Novel chromenyl-based 2-iminothiazolidin-4-one derivatives as tubulin polymerization inhibitors: Design, synthesis, biological evaluation and molecular modelling studies

Article abstract of DOI:10.1016/j.molstruc.2020.128847

Here-in, we present molecular design, chemical synthesis and evaluation of novel chromenyl-based 2-iminothiazolidin-4-one derivatives as tubulin polymerization inhibitors. The newly synthesized compounds were evaluated for their in vitro cytotoxicities against A549 (lung cancer), MDA-MB-231 and BT-471 (breast cancer), HepG2 (liver cancer) and HCT-116 (colon cancer) cell lines by MTT assay. Among the synthesized compounds, compound 12b showed excellent anticancer activity on MDA-MB-231 cell line with IC₅₀ value of 0.95 ± 1.88 μM and was verified to be safe in normal human bronchial epithelial cells (Beas-2B). Apoptosis induced by the lead 12b was observed using morphological observations, AO/EB and DAPI staining procedures. Further, dose-dependent increase in the depolarization of mitochondrial membrane was also observed through JC-1 staining. Annexin V-FITC/PI assay confirmed that 12b induced early apoptosis. Additionally, cell cycle analysis indicated that the MDA-MB-231 cells were arrested at sub-G2/M phase and also inhibited tubulin polymerization with IC₅₀ value of 3.54 ± 0.2 μM. Molecular docking simulations were employed to identify the important binding modes responsible for the tubulin inhibitory activity, thus supporting their effective anticancer potential.

Full text of DOI:10.1016/j.molstruc.2020.128847

Journal Pre-proof
Novel chromenyl-based 2-iminothiazolidin-4-one derivatives as tubulin polymerization
inhibitors: Design, synthesis, biological evaluation and molecular modelling studies
Dilep Kumar Sigalapalli, Venkatesh Pooladanda, Manasa Kadagathur, Sravanthi
Devi Guggilapu, Jaya Lakshmi Uppu, Chandraiah Godugu, Nagendra Babu Bathini,
Neelima D. Tangellamudi
PII:
S0022-2860(20)31172-8
DOI:
https://doi.org/10.1016/j.molstruc.2020.128847
MOLSTR 128847
Reference:
To appear in: Journal of Molecular Structure
Received Date: 27 April 2020
Revised Date: 4 July 2020
Accepted Date: 6 July 2020
Please cite this article as: D.K. Sigalapalli, V. Pooladanda, M. Kadagathur, S.D. Guggilapu, J.L.
Uppu, C. Godugu, N.B. Bathini, N.D. Tangellamudi, Novel chromenyl-based 2-iminothiazolidin-4-
one derivatives as tubulin polymerization inhibitors: Design, synthesis, biological evaluation and
molecular modelling studies, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/
j.molstruc.2020.128847.
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Novel Chromenyl-Based 2-Iminothiazolidin-4-one Derivatives as
Tubulin Polymerization Inhibitors: Design, Synthesis, Biological
Evaluation and Molecular Modelling Studies
Dileep, Manasa and Sravanthi Devi Guggilapu: Synthesis and characterization
Jaya Lakshmi Uppu: Tubulin assays.
Venkatesh Pooladanda, Chandraiah Godugu: MTT assay
Nagendra Babu Bathini: Concept
Neelima D. Tangellamudi: Concept, Writing, Reviewing and Editing

Graphical Abstract
Novel Chromenyl-Based 2-Iminothiazolidin-4-one Derivatives as
Tubulin Polymerization Inhibitors: Design, Synthesis, Biological
Evaluation and Molecular Modelling Studies
Dilep Kumar Sigalapalli,a Venkatesh Pooladanda,c Manasa Kadagathur,a Sravanthi Devi Guggilapu,a,d Jaya
Lakshmi Uppu,a Chandraiah Godugu,*c Nagendra Babu Bathini,*b and Neelima D. Tangellamudi,*a
[a] Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER),
Hyderabad 500037, India
[b] Fluoro-Agrochemicals, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
[c] Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research
(NIPER), Hyderabad 500037, India
[d] Present address: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney
Diseases, Laboratory of Bioorganic Chemistry, Bethesda, MD 20892, USA
aCorresponding author. E-mail: tdneelima@gmail.com

Novel Chromenyl-Based 2-Iminothiazolidin-4-one Derivatives as
Tubulin Polymerization Inhibitors: Design, Synthesis, Biological
Evaluation and Molecular Modelling Studies
Dilep Kumar Sigalapalli,a Venkatesh Pooladanda,c Manasa Kadagathur,a Sravanthi Devi Guggilapu,a,d Jaya
Lakshmi Uppu,a Chandraiah Godugu,*c Nagendra Babu Bathini,*b and Neelima D. Tangellamudi,*a
[a] Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER),
Hyderabad 500037, India
[b] Fluoro-Agrochemicals, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India
[c] Department of Regulatory Toxicology, National Institute of Pharmaceutical Education and Research
(NIPER), Hyderabad 500037, India
[d] Present address: National Institutes of Health, National Institute of Diabetes and Digestive and Kidney
Diseases, Laboratory of Bioorganic Chemistry, Bethesda, MD 20892, USA
aCorresponding author. E-mail: tdneelima@gmail.com
Abstract
Here-in, we present molecular design, chemical synthesis and evaluation of novel chromenyl-
based 2-iminothiazolidin-4-one derivatives as tubulin polymerization inhibitors. The newly
synthesized compounds were evaluated for their in vitro cytotoxicities against A549 (lung
cancer), MDA-MB-231 and BT-471 (breast cancer), HepG2 (liver cancer) and HCT-116
(colon cancer) cell lines by MTT assay. Among the synthesized compounds, compound 12b
showed excellent anticancer activity on MDA-MB-231 cell line with IC50 value of 0.95 ±
1.88 µM and was verified to be safe in normal human bronchial epithelial cells (Beas-2B).
Apoptosis induced by the lead 12b was observed using morphological observations, AO/EB
and DAPI staining procedures. Further, dose-dependent increase in the depolarization of
mitochondrial membrane was also observed through JC-1 staining. Annexin V-FITC/PI assay
confirmed that 12b induced early apoptosis. Additionally, cell cycle analysis indicated that
the MDA-MB-231 cells were arrested at sub-G2/M phase and also inhibited tubulin
polymerization with IC50 value of 3.54 ± 0.2 µM. Molecular docking simulations were
employed to identify the important binding modes resposible for the tubulin inhibitory
activity, thus supporting their effective anticancer potential.
KEY WORDS: 2-iminothiazolidin-4-one, chromenone, cytotoxicity, tubulin polymerization,
apoptosis, molecular modelling.

1. Introduction
Cancer, a complex and heterogeneous disease, that can be defined as growth of cells beyond
their usual boundaries is the cause for escalating number of deaths across the globe [1]. It is
characterized by unrestricted cell proliferation, division and metastasis that may be attributed
to irregular microtubule dynamics [2,3]. Microtubules are the structures with a key role in
cell formation, division, cell shape maintenance, intracellular transport, signalling etc [4,5].
The microtubules are comprised of α/β-tubulin heterodimers which fold and unfold to give
specific complexes [6]. Any alterations in the conformations of these complexes result in the
microtubule catastrophe. Microtubulin interacting drugs, are important agents in cancer
chemotherapy which alter microtubule dynamics to arrest cell division in mitosis and deploy
a crucial role as chemotherapeutic agents [7,8].
2-iminothiazolidin-4-one nucleus, one of the privileged scaffolds in modern medicinal
chemistry is of great pharmaceutical relevance with regard to its potential applications as
inhibitors of metalloproteinases [9], COX-2 [10], CDK-2 [11], and SHP-2 [12]. Apart from
the pharmacological activities as antidiabetic, anti-inflammatory, anticonvulsant,
antimicrobial and antipsychotic agents, 2-iminothiazolidin-4-one has been proven to be a
molecule of immense therapeutic interest as an anticancer agent and inhibitors of tubulin
assembly of the microtubules [13-17].
A series of 4-thiazolidinone-indolin-2-ones A and B were reportedly found to be significantly
cytotoxic against HT-29, H460, MDA-MB-231 and SMMC-7721 cancer cell lines
(IC50 = 0.025 μM, 0.075 μM, 0.77 μM, 1.95 μM respectively) [18]. A series of 2-imino
thiazolidinones C were reported to kill MDA-MB-231cancer cells at an IC50 between 0.3 and
6.43 μM [19]. In 2006, substituted 5-benzylidene-2-phenylimino-1,3-thiazolidin-4-one
analogs of D that can effectively induce apoptosis in various cancer cells, including
paclitaxel- and vinorelbine-sensitive and -resistant human lung cancer cells but not in normal
cells, have been reported [20]. Thus, 5-benzylidene-2-phenylimino thiazolidinone moiety has
been noted to be responsible for its anti-cancer potential.

Chart1: Chemical structures of thiazolidinone conjugates with potent anti-proliferative
activity on MDA-MB, SMMC-7721 and human lung cancer cells.
Chromene-2-ones and chromene-4-ones, collectively called chromenes, belonging to the
family of flavonoids, are known for their natural abundance in the form of quercetin, rutin
and luteolin which are known for their therapeutic properties against various diseases [21].
Yi-ming et.al reported novel chromene-2-ones as anticancer agents by targeting microtubule
depolymerization and G2-M arrest followed by apoptosis in cancer cell lines [22]. Recently
Rasha et.al synthesized new aryl chromene-2-ones as tubulin polymerization inhibitors [23].
Chromene-2-one-hydrazone derivatives were reported as tubulin targeted anticancer agents
and 7-Diethylamino chromene-2-one was also reported as a novel microtubule inhibitor with
antimitotic activity in multidrug resistant cancer cells [24]. RKS262 and TGX286 are some of
the chromene-2-one and chromene-4-one containing compounds respectively which are
reported for their vivid anticancer potential. Some of the representative structures of 2-
iminothiazolidin-4-one and chromenone core-containing molecules as anti-proliferative
agents are depicted in figure 1 [25-28].

Figure 1. Examples of bioactive 2-iminothiazolidin-4-one and chromenone containing anticancer agents.
Although chemotherapeutic drugs have different modes of action viz, cause DNA damage,
interfere with DNA synthesis and inhibit mitotic spindle, drugs that interfere with mitotic
spindle formation by tubulin polymerization inhibitors have ben the most successful
chemotherapeutic compounds currently used for anti-cancer treatment. In the light of these
previous reports on chrome-2-ones as tubulin polymerization inhibitors and anti-proliferative
activities of 5-benzylidene 2-iminothiazolidinone moiety in cancer research, and in
continuation with our consistent efforts towards the synthesis of cytotoxic NCEs [29-31], we
were encouraged to design and synthesize some lead molecules by incorporating chromenes
into 2-iminothiazolidinone framework (figure 1) with the primary aim of finding potent
newer molecules as tubulin polymerization inhibitors with a better therapeutic profile.
2. Material and Methods
2.1. Chemistry
The reagents and solvents used for the synthesis of designed molecules were obtained from
commercial suppliers. MERCK pre-coated silica gel 60-F-254 (0.5 mm) aluminium plates
used to perform analytical thin layer chromatography (TLC). Ultraviolet light was used to
visualize the spots on TLC plates. Column chromatography was performed using silica gel
60-120. 1H and 13C NMR spectra were recorded on Bruker 500 MHz instrument in CDCl3 or
DMSO-d6 solvents using tetramethylsilane (TMS) as the internal standard. Chemical shifts
for 1H and 13C are reported in parts per million (ppm) downfield from tetramethylsilane. 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). All IR spectra were recorded on a Perkin Elmer FT-IR spectrometer. HRMS
were determined with Agilent QTOF mass spectrometer 6540 series instrument. Melting
point was determined with the help of Stuart advanced melting point apparatus. The names of
all the compounds given in the supporting information were taken from ChemBioDraw Ultra,
Version 12.0.
2.2. Biology
Cell culture
Human cancer cell lines such as lung (A549), breast (MDA-MB-231 and BT-474), liver
(HepG2), colon (HCT-116) and normal human bronchial epithelial cells (Beas-2B) were
obtained from National Centre for Cell Science, Pune, India. DMEM and RPMI-1640
Medium (Sigma-Aldrich, USA) was used for the culture and growth of procured cell lines.
10% fetal bovine serum stabilized with 1% antibiotic-antimycotic solution (Sigma-Aldrich,
USA) was supplied to the cells in incubator at 37 C. After reaching 80-90% of confluency,
cells were further sub-cultured using 0.25% trypsin/1 mM EDTA solution for additional
passage.
MTT assay
The anti-proliferative activity of all newly synthesized compounds was determined by using
MTT assay, where the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) by mitochondrial succinate dehydrogenase was measured. In brief, the
cells were allowed for seeding in 96-well plates (4000 cells per well) using 100 µL of
medium. Further, the cells were allowed to grow overnight for attachment onto the wells.
Then, different concentrations of the synthesized compounds were used to treat cells for a
period of 72 h. Later, 100 µL of MTT (0.5 mg/mL) was added and incubated at 37 C for 4
h. After that, MTT reagent was aspirated and the formazan crystals formed were dissolved in
DMSO (200 µL) for the period of 20 min at 37 C. Finally, the formed formazan was
measured by using a spectrophotometric microtiter plate reader at 570 nm wavelength.
Initially, anti-proliferative activity of the synthesized compounds was screened by MTT assay
at 30 µM concentration. Further, the compounds with IC50 value <30 µM was used for the
dose dependent studies at different concentrations ranging from 0.78 µM to 30 µM.
2.3. Molecular modelling studies
Maestro10.4 of Schrödinger suite 2015-4 was used to perform molecular modelling studies of
the corresponding chromenyl-based 2-iminothiazolidin-4-one derivatives. Maestro Molecule
Builder was employed to draw the 3D structures of the compounds and further optimized by

means of LigPrep module of Schrödinger. Finally, 3D structures of the designed compounds
were docked at the active site of tubulin protein.
3. Results and Discussion
3.1. Chemistry
The structural diversity was accomplished by introducing different substituted benzyl or
phenethyl moieties on the 2-imino group of thiazolidin-4-one with chromene-4-one at 5
position (6a-w). This chromene-4-one side arm was designed to act as potential bioactive
moiety imparting certain physicochemical properties like lipophilicity and steric effect [32].
The substituents have been also swapped to check if the less bulkier moieties on 5-position
would have any positive effect. However, owing to the presence of α,β-unsaturated lactone
system introducing a chromene-2-one on the 2-imino group of thiazolidin-4-one was more
convenient than chromene-4-one. Thus 12a-f were designed by introducing the chromene-2-
one as a 2-imino substituent and a benzylidine appendage at 5-position (scheme 1).
O
HN
S
N
Z
Y
Y and Z = substituted phenyl, benzyl, phenethyl, chromen-2-one, chromene-4-one
O
O
O
HN
HN
R2
S
N
S
N
n
O
R3
6a-w
O
O
R1
12a-f
Scheme 1: Design of novel chromene-4-one/ chromene-2-one based molecular hybrids of 2-iminothiazolidin-4-
one
The multi-step reaction protocols used to arrive at 6a-w and 12a-f series are provided in
Scheme 2a and 2b. 2-Iminothiazolidin-4-one (5) moiety was synthesized in good to excellent
yields by cyclization of corresponding N-benzyl/phenethyl-2-chloroacetamide (4) which was
obtained by chloroacetylation of commercially available benzyl/phenethylamine (3) [33]. The
required appendage 3-formylchromone (2) was synthesized from readily available o-
hydroxyacetophenone (1) under Vilsmeier-Haack reaction conditions (POCl3 in DMF)
[34,35]. On the same note, 2-((2-oxo-2H-chromen-4-yl)imino)thiazolidin-4-one (10) was

obtained by cyclization of chloroacetamide derivative (9), that was synthesized by
chloroacetylation of the corresponding 4-aminocoumarin (8). 4-Aminocoumarin (8) was
obtained by simple amination of hydroxycoumarin (7) [36]. Finally, 2-iminothiazolidin-4-one
(5 or 10) was coupled with chromone-3-carbaldehyde/benzaldehyde using sodium acetate
in ethanol at 80 C to furnish the designed target chromenyl-based 2-iminothiazolidin-4-
ones (6a-w and 12a-f) in moderate to very good yields (scheme 2a and 2b) [37]. The
structures of the obtained compounds (6a-w and 12a-f) were established on the basis of
1
HRMS, 1H and 13C NMR spectroscopy. The H NMR spectrum of representative compound
12b showed a broad singlet of thiazoldinone N-H proton at δ 13.02, singlet of
chlorobenzylidene proton at δ 7.74. The sharp singlet of 2H-chromene proton appeared at δ
13
6.11 and rest all protons appeared in the range of δ 7.70-7.35. In the C NMR spectrum of
12b, the carbonyl carbons of thiazoldinone and chromene-2-one appeared at δ 167.72 and
161.21, respectively. The imine carbon of thiazoldinone appeared at δ 158.47. The third
carbon of chromene-2-one moiety appeared at δ 101.65 and the remaining carbons appeared
1
in the range of δ 154.35-116.95. Nearly similar patterns were noticed in H and 13C NMR
spectra of all the compounds (12a-f). The HRMS (ESI) of compounds (6a-w and 12a-f)
showed characteristic [M+H]+ corresponding peaks equivalent to their molecular formulae.
The relevant spectral data is furnished in supporting information.

Scheme 2a. Synthesis of 2-iminothiazolidin-4-one and chromene-4-one hybrids (6a-w).
O
NH2
O
OH
HN
i
ii
Cl
O
O
O
O
O
8
7
9
iii
O
O
O
HN
HN
S
N
R
311
S
N
iv
R3
O
O
O
O
12
10
R3= H; 3-Cl; 4-Br; 4-Cl; 3,4-di-OCH3; 3,4,5-tri-OCH3
12d: R3= 4-chloro
12a: R3= H
12e: R3= 3,4-dimethoxy
12f: R3= 3,4,5-trimethoxy
12b: R3= 3-chloro
12c: R3= 4-bromo
Reagents and conditions: i) ammonium acetate/140 °C, 3 h ii) DMF/ClCOCH2Cl/0 °C - rt, 4
h (iii) NH4SCN/ ethyl alcohol/reflux 6 h iv) ethyl alcohol/ piperidine/ reflux 8 h
Scheme 2b. Synthesis of 2-iminothiazolidin-4-one and chromene-2-one hybrids (12a-f).

The plausible mechanism of the heterocyclization step and the theoretical existence of
tautomeric forms of key intermediate 2-iminothiazoldin-4-ones (5 and 10) have been shown
in scheme 3.
Scheme 3: Mechanistic pathway for 2-iminothiazolidin-4-ones (5 and 10) and its tautomers.
Further, the stereochemistry was assigned for one of the representative compounds 12f by
detailed NOE studies. The NOESY experiment was performed for compound 12f in DMSO-
d6 at 30 . According to reported literature, in silico DFT calculations and NOESY studies
revealing the presence of Nuclear Overhauser Effect between H10-H9, H7-H8 and H10-H8
confirms the Z-geometry at the double bond as depicted in figure S1, S2 and table S2
(Supporting information) [20, 38-41].
3.2. Biological Screening
3.2.1. In vitro anti-proliferative activity
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was employed to
screen the anti-proliferative activity of all the synthesized compounds (6a-w and 12a-f) on
various human cancer cell lines such as lung cancer (A549), breast cancer (MDA-MB-231
and BT-474), liver cancer (HepG2) and colon cancer (HCT-116). The IC50 (μM) values of
investigational compounds and reference standard (nocodazole) are depicted in table 1.
Table 1. In vitro anticancer activity of compounds 6a-w and 12a-f.
IC50 (µM)a
A549b
>30
MDA-MB-231c
>30
BT-474d
>30
HepG2e
>30
HCT-116f
>30
Beas-2Bg
S. No
Compound
1
2
6a
6b
6c
6d
6e
6f
-
-
-
-
-
-
-
-
-
-
>30
>30
>30
>30
>30
3
19.43 ± 0.53
23.80 ± 1.13
>30
16.90 ± 0.55
26.10 ± 1.57
>30
18.15 ± 1.12
>30
>30
>30
4
18.12 ± 1.07
>30
24.72 ± 0.64
>30
5
>30
6
27.11 ± 0.54
21.42 ± 0.80
16.01 ± 2.18
19.21 ± 1.19
17.38 ± 0.80
>30
19.10 ± 2.10
20.86 ± 0.33
17.88 ± 1.08
>30
>30
>30
7
6g
6h
6i
>30
28.98 ± 0.42
>30
27.01 ± 2.14
>30
8
26.41 ± 2.31
17.93 ± 2.90
18.79 ± 1.42
9
18.54 ± 1.97
>30
12.03 ± 1.11
>30
10
6j
21.25 ± 0.58

11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
6k
6l
26.89 ± 0.70
>30
29.21 ± 0.23
21.47 ± 1.12
>30
>30
>30
>30
>30
25.74 ± 0.13
>30
-
-
6m
6n
>30
>30
>30
>30
-
26.22 ± 0.76
>30
20.47 ± 1.30
11.89 ± 1.19
19.07 ± 1.08
>30
24.80 ± 0.23
>30
18.23 ± 0.16
15.22 ± 1.158
25.28 ± 0.98
28.90 ± 0.57
25.01 ± 1.24
>30
19.09 ± 0.31
24.01 ± 0.87
28.848 ± 1.54
20.59 ± 2.01
24.12 ± 0.93
20.98 ± 0.50
>30
-
6o
-
6p
22.17 ± 0.98
26.19 ± 1.55
20.39 ± 0.90
19.23 ± 1.53
>30
17.98 ± 0.10
>30
-
6q
-
6r
19.88 ± 0.12
16.84 ± 0.82
>30
29.02 ± 0.30
>30
-
6s
-
6t
>30
>30
-
6u
>30
14.46 ± 0.18
10.27 ± 2.52
>30
>30
>30
12.79 ± 2.41
16.13 ± 1.81
>30
-
6v
>30
>30
15.46 ± 1.42
>30
-
6w
20.34 ± 0.85
>30
>30
-
12a
12b
12c
12d
12e
12f
Nocodazole
>30
26.98 ± 1.76
1.22 ± 0.08
4.65 ± 0.66
23.94 ± 1.37
>30
>30
>30
-
1.28 ± 0.98
15.04 ± 1.77
8.68 ± 0.11
25.90 ± 0.19
23.58 ± 0.49
0.68 ± 0.44
0.95 ± 1.88
13.77 ± 0.98
>30
30.83 ± 1.48
>30
10.14 ± 0.81
19.04 ± 1.01
19.14 ± 0.12
17.52 ± 0.03
>30
28.06 ± 1.24
-
-
-
-
8.02 ± 0.96
>30
28.76 ± 0.18
>30
21.01 ± 0.75
1.44 ± 0.14
16.29 ± 1.01
0. 99 ± 1.87
2.68 ± 1.24
0.87 ± 1.44
-
f
a 50% Inhibitory concentration after 72 h of drug treatment. b Human lung cancer. c, d Human breast cancer. e Human liver cancer. Human
colon cancer. g Humannormal bronchial epithelium.
The preliminary screening results indicated that some of the synthesized compounds exhibit
moderate to potent anti-proliferative activity against the tested cancer cells with IC50 values
in the range of 0.95 ± 1.88 to 16.13 ± 1.81 μM. From the observation of IC50 values of the
tested compounds, it is evident that some of the synthesized compounds are sensitive towards
lung (A549) and breast (MDA-MB-231 and BT-474) cancer cell lines and some are moderate
active on liver (HepG2) and colon (HCT-116) cancer cells. From the MTT assay results we
observed that, while compounds 6o, 6u, 6v, 12b, 12c and12d were the most active, the
remaining are moderately active to inactive against the tested cancer cells. Fascinatingly, the
compound 12b was found to be most active in lung (A549) and breast (MDA-MB-231 and
BT-474) cell lines and showed discernible anti-proliferative activity with IC50 of 0.95 ± 1.88
μM towards MDA-MB-231 cells. Gratifyingly, the compound 12b was found to be 28-fold
less active towards normal human bronchial epithelial cells (Beas-2B) with IC50 of 28.06 ±
1.24 μM in contrast to MDA-MB-231 cells, which portrays the selectivity of our synthesized
compounds towards cancerous cells. The promising anti-proliferative activity of compound

12b on MDA-MB-231 cells motivated us to further proceed with the mechanistic
investigation of cancer cell growth inhibition at cellular level.
2.2.2. Cell cycle analysis
The cytotoxic agents display their activity by arresting the progression of cells at a specific
phase in cell cycle. So, cell distribution at different phases of cell cycle was performed by
flow cytometry to figure out which phase has been arrested by compound 12b. MDA-MB-
231 cells are treated with different concentrations (0.5, 1, 2.5 μM) of 12b for about 24 h,
followed by fixation with ethanol and staining by propidium iodide and analysed using flow
cytometer.
From figure 2, it is clear that there is dose dependent increase in cell population in G2/M
phase from 38.62 in control to 59.44, 60.28 and 65.63in 0.5μM, 1μM and 2.5μM,
respectively, indicating the arrest at G2/M phase of cell cycle by 12b in MDA-MB-231 cells.
Figure 2. Effect of 12b on cell cycle progression of MDA-MB-231. Cells were treated with 12b (0.5, 1 and 2.5
μM) and cell cycle analysis was performed by flow cytometric analysis using PI staining after 24 h of
incubation.
3.2.3. Effect on tubulin polymerization
Microtubules are one of the most important components of the eukaryotic cytoskeleton
involved in several crucial cellular functions [42,43]. As per the previous literature reports
[13,24], 2-iminothiazolidin-4-ones and chromene derivatives are known anti-proliferative

agents that cause inhibition of tubulin polymerization. In the present investigation, a
significant inhibition of G2/M phase induced by 12b, led us to explore its microtubule
inhibitory functional aspects. The efficacy of compound 12b was analysed by studying its
effect on tubulin polymerization in vitro, with nocodazole as the positive control. Screening
at concentrations ranging between 0.31-5 µM revealed that 12b inhibited the tubulin
polymerization in a concentration dependent manner in comparison to nocodazole. At 5 µM
concentration, 12b led to 61.79% percentage of tubulin polymerization inhibition with IC50
value of 3.54 ± 0.2 µM. Taken together, the results help us conclude that tubulin might be the
molecular target of 2-iminothiazolidin-4-one derivatives.
Figure 3. Effect of 12b on tubulin polymerization. 12b inhibits the tubulin polymerization in concentration
dependent. Here, Noc represents the nocodazole. Data represents as mean ± SEM (n=3). ***p<0.01 and
****p<0.001 are significantly different from nocodazole.
3.2.4. Apoptosis induction studies
In order to investigate the apoptotic-inducing efficiency of 12b on MDA-MB-231, we have
performed AO/EB, DAPI, DCFDA, JC-1 staining, clonogenic growth inhibition and Annexin
V binding assays and summarized the results in the description below.
3.2.4.1. Morphological observations using phase contrast microscopy
To uncover the efficiency of the active compound on cellular morphology, MDA-MB-231
cells were treated with 12b at different concentrations of 0.5, 1 and 2.5 μM for about 48 h.
The cells were observed for the changes under phase contrast microscope and the images
were captured. From the figure 4, it can be inferred that, increase in concentration of
compound resulted in steep decrease in cell viability as compared to control (untreated cells).
Additionally, at the higher concentration of 2.5 μM, almost complete diminution of cell
number was observed.

Figure 4. Morphological changes observed in MDA-MB-231cells after treatment with 12b for 48 h. Images
were captured at 200X magnification under phase contrast microscope.
3.2.4.2. AO/EB staining
AO/EB is a dye which efficiently diffuses into the cell membranes of live cells and gives
green stain to the nuclei. AO/EB staining assay was performed on MDA-MB-231 following
the treatment of cells with 12b at different concentrations to able to distinguish the live cells
from the deceased ones.
Figure 5. AO/EB staining of 12b. Cells were treated with 12b in the concentrations of 0.5, 1 and 2.5 µM and
compared with control (DMSO treatment). Images were captured at 200X magnification under fluorescent
microscope.
12b was found to cause decrease in live-cell population in dose dependent manner. The assay
further enabled us to clearly visualize other characteristic apoptotic features such as cell
shrinkage and membrane blebbing (figure 5).

3.2.4.3. DAPI staining
The in vitro cytotoxic agents also induce apoptosis by causing nuclear damage and chromatin
condensation which can be visualized by a fluorescent stain DAPI (4',6-diamidino-2-
phenylindole). While the dye permeates less efficiently into live cell membrane, it has
impeccable ability to stain apoptotic cells due to the condensation of nucleus, which is a
distinctive feature of the dye exploited in this assay. Thus, DAPI staining was performed on
MDA-MB-231 cells following the treatment of cells with different concentrations of 12b.
From figure 6 we inferred that in comparison to control, the nucleus of the cells treated with
12b took the blue stain, displayed chromatin condensation and horse shoe shaped nuclei in a
dose dependent manner.
Figure 6. Nuclear morphology of cancer cells after DAPI staining. MDA-MB-231 cells were treated with
different concentrations of 12b for 48 h and stained with DAPI. The images were captured by fluorescence
microscope at 200X.
3.2.4.4. DCF fluorescence
ROS (reactive oxidative species) generation is a consistent property in most of the cytotoxic
agents. The ROS generation can be analysed in cancer cells using DCF-DA (2’,7’-
dichlorofluorescin diacetate) assay. To quantify the ability of ROS generation by 12b, DCF-
DA assay was performed on MDA-MB-231 cells treated with 12b and results are
summarized in figure 7. 12b induced significant generation of ROS and the clear
concentration dependency and quantification in treated cells as compared to control is
represented in bar diagram.

Figure 7. 12b enhances ROS accumulation in cancer cells. (A) MDA-MB-231 cells were treated with 12b (0.5,
1 and 2.5 µM) for 48 h (B) Relative DCF fluorescence intensity was measured and represented as bar chart.
3.2.4.5. Mitochondrial membrane potential (ΔΨm)
The mitochondria play a vital role in different cellular process and are essential for healthy
functioning of cell. Decrease in the mitochondrial membrane potential is observed as a result
of cellular oxidative stress mediated by ROS generation via inhibition of electron transport
chain (ETC). Any disruption in mitochondrial membrane potential is an apparent impact of
early apoptosis.
Apparently, ROS generation by 12b was confirmed by DCF-DA staining assay and JC-1
staining was performed to further quantify and analyse the mitochondrial membrane potential
in MDA-MB-231 cells. JC-1 is a lipophilic, cationic dye that stains normal polarised
mitochondria in red because of J-aggregates, and depolarised mitochondria in green color due
to the presence of J monomers. From the figure 8, it is evident that, with increase in
concentration of 12b, there is a steep fall in number of healthy mitochondria (P1:56.91 in 2.5
μM) and augmentation in depolarised mitochondria (P2:43.91 in 2.5 μM) indicating the
collapse of mitochondrial membrane potential (Dψm) in MDA-MB-231 cells.

Figure 8. Increased loss of mitochondrial membrane potential (MMP) by 12b in MDA-MB-231 cells.
3.2.4.6. AnnexinV/Propidium iodide (PI) dual staining assay
Cells were incubated with JC-1 and subjected to flow cytometer for measuring JC-1
aggregates and monomers. Flow cytometric analysis was performed at the concentration of
0.5, 1 and 2.5 µM. Induction of apoptosis by 12b in MDA-MB-231 cells was confirmed by
various staining assays. Therefore to further quantify the extent of apoptosis on MDA-MB-
231 cells by 12b, annexin V –FITC/propidium iodide dual staining assay was performed.
This assay has an added advantage of differentiating live (Q1-LL; AV-/PI-), early apoptotic
(Q2-LR; AV+/PI-), late apoptotic (Q3-UR; AV+/PI+) and necrotic cells (Q4-UL; AV-/PI+).
From the results of figure 9, it can be observed that there is gradual increase in early
apoptotic cells in LR quadrant from 2.06 in control to 10.6 in 0.5 μM, 26.49 in 1 μM and
62.75 in 2.5 μM. This reveals the concentration dependent apoptosis induction by 12b in
MDA-MB-231 cells.

Figure 9. Effect of 12b on apoptotic cell death in MDA-MB-231 cells after 48 h. 12b 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.
3.2.4.7. Clonogenic growth inhibition assay
Colony forming ability of a cancer determines its capability of proliferation and development
into solid tumors. A clonogenic assay was performed on MDA-MB-231 cells treated with 12b
at various concentrations (0.5, 1, 2.5 μM) and compared with untreated cells (control). The
formation of colonies was observed after 12 days, with an apparent reduction in concentration
of treated cells indicating the potency of compound in inhibiting the colony formation (figure
10).
Figure 10. Effect of 12b on clonogenic growth of MDA-MB-231 cells. Cells were treated with 12b (0.5, 1 and
2.5 µM) and incubated for 48 h. Further, cells were grown for 12 days and the colonies formed were fixed and
stained with 1% crystal violet and photographed using digital camera. (A) Clonogenic growth inhibition of 12b
on MDA-MB-231 cells. (B) Percentage colonies of MDA-MB-231 cells counted automatically by Vilber Fusion
Fx software and data quantified by GraphPad Prism 6.01. The values were expressed as mean ± S.E.M of three
similar independent experiments, ***p<0.001 and ****p<0.0001 versus control.

4. Structure-Activity Relationship (SAR)
The substitutions over benzyl/phenethyl moities on thiazolidinone, and chromenone seem to
play a significant role in imparting biological potency. 1) Compounds 6a-c and 6l-n with
unsubstituted benzyl/phenethyl group on 2-iminothiazolidin-4-one were nearly inactive at 30
μM; 2) The presence of halogen (chloro and fluoro) on benzyl/phenethyl group of
thiazolidinone (6h-j and 6s) and chromene-4-one (6o) containing compounds resulted in
good anti-proliferative activity; 3) Compounds containing electron donating groups such as
methoxy and hydroxyl on the benzyl group of thiazolidinone (6d-g and 6k) are nearly less
active; 4) Insertion of halogens, chlorine and bromine had a significant effect on IC50 values
than the corresponding non-halogenated analogues. Specifically, insertion of chlorine resulted
in a significant increase in cytotoxic activity compared to the analogue that has bromine.
Further, the position of the halogen seemed to be important, as compound 12b, with chlorine
in meta- position of the phenyl is the most potent with an IC50 value of 0.9 ± 1.88μM while
12c with bromine on para- position was the next active compound (13.77 ± 0.98 μM).
Surprisingly, 12d with chlorine in the para- position on the phenyl, however, was less active
against MDA-MB cells. Since addition of halogen substituents increase the lipophilicity of a
molecule, it might probably assume a bigger size and is more polarized. Overall, the
reasonably good IC50 values of 12b and 12c with chloro and bromo substitutents respectively
point towards hydrophobicity and lipophilicity both of which describe the tendency to
interact within themselves and other neighbouring moieties; 5) Overall, the N-chromenyl-2-
one 2-iminothiazolidin-4-ones (scheme 2b) showed greater cytotoxicity than N-
benzyl/phenethyl 2-iminothiazolidin-4-ones (scheme 2a).
5. Molecular modelling studies
Maestro 10.4 of Schrödinger suite 2015-4 was used to perform molecular modelling studies
of the corresponding chromenyl-based 2-iminothiazolidin-4-one derivatives. Maestro
Molecule Builder was employed to draw the 3D structures of the compounds and further
optimized by means of LigPrep module of Schrödinger. Finally, 3D structures of the designed
compounds were docked at the active site of tubulin protein.
5.1. Docking simulation study and Prime MM/GBSA binding energy calculations
The crystal structure coordinates of α/β-tubulin were retrieved from RCSB protein data bank
(PDB ID: 1SA0) [44]. Molecular docking simulation studies were performed to elucidate the
binding mode and type of interactions of 12b with α/β-tubulin using GLIDE docking module
of Schrödinger suite [45]. This molecular docking simulation study suggested that the top

ranked conformation of 12b was well accommodated inside the active site of tubulin protein.
Figure 11 illustrates the detailed analysis of 12b-enzyme complex, which reveals the key
interactions that appear to play a vital role in the binding of 12b in the active site of tubulin.
Figure 11. (a) Predicted binding pose of 12b (ball and stick); (b) Pattern of interactions in the active pocket of
α/β tubulin. The red dashed lines represent hydrogen bonds.
12b has shown four hydrogen bond interactions with the active site residues Asn101, Thr179,
Val181 and Lys352. In general, 12b demonstrated a higher H-bond score against the
colchicine binding site of α/β-tubulin (Glide H-bond score -0.420 kcal/mol) approximately
similar affinity was also observed with the bound ligand (Glide H-bond score -0.511
kcal/mol). The oxygen atom of keto functional group of thiazolidinone moiety has shown H-
bond interaction with amino group of Val181, Lys352 with a distance of 3.79 Å and 3.95 Å,
respectively. The nitrogen atom of thiazolidinone ring has established one H-bond interaction
with the back bone carboxylic acid group of Thr179 (d = 2.70 Å). Similarly, the oxygen atom
of keto functional group of chromene scaffold had shown hydrogen bond contact with
Asn101 (d = 1.86 Å). Furthermore, 12b has shown several hydrophobic interactions with the
key amino acid residues of α/β tubulin, e.g., Ala180, Val181, Tyr224, Cys241, Leu248,
Ala250, Leu255, Ala316, Ala317, Val318, Ala354 and Ile378, these interactions further
stabilized the binding of the 12b in the active site of tubulin.
Figure 12 demonstrates the superimposition of co-crystallized ligand and best docked pose of
12b in the colchicine binding site of α/β-tubulin. Additionally, binding energy calculations

were also performed on the 12b-enzyme complex. From the performed studies, we confirmed
that 12b has good binding energy (-49.222 KCal/mol) similar to the nocodazole (-52.761
KCal/mol) and bound ligand (-64.325 KCal/mol), signifying stable ligand-protein complex
formation, leading to the stronger binding to the target tubulin protein.
Figure 12. Superimposition of 12b (maroon colour stick) and co-crystalized ligand (green colour stick) in the
colchicine binding site of tubulin.
5.2. In silico ADME/T studies
QikProp program of Schrödinger software was used to assess the drug like properties of 12b.
Some of the computed ADME/T parameters are shown in table 2 and their recommended
ranges are mentioned. ADME/T prediction studies reveal that, 12b conforms to the Lipinski's
rule of five and has appropriate logP value. Additionally, the recommended ranges of
physico-chemical descriptors were also not violated.
Table 2. ADME/T profile of 12b.
S. No.
ADME/T Parameters
Rule of five
Recommended Values or
Range
12b
No
1
Maximum is 4
violation
90.477
2
3
PSA (Van der Waals surface area of 7.0 – 200.0
polar nitrogen and oxygen atoms and
carbonyl carbon atoms)
SASA (Total solvent accessible surface 300.0 – 1000.0
area)
623.357
4
5
6
Dipole Moment
Molecular Volume
Donor HB
1.0 – 12.5
8.328
500.0 – 2000.0
0.0 – 6.0
1080.568
1.000

7
8
Acceptor HB
2.0 – 20.0
6.000
0.213
QPlogKhsa (Prediction of binding to -1.5 – 1.5
human serum albumin)
9
QPlogPo/w (Predicted octanol/water -2.0 – 6.5
partition coefficient)
3.376
10
11
QPpolrz (Predicted polarizability in 13.0 – 70.0
cubic angstroms)
38.004
-0.855
QPlogBB
partition coefficient)
QPlogKp (Predicted skin permeability)
(Predicted
brain/blood -3.0 – 1.2
12
13
-8.0 – -1.0
-2.483
-6.233
QPlogHERG (Predicted IC50 value for concern below -5
blockage of HERG K+ channels.)
14
15
QPPCaco (Predicted apparent Caco-2 <25 is poor, >500 great
cell permeability in nm/sec.)
438.114
93.993
POA (Predicted human oral absorption >80% is high <25% is poor
on 0 to 100% scale)
6. Conclusion
In conclusion, we have successfully synthesized, characterized and evaluated different series
of chromenyl based 2-iminothiazolidin-4-one derivatives (6a-w and 12a-f) for their anti-
cancer potential. Anti-proliferative activities of the synthesized compounds were evaluated in
A549 (lung cancer), MDA-MB-231 and BT-474 (breast cancer), HepG2 (liver cancer) and
HCT-116 (colon cancer) cell lines using MTT assay. Amongst the synthesized compounds,
12b showed excellent anti-proliferative activity on all the tested cell lines, particularly in
breast cancer cell lines MDA-MB-231, BT-474 (IC50 of 0.95 ± 1.88 and 1.22 ± 0.08 μM,
respectively) and lung cancer cell line A549 (IC50 = 1.28 ± 0.98 μM). The cell cycle analysis
disclosed that 12b showed significant G2/M phase arrest in MDA-MB-231cells. Additionally,
12b significantly inhibited the tubulin polymerization with IC50 value of 3.54 ± 0.2 µM.
Further, various mechanistic studies for cell growth inhibition performed on MDA-MB-231
cells, revealed that 12b caused apoptosis-mediated cell death in MDA-MB-231 cells. The
molecular modelling studies inferred that 12b binds at the colchicine binding site of the
tubulin with prominent binding affinity. Furthermore, we have studied physico-chemical and
ADMET properties of 12b, which are in appreciative range. Thus from a practical stand
point, chromenyl-2-one based 2-iminothiazolidin-4-one derivatives may lead to the
development of potential therapeutic agents with the ability to act on tubulin protein for
various aggressive cancers.

Acknowledgment
The authors are thankful to NIPER-Hyderabad for facilities and Department of
Pharmaceuticals (DoP), Ministry of Chemicals & Fertilizers, Govt. of India, New Delhi, for
the award of NIPER fellowship.
Conflict of Interest
The authors declare no conflict of interest.
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