Design and synthesis of novel 4-thiazolidinone derivatives with promising anti-breast cancer activity: Synthesis, characterization, in vitro and in vivo results

Article abstract of DOI:10.1016/j.bioorg.2020.104276

Novel lead compounds as anticancer agents with the ability to circumvent emerging drug resistance have recently gained a great deal of interest. Thiazolidinones are among such compounds with well-established biological activity in the field of oncology. Here, we designed, synthesized and characterized a series of thiazolidinone structures (8a-8k). The results of anti-proliferative assay led to the discovery of compound 8j with a high potent cytotoxic effect using colon, liver and breast cancer cells. Furthermore, MDA-MB-231 and 4T1 cell lines were used to represent triple negative breast cancer (TNBC). Next, a number of in vitro and in vivo evaluations were carried out to demonstrate the potential activity against TNBC and also elucidate the possible mechanism of cell death induction. Our in vitro outcomes exhibited an impressive anticancer activity for compound 8j toward MDA-MB-231 cells through inducing apoptosis and a remarkable anti-metastatic feature via suppressing MMP-9 expression as well. Consistently, the in vivo and immunohistopathologic evaluations demonstrated that this compound significantly inhibited the 4T1 induced tumor growth and its metastasis to the lung. Altogether, among numerous thiazolidinone derivatives, compound 8j might represent a promising anticancer agent for TNBC, which is a major concern in the developed and developing countries.

Full text of DOI:10.1016/j.bioorg.2020.104276

BioorganicChemistry104(2020)104276
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design and synthesis of novel 4-thiazolidinone derivatives with promising
anti-breast cancer activity: Synthesis, characterization, in vitro and in vivo
results
Raheleh Tahmasvanda,b, Peyman Bayatc, Seyyed Mahmood Vahdaniparastc, Soudeh Dehghanib,
⁎
⁎
Zahra Kooshafarb, Sepideh Khaleghia, Ali Almasiradc, , Mona Salimib,
a Department of Medical Biotechnology, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
b Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, Iran
c Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
TNBC
Novel lead compounds as anticancer agents with the ability to circumvent emerging drug resistance have re-
cently gained a great deal of interest. Thiazolidinones are among such compounds with well-established bio-
logical activity in the field of oncology. Here, we designed, synthesized and characterized a series of thiazoli-
dinone structures (8a-8k). The results of anti-proliferative assay led to the discovery of compound 8j with a high
potent cytotoxic effect using colon, liver and breast cancer cells. Furthermore, MDA-MB-231 and 4T1 cell lines
were used to represent triple negative breast cancer (TNBC). Next, a number of in vitro and in vivo evaluations
were carried out to demonstrate the potential activity against TNBC and also elucidate the possible mechanism
of cell death induction. Our in vitro outcomes exhibited an impressive anticancer activity for compound 8j
toward MDA-MB-231 cells through inducing apoptosis and a remarkable anti-metastatic feature via suppressing
MMP-9 expression as well. Consistently, the in vivo and immunohistopathologic evaluations demonstrated that
this compound significantly inhibited the 4T1 induced tumor growth and its metastasis to the lung. Altogether,
among numerous thiazolidinone derivatives, compound 8j might represent a promising anticancer agent for
TNBC, which is a major concern in the developed and developing countries.
Thiazolidinone
Apoptosis
Metastasis
BALB/c
1. Introduction
distant regions is prohibited [5,6]. This concern highlights an im-
perative need for novel therapies and causes that a great deal of at-
According to the statistics reported by WHO in Feb 2017, breast
cancer ranks fifth among top causes of mortality due to cancer, which is
growing both in the developed and the developing countries. Among
different types of breast cancer, 266,120 new cases were diagnosed as
the invasive subtypes (triple negative breast cancer, TNBC) in women in
2018 [1]. In this subtype of breast cancer, mortality frequently results
from spreading of the tumor to other organs, named metastasis [2].
Treatment of the invasive breast cancer is also quite challenging be-
cause of its aggressive features [3,4]. A plentiful body of evidence re-
vealed that promising outcome will be achieved, if the metastasis to the
tention has been drawn towards the synthesis of novel anticancer
compounds capable of inhibiting metastasis.
Apoptosis is a programmed cell death which plays a decisive role in
the physiological process [7]. Apoptosis is a subject that has been ex-
tensively studied among biologists and its understanding is crucial in
disease condition so as it both gives information about pathogenesis
and leaves clues on treatment of a disease. Cancer cells receive no signal
of apoptosis due to the imbalance between cell division and cell death
leading to the uncontrolled cell growth [8]. Besides, failure in apoptosis
renders the tumor cells resistant to metastasis-associated death by
Abbreviations: DMEM, Dulbecco's minimum essential medium; DMSO, Dimethyl sulfoxide; ECL, Enhanced chemiluminescence; FBS, Fetal bovine serum; GAPDH,
Glyceraldehyde phosphate dehydrogenase; H&E, Hematoxylin and eosin; HRP/DAB, Horseradish peroxidase/3,3'-diaminobenzidine; IC50, half maximal inhibitory
concentration; IHC, Immunohistochemistry; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; MMP-9, Matrix metalloproteinase-9; MVD,
Microvessel density; NCBI, National cell bank of Pasteur Institute of Iran; PBS, Phosphate buffered saline; PFA, Paraformaldehyde; PI, Propidium iodide; PPAR-γ,
Peroxisome proliferator-activated receptor gamma; PS, Phosphatidyl serine; PVDF, Polyvinylidene difluoride; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel
electrophoresis; SI, Selectivity index; TNBC, Triple negative breast cancer
⁎ Corresponding authors.
E-mail addresses: almasirad.a@iaups.ac.ir (A. Almasirad), salimimona@pasteur.ac.ir (M. Salimi).
https://doi.org/10.1016/j.bioorg.2020.104276
Received 22 June 2020; Received in revised form 8 August 2020; Accepted 9 September 2020
Availableonline12September2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

R. Tahmasvand, et al.
BioorganicChemistry104(2020)104276
Scheme 1. Structural design of compounds (8a-8k).
enhancing tumor progression [9]. In this regard, compelling evidence
indicated that inducing apoptosis is one of the mechanisms by which
the chemotherapeutic agents act. Moreover, many of toxic side effects
induced by chemotherapeutics have roots in sensitivity of the normal
cells to apoptosis [10]. Therefore, continuing interest over the years has
been allocated to design chemical derivatives that their underlying
mechanism would be apoptosis induction in order to fight cancer cells
with nominal side effects on normal cells.
2. Results
2.1. Chemistry
A diverse array of derivatives (8a-8k) were synthesized according to
Scheme 2 and then characterized by physical and spectral data (IR,
1HNMR, 13CNMR, Mass). The hydrazide 7 was prepared as described in
our previous study, starting by dissolving the thiosalicylic acid and
bromobenzene in DMF, adding NaHCO3 plus Cu powder and refluxing
the mixture for 7 h to give compound 5 [18–20]. Compound 6 was
synthesized by esterification of compound 5 with methanol in the
presence of sulfuric acid and then boiling the solution for 24 h. The
treatment of hydrazine hydrate at room temperature with 6 in ethanol
for about 8 h was applied to synthesize 7 as the key intermediate. The
hydrazide 7 and corresponding aromatic aldehydes were refluxed in
toluene and then thioglycolic acid was added and refluxed again to give
compounds 8a-8k.
Among chemical derivatives, thiazolidinone as a privileged struc-
ture has been well recognized in medicinal chemistry owing to its di-
verse biological activity including anticancer [11]. Several reports have
revealed that thiazolidinones induce apoptosis through activating
PPAR-γ [12,13]. Interestingly, researchers from time to time modified
the thiazolidinone scaffold to potentiate its efficacy and lessen the re-
lated toxicity [14]. On the other hand, in our previous studies, we re-
ported the synthesis of new hydrazone compounds possessing diphenyl
thioether moiety (compound 1 in Scheme 1) with noteworthy anti-
cancer activity [15,16]. Keeping in mind these data along with knowing
that thiazolidinone is an important anti-cancer pharmacophore (com-
pound 2, scheme 1) [17], herein, new hybrid molecules bearing di-
phenyl thioether ring and thiazolidinone heterocycle were designed
with aim of exploring novel and potential chemical entities useful in
treatment of invasive triple negative breast cancer via inducing apop-
tosis (compounds 8a-8k) (Scheme 1). The substituted phenyl and their
bioisosteric replacements were selected as the first major factor in de-
signing the thiazolidinone compounds. In order to evaluate whether the
phenyl group plays an essential role in anticancer activity, we replaced
the phenyl group with its bioisosteres; thienyl, furyl and pyridyl at
different positions. Moreover, a diverse of the substituted phenyl deri-
vatives containing electron withdrawing as well as electron donating
groups at the para position of the phenyl were designed. Besides it and
based on our previous experience indicating the potential role of nitro
substitution in inhibiting the growth of cancer cells, we also introduced
a nitro moiety in the thienyl and furyl rings [15,16].
2.2. Compound 8j inhibited breast cancer cell growth in vitro
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) assay is an usual approach in anti-cancer drug evaluation and
considered as a primary screening test to determine cytotoxicity of the
potential medicinal agents. To do it, the percentage of cell cytotoxicity
of the thiazolidinone derivatives (8a-8k) was determined against MDA-
MB-231, HT-29 and HepG2 cell lines at 72 h (Table 1). For N-(2-(5-
nitrofuran-2-yl)-4-oxothiazolidin-3-yl)-2-(phenylthio) benzamide (8j)
inducing a cytotoxicity more than 60%, quantification of its ability to
inhibit the cell growth was performed by determining the IC50 value
against a panel of cancer cells (MDA-MB-231, HepG2 and HT-29) to
ensure its cytotoxic impact after 24, 48 and 72 h of incubation. As the
results shown in Table 2, compound 8j exhibited a time and con-
centration-dependent cytotoxic activity towards different cancer cell
lines; however, MDA-MB-231 cells as an in vitro model of TNBC was our
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R. Tahmasvand, et al.
BioorganicChemistry104(2020)104276
Scheme 2. Synthesis of compounds (8a-8k). Reagents and experimental conditions were as follows: (a) Na2CO3, Cu powder, DMF, reflux, 7 h; (b) H2SO4, CH3OH,
Reflux, 24 h; (c) Hydrazine hydrate, EtOH, Stir, 8 h; (d) ArCHO, toluene, reflux, 3 h; (e) Thioglycolic acid, ZnCl2, toluene, reflux, 24–48 h.
Table 1
Table 2
Cytotoxicity screening for compounds 8a-8k following treatment at 10 µM for
IC50 values for cytotoxic activity of compound 8j towards human cancer cell
72 h, towards human cancer cell lines.a.
lines.a.
Compounds
Cytotoxocity (% of Control/Vehicle)
IC50 (µM)
MDA-MB-231
HepG2
HT-29
Cell lines/time
24 h
48 h
72 h
8a
34.92
38.48
43.08
35.92
24.76
10.33
13.06
6.1
4.37
3.13
7.04
4.86
8.46
0.81
1.04
24.66
11.07
7.37
1.00
2.76
ND
MDA-MB-231
HepG2
58.84
7.0
1.13
1.08
1.17
4.1
7.1
10.9
—
1.08
1.08
1.9
1.15
1.13
1.16
8b
5.5
2.23
5.4
8c
1.38
4.89
9.15
4.52
6.58
2.18
ND
1.39
HT-29
16.37
—
1.13
6.5
8d
29.84
15.16
18.53
23.31
16.13
11.37
91.44
9.22
1.66
0.98
2.73
2.11
1.85
1.88
0.35
2.34
1.46
1.59
0.5
HGF-1
13.51
1.12
8e
a
8f
Values were determined at least three independent experiments each per-
SEM.
8g
formed in triplicate and expressed as mean
8h
2.38
8i
16.53
93.02
28.74
3.1
5.19
0.94
5.98
ND
compound 8j activity between normal fibroblastic cell and cancer cell
8j
74.29
2.09
2.71
74.74
0.54
(SI greater than 7).
8k
3.25
5.2
0.7
0.87
0.7
Control/Vehicle
Doxorubicin
1.14
2.71
95.96
0.23
90.16
0.58
2.3. Compound 8j induced cell apoptosis
a
Values were determined at least three independent experiments each per-
SEM. ND: Not Determined.
Apoptosis induced by compound 8j in tumor cells was quantita-
tively determined by flow cytometry. Phosphatidylserine (PS) in the
inside of the cell membrane is translocated to the outside in the early
stages of apoptosis and forms a complex with annexin V. Thus, annexin
V fluorescence is directly proportional to the population of apoptotic
cells, while PI stains the cells undergoing necrotic changes. Cells stained
with Annexin V-FLUOS and PI are distinguished as necrotic cells (Q1,
annexin-/PI + ), late apoptotic cells (Q2, annexin+/PI + ), intact cells
formed in triplicate and expressed as mean
ultimate goal. Given the results, compound 8j with the IC50 value of
1.9 µM revealed a high potential in cell growth inhibition of MDA-MB-
231 cells at 72 h. Interestingly, this compound exerted a certain extent
viability toward the normal cell line, HGF-1 (IC50 = 13.5 µM) at 72 h
(Table 2). Moreover, selectivity index (SI) manifested a selection of
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R. Tahmasvand, et al.
BioorganicChemistry104(2020)104276
Table 3
Percentage of MDA-MB-231 cells in each state after treatment with compound 8j at 48 h.a
Concentration
Vital cells (%)An-/PI-
Early apoptosis (%) An+/PI-
Late apoptosis (%) An+/PI+
Necrosis (%)An-/PI+
2 µM
96.33
94.23
79.7
0.33
0.40**
1.04****
0.10
1.05
2.25
3.4
0.30
0.31*
0.085**
0.14
0.58
0.7
0.023
0.044
1.7
0.09
4 µM
2.8
0.23**
8 µM
2.48
0.33
0.37***
0.024
13.46
1.03
0.3****
Control/vehicle
98.46
0.82
0.02
a
The data presented are the mean
SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****P < 0.0001 compared with control.
Caspases are believed to execute apoptosis through processing of
their substrates, which leads to the morphological changes associated
with apoptosis including membrane blebbing, DNA degradation and
chromatin condensation [22]. Hence, caspase-3 activity was de-
termined following treatment of MDA-MB-231 cells with 2 and 4 µM
concentrations of compound 8j at 48 h. Since the caspase-3 antibody
applied for our Western blotting analysis could not well detect the ac-
tive 17-kDa subunit, the activation/processing of caspase-3 was as-
sessed by cleavage of the 32-kDa precursor form. As shown in Fig. 3,
after treatment with 4 µM concentration of compound 8j, the expres-
sion level of pro caspase-3 decreased compared to that in the control
cells, whereas no obvious change was found following treatment with
2 µM of compound 8j. Overall, our results indicated that compound 8j
induced apoptosis by regulating pro-caspase 3.
2.4. Evaluating the cell cycle progression following compound 8j treatment
To further explore whether the anticancer activity of compound 8j
is via cell cycle arrest, we analyzed the treated and non-treated MDA-
MB-231 cells after 48 h. MDA-MB-231 cells treated with compound 8j
at different concentrations, 2 and 4 µM, exhibited a typical DNA pattern
Fig. 1. Annexin-V/PI flowcytometric analysis to quantify compound 8j-induced
apoptosis in MDA-MB-231 cells. Dot plot of MDA-MB-231 cells treated with
compound 8j at (A) 2 µM (B) 4 µM (C) 8 µM (D) Control/Vehicle for 48 h. The
results shown are representatives of three independent experiments. Quadrant
3, living cells An−/PI−; Quadrant 4, early apoptotic cells An+/PI−;
Quadrant 2, late apoptotic cells An+/PI+; Quadrant 1, necrotic cells An−/PI
+.
representing G0/G1, S, and G2/M phases of the cell cycle (Fig. 4). MDA-
MB-231 cells displayed a higher G0/G1 population (73.4%) compared
with 62.87% in the control when treated with 2 µM concentration of
compound 8j. The accumulation of cells in G0/G1 phase was aug-
mented by increasing the concentration to 4 µM by 77.7%. While
concomitantly the proportion of cells in S phase decreased from 33.5%
in the untreated cells to 24.60% and 23.17% in the cells treated with
compound 8j at 2 and 4 µM concentrations, respectively. Similarly, G2/
M cells percentage decreased from 7.1% in the control cells to 5.08%
and 5.1% in the treated cells. These findings indicated that cells did not
enter into the S phase and corroborated that the compound 8j affected
cell cycle progression before the G1/S checkpoint. The occurrence of
cell growth arrest at G1/S phase along with the increase in G0/G1
population following treatment with this compound at the two men-
tioned concentrations further explored the apoptotic effect of com-
pound 8j in the breast cancer cells.
(Q3, annexin-/PI-) and early apoptotic cells (Q4, annexin +/PI-). As
illustrated in Table 3, 2 µM concentration of compound 8j caused no
apoptosis induction in MDA-MB-231 cells after 48 h of treatment.
However, cell population at the early and late stages of apoptosis in-
creased gradually in a concentration-dependent manner following
treatment of MDA-MB-231 cells with 4 and 8 µM concentrations. As
depicted in Table 3, the percentage of early apoptotic cells increased
from 0.82% in the untreated cells to 2.25% and 3.4% in the cells treated
with 4 and 8 µM of compound 8j, respectively, at 48 h. Furthermore,
apoptotic cell percentages in the late stage were 0.7% at 4 µM and
2.48% at 8 µM concentrations vs. 0.33% for the control cells. A sig-
nificant increased number of annexin V positive cells were visualized
following compound 8j treatment at 4 µM compared with the control
cells (Fig. 1). Due to the highest rate of necrosis upon treating with the
8 µM, we excluded it and focused our further experiments on the other
two concentrations.
2.5. Compound 8j promoted anchorage-independent growth
In order to verify the inhibitory effect of compound 8j on the an-
chorage-independent growth and self-renew of breast cancer cells, soft
agar colony formation assay as a hallmark of cancer cells was carried
out and defined as the capability of the cells to independently grow on a
solid surface [23]. The results demonstrated that upon treatment of the
cells with compound 8j at 4 µM as the most effective concentration for
48 h, significantly less colonies were formed when compared to the
One of the features in process of apoptosis is morphologic changes
[21]. In this regard, DAPI staining displays the nuclei possessing cyto-
pathic marks typical of apoptosis including DNA condensation and
fragmentation into apoptotic bodies. To find out whether compound 8j
caused nuclear shrinkage and DNA fragmentation, MDA-MB-231 cells
were incubated with 2 and 4 µM concentrations for 48 h, and then
stained with DAPI (Fig. 2). In the control, the living cells presented
homogeneous nuclei staining in spots. The nucleus of the 4 µM -treated
cells demonstrated a condensed blue-fluorescence with the fragmented
apoptotic bodies.
control cells (237.3
14.11 vs. 303.2
7.3) (Fig. 5).
2.6. Anti-invasive property of compound 8j
To explore the effect of compound 8j on the metastatic feature of
highly invasive TNBC cells, we performed matrigel invasion test on
MDA-MB-231 cells treated with the 4 µM concentration of compound 8j
based on our apoptosis induction evaluation. We found out that
4

R. Tahmasvand, et al.
BioorganicChemistry104(2020)104276
Fig. 2. Nuclear staining assay by using DAPI on human breast cancer, MDA-MB-231 cells in the presence and absence of compound 8j. (A) Untreated and treated cells
with (B) 2 µM and (C) 4 µM concentrations for 48 h. The morphological changes were observed using fluorescent microscope (20X).
Fig. 3. Expression of procaspase-3 in MDA-MB-231
cells induced by 2 and 4 µM concentrations of
compound 8j for 48 h. GAPDH was used as internal
control. (A) Relative level of procaspase-3 expres-
sion was calculated and normalized to the loading
control. (B) Corresponding protein levels were as-
sessed using densitometry. Each value represents
the mean
SEM of three independent experi-
ments, one-way ANOVA analysis with Tukey post
test was performed (**p < 0.01 comparing to the
control/vehicle cells).
Fig. 4. Cell cycle phase distribution of MDA-MB-231 cells treated with com-
pounds 8j for 48 h. The data presented are the mean SEM of three in-
dependent experiments of MDA-MB-231 cell cycle phase. Differencesꢀinꢀthe
dataꢀwas comparedꢀwith controlꢀusing two way ANOVAꢀ(***
p < 0.001,
****p < 0.0001).
compound 8j diminished the invasive ability of MDA-MB-231 cells
compared to that of the control cells after 48 h of exposure (Fig. 6).
Fig. 5. Visualization of soft agar colony formation in (A) untreated and (B)
treated MDA-MB-231 cells with 4 µM concentration of compound 8j for 48 h.
(C) Quantification of colonies in the assay described in (A, B) performed using
Image J software of a representative experiment by unpaired t test,**p < 0.01.
2.7. Compound 8j down-regulated MMP-9 mRNA expression level
It has been reported that MMPs are closely associated with tumor
growth, invasion and metastasis [24,25]. Among MMPs family, MMP-9
contributes to breast cancer metastasis [26] as well as tumor angio-
genesis [27]. Thus, we aimed to ascertain that the anti-metastatic effect
of compound 8j is through down regulating the mRNA expression of
MMP-9. Our findings showed that expression level of MMP-9 after
treatment with compound 8j (4 µM) was significantly diminished
compared to the untreated cells (Fig. 7). Notably, MDA-MB-231 cells
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BioorganicChemistry104(2020)104276
Fig. 8. Effect of compound 8j at different doses (1 and 10 mg/kg/day) on tumor
growth during the 3 weeks treatment. Data are expressed as mean
SEM,
n = 8 mice per group. Data in the treated groups was compared with the
control group at each week using two way ANOVAꢀ(*p < 0.05, **p < 0.01,
***p < 0.001, ****p < 0.0001).
Fig. 6. Effect of compound 8j on the invasiveness of MDA-MB-231 cells. The
cells were cultured in the presence of 4 µM concentration of the compound for
48 h within a matrigel invasion chamber. (A, B) Microphotographs of filters in
the untreated and treated cells and (C) Quantitative analysis of the invaded
cells. Differencesꢀinꢀthe invasivenessꢀwas comparedꢀwith controlꢀusing un-
pairedꢀtꢀtestꢀ(**p < 0.01).
suppressed the growth of mammary tumor and 10 mg/kg of compound
8j seems to be more effective than that of 1 mg/kg in restraining the
tumor growth. Notably, no momentous body weight loss was found in
the compound-treated mice during the experimental period.
The tumor sections were then subjected to histopathological ana-
lysis (H & E staining) to find out the intratumor potential of compound
8j. The findings clearly demonstrated that the tumors treated with
compound 8j at 10 mg/kg/day underwent intratumor spaces owing to
the cell necrosis; however, tumor tissues in the 1 mg/kg of compound
8j-treated and control groups exhibited an intact structure with the
large and dark-blue nucleus (Fig. 9 A, B, C). To further confirm the
impact of compound 8j on inhibition of the cell proliferation in vivo,
immunohistochemical analysis was carried out to evaluate the expres-
sion level of a representative marker of proliferation, Ki-67 [28]. Ki-67
had a reduced expression in 10 mg/kg of compound 8j- treated group
(35%), whereas it was highly expressed in the control group (60%)
(Fig. 9D, E). Since cancers that express CD-31 as an endothelial cell
marker are able to spread to other organs, in the current study, tumor
sections were also stained with an anti-CD31 antibody to observe mi-
crovessels. Compound 8j at dose of 10 mg/kg/day led to a statistically
significant reduction in tumor microvessel density (MVD = 31.61
Fig. 7. MDA-MB-231 cells were incubated with compound 8j (4 µM) for 48 h.
The cells were subsequently assayed for MMP-9 mRNA expression by quanti-
tative Real-time PCR. Results are presented as the mean
SEM of three in-
dependent experiments. Unpaired t test analysis (*p < 0.05) was carried out
comparing to the MDA-MB-231cells.
revealed a basal level of MMP-9 mRNA expression.
2.8. Primary tumor growth was inhibited by compound 8j
We next applied orthotopic breast tumor model to verify the anti-
tumor activity of compound 8j on breast tumor growth and metastasis,
since this model has been designated as a well-established prototype to
mimic human breast cancer in an immune-competent situation. To
create this model, 4T1 cells were inoculated into the mammary fat pad
of female BALB/c mice. Compound 8j (1 and 10 mg/kg) was in-
traperitoneally administered daily for 20 days, once tumor was palp-
able. A reduction in tumor size was dramatically observed after 2, 3 and
4 weeks of compound 8j treatment. A 33% and 60% reduction in the
tumor growth was distinguished following 1 and 10 mg/kg of com-
pound 8j treatment, respectively, at the termination of experiment. On
the other hand, volume of the tumor in the control group increased
rapidly after a 4-week investigation, while the growth of the tumors
treated with 1 and 10 mg/kg of compound 8j showed a slowly upward
trend. The tumor volume values were: control group
Fig. 9. Effect of daily administration of compound 8j on solid tumors in BALB/c
mice injected with 4T1 cells. The mice were killed 34 days after cell injection,
and tumor sections were evaluated by (A, B, C) H&E staining and im-
munostaining detection of (D, E) Ki-67 and (F, G) CD 31 (original magnification
×200 and ×400).
(401.6
(157.5
19.13 mm3) and treated groups (272.4
56.47 mm3) and
51.68 mm3) for 1 and 10 mg/kg, respectively at the fourth
week of treatment (Fig. 8). Indeed, compound 8j dose-dependently
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R. Tahmasvand, et al.
BioorganicChemistry104(2020)104276
with anticancer features derived from hydrazides by reacting with
thioglycolic acid become a promising research area [30]. Hence, we
divulged the utility of this efficient pharmacophore to design a series of
compounds possessing antitumor activity (8a-8k).
Structures of the synthesized compounds were confirmed by spec-
tral analysis data. In FT-IR spectra, NH signals were found over
3100 cm−1 and two carbonyl groups were shown as two separate ab-
sorptions between 1664 and 1714 cm−1. In proton NMR, the proton
chemical shift of NH was present at 8.02–10.91 ppm. Thiazolidinone
ring closure was confirmed by finding the Ar-CH as a singlet between
5.97 and 6.27 ppm and two different S-CH doublets in the range of
3.59–4.03 ppm along with a coupling constant about 16.0 Hz. In 13C
NMR, the two carbonyl moieties were detected from 165 to 170 ppm.
The Ar-CH and S-CH peaks found near to 60.0 and 30.0 ppm, respec-
tively. Signals at 163.35 and 161.41 ppm are related to the coupling of
fluorine with C4-fluorophenyl (J = 244.4 Hz), whereas signals at
115.43 and 115.26 ppm belong to C3-, C5-fluorophenyl (J = 21.4 Hz).
The coupling of fluorine with C2, C6 and C1 was not seen due to the
small coupling constants. Exact mass of all the compounds were figured
out by mass spectrometry. The elemental analysis results revealed the
acceptable purity of the compounds.
Our preliminary study displayed that among different derivatives
with thiazolidinone structure, compound 8j bearing nitrofuran group
induced a remarkable cytotoxicity on MDA-MB-231 cells as an in vitro
model of TNBC, the most challenging subtype of breast cancer. Such
cytotoxicity of the compounds is attributed to the presence of the
thiazolidinone ring bearing substituted phenyl or its bioisosteres.
Comparing the cytotoxic activity of compounds 8a-8k, bioisisteric re-
placement of phenyl (compound 8a) with some heteroaromatic rings
(2- or 3-pyridyl-, 2-furyl- or 2-thienyl-) led to a deteriorative effect on
the cytotoxic activity. Moreover, presence of halogens as non-polar
electron withdrawing groups on para position of the phenyl ring was
tolerable, although it was resulted in decreasing the activity in a way
that by increasing the atomic radius and decreasing the electro-
negativity of halogens, the effect became weak (compounds 8b, 8c and
8d). In contrast to halogens, insertion of nitro as a polar and strong
electron withdrawing group (8e) reduced the activity. Thus, it can be
assumed that a hydrophobic pocket in the receptor is near to the para
position of phenyl ring. Notably, substitution on the phenyl ring was
not beneficial to create cytotoxic compounds. The most potent com-
pound obtained by adding the nitro group on the 2 position of the furan
ring (compound 8j), which may be attributed to different orientation of
nitro group in comparison to 8e. The more strong cytotoxic activity of
8j than that of its analogue 8k may be associated with the plausible
hydrogen bonding of oxygen in furan compared with sulfur in the
thiophen ring. These findings are in agreement with other studies cor-
roborating the presence of nitrofuran as an important pharmacophore
in various derivatives with anticancer activity [31–34]. Hence, com-
pound 8j was selected for further investigation.
Fig. 10. Compound 8j (10 mg/kg/day) inhibited metastasis to the lungs of
BALB/c mice inoculated with 4T1 cells. (A, B) H&E staining was performed on
the lungs. Upon treatment, lungs were sampled to evaluate the extent at which
the cells had metastasized. Lung sections were stained with antibody raised
against (C, D) Ki-67 and (E, F) CD31 and then with 1,3-diaminobenzidine (DAB)
and counterstained with hematoxylin. Representative images of the im-
munohistochemical analysis are shown. All photomicrographs are at ×200
magnification.
3.97%) compared to the control with MVD equivalent to
53.64
1.42% (Fig. 9F, G).
2.9. Compound 8j suppressed the metastasis to lung
Given the results obtained in the matrigel and MMP-9 determination
assays for compound 8j, we next studied its anti-metastatic activity in
an in vivo setting. To do it and to find out whether compound 8j
treatment would have therapeutic efficacy in controlling the outgrowth
of metastatic tumor, the number of metastatic nodules in the lung was
counted through microscopy in a 4T1 spontaneous lung metastasis
model 34 days post cell injection. Our H&E results indicated that mice
in the control group developed lung metastasis; however, the number of
metastatic lung foci significantly decreased in the 10 mg/kg of com-
pound 8j-treated group (Fig. 10 A, B). Moreover, to further verify the
histopathologic findings, immunohistochemistry of the lung sections
were carried out using anti-Ki-67 antibody to report the proliferation
index. Compound 8j diminished the proliferation index at dose of
10 mg/kg/day compared to the control (Fig. 10C, D). The extent of
pulmonary metastasis in the treated group was also assessed by de-
termining microvessel density using anti-CD31 anti body (Fig. 10E, F).
The outcomes demonstrated that compound 8j contributed to the re-
In the current study, we reported for the first time that compound 8j
could significantly inhibit growth of the breast cancer in vitro and in vivo
with induction of apoptosis as a main mechanism of action suggesting
that it could be a promising anticancer therapeutic agent. In vitro cy-
totoxicity assay exhibited that this compound could strongly kill the
breast cancer cells with a low IC50 value (1.9 µM) after 72 h of treat-
ment. Based on our literature survey, anti-cancer agents possessing SI
value of more than 2 toward normal cell line indicate a best compro-
mise between a strong activity and high selectivity [35]. Consistently,
our findings represent a desirable selectivity along with a powerful
activity for compound 8j on a triple negative breast cancer cell line.
Another feature of the cancer cells is their anchorage-independent
growth, thus, if a chemical compound is able to inhibit this property, it
will successfully transform the cancer cells to the normal cells [36].
Herein, we visualized a significant (p < 0.01) reduction in terms of
colony formation in the MDA-MB-231 treated cells with compound 8j
compared to the control cells, demonstrating efficacy of compound 8j
duction in lung angiogenesis (39.28
4.4% vs 59.8
4.3% in the
control lungs) corroborating its anti-metastatic property.
3. Discussion
Currently, cancer is known as a developing ecosystem in which
cancer cells effectively interact with their microenvironment. The
emerging drug resistance to chemotherapeutic agents used in the
treatment of cancer in general and breast cancer in particular due to
tumor heterogeneity has necessitated the designing of novel agents [1].
Many interesting properties were reported for compounds containing
hydrazide-hydrazone moiety, among them, anti- cancer activity has
been recently gained increasing attention [29]. To date, thiazolidinones
7

R. Tahmasvand, et al.
BioorganicChemistry104(2020)104276
to kill cancer cells.
On the other hand, increased expression and activity of proteolytic
enzymes including MMPs are associated with the tumor cells metastasis
[50]. It was reported that high expression level of MMP-9 in breast
cancer contributes to high rate of lymph node metastasis, distant me-
tastasis and poorer relapse- free survival [51–54]. Thus, we investigated
the influence of compound 8j on MMP-9 mRNA expression as our
subsequent study. Our findings demonstrated that the level of MMP-9
mRNA expression in the compound 8j-treated cells was lower than that
in the control cells. The obtained data were in line with the findings of
matrigel assay and suggest a further anti-metastatic role for compound
8j, which seems not to be farfetched due to the presence of thiazoli-
dinone and nitro furan moieties together that augmented the anti-me-
tastatic potential of this compound, even more than that of single
moiety alone [39].
Apoptosis plays a decisive role in cancer development and is sup-
posed to be a mechanism against cancer progression by eliminating the
mutated neoplastic cells [37,38]. Thiazolidinone derivatives exert the
apoptotic effect through activating PPARγ [13]. Furthermore, ni-
trofuran derivatives have a checked history as the anti-proliferative
agents with ability to inhibit topoisomerase II [39]. On the other side,
after an un-repairable DNA damage as a result of topoisomerase in-
hibition, cells undergo apoptosis [40]. In light of these considerations,
we decided to find out whether the cytotoxic effect of compound 8j was
related to apoptosis induction by employing caspase-3 activity along
with annexin/PI and DAPI staining assays. Annexin binding test is an
assay in which early detection of apoptosis would be possible before
loss of membrane integrity [41] and subsequently distinguishes the
apoptotic from necrotic cells. Our annexin/PI findings apparently de-
monstrated that a significant percentage of MDA-MB-231 cells under-
went apoptosis process following treatment with compound 8j in a
concentration-dependent manner; however, the higher concentration
(8 µM) was excluded for further evaluation due to the presence of a
large population of necrotic cells. On the other hand, one of the hall-
mark signs of apoptotic mode of cell death is nuclear fragmentation
[42]. Hence, fluorescent DNA-binding agent (DAPI) was applied to vi-
sualize the cellular morphological changes associated with apoptosis.
We found out the condensed nucleus and fragmented apoptotic bodies
upon treatment with compound 8j at 4 µM concentration indicating the
apoptotic potential of compound 8j.
In vivo animal model investigation was used to verify the reliability
of the in vitro results to address the various issues related to anti-breast
cancer properties. In our in vivo study, the anti-tumor and anti-meta-
static effects of compound 8j were evaluated in the 4T1 tumor-bearing
mouse model that mimics initiation and progression of human triple
negative breast cancer in an immune competent condition [55–57]. We
found that daily i.p. administration of compound 8j at doses of 1 and
10 mg/kg for 4 weeks (20 days) could remarkably suppress the growth
of breast cancer tumor. It is of note that no considerable difference was
detected on the body weight between the control and treated groups
implying no obvious toxicity of the compound to the animals. As our
results showed, treatment with compound 8j at dose of 10 mg/kg/day
displayed a potent tumor growth inhibition. Noteworthy is mentioning
that a significant drop in tumor volume was visualized upon treatment
with 1 and 10 mg/kg of compound 8j in the second week of treatment
demonstrating a rapid onset of action with a good penetration into the
tumor. However, increase in the tumor volume followed a slight trend
in the treated groups within the second and third weeks. Further vali-
dation by H&E staining displayed that after compound 8j administra-
tion at 10 mg/kg, large areas of tumor cell death were visualized
compared with the vehicle-control group. Moreover, tumor sections
were stained for the proliferation marker, Ki-67 to verify the anti-tumor
effect of compound 8j. As expected, tumor from the treated group at
10 mg/kg demonstrated less proliferative activity. Accumulating evi-
dence shows that microvessel density (MVD) is an essential prognostic
biomarker correlates with tumor growth and development [58,59]. In
other words, increased expression of CD31 and VEGF in tumor sections
is connected with angiogenesis [60]. Evidently, by increasing angio-
genesis, adequate blood and nutrients are provided for tumor growth
and metastasis. In this study, we noted that CD31 expression was di-
minished in the compound 8j-treated mice (10 mg/kg) compared with
the untreated ones, which was accompanied by the H&E results.
Since metastasis is a major challenge in cancer treatment [61] and
given that 4T1 cells are highly invasive and thereby primary tumor
metastasizes to the lungs after establishment for 3 weeks in BALB/c
mice [55,62], herein, we also focused on anti-metastatic potential of
compound 8j in our metastatic mouse model of breast cancer. In line
with our transwell assay that revealed a strong inhibition of cell inva-
sion following compound 8j treatment, histopathological assay was
carried out on the lung sections obtained from the treated and non-
treated mice. Results exhibited a lower number of pulmonary nodules
in the compound 8j-treated mice at 10 mg/kg. In addition, IHC findings
of the lungs demonstrated the inhibition of Ki-67 and CD31 expressions,
which corroborates our in vitro results suggesting an anti-metastatic role
for compound 8j. Interestingly, the IHC outcomes associating with
CD31 marker verified our in vitro results related to the MMP-9 mRNA
expression. Given that MMP-9 contributes to regulating tumor angio-
genesis through VEGF release from ECM store [63,64], we suggest an
anti-angiogenic role for compound 8j through inhibition of MMP-9
expression. However, further experiments will be required to unravel in
greater detail the underlying mechanism.
As a part of our study to evaluate the role of compound 8j in acti-
vating the process of apoptosis, we carried out Western blotting to
detect cleaved caspase-3. Caspase-3 is one the members of the cysteine
protease family and considered as a vital mediator to execute the pro-
grammed cell death apoptosis [43]. The activated caspase-3 proteoly-
tically degrades the PARP, which in turns inactivates its DNA-repairing
abilities during apoptosis [1,44]. Keeping in mind that MDA-MB-231
cells exhibited apoptotic bodies following treatment with compound 8j,
we next decided to determine the cleavage of caspase-3 for further
confirmation. Our results clearly revealed a reduced level of procas-
pase-3 expression corroborating an apoptotic activity of compound 8j
at 4 µM concentration after 48 h of incubation. Combined, the above
results suggest an apoptosis induction mechanism for anticancer ac-
tivity of the tested compound.
Moreover, a potentially strategy to control tumor growth is mod-
ulating the cell cycle progression in cancer [45]. On the other hand,
apoptosis is triggered by arresting in cell cycle [46], thus, we were
encouraged to evaluate the cell cycle profile upon compound 8j treat-
ment. Our findings indicated that compound 8j was able to induce cell
cycle arrest at G1/S phase in a concentration-dependent manner at 48 h
in MDA-MB-231 cells. These results along with accumulation of the
cells in G0/G1 phase after treating with this compound, validated the
apoptotic effect of compound 8j.
In terms of breast cancer, anti-metastatic activity of thiazolidinone
derivatives has been reported in MDA-MB-231 cells [23]; however, the
effect of compound 8j as a novel synthesized thazolidinone on MDA-
MB-231 cells invasion remained uninvestigated. Indeed, this study at-
tempted to uncover the anti-metastatic role of this compound on breast
cancer by performing matrigel assay on MDA-MD-231 cell line as a
suitable model of metastatic breast cancer. One approach for anti-me-
tastatic therapy is disturbing the one or more steps involved in cancer
metastasis including cell adhesion and invasion [47]. In metastasis
process, tumor cells first traverse through the basal membrane and then
enter the bloodstream; however, invasion via the basal membrane is a
vital step [48,49]. Therefore, we evaluated anti-invasive property of
compound 8j using a transwell membrane system. Our outcomes
showed that the number of MDA-MB-231 invaded cells significantly
decreased following compound 8j treatment at 4 µM concentration
compared to the untreated cells corroborating the ability of this com-
pound to successfully inhibit the cells penetrating the basal membrane.
8

R. Tahmasvand, et al.
BioorganicChemistry104(2020)104276
4. Conclusions
424 (M+, 6), 2229 (8), 213 (100), 184 (43), 139 (10), 93 (66), 77 (5).
Anal. Calcd. for C22H17FN2O2S2: C, 62.25; H, 4.04; F, 4.48; N, 6.60.
Found: C, 61.94; H, 4.28; N, 6.73.
Development of novel synthetic analogues possessing thiazolidinone
structure with anticancer properties, are topics of growing interest in
medicinal chemistry. In the current study, compound 8j as a new
thiazolidinone derivative exhibited a promising anticancer and anti
-metastatic activity in the in vitro and in vivo models of TNBC. The
potential effect of compound 8j is most likely through induction of
apoptosis with G1/S arrest and inhibition of angiogenesis as well. These
findings are owed not only to the thiazolidinone scaffold but also to the
nitrofuran moiety.
N-(2-(4-chlorophenyl)-4-oxothiazolidin-3-yl)-2-(phenylthio)
benzamide (8c). Yield 75%; m.p = 208–209 °C (EtOH). IR (KBr): υ
cm−1 3210 (NH), 1714(C]O), 1652(C]O).1H NMR (500 MHz,
DMSO‑d6): δ 10.74 (s, 1H, NH), 7.52 (d, J = 8.4 Hz, 2H, aromatic, H4,
H6), 7.45–7.29 (m, 9H, H5, H2′, H3′, H5′, H6′, H2, H3, H5, H6-chlor-
ophenyl), 7.26 (t, J = 7.5 Hz, 1H, aromatic, H4′), 6.97 (d, J = 7.9 Hz,
1H, aromatic, H3), 5.97 (s, 1H, Ar-CH), 3.96 (d, J = 16.0 Hz, 1H, S-CH),
3.84 (d, J = 15.8 Hz, 1H, SCH). 13C NMR (126 MHz, DMSO‑d6): δ
168.78, 165.95, 137.24, 136.34, 133.63, 133.55, 132.94, 131.29,
130.05, 130.00, 129.89, 129.65, 128.51, 128.31, 128.17, 126.11,
60.87, 29.27. MS: m/z (%) 442(M++2, 1.9), 440 (M+, 5.8), 213 (100),
184 (44), 152 (9), 33 (66). Anal. Calcd. for C22H17ClN2O2S2: C, 59.92;
H, 3.89; N, 6.35. Found: C, 59.67; H, 4.08; N, 6.52.
5. Experimental
5.1. Chemicals
Chemicals were purchased from Merck and Sigma/Aldrich
Companies. Melting points were taken on an electrothermal IA 9300
capillary melting-point apparatus (Ontario, Canada) and are un-
corrected. 1H NMR spectra were obtained using a Bruker FT-400 or FT-
500 spectrometers (Bruker, Rheinstetten, Germany). Mass spectra were
obtained using a 5973 Network Mass Selective Detector at 70 eV
(Agilent technology). FT-IR spectra were recorded using a Shimadzu
FT-IR 8400S spectrographs (KBr disks). Elemental analysis was carried
N-(2-(4-bromophenyl)-4-oxothiazolidin-3-yl)-2-(phenylthio)
benzamide (8d). Yield 95%; m.p = 198–199 °C (1,4-dioxan); IR (KBr):
υ cm−1 3122 (NH), 1695(C]O), 1664(C]O). 1H NMR (500 MHz,
CDCl3): δ(ppm) 8.08 (s, 1H, NH), 7.65 (dd, J = 7.5,1.3 Hz,1H, aro-
matic, H6), 7.42 (d, J = 8.4 Hz, 2H, aromatic, H2′, H6′), 7.32–7.24 (m,
7H, H4, H5, H4′, H2-, H3-, H5-, H6-bromophenyl), 7.19–7.17 (m, 2H,
aromatic, H3′, H5′), 7.14 (dd, J = 7.91,1.1 Hz, 1H, aromatic, H3), 5.95
(s, 1H, Ar-CH), 3.96 (d, J = 16.0 Hz, 1H, S-CH), 3.80 (d,J = 16.0 Hz,
1H, S-CH). 13C NMR (75 MHz, CDCl3): δ 169.62, 166.01, 136.11,
135.46, 133.87, 132.84, 132.38, 132.20, 131.75, 129.85, 129.43,
127.95, 127.09, 123.74, 62.17, 30.05. MS: m/z(%) 486(M+ +2,1.7),
484 (M+, 1.6), 213 (100), 184 (73), 152 (13), 93 (96), 77 (8), 51 (7).
Anal. Calcd. for C22H17BrN2O2S2: C, 54.44; H, 3.53; N, 5.77. Found: C,
54.15; H, 4.08; N, 5.44.
out with
a Perkin-Elmer Model 240-c apparatus (Perkin Elmer,
Norwalk, CT, USA) at Faculty of Science, University of Tehran. The
results of the elemental analyses (C, H, N) were within
calculated amounts.
0.4% of the
5.2. General procedures for the synthesis of compounds 8a-8k
N-(2-(4-nitrophenyl)-4-oxothiazolidin-3-yl)-2-(phenylthio)ben-
zamide (8e). Yield 70%; m.p = 195–196 °C (EtOH); IR (KBr):υ cm−1
3143(NH), 1691(C]O), 1673(C]O),1537, 1348(NO2). 1H NMR
(500 MHz, DMSO‑d6): δ 10.99 (bs, 1H, 1H, NH), 8.15 (d, J = 8.2 Hz,
2H, H3, H5-nitrophenyl), 7.76 (d, J = 8.3 Hz, 2H, H2-, H6-nitrophenyl),
7.49–7.15 (m, 8H, H4, H5, H6, H2′, H3′, H4′, H5′, H6′), 6.99 (d,
J = 7.9 Hz, 1H, aromatic, H3), 6.14 (s, 1H, Ar-CH), 4.01 (d,
J = 15.9 Hz, 1H, S-CH), 3.87 (d, J = 15.9 Hz, 1H, S-CH). 13C NMR
(126 MHz, DMSO‑d6): δ 168.71, 166.10, 147.59, 146.04, 133.93,
132.42, 131.23, 130.47, 129.49, 129.28, 129.14, 128.09, 128.01,
127.58, 126.35, 123.61, 60.48, 29.24. MS: m/z (%) 451 (M++6), 213
(100), 184 (95), 152 (19), 93 (60), 77(13), 57(14), 51 (9). Anal. Calcd.
for C22H17N3O4S2: C, 58.52; H, 3.80; N, 9.31. Found: C, 58.89; H, 3.47;
N, 8.93.
In a Dean-Starck trap, the hydrazide (7) 0.8 mmol and corre-
sponding aromatic aldehydes 0.8 mmol were refluxed in toluene about
3 h. After the end of the reaction, thioglycolic acid 2.4 mmol was added
and the mixture was refluxed 24–48 h. Finally, following checking the
reaction by TLC, toluene was evaporated and the remaining was neu-
tralized by NaHCO3 10% solution. The resulting precipitate was pur-
ified by crysallization in ethanol or 1,4-Dioxan to give the target
compounds 8a-8k [65,66].
N-(4-oxo-2-phenylthiazolidin-3-yl)-2-(phenylthio)benzamide
(8a). Yield 70%; m.p = 231–232 °C (1,4-Dioxan); IR (KBr): υ cm−1
3155(NH), 1695(C]O), 1670(C]O).1H NMR (500 MHz,CDCl3): δ ppm
8.02 (s,1H,NH), 7.57 (d, J = 7.6 Hz,1H, aromatic, H6), 7.45–7.43 (m,
2H, aromatic, H2′,H6′), 7.34–7.32 (m, 3H, aromatic, H4, H3′, H5′),
7.29–7.28 (m, 3H, aromatic, H5, H2-phenyl, H6-phenyl), 7.26–7.25 (m,
3H, H3,H4, H5-phenyl), 7.24–7.18 (t, 1H, aromatic, H4′), 7.07 (d,
N-(4-oxo-2-(pyridin-2-yl)thiazolidin-3-yl)-2-(phenylthio)benza-
mide (8f). Yield 95%; m.p = 203–204 °C (1,4-Dioxan); IR (KBr):υ
cm−1 3184(NH), 1712(C]O), 1679(C]O). 1H NMR (500 MHz,CDCl3):
δ(ppm) 8.59 (bs, 1H, NH), 8.55 (d, J = 4.7 Hz,1H, H6-pyridyl),
7.70–7.65 (m, 2H, aromatic, H6-, H4-pyridyl), 7.34 (d, J = 7.4 Hz, 1H,
H3-pyridyl), 7.31–7.20 (m, 8H, aromatic, H4, H5, H2′, H3′, H4′, H5′, H6′,
H5-pyridyl), 7.07 (d, J = 7.6 Hz, 1H, aromatic, H3), 6.17 (s, 1H, Ar-
CH), 3.86 (d, J = 15.8 Hz, 1H, S-CH), 3.71 (d, J = 15.8 Hz, 2H, S-CH).
13C NMR (126 MHz, CDCl3): δ 170.43, 166.23, 157.51, 150.06, 137.43,
134.13, 134.11, 133.20, 132.38, 131.84, 131.77, 129.57, 129.43,
128.05, 126.86, 123.83, 122.35, 62.71, 29.53. MS: m/z(%) 408 (M+
+1, 13), 407(M+, 6), 229 (9), 213(100), 184(51), 179 (38), 152 (9),
93(45), 77 (60, 51 (10). Anal. Calcd. for C21H17N3O2S2: C, 61.90; H,
4.21; N, 10.31. Found: C, 62.12; H, 4.10; N, 10.02.
J
= 8.0 Hz, 1H, aromatic, H3), 6.10 (s,1H, Ar-CH), 3.87 (d,
J = 15.9 Hz, S-CH), 3.71 (d, J = 15.9 Hz, S-CH). 13C NMR (126 MHz,
DMSO‑d6): δ 170.02, 166.20, 137.22, 136.78, 133.88, 132.80, 132.53,
131.95, 131.64, 129.77, 129.65, 129.27, 129.16, 128.31, 128.24,
126.69, 62.92, 30.25. MS: m/z (%) 406 (M+, 3), 230 (2.3), 213 (100),
184(62), 152 (7), 93 (95), 77 (13), 51 (9). Anal. Calcd. for
C
22H18N2O2S2: C, 65.00; H, 4.46; N, 6.89. Found: C, 65.31; H, 4.78; N,
6.58.
N-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-2-(phenylthio)
benzamide (8b). Yield 98%; m.p = 218–219 °C (4-Dioxan); IR(KBr):
νcm−1 3159(NH), 1710(C]O), 1668(C]O).1H NMR (500 MHz,
DMSO‑d6): δ 10.72 (s, 1H, NH), 7.56–7.52 (m, 2H, aromatic, H4, H6),
7.39 (bs, 3H, aromatic, H5, H2′, H6′), 7.37–7.30 (m, 4H, aromatic, H3′,
H5′, H2-fluorophenyl, H6- fluorophenyl), 7.25 (t, J = 7.3 Hz, 1H, aro-
matic, H4′), 7.17 (t, J = 8.5 Hz, 2H, H3-fluorophenyl, H5- fluor-
ophenyl), 6.96 (d, J = 7.8 Hz, 1H, aromatic, H3), 5.98 (s, 1H, Ar-CH),
N-(4-oxo-2-(pyridin-3-yl)thiazolidin-3-yl)-2-(phenylthio)benza-
mide (8g). Yield 90%; m.p = 168–169 °C (1,4-Dioxan). IR (KBr): υ
cm−1 3161 (NH), 1714 (C]O), 1674 (C]O). 1H NMR (500 MHz,
CDCl3): δ(ppm) 9.52(s, 1H, NH), 8.81 (s, 1H, H2-pyridyl), 8.51(dd,
J = 5.1,1.3 Hz, 1H, H6-pyridyl), 8.02 (d, J = 7.7 Hz, 1H, H4-pyridyl),
7.61 (d, J = 7.6 Hz, 1H, aromatic, H6), 7.37–7.36 (m, 1H, aromatic,
H4), 7.29–7.22 (m, 6H, aromatic, H2′, H3′, H4′, H5′, H6′, H5-pyridyl),
7.21–7.15 (t, J = 7.5 Hz, 1H, aromatic, H5), 7.01(d, J = 7.8 Hz, 1H,
3.95 (d, J = 16.0 Hz, 1H, S-CH), 3.83 (d, J = 15.9 Hz, 1H, S-CH). 13
C
NMR (126 MHz, DMSO‑d6): δ 168.76, 165.93, 163.35, 161.41, 136.41,
134.27, 133.61, 132.99, 131.27, 130.32, 130.26, 129.96, 129.66,
128.31, 128.17, 126.07, 115.43, 115.26, 60.91, 29.31. MS: m/z (%)
9

R. Tahmasvand, et al.
BioorganicChemistry104(2020)104276
aromatic, H3), 6.19 (s, 1H, Ar-CH), 3.86 (d, J = 12.6 Hz, 1H, S-CH),
3.75 (d, J = 12.6 Hz, 1H, S-CH). 13C NMR (75 MHz, CDCl3): δ 169.44,
156.70, 149.72, 149.10, 137.22, 136.62, 133.94, 133.74, 132.63,
132.54, 131.65, 131.04, 129.50, 128.76, 128.07, 126.39, 123.82,
60.43, 30.11. MS: m/z(%) 407 (M+, 5), 230 (2), 213 (100), 184 (95),
152(18), 93 (36), 77 (7), 51 (8), 46 (9). Anal. Calcd. for C21H17N3O2S2:
C, 61.90; H, 4.21; N, 10.31. Found: C, 61.64; H, 4.25; N, 10.14.
N-(2-(furan-2-yl)-4-oxothiazolidin-3-yl)-2-(phenylthio)benza-
mide (8h). Yield 65%; m.p = 178–180 °C (1,4-Dioxan). IR (KBr): υ
cm−1 3148(NH), 1701(C]O), 1682(C]O). 1H NMR (500 MHz,
DMSO‑d6): δ 10.87 (bs, 1H, NH), 7.73 (bs, 1H, aromatic, H6), 7.42 (bs,
5H, aromatic, H2′, H3′, H5′, H6′, H5-furyl), 7.36 (t, J = 7.7 Hz, 1H,
aromatic, H4), 7.26 (t, J = 7.5 Hz, 1H, aromatic, H5), 6.97 (d,
J = 8.0 Hz, 1H, aromatic, H3), 6.59 (s, 1H, H4-furyl), 6.44 (s, 1H, H3-
furyl), 6.01 (s, 1H, Ar-CH), 3.89 (d, J = 16.0 Hz, 1H, S-CH), 3.79 (d,
J = 15.9 Hz, 1H, S-CH). 13C NMR (126 MHz, DMSO‑d6): δ 168.19,
166.01, 149.94, 144.22, 136.95, 133.27, 133.35, 133.01, 132.90,
131.35, 129.74, 129.65, 128.44, 125.89, 110.67, 110.42, 54.56, 28.86.
MS: m/z (%) 397 (M++1, 8), 396 (M+, 11), 229 323 (4.5), 229 (14),
213 (100), 184(38), 152(8), 93(81), 77 (4.8), 51 (8.5), 46 (12). Anal.
Calcd. for C20H16N2O3S2: C, 60.59; H, 4.07; N, 7.07. Found: C, 60.48; H,
3.79; N, 6.83.
5.3. Cell culture
Adenocarcinoma breast cancer (MDA-MB-231, C578), human colon
carcinoma (HT-29, C466), human hepatocyte carcinoma (HepG2,
C158), mouse mammary tumor (4T1, C604) and human gingival fi-
broblast (HGF-1, C165) cell lines were obtained from National Cell
Bank of Pasteur Institute of Iran (NCBI). Cells were grown in Dulbecco's
Minimum Essential Medium (DMEM) (Gibco-BRL, Rockville, IN) con-
taining 10% (v/v) Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100
units/ml penicillin G, and 100 µg/ml streptomycin (Gibco-BRL,
Rockville, IN). Then, cells were maintained in a humidifier atmosphere
of 5% CO2 at 37 °C.
5.4. Cell cytotoxicity assay
In this test, MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium bromide) is reduced to the insoluble purple-blue formazan
crystals precipitated by the living cells [67]. Briefly, chemical com-
pounds were solubilized in DMSO (Dimethyl Sulfoxide) and then di-
luted with medium to the desired concentrations (0.1–50 µM), whereas
DMSO final concentration was maintained at < 0.5%. Cell cytotoxicity
assay was carried out in two stages: a preliminary screening at a unique
concentration (10 µM) of compounds at 72 h (8a-8k) and an IC50 de-
termination test with the increasing concentrations for the selected
compounds with the percentage of cytotoxicity more than 60% at 24,
48 and 72 h. Tested cells were plated in 96-well plates at a density of
1–5 × 103 cells/well for (MDA-MB-231), 3–7 × 103 cells/well for (HT-
29 and HepG2) and 5 × 103 for HGF-1 in the proper culture medium.
Twenty-four hours following seeding, cells were exposed to the inter-
esting compounds for 24, 48 and 72 h. After completion of the in-
cubation time, 20 µl of MTT dye solution (5 mg/ml in PBS) was added
to each well and the plates were further incubated at 37 °C for 4 h. The
visible formazan crystals formed by mitochondrial succinate dehy-
drogenase in the live cells, were then solubilized in DMSO and the
optical density was quantified at 570 nm by using an ELISA plate reader
(Epoch2, Biotek, USA). Relative cell cytotoxicity was calculated com-
pared to the non-treated cells.
N-(4-oxo-2-(thiophen-2-yl)thiazolidin-3-yl)-2-(phenylthio)ben-
zamide (8i). Yield 60%; m.p = 180–182 °C (1,4-Dioxan). IR (KBr): υ
cm−1 3158 (NH), 1713 (C]O), 1673 (C]O). 1H NMR (500 MHz,
DMSO‑d6): δ 10.82 (s, 1H, NH), 7.65 (d, J = 4.9 Hz, 1H, aromatic, H6),
7.41 (bs, 5H, aromatic, H2′, H3′, H5′, H6′, H4), 7.39–7.34 (m, 2H, aro-
matic, H5, H5-thienyl), 7.25 (d, J = 8.8 Hz, 2H, aromatic, H3, H4′), 6.97
(t, J = 7.0 Hz, 2H, H3, H4-thienyl), 6.26 (s, 1H, Ar-CH), 3.91 (d,
J = 16.4 Hz, 1H, S-CH), 3.86 (d, J = 16.1 Hz, 1H, S-CH). 13C NMR
(126 MHz, DMSO‑d6): δ 168.25, 166.01, 142.81, 141.71, 133.17,
132.63, 131.33, 130.88, 129.74, 129.69, 129.12, 128.99, 128.39,
128.27, 126.76, 125.92, 57.07, 29.41. MS: m/z(%) 412 (M+,3),
229(16), 213(100), 184 (69), 152 (13), 93(96), 77 (6), 51 (7). Anal.
Calcd. C20H16N2O2S3: C, 58.23; H, 3.91; N, 6.79. Found: C, 57.91; H,
3.77; N, 6.68.
N-(2-(5-nitrofuran-2-yl)-4-oxothiazolidin-3-yl)-2-(phenylthio)
benzamide (8j). Yield 47%; m.p = 156–157 °C (1,4-Dioxan). IR(KBr):
υ
cm−1 3158 (NH), 1713 (C]O), 1673 (C]O).1H NMR
(400 MHz,DMSO‑d6): δ(ppm) 10.82 (s, 1H, NH), 7.61 (bs,1H, aromatic,
H6), 7.49 (d, J = 7.2 Hz,1H, H4-furyl), 7.38–6.99 (m,9H,Ar,furan), 6.09
(s,1H,Ar-CH), 3.97–3.81(2d, J = 16.0 Hz, 2H, S-CH2). 1H NMR
(500 MHz, DMSO‑d6): δ 10.87 (s, 1H, NH), 7.64 (bs, 1H, H4-furyl), 7.49
(d, J = 6.5 Hz, 1H, aromatic, H6), 7.42–7.30 (m, 6H, aromatic, H4, H5,
H2′, H3′, H5′, H6′), 7.09–7.01 (m, 3H, aromatic, H3, H4′, H3-nitrofuryl),
6.10 (s, 1H, Ar-CH), 3.98 (d, J = 15.9 Hz, 1H, S-CH), 3.85 (d,
J = 15.9 Hz, 1H, s-CH). 13C NMR (126 MHz, DMSO‑d6): δ 168.04,
166.02, 154.50, 151.59, 136.61, 135.57, 132.99, 132.76, 131.49,
131.27, 129.96, 129.70, 128.33, 126.14, 113.87, 113.63, 54.35, 28.69.
MS: m/z(%) 441 (M+, 6), 213(100), 184 (50), 152(9), 93 (22), 77 (5),
69 (3), 51 (10). Anal. Calcd. C20H15N3O5S2: C, 54.41; H, 3.42; N, 9.52.
Found: C, 54.57; H, 3.29; N, 9.73.
5.5. Annexin V/PI assay
Apoptosis was tracked by an Annexin V/PI kit obtained from Roche
Applied Science (Indianapolis, IN, USA) according to the manufac-
turer's instructions with minor modification [68]. This experiment was
performed to quantify the percentage of live, apoptotic and necrotic
cells upon compounds treatment. MDA-MB-231 cells were seeded at a
density of 5 × 104 cells/well and exposed to 2, 4 and 8 µM con-
centrations of compound 8j for 48 h. Following washing the cells with
PBS, cells were gently re-suspended in 100 μl of binding buffer. After-
wards, annexin V-FLUOS (0.5 µl) and PI (0.5 μl) solutions were added
and then the tubes were incubated for 10 min in the dark before being
analyzed by flow cytometry.
N-(2-(5-nitrothiophen-2-yl)-4-oxothiazolidin-3-yl)-2-(phe-
nylthio)benzamide (8k). Yield 50%, m.p = 185–186 °C (1,4-Dioxan);
IR (KBr): υ cm−1 3157 (NH), 1710 (C]O), 1668 (C]O), 1510, 1348
(NO2). 1H NMR (500 MHz, DMSO‑d6): δ 10.91 (s, 1H, NH), 7.95 (d,
J = 4.4 Hz, 1H, H4-nitrothienyl), 7.48 (d, J = 7.6 Hz, 1H, aromatic,
H6), 741–7.34 (m, 7H, H4, H5, H2′, H3′, H5′, H6′, H3-nitrothienyl), 7.31
(t, J = 7.5 Hz, 1H, aromatic, H4′), 7.01 (d, J = 8.0 Hz, 1H, aromatic,
H3), 6.27 (s, 1H, Ar-CH), 4.03 (d, J = 16.0 Hz, 1H, S-CH), 3.88 (d,
J = 16.1 Hz, 1H, S-CH). 13C NMR (126 MHz, DMSO‑d6): δ 168.04,
166.08, 151.64, 151.13, 136.22, 133.74, 133.44,132.68, 131.47,
130.30, 129.65, 129.49, 128.49, 128.28, 128.21, 126.32, 56.88, 29.07.
MS: m/z(%) 457 (M+, 6), 213 (100), 184 (47), 152(8),93 (32), 77 (5),
69 (6), 51(6.5), 46 (9). Anal. Calcd. C20H15N3O4S3 C, 52.50; H, 3.30; N,
9.18. Found: C, 52.23; H, 3.53; N, 8.98.
5.6. Cell cycle distribution analysis
The effect of compound 8j on cell cycle progression was assessed by
a standard Propidium Iodide (PI) staining procedure followed by flow
cytometry analysis [67]. MDA-MB-231 cells were exposed to 2 and
4 µM concentrations of compound 8j for 48 h. The cells were then
gathered and fixed using 70% ice-cold ethanol at −20 °C for 24 h.
Following treating the cells with RNase A (20 mg/ml) and PI (1 mg/ml)
for 15 min at 37 °C, the samples were analyzed with PARTEC flow
cytometer (PARTEC GmbH, Munster, Germany) using FlowJo software.
Cell populations were distributed into three subsets of cells representing
G0/G1, S and G2/M phases.
10

R. Tahmasvand, et al.
BioorganicChemistry104(2020)104276
5.7. DAPI staining
5.11. Determination of MMP-9 mRNA level
Cell nuclear morphology was investigated by using fluorescence
microscopy upon DAPI staining. MDA-MB-231 cells were seeded in 24-
well plates and treated with 2 and 4 µM concentrations of compound 8j
for 48 h. After staining the cells with a DAPI solution (2.5 μg/ml) for
15 min at 37 °C, the cells were washed with PBS and methanol and the
nuclear changes were observed by a fluorescence microscope (Nikon
eclipse TS100) [69].
mRNA expression of MMP-9 (Matrix Metalloproteinase-9) was as-
sessed by quantitative real-time polymerase chain reaction (qRT-PCR)
[73]. To do it, Trizol (Invitrogen, USA) was applied to extract total RNA
from the cells. Then, RNA was reverse-transcribed using the Superscript
First-Strand Synthesis System (ThermoFisher Scientific, USA). Primers
for MMP-9 and GAPDH as a housekeeping gene were designed by Al-
leleID6 software (PREMIER Biosoft International, USA). All primers
were checked against the GenBank database to ensure that no simila-
rities remained with other known human DNA sequences
5.8. Transwell invasion assay
5.12. In vivo study
The Transwell assay was conducted to explore the cell invasion
capability [70]. In the current study, invasion assay was performed
with the 24-well Transwell culture chamber with 8 µm pores (SPL Life
Sciences, Korea) coated with sixty microliters of diluted matrigel
(0.5 mg/ml, BD Biosciences, San Jose, USA) on the inner surface. Un-
treated and treated MDA-MB-231 cells with 4 µM concentration of
compound 8j for 48 h, were then starved and suspended in 200 μl of
serum-free medium and implanted into the upper section of the
Transwell culture chamber. Afterwards, 750 μl of the culture medium
supplemented with 10% FBS was added to the lower compartment in
order to attract cells. Upon incubating at 37 °C for 24 h, non-invaded
cells were removed from the top of the matrigel using a cotton-tipped
swab and the cells on the underside of the membrane were washed and
fixed in 4% PFA and stained for 20 min in crystal violet solution
(0.5 mg/ml). Finally, four random fields of the penetrated cells in each
well were counted by using a microscope at 10X magnification in order
to assess the invasiveness.
Thirty two female BALB/c mice (5–7 weeks, 18–20 g weights) were
obtained from the National Animal Center (Pasteur Institute of Karaj)
and maintained under standard conditions of 12/12-h light–dark cycle,
with food and water provided ad libitum. Animals were treated in ac-
cordance with the guidelines approved by the animal ethics committee
of
Tehran
Medical
Sciences,
Islamic
Azad
University
(IR.IAU.PS.REC.1398.104). Mice were subcutaneously implanted with
106 cells/50 µl of exponentially 4T1 cells at the mammary fat pad
[15].Then the mice bearing tumor were randomly assigned into four
groups (n = 8). Ten days after cancer cell inoculation, treatments were
initiated five days a week and lasted for 4 weeks. To explore the anti-
tumor effect of compound 8j, mice were intra-peritoneally injected by
vehicle alone (DMSO) and two doses of the compound (1 and 10 mg/
kg/day) for 20 days. Mice received no treatment in the negative control
group. Following measuring the tumor volumes in two dimensions
thrice a week by a digital caliper during the treatment period, they were
calculated according to the formula: (length × width2)/2. Daily mon-
itoring of the mice was conducted by the same observer to check
toxicity including weight loss, discomfort and death. The animals were
anesthetized with 60 mg/kg of sodium pentobarbital and then chest
was surgically opened and concurrently perfused by 0.9% saline. Fol-
lowing removing the tumors and lungs from the animals, samples were
placed in 10% formalin for fixation and histopathological analysis.
5.9. Soft agar colony formation assay
For this experiment, treated cells with 4 µM concentration of com-
pound 8j for 48 h were seeded in 6-well plates at a density of 5000
cells/well. Following suspending the cells in DMEM containing 0.35%
low-melting agarose, the cells were plated onto solidified 0.5% agarose
having DMEM in 6-well culture plates and allowed to grow for 22 days
in 5% CO2 at 37 °C. After washing and air-drying the cells, the colonies
were manually counted in five fields [71]. Phase contrast micrographs
of the colonies were taken with a Nikon microscope (Nikon, Tokyo,
Japan).
5.13. Histology and immunohistochemistry
Mice were euthanized and then after tissues were perfused, harvested
and fixed in 10% neutral buffered formalin solution. The tissues were
subsequently embedded in paraffin, sectioned longitudinally at 5 µm and
stained with hematoxylin and eosin (H&E). For immunohistochemistry,
the tissue sections were prepared in the same manner as those used for H&
E staining [71]. In addition, tissue sections were assessed for angiogenesis
and proliferation using common markers, CD31 (Abcam, ab28364, Cam-
bridge, UK, 1:50 dilution), and Ki-67 (Abcam, ab15580, Cambridge, UK,
1:200 dilution). Briefly, upon de-paraffinization and antigen retrieving the
sections by boiling in 10 mM sodium citrate for 30 min, the slides were
incubated with the primary antibodies diluted in blocking buffer and kept
at 4˚C overnight. Then, the sections were washed with PBS and incubated
with biotinylated secondary antibody using an HRP/DAB (Horseradish
peroxidase/3,3'-diaminobenzidine) detection IHC kit (Abcam, ab64264,
Cambridge, UK) according to the manufacturer’s instructions and finally
analyzed by an expert pathologist. Images were taken using a Carl Zeiss
AxioImager microscope and Image M1 Software (Carl Zeiss, Jena, Ger-
many) (magnification, 200 and 400×). The percentage of positive tumor
cells within 5 high-randomly fields were accounted for IHC expression
level of Ki-67. For microvessel density, brown-stained vessels were
counted in the selected fields.
5.10. Western blotting
MDA-MB-231 cells (5 × 104 per well) were seeded in 6-well plates
and treated with 2 and 4 µM concentrations of compound 8j for 48 h.
Total protein was extracted using ice-cold lysis buffer consisting of Tris
62.5 mM (pH 6.8), DTT 50 mM, SDS 10%, glycerol and the protease
inhibitor [69]. The protein concentration was determined by Bradford
assay [72]. Almost 40 µg of protein was loaded onto the 15% sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sepa-
rated electrophoretically and transferred onto the PVDF membrane (GE
Health Care Life Sciences, Buckinghamshire, UK). Membranes were
then blocked in 1% (w/v) casein for overnight and probed with anti-
bodies targeting caspase-3 (Abcam, ab13847, Cambridge, UK) at 1:500
dilution. After washing, the membranes were incubated with the sec-
ondary antibody conjugated with horseradish peroxidase (Cell Sig-
naling Technology, 7074S, Beverly, MA) at 1:8000 dilution for two
hours at room temperature. GAPDH (Glyceraldehyde phosphate dehy-
drogenase) (Abcam, ab37168, Cambridge, UK) was used to confirm
equal loading in wells. Following washing steps, the blots were visua-
lized with the ECL (Enhanced Chemiluminescence) advance Western
blotting detection kit obtained from GE Healthcare Life Sciences
(Buckinghamshire, UK) to compare the amount of proteins in each lane.
The black values were determined using Image J software.
5.14. Statistical analysis
All data were analyzed using the Graph Pad Prism 6.0 Software. For
all the measurements, unpaired t-test and one-way ANOVA followed by
11

R. Tahmasvand, et al.
BioorganicChemistry104(2020)104276
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Declaration of competing interest
[16] S. Tavakolfar, E. Mousavi, A. Almasirad, A. Amanzadeh, S.M. Atyabi, P. Yaghamii,
S. Samiee-Sadr, M. Salimi, In vitro anticancer effects of two new potent hydrazide
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
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Acknowledgments
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We acknowledge Dr. Ahad Mohammadnejad of the Department of
Cancer Biology, Research Center, Cancer Institute of Iran, Tehran
University of Medical Sciences for excellent technical support. This
research was financially supported by the Iran National Science
Foundation grant No 96010968 and Pasteur Institute of Iran for pro-
viding materials and research facilities to prepare this valuable docu-
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