Pt(IV) complexes conjugating with chalcone analogue as inhibitors of microtubule polymerization exhibited selective inhibition in human cancer cells

Article abstract of DOI:10.1016/j.ejmech.2018.01.075

Six novel of Pt(IV) complexes comprising chalcone analogues were synthesized and evaluated for anti-proliferative activity using MTT assay. In vitro evaluation revealed that all Pt(IV) complexes showed better and more potent activity against three human cancer cells including CDDP resistant cells than that of their corresponding mother Pt(II) species. Among them, two representative complexes, 14 and 17, exhibited better cell selectivity between cancer cells and normal cells than CDDP. Molecular docking study indicated that complexes 14 and 17 could bind to the colchicine site of tubulin. Moreover, complexes 14 and 17 also remarkably displayed inhibition of cell migration against HUVEC cells in vitro. Molecular mechanism studies suggested that 14 and 17 induced production of reactive oxygen species (ROS), cell cycle arrest at the G2/M phase, and mitochondria-mediated apoptosis by regulating the expression of Bcl-2 family members.

Full text of DOI:10.1016/j.ejmech.2018.01.075

European Journal of Medicinal Chemistry 146 (2018) 435e450
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Pt(IV) complexes conjugating with chalcone analogue as inhibitors of
microtubule polymerization exhibited selective inhibition in human
cancer cells
,
,
, *
Xiaochao Huang a 1, Rizhen Huang a 1, Zhimei Wang a, Lingxue Li a, Shaohua Gou a
,
Zhixin Liao a, Hengshan Wang b
,
**
a Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing 211189, China
b State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), School of Chemistry and
Pharmaceutical Sciences of Guangxi Normal University, Guilin 541004, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Six novel of Pt(IV) complexes comprising chalcone analogues were synthesized and evaluated for anti-
proliferative activity using MTT assay. In vitro evaluation revealed that all Pt(IV) complexes showed
better and more potent activity against three human cancer cells including CDDP resistant cells than that
of their corresponding mother Pt(II) species. Among them, two representative complexes, 14 and 17,
exhibited better cell selectivity between cancer cells and normal cells than CDDP. Molecular docking
study indicated that complexes 14 and 17 could bind to the colchicine site of tubulin. Moreover, com-
plexes 14 and 17 also remarkably displayed inhibition of cell migration against HUVEC cells in vitro.
Molecular mechanism studies suggested that 14 and 17 induced production of reactive oxygen species
(ROS), cell cycle arrest at the G2/M phase, and mitochondria-mediated apoptosis by regulating the
expression of Bcl-2 family members.
Received 25 May 2017
Received in revised form
17 September 2017
Accepted 23 January 2018
Available online 4 February 2018
Keywords:
Anticancer activity
Pt(IV) complex
Tubulin polymerization
Apoptosis
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
resistance of CDDP [10,11]. In contrast to their Pt(II) counterparts,
Pt(IV) complexes with a low-spin, d6 octahedral geometry dis-
Cisplatin (CDDP), carboplatin and oxaliplatin (OXP) (Fig. 1) as
Pt(II)-based anticancer drugs approved by FDA have been widely
used for the treatment of a variety of cancers in the clinical [1e5].
However, the development of drug resistance, side effects and high
toxicity have rendered CDDP and its analogues suboptimum for
clinical applications [6e9]. Therefore, great efforts have been
devoted to the design of novel and effective platinum based drugs
for overcoming the above mentioned shortcomings [10,11]. In the
past several decades, thousands of Pt(II) complexes have been
synthesized and screened for anticancer activity, but only a small
number of compounds were used in clinical trials and most of them
turned out to be invalid [12]. Hence, use of Pt(IV) complexes as
prodrugs is presently one of strategies to overcome the drawbacks
including low bioavailability, severe side effects and acquired
playing kinetic inertness are expected to exhibit great promise in
the search for the next generation of platinum drugs, because they
can be easily reduced to their Pt(II) equivalents by intracellular
reducing molecules such as ascorbic acid or glutathione [11,13]. So
far, there have been several reports on Pt(IV) complexes including
those with active pharmacophores, which were used as prodrugs to
improve the antitumor activity and overcome the side effect of
CDDP (Fig. 2) [14e19].
Microtubules, formed through polymerization of
a- and b-
tubulin heterodimers, are crucial to a large number of fundamental
cell functions, such as cell replication, maintenance of cell shape,
intracellular transport and migration [20e22]. Since microtubules
play a major role in mitosis, they have been recognized as an
important target for the development of novel antitumor agents in
anticancer chemotherapy (Fig. 3) [23e25]. Generally speaking, the
inhibitors of microtubule can be divided into two major types:
microtubule destabilizers including combretastatin A-4, colchicine,
and vinca alkaloids, and microtubule stabilizers like taxanes
[26,27]. Up to now, the inhibitors of tubulin including taxanes,
* Corresponding author.
** Corresponding author.
E-mail addresses: sgou@seu.edu.cn (S. Gou), whengshan@163.com (H. Wang).
Co-first author: These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2018.01.075
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

side effects, poor solubility, the development of drug resistance,
low oral bioavailability and complicated synthesis have always
limited these compounds' using in the clinical treatment of solid
tumors. Recent studies suggested that the combination of platinum
anticancer agents with tubulin inhibitors including paclitaxel and
docetaxel can result in the improvement of therapeutic efficacy
[30]. In recent years, chalcone and its analogues have been found to
exhibit potent cytotoxicity against a large number of human cancer
cells including multidrug resistant cancer cell lines and strongly
inhibit tubulin polymerization by binding to the colchicine binding
site [31e36]. In addition, the chalcone derivatives 1a and 2a (Fig. 3),
served as inhibitor of microtubule polymerization, displayed good
Fig. 1. FDA approved Pt(II) anticancer agents.
vinca alkaloids and paclitaxel are forceful as anti-mitotic agents,
and they have been widely used against certain types of malig-
nancies in the past decades [28,29]. Unfortunately, high toxicity,
Fig. 2. Chemical structures of some Pt(IV) complexes prodrugs.

X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
437
antitumor activity against human cancer cell lines [37e39]. More-
over, Schobert and co-workers recently showed that the Pt(II)
complex conjugating with 2a and its derivatives exhibited moder-
ate antitumor activities against diverse human cancer cells owing
to the different mode of actions such as DNA-targeting Pt(II) com-
plexes and tubulin-targeting chalcone moieties [39]. Hence, a
combination of tubulin inhibitors with cytotoxic DNA damaging
platinum-based agents can be considered as a fascinating strategy
for targeting tubulin and DNA, and at least in theory, can effectively
improve the antitumor activities of platinum drugs and overcome
their adverse side effects. However, up to now, no example of a
prodrug containing both CDDP and the chalcone derivative has
been reported as an inhibitor of tubulin polymerization. In this
paper, a series of Pt(IV) prodrugs containing chalcone derivatives as
an axial ligand in the octahedrally geometric Pt(IV) atom were
designed, synthesized and evaluated for antitumor activity. Among
these Pt(IV) complexes, complexes 14 and 17 displayed excellent
anticancer activity against the tested cancer cell lines including
CDDP-resistant cell line A549. The mechanism action of the potent
Pt(IV) complexes 14 and 17 was investigated. In short, these studies
obviously suggested that the anti-proliferative activity of the
typical complex was significantly more potent than that of CDDP
due to the synergism of conjugating DNA damaging platinum-
based agents and tubulin-targeting chalcone moieties.
the connectivity of the aliphatic chain [(CH2)3 or (CH2)4] to the
chalcone moiety [the proton signals at dH 2.53e2.45 (m, 4H),
2.13e2.03 (m, 2H) for compound 7 and 2.48e2.36 (m, 4H),
1.83e1.73 (m, 4H) for compound 8 ], and its molecular formula was
established as C25H29NO8 (7) and C26H31NO8 (8) on the basis of the
HR-ESI-MS data (m/z 472.18732 and 486.20171, [MþH]þ), respec-
tively. Furthermore, 13C NMR resonances for carbonyl group were
observed at dC 189.43, 173.54 and 170.59 (7) and 189.45, 173.80 and
170.99 (8). For compounds 9 and 10, the molecular formula was
assigned as C24H27NO8 and C25H29NO8 on the basis of the HR-ESI-
MS data (m/z 456.16883 (9) and 470.22474 (10), [M-H]-), and the
1H NMR spectroscopic data of compounds 9 and 10 displayed a
single peak at dH 11.90 (9) and 11.99 (10) suggested the presence of
carboxylic acid group (-COOH), respectively. All the HR-ESI-MS
spectra of the Pt(IV) complexes (14e19) gave main peaks corre-
sponding to [M-H]- or [MþCl]- ions, which were composed of a few
isotopic peaks due to the presence of platinum isotopes, and
characteristic resonances could be observed at d 544.37 (14), 412.26
(15), 1000.02 (16), 544.67 (17), 414.26 (18) and 996.61 (19) in 195Pt
NMR, suggested the bonding between the Pt(IV) ions and the li-
gands. In addition, 1H NMR spectroscopic data of 14 and 17 were
similar to 9 and 10, except for characteristic resonances at
d
6.52e5.70 (m, 6H) for 14 and 6.45e5.76 (m, 6H) for 17 of (NH3)2 in
1H NMR and were assigned to the CDDP core, and a single peak
(-COOH) disappears compared to 9 and 10, respectively. For com-
plexes 15 and 18, characteristic resonances at dH 9.60e9.56 (m, 1H),
8.14 (s, 1H), 7.79 (s, 1H) and 7.73e7.70 (m, 1H) for 15 and 9.64e9.59
(m, 1H), 8.13 (s, 1H), 7.78 (s, 1H) and 7.73e7.71 (m, 1H) for 18 of
(NH2)2 in 1H NMR and were assigned to the DACHPt core, and
resonances at dH 2.76e2.70 (m, 2H), 2.19e2.04 (m, 1H), 1.55e1.48
(m, 3H), 1.31e1.06 (m, 3H) for 15 and 2.75e2.69 (m, 2H), 2.19e2.03
(m, 2H), 1.55e1.46 (m, 3H), 1.32e1.04 (m, 3H) for 18 of (CH2)4 or
(NH2CH)2 in 1H NMR and were assigned to the DACHPt core,
respectively. The 1H and 13C NMR spectroscopic data of 16 and 19
were similar to 15 and 18, except for characteristic resonances at dC
163.82 (16) and 163.69 (19) of the carbonyl group in 13C NMR and
were assigned to the OXP core compared to 15 and 18. Moreover,
the far-IR spectrum of the target Pt(IV) complexes 14e19 displayed
characteristic absorption bands of Pt-Cl in the range from 321 to
361 cmꢀ1, and the Pt-N stretches in the region between 434 and
456 cmꢀ1. In addition, in all complexes the Pt-O stretches in the
range 525e575 cmꢀ1 were also observed.
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of target compounds
The general steps to synthesize the target compounds have been
outlined in Scheme 1. Pt(IV) complexes 11e13 were prepared ac-
cording to the reported procedures [18,40]. First, by Claisen-
Schmidt condensation of 1 with 2 in the presence of 50% KOH in
CH3OH to produce the key intermediate 3, and then treating with
Fe powder and NH4Cl in ethanol/water easily yielded compound 4.
Subsequently, intermediate 7 or 8 was achieved by the formation of
amide bond between 5 or 6 and 4, respectively, in the presence of
HOBT/EDCI, then 7 or 8 followed by its hydrolysis with aluminum
hydroxide in the presence of THF/H2O to obtain the acid 9 or 10.
Finally, the synthesis of target compounds (14e19) obtained by
esterification reactions between 9, or 10 and Pt(IV) precursor
complexes 11e13, respectively, in the presence of TBTU/Et3N.
2.2. Biological evaluation
2.1.2. Characterization
The chemical structures of these target Pt(IV) complexes were
confirmed by IR, 1 H NMR, 13 C NMR, 195 Pt NMR, elemental analysis
and high resolution mass spectra (HR-MS). The [MþH]þ parent ions
of compounds 3, 4, 7, 8 and the [MþCl]- parent ions of compound 17
and the [M-H]- parent ions of compounds 9, 10, 14, 15, 16, 18 and 19
were observed using HR-ESI-MS. The IR spectrum showed charac-
teristic absorption bands for 1653 cmꢀ1 (C¼O) and 1577 and
1457 cmꢀ1 (aromatic ring) and 1354 cmꢀ1 (NO2) (3) and
1650 cmꢀ1 (C¼O) and 3444 and 3358 cmꢀ1 (NH2) (4) functional-
2.2.1. Cytotoxicity test
The cytotoxic activity of compounds 9e10 and 14e19 was
evaluated in vitro against three human cancer cell lines (HepG-2,
hepatoma; SK-OV-3, ovarian; NCI-H460, lung), and two human
normal cell lines (HL-7702, liver; BEAS-2B, lung) with CDDP, OXP,
DACHPt, and compound 4 as reference controls using the MTT
assay. The biological results of the compounds are summarized in
Table 1. As shown in Table 1, compounds 9 and 10 showed lower
cytotoxicity against the tested cancer cells than that of positive
drug compound 4. However, complexes 14e16 and 17e19, the
Pt(IV) derivatives of CDDP, DACHPt or OXP with one chalcone de-
rivative 9 or 10 ligand in the axial position, displayed significant
antitumor activity against all tested cell lines. Complexes 14 and 17,
Pt(IV) derivatives of CDDP, owned up to 2.71-fold and 5.54-fold
increased cytotoxicity compared with CDDP in SK-OV-3 cells,
significantly exhibiting more effective antitumor activity than
compound 4. It was also noticed that complexes 14 and 17 showed
high sensitivity toward HepG-2 and NCI-H460 cancer cells with IC50
ities. In the 1H NMR spectrum (3), the proton signals at
d 7.75 (d,
J ¼ 15.3 Hz, 1H) and 7.43 (d, J ¼ 15.6 Hz, 1H) suggested the presence
of one trans-substituted double bond segment, and the character-
istic resonances of four methoxy [dH 4.02 (3H, s), 3.96 (6H, s), 3.95
(3H, s)] groups were also observed. The 1H NMR spectroscopic data
of compound 4 was very similar to compound 3. We could observe
characteristic resonances by 13C NMR at dC 188.42 (3) and 189.42 (4)
were assigned to the carbonyl group, respectively. In addition, the
IR and 1H NMR spectroscopic data of compounds 7 and 8 were
similar to compound 4, except for the presence of an amide bond
[the proton signals at dH 8.81 (s, 1H) for compounds 7 and 8], and
values of 2.23, 4.65 and 0.97, 3.66
two normal cells HL-7702 and BEAS-2B with IC50 values of 24.56,
mM, but low cytotoxicity toward

438
X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
Scheme 1. Synthetic route of the Pt(IV) complexes. Reagents and conditions: (a) 50% KOH/H2O, MeOH, 0 ꢁC; (b) Fe, NH4Cl, EtOH, 85 ꢁC; (c) EDCI, DMAP, CH2Cl2, room temperature;
(d) LiOH$H2O, THF/H2O, room temperature; (e) TBTU, Et3N, DMF, room temperature.
34.47 and 33.16, 35.14
m
M compared with the positive drug CDDP
the efficacies of CDDP for different human cancer cells. According to
the above biological results, we further evaluated the cytotoxicity of
complexes 14e17 against CDDP sensitive and resistant cancer cells
(human lung epithelial cells A549 and A549/CDDP)). The biological
results of the compounds are shown in Table 2 and Fig. 4 A. As
shown in Table 2, the IC50 value of CDDP against A549/CDDP
(8.51, 10.42 M), respectively. Therefore, the selectivity index of
m
complexes 14 and 17 were calculated out as 11.05, 7.41 and 34.19,
9.34, respectively, much higher than that of CDDP (0.94 and 0.93).
The similar trend was also observed in complexes 15e16 and
18e19. Interestingly, complexes 14e16 have a different carbon
chain from complexes 17e19, the corresponding compounds dis-
played different antitumor activities that are enhanced with an
increase of the carbon chain length. Moreover, all Pt(IV) complexes
showed obvious lower cytotoxicity than those of their corre-
sponding Pt(II) positive controls (CDDP, DACHPt and OXP) against
two human normal cells, suggesting that these Pt(IV) complexes
displayed better cell selectivity between cancer cells and normal
liver cells than their mother Pt(II) agents.
resistant cells was increased to 42.51 mM. However, the activity of
complexes 14 and 17 was not obviously changed for the CDDP
resistant cancer cells when compared with the CDDP sensitive cells.
IC50 values of 14 and 17 against CDDP resistant A549 cells were 4.25
and 3.05 mM, respectively. It was much significant to discover that
complexes 14 and 17 had much lower resistance factors (1.15 and
1.26) than that of CDDP (4.95). Moreover, other Pt(IV) complexes
15e16 and 18e19 also exhibited obvious anticancer activities
against the CDDP resistant A549/CDDP cells comparable to those of
DACHPt and oxaliplatin with small resistance factors. The results
indicated that these Pt(IV) complexes might be useful in the
treatment of drug refractory cancer resistance to other platinum
2.2.2. Antitumor activity of target complexes against CDDP
resistant cancer cell line
Drug resistance is a major therapeutic problem that confined

X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
439
Table 1
Cytotoxic effects of Pt(IV) complexes on human cancer and normal cell lines.
Comp.
IC50 (m
M)d
HepG-2
SK-OV-3
NCI-H460
HL-7702
BEAS-2B
SIe
SIf
4
9
10
14
15
16
17
18
10.88 2.11
35.04 3.08
25.85 3.07
2.23 0.39
6.25 1.28
13.12 2.13
0.97 0.25
3.81 0.91
10.21 1.01
9.05 1.23
10.50 2.01
15.58 3.13
5.46 1.03
20.21 2.15
15.43 2.35
2.75 1.06
6.78 1.89
9.85 2.54
2.11 0.38
5.56 0.67
8.52 1.19
7.46 1.48
9.03 0.81
12.06 1.66
8.43 2.17
45.09 3.43
35.43 2.91
4.65 1.12
5.29 2.07
14.22 2.03
3.66 0.77
4.61 1.06
10.38 2.35
11.21 1.71
9.58 1.16
16.09 2.44
15.07 2.73
55.38 3.29
65.43 3.26
24.65 2.13
37.29 3.15
41.22 2.94
33.16 2.51
44.61 4.02
48.54 3.61
8.51 3.61
7.61 1.83
10.25 1.95
18.43 2.38
48.39 3.16
48.42 3.18
34.47 3.11
45.43 3.73
44.91 4.01
35.14 2.38
41.69 3.05
49.08 2.33
10.42 1.92
8.55 1.36
1.39
1.58
2.53
11.05
5.97
3.14
34.19
11.70
4.75
0.94
0.72
0.66
2.18
1.08
1.37
7.41
8.59
3.16
9.34
9.04
4.72
0.93
0.89
0.75
19
CDDPa
DACHPtb
OXPc
12.13 2.41
a
Cisplatin.
b
Dichloro(1R,2R-diaminocyclohexane)platinum(II).
Oxaliplatin.
c
d
e
f
In vitro cytotoxicity was determined by MTT assay upon incubation of the live cells with the compounds for 72 h.
Selectivity Index ¼ IC50(HL-7702)/IC50(HepG-2).
Selectivity Index ¼ IC50 (BEAS-2B)/IC50 (NCI-H460).Mean values based on three independent experiments.
Table 2
drugs.
Biological activity of Pt(IV) complexes against cisplatin sensitive and resistant cancer
cells (A549 and A549/CDDP).
2.2.3. Cellular uptake
Comp.
IC50
(
m
M)d
Because Pt(IV) complexes 14 and 17 exhibited better cytotox-
icity, they were further typically selected to carry out the cellular
uptake test in HepG-2 cells by using the inductively coupled plasma
mass spectrometry (ICP-MS). As shown in Fig. 4 B and Table 3,
A549
A549/CDDP
R Fe
4
9
10
14
15
16
17
18
5.61 1.36
27.11 2.81
15.51 2.33
3.69 0.57
6.83 1.01
10.15 0.38
2.42 0.25
4.50 1.09
8.51 1.61
8.59 1.52
8.65 1.31
11.95 1.43
6.55 2.08
32.11 3.09
19.82 1.89
4.25 0.34
8.23 1.27
12.05 2.04
3.05 0.53
6.01 1.15
10.29 1.07
42.51 3.15
21.29 2.68
23.76 3.34
1.17
1.18
1.28
1.15
1.20
1.19
1.26
1.34
1.21
4.95
2.46
1.99
treating HepG-2 cells with complexes 14 and 17 (10.0 and 20.0 mM)
for 24 h resulted in an abundant increase in the content of cellular
platinum at a dose-dependent manner, showing facile internali-
zation of the complexes within 24 h. Especially, the uptake of two
complexes was remarkably higher than that of CDDP. After exposed
19
to 20.0 mM of complex 17 for 24 h, the concentration of cellular
CDDPa
DACHPtb
OXPc
platinum rose to 605 ng/106 cells, which was two times as much as
that of CDDP. Based on the data from both cytotoxicity assay and
cellular uptake tests, it is concluded that the improved cellular
uptake can lead to the enhance of the antitumor activity.
a
Cisplatin.
b
Dichloro(1R,2R-diaminocyclohexane)platinum(II).
c
Oxaliplatin.
d
In vitro cytotoxicity was determined by MTT assay upon incubation of the live
cells with the compounds for 72 h.
RF (resistant factor) is defined as IC50 in A549CDDP/IC50 in A549. Mean values
based on three independent experiments, and the results of the representative
experiments are shown.
2.2.4. Cell viability
The effect of complexes 14 and 17 on cell proliferation after 72 h
treatment were checked in HepG-2 cancer cells using the MTT
assay. The biological results of the complexes 14 and 17 are shown
in Fig. 4 C. Our results indicated that the Pt(IV) complexes 14 and 17
e
Fig. 4. (A) IC50 values of target compounds in A549 and A549/CDDP cancer cells. (B) Intracellular accumulation of CDDP, 14 and 17 (10, 20 mM) in Bel-7404 cells after 24 h. Each value
is in nanograms of platinum per 106 cells. (C) In vitro cytotoxicity of 14, 17 and CDDP to HepG-2 cancer cells at the same concentration for 72 h. Results are expressed as the
mean SD for three independent experiments. P < 0.05.

440
X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
Table 3
controls. As shown in Fig. 7, in the control group, 5.86% of the HepG-
2 cells were in the G2/M phase. It was observed that 21.66% and
19.86% proportion of cells were in the G2/M phase, when treated
Cellular uptake of Pt(IV) complexes 14 and 17 in HepG-2 cells after 24 h of
incubation.
Pt content (ng/106 cells)
with 10
mM CDDP and compound 4 for 48 h, respectively. Notably,
after treatment with CDDP/4 mixture at 10
mM, the percentage of
Complexes
HepG-2
14 (10
14 (20
17 (10
17 (20
Cisplatin (10
Cisplatin (20
m
m
m
m
M)
M)
M)
M)
235 23
526 54
275 31
605 62
173 19
301 36
cells in the G2/M phase increased to 76.14%. Interestingly, it was
significant to observe that complexes 14 and 17 were also effective
in arresting the cell cycle at G2/M phase. After treatment with
10 mM complexes 14 and 17 for 48 h, the percentage of cells in the
m
m
M)
M)
G2/M phase increased to 79.77% and 85.13%, respectively. In short,
these results demonstrated that complexes 14 and 17 could induce
cell cycle arrest at the G2/M phase.
The experiments were performed three times, and the results of the
representative experiments are shown.
2.2.8. Molecular docking
more effectively inhibited cell proliferation in HepG-2 cells than
To gain better understanding on the potency of the synthesized
Pt(IV) complexes 14 and 17, we proceeded to examine the inter-
action of 14 and 17 with tubulin crystal structure (PDB code: 1SA0)
using Sybyl-X 2.0 on a Windows workstation. The binding modes of
compounds 14, 17 and 4 in the colchicine binding site of tubulin are
depicted in Fig. 8 and the docking scores obtained are shown in
Table 4. The Surflex docking scores are 8.70 for 14, 11.11 for 17 and
6.59 for 4, where higher scores indicate greater binding affinity. As
the biological activity studies suggested that complexes 14 and 17
were potent, hence, the docking detail of these two compounds
were examined for their interactions in ATP binding pocket of
tubulin.
Fig. 8 A shows that the interacting mode of compound 14 with
the chalcone moiety in the binding site is surrounded by THR327,
ALA326, THR181, GLY45 and ASN76. In particular, compound 14
forms five hydrogen bonds with the polar amino acids ASP150,
SER151, LEU175, GLU43 and VAL179, suggesting a probable strong
electrostatic interaction with the protein. In addition, the hydro-
phobic moiety of 14 is well embedded in a pocket interacting with
several hydrophobic residues making 14 bind tightly to tubulin. Not
surprisingly, the accommodation of complex 17 in the binding site
is similar to 14 (Fig. 8B). Also in this case, docking simulations
showed that the chalcone moiety of compound 17 like 14 can also
be accommodated in the same hydrophobic groove, adopting an
energetically stable conformation. Moreover, the chalcone moiety
of compound 17 forms eight hydrogen bonds with the polar amino
acids GLU43, VAL179, ASN269, ASP272 and THR327. In addition, the
crucial electrostatic interactions between the ammine from the
Pt(IV) unit and GLN172, THR327 and ASP272 residues from the
that of CDDP under the same condition (10 and 20
tively. (Fig. 4 C).
mM), respec-
2.2.5. Complexes 14 and 17 induced apoptosis in HepG-2 cells
It is well-known that most metal complex anticancer drugs
generally kill tumor cells by activating apoptosis [41,42]. Since
complexes 14 and 17 were also found to exhibit broad spectrum
antitumor activity against all tested human cancer cells and the
best activity against HepG-2 cancer cells in vitro, they were chosen
to be further investigated on the mechanism of action. The induc-
tion of apoptosis of HepG-2 cells by complexes 14 and 17 was
studied using FITC-Annexin V staining, in which Q1, Q2, Q3, and Q4
represent four different cell states: necrotic cells, late apoptotic or
necrotic cells, apoptotic cells and living cells, respectively. HepG-
2 cells were treated with 14, 17, 4, CDDP and CDDP/4 mixture under
the same concentration (10 mM) for 24 h, respectively. As shown in
Fig. 5, the percentage of cell apoptosis was 0.18% for the control, and
cell apoptosis was added up to 16.62% and 19.99%, respectively, at
10
mM of CDDP and 4, while cells treated with CDDP/4 mixture
(10
mM), the percentage of cell apoptosis was increased to 30.99%.
However, the percentage of cell apoptosis was added up to 41.21%
and 47.01%, respectively, when cells were treated with Pt(IV)
complexes 14 and 17 at 10
suggested that complexes 14 and 17 could effectively induce
apoptosis in HepG-2 cancer cells at the concentration of 10 M.
mM. Taken together, these results clearly
m
Interestingly, complex 17 was found to be more effectively inducing
apoptosis in human HepG-2 cancer cells than complex 14 under the
same condition, hinting that butyl carbon chain in 17 can improve
the antitumor activity better than propanyl carbon chain in 14. The
result is compatible to the data obtained from cytotoxicity assay
and cellular uptake tests.
neighboring
a-subunit were observed in the binding pocket.
Similar interactions with ASP272 or THR327 were observed in the
binding mode of compound 4 (Fig. 8C). It is interesting to note that
the crucial electrostatic interactions between the methoxy group of
the 3,4,5-trimethoxy-phenyl unit and residue VAL179 of the
2.2.6. AO/EB double staining
Apoptosis was further investigated by acridine orange (AO) and
ethidium bromide (EB) double staining. Viable cells stained by AO
and EB exhibited bright green fluorescence, while the apoptotic
cells stained by AO and EB displayed bright orange fluorescence.
HepG-2 cells were treated with CDDP/4, 14 and 17, respectively, at
neighboring
a-subunit are observed in the binding pocket,
demonstrating a plausible competitive mechanism of action at the
colchicine site.
2.2.9. Complexes 14 and 17 inhibited the polymerization of tubulin
in vitro
the same concentration (10 mM) for 24 h, and untreated HepG-
2 cells were used as a negative control and cells treated with CDDP
and 4 were used as positive controls. As shown in Fig. 6, complexes
14 and 17 were also confirmed to be able to induce apoptosis in
HepG-2 cells.
To further investigate whether the antitumor activities of these
Pt(IV) complexes were related to the interaction with the micro-
tubule system, the effect of complexes 14 and 17 on the polymer-
ization of purified tubulin were evaluated at a concentration of
10
ence. As illustrated in Fig. 9, paclitaxel (10
ulate tubulin polymerization, while CA-4 (10
(10 M) effectively inhibited tubulin polymerization as expected.
For complexes 14 and 17, an obvious inhibition of polymerization
was observed at the indicated concentration. More importantly,
complex 17 exhibited more significant inhibition tubulin
m
M. Compound 4, paclitaxel and CA-4 were served as a refer-
M) was found to stim-
M) or compound 4
2.2.7. Cell cycle analysis
m
To study the effect of the synthetic Pt(IV) complexes on the cell
growth and division, we used flow cytometry to evaluate the cell
cycle distribution of HepG-2 cells following a 48 h treatment with
m
m
CDDP/4, 14 and 17 at 10
mM. Untreated cells were used as a negative
control, and cells treated with CDDP and 4 were used as positive

X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
441
Fig. 5. Representative flow cytometry histograms of apoptotic HepG-2 cells after 24 h treatment with CDDP, 4, CDDP/4, 14 and 17 at the indicated concentrations. The cells were
harvested and labeled with annexin-V-FITC and PI, and analyzed by flow cytometry. The experiments were performed at least three times, and the results of the representative
experiments were shown.
polymerization compared to complex 14, which indicated the
rational relationship between the inhibition of tubulin and the
corresponding antitumor activity.
including the reduction of mitochondrial membrane potential
(MMP) and the release of cytochrome c (Cyt c) [45,46]. To further
investigate whether apoptosis induced by 14 and 17 was related to
mitochondrial dysfunction, flow cytometry analysis was used to
detect the MMP using JC-1 staining. As shown in Fig. 11, treatment
of HepG-2 cells with 4, CDDP/4 mixture, 14 and 17, respectively,
2.2.10. Complexes 14 and 17 triggered ROS generation
Reactive oxygen species (ROS) production are highly harmful
elements to cells as they initiate oxidative stress and ultimately
cause apoptosis in cancer cells [43,44]. In order to determine
whether Pt(IV) complexes 14 and 17 inducing apoptosis was ROS-
dependent, HepG-2 cells were treated with 4, CDDP/4 mixture, 14
caused a decrease the MMP level at the same concentration (10 mM)
for 24 h. These data clearly suggested that both 14 and could
effectively induced MMP collapse and eventually triggered
apoptotic cell death. Notably, complex 17 was found to be more
effectively inducing MMP collapse in human HepG-2 cancer cells
than complex 14 under the same condition. The results were
consistent well with the previous experimental data, indicating
that butyl carbon chain in 17 can enhance the antitumor activity of
the target compound better than propanyl carbon chain in 14.
and 17 at the same concentration (10 mM) for 24 h, the fluorescent
probe 2,7-dichlorofluorescein diacetate (DCF-DA) was used to
detect by flow cytometry. As shown in Fig. 10, the results suggested
that complexes 14 and 17 induced the production of significant
amounts of ROS in HepG-2 cells. After exposure to 10 mM of com-
plexes 14 and 17 for 24 h, the ROS level was 68.1% and 71.6%,
respectively, which were four times as great as those of the control
and CDDP. Taken together, these results proved that complexes 14
and 17 caused oxidative imbalance in HepG-2 cells.
2.2.12. Complexes 14 and 17 inhibited the migration of HUVEC cell
in vitro
Metastasis was an important event in later period of cancer
progression. Hence, the inhibition of metastasis was critical for
efficient cancer treatment. Moreover, migration was a major step
during metastasis. Therefore, in this study, we evaluated the inhi-
bition effect of complexes 14 and 17 on cell migration in vitro by
wound healing assay. As expected, complexes 14 and 17 strongly
2.2.11. Complexes 14 and 17 caused mitochondrial dysfunction
through decreasing the mitochondrial membrane potential (MMP)
Mitochondrial dysfunction plays an important role in the pro-
gression of apoptosis, which is demonstrated by several vital events

442
X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
Fig. 6. AO/EB double staining was used to detect the apoptosis after HepG-2 cells were exposed at the same concentration of CDDP, 4, CDDP/4, 14 and 17 for 24 h. Untreated cells
were used as a negative control and cells treated with CDDP and 4 were used as positive controls for 24 h, respectively. The experiments were performed at least three times, and
the results of the representative experiments were shown.
suppressed the migration of HUVEC cells compared with CDDP
corresponding Pt(II) complexes against two normal human cells. In
addition, all Pt(IV) complexes exhibited certain capability to over-
come CDDP drug resistance with low resistance indices when
treated with CDDP sensitive cells A549 and CDDP resistant cells
A549/CDDP. Two typical compounds, 14 and 17 with different alkyl
chain linkers, were chosen for further biological study. Complexes
14 and 17 displayed different antitumor activities and cellular up-
take, which can be attributed to the difference of their carbon chain
length. It was noted that these two complexes significantly caused
cell cycle arrest at G2/M phases and effectively induced cell
apoptosis when treated with HepG-2 cells, and they could effec-
tively inhibit cell migration against HUVEC cells in vitro. Moreover,
the results of tubulin polymerization assay and molecular docking
analysis indicated that complexes 14 and 17 could inhibit tubulin
polymerization and bind to the colchicine site of tubulin. Molecular
mechanism studies suggested that complexes 14 and 17 caused
apoptotic cell death of human cancer cells HepG-2 through the
mitochondrial mediated pathway by releasing mitochondrial Cyt c,
down-regulating Bcl-2, up-regulating Bax, which in turn proteo-
lytically activated downstream caspase-9 and caspase-3. Taken
together, our study indicated that Pt(IV) anticancer prodrugs con-
taining a tubulin inhibitor moiety can effectively enhance or inhibit
tubulin polymerization that may be a promising approach for
multiple targeted cancer therapy.
after 24 h under the same concentration (10
detail is shown in Fig. 12.
mM), respectively. The
2.2.13. Complexes 14 and 17induced apoptosis via the regulation of
apoptosis-related protein expression
Bcl-2 family members are essential components of mitochon-
drial stress-induced cellular apoptosis [47]. Previously, many re-
ports proposed that metal complexes induced mitochondria
mediated apoptosis in cancer cells through the regulation of Bcl-2
family proteins [41,48,49]. Therefore, the expression of apoptosis-
related proteins was also investigated for complexes 14 and 17.
Western blot assay demonstrated that these two complexes up-
regulated the expression of Bax (pro-apoptotic protein) and
correspondingly down-regulated the expression of Bcl-2 (anti-
apoptotic protein) at the indicated concentration (Fig. 13). The ratio
of Bcl-2/Bax was decreased, resulting in the release of Cyt c. Sub-
sequently, Cyt c caused activation of downstream caspase-9 and -3.
In short, these results proved that complexes14 and 17 could induce
mitochondria mediated apoptosis in HepG-2 cells through regu-
lating the expression of Bcl-2 family members. It was noted again
that the change of the expression levels of these proteins for
complex 17 was more remarkable than complex 14, suggesting that
the length of carbon chain played an important role in anticancer
activity.
4. Experimental section
3. Conclusions
All chemicals and solvents were analytical reagent grade and
commercially available, and used without further purification un-
less noted specifically. Column chromatography was performed
using silica gel (200e300 mesh). Mass spectra were measured on
an Agilent 6224 TOF LC/MS instrument. Elemental analyses of C, H,
and N used a Vario MICRO CHNOS elemental analyzer (Elementary).
Infrared spectra were obtained on a TENSOR27 PMA 50 FT-IR
In summary, six Pt(IV) prodrugs derived from CDDP, DACHPt and
OXP with two chalcone analogues as an inhibitor of tubulin were
designed and synthesized. In vitro evaluation revealed that all the
resulting Pt(IV) complexes not only displayed better anticancer
activities than their mother Pt(II) counterparts against three tested
human cancer cells, but also indicated less toxic than their

X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
443
Fig. 7. Effects of Pt(IV) complexes 14 and 17 on cell cycle phase arrest in HepG-2 cells. Cells were treated with 10 mM of 14 and 17 for 48 h. Untreated cells were used as a negative
control and cells treated with CDDP and 4 were used as positive controls for 48 h, respectively. Then the cells were fixed and stained with PI to analyze DNA content by flow
cytometry. The experiments were performed three times, and the results of the representative experiments are shown.
spectrometer and far infrared spectra were obtained on
a
for C19H19NO7: C, 61.12; H, 5.13; N, 3.75; found: C, 61.23; H, 5.24; N,
3.51. HR-MS (m/z) (ESI): calcd for C19H19NO7 [MþHþ]: 374.12398;
found: 374.11838.
NEXUS870 FT-IR spectrometer. 1H NMR, 13C NMR and 195Pt NMR
spectra were recorded in CDCl3 or DMSO‑d6 with a Bruker 300, 400
or 600 MHz NMR spectrometer.
Synthesis of compound 4. To a solution of compound 3 (1.86 g,
5.0 mmol) in ethanol (30 mL)/water (3 mL) was added iron powder
(1.4 g, 25.00 mmol) and NH4Cl (161 mg, 3.0 mmol). The reaction
was stirred at 85 ꢁC for 2 h and monitored by TLC. After completion
of reaction, the mixture was cooled to room temperature and
filtered through Celite. The filtered cake was washed with CH2Cl2
(50 mL), and the filtrate was concentrated under pressure. The
residue was dissolved in dichloromethane (50 mL), washed with
water (100 mL), dried over anhydrous Na2SO4 overnight, then the
filtered solution was concentrated under pressure. The crude
product was purified by chromatography on silica gel eluted with
CH2Cl2/MeOH (V:V ¼ 100:1) to give the desired compound as a
4.1. General procedure for the preparation of compounds 14e16
and 17e19
Synthesis of compound 3. An aqueous solution of 50% KOH
(20 mL) was added dropwise to a stirred solution of 30,40,50-tri-
methoxyacetophenone (1, 2.1 g, 10.0 mmol) and 4-methoxy-3-
nitrobenzaldehyde (2, 1.8 g, 10.0 mmol) in methanol (35 mL) at
0 ꢁC. The resulting mixture was stirred at the same temperature for
overnight and monitored by TLC. After completion of reaction, the
mixture was poured into water and adjusted to pH ¼ 2 with 2 N
HCl. The precipitated solid was filtered, washed with water and
dried to offer the crude product which was purified by chroma-
tography on silica gel eluted with petroleum ether/ethyl acetate
(V:V ¼ 4:1, 3:1, 2:1) to give the desired compound as a yellow solid
yellow solid (yield: 1.6 g, 93.6%). 1H NMR (400 MHz, CDCl3)
d 7.72
(d, J ¼ 15.5 Hz, 1H), 7.30 (d, J ¼ 15.9 Hz, 1H), 7.26 (s, 2H), 7.03 (d,
J ¼ 8.7 Hz, 2H), 6.80 (d, J ¼ 7.9 Hz, 1H), 3.94 (d, J ¼ 2.7 Hz, 9H), 3.89
(s, 3H). 13 C NMR (100 MHz, CDCl3)
d 189.42, 153.10, 149.72, 145.40,
142.21, 136.62, 133.96, 127.97, 121.02, 119.27, 113.29, 110.24, 106.00,
78.08, 77.41, 60.97, 56.39, 55.62. IR (KBr): 3444, 3358, 2939, 2834,
1650, 1575, 1510, 1461, 1411, 1345, 1265, 1153, 1124, 1025, 918,
788 cmꢀ1. Elemental analysis calcd (%) for C19H21NO5: C, 66.46; H,
6.16; N, 4.08; found: C, 66.35; H, 6.32; N, 3.91. HR-MS (m/z) (ESI):
calcd for C19H21NO5 [MþHþ]: 344.14980; found: 344.14445.
Synthesis of compounds 7 and 8. To a solution of compound 5 or
(yield: 2.65 g, 71.0%). 1H NMR (300 MHz, CDCl3)
d
8.17 (d, J ¼ 2.0 Hz,
1H), 7.80e7.73 (m, 2H), 7.43 (d, J ¼ 15.6 Hz, 1H), 7.28 (s, 2H), 7.15 (d,
J ¼ 8.8 Hz, 1H), 4.02 (s, 3H), 3.95 (d, J ¼ 4.6 Hz, 9H). 13C NMR
(100 MHz, CDCl3)
d 188.42, 154.12, 153.22, 142.80, 141.62, 139.96,
134.50, 133.13, 127.64, 124.71, 121.95, 113.88, 106.17, 60.99, 56.79,
56.47. IR (KBr): 3451, 3065, 2996, 2952, 1653, 1577, 1530, 1457, 1414,
1353, 1276, 1161, 1128, 1002, 771 cmꢀ1. Elemental analysis calcd (%)

444
X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
Fig. 8. Molecular modeling of 14, 17 and 4 in complex with tubulin (PDB code: 1SA0ISAO). Illustrated are the proposed binding mode and interaction between tubulin and selected
compounds, (A) compound 14, (B) compound 17, (C) compound 4. The compounds and important amino acids in the binding pockets are shown in stick model, whereas tubulin is
depicted in the ribbon model. The Mg2þ-ion is shown as a purple sphere.
Table 4
Docking scores (kcal/mol) for all studied compounds.
Compd.
Score
Crash
Polar
D-
Score
PMF-
Score
G-
Score
Chem-
Score
CScore
4
14
17
6.59
8.70
11.11
ꢀ3.94
ꢀ9.15
ꢀ8.46
3.63
6.54
7.27
ꢀ127.595
ꢀ282.292
ꢀ249.353
ꢀ21.857
ꢀ12.206
ꢀ18.992
ꢀ245.693
ꢀ436.320
ꢀ443.562
ꢀ32.025
ꢀ39.748
ꢀ44.484
3
3
3
6 (3.75 mmol) in dry CH2Cl2 (30 mL) was added EDCI (959 mg,
5.0 mmol), DMAP (610 mg, 5.0 mmol) and compound 4 (860 mg,
2.5 mmol). The resulting mixture was stirred at room temperature
for overnight and monitored by TLC. After completion of reaction,
the mixture was diluted with CH2Cl2 (100 mL) and washed with
water (100 mL). The organic phase was dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
crude product which was purified by chromatography on silica gel
eluted with petroleum ether/ethyl acetate (V:V ¼ 5:1, 4:1, 3:1) to
give compound 7 or 8.
55.96, 51.63, 36.65, 32.96, 20.63. IR (KBr): 3443, 3292, 2944, 1731,
1656, 1581, 1541, 1494, 1462, 1438, 1413, 1351, 1309, 1268, 1157, 1130,
1071, 1021, 1005, 799 cmꢀ1. Elemental analysis calcd (%) for
C
25H29NO8: C, 63.68; H, 6.20; N, 2.97; found: C, 66.56; H, 6.39; N,
2.84. HR-MS (m/z) (ESI): calcd for C25H29NO8 [MþHþ]: 472.19714;
found: 472.18732.
Compound 8. 1.05 g, 86.8% yield as a yellow solid. 1H NMR
(300 MHz, CDCl3)
d
8.81 (s, 1H), 7.77 (d, J ¼ 15.6 Hz, 2H), 7.40 (d,
J ¼ 15.6 Hz, 1H), 7.43e7.28 (m, 3H), 6.91 (d, J ¼ 8.5 Hz, 1H), 3.95 (s,
6H), 3.94 (d, J ¼ 1.7 Hz, 6H), 3.68 (s, 3H), 2.48e2.36 (m, 4H),
Compound 7. 950 mg, 80.5% yield as a yellow solid. 1H NMR
1.83e1.73 (m, 4H). 13C NMR (100 MHz, CDCl3)
d 189.45, 173.80,
(300 MHz, CDCl3)
d
8.81 (s, 1H), 7.81e7.74 (m, 2H), 7.39 (d,
170.99, 153.10, 149.66, 144.88, 142.31, 133.81, 128.18, 128.11, 125.82,
120.31, 118.37, 110.01, 106.11, 60.95, 56.44, 55.96, 51.56, 37.54, 33.72,
24.95, 24.42. IR (KBr): 3422, 3311, 2944, 1735, 1660, 1581, 1533,
J ¼ 15.6 Hz, 1H), 7.30 (d, J ¼ 8.5 Hz, 1H), 7.27 (s, 2H), 6.91 (d,
J ¼ 8.5 Hz, 1H), 3.95 (s, 6H), 3.94 (d, J ¼ 2.9 Hz, 6H), 3.69 (s, 3H),
2.53e2.45 (m, 4H), 2.13e2.03 (m, 2H). 13C NMR (100 MHz, CDCl3)
1493, 1441, 1415, 1344, 1263, 1159, 1125, 1071, 1025, 1001, 801 cmꢀ1
.
d
189.43, 173.54, 170.59, 153.10, 149.70, 144.86, 142.30, 133.80,
Elemental analysis calcd (%) for C26H31NO8: C, 64.32; H, 6.44; N,
128.15, 128.09, 125.81, 120.31, 118.43, 110.04, 106.10, 60.95, 56.43,
2.88; found: C, 66.45; H, 6.57; N, 2.65. HR-MS (m/z) (ESI): calcd for

X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
445
over anhydrous Na2SO4 and concentrated under reduced pressure.
The residue was purified on silica gel column eluted CH2Cl2/MeOH
(V:V ¼ 80:1, 60:1, 40:1) to give the desired product as a yellow solid.
Compound 14. 85 mg, 32.8% yield as a yellow solid.1H NMR
(400 MHz, DMSO-d6)
d 9.12 (s, 1H), 8.29 (s, 1H), 7.76e7.63 (m, 3H),
7.39 (s, 2H), 7.12 (d, J ¼ 8.6 Hz, 1H), 6.52e5.70 (m, 6H), 3.89 (s, 9H),
3.76 (s, 3H), 2.44e2.38 (m, 2H), 2.34e2.27 (m, 2H), 1.82e1.78 (m,
2H). 13C NMR (100 MHz, DMSO-d6)
d 188.39, 180.61, 172.03, 153.35,
152.72, 144.64, 142.30, 133.72, 128.04, 127.39, 126.51, 123.75, 120.32,
111.89, 106.57, 60.67, 56.71, 56.49, 56.44, 36.08, 35.92, 22.25. 195 Pt
NMR (129 MHz, DMSO-d6)
1582,1530, 1485, 1417, 1338, 1287, 1260, 1070, 1025, 982, 800, 575,
527, 453, 344 cmꢀ1
Elemental analysis calcd (%) for
d 544.37. IR (KBr): 3422, 2923, 1641,
.
C
24H32Cl3NO8Pt: C, 36.40; H, 4.07; N, 5.31; found: C, 36.53; H, 4.24;
N, 5.12. HR-MS (m/z) (ESI): calcd for C24H32Cl3NO8Pt [M-Hþ]:
790.95900; found: 790.08831.
Compound 15. 135 mg, 47.4% yield as a yellow solid.1H NMR
(400 MHz, DMSO-d6)
d 9.60e9.56 (m, 1H), 9.13 (s, 1H), 8.29 (s, 1H),
Fig. 9. Effects of complexes 14 and 17 on microtubule dynamics. Polymerization of
tubulin at 37 ꢁC in the presence of paclitaxel (10
M), CA-4 (10 M), 4 (10 M), 14
(10 M) and 17 (10 M) were monitored continuously by recording the absorbance at
340 nm over 60 min. The reaction was initiated by the addition of tubulin to a final
concentration of 3.0 mg/mL.
8.14 (s, 1H), 7.79 (s, 1H), 7.73e7.70 (m, 1H), 7.69e7.63 (m, 2H), 7.45
(d, J ¼ 10.2 Hz, 1H), 7.38 (s, 2H), 7.12 (d, J ¼ 8.7 Hz, 1H), 3.89 (s, 9H),
3.75 (s, 3H), 2.76e2.53 (m, 2H), 2.44 (t, J ¼ 7.2 Hz, 2H), 2.33 (t,
J ¼ 7.4 Hz, 2H), 2.19e2.04 (m, 2H), 1.85e1.78 (m, 2H), 1.55e1.48 (m,
m
m
m
m
m
3H), 1.31e1.06 (m, 3H). 13C NMR (100 MHz, DMSO-d6)
d 188.39,
183.23, 171.85, 153.35, 152.71, 144.62, 142.30, 133.72, 128.03, 127.40,
C26H31NO8 [MþHþ]: 486.21279; found: 486.20171.
126.51, 123.77, 120.33, 111.90, 106.57, 63.86, 62.73, 60.67, 56.70,
195
Synthesis of compounds 9 and 10. To a solution of compound 7
(900 mg, 1.91 mmol) or 8 (925 mg, 1.91 mmol) in THF/H2O (30/
5 mL) at 0 ꢁC was added lithium hydroxide (241 mg, 5.73 mmol),
the resulting mixture was stirred at the same temperature for 2 h.
After completion of reaction, the mixture was adjusted to pH ¼ 2
with 2 N HCl solution, then 100 mL of CH2Cl2was added and
washed with water (100 mL). The organic phase was dried over
anhydrous Na2SO4 and concentrated under reduced pressure to
obtain the target product.
56.48, 37.18, 35.80, 31.38, 31.33, 24.07, 24.03, 22.21.
(129 MHz, DMSO-d6)
Pt NMR
d
412.86. IR (KBr): 3447, 3209, 2939, 1642,
1624,1581,1535,1495,1454,1413,1341,1308,1266,1230,1158,1127,
1024, 808, 591, 537, 434, 347, 330 cmꢀ1. Elemental analysis calcd (%)
for C30H40Cl3N3O8Pt: C, 41.32; H, 4.62; N, 4.82; found: C, 41.21; H,
4.84; N, 4.63. HR-MS (m/z) (ESI): calcd for C30H40Cl3N3O8Pt [M-Hþ]:
869.14505; found: 870.16873.
Compound 16. 165 mg, 56.7% yield as a yellow solid.1H NMR
(400 MHz, DMSO-d6)
d 9.19 (s, 1H), 8.54e8.26 (m, 4H), 7.74e7.63
Compound 9. 795 mg, 91.2% yield as a yellow solid. 1H NMR
(m, 4H), 7.39 (s, 2H), 7.12 (d, J ¼ 8.6 Hz, 1H), 3.89 (d, J ¼ 3.4 Hz, 9H),
3.76 (s, 3H), 2.59e2.50 (m, 2H), 2.40e2.34 (m, 4H), 2.11e2.01 (m,
2H), 1.80e1.74 (m, 2H), 1.55e1.37 (m, 4H), 1.25e1.10 (m, 2H). 13C
(400 MHz, DMSO-d6)
d 11.90 (s, 1H), 9.27 (s, 1H), 8.26 (s, 1H),
7.76e7.65 (m, 3H), 7.40 (s, 2H), 7.14 (d, J ¼ 8.6 Hz, 1H), 3.90 (d,
J ¼ 2.9 Hz, 9H), 3.77 (s, 3H), 2.43 (t, J ¼ 7.3 Hz, 2H), 2.30 (t, J ¼ 7.3 Hz,
NMR (100 MHz, DMSO-d6)
d 188.39, 180.79, 171.78, 163.82, 153.36,
2H), 1.85e1.79 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
d 188.35,
152.86, 144.62, 142.30, 133.72, 127.98, 127.39, 126.61, 124.02, 120.32,
174.68, 171.63, 153.36, 152.92, 144.57, 142.31, 133.73, 128.00, 127.42,
126.63, 124.18, 120.34, 111.91, 106.58, 60.65, 56.67, 56.42, 35.55,
33.45, 21.05. IR (KBr): 3452, 3307, 2944, 1741, 1659, 1566, 1532,
111.94, 106.57, 62.03, 61.85, 60.67, 56.71, 56.45, 36.36, 35.66, 31.34,
195
31.05, 24.08, 24.00, 21.93.
Pt NMR (129 MHz, DMSO-d6)
d
1000.02. IR (KBr): 3450, 3416, 3201, 2945, 1722, 1655, 1584, 1536,
1500, 1456, 1416, 1350, 1257, 1221, 1154, 1125, 1025, 807, 571, 525,
456, 352, 321 cmꢀ1
Elemental analysis calcd (%) for
1494, 1445, 1356, 1315, 1272, 1161, 1127, 1027, 1006, 767 cmꢀ1
.
Elemental analysis calcd (%) for C24H27NO8: C, 63.01; H, 5.95; N,
.
3.06; found: C, 63.17; H, 6.09; N, 2.85. HR-MS (m/z) (ESI): calcd for
C32H40ClN3O12Pt: C, 43.22; H, 4.53; N, 4.73; found: C, 43.35; H, 4.67;
N, 4.51. HR-MS (m/z) (ESI): calcd for C32H40ClN3O12Pt [M-Hþ]:
887.18700; found: 888.22014.
C
24H27NO8 [M-Hþ]: 456.16584; found: 456.16883.
Compound 10. 850 mg, 94.4% yield as a yellow solid. 1H NMR
(300 MHz, DMSO-d6)
d 11.99 (s, 1H), 9.19 (s, 1H), 8.27 (s, 1H),
General synthesis of compounds 17e19. To a solution of com-
pound 10 (150 mg, 0.318 mmol), TBTU (154 mg, 0.478 mmol), and
Et3N (48 mg, 0.478 mmol) in dry DMF (3 mL), compound 11, 12, or
13 (0.318 mmol) was added in portions and the mixture was stirred
at room temperature overnight. After completion of reaction, the
whole mixture was added to CH2Cl2 (150 mL), and then extracted
twice with water (100 mL). The organic phase was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The
residue was purified on silica gel column eluted CH2Cl2/MeOH
(V:V ¼ 80:1, 60:1, 40:1) to give the desired product as a yellow solid.
Compound 17. 65 mg, 25.4% yield as a yellow solid. 1H NMR
7.74e7.62 (m, 3H), 7.39 (s, 2H), 7.13 (d, J ¼ 8.5 Hz, 1H), 3.90 (s, 9H),
3.77 (s, 3H), 2.39 (d, J ¼ 6.1 Hz, 2H), 2.25 (t, J ¼ 6.1 Hz, 2H), 1.67e1.49
(m, 4H). 13C NMR (100 MHz, DMSO-d6)
d 188.35, 174.89, 171.94,
153.36, 152.82, 144.59, 142.31, 133.74, 128.07, 127.42, 126.51, 124.01,
120.33, 111.88, 106.57, 60.65, 56.67, 56.42, 36.13, 33.92, 25.19, 24.60.
IR (KBr): 3421, 2939, 1725, 1656, 1583, 1534, 1502, 1461, 1341, 1310,
1263, 1158, 1126, 1024, 1000, 772 cmꢀ1. Elemental analysis calcd (%)
for C25H29NO8: C, 63.68; H, 6.20; N, 2.97; found: C, 63.81; H, 6.35; N,
2.74. HR-MS (m/z) (ESI): calcd for C25H29NO8 [M-Hþ]: 470.118149;
found: 470.22474.
General synthesis of compounds 14e16. To a solution of com-
pound 9 (150 mg, 0.328 mmol), TBTU (158 mg, 0.492 mmol), and
Et3N (50 mg, 0.492 mmol) in dry DMF (3 mL), compound 11, 12, or
13 (0.328 mmol) was added in portions and the mixture was stirred
at room temperature for overnight. After completion of reaction,
the whole mixture was added to CH2Cl2 (150 mL), and then
extracted twice with water (100 mL). The organic phase was dried
(400 MHz, DMSO-d6)
d 9.18 (s, 1H), 8.29 (s, 1H), 7.73e7.63 (m, 2H),
7.65 (d, J ¼ 15.5 Hz, 1H), 7.38 (s, 2H), 7.12 (d, J ¼ 8.7 Hz, 1H),
6.45e5.76 (m, 6H), 3.89 (d, J ¼ 4.3 Hz, 9H), 3.76 (s, 3H), 2.40 (t,
J ¼ 6.8 Hz, 2H), 2.28 (t, J ¼ 7.1 Hz, 2H), 1.65e1.51 (m, 4H). 13C NMR
(100 MHz, DMSO-d6) d 188.39, 181.03, 174.90, 172.11, 153.36, 152.73,
144.65, 142.30, 133.73, 128.10, 127.38, 126.40, 123.86, 120.32, 111.91,
106.57, 60.67, 56.71, 56.49, 36.57, 36.25, 25.54, 25.29.
195
Pt NMR

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X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
Fig. 10. Assessment of the ROS production in HepG-2 cells after 24 h incubations with 14 and 17, cells were stained with DCF-DA and analyzed by flow cytometry. The experiments
were performed three times, and the results of the representative experiments were shown.
(129 MHz, DMSO-d6)
1583, 1532, 1486, 1413, 1340, 1261, 1157, 1124, 988, 935, 808, 573,
529, 454, 344 cmꢀ1
Elemental analysis calcd (%) for
d
544.67. IR (KBr): 3422, 3228, 2926, 1649,
Compound 19. 135 mg, 47.2% yield as a yellow solid. 1H NMR
(400 MHz, DMSO-d6) 9.20 (s, 1H), 8.55e8.50 (m, 1H), 8.32 (d,
J ¼ 30.3 Hz, 3H), 7.73e7.63 (m, 4H), 7.38 (s, 2H), 7.12 (d, J ¼ 8.7 Hz,
1H), 3.89 (s, 9H), 3.76 (s, 3H), 2.61e2.50 (m, 2H), 2.37 (d, J ¼ 6.6 Hz,
2H), 2.31 (t, J ¼ 6.4 Hz, 2H), 2.12e2.01 (m, 2H), 1.55e1.47 (m, 4H),
1.45e1.35 (m, 4H), 1.21e1.01 (m, 2H). 13C NMR (100 MHz, DMSO-d6)
d
.
C
25H34Cl3N3O8Pt: C, 37.26; H, 4.25; N, 5.21; found: C, 37.38; H, 4.38;
N, 5.01. HR-MS (m/z) (ESI): calcd for C25H34Cl3N3O8Pt [MþClꢀ]:
840.06971; found: 840.09689.
Compound 18. 145 mg, 51.6% yield as a yellow solid. 1H NMR
d 188.40, 181.43, 172.02, 163.69, 153.36, 152.76, 144.65, 142.30,
(400 MHz, DMSO-d6)
d
9.62 (t, J ¼ 9.6 Hz, 1H), 9.18 (s, 1H), 8.29 (s,
133.72, 128.05, 127.37, 126.45, 123.92, 120.30, 111.90, 106.56, 62.04,
1H), 8.13 (s, 1H), 7.78 (s, 1H), 7.73e7.71 (m, 1H), 7.69e7.63 (m, 2H),
7.44 (d, J ¼ 10.3 Hz, 1H), 7.38 (s, 2H), 7.12 (d, J ¼ 8.7 Hz, 1H), 3.89 (s,
9H), 3.76 (s, 3H), 2.75e2.69 (m, 2H), 2.40 (d, J ¼ 6.7 Hz, 2H), 2.29 (t,
J ¼ 6.6 Hz, 2H), 2.19e2.03 (m, 2H), 1.60e1.58 (m, 4H), 1.55e1.46 (m,
61.94, 60.67, 56.69, 56.45, 36.81, 36.09, 31.39, 31.06, 25.45, 25.12,
195
24.07, 23.98.
Pt NMR (129 MHz, DMSO-d6) d 996.61. IR (KBr):
3423, 3179, 2937, 1728, 1657, 1582, 1534, 1502, 1487, 1453, 1414,
1342, 1262, 1157, 1126, 1023, 806, 574, 455, 358, 321 cmꢀ1
.
3H), 1.29e1.04 (m, 3H). 13C NMR (100 MHz, DMSO-d6)
d
188.39,
Elemental analysis calcd (%) for C33H42ClN3O12Pt: C, 43.88; H, 4.69;
N, 4.65; found: C, 43.76; H, 4.89; N, 4.46. HR-MS (m/z) (ESI): calcd
for C33H42ClN3O12Pt [M-Hþ]: 902.23400; found: 902.23301.
183.49, 172.06, 153.36, 152.69, 144.64, 142.30, 133.73, 128.07, 127.38,
126.39, 123.84, 120.32, 111.90, 106.57, 63.91, 62.67, 60.67, 56.70,
56.47, 37.58, 36.17, 31.41, 31.30, 25.56, 25.17, 24.08, 24.01. 195 Pt NMR
(129 MHz, DMSO-d6)
1583, 1532, 1502, 1486, 1453, 1413, 1340, 1262, 1158, 1126, 1022, 810,
574, 491, 438, 345 cmꢀ1
Elemental analysis calcd (%) for
C31H42Cl3N3O8Pt: C, 42.02; H, 4.78; N, 4.74; found: C, 42.14; H, 4.90;
N, 4.51. R-MS (m/z) (ESI): calcd for C31H42Cl3N3O8Pt [M-Hþ]:
883.16070; found: 884.18738.
d 414.16. IR (KBr): 3423, 3197, 2932, 1650,
4.2. Cell culture and maintenance
.
All human cancer cell lines and two human normal cell lines in
this study were purchased from China Life Science Collage
(Shanghai, PRC). Culture medium Dulbecco's modified Eagle me-
dium (DMEM), fetal bovine serum (FBS), phosphate buffered saline

X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
447
Fig. 11. Complexes 14 and 17 decreased the MMP of HepG-2 cells. The HepG-2 cells were treated with 14 and 17 at the indicated concentration for 24 h followed by incubation with
the fluorescence probe JC-1 for 30 min, and then the cells were detected by flow cytometry. The experiments were performed three times, and the results of the representative
experiments were shown.
Fig. 12. Complexes 14 and 17 inhibited the migration of HUVEC cells in vitro. At the same concentration of CDDP, complexes 14 and 17 suppressed HUVEC cells migration. The
experiments were performed three times, and the results of the representative experiments were shown.
(PBS, pH ¼ 7.2), and Antibiotice-Antimycotic came from KeyGen
supplemented with 10% FBS, 100 units/ml of penicillin and 100 g/
Biotech Company (China). Cell lines were grown in the
mL of streptomycin in a humidified atmosphere of 5% CO2 at 37 ꢁC.

448
X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
Fig. 13. Western blot analysis of Cyt c, Bax, Bcl-2, caspase-9 and caspase-3 after treatment of HepG-2 cells with 14 and 17 at the indicated concentration for 48 h. b-Actin antibody
was used as reference control. The experiments were performed three times, and the results of the representative experiments were shown.
4.3. Cytotoxicity assay
were removed from the crystal structure. Subsequently, the protein
was prepared by using the Biopolymer module implemented in
Sybyl. The polar hydrogen atoms were added. The automated
docking manner was applied in the present work. Other parame-
ters were established by default to estimate the binding affinity
characterized by the Surflex-Dock scores in the software. Surflex-
Dock total scores, which were expressed in -log10 (Kd) units to
represent binding affinities, were applied to estimate the ligand-
receptor interactions of newly designed molecules. A higher score
represents stronger binding affinity. The optimal binding pose of
the docked compounds was selected based on the Surflex scores
and visual inspection of the docked complexes.
The anticancer activity of the title compounds were dissolved in
DMF and evaluated in three human cancer cells (HepG-2, SK-OV-3
and NCI-H460) and two human normal cells (HL-7702 and BEAS-
2B) and CDDP-resistance cells (A549/CDDP), respectively. About
1.0 ꢂ 105 cells/mL cells, which were in the logarithmic phase, were
grown in each well of 96-well plates and incubated for 12 h at 37 ꢁC
in 5% CO2. Complexes at five different concentrations (2.5, 5, 10, 20
and 50 mM) were also added to the test well and then the cells were
incubated at 37 ꢁC in a 5% CO2 atmosphere for 72 h. An enzyme
labeling instrument was used to read absorbance with 570/630 nm
double wavelength measurement. Cytotoxicity was detected on the
percentage of cell survival compared with the negative control. The
final IC50 values were calculated by the Bliss method (n ¼ 5). All of
the tests were repeated in triplicate.
4.6. Flow cytometry analysis of cell cycle distribution
HepG-2 cells were grown on 6-well plates and treated with 4,
CDDP, 14, 17 and CDDP/4 at the same concentrations (10 mM) and
maintained with of the proper culture medium in 5% CO2 at 37 ꢁC
for 48 h. After completion of incubation, cells were harvested and
washed three times with ice-cold PBS, fixed with ice-cold 70%
4.4. Cell uptake
HepG-2 cells were grown in each well of 96-well plates. After
the cells reached about 80% confluence, 10 and 20 mM of CDDP, 14
ethanol at ꢀ20 ꢁC for overnight. The cells were treated with 100
mg/
mL RNase A for 30 min at 37 ꢁC after washed with twice ice-cold
PBS, and finally stained with 1 mg/mL propidium iodide (PI) in
the dark at 4 ꢁC for 30 min. Analysis was performed with the system
software (Cell Quest; BD Biosciences).
and 17 were added and the plates were also incubated for 24 h at
37 ꢁC in 5% CO2, respectively. After completion of 24 h incubation,
cells were collected and washed three times with ice-cold PBS, then
centrifuged at 1000 ꢂ g for 10 min and resuspended in 2 mL PBS. A
volume of 100 mL was taken out to examine the cell density. The
remaining cells were digested by HNO3 (200 m
L, 65%) at 65 ꢁC for
4.7. Apoptosis analysis
12 h. The Pt level in cells were detected by ICP-MS.
Apoptosis was also evaluated by flow cytometric analysis of
annexin V/PI staining. HepG-2 cells were grown in each well of six-
well plates at the density of 5.0 ꢂ 104 cells/mL of the DMEM me-
dium with 10% FBS to the final volume of 2 mL. The plates were
incubated for overnight and treated with 4, CDDP, 14, 17 and CDDP/
4.5. Molecular docking
All the docking studies were carried out using Sybyl-X 2.0 on a
windows workstation. The crystal structure of the tubulin in
complex with colchicine was retrieved from the RCSB Protein Data
Bank (PDB:1SA0.pdb) [50]. The synthetic chalcone analogues, Pt(IV)
complexes (14 and 17) including the parent compound 4, were
selected for the docking studies. The 3D structures of these selected
compounds were first built using Sybyl-X 2.0 sketch followed by
energy minimization using the MMFF94 force field and Gasteiger-
Marsili charges. We employed Powell's method for optimizing the
geometry with a distance dependent dielectric constant and a
termination energy gradient of 0.005 kcal/mol. All the selected
compounds were automatically docked into the colchicine binding
pocket of tubulin by an empirical scoring function and a patented
search engine in the Surflex docking program. Before the docking
process, the natural ligand was extracted; the water molecules
4 at the same concentration (10
mM) for 24 h. Briefly, cells were
harvested and washed with twice ice-cold PBS, and then suspended
cells in the annexin-binding buffer at
a
concentration of
L of annexin V-
5 ꢂ 105 cells/ml. cells were then incubated with 5
m
FITC and 5 m
L of PIin the dark at 4 ꢁC for 30 min. The cells were
detected by system software (Cell Quest; BD Biosciences).
4.8. AO/EB double staining
HepG-2 cells were grown in each well of six-well plates at the
density of 5.0 ꢂ 104 cells/mL of the DMEM medium with 10% FBS to
the final volume of 2 mL. The plates were incubated for overnight
and treated with 4, CDDP, 14, 17 and CDDP/4 at the same

X. Huang et al. / European Journal of Medicinal Chemistry 146 (2018) 435e450
449
concentration (10
cover slip with monolayer cells were inverted on a glass slide with
20 L of AO/EB stain (100 mg/mL). Fluorescence was read on a
m
M) for 24 h. After the treatment period, the
4.13. Tubulin polymerization assay in vitro
m
Tubulin polymerization assay was monitored by the change in
optical density at 340 nm using modification of methods
Nikon ECLIPSETE2000-S fluorescence microscope (OLYMPUS Co.,
a
Japan).
described by Jordan et al. [28] Purified brain tubulin polymerization
kit was purchased from Cytoskeleton (BK006P, Denver, CO). The
final buffer concentrations for tubulin polymerization contained
80.0 mM piperazine-N,N0-bis(2-ethanesulfonic acid) sequisodium
4.9. Mitochondrial membrane potential (MMP) assay
salt (pH 6.9), 2.0 mM MgCl2, 0.5 mM ethylene glycol bis(b-amino-
The MMP was measured with the lipophilic cation probe JC-1
(Beyotime, Haimen, China, Molecular Probe), as previously
described [51]. HepG-2 cells were seeded into six-well plates and
ethyl ether)-N,N,N0,N'-tetraacetic acid (EGTA), 1 mM GTP, and 10.2%
glycerol. Test compounds were added at the indicated concentra-
tion (10 mM), and then all components except the purified tubulin
treated with the same concentration (10 mM) of the test compounds
were warmed to 37 ꢁC. The reaction was initiated by the addition of
tubulin to a final concentration of 3.0 mg/mL. Paclitaxel, CA-4 and
compound 4 were used as positive controls under similar experi-
mental conditions. The optical density was measured for 1 h at
1 min intervals in BioTek's Synergy 4 multifunction microplate
spectrophotometer with a temperature controlled cuvette holder.
Assays were performed according to the manufacturer's in-
structions and under conditions similar to those employed for the
tubulin polymerization assays described above [52,53].
for 24 h. After incubation for 24 h, the JC-1 fluorescent probe was
added 20 min after replacing with fresh medium. Cells were
collected at 2500 rpm and washed twice with ice-cold PBS and then
the MMP were examined by flow cytometer, respectively. The
emission fluorescence for JC-1 was detected by 530 and 590 nm,
under the excitation wavelength at 488 nm, respectively.
4.10. ROS assay
The production of ROS was examined by flow cytometry using
DCFH-DA (Molecular Probe, Beyotime, Haimen, China), as previ-
ously described [45,51]. HepG-2 cells were grown into six-well
4.14. Statistical analysis
All statistical analysis was performed with SPSS Version 10. Data
was analyzed by one-way ANOVA. Mean separations were per-
formed using the least significant difference method. Each experi-
ment was replicated thrice, and all experiments yielded similar
results. Measurements from all the replicates were combined, and
treatment effects were analyzed.
plates and treated with the same concentration (10 mM) of the
test compounds for 24 h. On the following treatment, cells were
harvested at 2000 rpm and washed twice with ice-cold PBS, and
then resuspend cells in 10 mM DCFH-DA dissolved in cell free
medium at 37 ꢁC for 30 min in dark, and then washed twice with
PBS. Cellular fluorescence was detected by flow cytometry at an
excitation of 485 nm and an emission of 538 nm, respectively.
Acknowledgments
4.11. Migration assay
We are grateful to the National Natural Science Foundation of
China (Grant No. 21571033) and the New Drug Creation Project of
the National Science and Technology Major Foundation of China
(Grant No. 2015ZX09101032) for financial aids to this work. The
authors would also like to thank the Fundamental Research Funds
for the Central Universities (Project 2242016K30020) for supplying
basic facilities to our key laboratory. We also want to express our
gratitude to the Priority Academic Program Development of Jiangsu
Higher Education Institutions for the construction of fundamental
facilities (Project 1107047002). Huang is grateful to the Innovation
Program of Jiangsu Province postgraduate education (Project
KYLX16_0263). The research was also supported by the Scientific
Research Foundation of Graduated School of Southeast University
(YBJJ1677).
The migration effects of complexes 14 and 17 on HUVEC cells
were detected by wound-heal assay. HUVEC cells were grown into
six-well plates and treated with at the same concentration (10 mM)
of the test complexes 14 and 17for 24 h, respectively. The extent of
wound heal was observed after 24 h by imaging with fluorescence
microscope.
4.12. Western blot analysis
Western blot analysis was performed as described previously
[45,51]. HepG-2 cells were treated with the same concentration
(10 mM) of the test complexes 14 and 17 for 48 h, respectively. After
incubation for 48 h, cells were collected, centrifuged, and washed
twice with ice-cold PBS. The pellet was then resuspended in lysis
buffer. After the cells were lysed on ice for 30 min, lysates were
centrifuged at 20000 g at 4 ꢁC for 5 min. The protein concentration
in the supernatant was examined using the BCA protein assay re-
agents (Imgenex, USA). Equal amounts of protein per line were was
separated on 12% SDS polyacrylamide gel electrophoresis and
transferred to PVDF Hybond-P membrane (GE Healthcare). Mem-
branes were incubated with 5% skim milk in Tris-buffered saline
with Tween 20 (TBST) buffer for 1 h and then the membranes being
gently rotated overnight at 4 ꢁC. Membranes were then incubated
with primary antibodies against Bcl-2, Bax, Cyt c, caspase-9, cas-
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.01.075.
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