 0.23 µM. The cell cycle analysis disclosed that compound G37
showed sub-G1 and significant G2/M phase arrest in HCT-15 cells. Further, compound G37 impaired
the morphological changes by inducing intracellular ROS and increased the loss of mitochondrial
membrane potential, thus causing the apoptosis-mediated cell death in HCT-15 cells. The molecular
modeling studies inferred that compound G37 binds at the colchicine binding site of the tubulin with
prominent binding affinity, stable ligand-protein complexation, good PSA as well as appreciative logP
and physico-chemical descriptors. Taking everything into account, benzyl/phenethyl thiazolidinone-
indole hybrids presented in this work signify the exciting development of new anti-proliferative agents
through modifications in their chemical structure.
ACKNOWLEDGMENT
Authors are thankful to CSIR-IICT and dept. of pharmaceuticals (DoP), Ministry of Chemicals &
Fertilizers, Govt. of India, New Delhi, for the support (IICT Communication No #
IICT/Pubs./2019/216).
CONFLICT OF INTEREST STATEMENT
The authors have declared no conflict of interest.
ORCID
Bathini Nagendra Babu: 0000-0001-7378-0878
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Graphical Abstract
Discovery of certain benzyl/phenethyl thiazolidinone-indole hybrids
as potential anti-proliferative agents: Synthesis, molecular modeling
and tubulin polymerization inhibition study
Dilep Kumar Sigalapallia,b, Venkatesh Pooladandac, Priti Singhb, Manasa Kadagathurb, Sravanthi Devi
Guggilapub,†, Jaya Lakshmi Uppuc, Neelima D. Tangellamudib, Pavan Kumar Gangireddyb, Chandraiah
Godugu*c, Bathini Nagendra Babu*a
aFluoro-Agrochemicals, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research
(NIPER), Hyderabad 500037, India
cDepartment of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research
(NIPER), Hyderabad 500037, India
a Corresponding author. Tel.: +91-40-27193157; E-mail: bathini@iict.res.in, chandragodugu@gmail.com

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Research Highlights
 The new benzyl/phenethyl thiazolidinone-indole hybrids were synthesized.
 Synthesized compounds displayed prominent antiproliferative activity on different human
cancer cell lines.
 Compound G37 induced apoptosis and cell cycle arrest in G2/M phase in HCT-15 cancer cells.
 G37 effectively inhibited polymerization of tubulin in a cell-free assay.
 G37 was almost 10 times more selective on HCT-15 cells compared to L132 cells.
 Further, a molecular docking analysis of lead compounds was performed against the tubulin
protein.