In vitro and in vivo anti-tumor activity of two gold(III) complexes with isoquinoline derivatives as ligands

Article abstract of DOI:10.1016/j.ejmech.2018.11.047

Two gold(III) complexes of isoquinoline derivatives: [Au(L¹)Cl₂] (Au1) and [Au(L²)Cl₂] (Au2) have been prepared and characterized. Au1 and Au2 exhibited greater cytotoxicity than their corresponding ligands and

Full text of DOI:10.1016/j.ejmech.2018.11.047

European Journal of Medicinal Chemistry 163 (2019) 333e343
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
In vitro and in vivo anti-tumor activity of two gold(III) complexes with
isoquinoline derivatives as ligands
Taj-Malook Khan a, Noor Shad Gul a, Xing Lu a, Jian-Hua Wei a, Yan-Cheng Liu a,
,
,
, **
Hongbin Sun a *, Hong Liang a ***, Chris Orvig b, Zhen-Feng Chen a
a State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, 15
Yucai Road, Guilin 541004, PR China
b Department of Chemistry, Faculty of Science, The University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T1Z1, Canada
a r t i c l e i n f o
a b s t r a c t
Two gold(III) complexes of isoquinoline derivatives: [Au(L1)Cl2] (Au1) and [Au(L2)Cl2] (Au2) have been
prepared and characterized. Au1 and Au2 exhibited greater cytotoxicity than their corresponding ligands
and cisplatin against T-24 cells. Both complexes arrested cell cycle at S-phase by upregulation of p53, p27,
and p21, and downregulation of cyclin A and cyclin E. The depolarization of the mitochondrial membrane
potential, generation of ROS, and stimulated Ca2þ release activated the caspase cascade and ultimately
caused apoptosis by increasing the levels of Bax and Bak, and decreasing the levels of Bcl-2 and Bcl-xl.
Cell apoptosis was achieved via mitochondria mediated pathways. The in vivo studies of Au1 and Au2
demonstrated that they were safer than cisplatin and could effectively inhibit tumor growth.
© 2018 Elsevier Masson SAS. All rights reserved.
Article history:
Received 23 August 2018
Received in revised form
17 November 2018
Accepted 20 November 2018
Available online 23 November 2018
Keywords:
Isoquinoline
Gold(III) complexes
Cell apoptosis
Mitochondrial dysfunction
Antitumor activity
1. Introduction
physicians, the anticancer properties of gold complexes have been
reported only very recently [10,11].
Metal complexes have been used for the treatment of many
human diseases. Among the metal complexes, cisplatin, carboplatin
and oxaliplatin are the most prominent anticancer agents in the
treatment of solid tumor [1e3]. However, drug resistance and
adverse effects limit the use of platinum containing drugs [4,5].
Therefore, over the past few decades a great deal of efforts has been
invested in the search of new metal-based anticancer drugs that
might show innovative action mechanism and, accordingly offer
better pharmacological activity over the clinically well-established
platinum-based drugs (cisplatin, oxaliplatin and carboplatin)
[6e9]. In line with this endeavor, numerous platinum and non-
platinum complexes were synthesized, characterized and evalu-
ated as potential anticancer drugs. Although the phenomenon of
using gold in medicine dates back to ancient Arabic and Chinese
Recently gold complexes have been used for the treatment of
rheumatoid arthritis, cancer, tuberculosis, HIV, and other infectious
diseases [12e15]. Many gold complexes have been found to inhibit
the proliferation of tumor cells and were shown to possess strong
cytotoxicity and promising therapeutic properties that circumvent
cisplatin resistance [16e19]. The induction of auranofin, which is
one of the leading gold complex, and its analogues as anti-tumor
agents in clinical practice highlight the potential utility of gold-
based agents [20]. The mode of action of gold complexes remains
unclear but mechanistic studies have suggested that DNA is not the
primary target as with cisplatin, rather they induce cytotoxicity by
disrupting the mitochondrial membrane potential, interfering with
DNA-protein interactions, inhibiting protein synthesis as a result of
ROS generation, releasing of free Ca2þ, and activating the caspase
cascade, ultimately resulting in cell death via apoptosis [21,22].
To enhance cytotoxicity and to overcome drug resistance, metal
ions are often coordinated with biologically active ligands that can
be used as a novel strategy [23]. In this regard, isoquinoline, an
alkaloid that is abundant in nature and possess potential anticancer
activity [24]. Transition metal complexes of isoquinoline (lir-
iodenine) were more potent than the free ligand and demonstrated
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: hongbinsun@cpu.edu.cn (H. Sun), hliang@gxnu.edu.cn
(H. Liang), chenzf@gxnu.edu.cn (Z.-F. Chen).
https://doi.org/10.1016/j.ejmech.2018.11.047
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

334
T.-M. Khan et al. / European Journal of Medicinal Chemistry 163 (2019) 333e343
promising anticancer activity. Notably, liriodenine is the active
ingredient of certain traditional Chinese medicines (TCM) [25].
Also, papaverine significantly inhibited the replication of HIV and
reduced cell growth in H9 cells [26]. Chelidonine has already been
recognized as an anti-proliferative agent, a strong telomerase in-
hibitor, and an inducer of apoptosis [27]. Another derivative of
isoquinoline 3,4-Dihydroisoquinoline (thalpetaline) has been iso-
lated from the Chinese medicinal herb (T. petaloideum), showing
effectiveness in the treatment of cancer, skin infections, and hep-
atitis [28,29]. Recently, we reported two organometallic gold(III)
complexes similar to tetrahydroisoquinoline (THIQ) that induce ER-
stress-mediated apoptosis and pro-death autophagy in A549 can-
cer cells [30].
2.1.1. Crystal structure
Crystal structures and refinement details are shown in Table 1.
Selected bond length and angles are listed in Tables S1 and S2. Au1
and Au2 have distorted square planner geometry (Fig. 1). The
central Au(III) ions are coordinated with bidentate ligand as N^N-L1
and N^N-L2 by the heterocyclic nitrogen of isoquinoline and two
chlorine atoms (Fig. 1). In the reaction of complexes formation, the
-NH2 of the ligand has taken place deprotonation and lost one H. In
Au1, the bond lengths of AueN1 and AueN2 are 2.008 Å and
2.034 Å, whereas the bond lengths of AueCl1 and AueCl2 are
2.3218, 2.2659 Å. In Au2, the bond lengths of AueN1, AueN2 are
2.002 Å and 2.053 Å and the bond lengths of AueCl1, AueCl2 are
2.3243 and 2.281 Å.
Herein, we report the synthesis of [6,7-dimethoxy-1-(2-
aminophenyl)-3,4-dihydroisoquinolines] (H-L1), [6-methoxy-1-(2-
aminophenyle)-3-4-dihydroisoquinolines (H-L2), and their gold(-
III) complexes: [Au(L1)Cl2] (Au1) and [Au(L2)Cl2] (Au2). We
explored their in vitro and in vivo antitumor activity and investi-
gated their action mechanisms.
2.2. Studies of stability in TBS of Au1 and Au2
The stability of Au1 and Au2 is very important for their bio-
logical function. TBS (10 mM tris-HCl, pH 7.40) was used for sta-
bility studies. Au1 and Au2 were dissolved in DMSO and diluted
with TBS to a final concentration of 3.0 ꢁ 10ꢀ5 M. The absorption
spectrum was recorded after 0, 4, 12, and 24 h at room temperature.
The spectra and absorption peaks clearly indicated that the com-
plexes are stable in TBS as shown in Figs. S13 and S14. Furthermore,
the HPLC analysis of Au1, Au2, and the corresponding ligand
demonstrated that all of these compounds have no significant
degradation in the stock solution in DMSO (Figures. S15ꢀS18).
2. Results and discussion
2.1. Chemistry
For the synthesis of isoquinoline, various methods have been
followed using benzoic acid and 3,4-dimethoxyphenethyleamine
as the starting materials. Dihydroisoquinoline [6,7-dimethoxy-1-
2.3. Uptake and distribution of gold in T-24 cells
(2-aminophenyle)-3,4-dihydroisoquinolines
(H-L1)
and
6-
methoxy-1-(-2-aminophenyle)-3,4 dihydroisoquinoline (H-L2)
were obtained by the Bischler-Napieralski reaction (Scheme 1) [31].
Reaction of KAuCl4 and isoquinoline ligands H-L1 and H-L2 in
presence of methanol or ethanol and dichloromethane under
refluxing conditions yielded crystals of their gold(III) complexes.
Using ESI-MS, 1H NMR and 13C NMR spectroscopy, the gold(III)
complexes and their corresponding ligands were characterized
(Supporting Information, Figures S1ꢀS12) with the formula:
[Au(L1)Cl2] (Au1) and [Au(L2)Cl2] (Au2).
Metal distribution in different organelles have a direct rela-
tionship with different cellular pathways [32,33]. In the present
work, the subcellular distribution of gold was investigated in
T24 cells. The gold content was measured in the membrane frac-
tion, cytosolic fraction, cytoskeletal fraction, and nuclear fraction
(Fig. 2). In the cytosolic fraction (2163 and 885 ng) and membrane
fraction (2072, 977 ng), the amount of gold was the greatest. The
accumulation of gold upon treatment with Au1 was greater than
that upon treatment with Au2 or the control. Due to the absorption
Scheme 1. Synthetic route for preparation of [6,7-dimethoxy-1-(2-aminophenyl)-3,4-dihydroisoquinolines] (H-L1), [6-methoxy-1-(2-aminophenyle)-3,4-dihydroisoquinolines (H-
L2), and their gold(III) complexes Au1 and Au2. Chemical reagents: (a) SOCl2, Benzene (room temperature, 24 h) yield: 80e95%; (b) POCl3, CH3CN (80 ꢂC, 5 h), yield: 70e80%; (c)
SnCl2$2H2O, EtOAc, (80 ꢂC, 10e22 h), yield: 60e70%; (d) H-L1, KAuCl4, methanol/dichloromethane (1:1) (60e70 ꢂC, 24 h), yield: 85e95% (e) H-L2, KAuCl4, methanol/dichloromethane
(1:1) (60e70 ꢂC, 24 h) yield: 70e80%.

T.-M. Khan et al. / European Journal of Medicinal Chemistry 163 (2019) 333e343
335
Table 1
Crystal data and structure refinement details summery for Au1 and Au2.
Compound
Au1
Au2
Formula
M
C17H17AuCl2N2O2
549.20
C16H15AuCl2N2O
519.17
Cryst. Syst.
Space group
a (Å)
b (Å)
c (Å)
Monoclinic
P21/c
9.297 (5)
8.811 (4)
22.272 (10)
90.00
104.878 (18)
90.00
1763.3 (15)
4
2.069
1040
Orthorhombic
Pbca
13.5520(14)
7.7992(8)
29.960(3)
90.00
90.00
90.00
3166.6 (6)
8
2.178
a
b
g
(ꢂ)
(ꢂ)
(ꢂ)
V/(Å3)
Z
Dc (g cmꢀ3
F (000)
)
1968
q
range for data collection (ꢂ)
1.892e25.350
13054/3226
1.111
1.359e25.348
10265/2872
0.861
Reflections collected/unique
Goodness-of-fit on F2
Rint
0.0379
0.0335
Final R indices [I > 2
R indices (all data)
s
(I)]
R1 ¼ 0.0302 wR ¼ 0.0829
R1 ¼ 0.0309 wR ¼ 0.1041
R1 ¼ 0.0380 wR ¼ 0.0866
R1 ¼ 0.0439 wR ¼ 0.1159
Fig. 1. Crystal structures of Au1 and Au2 with atom label.
and distribution of metals in different fraction of cells, it is neces-
sary to carry out a deeper biological study and investigate the
possible action mechanism [34].
2.4. In vitro cytotoxicity
The MTT assay was utilized to determine the in vitro cytotoxicity
of Au1, Au2, free ligands, and a gold(III) salt against a series of
human tumor cells. As listed in Table 2, the Au1 and Au2 showed
considerable cytotoxicity towards T-24 cells as compared with the
other cells in the panel, including SK-OV-3, BEL-7402, A549,
MGC80-3, BEL-7404, HeLa, and HL-7702 normal liver cells. More-
over, Au1 and Au2 possessed higher cytotoxicity than their corre-
sponding ligand, gold(III) salt, and cisplatin, which was taken as the
reference. The results suggest a synergistic effect between the
ligand and the Au(III), which enhanced the biological activity. The
cytotoxicity of both complexes was much lower than that of
cisplatin against HL-7702 (human normal liver cell). The cytotox-
icity is dependent on certain structural features of the gold(III)
complexes, as Au1 (IC50 2.52 0.34
cytotoxicity having two methoxy groups in the isoquinoline,
whereas Au2 (IC50 5.07 0.23 M) has only one methoxy group and
mM) possessed the highest
m
a resultingly lower cytotoxicity. The difference in cytotoxicity might
be attributed to the basicity of the isoquinoline ligand. Both Au1
and Au2 were used to investigate the potential anticancer
mechanism.
Fig. 2. Determination of gold content by ICP-MS in different fractions of T-24 cells after
10 h treatment of Au1 and Au2. Data are shown as mean standard deviation of three
independent trials.

336
T.-M. Khan et al. / European Journal of Medicinal Chemistry 163 (2019) 333e343
Table 2
IC50 values of Au1 and Au2 and their corresponding ligands and gold(III) salt against seven tumor cells and one normal cell.
Compd
SK-OV-3a
A-549a
MGC80-3a
BEL-7402a
HeLaa
BEL-7404a
T-24a
HL-7702a
K2AuCl4
H-L1
76.15 0.63
52.5 0.51
49.47 0:34
11.04 0:82
15.04 0:73
18.03 0:75
61.31 0.23
>100
80.01 0:41
12.16 0:76
35.73 0:25
19.02 0:63
82.52 0.51
>100
90.33 0:48
10.05 0:32
13.06 0:91
6.05 0:43
95.03 0.51
80.50 0.42
>100
26.52 0:15
9.68 0:67
15.42 0:71
50.43 0.45
>100
>100
6.78 0:71
8.5 0:34
7.58 0:82
69.35 0.21
64.20 0.43
80.02 0:37
>100
11.72 0:12
23.23 0:35
49.17 0.34
24.02 0.53
60.04 0:51
2.52 0:34
5.07 0:23
27.5 0:47
89.44 0.51
>100
90.0 0:34
>100
>100
16.61 0:42
H-L2
Au1
Au2
Cisplatin
a
SK-OV-3-ovarian, A-549-lung, MGC80-3-gastric, BEL-7402-liver, HeLa-cervical, BEL-7404-liver, T-24-bladder, HL-7702-Human normal liver cell.
2.5. Assessment of cell cycle
Cell cycle progression gives rise to cell division and is an
important feature of cancer cells. There are primarily three phases
that are most important for anticancer studies: G0/G1, S and G2
phases [34]. The effects of Au1 and Au2 on cell cycle progression of
T-24 were investigated by propidium iodide staining as the content
of DNA could easily be determined using flow cytometry. Cells were
treated with Au1 (1.25, 2.50, 5.00 mM) and Au2 (2.5, 5.0, 10.0 mM)
for 24 h, and the proportion of the cells in S-phase increased from
24.70% in the control, to 61.41% and 59.19% after Au1 and Au2
treatment, respectively. The population of cells in the control was
mostly in G0/G1 and G2 phases. The accumulation of cells in S
phase increased as compared to in G2 phase as shown in Fig. 3A.
Further investigation of the protein levels of cell cycle related
proteins was carried out by western blot (Fig. 3B). The expression
level of cyclin A and cyclin E were significantly decreased, whereas
little change was observed in cdk2, cdk4 and cdk6. A dose depen-
dent decrease was caused by the treatment of A1 and Au2. As
shown in the immunoblot, by limiting the amount of cyclin A and E
of S phase, the formation of cyclin-cdk complexes was inhibited,
causing cell cycle arrest in S phase. Replication of DNA occurs in this
phase, and therefore the cellular action of these complexes could
interfere with the formation of DNA-protein complexes and inhibit
protein synthesis. Additionally, the upregulation of cell cycle con-
trol inhibitor of cyclin-cdk complexes (P53, P27 and P21) leads to
cell cycle arrest in S phase. In particular, the over expression of P53
and P27 attenuates the proliferation of cancer cells and induces cell
apoptosis by the activation of Bax through different inhibitory
signals.
2.6. Determination of apoptosis
Programmed cell death occurs via apoptosis, and it is required
for the normal development and is involved in the eradication of
cells having significant DNA damage. This process is often studied
for the evaluation of anticancer drug activities [35]. Using annexin
V and propidium iodide staining, the extent of apoptosis caused by
Au1 and Au2 was investigated using flow cytometry. T-24 cells
were treated with Au1 (1.25, 2.5, 5.0 mM) and Au2 (2.5, 5.0, 10 mM)
for 24 h, and a dose-dependent increase in early and late apoptosis
was observed. It was strongly suggested by the percentages of
apoptotic cells that the extent of apoptosis caused by Au1 was
greater than that of Au2, as the highest concentration of Au1 was
5 mM and Au2 was 10 mM (Fig. 4A). By means of different stimuli,
the intrinsic pathway plays a vital role in the regulation of apoptosis
[36]. Using western blotting, the expression level of apoptosis
related proteins were investigated upon treatment with Au1 and
Au2 and were consistent with activation of the intrinsic mito-
chondrial pathway (Fig. 4B). The up-regulation of pro-apoptotic
proteins Bax and Bak, and down-regulation of anti-apoptotic
Fig. 3. (A) Cell cycle arrest induced by Au1 and Au2 in T-24 cells after 24 h treatment
as compared with the negative control. The histogram showed the percentages of cells
in different cell cycle phases. (B) Western blot of Cyclin A, Cyclin E, cdk2, cdk4, cdk6,
P27, P53 and P21 levels after 24 h treatment of T-24 with Au1 and Au2.

T.-M. Khan et al. / European Journal of Medicinal Chemistry 163 (2019) 333e343
337
and changes in the level of Ca2þ might be involved in the induction
mechanism of apoptosis caused by Au1 and Au2.
2.8. ROS generation
Recent studies have shown that ROS generation has dramatic
effects on cancer cell integrity by damaging the DNA through
oxidative stress and causing mitochondrial dysfunction, leading to
apoptosis and cell death [40,41]. T-24 cells were inoculated with
Au1 (5 mM) and Au2 (10 mM) for 24 h, and the generation of ROS
was investigated in treated cells using flow cytometry. It has been
shown that the level of ROS generation is higher in cells treated
with these complexes than in untreated cells. These findings sug-
gested that Au1 and Au2 activated ROS-induced apoptosis in T-
24 cells (Fig. 6).
2.9. Depolarization of mitochondrial membrane potential
Depolarization of the mitochondrial membrane potential is
treated as mitochondrial malfunction and can lead to the activation
of intrinsic apoptotic pathway [42]. To investigate mitochondrial
membrane potential upon treatment with Au1 (1.25, 2.5, 5
mM) and
Au2 (2.5, 5.0, 10 M), the JC-1 kit was used. A dose dependent
m
reduction in the mitochondrial membrane polarization was
observed upon treatment with both complexes. In particular, Au1
led to higher damage to the membrane as compared to Au2 with
the negative control taken as the reference (Fig. 7). The extent of
mitochondrial membrane potential disruption upon treatment
with Au1 was 67.8%, whereas that with Au2 was lower, thus
demonstrating that Au1 and Au2 induced mitochondria-mediated
apoptosis in T-24 cells.
2.10. Caspase-3, 8, 9 activations
The activation of caspases plays a vital role in apoptosis, and is
implicated in numerous diseases especially cancer [43,44]. T-
24 cells were exposed to the different concentrations of Au1 and
Au2 for 24 h. The ratio of caspase-3 was increased from 1.60% in
control cells to 53.7% and 40% upon treatment with Au1 and Au2,
respectively. Similarly, the ratio of caspase-8 was increased to 44.9%
and 30.3%, whereas the activation of caspase-9 increased to 42.5%
and 28.0%, respectively (Fig. 8). The activation of caspases provides
evidence that Au1 and Au2 induces of apoptosis through the cas-
pase pathway.
2.11. In vivo anticancer activity
Fig. 4. (A) Induction of apoptosis in T-24 cells after Au1 and Au2 treatment for 24 h.
Apoptosis was studied using PI and Annexin V staining and FACS analysis. (B) Analysis
of Bax, Bak, Bcl-xl, Bcl-2 levels by Western Blotting after 24 h treatment with Au1 and
Au2.
To further investigate in vivo anticancer activities, Au1 and Au2
were used in a xenograft model in mice with the T-24 cell lines. The
mice were divided randomly into four groups: cisplatin-treated,
negative control, Au1 treated, Au2 treated groups. When the tu-
mor volume was 80e100 mm3, the mice were inoculated with
cisplatin (2 mg/kg), Au1 and Au2 (5.0, 10.0 mg/kg), and a vehicle
control with 5% DMSO in saline (v/v) intraperitoneally (Figs. 9 and
10 and Table S3 Supporting information). From Figs. 9B and 10B, it
can be seen that there were no large changes in the body weight of
Au1, Au2 treated and control groups, whereas the cisplatin treated
group had a significant weight loss during the fourteen days of
treatment. The inhibition of tumor growth by Au1 (13.0, 37.1%) and
Au2 (9.0, 21.0%) were higher than that of control, but lower than
cisplatin which was 46.3% (Figs. 9C and 10C). From the above ex-
periments, it is suggested that Au1 and Au2 have no significant
toxicity as compared with cisplatin. The results showed Au1 and
Au2 exhibited higher in vivo safety than cisplatin, and can inhibit
tumor growth effectively.
proteins Bcl-2 and Bcl-xl, demonstrated the involvement of Bcl-2
family proteins, which are important for regulating mitochondrial
membrane potential [37]. From the above results, it was concluded
that Au1 and Au2 triggered apoptosis through the mitochondria
mediated pathway.
2.7. Intracellular Ca2þ release
The release of Ca2þ inside the cells is recognized as it alters the
polarization of the mitochondrial membrane (
Dѱ) and thus has
been involved in cell apoptosis and cell death [38,39]. T-24 cells
were treated with Au1 (5 mM) and Au2 (10 mM) for 24 h, and flow
cytometry studies showed an increase in the release of Ca2þ as
compared with control (Fig. 5). It was suggested that the release

338
T.-M. Khan et al. / European Journal of Medicinal Chemistry 163 (2019) 333e343
Fig. 5. Effects of Au1 (2.5 mM) and Au2 (5.0 m
M) on the production of Ca2þ in T-24 cells after 24 h treatment using Fluo-3 AM staining and flow cytometry.
Fig. 6. Flow cytometric analysis of T-24 treated with Au1 and Au2 for 24 h for the measurement of reactive oxygen species.
2.12. Structure-activity relationships (SARs)
3. Conclusion
From the studies of biological activity and anticancer mecha-
nism, the following SARs trend could be established with the li-
gands and complexes. Au1 has two methoxy groups whereas Au2
had only one methoxy group in the ligand. Cytotoxicity studies
showed that Au1 was more effective than Au2 against T-24 cells
because of the two electron donor group. Similarly, the study of the
anticancer mechanism of both complexes Au1 and Au2 provide an
understanding of their different biological performance. For
example, Au1 triggered apoptosis more effectively, produced
greater damage to the mitochondrial membrane, and achieved a
greater extent of activation of the caspase pathway as compared
with Au2. Additionally, the xenograft model of mice having T-
24 cells also presented the same SARs trend with respect to Au1
and Au2. Both Au1 and Au2 contained electron-donating methoxy
groups, however Au1 has two methoxy groups at the carbon-6 and
carbon-7 position of the isoquinoline, whereas Au2 has only one
methoxy group, and the more basic isoquinoline led to the more
effective complex. The results are similar to the findings of Steglich
and T. Srinivasa, in which the anticancer mechanism of a complex is
associated with the basic nature of amine ligands. Complexes
having more basic ligands will have greater cytotoxicity [45e47].
We have synthesized and characterized two new gold(III)
complexes with isoquinoline derivatives as ligands. Both Au1 and
Au2 exhibited higher cytotoxicity and specificity towards T-24 cells,
and less toxicity towards normal HL-7702 cells. These complexes
arrested cell cycle in S phase and triggered apoptosis via depolar-
ization of the mitochondrial membrane potential. In the case of
Au1, it produced significant damage to the mitochondrial mem-
brane by increasing the generation of ROS, the release of Ca2þ, and
the activation of caspases-3, -8, -9, whereas Au2 caused the same
effects but to a lesser extent. Induction of cell apoptosis occurred
via the mitochondrial pathway. Au1 and Au2 demonstrated better
in vivo safety than cisplatin and can effectively inhibit tumor
growth in a xenograft model of mice bearing T-24 cells.
4. Experimental
4.1. Materials and instruments
Chemical reagents were used without additional purification
unless stated specifically. All were commercially available. All
spectroscopic experiments were conducted at room temperature.

T.-M. Khan et al. / European Journal of Medicinal Chemistry 163 (2019) 333e343
339
Fig. 7. Mitochondrial membrane potential damage caused by Au1 and Au2 analysis by flow cytometry with different concentrations for 24 h.
Fig. 8. Activation of caspase-3/-8/-9 analyzed by flow cytometry using the CaspGLOW™ staining kit after 24 h treatment with Au1 and Au2 in T-24 cells.
ESI-MS was performed on a Bruker HCT Electrospray Ionization
Mass Spectrometer. A Bruker AV-500 NMR Spectrometer was used
to obtain NMR spectra. For the stability and purity of these com-
plexes, HPLC was used on a regular basis. Pure Au1 and Au2 were
used in biological and biophysical experiments. FACS Aria ІІ Flow
Cytometer was used for all flow cytometry assays (BD Biosciences,
San Jose, USA). Propidium Iodide (PI), RNAse A, fixative solution,
and apoptosis detection kit were obtained from BD Biosciences. JC-
1 mitochondrial membrane potential (MMP) detection kit was
obtained from Beyotime Institute of Biotechnology (China).
Caspase-3, -8, -9 activation kit was purchased from BioVision.
Chinese Academy of Sciences, Institute of Biochemistry and Cell
Biology provided human normal liver cell line HL-7702 and tumor
cell lines, including A549, SK-OV-3, MGC80-3, T-24, BEL-7404,

340
T.-M. Khan et al. / European Journal of Medicinal Chemistry 163 (2019) 333e343
Fig. 9. Xenograft model of T-24 cell line showing in vivo anticancer activity. (A) Effect of Au1, cisplatin on tumor growth. For control, 5% DMSO in saline was used. Tumor volume was
calculated in the form of T/C % values in mm. (B). Relative body weight from day 0e14. On day 0, the body weight was considered 100%. (C). Inhibition of tumor growth in treated
cells and control. (D) Photograph of control and treated cells with Au1 and cisplatin.
HepG2, T-24. All antibodies, which include cdk2, cdk4, cdk6, cylin E,
cyclin A, Bax, Bak, Bcl-xl, Bcl-2, P53, P27, P21, were purchased from
Abcam.
block crystals were harvested by slow evaporation of the filtrate
after filtration of the dark red reaction mixture.
4.2.2.1. NMR data for Au1. 1H NMR (500 MHz, (CD3)2SO):
d3.06 (t,
4.2. Synthesis and characterization of H-L1, H-L2, Au1 and Au2
J ¼ 7.0 Hz, 2H), 3.69 (s, 3H), 3.98 (s, 3H), 4.58 (brs, 2H), 6.89 (s, 1H),
7.02 (t, J ¼ 7.3 Hz,1H), 7.21 (s, 1H), 7.42 (d, J ¼ 7.9 Hz, 1H), 7.48 (t,
J ¼ 7.7 Hz, 1H), 7.58 (d, J ¼ 8.1 Hz, 1H), 7.89 (s, 1H).13C NMR
4.2.1. Synthesis of H-L1 and H-L2
L1 and L2 preparation were carried out by a reported procedures
(via intermediate products 1 and 2) described by Nussbaun [45].
The characterization data for H-L1 and H-L2 are given below.
(125 MHz, (CD3)2SO): d27.8, 53.4, 56.4, 56.9, 111.5, 117.1, 119.4, 120.6,
120.7, 127.7, 134.6, 135.3, 135.8, 147.8, 152.4, 154.4, 163.3. ESI-MS m/
z: 549.0 [M þ H]þ.
(a) Data for H-L1: 1H NMR (500 MHz, (CD3)2SO)
d2.66 (t,
4.2.2.2. NMR data for Au2. 1H NMR (400 MHz, CDCl3)
d3.07 (t,
J ¼ 7.3 Hz, 2H), 3.61 (s, 3H), 3.71 (t, J ¼ 7.3 Hz, 2H), 3.86 (s, 3H), 5.91
(s, 2H), 6.57e6.60 (m, 1H), 6.70 (s, 1H), 6.80 (dd, J ¼ 0.5,8.0 Hz, 1H),
J ¼ 6.8 Hz, 2H), 3.92 (s, 3H), 4.67 (t, J ¼ 6.3 Hz, 2H), 6.53 (brs, 1H),
6.83e6.88 (m, 2H), 6.92 (t, J ¼ 7.6 Hz, 1H), 7.18 (d, J ¼ 8.0 Hz, 1H),
7.27 (m, 1H), 7.35e7.39 (m, 1H), 7.41 (d, J ¼ 8.7 Hz, 1H). 13C NMR
6.99 (s, 1H), 7.08e7.14 (m, 2H).13C NMR (125 MHz, (CD3)2SO)
d26.1,
47.6, 56.6, 111.8, 112.5, 115.6, 116.9, 121.0, 122.1, 130.4, 131.3, 133.3,
147.5, 148.9, 151.5, 166.7. H-RMS(EI): Calcd for C17H18N2O2, m/z
283.1402, Found m/z 283.1435.
(125 MHz, (CD3)2SO)
d 29.1, 52.7, 56.2, 113.3, 120.4, 120.6, 127.8,
134.2, 135.0, 136.6, 142.8, 152.1, 164.4. ESI-MS m/z: 519.0 [M þ H]þ.
(b) Data for H-L2: 1H NMR (500 MHz, (CD3)2SO)
d2.72 (t,
J ¼ 7.2 Hz, 2H), 3.73 (t, J ¼ 7.2 Hz, 2H), 3.83 (s, 3H), 5.83 (s, 2H), 6.58
(t, J ¼ 7.1 Hz, 1H), 6.79 (d, J ¼ 8.0 Hz, 1H), 6.85 (dd, J ¼ 2.5, 8.6 Hz,
1H), 6.93 (d, J ¼ 2.3 Hz, 1H), 7.04e7.14 (m, 3H). 13C NMR (125 MHz,
4.3. Single X-ray crystallography
A crystal was placed on a thin glass fiber for data collection.
Applying the multi-scan program SADABS for absorption correc-
tion, a super Nova CCD diffractometer with graphite mono-
(CD3)2SO) d26.7, 47.2, 55.9, 112.3, 113.4, 115.4, 116.5, 121.1, 122.7,
129.8, 130.0, 131.0, 141.4, 148.4, 161.3, 166.4. H-RMS(EI): Calcd for
16H16N2O, m/z 253.1296, Found m/z 253.1332.
C
chromatic Mo-K
a
radiation (
l
¼ 0.71073 Å) was used at room
temperature. For crystal structure refinement, SHELX-97 was used
[48,49]. Differential Fourier Transforms were used to locate non-
hydrogen atoms. Full-matrix squares was used for the final
refinement with anisotropic thermal parameters of non-hydrogen
atoms on F2. The addition and adjustment of hydrogen atoms
4.2.2. Synthesis of Au1 and Au2
To a solution of KAuCl4 (0.1 mmol, 41 mg) dissolved in 25 mL of
methanol or ethanol and dichloromethane (1:1), H-L1 and H-L2
were added and stirred at 65e70 ꢂC for 24 h. After one week, shiny

T.-M. Khan et al. / European Journal of Medicinal Chemistry 163 (2019) 333e343
341
Fig. 10. The in vivo antitumor activity of Au2 in mice bearing a T-24 xenograft. (A) Tumor volume growth trend of the control group, Au2-treated groups, and cisplatin group. Tumor
volumes were tracked by the mean tumor volume (mm3) SD (n ¼ 6) and calculated as relative tumor growth rate [T/C%] values. (B) Body weight change (presented as % change
from initial weight). (C) Tumor growth inhibition rate vs vehicle control. (D) Photographs of tumor from treated groups and vehicle group.
were taken theoretically on selected atoms. All refinement and
4.6. Cell cycles studies via flow cytometry
crystallographic data are listed in Table 1.
The T-24 cells were cultured in 70 mm flat-bottomed plates.
Cells were treated with Au1 and Au2 and incubated for 24 h. Using
70% ethanol for fixation after treatment and harvesting, the sam-
ples were stored overnight at ꢀ20 ꢂC. The cells were resuspended
with PI and RNAse and analyzed by FACS. Mod Fit software was
used for calculation of cell cycle.
4.4. Cell culturing and treatment
All cancerous cell line and human normal liver cell line were
cultured in DMEM (Dulbecco's modified eagle medium). The me-
dium was supplemented with heat inactivated 10% fetal bovine
serum, 1% penicillin, and 100 mg/mL streptomycin. Cells were
cultured in a humidified atmosphere with 5% CO2 at 37 ꢂC. DMSO
was used to prepare the stock solution (2 mM), and serial dilutions
were carried out to obtain the required working solution of Au1
and Au2 in PBS.
4.7. Assessment of apoptosis by flow cytometry
Induction of apoptosis by Au1 and Au2 in T-24 cell line was
measured by flow cytometry using annexin V and propidium iodide
staining. T-24 cells were cultured in six well plates. The cells were
collected and washed three times with PBS after the 24 h treatment
4.5. Evaluation of cytotoxicity
For the investigation of cytotoxicity of Au1 and Au2, the MTT
assay was utilized. Cells were cultured in a 96 well flat-bottomed
plate at a density of 4 ꢁ 103 for 24 h. After 24 h the cells were
with Au1 and Au2. The cells were resuspended with 500 mL of
1 ꢁ binding buffer at a concentration of 1 ꢁ 107 cells/mL. 5
mL
annexin V and 5 mL PI were added to the cells and incubated for
treated with different concentrations of Au1 (1.25, 2.5, 5.0 mM), Au2
20 min in the dark at 30 ꢂC. The cells were analyzed by FACS. In this
period, the cells were protected from light.
(2.5, 5.0, 10.0 mM). Cisplatin was dissolved in DMSO and diluted
with PBS. DMEM with 1% DMSO were added to medium as the
control. The 96 well plate was incubated for an additional 48 h after
treatment with the complexes. The MTT solution was added into
each well at the end of each incubation period and incubated
further for 4 h. Before measuring the absorbance at 490/650 nm
with a micro-plate reader, the supernatant was discarded and
DMSO was added to dissolve the formazan crystals. The cytotoxicity
was estimated by comparing the treated cells with control at same
wavelength. By using the Bliss method (n ¼ 5), the IC50 was calcu-
lated and interpreted as the sensitivity of the cells to the metal
complexes and ligands.
4.8. Exploration of caspases activity
Caspase-3, -8, -9 activities were measured using CaspGLOW™
staining kit. After treatment with Au1 and Au2 of various concen-
trations, the cells were collected and washed with PBS three times.
1 mL of FITC-LEHD-FMK was added and incubated for 30 min. Cells
were then analyzed using FACS, and the percent changes were
established with reference to the control.

342
T.-M. Khan et al. / European Journal of Medicinal Chemistry 163 (2019) 333e343
4.9. Intracellular Ca2þmeasurement
Laboratory Animal Resources of National Institute for Food and
Drug Control Beijing (NIFDC). All animals’ treatment and experi-
mental design were performed according to the rules and regula-
tions of Animal Care and Ethics Committee of the institute. All
animals were housed in a suitable environment: temperature
(25e28 ꢂC), humidity (60e75%), and constant photoperiod of light
and dark (12 h light, 12 h dark). 5 ꢁ 106 T-24 cells were injected into
the right flank of the mice. The mice were sacrificed when the
xenograft approached 1100 mm3 and translocated into the right
side of the female nude mice in small pieces of about 2 mm3. All the
mice were randomized for treatment groups (n ¼ 6 per group) and
vehicle control when the tumor grew to 100e260 mm3. The sub-
sequent treatments have been established for the T-24 xenograft
model: (a) 5% v/v DMSO/saline infusion for vehicle control. (b) 5
and 10 mg/kg each of Au1 and Au2 were injected in intervals of two
days, (DMSO 5% in saline v/v). (d) Cisplatin, 2 mg/kg dissolved in
saline was used as the positive control. After 14 days of treatment,
all mice were sacrificed, and the tumor was dissected and weighed.
Tumor volume was calculated as [V¼(w2 ꢁ l/2)] where w is the
width and l is the length of tumor in millimeters [48,49]. The in vivo
antitumor activity was determined by two indicators [50]: Tumor
relative increment rate (T/C % ¼ TRTV/CRTV ꢁ 100%) and tumor
growth inhibition rate [IR % ¼ (Wc -Wt)/Wc ꢁ 100%], where as TRTV
and CRTV are relative tumor volume of treated group and control
group, respectively. Wt and Wc represent the average tumor weight
of treated group and vehicle-controlled group respectively
[50e52].
The level of free intracellular Ca2þ in Au1 and Au2-treated T-
24 cells were determined by a fluorescent Fluo-3 AM dye, which is
able to cross the cell membrane and cut by intracellular esterase
into Fluo-3. Fluo-3 combined with Ca2þ has a strong fluorescence at
488 nm. T-24 cells were treated with Au1 and Au2 at different
concentrations for 24 h. After the exposure the cells were har-
vested, washed with PBS and incubated with Fluo-3 AM (5.0 mM)
for 30 mints in dark. The detection of intracellular free Ca2þ has
been conducted on Flow cytometer with the excitation wavelength
of 525 nm.
4.10. Investigation of mitochondrial membrane potential (MMP)
dysfunction
JC-1 is a lipophilic fluorescent dye, and was used to measure the
mitochondrial membrane potential. After the treatment of the cells
with given complexes at different concentration, the cells were
incubated with JC-1 for 20 min. The mitochondrial membrane po-
tential was determined using flow cytometry with orange excita-
tion wavelengths and green emission wavelengths. Mitochondrial
membrane damage could be correlated with the orange emission of
the dye because of its mitochondrial membrane potential depen-
dent accumulation in mitochondria. Green fluorescent represents
the monomeric form of dye present in the cytosol upon depolari-
zation of the mitochondrial membrane potential.
4.11. Gold uptake in T-24 cells
Acknowledgement
T-24 cells were cultured in a 100 mm dish and treated with
10 mM of Au1 and Au2 for 10 h. After incubation with the com-
This work was supported by the National Natural Science
Foundation of China (Grants 81473102, 21431001), IRT_16R15, and
Natural Science Foundation of Guangxi Province of China (Grant
2016GXNSFGA380005) as well as “BAGUI Scholar” program of
Guangxi Province of China.
plexes, the cells were collected, washed with PBS, and fractionated
using the Fraction PERP kit. The four isolated fractions, including
the cytosol, nucleus, cytoskeleton and membrane fractions, were
digested with 5% HNO3 (5 mL) and diluted with double-distilled
water. Finally, the content of gold was determined using induc-
tively coupled plasma-mass spectrometry (ICP-MS). The experi-
ment was repeated three times and the results are reported as
mean SD.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2018.11.047.
4.12. Western blotting
References
Cells were grown in a 100 mm Petri dish and exposed to Au1
and Au2 for 24 h. After inoculation, cells were harvested, washed
and lysed with lysis buffer containing protease inhibitor. An
appropriate concentration SDS-polyacrylamide gel was made and
[1] T.W. Hambley, Platinum binding to DNA: structural controls and conse-
quences, Dalton Trans. (2001) 2711e2718.
€
[2] P. Kopf-Maier, Complexes of metals other than platinum as antitumor agents,
Eur. J. Clin. Pharmacol. 47 (1994) 1e16.
[3] T.T. Tavares, G.C. Azevedo, A. Garcia, A.G. Carpanez, P.M. Lewer, D. Paschoal,
B.L. Müller, H.F. Dos Santos, R.C. Matos, H. Silva, Gold(I) complexes with aryl-
thiosemicarbazones: molecular modeling, synthesis, cytotoxicity and TrxR
inhibition, Polyhedron 132 (2017) 95e104.
[4] S. Dasari, P.B. Tchounwou, Cisplatin in cancer therapy: molecular mechanisms
of action, Eur. J. Pharmacol. 740 (2014) 364e378.
[5] L. Galluzzi, L. Senovilla, I. Vitale, J. Michels, I. Martins, O. Kepp, M. Castedo,
G. Kroemer, Molecular mechanisms of cisplatin resistance, Oncogene 31
(2012) 1869e1883.
[6] D. Wang, S.J. Lippard, Cellular processing of platinum anticancer drugs, Nat.
Rev. Drug Discov. 4 (2005) 307e320.
[7] Z.-F. Chen, Y.-C. Liu, K.-B. Huang, H. Liang, Alkaloid-metal based anticancer
agents, Curr. Top. Med. Chem. 13 (2013) 2104e2115.
[8] L. Cattaruzza, D. Fregona, M. Mongiat, L. Ronconi, A. Fassina, A. Colombatti,
D. Aldinucci, Antitumor activity of gold(III)-dithiocarbamato derivatives on
prostate cancer cells and xenografts, Int. J. Canc. 128 (2011) 206e215.
loaded with total protein (50 mg) extracts. After transferring to
polyvinylidene fluoride (PVDF) membranes, the membrane was
blocked with 5% BSA in TBST buffer and incubated overnight with
corresponding primary antibodies at 4 ꢂC. Primary antibodies,
including cyclin E, cyclin A, cdk2, cdk4, cdk6, P53, P27, P21, Bax, Bak,
Bcl-2, Bcl-xl, were purchased from Abcam. Before incubation with
secondary antibodies conjugated with horseradish peroxidase for
2 h, the membrane was washed with TBST. The immunoreactive
signal were detected by using an enhanced chemoluminance kit
(Pierce ECL western blotting substrate).
4.13. In vivo antitumor activity toward T-24 human urinary bladder
carcinoma
ꢀ
ꢀ
ꢀ
[9] A. Casado-Sanchez, C. Martín-Santos, J.M. Padron, R. Mas-Balleste, C. Navarro-
ꢀ
Ranninger, J. Aleman, S. Cabrera, Effect of electronic and steric properties of 8-
substituted quinolines in gold(III) complexes: synthesis, electrochemistry,
stability, interactions and antiproliferative studies, J. Inorg. Biochem. 174
(2017) 111e118.
Institute of Biomedical Engineering affiliated with Chinese
Academy of Medical Sciences Tianjin (China) supported the in vivo
anti-cancer studies. Nude mice, BALB/c (aged 5e6 weeks, body
weight 17e20 g, male) were obtained from the Institute for
ꢁ
ꢀ
ꢁ ꢀ
[10] T. Lazarevic, A. Rilak, Z.D. Bugarcic, Platinum, palladium, gold and ruthenium
complexes as anticancer agents: current clinical uses, cytotoxicity studies and
future perspectives, Eur. J. Med. Chem. 142 (2017) 8e31.

T.-M. Khan et al. / European Journal of Medicinal Chemistry 163 (2019) 333e343
343
[11] A. Nandy, T. Samanta, S. Mallick, P. Mitra, S.K. Seth, K.D. Saha, S.S. Al-Deyab,
J. Dinda, Synthesis of gold(III))gold(I)eNHC through disproportionation: the
role of gold(I)eNHC in the induction of apoptosis in HepG2 cells, New J. Chem.
40 (2016) 6289e6298.
[12] E.R. Tiekink, Gold compounds in medicine: potential anti-tumour agents, Gold
Bull. 36 (2003) 117e124.
[32] S. Petoud, G. Muller, E.G. Moore, J. Xu, J. Sokolnicki, J.P. Riehl, U.N. Le,
S.M. Cohen, K.N. Raymond, Brilliant Sm, Eu, Tb, and Dy chiral lanthanide
complexes with strong circularly polarized luminescence, J. Am. Chem. Soc.
129 (2007) 77e83.
ꢀ
[33] I. Romero-Canelon, L. Salassa, P.J. Sadler, The contrasting activity of iodido
versus chlorido ruthenium and osmium arene azo-and imino-pyridine anti-
cancer complexes: control of cell selectivity, cross-resistance, p53 depen-
dence, and apoptosis pathway, J. Med. Chem. 56 (2013) 1291e1300.
[34] Y.-P. Ho, K.K. To, S.C. Au-Yeung, X. Wang, G. Lin, X. Han, Potential new anti-
tumor agents from an innovative combination of demethylcantharidin, a
modified traditional Chinese medicine, with a platinum moiety, J. Med. Chem.
44 (2001) 2065e2068.
[13] E. Tiekink, Anti-cancer potential of gold complexes, Inflammopharmacology
16 (2008) 138e142.
[14] J.M. Madeira, S.M. Schindler, A. Klegeris, A new look at auranofin, dextro-
methorphan and rosiglitazone for reduction of glia-mediated inflammation in
neurodegenerative diseases, Neural Regener. Res. 10 (2015) 391e393.
[15] M. Porchia, M. Pellei, M. Marinelli, F. Tisato, F. Del Bello, C. Santini, New in-
sights in Au-NHCs complexes as anticancer agents, Eur. J. Med. Chem. 146
(2018) 709e746.
[16] S. Tian, F.-M. Siu, S.C. Kui, C.-N. Lok, C.-M. Che, Anticancer gold(I)ephosphine
complexes as potent autophagy-inducing agents, Chem. Commun. 47 (2011)
9318e9320.
[35] C.-C. Chou, J.-S. Yang, H.-F. Lu, S.-W. Ip, C. Lo, C.-C. Wu, J.-P. Lin, N.-Y. Tang, J.-
G. Chung, M.-J. Chou, Quercetin-mediated cell cycle arrest and apoptosis
involving activation of a caspase cascade through the mitochondrial pathway
in human breast cancer MCF-7 cells, Arch Pharm. Res. 33 (2010) 1181e1191.
[36] C. Trejo-Solís, G. Palencia, S. Zúniga, A. Rodríguez-Ropon, L. Osorio-Rico,
ꢀ
[17] B. Desoize, Metals and metal compounds in cancer treatment, Anticancer Res.
24 (2004) 1529e1544.
S.T. Luvia, I. Gracia-Mora, L. Marquez-Rosado, A. Sanchez, M.E. Moreno-García,
Casilgly induces apoptosis in glioma C6 cells in vitro and in vivo through
[18] C. Frank Shaw III, Gold-based therapeutic agents, Chem. Rev. 99 (1999)
2589e2600.
[19] K. Czerwinska, M. Golec, M. Skonieczna, J. Palion-Gazda, D. Zygadło, A. Szlapa-
caspase-dependent and caspase-independent mechanisms, Neoplasia 7
(2005) 563e574.
[37] C. Soldani, A. Scovassi, Poly (ADP-ribose) polymerase-1 cleavage during
apoptosis: an update, Apoptosis 7 (2002) 321e328.
ꢀ
Kula, S. Krompiec, B. Machura, A. Szurko, Cytotoxic gold(III) complexes
incorporating a 2, 20: 60, 200-terpyridine ligand frameworkethe impact of the
[38] M.H. Kang, C.P. Reynolds, Bcl-2 inhibitors: targeting mitochondrial apoptotic
pathways in cancer therapy, Clin. Canc. Res. 15 (2009) 1126e1132.
[39] D.R. Laver, Ca2þ stores regulate ryanodine receptor Ca2þ release channels via
luminal and cytosolic Ca2þ sites, Biophys. J. 92 (2007) 3541e3555.
substituent in the 40-position of
a terpy ring, Dalton Trans. 46 (2017)
3381e3392.
[20] A. Trommenschlager, F. Chotard, B. Bertrand, S. Amor, L. Dondaine, M. Picquet,
P. Richard, A. Bettaïeb, P. Le Gendre, C. Paul, Gold(I)eBODIPYeimidazole
bimetallic complexes as new potential anti-inflammatory and anticancer
trackable agents, Dalton Trans. 46 (2017) 8051e8056.
ꢀ
[40] F. Chauvigne, M. Boj, R.N. Finn, J. Cerda, Mitochondrial aquaporin-8-mediated
hydrogen peroxide transport is essential for teleost spermatozoon motility,
Sci. Rep. 5 (2015) 7789, https://doi.org/10.1038/srep07789.
[41] K. Sinha, J. Das, P.B. Pal, P.C. Sil, Oxidative stress: the mitochondria-dependent
and mitochondria-independent pathways of apoptosis, Arch. Toxicol. 87
(2013) 1157e1180.
[42] S. Zheng, X. Li, Y. Zhang, Q. Xie, Y.-S. Wong, W. Zheng, T. Chen, PEG-nanolized
ultrasmall selenium nanoparticles overcome drug resistance in hepatocellular
carcinoma HepG2 cells through induction of mitochondria dysfunction, Int. J.
Nanomed. 7 (2012) 3939e3949.
^
[21] A. Garcia, R.C. Machado, R.M. Grazul, M.T.P. Lopes, C.C. Correa, H.F. Dos Santos,
M.V. de Almeida, H. Silva, Novel antitumor adamantaneeazole gold(I) com-
plexes as potential inhibitors of thioredoxin reductase, J. Biol. Inorg. Chem. 21
(2016) 275e292.
[22] C.T. Lum, A.S.-T. Wong, M.C. Lin, C.-M. Che, R.W.-Y. Sun, A gold(III) porphyrin
complex as an anti-cancer candidate to inhibit growth of cancer-stem cells,
Chem. Commun. 49 (2013) 4364e4366.
[23] X. Yang, S. Yang, H. Chai, Z. Yang, R.J. Lee, W. Liao, L. Teng, A novel isoquinoline
derivative anticancer agent and its targeted delivery to tumor cells using
transferrin-conjugated liposomes, PloS One 10 (2015), https://doi.org/
10.1371/journal. pone.0136649 e0136649.
[24] A. Turano, G. Scura, A. Caruso, C. Bonfanti, R. Luzzati, D. Bassetti, N. Manca,
Inhibitory effect of papaverine on HIV replication in vitro, AIDS Res. Hum.
Retrovir. 5 (1989) 183e192.
[25] M.Z. El-Readi, S. Eid, M.L. Ashour, A. Tahrani, M. Wink, Modulation of multi-
drug resistance in cancer cells by chelidonine and chelidonium majus alka-
loids, Phytomedicine 20 (2013) 282e294.
[43] S.J. Riedl, Y. Shi, Molecular mechanisms of caspase regulation during
apoptosis, Nat. Rev. Mol. Cell Biol. 5 (2004) 897e1000.
[44] C. Stroh, K. Schulze-Osthoff, Death by a thousand cuts: an ever-increasing list
of caspase substrates, Cell Death Differ. 5 (1998) 997e1000.
[45] F. von Nussbaum, B. Miller, S. Wild, C.S. Hilger, S. Schumann, H. Zorbas,
W. Beck, W. Steglich, Synthesis of 1-(2-aminophenyl)isoquinolines and the
biological activity of their cis-dichloro platinum(II) complexes, J. Med. Chem.
42 (1999) 3478e3485.
[46] Y.P. Ho, S.C. Au-Yeung, K.K. To, Platinum-based anticancer agents: innovative
design strategies and biological perspectives, Med. Res. Rev. 23 (2003)
633e655.
[26] C.-Y. Kuo, M.-J. Wu, Y.-H. Kuo, Synthesis and antitumor activity of cis-
ꢀ
dichloroplatinum(II)
complexes
of
1-(2-aminophenyl)-1,2,3,4-
[47] T. Srinivasa Reddy, Steven H. Priver, Vijay V. Rao, Nedaossadat Mirzadeh,
tetrahydroisoquinolines, Eur. J. Med. Chem. 41 (2006) 940e949.
[27] S.K. Mantena, S.D. Sharma, S.K. Katiyar, Berberine, a natural product, induces
G1-phase cell cycle arrest and caspase-3-dependent apoptosis in human
prostate carcinoma cells, Mol. Canc. Therapeut. 5 (2006) 296e308.
[28] Y.-C. Liu, Z.-F. Chen, L.-M. Liu, Y. Peng, X. Hong, B. Yang, H.-G. Liu, H. Liang,
C. Orvig, Divalent later transition metal complexes of the traditional Chinese
medicine (TCM) liriodenine: coordination chemistry, cytotoxicity and DNA
binding studies, Dalton Trans. (2009) 10813e10823.
[29] K.-B. Huang, Z.-F. Chen, Y.-C. Liu, X.-L. Xie, H. Liang, Dihydroisoquinoline
copper(II) complexes: crystal structures, cytotoxicity, and action mechanism,
RSC Adv. 5 (2015) 81313e81323.
[30] K.-B. Huang, F.-Y. Wang, X.-M. Tang, H.-W. Feng, Z.-F. Chen, Y.-C. Liu, Y.-N. Liu,
H. Liang, Organometallic gold(III) complexes similar to tetrahydroisoquinoline
(THIQ) induce ER-stress-mediated apoptosis and pro-death autophagy in
A549 cancer cells, J. Med. Chem. 61 (2018) 3478e3490.
Suresh K. Bhargava, Gold(I) and gold(III) phosphine complexes: synthesis,
anticancer activities towards 2D and 3D cancer models, and apoptosis
inducing properties, Dalton Trans. 47 (2018) 15312e15323.
€
€
[48] G.M. Sheldrick, SADABS, Version 2.05, University of Gottingen, Gottingen,
Germany, 2002.
[49] G.M. Sheldrick, SHELXS-97, Program for Solution of Crystal Structures, Uni-
€
€
versity of Gottingen, Gottingen, Germany, 1997.
[50] H.-Y. Lee, A.-C. Tsai, M.-C. Chen, P.-J. Shen, Y.-C. Cheng, C.-C. Kuo, S.-L. Pan, Y.-
M. Liu, J.-F. Liu, T.-K. Yeh, Azaindolylsulfonamides, with a more selective
inhibitory effect on histone deacetylase 6 activity, exhibit antitumor activity
in colorectal cancer HCT116 cells, J. Med. Chem. 57 (2014) 4009e4022.
[51] Y. Wang, J. Xiao, H. Zhou, S. Yang, X. Wu, C. Jiang, Y. Zhao, D. Liang, X. Li,
G. Liang, A novel monocarbonyl analogue of curcumin, (1E, 4E)-1,5-bis (2,3-
dimethoxyphenyl) penta-1, 4-dien-3-one, induced cancer cell H460
apoptosis via activation of endoplasmic reticulum stress signaling pathway,
J. Med. Chem. 54 (2011) 3768e3778.
[31] N. Sotomayor, E. Domínguez, E. Lete, Bischler-Napieralski cyclizationꢀN/C-
alkylation sequences for the construction of isoquinoline alkaloids. Synthesis
of protoberberines and benzo [c] phenanthridines via C-2 ‘-functionalized 3-
arylisoquinolines, J. Org. Chem. 61 (1996) 4062e4072.
[52] D. Palanimuthu, S.V. Shinde, K. Somasundaram, A.G. Samuelson, In vitro and
in vivo anticancer activity of copper bis(thiosemicarbazone) complexes,
J. Med. Chem. 56 (2013) 722e734.