Synthesis and evaluation of in vitro cytotoxic effects of triazol/spiroindolinequinazolinedione, triazol/indolin-3-thiosemicarbazone and triazol/thiazol-indolin-2-one conjugates

Article abstract of DOI:10.1007/s40199-020-00364-7

Purpose: Cancer as one of the major diseases with high mortality rates threats human life in the world. Subsequently, the design new potent anticancer agents has attracted much attention in the area of synthetic and medicinal chemistry. In this study, new triazol-linked spiroindolinequinazolinone, thiazol-oxindole and oxindole-thiosemicarbazone conjugates were synthesized and evaluated for their in vitro cytotoxic activity toward different cancer lines. Methods: Some new triazol-linked oxindoles and spirooxindoles conjugates were synthesized. The synthesized compounds were tested for their in vitro cytotoxic activity toward cancer lines including A375, PC3, LNCaP, MDA MB231 and normal cells HDF (human dermal fibroblast). Results: Among all synthesized compounds, the triazol-linked oxindol-thiosemicarbazone conjugate 10b showed the highest cytotoxic activity against different cancer cells. By using quantitative real time PCR (qRT-PCR), it was found that 10b is able to induce apoptosis by alteration of Bax, Bcl2 balance (i.e. by up regulation of Bax and down regulation of Bcl-2 mRNA expression levels). The DAPI staining was used to show the death of cancer cells in the presence of 10b. Interestingly, 10b suppressed the migration of LNCaP cancer cells by up-regulation of epithelial markers (E-cadherin) and down-regulation of mesenchymal markers (vimentin). Conclusion: Our findings revealed that the compound 10b may be a new potent candidate with multiple biological activities to design therapeutic agents against different cancers. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s40199-020-00364-7

DARU Journal of Pharmaceutical Sciences
https://doi.org/10.1007/s40199-020-00364-7
RESEARCH ARTICLE
Synthesis and evaluation of in vitro cytotoxic effects
of triazol/spiroindolinequinazolinedione,
triazol/indolin-3-thiosemicarbazone
and triazol/thiazol-indolin-2-one conjugates
Saeed Nazari1 & Fatemeh Safari2 & Mohammad Barasm Mamaghani1 & Ayoob Bazgir1
Received: 5 May 2020 /Accepted: 29 July 2020
#
Springer Nature Switzerland AG 2020
Abstract
Purpose Cancer as one of the major diseases with high mortality rates threats human life in the world. Subsequently, the design
new potent anticancer agents has attracted much attention in the area of synthetic and medicinal chemistry. In this study, new
triazol-linked spiroindolinequinazolinone, thiazol-oxindole and oxindole-thiosemicarbazone conjugates were synthesized and
evaluated for their in vitro cytotoxic activity toward different cancer lines.
Methods Some new triazol-linked oxindoles and spirooxindoles conjugates were synthesized. The synthesized compounds were
tested for their in vitro cytotoxic activity toward cancer lines including A375, PC3, LNCaP, MDA MB231 and normal cells HDF
(human dermal fibroblast).
Results Among all synthesized compounds, the triazol-linked oxindol-thiosemicarbazone conjugate 10b showed the highest
cytotoxic activity against different cancer cells. By using quantitative real time PCR (qRT-PCR), it was found that 10b is able to
induce apoptosis by alteration of Bax, Bcl2 balance (i.e. by up regulation of Bax and down regulation of Bcl-2 mRNA expression
levels). The DAPI staining was used to show the death of cancer cells in the presence of 10b. Interestingly, 10b suppressed the
migration of LNCaP cancer cells by up-regulation of epithelial markers (E-cadherin) and down-regulation of mesenchymal
markers (vimentin).
Conclusion Our findings revealed that the compound 10b may be a new potent candidate with multiple biological activities to
design therapeutic agents against different cancers.
.
.
.
.
Keywords Spirooxindole containing triazole Spiroindolinequinazoline-dione Oxindole containing thiazole Isatin
Apoptosis Anticancer activity
.
Introduction
Cancer as an uncontrollable growth of abnormal cells is con-
sidered as one of the major diseases with high mortality rates
which threats human life in the world [1]. Most of the clini-
cally available anticancer chemotherapeutic agents cannot dis-
tinguish between the normal and cancerous cells. Moreover,
they have unwanted side effects and drug resistance [2, 3].
Subsequently, the design, development and synthesize new
potent anticancer agents has attracted much attention in the
area of synthetic and medicinal chemistry.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s40199-020-00364-7) contains supplementary
material, which is available to authorized users.
* Fatemeh Safari
fsafari@guilan.ac.ir
* Ayoob Bazgir
a_bazgir@sbu.ac.ir
1
Isatin is endogenously found in human and other mamma-
lian tissues and has been observed as a privileged scaffold in
many natural products and alkaloids [4]. Isatin has become
known as an important moiety that is endowed with many
Department of Chemistry, Shahid Beheshti University, General
Campus, Tehran 1983963113, Iran
2
Department of Biology, Faculty of Science, University of Guilan,
Rasht, Iran

DARU J Pharm Sci
promising biological properties [4–8], mainly anticancer
activity [9]. Spirotryprostatin A (I), Spirotryprostatin B
(II) and Strychnofoline are natural spirooxindoles
possessing anticancer activities (Fig. 1) [10]. The FDA
has been approved Sunitinib (Sutent®) (III), a 5-fluoro-
3-substituted oxindole derivative, for treating advanced
renal cell carcinoma (RCC) and gastrointestinal stromal
tumors (GIST) [11]. Nintedanib (Ofev®) (IV) in combi-
nation with docetaxel for the patients with non-small cell
lung cancer is in first-line chemotherapy [12].
Nintedanib was tested against colorectal cancer [13]
and breast cancer [14].
Recently, molecular conjugation and hybridization
have reported as efficient and applicable tools for devel-
opment of new potentially active molecules [15]. The
strategy is extremely interesting and can minimize the
drug resistance and risk of drug-drug interactions [16].
Therefore, a lot of efforts have been focused on anticancer
potential of isatin-based hybrid or conjugate molecules
such as isatin-thiazol hybrids (V) [17], isatin–
benzothiazole analogs (VI) [18], isatin-thiazolidinone
(VII) [19], isatin-benzimidazole (VIII) [20] and N-
indolylmethyl spiroindoline-quinazolines (IX) [21]. The
outstanding structural properties of 1,2,3-triazoles like hy-
drogen bonding ability, moderate dipole character, and
stability and rigidity under in vivo conditions make their
enhanced biological activities [22]. Likewise,
thiosemicarbazones have significant antitumor activities
[23]. Their antitumor activity is because of an inhibition
of DNA formation produced by the moderation in the re-
ductive reaction of ribonucleotides to deoxyribonucleo-
tides [23]. In the recent years the attention of organic
and medicinal chemists has attracted to design the 1,2,3-
triazoles or thiosemicarbazone conjugate or hybrid with
isatin derivatives as new anticancer candidate drugs
[24–30]. For example, the 1,2,3-triazole tethered
nitroimidazole-isatin conjugates [25], and spirooxindole-
derived morpholine-fused-1,2,3-triazoles [26] displayed a
good anti-proliferative activity against selected human
cancers. The potential anticancer activity of triazole-
linked indole and oxindole glycoconjugates was evaluated
by Babu’s research group [27]. The oxindole-
thiosemicarbazones (X) with anti-cancer activity was syn-
thesized and the effectiveness of aromatic/hydrophobic
properties at the N4 position of the thiosemicarbazone
was investigated [28]. The 4-thiazolidinone-indoles
showed anti-proliferative activities against human tumor
cells [29]. Inspired by the above reports and as a continu-
ation of our previous work on the development of new
methods for oxindole and spirooxindole synthesis
[31–33], we report herein the synthesis and in vitro cyto-
toxic evaluation of 1,2,3-triazol/spiroindolinequinazoline-
dione, 1,2,3-triazol/thiazol-indolin-2-one and 1,2,3-triazol/
indolin-3-thiosemicarbazone conjugates (Fig. 1).
Fig. 1 Design of target molecules
based on reported isatin-based
molecules with cytotoxic
potential

DARU J Pharm Sci
Results and discussion
Chemistry
isolated yield after 5 h. It confirms the use of ultrasound irra-
diation leads to the higher yield at shorter reaction time
(Scheme 1). Therefore, ultrasonic irradiation conditions was
selected for the final step.
The synthesis pathway of 1,2,3-triazol/spiro[indoline-3,2′-
quinazoline]-dione conjugate 6 has been shown in
Scheme 1. The product 6 was synthesized by a sequential
reaction starting from isatin 1. The cyclization and Cu-
catalyzed click chemistry are the key steps for the operation-
ally simple synthesis of 6. First, N-propargyl isatins 2 were
synthesized by the nucleophilic reaction of isatins 1 and pro-
pargyl bromide in the presence of anhydrous potassium car-
bonate. The subsequent P-toluenesulfonic acid (p-TSA) cata-
lyzed nucleophilic condensation reaction of 2 with 2-
aminobenzamide 3 in EtOH under reflux conditions afforded
1-(prop-2-yn-1-yl)-1′H-spiro[indoline-3,2′-quinazoline]-
2,4′(3′H)-diones 4 [31]. The target triazol-liked
spiro[indoline-quinazoline]-diones 6 were obtained by the
Cu-catalyzed click reaction of 4 and aryl or alkyl azids 5 under
ultrasonic irradiation in t-BuOH-H2O for 1 h (Scheme 1). To
delineate the role of ultrasound, the final step of 6a synthesis
was done without ultrasonic irradiation at the same tempera-
ture in t-BuOH-H2O. The product 6a was obtained in 72%
The potential anticancer activities of thiazole skeleton
[34] encouraged us to synthesize a series of 1,2,3-
triazol-linked thiazole-oxindole conjugates 9
(Scheme 2). The 1H-1,2,3-triazol-indoline-2,3-diones 7
were obtained by the Cu-catalyzed click reaction of 2
and phenacyl azides 5 under ultrasonic irradiation in t-
BuOH-H2O at room temperature. Then, 1,2,3-triazol-
linked thiazole-oxindole conjugates 9 were synthesized
by a three-component reaction of 7, thiosemicarbazid
and phenacyl bromides 8 in isopropyl alcohol under ul-
trasonic irradiation at 65 °C for 1 h.
So far, there is not any report to evaluate the possible cy-
totoxic effects of indolinone and thiosemicarbazone conjugate
molecule. Therefore, the 1,2,3-triazol-linked oxindol-
thiosemicarbazone conjugates 10 were synthesized by the re-
action of 7 and thiosemicarbazid in isopropyl alcohol under
ultrasonic irradiation at 65 °C for 1 h (Scheme 2). All com-
pounds 6, 9 and 10 are stable solids whose structures were
established by IR, Mass, and 1H, and 13C NMR spectroscopy.
Scheme 1 Synthesis of 1,2,3-
triazol/spiro[indoline-3,2′-
quinazoline]-diones 6

DARU J Pharm Sci
Scheme 2 Synthesis of triazol-
linked thiazole-oxindole conju-
gates 9
In vitro cytotoxic evaluation
against LNCaP and PC3, respectively. The cytotoxicity eval-
uation of triazol-linked oxindol-thiosemicarbazone conjugates
10a-c suggested that the compounds activity is strongly influ-
enced by the nature of the substituents in the phenyl ring of
triazole moiety. The compound 10a with methoxy group was
inactive while 10c with nitro group showed good activity
against MDA-MB231 and PC3. Interestingly, compound
10b having Br demonstrated the highest cytotoxic activity
against A375 cell line (IC50 = 25.91 μM), MDA-MB-231 cell
line (IC50 = 18.42 μM), PC3 cell line (IC50 = 15.32 μM) and
LNCaP cell line (IC50 = 29.23 μM). Therefore, 10b was se-
lected for further study.
Then, DAPI staining was used to show clear morphological
changes and chromatin fragmentation within the nucleus of
A375, MDA-MB-231, PC3 and LNCaP treated cells with
10b. It was found, their morphology is not altered in untreated
cells (or controls). The images of inverted fluorescence mi-
croscopy reveal that compound 10b led to cell death of cancer
cells (Fig. 2).
To prove the cancer cell death, we used the qRT-PCR
method to test the expression levels of Bax and Bcl-2
mRNAs. The balance of Bcl-2 protein and pro-apoptotic
Bax prevents cytochrome c translocation from the mitochon-
dria to cytoplasm and represents the apoptosis resistance. This
balance is compromised by down-regulation of Bcl-2 and/or
up-regulation of Bax and contributes to apoptosis. The results
showed the apoptosis of LNCaP cancer cells (Fig. 3a and b).
We used wound healing assay to find out the effect of 10b
on the cancer cells migration. In this regard, the LNCaP cells
were scratched with a pipette tip and then treated with 10b.
The cells migration into wound was assessed at 0 and 24 h
In an initial step, the MTT colorimetric assay was used to
evaluate the cytotoxic potential of synthesized compounds
6a-q, 7a-c, 9a-i and 10a-c against cancer cell lines such as
A375 cells, PC3 cells, LNCaP cells, MDA-MB-231 cells, and
normal cell line HDF (human dermal fibroblast).
The results is indicated in term of inhibitory concentration
of 50% growth (IC50) at 48 h (Table 1) The Etoposide and
DMSO (1%) were used as a positive and a negative control,
respectively. As can be seen in Table 1, the most of com-
pounds 6 showed inhibitory effects against A375 cancer cell
line. The compounds 6f, 6i and 6m showed cytotoxicity
against two cancer cell lines. However, their IC50 values were
higher than IC50 values of Etoposide. Notably, the compounds
6q was able to inhibit cell growth in A375 and LNCaP cancer
cell lines with IC50 values that was close to the response rates
for Etoposide. The results revealed that the type of substituent
on isatin and phenyl ring of triazol moiety considerably influ-
ences cytotoxic potential of conjugate molecules 6. As evident
from the activity results, on in vitro evaluation of the interme-
diates 1H-1,2,3-triazol-indoline-2,3-diones 7, compounds 7a
and 7c having 4-methoxy or nitro group in the phenyl ring of
triazol moiety showed moderate to good activity against tested
cancer cell lines. The cytotoxicity comparison of the triazol-
linked thiazole-oxindoles 9 with triazol-indoline-2,3-dione in-
termediate 7 showed that the conjugation of hydrazino thia-
zole moiety to triazol-indoline-2,3-dione 7 did not improve
the cytotoxicity effect in the most of compounds 9 and they
were inactive against cancer cell lines. Compared to com-
pounds 7, the compounds 9a and 9d showed better activity

DARU J Pharm Sci
Table 1 In vitro cytotoxic activities of 6, 7, 9, and 10 against A375, MDA-MB-231, LNCaP, PC3 cells and normal cell HDF. Data represent mean
SD of three independent experiments
Compounds
IC50 (μM)
A375
MDA-MB231
PC3
LNCaP
HDF
6a
42.40 + 0.002
50.26 + 0.008
50.11 + 0.005
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6b
6c
>100
>100
>100
>100
>100
>100
6d
6e
>100
>100
>100
>100
>100
>100
>100
6f
50.01 + 0.006
>100
>100
>100
47.50 + 0.007
>100
6g
>100
>100
6h
6i
>100
>100
>100
>100
37.95 + 0.005
56.54 + 0.005
50.78 + 0.006
50.27 + 0.001
57.32 + 0.001
>100
>100
52.20 + 0.008
>100
>100
6j
>100
>100
6k
6l
>100
>100
>100
>100
>100
>100
6m
6n
6o
58.45 + 0.004
>100
>100
>100
>100
>100
50.05 + 0.001
56.80 + 0.008
27.74 + 0.001
35.86 + 0.002
>100
>100
>100
>100
6p
6q
7a
>100
>100
>100
>100
>100
28.07 + 0.004
59.42 + 0.001
>100
51.76 + 0.001
>100
>100
7b
7c
>100
37.69 + 0.002
46.33 + 0.001
>100
51.05 + 0.002
>100
72.01 + 0.003
>100
>100
9a
45.01 + 0.012
>100
9b
9c
55.10 + 0.005
>100
>100
>100
>100
>100
9d
9e
>100
>100
62.95 + 0.010
>100
>100
>100
>100
>100
9f
>100
>100
>100
>100
9g
>100
>100
>100
>100
9h
9i
>100
>100
>100
>100
>100
>100
>100
51.34 + 0.006
>100
10a
10b
10c
Etoposide
>100
>100
>100
25.91 + 0.005
>100
18.42 + 0.002
32.74 + 0.003
31.02 + 0.051
15.32 + 0.002
57.98 + 0.012
30 + 0.037
29.23 + 0.003
>100
24.46 + 0.019
31.21 + 0.005
after treatment. It was found, 10b has potential inhibitory ac-
tivity on LNCaP cells migration (compared to untreated cells
as control) (Fig. 4).
Previously, the anti-tyrosine kinase activity of
indolinones [37, 38] and inhibitory effects of
thiosemicarbazones on DNA replication by suppression of
ribonucleotide reductase activity were reported [23, 39,
40]. Notably, it seems that 10b may involve to inhibit tyro-
sine kinases activity or/and DNA synthesis process.
Moreover, we showed inhibition effects of 10b on cell mi-
gration. Thus, it supports this idea that 10b may have anti
tyrosine activity. Most of tyrosine kinases regulate the cell
motility. There are some potent candidates as mitogen-
activated protein kinase (MAPK) cascade activated by epi-
dermal growth factor receptor (EGFR). Proliferation and
Eventually, we examined the expression rate for mRNAs
of two genes as potent markers of EMT (E-cadherin,
vimentin) to validate the impact of 10b on cancer cell migra-
tion [35, 36] (Fig. 5a). The expression levels of E-cadherin
and vimentin mRNA were evaluated by qRT-PCR method.
As can be seen in Fig. 5b and c, the E-cadherin expression was
significantly increased, while vimentin expression was signif-
icantly reduced in 10b-treated LNCaP cells. This indicates
that 10b is able to inhibit EMT.

DARU J Pharm Sci
Fig. 2 Inverted fluorescent
microscopy images of chromatin
damages occurrence in the
nucleus of treated cells with 10b
compound and DMSO (1%),
which have been stained with
DAPI in MDA-MB 231, A375,
LNCaP, and PC3 cancer cell
lines. The experiments were per-
formed three times (original mi-
croscope magnification, 40X,
Scale bar, 10 μm)
Fig. 3 (a), (b): In treated LNCaP
cells with 10b compound (as
sample) and no treated cells (as
control), relative expression of
Bax mRNA and relative
expression of Bcl-2 mRNA were
shown. Data represent mean SD
of three independent experiments.
p < 0.05 was considered to be
statistically significant

DARU J Pharm Sci
Fig. 4 Effects of compounds 10b
on the migration of LNCaP cells
in different time (0 and 24 h after
treatment; concentration of 10b
compound was 29.23 μM).
Images were obtained using
phase-contrast microscopy. Scale
bars represent 50 μm
Fig. 5 (a): Schematic model of epithelial-mesenchymal transition
(EMT). (b, c): Relative expression of E-cadherin and Vimentin mRNAs
as two potential markers of EMT in treated LNCaP cells with 10b
compound (compared to control) were shown. Data represent mean
SD of three independent experiments. p < 0.05 was considered to be
statistically significant

DARU J Pharm Sci
migration are outcomes of EGFR activity that are tightly
regulated by MAPKs. Thus, the exploring of the possible
effects of 10b on related tyrosine kinases in MAPK cascade
was suggested. Also, in vivo experiments will be required
to find the side effects of 10b.
Synthesis of spiro[indoline-3,2′-quinazoline]-diones 4
A mixture of N-propargyl isatins 2 (1 mmol),2-
aminobenzamide 3 (1 mmol) and P-TSA (10 mol%) in
EtOH (5 mL) was stirred at reflux conditions for 10 h. Then,
water (15 mL) was added and stirred for 15 min. The solid
residue was filtered and washed with water. Then, the solid
was crystallized with EtOH to afford the pure product.
Conclusion
Synthesis of compound 6
In conclusion, in vitro cytotoxic activity of some new synthe-
sized triazol-linked spiroindolinequinazolinone 6, thiazol-
oxindole 7 and oxindole-thiosemicarbazone conjugates 9
were evaluated toward different cancer lines. The synthesized
compounds 6, 7, and 9 showed IC50 values higher than IC50
value of Etoposide as positive control. Among three synthe-
sized triazol-linked oxindol-thiosemicarbazone 10a-c, com-
pound 10b displayed promising antitumor activity against all
tested cancer cell lines (IC50 = 15.32–29.23 μ) compared to
the Etoposide. It showed the importance of bromophenyl
group substitution in triazol/indolin-3-thiosemicarbazone se-
ries 10 in inducing more cytotoxic effects on cancer cells. The
results revealed that the compound 10b induces apoptosis and
has EMT inhibition activity. Therefore, the compound 10b as
an indolinone and thiosemicarbazone conjugate molecule
could be a novel active agent with multiple activities to design
anticancer therapeutic strategies. Our current study reports
that conjugation of indolinone, thiosemicarbazone and tri-
azole moieties can be considered as a new therapeutic agent
against different cancer cells.
A mixture of spiro[indoline-3,2′-quinazoline]-diones 4
(1 mmol), azide 5 (1 mmol), CuSO4.5H2O (15 mol%) and
sodium ascorbate (30 mol%) in t-BOH/H2O (2 mL, 1:1) was
sonicated at room temperature for 1 h. Then, water (5 mL) was
added and stirred for 15 min. The solid residue was filtered
and washed with water. Then, the solid was crystallized with
DMF/H2O to afford the pure product.
Synthesis 1H-1,2,3-triazol-indoline-diones 7
A mixture of N-propargyl isatins 2 (1 mmol), azide 5
(1 mmol), CuSO4.5H2O (15 mol%) and sodium ascorbate
(30 mol%) in t-BOH/H2O (2 mL, 1:1) was sonicated at room
temperature for 1 h. Then, water (3 mL) and EtOAc (3 mL)
was added and stirred for 15 min. The aqueous layer was
extracted with EtOAc for three times. The combined organic
layers were washed with brine, dried over MgSO4 and evap-
orated to give the pure product.
Synthesis of triazol/thiazol-indolinones 9
Experimental
A mixture of 7 (1 mmol), thiosemicarbazid (1 mmol) and
phenacyl bromides (1 mmol) in iso-PrOH (2 mL) was soni-
cated at 65 °C for 1 h. Then, water (5 mL) was added and
stirred for 15 min. The solid residue was filtered and washed
with water and EtOH to afford the pure product.
General information
Melting points were determined on a melting point apparatus
and are uncorrected. IR spectra were taken with a Bomem FT-
IR MB spectrometer. The NMR spectra were recorded on a
BRUKERDRX-300AVANCE spectrometer. Mass spectra
were recorded on an Agilent 5975C VL MSD with Tripe-
Axis Detector operating at an ionization potential of 70 eV.
Elemental analyses were performed using a Heraeus CHN–
O–Rapid analyzer. All chemicals were purchased from Merck
or Aldrich and were used without further purification.
Synthesis of triazol/indolin-thiosemicarbazones 10
A mixture of 7 (1 mmol) and thiosemicarbazid (1 mmol) in
iso-PrOH (2 mL) was sonicated at 65 °C for 1 h. Then, water
(5 mL) was added and stirred for 15 min. The solid residue
was filtered and washed with water and EtOH to afford the
pure product.
Synthesis of N-propargyl isatins 2
Cell lines and cell culture
A mixture of isatins (1 mmol), propargyl bromide (1 mmol)
and Na2CO3 (2 mmol) in DMF (3 mL) was stirred at room
temperature for 3 h. Then, water (5 mL) was added and stirred
for 15 min. Then, the solid residue was filtered and crystal-
lized with EtOH to afford the pure product.
Human malignant melanoma cells (A375), human prostate
cancer cells (PC3 cells, LNCaP cells), human breast cancer
cells (MDA-MB-231) and normal cells HDF (human dermal
fibroblast) were received from Pasture Institute, Tehran, Iran.
A375, PC3, LNCaP and HDF cell lines were grown in

DARU J Pharm Sci
DMEM medium and MDA-MB-231 cell line was grown in
RPMI 1640 medium. All media contains 10% fetal bovine
serum (FBS; Bioidea BI 201, Iran), penicillin G, streptomycin
100 μg/mL and 1% L-Glutamine. The cells were cultured and
incubated under humidified 5% CO2 atmosphere at 37 °C.
RNA extraction, cDNA synthesis and quantitative real-
time PCR (qRT-PCR)
For quantitative real-time RT-PCR analysis, after 48 h of treat-
ment with 29.23 μM of related drug (10b), LNCaP cells was
lysed and the total RNA was extracted using 500 μL of
Trizol® reagent according to the protocol provided by the
manufacturer (Invitrogen Life Technologies, Carlsbad, CA,
USA) followed by reverse transcription into cDNA according
to manufactures protocol (ReveretAid M-Mulv reverse tran-
scriptase kit, Thermo Fisher Scientific, MA, USA). Real-time
RT-PCR was then performed to amplify cDNA using SYBR
Green dye universal master mix (Bioron GmbH, Germany),
on a Light Cycler 480 (Roche) using the primers for GAPDH-
F: 5′-CAA GGT CAT CCA TGA CAA CTTTG-3′, R:5′-
GTCCACCACCCTGTTGCTGTAG-3′; Bax-F:5′-GTCG
C C C T T T T C T A C T T T G C C - 3 ′ , R : 5 ′ - C T C C
CGCCACAAAGATGGTCA-3′and Bcl-2-F: 5′-CCCC
TCGTCCAAGAATGCAA-3′, R: 5′- TCTCCCGG
TTATCGTACCCTG-3′ for 40 cycles. Data represent aver-
aged copy number normalized to the GAPDH housekeeping
gene. Primer synthesis was done by Pishgam Biotech Co.
Tehran, Iran. The negative control reaction was set as a reac-
tion similar to the above but with deionized water instead of
cDNA. Thermal conditions of the PCR consisted of primary
denaturation at 94 °C for 2 min, 45 cycles of denaturation at
94 °C for 30 s, annealing at 59 °C for 30 s, amplification at
72 °C for 30 s. Primers were used for E-cadherin–F: 5′-GCCG
AGAGCTACACGTTCAC-3′, R: 5′- CAGGCGTA
GACCAAGAAATG-3′and for vimentin-F: 5′-CTAC
GTCCACCCGCACCTAC-3′; R: 5′- CCAGCGAG
AAGTCCACCGAG-3′ with the following conditions: 95 °C
for 90 s; followed by 45 cycles of 95 °C for 30 s, 60 °C for 30 s
(E-cadhein) and 57 °C for 30 s (vimentin), and 72 °C for 30 s.
All reactions were triplicated.
MTT assay
The effect of compounds treatment on the viability of cancer
cell lines was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide or MTT assay (MTT assay kit,
Bio IDEA, CatNo:BI1017, Iran) based on the ability of live
cells to cleave the tetrazolium ring to a molecule that absorb at
490 nm as per the manufacturer’s instructions [41]. Etoposide
(was kindly provided by Dr. A. Foroumadi, Tehran medical
Science University, Iran) and DMSO was used as positive and
negative controls, respectively. Briefly, cells were plated in
96-well culture plates (5 × 103 cells/well). After 24 h incuba-
tion, the cells were treated with different concentrations of the
compounds. After 48 h at 37 °C, the medium was removed
and 100 μL of MTT reagent (1 mg/mL) was added to each
well, and cells were further incubated at 37 °C for 4 h. The
MTT solution was removed, 50 μL of DMSO was added to
each well to dissolve formazan crystals, and the plates were
gently shaken for 10 min, followed by reading with an ELISA
plate reader (BiotekELx 800, USA). The 50% inhibition con-
centration (IC50) was defined as the concentration that
inhibited cell proliferation by 50% when compared to
DMSO treated cells (as negative control).
DAPI staining assay
DAPI staining assay was used to determine chromatin chang-
es. Related cancer cell lines were seeded in six well plates (5 ×
104 cells/well) containing 12 mm cover-slips and subsequent-
ly treated for 10b compound (Sample or treated cells) and
DMSO (Control or untreated cells) for 24 h. Cells then were
fixed with 3.7% paraformaldehyde, permeabilized in 0.5% (w/
v) Triton X-100, 1% BSA (w/v) for 5 min, washed in PBS,
and stained with DAPI (Sigma-Aldrich, USA). All images
were taken by an inverted fluorescent microscope (Nikon
Eclipse Ti-E).
Acknowledgments Financial support of this research from Shahid
Beheshti University, University of Guilan and the Iran National Science
Foundation (INSF) is gratefully acknowledged.
Authors’ contributions SN and MBM synthesis of the title compounds.
FS supervision of the pharmacological part and collaboration in manu-
script preparations. AB supervision of the chemistry part, designing of
title compounds and manuscript preparation.
Compliance with ethical standards
Wound-healing migration assay
Conflict of interest The authors declare that they have no conflict of
interest.
The LNCaP cells were seeded in culture medium onto 6-well
plates at a density of 4 × 105 cells per well. The confluent
monolayer of cells was scratched with a fine pipette tip, and
cell migration into the wound was visualized and scored by
measuring the size of the initial wound and comparing it to the
size of the wound after 24 h by microscopy.
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