Design and synthesis of isatin/triazole conjugates that induce apoptosis and inhibit migration of MGC-803 cells

Article abstract of DOI:10.1016/j.ejmech.2016.08.065

A series of new isatin/triazole conjugates were designed based on the hypothesis that the ester-linked compounds could be enzymatically hydrolyzed by cellular esterases inside the cells. These compounds showed moderate to good growth inhibition toward the

Full text of DOI:10.1016/j.ejmech.2016.08.065

European Journal of Medicinal Chemistry 124 (2016) 350e360
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design and synthesis of isatin/triazole conjugates that induce
apoptosis and inhibit migration of MGC-803 cells
,
Bin Yu* 1, Sai-Qi Wang 1, Ping-Ping Qi, Dong-Xiao Yang, Kai Tang, Hong-Min Liu**
School of Pharmaceutical Sciences & Collaborative Innovation Center of New Drug Research and Safety Evaluation, Zhengzhou University, Zhengzhou
450001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new isatin/triazole conjugates were designed based on the hypothesis that the ester-linked
compounds could be enzymatically hydrolyzed by cellular esterases inside the cells. These compounds
showed moderate to good growth inhibition toward the tested cancer cells, exerted selective inhibition
toward MGC-803 cells and were less toxic to normal cells HL-7702 and GES-1. Of these compounds,
Received 20 March 2016
Received in revised form
25 August 2016
Accepted 26 August 2016
Available online 28 August 2016
compound 5a showed the best inhibitory activity against MGC-803 cells (IC50 ¼ 9.78
mM), induced
apoptosis through multiple mechanisms, as well as inhibited migration of MGC-803 cells.
© 2016 Elsevier Masson SAS. All rights reserved.
Keywords:
Isatin
Triazole
Cytotoxicity
Apoptosis
Migration inhibition
LSD1 inactivation
1. Introduction
solubility and therefore has been extensively used in the design of
bioactive molecules [16e18].
Isatin, a well-known natural indole derivative found in many
plants, has also been observed as a metabolite of adrenaline in
human bodies [1]. Recent studies indicated that isatin itself is
capable of inhibiting growth of SH-SY5Y cells and inducing
apoptosis via the intrinsic apoptotic pathway [2,3]. Isatin-based
structural modifications have been highly pursued in last decades
because isatin derivatives have been found to possess diverse and
promising biological activities, and some of these isatin derivatives
have shown therapeutical effects in preclinical investigations
[4e9]. Representative examples are SAR405835 [10], CFI-400945
[11], and KAE609 [12] (Fig. 1), which are currently undergoing
clinical assessment for the treatment of cancer and malaria,
respectively. In addition to the biological functions, isatin and its
derivatives have also found synthetic utilities in the construction of
spirooxindole frameworks due to the high reactivity of C3 carbonyl
group [13e15]. On the other hand, the 1,2,3-triazole group has
proven to be metabolically stable and able to decrease the aqueous
As part of our efforts toward the identification of potent anti-
cancer agents, we previously introduced the isatin motif to the
steroid nucleus, generating a large library of structurally new ste-
roidal oxindoles with good antiproliferative activity [19e22].
Intriguingly, a series of steroid/isatin conjugates were identified as
the first steroid-based lysine specific demethylase 1 (LSD1) in-
hibitors, which inactivated LSD1 at low micromolar levels [23]. The
shortlisted compound I (Fig. 2) was found to be able to inhibit
growth of cancer cells and induce apoptosis. The carboxylic ester
group is believed to be able to be hydrolyzed by cellular esterases
inside the cells, giving the free carboxylic acid and alcohol. The
characteristics of the ester group make it widely used in the design
of prodrugs [24e26] and fluorescent probes such as the DCFH-DA
for cellular ROS detection [27]. Evidently, both parts of compound
I are linked through the ester group and therefore could be
potentially enzymatically hydrolyzed to afford the DHEA and
another fragment (the isatin/triazole conjugate). It is well-known
that DHEA inhibits growth of cancer cells through multiple mech-
anisms [28e32]. Therefore, the question remains whether the
compound I exerted the good cytotoxicity alone or through the
synergistic effects of both components. The hypothesis promoted
us to investigate the anticancer potential of the isatin/triazole
fragment by varying the R group (Fig. 2). We herein report the
* Corresponding author.
** Corresponding author.
E-mail addresses: zzuyubin@hotmail.com (B. Yu), liuhm@zzu.edu.cn (H.-M. Liu).
Bin Yu and Sai-Qi Wang contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2016.08.065
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

B. Yu et al. / European Journal of Medicinal Chemistry 124 (2016) 350e360
351
Fig. 1. Representative examples of isatin-derived oxindole derivatives.
Fig. 2. Identification of isatin/triazole conjugates with anticancer potential.
cytotoxicity of the isatin/triazole conjugates against several cancer
cells and the possible modes of action, hoping to provide a basis for
the development of isatin-based anticancer agents.
of K2CO3 gave compound 2, which then reacted with azido com-
pounds 4aeg, affording compounds 5aeg. Compounds 4aeg were
synthesized from compounds 3aeg and sodium azide through the
nucleophilic substitution. Compound 2 and compounds 4aeg were
then subjected to the Cu-catalyzed Click reactions, affording com-
pounds 5aeg. Deprotection of compound 5g in the presence of TFA
generated compound 5h bearing a terminal amine salt.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 5aeh is operationally simple using
the Cu-catalyzed click chemistry as the key step (Scheme 1). 4, 7-
Dichloroisatin 1 reacted with propargyl bromide in the presence
2.2. Cytotoxicity evaluation
With these compounds in hand, we next tested their
Scheme 1. Synthesis of compounds 5aeh. Reagents and conditions: (a) Propargyl bromide, K2CO3, DMF, rt; (b) NaN3, DMSO, rt-50 ꢀC; (c) Sodium ascorbate, CuSO4$5H2O, THF/H2O
(1/1), rt; (d) TFA, DCM, rt.

352
B. Yu et al. / European Journal of Medicinal Chemistry 124 (2016) 350e360
cytotoxicity against several cancer cell lines (TE-1, MCF-7, SW780,
and MGC-803) using the CCK-8 assay. Also, the cytotoxic evaluation
of compounds 5aeg against normal cells HL-7702 and GES-1 was
carried out to explore the toxicity and selectivity. In the assay, the
well-known anticancer drug 5-flurouracil (5-FU) was used as the
control. However, we failed to test the inhibitory effect of com-
pound 5h toward the tested cancer cell lines because of its poor
solubility observed.
As shown in Table 1, compounds 5aeg exhibited moderate to
good inhibitory effect toward the tested cancer cell lines. Interest-
ingly, compared to compounds 5beg with different terminal car-
boxylic amide groups, compound 5a bearing an enzymatically
hydrolyzable ester group showed the best inhibition against all the
toward MGC-803 cells is due to the LSD inactivation remains un-
clear and needs to be investigated further, although the structurally
similar compound I (Fig. 2) inhibits LSD1 at low micromolar levels.
Furthermore, compounds 5aeg were less sensitive to normal cells
over cancer cells, showing certain selectivity between normal and
cancerous cells. Also, compounds 5aeg were less toxic toward
normal cells than 5-FU. The low toxicity and selectivity warrant
their further development for identifying more potent isatin-based
anticancer agents.
2.3. In silico prediction of drug-like properties of compounds 5aeh
Molecular properties of designed compounds 5aeh were
calculated using the free molecular calculation services provided by
Molinspiration (http://www.molinspiration.com). As shown in
Table 2, compounds 5aeh showed favorable drug-like properties
that are in line with the Lipinski's rule of five [36].
tested cancer cell lines with the IC50 values less than 26
mM,
possibly suggesting that a terminal carboxylic acid group for this
series of compounds is beneficial for the activity. This finding gives
us directions for further structural modifications. Compound 5a
displayed good inhibition against TE-1 and MGC-803 with the IC50
values of 14.22 and 9.78 mM, respectively, comparable to that of 5-
2.4. Morphological changes of MGC-803 cells induced by compound
FU. For compounds 5aeg, the moderate to weak inhibitory effect
was observed toward TE-1, MCF-7 and SW780 with the IC50 values
5a
ranging from 25 to >128 mM. It should be noted that compound 5f
Morphological changes of cancer cells are always associated
with the growth inhibition induced by cytotoxic agents [37,38].
Inspired by the good inhibition of compound 5a against MGC-
803 cells, we then investigated whether compound 5a was able to
induce morphological changes. After being incubated with 5a for
with a terminal morpholine group showed significantly decreased
inhibition against all the tested cancer cells, compared to the
structurally similar compound 5d. The underlying mechanism re-
mains unknown. More intriguingly, compounds 5aeg were more
sensitive to MGC-803 cells, indicating that this series of compounds
may exert the inhibitory effect by targeting overexpressed cancer-
related proteins in MGC-803 cells. It has been proved that LSD1 is
overexpressed in MGC-803 cells, and we have successfully
designed three series of selective LSD1 inhibitors, which potently
inhibited growth of MGC-803 cells in vitro and in vivo [33e35].
However, whether the selective inhibition of compounds 5aeg
24 h at different concentrations (0, 5, 10, 20 mM), the morphological
changes of MGC-803 cells were recorded using an inverted mi-
croscope. As shown in Fig. 3A, significant changes of cell
morphology such as rounding up and cell debris were observed,
especially at high concentrations. After staining with Hoechst
33258, remarkable nuclear changes including the chromatin
condensation, nuclear fragmentation and condensation were also
Table 1
Preliminary in vitro cytotoxicity of compounds 5aeh against the tested cancerous and normal cell lines.
Compound
R
IC50
(mM)
TE-1
MCF-7
SW780
MGC-803
HL-7702
GES-1
5a
14.22 4.09
25.19 4.36
18.86 0.67
25.21 3.65
9.78 1.67
40.27 10.00
35.97 6.32
5b
5c
51.8 2.80
93.33 16.56
26.97 7.97
17.40 2.44
17.48 5.95
83.92 12.25
24.87 4.11
47.429 7.32
24.87 2.00
30.61 14.03
38.46 4.80
5d
5e
5f
40.62 10.23
51.60 12.77
118.93 18.75
34.61 6.59
62.35 4.66
146.40 36.75
29.69 5.08
48.22 8.90
112.74 22.10
18.67 4.55
14.75 1.27
51.70 14.60
39.29 7.13
>128
50.693 6.32
88.72 10.73
>128
>128
5g
39.25 4.58
46.85 5.65
>128
22.04 6.68
41.96 10.12
84.19 7.83
5ha
e
e
e
e
e
e
5-FU
e
17.32 3.98
13.19 0.81
6.99 2.60
10.64 2.96
12.85 1.22
13.75 3.41
a
The cytotoxicity of compound 5h was not determined because of its poor solubility in the CCK-8 assay.

B. Yu et al. / European Journal of Medicinal Chemistry 124 (2016) 350e360
353
Table 2
Molecular properties of compounds 5aeh.a
Compound
MW
nviolations
natoms
nON
nOHNH
nrotb
miLogP
TPSA
MV
Desirable value
5a
5b
5c
5d
5e
<500
e
0
0
0
0
0
0
2
0
0
e
<10
8
8
8
8
8
9
11
9
4
<5
0
1
1
0
0
0
0
2
ꢁ10
6
5
5
4
4
4
6
4
<5
<140
96.10
98.89
98.89
90.10
90.10
99.34
119.65
106.71
65.72
e
437.28
436.30
430.25
422.27
408.25
424.24
523.38
424.27
130.08
29
29
29
28
27
28
35
28
9
3.67
2.92
2.71
2.17
1.66
1.10
2.19
ꢂ2.11
ꢂ0.59
351.05
354.47
335.88
338.02
321.22
330.21
428.16
334.60
96.91
5f
5g
5h
5-FU
2
0
a
MW: Molecular weight; nviolations: Number of violations; natoms: Number of atoms; nON: Number of hydrogen bond acceptors; nOHNH: Number of hydrogen bond
donors; nrotb: Number of rotatable bonds; miLogP: LogP value predicted by Molinspiration; TPSA: Topological polar surface area; MV: Molecular volume.
Fig. 3. Morphological changes (A) and nuclear degradation (B) of MGC-803 cells induced by compound 5a. Scale bar is 50 mm.
observed (Fig. 3B).
apoptotic cells was about 1.3% for the control group. When treated
with high concentration (20 M) of compound 5a, around 22.5% of
m
apoptosis rate was observed. While the late apoptosis rate of MGC-
803 cells was not changed significantly with increasing
concentrations.
2.5. Cell cycle distribution assay by flow cytometry
Targeting the cell cycle of tumor cells has been recognized as a
promising strategy for cancer therapy [39]. In this study, compound
5a was chosen to investigate the effect of our designed conjugates
on the cell cycle of MGC-803 cells. After treating MGC-803 cells
2.7. Measurement of intracellular ROS levels
with compound 5a at different concentrations (0, 5, 10, 20 mM) for
Studies above showed that compound 5a can arrest the cell
cycle at G2/M phase and induce the apoptosis concentration-
dependently. However, the precise mechanisms of action remain
unclear. Oliveira et al. reported that Uncaria tomentosa extract
containing oxindole alkaloids triggered apoptosis of HT29 cells
through the ROS-mediated caspase activation and DNA repair [40].
More recently, Shankaraiah and co-workers reported that
spirooxindole-derived morpholine-fused-1,2,3-triazoles can in-
crease ROS levels in A549 cells and decrease the mitochondrial
membrane potential, finally leading to apoptosis of A549 cells [41].
These studies suggest that isatin-derived compounds could have
the potential of increasing cellular ROS levels. From the structural
point of view, we reasoned that treatment of MGC-803 cells with
the designed isatin/triazole conjugates could also induce cellular
ROS production, which then contributed to the apoptosis of cancer
cells.
24 h, cells were then fixed and stained with PI for flow cytometry
analysis. As shown in Fig. 4, compound 5a concentration-
dependently arrested cell cycle at G2/M phase, accompanied with
decrease of cells at G0/G1 phase. Specifically, the percentage of cells
at G2/M phase for the high concentration group (20
32.26%, about 12% higher than that of the control group.
mM) was
2.6. Apoptosis detection by flow cytometry
The effect of compound 5a on the apoptosis was investigated
using the propidium iodide (PI) and Annexin V-FITC biparametric
cytofluorimetric analysis. After treatment with different concen-
trations of compound 5a (0, 5, 10 and 20
mM) for 24 h, MGC-
803 cells were stained with PI and FITC, and then analyzed by the
flow cytometry. As illustrated in Fig. 5, compound 5a induced
apoptosis of MGC-803 cells in a concentration-dependent manner,
especially the early apoptosis. Specifically, the percentage of
To investigate whether compound 5a is capable of inducing ROS
generation
in
MGC-803
cells,
the
DCFH-DA
(2,7-

354
B. Yu et al. / European Journal of Medicinal Chemistry 124 (2016) 350e360
Fig. 4. Effect of compound 5a on the cell cycle of MGC-803 cells. *P < 0.05 and **P < 0.01.
Fig. 5. Compound 5a induced apoptosis of MGC-803 cells in a dose-dependent manner. **P < 0.01.
dichlorodihydrofluorescein diacetate) assay was carried out to
determine the cellular ROS levels. Principally, the non-fluorescent
DCFH-DA is enzymatically hydrolyzed by cellular esterases to
DCFH, followed by the ROS-mediated oxidation to form the highly
fluorescent DCF [27]. The green fluorescence intensity correlates
with cellular ROS levels. Therefore, the fluorescence intensity is
used to reflect intracellular ROS levels. After treatment with com-
analyzed using the flow cytometry. As shown in Fig. 6, compound
5a concentration-dependently enhanced the green fluorescence
intensity. The cells with green fluorescence accounted for 19.3%,
significantly higher than that of the control group (2.84%).
2.8. Expression changes of key proteins induced by compound 5a
pound 5a at different concentrations (0, 5, 10, 20
DCFH-DA treatment for 30 min, the fluorescence intensity was
mM), following
It has been widely accepted that the mitochondrial dysfunction
is always associated with increased ROS levels, which then induces

B. Yu et al. / European Journal of Medicinal Chemistry 124 (2016) 350e360
355
Fig. 6. Compound 5a induced ROS production in MGC-803 cells. *P < 0.05 and **P < 0.01.
apoptosis of cancer cells through multiple mechanisms [42]. The
mitochondrial dysfunction always causes the decrease of mito-
chondrial membrane potential, followed by release of cytochrome c
from the mitochondria to the cytoplasm and caspase activation,
finally leading to the apoptosis of cancer cells. In this study,
Expression changes of key proteins were detected by the Western
blot analysis. As shown in Fig. 7, compound 5a indeed inhibited
expression of LSD1 in a concentration-dependent manner, which
may explain our aforementioned hypothesis that the selective in-
hibition of MGC-803 cells is at least in part attributed to the inac-
tivation of LSD1. Moreover, compound 5a activated pro-apoptotic
protein Bax and inhibited expression of anti-apoptotic proteins Bcl-
2 and Mcl-1. The activation of pro-apoptotic protein and inactiva-
tion of anti-apoptotic proteins finally led to the mitochondrion-
mediated intrinsic apoptosis. We can also find that PARP was
cleaved after compound 5a treatment, generating the 89 kDa
fragment. The cleavage of PARP is possibly attributed to the caspase
activation, especially the caspase-3 and caspase-7.
Next, we further examined the expression changes of caspases
by the Western blot analysis. As shown in Fig. 8, caspase-9 and
caspase-8 were cleaved by compound 5a, which then activated
downstream proteins caspase-3, caspase-6 and caspase-7, finally
leading to apoptosis of MGC-803 cells. Taken together, compound
5a induced apoptosis of MGC-803 cells at least through three
pathways, namely the mitochondrion-mediated intrinsic apoptotic
pathway, the death receptor-mediated extrinsic apoptotic pathway,
as well as the LSD1 inactivation.
using the high content screening system. As shown in Fig. 9,
compound 5a indeed inhibited migration of MGC-803 cells even at
low concentration (2 mM), although the inhibition was not signifi-
cant. Intriguingly, the inhibition was concentration-independent;
the underlying mechanism remains unclear and needs to be
further explored.
3. Conclusions
Following our previous work, we designed a series of new isatin/
triazole conjugates that possessed moderate to good growth inhi-
bition against the tested cancer cells and exerted selective inhibi-
tion against MGC-803 cells. Of these compounds, compound 5a was
the most potent one against MGC-803 cells (IC50 ¼ 9.78
less toxic to normal cells HL-7702 and GES-1 (IC50 ¼ 40.27 and
35.97 M, respectively). Besides, compound 5a caused morpho-
mM) and
m
logical changes of MGC-803 cells, induced cell cycle arrest at G2/M
phase, cellular ROS generation and migration inhibition of MGC-
803 cells in a concentration-dependent manner. Compound 5a
induced apoptosis through the mitochondria-mediated intrinsic
apoptotic pathway, the death receptor-mediated extrinsic
apoptotic pathway, as well as the LSD1 inactivation. These conju-
gates described here may serve as bioactive fragments for devel-
oping more potent cytotoxic agents. Further modifications will
focus on installation of other functional groups on the phenyl ring,
replacement of triazole linker with other heterocycles, as well as
further conversions based on the highly reactive C3 carbonyl group
of isatin core.
2.9. Migration inhibition of MGC-803 cells by compound 5a
4. Experimental section
From above studies, we can conclude that compound 5a is
capable of inducing apoptosis of MGC-803 cells through multiple
mechanisms. We next investigated whether compound 5a could
inhibit migration of cancer cells. After incubation at different con-
4.1. General
Reagents and solvents were used directly without special
treatment. Thin layer chromatography (TLC) was carried out on
glass plates coated with silica gel and visualized by UV light
centrations (0, 2, 4, 8
mM) for 24 h, MGC-803 cells were stained with
Hoechst 33258, and migrated cells were detected and numbered

356
B. Yu et al. / European Journal of Medicinal Chemistry 124 (2016) 350e360
Fig. 7. Expression changes of key apoptotic proteins induced by compound 5a.
Fig. 8. Caspase cascade activation induced by compound 5a in MGC-803 cells. *P < 0.05 and **P < 0.01.
(254 nm). Melting points were determined on an X-5 micromelting
apparatus and are uncorrected. All the NMR spectra were recorded
with a Bruker DPX 400 MHz spectrometer with TMS as an internal
standard in CDCl3 or DMSO-d6. Chemical shifts are given as ppm
d

B. Yu et al. / European Journal of Medicinal Chemistry 124 (2016) 350e360
357
Fig. 9. Migration inhibition induced by compound 5a. **P < 0.01.
values relative to TMS. High-resolution mass spectra (HRMS) of all
final compounds were recorded on a Waters Micromass Q-T of
Micromass spectrometer by ESI. CAUTION: Particular care should
be taken to avoid grinding sodium azide and acidifying mixtures
containing sodium azide because the azide ion can react with acids
to form the extremely explosive, volatile and toxic hydrazoic acid.
Besides, sodium azide can also form highly explosive salts with
many transition metals. Halogenated solvents such as dichloro-
methane and chloroform should be avoided for sodium azide-
involved reactions and subsequent work-up procedures because
such solvents can form highly explosive di- and triazidomethanes
with sodium azide [43].
combined organic layers were washed with H2O for several times to
remove the DMSO, and then washed with brine, dried over MgSO4
and evaporated to give the corresponding products 4aeg. Com-
pounds 4d and 4e were known compounds [44] and therefore not
characterized in this work.
4.3.1. Compound 4a
Compound 4a, light yellow liquid, 61%; 1H NMR (400 MHz,
CDCl3)
1.78e1.62 (m, 2H), 1.62e1.14 (m, 6H); 13C NMR (100 MHz, CDCl3)
167.73, 74.55, 50.52, 31.43, 25.19, 23.54.
d 4.97e4.71 (m, 1H), 3.82 (s, 2H), 1.93e1.77 (m, 2H),
d
4.3.2. Compound 4b
Compound 4b, brown solid, yield: 55%, m.p.: 82e84 ꢀC; 1H NMR
4.2. Synthesis of compound 2
(400 MHz, CDCl3) d 6.08 (s, 1H), 3.92 (s, 2H), 3.82e3.61 (m, 1H), 1.85
To a solution of 4,7-dichloroisatin (0.68 mmol, 1.0 eq) in DMF
(4 mL) was added K2CO3 (0.68 mmol, 1.0 eq) at room temperature,
the mixture was stirred for about 30 min, and then the propargyl
bromide (0.75 mmol, 1.1 eq) was added dropwise. The mixture was
stirred overnight at room temperature. Upon completion, EtOAc
and H2O were added. The aqueous layer was extracted with EtOAc
for several times; the combined organic layers were washed with
H2O for several times to remove the DMF, and then washed with
brine, dried over MgSO4 and evaporated to give the products.
Compound 2, saffron solid, yield: 76%, m. p.: 151.8e153.3 ꢀC,
Rf ¼ 0.52 (petroleum ether/ethyl acetate ¼ 2/1). 1H NMR (400 MHz,
(m, 2H), 1.75e1.59 (m, 2H), 1.56 (m, 2H), 1.40e1.23 (m, 2H), 1.12 (m,
2H); 13C NMR (100 MHz, CDCl3)
23.75.
d 164.42, 51.80, 47.27, 31.95, 24.40,
4.3.3. Compound 4c
Compound 4c, white solid, yield: 63%, m.p.: 83e86 ꢀC; 1H NMR
(400 MHz, CDCl3)
d
7.94 (s, 1H), 7.47 (d, J ¼ 7.7 Hz, 2H), 7.29 (dd,
J ¼ 10.8, 5.1 Hz, 2H), 7.09 (t, J ¼ 7.4 Hz, 1H), 4.08 (s, 2H); 13C NMR
(100 MHz, CDCl3)
d 163.45, 135.70, 128.12, 124.05, 119.03, 51.99.
CDCl3)
d
7.51 (d, J ¼ 8.7 Hz, 1H), 7.10 (d, J ¼ 8.7 Hz, 1H), 4.94 (d,
4.3.4. Compound 4f
Compound 4f, light yellow solid, yield: 11%, m.p.: 69e71 ꢀC; 1H
J ¼ 2.4 Hz, 2H), 2.34 (t, J ¼ 2.4 Hz, 1H); 13C NMR (100 MHz, CDCl3)
d
178.68, 157.00, 146.19, 140.48, 133.05, 126.79, 117.16, 116.11, 77.22,
NMR (400 MHz, CDCl3)
d
3.94 (s, 2H), 3.70 (t, J ¼ 4.8 Hz, 4H), 3.66 (d,
J ¼ 5.1 Hz, 2H), 3.43e3.36 (m, 2H); 13C NMR (100 MHz, CDCl3)
73.27, 31.94.
d
164.76, 65.75, 65.42, 49.61, 44.53, 41.29.
4.3. General procedure for the synthesis of compounds 4aeg
4.3.5. Compound 4g
To a solution of bromide 3aeg (1.0 eq) in DMSO was added NaN3
(1.2 eq) at room temperature. Then the reaction mixture was
heated to 50 ꢀC and kept for 3 h. Upon completion, EtOAc and H2O
were added. The aqueous layer was extracted with EtOAc; the
Compound 4g, white solid, yield: 74%, m.p.: 118e120 ꢀC; 1H
NMR (400 MHz, CDCl3)
d
3.88 (s, 2H), 3.55 (d, J ¼ 5.2 Hz, 2H), 3.39 (s,
4H), 3.30 (d, J ¼ 5.2 Hz, 2H), 1.41 (s, 9H); 13C NMR (100 MHz, CDCl3)
d
164.78, 153.85, 153.41, 79.55, 49.79, 43.99, 40.86, 27.33.

358
B. Yu et al. / European Journal of Medicinal Chemistry 124 (2016) 350e360
4.4. General procedure for the synthesis of compounds 5aeg
NMR (100 MHz, DMSO-d6) d 178.65, 164.32, 158.15, 146.44, 139.81,
130.28, 125.70, 117.60, 114.64, 65.90, 65.81, 50.59, 44.71, 41.85,
37.52.; HRMS (ESI): m/z calcd for C17H16Cl2N5O4 (MþH)þ, 424.0579;
found, 424.0582.
To a solution of azide (1.0 eq) and alkyne (1.0 eq) in THF/H2O (1/
1) were added soduim ascorbate (0.5 eq) and CuSO4$5H2O (0.4 eq)
at room temperature. The mixture was then stirred for 5e8 h at
room temperature. Upon completion, the mixture was filtered and
washed with H2O to give the products.
4.4.7. Compound 5g
Compound 5g, yellow solid, yield: 73%, m. p.: 203.1e204.4 ꢀC,
Rf ¼ 0.09 (PE/acetone ¼ 2/1); 1H NMR (400 MHz, DMSO-d6)
d 8.08
4.4.1. Compound 5a
(s, 1H), 7.61 (d, J ¼ 8.7 Hz, 1H), 7.19 (d, J ¼ 8.7 Hz, 1H), 5.45 (s, 2H),
Compound 5a, yellow solid, yield: 50%, m. p.: 163.1e164.7 ꢀC,
5.30 (s, 2H), 3.66e3.35 (m, 8H), 1.41 (s, 9H); 13C NMR (100 MHz,
Rf ¼ 0.25 (PE/acetone ¼ 2/1); 1H NMR (400 MHz, DMSO-d6)
d 8.18
DMSO-d6)
d 178.64, 164.31, 158.15, 153.73, 146.44, 139.80, 130.28,
(s, 1H), 7.62 (d, J ¼ 8.7 Hz, 1H), 7.19 (d, J ¼ 8.7 Hz, 1H), 5.34 (s, 2H),
125.70, 117.60, 114.64, 79.22, 50.70, 43.97, 41.30, 37.51, 27.99; HRMS
(ESI): m/z calcd for C22H24Cl2N6NaO5 (MþNa)þ, 545.1083; found,
545.1069.
5.31 (s, 2H), 4.81e4.63 (m, 1H), 1.78e1.65 (m, 2H), 1.57 (m, 2H),
1.49e1.11 (m, 6H); 13C NMR (100 MHz, DMSO-d6)
d 178.54, 166.33,
158.05, 146.30, 142.96, 139.73, 130.21, 125.63, 124.30, 117.50, 114.55,
73.43, 50.52, 37.35, 30.64, 24.60, 22.67; HRMS (ESI): m/z calcd for
C
4.5. Synthesis of compound 5h
19H18Cl2N4NaO4 (M þ Na)þ, 459.0603; found, 459.0599.
Compound 5g (30 mg, 0.057 mmol,1.0 eq) was dissolved in DCM
(2 mL), followed by addition of TFA (166.8 mg, 1.72 mmol, 30 eq).
The resulting solution was kept at rt for 2 h. Then the mixture was
diluted with DCM. The organic phase was washed with saturated
Na2CO3 and H2O twice, drived over MgSO4 and evaporated under
vacuum to give compound 5h. Orange-red solid, yield: 31%, m.p.:
138.1e139.5 ꢀC, Rf ¼ 0.16 (DCM/MeOH ¼ 2/1); 1H NMR (400 MHz,
4.4.2. Compound 5b
Compound 5b, orange solid, yield: 84%, m. p.: 269.3e270.5 ꢀC,
Rf ¼ 0.53 (PE/acetone ¼ 1/1); 1H NMR (400 MHz, DMSO-d6)
d 8.22
(d, J ¼ 7.5 Hz, 1H), 8.12 (s, 1H), 7.62 (d, J ¼ 8.7 Hz, 1H), 7.19 (d,
J ¼ 8.7 Hz, 1H), 5.29 (s, 2H), 5.00 (s, 2H), 3.57e3.41 (m, 1H), 1.69 (m,
4H), 1.53 (m, 1H), 1.41e0.99 (m, 5H). 13C NMR (100 MHz, DMSO-d6)
d
178.65, 164.05, 158.16, 146.45, 142.72, 139.81, 130.27, 125.68,
DMSO-d6)
d
8.98 (brs, 2H), 8.05 (s, 1H), 7.62 (d, J ¼ 8.7 Hz, 1H), 7.19
124.37, 117.58, 114.64, 51.72, 47.77, 37.48, 32.19, 25.08, 24.31; HRMS
(ESI): m/z calcd for C19H20Cl2N5O3 (M þ H)þ, 436.0943; found,
436.0945.
(d, J ¼ 8.7 Hz, 1H), 5.49 (s, 2H), 5.31 (s, 2H), 3.92e3.09 (m, 8H); 13
C
NMR (100 MHz, DMSO-d6)
d 178.64, 164.50, 158.17, 146.44, 139.82,
130.30, 125.71, 124.62, 117.58, 114.64, 50.53, 42.50, 41.32, 38.38,
37.48; HRMS (ESI): m/z calcd for C17H17Cl2N6O3 (MþH)þ, 423.0734;
found, 423.0737.
4.4.3. Compound 5c
Compound 5c, yellow solid, yield: 64%, m. p.: 213.6e214.9 ꢀC,
Rf ¼ 0.10 (PE/acetone ¼ 2/1); 1H NMR (400 MHz, DMSO-d6)
d 10.45
4.6. Cytotoxic evaluation
(s, 1H), 8.21 (s, 1H), 7.63 (d, J ¼ 8.7 Hz, 1H), 7.56 (d, J ¼ 7.9 Hz, 2H),
7.32 (t, J ¼ 7.7 Hz, 2H), 7.19 (d, J ¼ 8.7 Hz, 1H), 7.08 (t, J ¼ 7.3 Hz, 1H),
5.32 (s, 2H), 5.29 (s, 2H); 13C NMR (100 MHz, DMSO-d6)
d 178.64,
All the human carcinoma cell lines were purchased from the Cell
Bank of the Chinese Academy of Science (Shanghai, China) and
cultured in at 37 ꢀC in a humidified atmosphere containing 5% CO2.
The CCK-8 method was used to investigate the cytotoxicity of
synthesized compounds against human breast, bladder, esophageal
and gastric cancer cell lines (MCF-7, SW-780, TE-1 and MGC-803)
and two normal cells (HL-702 and GES-1). In brief, cells were
collected during the exponential growth phase following trypsini-
164.04, 158.17, 146.46, 139.83, 138.33, 130.29, 128.87, 125.70, 123.76,
119.17, 117.58, 114.65, 52.27, 37.50; HRMS (ESI): m/z calcd for
C
19H14Cl2N5O3 (M þ H)þ, 430.0474; found, 430.0470.
4.4.4. Compound 5d
Compound 5d, orange solid, yield: 63%, m. p.: 205.6e206.8 ꢀC,
Rf ¼ 0.09 (PE/acetone ¼ 2/1); 1H NMR (400 MHz, DMSO-d6)
d 8.08
zation. 100
m
L Cells suspension (2 ꢃ 104 cells/mL) were seeded in
(s, 1H), 7.62 (d, J ¼ 8.7 Hz, 1H), 7.19 (d, J ¼ 8.7 Hz, 1H), 5.39 (s, 2H),
5.30 (s, 2H), 3.40 (s, 4H), 1.54 (d, J ¼ 24.8 Hz, 4H), 1.42 (s, 2H); 13C
96-well plates culture medium each well. After 24 h' incubation,
the medium was removed and replaced by 200
taining different concentrations of compounds mentioned above
(0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256 M). The control wells were added
with fresh medium. The plates were incubated at 37 ꢀC in 5% CO2
for 72 h. Thereafter, the cells were cultured for 4 h at 37 ꢀC with 7
mL medium con-
NMR (100 MHz, DMSO-d6)
d 178.65, 163.62, 158.14, 146.44, 139.80,
130.27, 125.69, 124.61, 117.60, 114.64, 50.74, 45.19, 42.42, 37.52,
25.72, 25.10, 23.76; HRMS (ESI): m/z calcd for C18H18Cl2N5O3
(M þ H)þ, 422.0787; found, 422.0784.
m
mL
of CCK-8 in each well. The mixture was shaken for 10 min at room
temperature. The absorbance was measured using a microplate
reader (BioTek Instruments, Inc., VT) at 450 nm. All experiments
were performed three times. IC50 values were calculated by soft-
ware SPSS 20.0 and showed by mean and SD.
4.4.5. Compound 5e
Compound 5e, yellow solid, yield: 53%, m. p.: 221.0e226.7 ꢀC,
Rf ¼ 0.28 (PE/acetone ¼ 1/1); 1H NMR (400 MHz, DMSO-d6)
d 8.08
(s, 1H), 7.62 (d, J ¼ 8.7 Hz, 1H), 7.19 (d, J ¼ 8.7 Hz, 1H), 5.30 (s, 4H),
3.48 (t, J ¼ 6.7 Hz, 2H), 3.29 (t, J ¼ 6.8 Hz, 2H), 1.89 (quintet, J ¼ 13.3,
6.6 Hz, 2H), 1.78 (quintet, J ¼ 13.3, 6.7 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6)
d
178.65, 163.60, 158.16, 146.46, 139.81, 130.28, 125.69,
4.7. Hoechst 33258 staining assay
124.48, 117.59, 114.66, 51.27, 45.75, 45.06, 37.50, 25.52, 23.67; HRMS
(ESI): m/z calcd for C17H15Cl2N5NaO3 (MþNa)þ, 430.0450; found,
430.0451.
Cells were seeded on glass slides placed in 24-well plates. After
growing to approximately 70% confluence, the cells were treated
with 0, 5, 10 and 20
washed with PBS for three times, and incubated with Hoechst
33258 staining buffer (10 g/mL Hoechst 3325 and 0.1% Triton X-
100) in dark for 20 min. Then cells were washed with PBS three
times, the slides seeded with the cells were mounted and analyzed
by Laser Scanning Confocal Microscope (Nikon A1R, Tokyo, Japan).
mM of compound 5a for 24 h. Then cells were
4.4.6. Compound 5f
Compound 5f, yellow solid, yield: 58%, m. p.: 205.7e207.0 ꢀC,
m
Rf ¼ 0.25 (PE/acetone ¼ 1/1); 1H NMR (400 MHz, DMSO-d6)
d
8.08
(s, 1H), 7.62 (d, J ¼ 8.7 Hz, 1H), 7.19 (d, J ¼ 8.7 Hz, 1H), 5.43 (s, 2H),
5.33 (s, 2H), 3.61 (s, 2H), 3.55 (s, 2H), 3.48 (s, 2H), 3.42 (s, 2H); 13C

B. Yu et al. / European Journal of Medicinal Chemistry 124 (2016) 350e360
359
4.8. Cell cycle distribution assay by flow cytometry
4.13. Statistical analysis
Cells were seeded in 6-well plates and treated with 0, 5, 10 and
M of compound 5a for 24 h. Then cells were collected and fixed
by 70% ethanol at 4 ꢀC overnight. The fixed cells were washed with
cold PBS and resuspended in 100 ul PBS containing 10 g/mL RNase
A and 50 g/mL PI for 20 min in dark. Samples were then analyzed
Data are presented as mean SD from three independent ex-
periments. SPSS statistics version 20.0 (IBM, New York City, NY) was
used to calculate IC50 and one-way ANOVA was employed to
determine statistical significance using GraphPad Prism 5 (Graph-
Pad Software Inc., San Diego, CA). *P < 0.05, **P < 0.01, as compared
with the control group.
20
m
m
m
for DNA content by flow cytometry (Becton, Dickinson and Com-
pany, NJ.).
Acknowledgment
4.9. Apoptosis detection by flow cytometry
We gratefully acknowledge the National Natural Science Foun-
dation of China (No. 21372206) and the Development Foundation of
Priority, Ministry of Education (No. 20134101130001) for the
financial support. Bin Yu is grateful for the financial support from
the Outstanding PhD Training Program of Zhengzhou University.
An Annexin V-FITC/PI kit (KeyGEN BioTECH, Nanjing, China) was
used to detect apoptosis. Cells were seeded in 6-well plates and
treated with 0, 5, 10 and 20 mM of compound 5a for 24 h. Then the
cells were collected and suspended in binding buffer containing
Annexin V-FITC (0.5 mg/mL) and PI (0.5 mg/mL) and incubated in
dark for 20 min and analyzed by flow cytometry (Becton, Dickinson
and Company, NJ).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.08.065.
4.10. Measurement of intracellular ROS levels
References
The redox-sensitive probe 2,7-dichlorodihydrofluorescein
diacetate was used to measure intracellular ROS levels. Cells were
[1] M. d'Ischia, A. Palumbo, G. Prota, Adrenalin oxidation revisited. New products
beyond the adrenochrome stage, Tetrahedron 44 (1988) 6441e6446.
[2] J. Song, L. Hou, C. Ju, J. Zhang, Y. Ge, W. Yue, Isatin inhibits proliferation and
induces apoptosis of SH-SY5Y neuroblastoma cells in vitro and in vivo, Eur. J.
Pharmacol. 702 (2013) 235e241.
[3] L. Hou, C. Ju, J. Zhang, J. Song, Y. Ge, W. Yue, Antitumor effects of isatin on
human neuroblastoma cell line (SH-SY5Y) and the related mechanism, Eur. J.
Pharmacol. 589 (2008) 27e31.
seeded and treated with 0, 5, 10, and 20
24 h, then cells were collected and incubated with DCFH-DA
(10
M) for 30 min at 37 ꢀC. Then cells were washed with serum
mM of compound 5a for
m
free medium for three times. And the fluorescence intensity was
determined by a flow cytometer.
[4] X. Jiang, Y. Sun, J. Yao, Y. Cao, M. Kai, N. He, X. Zhang, Y. Wang, R. Wang, Core
scaffold-inspired concise synthesis of chiral spirooxindole-pyranopyrimidines
with broad-spectrum anticancer potency, Adv. Synth. Catal. 354 (2012)
917e925.
[5] L. Hong, R. Wang, Recent advances in asymmetric organocatalytic construc-
tion of 3,30-Spirocyclic oxindoles, Adv. Synth. Catal. 355 (2013) 1023e1052.
[6] B. Yu, D.Q. Yu, H.M. Liu, Spirooxindoles: promising scaffolds for anticancer
agents, Eur. J. Med. Chem. 97 (2015) 673e698.
[7] Z. Zhang, X.J. Chu, J.J. Liu, Q. Ding, J. Zhang, D. Bartkovitz, N. Jiang, P. Karnachi,
S.S. So, C. Tovar, Z.M. Filipovic, B. Higgins, K. Glenn, K. Packman, L. Vassilev,
B. Graves, Discovery of potent and orally active p53-MDM2 inhibitors RO5353
and RO2468 for potential clinical development, ACS Med. Chem. Lett. 5 (2013)
124e127.
[8] B. Yu, Y.C. Zheng, X.J. Shi, P.P. Qi, H.M. Liu, Natural product-derived spiroox-
indole fragments serve as privileged substructures for discovery of new
anticancer agents, Anticancer Agents Med. Chem. (2016), http://dx.doi.org/
10.2174/1871520615666151102093825.
4.11. Western blot analysis
Cells were seeded and treated with 0, 5,10 and 20 mM of com-
pound 5a for 24 h, then cells were collected and lysed by radio
immunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH
7.5, 150 mM NaCl, 0.25% sodium deoxycholate, 0.1% Nonidet P-40,
0.1% Triton X-100) with the complete proteinase inhibitor cocktail
(Roche, Basel, Switzerland) for 30 min. After centrifugation of
12,000 rpm for 10 min at 4 ꢀC, supernatant was collected and the
protein concentration was detected using a bicinchonininc acid
(BCA) assay kit (Beyotmie Biotechnology, Haimen, China). After
added with loading buffer, cell lyses were boiled for 10 min at
100 ꢀC for SDS-polyacrylamide gel electrophoresis (PAGE). Proteins
were transferred to nitrocellulose (NC) membranes. The mem-
branes were blocked with 5% skim milk at room temperature for
2 h, and then incubated overnight at 4 ꢀC with primary antibodies.
After washing the membrane with TBST (TBS, 0.05% Tween-20)/TBS
three times (5 min per wash), blots were incubated with the sec-
ondary antibody (1:5000) at room temperature for 2 h. Finally, the
blots were washed in TBST/TBS. The antibody-reactive bands were
revealed by enhanced chemiluminescence (ECL) and exposed on
Kodak radiographic film.
[9] C.V. Galliford, K.A. Scheidt, Pyrrolidinyl-spirooxindole natural products as
inspirations for the development of potential therapeutic agents, Angew.
Chem. Int. Ed. 46 (2007) 8748e8758.
ꢀ
[10] S. Wang, W. Sun, Y. Zhao, D. McEachern, I. Meaux, C. Barriere, J.A. Stuckey,
J.L. Meagher, L. Bai, L. Liu, C.G. Hoffman-Luca, J. Lu, S. Shangary, S. Yu,
D. Bernard, A. Aguilar, O. Dos-Santos, L. Besret, S. Guerif, P. Pannier, D. Gorge-
Bernat, L. Debussche, SAR405838: an optimized inhibitor of MDM2ep53
interaction that induces complete and durable tumor regression, Cancer Res.
74 (2014) 5855e5865.
[11] B. Yu, Z. Yu, P.P. Qi, D.Q. Yu, H.M. Liu, Discovery of orally active anticancer
candidate CFI-400945 derived from biologically promising spirooxindoles:
success and challenges, Eur. J. Med. Chem. 95 (2015) 35e40.
[12] N.J. White, S. Pukrittayakamee, A.P. Phyo, R. Rueangweerayut, F. Nosten,
ꢀ
P. Jittamala, A. Jeeyapant, J.P. Jain, G. Lefevre, R. Li, B. Magnusson, T.T. Diagana,
F.J. Leong, Spiroindolone KAE609 for falciparum and vivax malaria, N. Engl. J.
Med. 371 (2014) 403e410.
4.12. Transwell assay
[13] G.S. Singh, Z.Y. Desta, Isatins as privileged molecules in design and synthesis
of spiro-fused cyclic frameworks, Chem. Rev. 112 (2012) 6104e6155.
[14] N.R. Ball-Jones, J.J. Badillo, A.K. Franz, Strategies for the enantioselective
synthesis of spirooxindoles, Org. Biomol. Chem. 10 (2012) 5165e5181.
[15] M. Xia, R.Z. Ma, Recent progress on routes to spirooxindole systems derived
from isatin, J. Heterocycl. Chem. 51 (2013) 539e554.
For the migration assay, 100
different concentrations of compound 5a and 10, 000 cells were
added to each upper chamber. In the lower chamber, 500 L me-
dium with 20% FBS was used as chemoattractant. After incubation
for 24 h, both chambers were washed by PBS for three times. After
staining with Hoechst 33258 (10 mg/mL) and twice wash, migrated
cells were detected and numbered using high content screening
system (ArrayScan XTI, Thermo Fisher Scientific, MA).
mL medium containing 1% FBS,
m
[16] S.G. Agalave, S.R. Maujan, V.S. Pore, Click chemistry: 1,2,3-Triazoles as phar-
macophores, Chem. Asian J. 6 (2011) 2696e2718.
[17] A. Massarotti, S. Aprile, V. Mercalli, E. Del Grosso, G. Grosa, G. Sorba, G.C. Tron,
Are 1,4- and 1,5-Disubstituted 1,2,3-Triazoles good pharmacophoric groups?
ChemMedChem 9 (2014) 2497e2508.
ꢁ ꢁ
[18] D. Gonzalez-Calderon, M.G. Mejía-Dionicio, M.A. Morales-Reza, A. Ramírez-

360
B. Yu et al. / European Journal of Medicinal Chemistry 124 (2016) 350e360
Villalva, M. Morales-Rodríguez, B. Jauregui-Rodríguez, E. Díaz-Torres,
C. Gonzalez-Romero, A. Fuentes-Benítes, Azide-enolate 1,3-dipolar cycload-
348e356.
ꢁ
[33] X.W. Ye, Y.C. Zheng, Y.C. Duan, M.M. Wang, B. Yu, J.L. Ren, J.L. Ma, E. Zhang,
H.M. Liu, Synthesis and biological evaluation of coumarin-1,2,3-triazole-
dithiocarbamate hybrids as potent LSD1 inhibitors, MedChemComm 5 (2014)
650e654.
[34] Y.C. Zheng, Y.C. Duan, J.L. Ma, R.M. Xu, X. Zi, W.L. Lv, M.-M. Wang, X.W. Ye,
S. Zhu, D. Mobley, Y.Y. Zhu, J.W. Wang, J.F. Li, Z.R. Wang, W. Zhao, H.M. Liu,
Triazole-Dithiocarbamate based selective lysine specific demethylase 1 (LSD1)
inactivators inhibit gastric Cancer cell growth, invasion, and migration, J. Med.
Chem. 56 (2013) 8543e8560.
[35] L.Y. Ma, Y.C. Zheng, S.Q. Wang, B. Wang, Z.R. Wang, L.P. Pang, M. Zhang,
J.W. Wang, L. Ding, J. Li, C. Wang, B. Hu, Y. Liu, X.D. Zhang, J.J. Wang, Z.J. Wang,
W. Zhao, H.M. Liu, Design, synthesis, and structure-activity relationship of
novel LSD1 inhibitors based on pyrimidine-thiourea hybrids as potent, orally
active antitumor agents, J. Med. Chem. 58 (2015) 1705e1716.
dition in the synthesis of novel triazole-based miconazole analogues as
promising antifungal agents, Eur. J. Med. Chem. 112 (2016) 60e65.
[19] B. Yu, X.J. Shi, P.P. Qi, D.Q. Yu, H.M. Liu, Design, synthesis and biological
evaluation of novel steroidal spiro-oxindoles as potent antiproliferative
agents, J. Steroid Biochem. Mol. Biol. 141 (2014) 121e134.
[20] B. Yu, P.P. Qi, X.J. Shi, L.H. Shan, D.Q. Yu, H.M. Liu, Discovery of novel steroidal
pyraneoxindole hybrids as cytotoxic agents, Steroids 88 (2014) 44e52.
[21] B. Yu, X.N. Sun, X.J. Shi, P.P. Qi, Y.C. Zheng, D.Q. Yu, H.M. Liu, Efficient synthesis
of novel antiproliferative steroidal spirooxindoles via the [3þ2] cycloaddition
reactions of azomethine ylides, Steroids 102 (2015) 92e100.
[22] Y.L. Zhang, Y.F. Li, J.W. Wang, B. Yu, Y.K. Shi, H.M. Liu, Multicomponent as-
sembly of novel antiproliferative steroidal dihydropyridinyl spirooxindoles,
Steroids 109 (2016) 22e28.
[23] B. Yu, P.P. Qi, X.J. Shi, R. Huang, H. Guo, Y.C. Zheng, D.Q. Yu, H.M. Liu, Efficient
synthesis of new antiproliferative steroidal hybrids using the molecular hy-
bridization approach, Eur. J. Med. Chem. 117 (2016) 241e255.
[24] Y.H. Yang, H. Aloysius, D. Inoyama, Y. Chen, L.Q. Hu, Enzyme-mediated hy-
drolytic activation of prodrugs, Acta Pharm. Sin. B 1 (2011) 143e159.
[25] J. Rautio, H. Kumpulainen, T. Heimbach, R. Oliyai, D. Oh, T. Jarvinen,
J. Savolainen, Prodrugs: design and clinical applications, Nat. Rev. Drug Discov.
7 (2008) 255e270.
[36] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and
computational approaches to estimate solubility and permeability in drug
discovery and development settings, Adv. Drug Deliv. Rev. 46 (2001) 3e26.
[37] E. Ginsburg, D. Salomon, T. Sreevalsan, E. Freese, Growth inhibition and
morphological changes caused by lipophilic acids in mammalian cells, Proc.
Natl. Acad. Sci. U. S. A. 70 (1973) 2457e2461.
[38] Alexi I. Goranov, A. Gulati, N. Dephoure, T. Takahara, T. Maeda, Steven P. Gygi,
S. Manalis, A. Amon, Changes in cell morphology are coordinated with cell
growth through the TORC1 pathway, Curr. Biol. 23 (2013) 1269e1279.
[39] G.K. Schwartz, M.A. Shah, Targeting the cell cycle: a new approach to Cancer
therapy, J. Clin. Oncol. 23 (2005) 9408e9421.
[40] L.Z. de Oliveira, I.L.G. Farias, M. Rigo, W.G. Glanzner, P.B.D. Goncalves,
F.C. Cadona, I.B. Cruz, J.G. Farias, M.M.M.F. Duarte, L. Franco, G. Bertol, E. Colpo,
P.C. Brites, J.B.T. Rocha, D.B.R. Leal, Effect of Uncaria tomentosa extract on
apoptosis triggered by oxaliplatin exposure on HT29 cells, Evid. Based Com-
plement. Altern. Med. 2014 (2014) 1e10.
[26] P. Huang, D. Wang, Y. Su, W. Huang, Y. Zhou, D. Cui, X. Zhu, D. Yan, Combi-
nation of small molecule prodrug and nanodrug delivery: amphiphilic
drugedrug conjugate for Cancer therapy, J. Am. Chem. Soc. 136 (2014)
11748e11756.
€
[27] R.P. Rastogi, S.P. Singh, D.P. Hader, R.P. Sinha, Detection of reactive oxygen
species (ROS) by the oxidant-sensing probe 20,70-dichlorodihydrofluorescein
diacetate in the cyanobacterium Anabaena variabilis PCC 7937, Biochem.
Biophys. Res. Commun. 397 (2010) 603e607.
[28] S. Schulz, J.W. Nyce, Inhibition of protein isoprenylation and p21ras mem-
brane association by dehydroepiandrosterone in human colonic adenocarci-
noma cells in vitro, Cancer Res. 51 (1991) 6563e6567.
[29] R. Vegliante, E. Desideri, L. Di Leo, M.R. Ciriolo, Dehydroepiandrosterone
triggers autophagic cell death in human hepatoma cell line HepG2 via JNK-
mediated p62/SQSTM1 expression, Carcinogenesis 37 (2016) 233e244.
[30] Clinical development plan: dehydroepiandrosterone (DHEA), J. Cell. Biochem.
63 (1996) 86e99.
[41] K.R. Senwar, P. Sharma, T.S. Reddy, M.K. Jeengar, V.L. Nayak, V.G.M. Naidu,
A. Kamal, N. Shankaraiah, Spirooxindole-derived morpholine-fused-1,2,3-
triazoles: design, synthesis, cytotoxicity and apoptosis inducing studies, Eur.
J. Med. Chem. 102 (2015) 413e424.
[42] Y.H. Han, Y.M. Yang, S.Z. Kim, W.H. Park, Attenuation of MG132-induced HeLa
cell death by N-Acetyl cysteine via reducing reactive oxygen species and
preventing glutathione depletion, Anticancer Res. 30 (2010) 2107e2112.
ꢁ
[43] F. Gonzalez-Bobes, N. Kopp, L. Li, J. Deerberg, P. Sharma, S. Leung, M. Davies,
[31] A.G. Schwartz, L. Pashko, J.M. Whitcomb, Inhibition of tumor development by
dehydroepiandrosterone and related steroids, Toxicol. Pathol. 14 (1986)
357e362.
J. Bush, J. Hamm, M. Hrytsak, Scale-up of azide chemistry: a case study, Org.
Process Res. Dev. 16 (2012) 2051e2057.
[44] D. Lee, D. Kim, S. Lee, T. Kim, J. Kim, S. Kim, K.H. Liu, S. Lee, J.S. Bae, K.S. Song,
C.W. Cho, Y.K. Son, D.J. Baek, T. Lee, Efficient syntheses of 1,2,3-Triazoloamide
derivatives using solid-and solution-phase synthetic approaches, Molecules
20 (2015) 19984e20013.
[32] D. Safiulina, N. Peet, E. Seppet, A. Zharkovsky, A. Kaasik, Dehydroepiandros-
terone inhibits complex
I of the mitochondrial respiratory chain and is
neurotoxic in vitro and in vivo at high concentrations, Toxicol. Sci. 93 (2006)