Benzylidene thiazolidinediones: Synthesis, in vitro investigations of antiproliferative mechanisms and in vivo efficacy determination in combination with Imatinib

Article abstract of DOI:10.1016/j.bmcl.2020.127561

Thiazolidinedione (TZD) has been an interesting scaffold due to its proven antidiabetic activity and encouraging findings in anticancer drug discovery. We synthesised benzylidene thiazolidinedione derivatives which exhibited excellent antiproliferative effects in chronic myeloid leukemic cells K562 and the most active compounds 3t and 3x had GI₅₀ value of 0.9 and 0.23 μM respectively. Both the compound was found to arrest the growth of K562 cells in G0/G1 phase in a time and dose dependent manner. Further, western blot analysis revealed that 3t and 3x could also inhibit the expression of cell proliferation markers, PCNA and Cyclin D1 and compound 3x up-regulated apoptosis markers, cleaved PARP1 and activated caspase 3, which could be a possible mechanism for the excellent antiproliferative effects exhibited by these compounds. In vitro combination studies of 3t and 3x with Imatinib found to potentiate the antitumor effects of Imatinib. Further in vivo efficacy in K562 xenografts, of 3t and 3x alone and in combination with Imatinib was found to be promising and far better than control group and combination treatment was found to be more effective as compared to only Imatinib treated or test compound treated animals. Thus, our findings suggest that these compounds are promising antitumor agents and could help to enhance the anticancer effects of Imatinib and other tyrosine kinase inhibitors, when used in combination.

Full text of DOI:10.1016/j.bmcl.2020.127561

Bioorganic&MedicinalChemistryLetters30(2020)127561
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzylidene thiazolidinediones: Synthesis, in vitro investigations of
antiproliferative mechanisms and in vivo efficacy determination in
combination with Imatinib
⁎
Hardik Joshia, Vijay Patila, Kalpana Tilekara, Neha Upadhyaya, Vikram Gotab, C.S. Ramaaa,
a Department of Pharmaceutical Chemistry, Bharati Vidyapeeth’s College of Pharmacy, Navi Mumbai, India
b Tata Memorial’s Advance Centre in Treatment, Research and Education in Cancer (ACTREC), Kharghar Navi Mumbai, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Thiazolidinedione
PCNA
Thiazolidinedione (TZD) has been an interesting scaffold due to its proven antidiabetic activity and encouraging
findings in anticancer drug discovery. We synthesised benzylidene thiazolidinedione derivatives which exhibited
excellent antiproliferative effects in chronic myeloid leukemic cells K562 and the most active compounds 3t and
3x had GI50 value of 0.9 and 0.23 µM respectively. Both the compound was found to arrest the growth of K562
cells in G0/G1 phase in a time and dose dependent manner. Further, western blot analysis revealed that 3t and
3x could also inhibit the expression of cell proliferation markers, PCNA and Cyclin D1 and compound 3x up-
regulated apoptosis markers, cleaved PARP1 and activated caspase 3, which could be a possible mechanism for
the excellent antiproliferative effects exhibited by these compounds. In vitro combination studies of 3t and 3x
with Imatinib found to potentiate the antitumor effects of Imatinib. Further in vivo efficacy in K562 xenografts, of
3t and 3x alone and in combination with Imatinib was found to be promising and far better than control group
and combination treatment was found to be more effective as compared to only Imatinib treated or test com-
pound treated animals. Thus, our findings suggest that these compounds are promising antitumor agents and
could help to enhance the anticancer effects of Imatinib and other tyrosine kinase inhibitors, when used in
combination.
Cyclin D1
PARP1
Caspase3
Apoptosis
In vivo
In vitro
Cell cycle
Antiproliferative
Cancer is a dreadful multicellular disease which can arise from any
cell types and organs with multi-factorial etiology. Cancer cells often
obtain their proliferative advantage over their normal counterparts at
least, in part, because of their inability to undergo terminal differ-
entiation. They remain in the proliferative pool, providing themselves
with a growth advantage.1 Activation of nuclear receptors has been
identified as an approach to induce differentiation and inhibit pro-
liferation of cancer cells.2
PPARγ-dependent and PPARγ-independent properties of TZD con-
taining glitazones, Shiau et al. developed Δ2-PG, Δ2-TG, Δ2-CG, ben-
zylidene derivatives of Pioglitazone, Troglitazone and Ciglitazone , re-
spectively, lacking PPAR-γ activity (Fig. 1). These derivatives have a
double bond adjoining the terminal thiazolidine-2,4-dione ring (ben-
zylidene double bond) which abolishes/decreases ligand binding to
PPAR-γ.8 Both Troglitazone and Δ2-TG induced cytochrome C release
and DNA fragmentation in PC3 and LNCaP cells, attributing to the
growth inhibition by apoptosis. These results and further several re-
ports suggest that TZDs can induce apoptosis and cell cycle arrest in-
dependent of PPARγ activation.4,5,9,10 Interestingly, these TZD deriva-
tives have also been reported to possess anti-inflammatory effects,11,12
which could be of benefit to relieve the pain associated with cancer.
These findings initiated further research to assess the potential of
Thiazolidinediones, as promising new therapeutics for cancer.
Activation of PPARγ results in decreased concentration of serum
glucose in diabetes, which led to the development of PPARγ agonists,
thiazolidinediones (TZDs) containing glitazones such as Pioglitazone
that are in clinical use as antidiabetic drugs.3 Apart from established
metabolic actions, PPARγ agonists induces apoptosis, cell cycle arrest,
and terminal differentiation in several malignant cell lineages. During
last two decades, studies have reported antitumor effects of various
PPARγ ligands on almost every kind of tumor cell.4
Since more than a decade, we are exploring the therapeutic poten-
tial of TZDs as anticancer agents targeting various pathways in cancer.
In one of our reports, we had reported the derivatives of benzylidene
Some glitazones have also been inducing apoptosis by the intrinsic
pathway in a PPARγ- independent manner.5–7 To help discern the
⁎ Corresponding author.
E-mail address: sinharamaa@yahoo.in (C.S. Ramaa).
https://doi.org/10.1016/j.bmcl.2020.127561
Received 11 September 2020; Accepted 14 September 2020
Availableonline19September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

H. Joshi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127561
Fig. 1. Structures of most active compounds 3t and 3x, Pioglitazone (PG), Ciglitazone (CG) and their PPARγ inactive analogues 2-PG and 2-CG respectively.
TZD’s with antiproliferative effects in sub-micromolar range in panel of
cell lines13 and the promising antiproliferative effects exhibited by
these compounds gave us impetus to take this work further to find the
exact molecular mechanism involved in the antiproliferative effects of
these derivatives. These derivatives were designed as to circumvent the
toxicity concerns associated with marketed glitazones.14,15 We started
designing of our compounds with Pioglitazone by considering two
major modifications. Firstly, introduced of easily metabolizable amide
linkage as linker between lipophilic tail and central aryl ring. This
amide linkage may act as metabolic soft spot (diverting metabolism
from TZD ring) and cleave in the presence of amidases in liver, thus
rendering them inactive and improving their metabolic profile as
compared to Pioglitazone. Also, toxicity of glitazones has lately been
correlated with the full agonistic activity of the TZDs. Discovery of
novel PPARγ sparing TZDs such as MCC-555 or 2 derivatives similar to
that of reported by Shiau et al. might help in dodging the PPARγ related
side effects.6 Hence, secondly, we considered the addition of benzyli-
dene double bond in between central aryl ring and TZD ring, as this
may give rise to the compounds which may act by PPARγ independent
manner or partial agonistic manner.
(
13C NMR) and mass spectroscopy. The formation of Knoevenagel
product was confirmed on the basis of proton NMR wherein the ben-
zylidene proton exhibits a singlet signal in the range of at
δ
7.8–7.9 ppm. Formation of chloroacetylated product was confirmed by
the presence of singlet of –CH2 proton resonating at δ 4.0–4.8 ppm. The
IR spectra of the chloroacetylated aryl amines exhibited characteristic
band in the range of 1680–1660 cm−1 corresponding to (C]O)eNH.
The presence of resonance assigned to the eCH2 protons provide evi-
dence for formation of -CH2-O-linkage in the final product, these pro-
tons resonated in 4.64–5.60 ppm region as singlet. 13C NMR spectrum
showed characteristic peaks of –CH2-O in the range of 67 ppm and
carbonyl peaks between 167 ppm for all the moieties. Spectral details
have been included in Supplementary Section 3.
Toxicity to the untransformed hepatocytes was determined by MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
Details of the experimental methods are presented in Supplementary
Data. The TZDs, 3a-3y portrayed a differential inhibition; with MTT
reduction ranging between 70 and 93% (Fig. 2, Supplementary Table
S1). Since, all the compounds showed MTT reduction of above 65%
against the vehicle control, we could significantly state that none of the
compounds exerted any severe hepatotoxic effect. With this data, we
moved ahead and screened the molecules for their antiproliferative
potential.
To further study the structure activity relationship, in addition to
previously reported 3a-3j, we synthesized more derivatives 3k-3y by
retaining of the amide linkage and benzylidene double bond, and by
varying the aromatic (Ar) moiety (Scheme 1). We tried substituting
various electron donating and withdrawing groups on already active
molecules from previous series. In newly selected Ar group, we also
incorporated amines having mono substituted halogens like chlorine,
fluorine and bromine. Further, the derivatives 3a-3y were evaluated in
vitro to find out the molecular mechanism of antiproliferative effects
and in vivo efficacy.
The antiproliferative effects of 3a-3j have already been reported13
and have been included for comparison purpose in Table 1 below, 3k-3y
were evaluated in same panel of cell lines to find antiproliferative ac-
tivity.
Interestingly, all the molecules displayed considerable activity in
MCF7 (breast cancer), oral Cancer (GURAV) and chronic myeloid leu-
kemia cell line (K562). GI50 values of molecules 3k-3y for MCF7 cell
line were in the range of 28–74.5 μM and for GURAV GI50 were in the
range of 35.6–75 μM. However, lower range of GI50 (0.23–24 μM) were
found for K562 cell line, suggesting potential of these molecules in
chronic myeloid leukemia (CML). Few molecules also showed activity
in other cell lines. Molecule 3f from previous series was found to be
active on PC3 and MCF7 cell lines with GI50 values 28.18 μM and
25.11 μM respectively. Addition of methyl group on para position of
phenyl ring of compound 3f leads to 3k, which exhibited higher GI50
values on PC3 (> 100 μM) and MCF7 (61.2 μM) cell lines. However, 3k
showed moderate GI50 on K562 (45.8 μM) and GURAV (58.7 μM) cell
lines. Another interesting molecule is 3i from previous series. In the R
group of 3i, we substituted methyl group which led to 3 l. Compound 3i
The final derivatives were synthesised in three steps as described
earlier13 and detailed synthetic procedures has been included in
Supplementary Section 1.1. Molecular structures of 3k-3y were con-
firmed by using infrared (IR) spectroscopy, proton nuclear magnetic
resonance (1H NMR) spectroscopy, carbon nuclear magnetic resonance
had showed notable antiproliferative activity on HOP62 (GI50
-
2.29 μM)), PC3 (GI50-29.51 μM)) and MCF7 (GI50-29.51 μM)) cell lines.
However, methyl substituted derivative, 3 l exhibited higher GI50 va-
lues on HOP62 (GI50-81.4 μM), PC3 (GI50-58.3 μM)) and activity on
MCF7 (GI50-28.3 μM)) remained unchanged. It is also important to note
that, 3l exhibited substantial effect on K562 (GI50-10.7 μM)), GURAV
(GI50-60.9 μM)) and KB (GI50-75 μM)) cell lines, whereas 3i had not
shown activity on these three cell lines. Addition of methyl as R group
of 3f and 3i increased the activity in some cell lines and decreased
activity in others. Molecule 3t which has di-substituted fluorine showed
Scheme 1. Synthetic scheme for synthesizing 3a-3y. Reagents and conditions.
(A)Toluene, piperidinium benzoate, reflux 5–6 hrs., (B) Chloroacetyl chloride,
DCM, K2CO3, stir rt., (C) DMF, K2CO3- stir, rt. overnight.
2

H. Joshi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127561
MTT Reducꢀon in untransformed hepatocytes
100
90
80
70
60
50
40
30
20
10
0
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o 3p 3q 3r 3s 3t 3u 3v 3w 3x 3y
Fig. 2. Reduction of MTT by 3a-3y against control value of 100% in untransformed hepatocytes. Values are presented as Mean
SD. The error bars represent the
standard error in individual determinations.
significant GI50 value on K562 cell line. Addition of chlorine at para
position of R group of 3e leads to 3x, which was also found to be active
on K562 cell line with significant GI50 value of 0.23 μM. When com-
pared to Pioglitazone, almost 70% of the compounds exhibited better
antiproliferative effects in K562 cells.
in HOP62 cell line, they did not show greater or equal activities on
other selected lung cancer cell lines (A549, NCI-H266). Rest of the
compounds (3e, 3o, 3q, 3t, 3x, 3y) which were found to be active on
K562 cell line, showed activity at higher concentrations on selected
leukemic cell lines (Jurkat, MOLT-4, HL-60, U973). Molecule 3e was
found to be active on prostate cancer (PC3) cell line with lower GI50
value and was further evaluated on DU145 cell line. 3e exhibited GI50
value at 59.2 μM on DU145 cell line. Since all the synthesized com-
pounds found to exhibit discernible activity on K562, we continued our
work with this cell line.
In second level of antiproliferative assays, the promising compounds
from 3a to 3y were further treated with the other variants of lung
cancer (A549, NCI-226), prostate (DU145), breast (MDA-MB-435 –
triple negative breast cancer cell) and leukemic (Jurkat, Mot-4, HL60,
U937) cell lines, GI50 values are presented in Table 2.
Although compounds 3a, 3c and 3f exhibited significant GI50 values
To find the effect on phase distribution of cell cycle in K562 cells,
Table 1
GI50 values of 3 k-3y on panel of 6 cancer cell lines.
Compounds
Ar
HOP62 GI50 µM PC3 GI50 µM MCF7 GI50 µM K562 GI50 µM GURAV GI50 µM KB GI50 µM
3a
10H-Phenothiazin-10-yl
0.19
29.5
29.51
> 100
> 100
28.18
0.194
25.11
> 100
37.15
29.51
39.81
61.2
> 100
> 100
2.23
> 100
0.186
> 100
> 100
2.69
> 100
2.75
45.8
10.7
29.5
20.5
9.6
> 100
> 100
0.194
> 100
0.186
> 100
> 100
31.62
> 100
38.9
> 100
> 100
> 100
> 100
2.2
3b
1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl 0.186
> 100
2.2
3c
5-Methylthiazol-2-yl
2-Butyl-4-oxo-1,3-diazaspiro[4,4]non-1-en-3-yl
3-(Trifluromethyl) phenyl
Benzo[d]thiazol-2-yl
5-Nitrothiazol-2-yl
0.169
29.51
> 100
0.99
3d
> 100
2.51
3e
3f
28.18
> 100
> 100
29.51
> 100
> 100
58.3
> 100
> 100
> 100
> 100
> 100
> 100
75
3g
> 100
> 100
2.29
3h
1,2-Benzothiazol-3(2H)-one-1,1-dioxide
Pyridine-2-yl
3i
3j
5-Methylisoxazol-3-yl
4-Methyl-benzothiazol-2-yl
6-Methyl-pyridin-2-yl
4-Nitro-phenyl
> 100
> 100
81.4
3k
58.7
3l
28.3
60.9
3m
> 100
> 100
68
> 100
> 100
> 100
> 100
83.4
72.5
> 100
71.5
> 100
> 100
> 100
> 100
50.3
3n
3-Nitro-phenyl
58.5
3o
4-Fluoro-phenyl
54.6
56.8
3p
Phenyl
> 100
36.1
74.5
17.6
7.4
57
3q
4-Chlorophenyl
51.1
35.6
3r
4-Bromo-phenyl
45.4
86.6
55.1
23.4
20.7
0.9
54.4
32.1
3s
4-Bromo-2-methylphenyl
2,4-Diflurophenyl
> 100
> 100
> 100
75.2
> 100
> 100
> 100
> 100
> 100
> 100
> 100
< 0.1
NE
66.8
69.7
> 100
> 100
99.6
3t
65.8
61.4
3u
3-Chloro-4-methylphenyl
4-Bromo-2,6-diflurophenyl
2-Chloro-4-trifluromethylphenyl
4-Chloro-2-trifluromethylphenyl
2,6-Dichlorophenyl
–
61.8
19.6
23.1
16.3
0.23
6.7
71.4
3v
57.3
64.1
> 100
78.6
3w
> 100
> 100
> 100
< 0.1
NE
67.4
99.3
3x
49.7
86.8
> 100
> 100
< 0.1
NE
3y
61.7
56.8
Doxorubicin
Pioglitazone
< 0.1
NE
< 0.1
40.07
< 0.1
NE
–
GI50 values are presented as mean of three independent experiments. HOP62- Lung cancer, PC3-Prostate cancer, MCF7- Breast cancer, K562- acute myeloid leukemia,
Gurav- Oral cancer, KB-Oral cancer cell lines. Values are presented as mean of 3 independent experiments.*NE-not evaluated.
3

H. Joshi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127561
Table 2
GI50 values of selected compounds on variants of lung, prostate and leukemic cells.
Compounds
GI50 values in µM
A549
NCI-H226
Jurkat
Molt-4
HL60
U937
DU145
MDA-MB 435
3a
3c
3f
> 100
88.7
46.9
NE
82.1
57.8
26.5
NE
NE
NE
NE
NE
NE
NE
NE
59.2
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
3e
3o
3q
3t
26.5
59.9
> 100
34.6
28.9
21.9
47.3
53.5
42.7
40.3
32.5
> 100
73.3
61.1
52.4
47.8
56.7
97.6
49.1
63.9
46.3
35.6
67.5
60
> 100
NE
NE
NE
NE
NE
NE
NE
NE
NE
3x
3y
NE
NE
NE
NE
NE
NE
*NE- not evaluated. HOP62, A549, NCI-H266-non-small cell lung carcinoma cell line, Jurkat, Molt-4, HL60, U973- leukemic cell line, DU145- Prostrate cancer cell
line, MDA-MB-435 – breast triple negative cell line. Values are presented as mean of 3 independent experiments.
Fig. 3. Effect of treatment of 3t and 3x on cell cycle of K562 cells. The cells were treated with 3 concentrations 5 µM, 10 µM and 20 µM and the analysis was done a
three time points of 24 h, 48 h and 72 h. The untreated cells serve as negative control and DMSO treated cells as vehicle control. The error bars represent standard
errors of mean (SEM). The statistical analysis was performed with two tail students ‘t’ test, n = 3, P < 0.05.
4

H. Joshi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127561
flowcytometric analysis of compounds 3x and 3t was carried out. Cells
were incubated with three concentrations of test compounds Viz., 5 μM,
10 μM and 20 μM and samples were withdrawn at 24-, 48- and 72-hours
time points. Details of experimental methodology and statistical ana-
lysis have been included in Supplementary section (Supplementary
Tables S2–S7). At lower dose of 5 μM and 24 h of treatment with 3t and
3x, the K562 cells started to accumulate in G0/G1, S, and G2/M phases.
As the treatment time increased to 48 and 72 h and treatment con-
centrations increased to 10 µM and 20 µM, the cell distribution was
significantly altered in all the phases compared to the control cells.
Thus, as shown in Fig. 3, after exposure of K562 cells 3t and 3x, there
was dose dependent and time dependent effects on distribution of cells
in various phases. The number of cells in G0/G1 phase was found to
increase significantly in comparison with control cells. Also, a sub-
sequent decrease in the number of cells in the S and G2/M phases re-
lative to the control cells were observed. Thus, compounds 3t and 3x
causes the growth arrest of K562 cells in G0/G1 phase in dose and time
dependent manner. The G1 phase is considered a cell differentiation
phase. The arrest of the G1 phase for the cell cycle can be used as in-
dicator of induced differentiation.16,17 Effect of thiazolidinedione
treatment on distribution of cells in various phases of cell cycle have
been studied and reported previously. The glitazones like Pioglitazone,
Ciglitazone and Troglitazone were found to arrest the growth of cells in
G1/G0 phase in cancer cell lines from colon,18 ovarian,19 melanoma20
and adrenocortical21 cancers. Thus, our newly synthesized TZD ana-
logs, 3t and 3x leads to cell cycle arrest in G0/G1 phase, which is
consistent with the reported results.
factor of DNA polymerase, is not only essential for eukaryotic replica-
tion but also plays pivotal role in several DNA damage responsive
pathways. PCNA is a well-known marker for cell proliferation. Glita-
zones in the promotion of G1 cell-cycle arrest has been correlated to the
downregulation of cyclin D1 in several cell lines.25 Cyclin D1 is in-
volved in regulation of G1 to S phase transition in the cell cycle.
Overexpression of cyclin D1 in normal non cancerous cells leads to
malignant phenotype whereas downregulation of cyclin D1 in malig-
nant cells results in loss of the malignant phenotype.26 Treatment of
10 μM and 20 μM concentrations of compound 3t inhibited cyclin D1 at
48 and 72 h, however it did not inhibit PCNA at 48 hrs. Effect of 3t on
PCNA was observed at 20 μM at 72 h. Whereas, compound 3x inhibited
PCNA with 48 h and at 72 h treatments at both concentrations, 10 and
20 μM. 3x significantly inhibited cyclin D1 at 10 μM & 20 μM. Both the
molecules did not show any effect at 24 h (Fig. 4).
Accumulation of subG1 phase cells at higher doses suggests that the
compounds could be inducing apoptotic death of the cells. To de-
termine this, we studied the expression of apoptosis markers PARP127
and caspase 328,29 in the K562 cells treated with 3x and results were
compared with Vehicle (DMSO) control cells. Compound 3x was treated
at three concentrations of 5 µM, 10 µM and 20 µM, keeping DMSO
control for 72 hrs. Fig. 5 elucidates the action of 3x on apoptotic
markers. There was an increase in the expression of cleaved PARP1 and
activated caspase 3 with increasing concentration of 3x, confirming the
contribution of apoptotic death of cells to the antiproliferative effects
exerted.
Recently in phase II trial, the addition of thiazolidinedione
(Pioglitazone) to patients taking Imatinib leading to complete mole-
cular remission has been reported. Out of 24 assessable patients, the
cumulative incidence rate in the treated group reached 57% versus 27%
(P = 0.02) for a historical group of patients having received Imatinib
alone, thus indicating that clinical evidence of efficacy can be detected
even after very brief treatment and early analysis.30 Also, the addition
Based on cell cycle analysis and antiproliferative data we decided to
analyze the protein levels of proliferative cell nuclear antigen (PCNA)
and cyclin D1. The co-relation of these proteins and PPARγ in hema-
tological cancer is well explained.22–24 Moreover, many studies sub-
stantiate that PPARγ ligands inhibits the expression of these proteins by
PPARγ dependent or independent mechanisms. PCNA an auxiliary
Fig. 4. Western blot analysis of markers associated with cell proliferation (PCNA and Cyclin D1), in 3t and 3x-treated cells and controls. (A) K562 cells were treated
with 3t at concentrations of 10 µM and 20 µM for 24 h, 48 h and 72 h and subjected to Western blotting. (B) cells were treated with 3x at the concentrations of 10 µM
and 20 µM for 24 h, 48 h and 72 h and subjected to Western blotting. (C) Cyclin D1 and PCNA bands were quantified and normalized to actin bands. Data are shown
as means of three separate experiments, P < 0.05; bars, SD.
5

H. Joshi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127561
Fig. 5. Western blot analysis of 3x to
determine the expression of apoptosis
marker PARP1 and caspase 3. K562
cells were treated with 3x at con-
centrations of 5 µM, 10 µM and 20 µM
at 72 h of cell treatment and subjected
to western blotting to find the expres-
sion of apoptotic markers, PARP1 and
caspase 3 which were quantified and
normalized to actin bands. Data are
shown as the means of at least two se-
parate experiments P < 0.05; bars, SD.
of various TZDs to Imatinib, Nilotinib and Dasatinib has been found to
increase the activity of these tyrosine kinase inhibitors as evident by
latest reports.31,32 Thus, it was of an interest to find if our compounds
which are modified Pioglitazone derivatives, also potentiates the anti-
tumor effects of Imatinib when used in combination with it, hence in
vitro and in vivo efficacy was determined in K562 cell line and xenograft
model respectively. The in vitro combination study was performed by
treating K562 cells with Imatinib (1 µM), 3t (1 µM) and 3x (1 µM) alone
and the combination of Imatinib (1 µM) + 3t (1 µM) and Imatinib
(1 µM) + 3x (1 µM) by MTT assay as described above. The treatment of
K562 cells with Imatinib, 3t and 3x led to 71%, 62% and 65% inhibition
of proliferation, respectively. However, 3t and 3x in combination with
Imatinib led to 79% and 82% inhibition respectively. Thus, it was ob-
served that these compounds hold the capacity to potentiate the anti-
tumor effects of Imatinib.
The combination of treatment results in significant (p < 0.01) de-
crease in the tumor volume at 30 days. Single drug (without Imatinib)
oral dose administration of 3t and 3x also moderately inhibited the
tumor volume (p < 0.05) (Fig. 6, Supplementary Table S9). Our re-
sults suggest that 3t and 3x could be used along with Imatinib to
overcome resistance and enhance the activity in chronic myeloid leu-
kemia. However, clinical studies are required to validate the results.
Pre-clinical pharmacokinetic characteristics can help predict drug
behavior. The rapid HPLC method was developed and validated by
using rat plasma to study the pharmacokinetic behavior of 3t and 3x
and has been reported elsewhere.33 Both the compounds found to ex-
hibit comparable Pharmacokinetic behavior to the clinical TZD con-
taining glitazones, Pioglitazone.
In conclusion, a series of benzylidene thiazolidine-2,4-diones were
synthesized. All the derivatives were screened to determine their in vitro
cytotoxic activity against panel of tumor cell lines consisting of HOP62,
K562, GURAV, KB2, Hep G2, MCF7, PC3 and their variants. The com-
pounds 3c, 3e, 3o, 3q, 3t, 3x and 3y exhibited promising
With hopeful in vitro effects of combination were observed, we
proceeded further to determine in vivo efficacy of compounds 3t and 3x
alone and in combination with Imatinib. First the acute toxicity and
maximum tolerated dose was determined in healthy mice after oral
administration of increasing doses of 3t and 3x. All the animals were
observed for weight loss, behavioral changes and mortality. None of the
mice in any group showed decrease in weight or mortality. Dose was
gradually increased over the period if previous dose was found to be
well tolerated. Both the molecules were found to be well tolerated upto
2000 mg/kg. MTD results are summarized in Supplementary section,
Table S8. Further to assess the in vivo efficacy, tumors were induced by
subcutaneously injecting 6 × 106 K562 cells/100 μl/mouse mixed with
50% matrigel into the right rear flank of the mouse. All animal work
was approved by Institutional Animal Ethics Committee (IAEC/PR/
2014-2015/03). Treatment was initiated when the tumors were around
300 mm3 in size. Body weight and tumor size were measured every
other day until day 30 after treatment. Tumor size or volume was
calculated as V = (L × W2) × 0.52, where L is the length and W is the
width of the xenograft. The Dunnet’s test was used to determine the
significant reduction in tumor volume as compared to control. Tumor
Fig. 6. Graph of tumor volume vs. time in K562 xenografts treated intra peri-
toneally with 3t and 3x, alone at 200 mg/kg i.p. (up triangle and cross re-
spectively), 3t and 3x in combination with Imatinib, 100 mg/kg + 100 mg kg
Imatinib (asterisk and circle respectively), Imatinib 100 mg/kg (square), saline
(diamond), The Dunnet’s test was used to determine the significant reduction in
tumor volume as compared to control, n = 6. p < 0.05.
growth data are expressed as mean tumor volumes
SEM. For all
data, differences were considered significant at P ≤ 0.05. Encouraging
results were obtained when 3t and 3x were combined with Imatinib.
6

H. Joshi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127561
antiproliferative effects. Several molecules exhibited better anti-
proliferative profile in K62 cells as compared to non-benzylidene TZD
derivative, Pioglitazone proving that the benzylidene derivatives are
more effective anticancer agents. The most potent antiproliferative
compounds were 3t and 3x against K562 cells with GI50 value of 0.9 µM
and 0.23 µM respectively. Both the derivatives, further in flow cyto-
metric analysis, were found to exhibit growth arrest of K562 cells in
G0/G1 phase in time and dose dependent manner. The western blot
analysis revealed that 3t and 3x reduces the expression of cell pro-
liferation markers, PCNA and Cyclin D1. Additionally, antiapoptotic
effects were proved with the modulation of apoptosis marker proteins
PARP1 and caspase 3. Thus, excellent antiproliferative effects of 3t and
3x can be correlated with the dose dependent inhibition of expression of
these cell proliferation and apoptosis markers. Further the in vitro cell
line study and in vivo efficacy study in K562 xenografts in combination
with Imatinib was found to be promising, in line with the reported
effects of combination of TZD’s and tyrosine kinase inhibitors. The
compounds 3t and 3x also possesses the desirable pharmacokinetic
profile making them drug like molecules. Thus, our findings indicate
that these compounds are promising antitumor agents and could help to
enhance the anticancer effects of Imatinib and other tyrosine kinase
inhibitors when used in combination.
in medicinal chemistry: potential anticancer agents. Eur J Med Chem.
2014;87:814–833.
10. Grillier Vuissoz I, Mazerbourg S. PPARγ-independent Activity of Thiazolidinediones:
A Promising Mechanism of Action for New Anticancer Drugs? J Carcinog Mutagen.
2012;01(S8). doi:10.4172/2157-2518.S8-002.
11. Ma L, Pei H, Lei L, He L, Chen J, Liang X, et al. Structural exploration, synthesis and
pharmacological evaluation of novel 5-benzylidenethiazolidine-2,4-dione derivatives
as iNOS inhibitors against inflammatory diseases. Eur J Med Chem. 2015;92:178–190.
12. Ma L, Xie C, Ma Y, Liu J, Xiang M, Ye X, Zheng H, Chen Z, Xu Q, Chen T, Chen J, Yang
J, Qiu N, Wang G, Liang X, et al. Synthesis and biological evaluation of novel 5-
benzylidenethiazolidine-2,4-dione derivatives for the treatment of inflammatory
diseases. J Med Chem. 2011;54(7):2060–2068.
13. Patil V, Tilekar K, Mehendale-Munj S, Mohan R, Ramaa CS. Synthesis and primary
cytotoxicity evaluation of new 5-benzylidene-2,4-thiazolidinedione derivatives. Eur J
Med Chem. 2010;45(10):4539–4544.
14. Crunkhorn S. Bypassing the side effects of thiazolidinediones? Nat Rev Drug Discov.
2007;6(7):517.
15. Isley WL. Hepatotoxicity of thiazolidinediones. Exp Opin Drug Saf.
2003;2(6):581–586.
16. Rizzardi LF, Cook JG. Flipping the switch from G1 to S phase with E3 ubiquitin
ligases. Genes & Cancer. 2012;3(11-12):634–648.
17. Song E, Ma X, Li H, et al. Attenuation of Krüppel-Like Factor 4 Facilitates
Carcinogenesis by Inducing G1/S Phase Arrest in Clear Cell Renal Cell Carcinoma.
Müller R, ed. PLoS ONE. 2013;8(7):e67758. doi:10.1371/journal.pone.0067758.
18. Yoon J-K, Byeon HE, Ko SA, Park B-N, et al. Cell cycle synchronisation using thia-
zolidinediones affects cellular glucose metabolism and enhances the therapeutic ef-
fect of 2-deoxyglucose in colon cancer. Sci Rep. 2020;10(1) https://doi.org/10.1038/
s41598-020-61661-4.
19. Al-Alem L, Southard RC, Kilgore MW, Curry TE. Specific Thiazolidinediones Inhibit
Ovarian Cancer Cell Line Proliferation and Cause Cell Cycle Arrest in a PPARγ
Independent Manner. Agoulnik I, ed. PLoS ONE. 2011;6(1):e16179. doi:10.1371/
journal.pone.0016179.
Declaration of Competing Interest
20. Botton T, Puissant A, Bahadoran P, Annicotte J-S, Fajas L, Ortonne J-P, Gozzerino G,
Zamoum T, et al. In Vitro and in vivo anti-melanoma effects of ciglitazone. J Invest
Dermatol. 2009;129(5):1208–1218.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
21. Ferruzzi P, Ceni E, Tarocchi M, Grappone C, Milani S, et al. Thiazolidinediones in-
hibit growth and invasiveness of the human adrenocortical cancer cell Line H295R. J
Clin Endocrinol Metabol. 2005;90(3):1332–1339.
22. Body S, Esteve-Arenys A, Miloudi H, Recasens-Zorzo C, Tchakarska G, Moros A,
Bustany S, Vidal-Crespo A, Rodriguez V, Lavigne R, Com E, Casanova I, Mangues R,
Weigert O, Sanjuan-Pla A, Menéndez P, Marcq B, Picquenot J-M, et al. Cytoplasmic
cyclin D1 controls the migration and invasiveness of mantle lymphoma cells. Sci Rep.
2017;7(1) https://doi.org/10.1038/s41598-017-14222-1.
Acknowledgment
This work was funded by the Board of Research in Nuclear Sciences
(BRNS), India, grant ID 37B/30/2012.
23. Alao JP. The regulation of cyclin D1 degradation: roles in cancer development and
the potential for therapeutic invention. Mol Cancer. 2007;6(1):24. https://doi.org/
10.1186/1476-4598-6-24.
Appendix A. Supplementary data
24. Smetana K, Gyorkey F, Chan P-K, Tan E, Busch H. Proliferating cell nuclear antigen
(PCNA) and human malignant tumor nucleolar antigens (HMTNA) in nucleoli of
human hematological malignancies. Blut. 1983;46(3):133–141.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127561.
25. C. Blanquicett J. Roman C.M. Hart Thiazolidinediones as anti-cancer agents Cancer
Ther. 2008;6(A):25–34.
26. Prabhakar MM, Vasudevan K, Karthikeyan S, Baskaran N, Silvan S, Manoharan S.
Anti-cell proliferative efficacy of ferulic acid against 7, 12-dimethylbenz(a)
Anthracene induced hamster buccal pouch carcinogenesis. Asian Pac J Cancer Prev.
2012;13(10):5207–5211.
References
1. Andreeff M, Goodrich DW, Pardee AB. Proliferation. Holland-Frei Cancer Medicine
6th edition. Published online 2003. Accessed July 9, 2020. https://www.ncbi.nlm.
nih.gov/books/NBK13035/.
27. Qvarnström. γH2AX and cleaved PARP-1 as apoptotic markers in irradiated breast
cancer BT474 cellular spheroids. Int J Oncol. 2009;35(01).
28. Kumar S. Mechanisms mediating caspase activation in cell death. Cell Death Differ.
1999;6(11):1060–1066.
2. Sherman MH, Downes M, Evans RM. Nuclear receptors as modulators of the tumor
microenvironment. Cancer Preven Res. 2012;5(1):3–10.
29. Fischer U, Jänicke RU, Schulze-Osthoff K. Many cuts to ruin: a comprehensive update
of caspase substrates. Cell Death Differ. 2003;10(1):76.
3. Karak M, Bal N, Bal C, Sharon A. Targeting peroxisome proliferator-activated re-
ceptor gamma for generation of antidiabetic drug. CDR. 2013;9(4):275–285. https://
doi.org/10.2174/15733998113099990065.
30. Prost S, Relouzat F, Spentchian M, Ouzegdouh Y, Saliba J, Massonnet G, Beressi J-P,
Verhoeyen E, Raggueneau V, Maneglier B, Castaigne S, et al. Erosion of the chronic
myeloid leukaemia stem cell pool by PPARγ agonists. Nature.
4. Grommes C, Landreth GE, Heneka MT. Antineoplastic effects of peroxisome pro-
liferatoractivated receptor γ agonists. Lancet Oncol. 2004;5(7):419–429.
5. Feinstein DL, Spagnolo A, Akar C, et al. Receptor-independent actions of PPAR
thiazolidinedione agonists: Is mitochondrial function the key? Biochem Pharmacol.
2005;70(2):177–188.
2015;525(7569):380–383.
31. Prost S, Kirszenbaum M, Dantec ML, Rousselot P, LeBoulch P. Combination of an
anti-cancer agent such as a tyrosinekinase inhibitor and a STAT5 antagonist, pre-
ferably a thiazolidinedione, for eliminating hematologic cancer stem cells in vivo and
for preventing hematologic cancer relapse. Published online April 18, 2017. Accessed
July 11, 2020. https://patents.google.com/patent/US9623015B2/en.
32. Glodkowska-Mrowka E, Manda-Handzlik A, Stelmaszczyk-Emmel A, et al. PPARγ
ligands increase antileukemic activity of second- and third-generation tyrosine ki-
nase inhibitors in chronic myeloid leukemia cells. Blood Cancer J. 2016;6(1):e377.
33. Joshi H. Simple and rapid HPLC method determination of CSR1 and CSR2, new
Heterocyclic thiazolidinedione derivatives, in rat plasma. Int J Pharm Pharm Sci.
2016;8(8):273–276.
6. Joshi H, Pal T, Ramaa CS. A new dawn for the use of thiazolidinediones in cancer
therapy. Expert Opin Invest Drugs. 2014;23(4):501–510.
7. Weng J-R, Chen C-Y, Pinzone JJ, Ringel MD, Chen C-S. Beyond peroxisome pro-
liferator-activated receptor γ signaling: the multi-facets of the antitumor effect of
thiazolidinediones. Endocrine-Related Cancer. Published online June 2006:401-413.
doi:10.1677/erc.1.01182.
8. Shiau C-W, Yang C-C, Kulp SK, et al. Thiazolidenediones mediate apoptosis in
prostate cancer cells in part through inhibition of Bcl-xL/Bcl-2 functions in-
dependently of PPARγ. Cancer Res. 2005;65(4):1561–1569.
9. Asati V, Mahapatra DK, Bharti SK. Thiazolidine-2,4-diones as multi-targeted scaffold
7