Synthesis of Gold(I) Complexes Containing Cinnamide: In Vitro Evaluation of Anticancer Activity in 2D and 3D Spheroidal Models of Melanoma and in Vivo Angiogenesis

Article abstract of DOI:10.1021/acs.inorgchem.9b00281

A series of alkynylgold(I) phosphine complexes containing methoxy-substituted cinnamide moieties (3a-3c and 4a-4c) have been synthesized and characterized. All of the synthesized complexes were evaluated for their cytotoxicity against three human cancer c

Full text of DOI:10.1021/acs.inorgchem.9b00281

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Synthesis of Gold(I) Complexes Containing Cinnamide: In Vitro
Evaluation of Anticancer Activity in 2D and 3D Spheroidal Models of
Melanoma and In Vivo Angiogenesis
†
V. Ganga Reddy,† T. Srinivasa Reddy,*,† Steven H. Priver, Yutao Bai,‡ Shweta Mishra,§
́
Donald Wlodkowic,‡ Nedaossadat Mirzadeh,*,† and Suresh Bhargava*,†
†Centre for Advanced Materials & Industrial Chemistry, School of Science, RMIT University, G.P.O. Box 2476, Melbourne 3001,
Australia
‡Phenomics Laboratory, School of Science, RMIT University, Plenty Road, P.O. Box 71, Bundoora, Victoria 3083, Australia
§School of Pharmacy, Devi Ahilya Vishwavidyalaya, Takshila Parisar, Indore, Madhya Pradesh 452 001, India
S
* Supporting Information
ABSTRACT: A series of alkynylgold(I) phosphine complexes containing
methoxy-substituted cinnamide moieties (3a−3c and 4a−4c) have been
synthesized and characterized. All of the synthesized complexes were evaluated
for their cytotoxicity against three human cancer cell lines A549 (lung), D24
(melanoma), and HT1080 (fibrosarcoma) and the human embryonic kidney 293
cell line (Hek293T) as a proxy model for noncancer cells. Most of the
synthesized compounds showed antiproliferative activity against cancer cell lines
at low micromolar concentrations. Among these, complex 3c showed a broad
spectrum of anticancer activity with IC50 values in the range of 1.53−6.05 μM
against all tested cancer lines. Complex 3c possessed 20 times higher cytotoxicity
than the reference drug cisplatin against D24 melanoma cells and showed
significant anticancer activity in 3D spheroidal models of melanoma cells.
Mechanistic investigations of 3c activity indicate thioredoxin reductase inhibition
through steric and hydrogen-bonding interactions, followed by the induction of
oxidative stress and a mitochondrial pathway of cell death. Compound 3c also showed significant antiangiogenic properties in a
transgenic zebrafish Tg(fli1a:EGFP) in vivo model.
molecular hybridization approach. The facile coordination of a
gold-containing moiety to a biologically active molecule via a
propargyl group has been previously reported. In these studies,
gold complexes A−C (Figure 1) containing bioactive
chloroquine (used in the treatment of malaria) or flavonoids
(known for their antioxidant and anticancer properties) have
been prepared and their anticancer activities investigated.10,11
In this work, a similar approach has been followed to
develop gold complexes containing a cinnamide moiety
INTRODUCTION
■
The incidence of cancer worldwide is increasing, with 9.6
million deaths in 2018 according to World Health Organ-
ization statistics.1 Despite the presence of existing clinical
metal compounds (such as cisplatin, a platinum-based cancer
drug), further developments in the formulation of metal-based
drugs are necessary to overcome their high toxicity, which
result in unwanted side effects.2,3 The development of gold-
based therapeutic chemistry has emerged as a promising new
class of metal compounds in cancer therapy.4−6 However, the
poor bioavailability and lipophilic nature of gold complexes
remain as challenges for their successful clinical translation.
The incorporation of methoxy substituents into the
molecular framework of gold complexes has been shown
recently to be effective in balancing the lipophilicity and
hydrophilicity of the compound, a key factor responsible for
cellular uptake, pharmacokinetic properties, and intracellular
distribution.7,8 Furthermore, such modification can positively
influence the anticancer properties.9
Figure 1. Gold complexes containing bioactive chloroquine (A) and
flavonoids (B and C).
Various synthetic strategies have been adopted in developing
new gold molecules with enhanced anticancer properties and
bioavailability. One such strategy is combining the properties
of gold with other biologically active molecules through a
Received: January 31, 2019
© XXXX American Chemical Society
A
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Figure 2. Structures of the methoxy-substituted cinnamide compounds and their gold phosphine containing derivatives used in this investigation.
Scheme 1. Synthesis of Gold(I) Phosphine Complexes (3a−3c and 4a−4c) Containing Methoxy-Substituted Cinnamide
(Figure 2). Bioactive cinnamic acid derivatives are known for
their medicinal applications including their anticancer
potentials, which, although promising, have largely remained
underutilized. Generally, cinnamic acids are often employed as
an active moiety to enhance the anticancer activity of several
natural products such as tallimustine, howiinol A, and
acronycine, which already possess some anticancer proper-
ties.12 This cinnamoylation approach to enhancing the
anticancer activity of natural products and synthetic molecules
is well documented.13−16 For instance, trimethoxyrescinnamate
is better than reserpine in its efficacy in apoptosis induction in
cancer cells. In addition, introduction of the trimethoxycinna-
moyl functionality to fumagillin, a natural product isolated
from Asparagus fumagitis, leads to a compound (CKD731)
with 1000-fold more potent cancer cell growth inhibitory
activity than the parent compound. In spite of its rich
medicinal significance, cinnamoylation of metal complexes and
their anticancer potentials has yet to be explored. Therefore,
for the first time, we have linked gold(I) phosphine moieties
with propargylated cinnamides to investigate the effect of
cinnamoylation on the anticancer effects of the resulting metal
complexes.
ide (EDC), 1-hydroxybenzotriazole (HOBT), and N,N-
diisopropylethylamine (DIPEA) under a nitrogen atmos-
phere.17 Reactions of 2a−2c with [AuCl(PPh3)] or [AuCl-
(PTA)] under basic conditions conveniently give the desired
air- and moisture-stable gold(I) complexes 3a−3c and 4a−4c,
respectively, as shown in Scheme 1. The newly synthesized
compounds were characterized by multinuclear NMR spec-
troscopy (Figures S1−S15) and, in the case of 3a, by X-ray
crystallography.
Formation of the gold alkyne complexes 3a−3c and 4a−4c
was indicated by the disappearance of the terminal proton (δ
1
2.0−2.2) in the H NMR spectra. Complexes 3a−3c each
show a singlet resonance in their 31P NMR spectrum at about
at δ 40 and the PTA analogues 4a−4c at about δ −51. The
structure of 3a was also confirmed by X-ray diffraction. The
molecular structure of 3a is shown in Figure 3, with the
selected bond lengths and angles in the caption. As expected,
the gold atom in 3a is approximately linearly coordinated, and
the Au−P [2.2737(12) Å] and Au−C [1.986(4) Å] bond
lengths are similar to those in the gold alkynyl complexes B
To investigate the role of the cinnamide alkynes in the
biological spectrum of the gold species, a range of mono-, di-,
and trimethoxy-substituted cinnamides were used. We also
chose lipophilic (PPh3) and hydrophilic (PTA = 1,3,5-triaza-7-
phosphaadamantane) phosphine ligands to study the effect of
water solubility on the anticancer activities of this novel class of
gold complexes.
Herein, we report the synthesis of six gold(I) alkyne
compounds containing methoxy-substituted cinnamide moi-
eties (Figure 2), along with their luminescent and anticancer
properties and their mechanism of action.
RESULTS AND DISCUSSION
■
Figure 3. Molecular structure of 3a. Ellipsoids show 50% probability
levels. Selected bond lengths (Å) and angles (deg): Au1−P1
2.2737(12), Au1−C1 1.986(4), C1−C2 1.215(6), C2−C3
1.466(6), C3−N1 1.460(5), N1−C4 1.340(5), C4−C5 1.480(6),
C5−C6 1.340(6), O1−C4 1.239(5), O2−C13 1.420(6); P1−Au1−
C1 176.38(13), C2−C3−N1 111.5(4), C3−N1−C4 120.0(4).
Synthesis of Gold Complexes. The propargylated
cinnamides 2a−2c were synthesized according to a previously
reported method by reacting propargylamine with the
corresponding cinnamic acids (1a−1c) in the presence of N-
[3-(dimethylamino)propyl]-N′-ethylcarbodiimide hydrochlor-
B
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Figure 4. Electronic absorption spectra of complexes 3a−3c and 4a−4c (30 μM) in Tris-buffered saline (50 mM Tris, 4 mM NaCl, pH 7.4) over a
period of 72 h.
Table 1. Antiproliferative Activity of Alkynes (2a−2c) and Gold(I) Alkynyl Complexes (3a−3c and 4a−4c) Expressed as IC50
Values (μM) Obtained from Three Independent Experiments
compound
2a
A549 (lung)
>25
D24 (melanoma)
HT1080 (fibrosarcoma)
Hek293T (normal)
>25
>25
>25
2b
2c
>25
>25
>25
>25
>25
>25
>25
>25
3a
3b
3c
4a
4b
4c
15.5 1.16
10.61 1.77
6.05 0.21
>25
>25
>25
18.9 2.31
>25
5.69 0.38
2.46 0.31
2.3 0.15
1.53 0.12
13.59 0.65
17.32 2.52
8.93 0.78
7.90 0.56
14.67 2.65
23.5 1.68
2.01 0.12
3.42 0.26
1.62 0.31
6.59 0.35
10.44 0.68
3.96 0.34
3.89 0.23
10.65 1.13
0.63 0.12
8.14 0.86
7.25 1.24
7.51 0.57
11.46 1.17
9.88 0.32
4.67 0.37
3.36 0.32
8.96 0.87
4.78 0.58
[AuCl(PPh3)]
[AuCl(PTA)]
cisplatin
[2.2795(6) and 1.998(2) Å, respectively] and C [2.2829(6)
and 2.001(2) Å, respectively].11
4a−4c toward A549 (lung), D24 (melanoma), and HT1080
(fibrosarcoma) cancer cells were carried out using MTT assay.
Cisplatin was used as a positive control. As shown in Table 1,
the gold(I) complexes 3a−3c and 4a−4c triggered strong
cytotoxic effects, whereas [AuCl(PPh3)] and [AuCl(PTA)]
only showed moderate activity and the free alkynes 2a−2c
were inactive. These results clearly indicate that the gold(I)
alkynyl fragment enhances the anticancer activity in this series
of molecules. The triphenylphosphine-containing complexes
3a−3c displayed cell growth inhibition effects in all three
tested cancer cell lines, with IC50 values in the low micromolar
range (1.53−15.5 μM). The IC50 values of gold complex 3c
were better than those for a series of recently studied gold(I)
alkynyl complexes, which showed growth inhibition values in
the range of 2.65−51.78 μM against different cancer cells.18−21
Compound 3c was the most active complex in the series and
exhibited a 15-fold higher cytotoxicity than cisplatin against
D24 melanoma cells. The gold complexes containing the
water-soluble PTA ligand (4a−4c) were moderately active
toward D24 and HT1080 cancer cells, with IC50 values in the
range 3.96−17.32 μM. The cytotoxicity of the compounds was
also influenced by the number of methoxy groups present in
the alkyne moiety. Complexes 3c and 4c containing three
methoxy substituents showed the highest toxicity toward
Stability in Dimethyl Sulfoxide (DMSO) and Physio-
logical-like Conditions. To investigate the stability of the
synthesized gold complexes, the 31P NMR spectra of
complexes 3a−3c and 4a−4c in DMSO-d6 were measured
over a period of 72 h. From the results, no significant changes
were observed in the spectra, indicating that the complexes did
not undergo any reduction or participate in ligand-exchange
reactions with DMSO-d6 (Figures S16−S21). The stability of
the complexes was also monitored under physiological-like
conditions in a buffer solution (50 mM Tris, 4 mM NaCl, pH
7.4) over 72 h using UV−vis spectroscopy. As shown in Figure
4, no significant time-dependent spectral changes were
observed in the electronic spectra of complexes 3a−3c, 4a,
and 4b, indicating that the complexes were stable under
physiological-like conditions. Complex 4c did not show the
characteristic absorbance peak between 300 and 340 nm
attributed to electronic transitions of the alkyne to gold(I), but
the presence of an absorbance peak at 520 nm suggests
reduction of the complex to colloidal gold.
In Vitro Cytotoxicity. Proliferation assays for the alkyne
compounds 2a−2c, gold precursors [AuCl(PPh3)] and
[AuCl(PTA)], and gold(I) alkynyl complexes 3a−3c and
C
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Figure 5. Antitumor activity of 3c on 3D cellular spheroids of D24 cancer cells. (A) Cellular spheroids formed in 96-well plates treated with 3c for
3 days examined using phase-contrast light microscopy. The arrows indicate areas of extensive disaggregation. (B) Representative images of calcein
AM (live)/propidium iodide (dead) staining on D24 MCSs after treatment with different concentrations of the gold complex 3c.
Figure 6. Electronic absorption spectra of gold(I) alkynes 3a−3c and 4a−4c in the presence of increasing concentrations of CT-DNA (0−25 μM).
cancer cells compared to those containing only one (3a and
4a) or two (3b and 4b). The results from the proliferation
assays using human embryonic kidney cells indicated that the
newly synthesized compounds were moderately selective
toward tumor cells. For instance, complex 3c, the most active
compound among the series, displayed 5 times more selective
cytotoxicity toward D24 and HT1080 cells compared to
Hek293T cells.
3D Tumor Spheroidal Models. Tumor 3D spheroidal
models are considered better models for drug efficacy testing
than 2D monolayer cells because spheroids mimic to some
degree in vivo solid tumors with respect to the development of
an extracellular matrix, metabolism, nutritional concentration
gradients (oxygen and glucose), and outer layer to core cell
proliferation.22,23 These models also demonstrate higher drug
resistance and tumor-like penetration of drug molecules.
Herein, we investigated the cytotoxicity of the most active
compound 3c in D24 spheroids. Spheroids formed over 24 h
of incubation in 96-well Corning spheroidal microplates were
untreated or treated with different concentrations of the gold
complex for 3 days and examined for growth inhibition using
phase-contrast microscopy. As shown in Figure 5A, the
treatment with 3c led to disruption and disaggregation of the
spheroidal structure of D24 melanoma tumors in a
concentration-dependent manner, with extensive disaggrega-
tion at concentrations of 25 and 12.5 μM. Because the
spheroids were significantly disaggregated by the complex, the
survival conditions, following drug treatment, were investigated
using a live/dead viability assay. In this assay, the intracellular
esterases in living cells convert nonfluorescent cell-permeant
calcein AM into green fluorescent calcein. In contrast,
propidium iodide can only enter dead cells and emits 20-fold
higher fluorescence upon binding with nucleic acids. As shown
in Figure 5B, untreated spheroids emitted steady green
fluorescence from the live cells. However, the green
fluorescence was significantly weakened, and bright-red
fluorescence was observed when the spheroids were treated
with 12.5 and 25 μM concentrations of 3c, indicating that
severe cell damage had occurred. These results revealed that
complex 3c induced cell death in a dose-dependent manner in
a 3D spheroidal culture.
D
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Figure 7. DNA cleavage study of plasmid PBr322 (300 ng) with varying amounts of 3a−3c and 4a−4c (1, 5, and 10 μM). The incubation was
performed at 37 °C for 1 h, and the gel electrophoresed. The gel was then stained with SYBR safe for 30 min and photographed under UV light.
Lanes A and K: PBr322 DNA (15 ng). Lanes B−D: DNA + 1, 5, and 10 μM 3a, respectively. Lanes E−G: 1, 5, and 10 μM 3b, respectively. Lanes
H−J: 1, 5, and 10 μM 3c, respectively. Lanes L−N: 1, 5, and 10 μM 4a, respectively. Lanes O−Q: 1, 5, and 10 μM 4b, respectively. Lanes R−T: 1,
5, and 10 μM 4c, respectively.
DNA Binding Properties. To understand the interactions
between the synthesized gold complexes and DNA, the UV−
vis spectra of 25 μM solutions of 3a−3c and 4a−4c were
recorded in the absence and presence of increasing amounts of
calf-thymus DNA (CT-DNA; 5−25 μM). From the obtained
electronic spectral data (Figure 6), no significant changes in
the absorbance were observed for any of the complexes in the
presence of increasing concentrations of DNA except for 3a. In
this case, a slight decrease in the absorbance (hypochromism)
with an increase in the DNA concentration was observed,
indicating that the complex binds to DNA through
intercalation involving interactions between aromatic chromo-
phores and DNA base pairs.
Table 2. TrxR Inhibition Activity (EC50, μM) of Gold(I)
Alkynes 3a−3c and 4a−4c
compound
TrxR
GR
selectivity GR/TrxR (x-fold)
3a
3b
3c
4a
4b
4c
0.28 0.05
0.21 0.08
0.19 0.03
0.85 0.12
1.64 0.18
2.45 0.09
1.36 0.07
2.93 0.14
2.16 0.08
1.87 0.23
5.67 0.36
2.62 0.44
4.8
13.9
11.3
2.2
3.4
1.0
out a docking study with the crystallographic structure of TrxR
(PDB code: 2J3N). A selenocysteine residue present at the C-
terminal active site is essential for catalysis. The C and N
terminals are the flexible ends and the redox centers are
reactive toward electrophilic inhibitors, thus demonstrating a
target for antitumor drug development. It has been reported
that gold complexes inhibit TrxR by binding in the cavity
between the C- and N-terminal active sites.27
As shown in Figure 8 and Table 3, all of the compounds
bind in a similar mode with the TrxR enzyme at the principal
catalytic site through steric and hydrogen-bonding interactions.
The lipophilic compounds 3a−3c showed the best TrxR
inhibitory activity, indicated by the high moldock score
(−159.76 to −157.79) compared to compounds 4a−4c
(−117.58 to −108.66) containing the water-soluble PTA
ligand. These results, together with the biological experiments,
suggest that the compounds’ cytotoxicity is through TrxR
inhibition.
Reactive Oxygen Species (ROS). Recent reports suggest
that ROS (superoxide, hydrogen peroxide, and hydroxyl
radicals) play a major role in the mechanism of many
anticancer drugs through the initiation of programmed cell
death pathways.28,29 Inhibition of the antioxidant enzyme TrxR
may result in increased ROS accumulation, leading to DNA
damage and apoptosis in cancer cells.30 Therefore, we
determined the intracellular ROS levels in D24 cells after
treatment with different concentrations of complex 3c using
the ROS indicator 6-carboxy-2′,7′-dichlorodihydrofluorescein
diacetate (carboxy-H2DCFDA). As shown in Figure 9,
treatment with 1, 2, and 4 μM complex 3c in D24 melanoma
cells resulted in 1.5-, 2.3-, and 3.1-fold increases, respectively,
of ROS production compared to the control samples. These
data indicate the involvement of ROS-mediated cell death after
treatment with complex 3c.
DNA Cleavage Studies. It has been established that metal
ions can activate and initiate DNA endonucleolytic cleavage
reactions.24,25 Therefore, the nuclease activity of the gold(I)
alkynes was investigated using pBR322, a supercoiled plasmid
DNA. In agarose gel electrophoresis, the naturally occurring
supercoiled DNA (form I) moves faster than the relaxed open-
circular DNA (form II), also known as a nicked circular form
of DNA, whereas a linear form of DNA (form III), obtained
from the cleavage of both DNA strands, can migrate between
forms I and II. In this study, different concentrations of gold(I)
alkynes (1, 5, and 10 μM) were incubated with pBR322 DNA
and agarose gel electrophoresis was carried out. As shown in
Figure 7, complexes 3a−3c and 4a−4c showed minor or no
cleavage activity under the conditions adopted in this study.
Thioredoxin Reductase (TrxR) Inhibition. It has been
reported that TrxR represents a critical pharmacological target
for gold(I) complexes, and its overexpression in many tumor
cell lines is associated with poor cancer cell prognosis and drug
resistance.5,26 Therefore, we investigated the TrxR inhibition
ability of the gold(I) alkynes to determine possible relation-
ships between the TrxR activity and their cytotoxicity. In this
assay, we also used structurally and functionally similar
glutathione reductase (GR) as a reference enzyme besides
the target enzyme TrxR to determine the specificity of the
enzyme inhibition. The difference between TrxR and GR is the
presence of a cysteine fragment in the active site of GR,
whereas TrxR contains a selenocysteine fragment. Therefore, a
selective TrxR inhibitor would react with the selenolate in
TrxR and trigger lower inhibition against GR. The results,
shown in Table 2, indicated that the gold(I) alkynes 3a−3c
exhibited significant TrxR inhibitory activity with EC50 values
of 0.19−0.28 μM, whereas 4a−4c showed moderate inhibition
with EC50 values of 0.85−2.45 μM. Further, compounds 3a−
3c had higher selectivity toward TrxR (4.8−13.9-fold) with
moderate activity on GR.
Dissipation of the Mitochondrial Membrane Poten-
tial. Dissipation of the mitochondrial potential is known to
occur at the early stages of apoptosis. Enhanced ROS
generation can cause oxidative stress, thereby leading to
altered mitochondrial membrane potentials.31 The effect of
Molecular Docking. To better understand the binding
modes of compounds 3a−3c and 4a−4c with TrxR, we carried
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Figure 8. Pose orientation and binding interaction of 3a−3c and 4a−4c with TrxR (PDB code: 2J3N). Red denotes steric interactions and blue
hydrogen-bonding interactions.
Table 3. Docking Scores and Interactions of Compounds
3a−3c and 4a−4c with TrxR
docking
score
hydrogen-
bonding
compound (kcal mol−1
)
steric interactions
Pro 408, His 403
interactions
3a
3b
3c
4a
4b
−159.76
−158.58
−157.79
−117.58
−110.56
Ser 483, Val
484
Gly 470
Thr 412, Cys 475, Trp 407,
Ser 404, Thr 480, Glu 477
Glu 477, Thr 481, Tyr 405,
Ser 404
Leu 409, Trp 407, Val 474,
Glu 477
Thr 480, Tyr
405, Ser 404
Leu 409
Ser 491, Ile 492, Ser 404, Phe Gln 494, Tyr
406, Cys 475, Pro 408
405, Cys 475,
Leu 409
Figure 9. Effect of complex 3c on intracellular ROS accumulation.
Treated D24 cells were stained with the ROS indicator carboxy-
H2DCFDA and analyzed using flow cytometry.
4c
−108.66
Ser 483, Thr 481, Ser 404, Ala Ser 483, Val
494, Cys 475, Phe 406, Pro
408, Leu 409
484, Cys 475
Hoechst Staining. Apoptosis is a process of programmed
cell death that plays an important role in maintaining cellular
homeostasis and regular functioning of cells. Changes in the
morphological features, such as nuclear fragmentation,
chromatin condensation, cell shrinkage, and rupture of the
cell membrane, are hallmarks of apoptotic cells.33,34 In order to
investigate the role compound 3c plays in induced cell death,
Hoechst 33242 staining was carried out on D24 cells after
treatment with different concentrations of the compound for
48 h. As shown in Figure 11, the control cells were uniformly
stained without any morphological changes, whereas the 3c-
treated cells were significantly more stained because of nucleus
condensation. Moreover, increasing the concentration of 3c
from 1 to 4 μM resulted in a significantly higher number of
apoptotic cells. As seen from the bright-field images, cell
complex 3c on intracellular mitochondrial membrane
potentials (DΨm) was studied using a JC-1 fluorescent
ratiometric probe.32 In this assay, JC-1-mitochondrial-specific
cationic dye aggregates in the intact mitochondria and emits a
red fluorescence, whereas in depolarized mitochondria, the dye
forms monomers and emits a green fluorescence. Therefore,
changes in the mitochondrial membrane potential during
apoptosis are indicated by an increase in the green/red
fluorescence ratio. As shown in Figure 10, D24 cells after
treatment with complex 3c showed a decrease in the red
fluorescence and an increase in the green fluorescence in a
concentration-dependent manner. These results indicate that
the induction of apoptosis by 3c is associated with a
mitochondrial or intrinsic pathway.
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Figure 10. Effect of complex 3c on the mitochondrial membrane potential. D24 cells incubated with the complex at different concentrations were
stained with JC-1 and observed under a fluorescence microscope.
shrinkage was also observed after treatment with 3c, indicating
that apoptosis was involved in compound 3c-induced cell
death.
As shown in Figure 12, no detectable ISV inhibition was
observed in vehicle (0.1% DMSO)-treated embryos. In
contrast, compound 3c treatment resulted in the dose-
dependent inhibition of ISV sprouting (highlighted with
asterisks in Figure 12B′−D′). Partial inhibition of the ISV
formation was observed after treatment with 0.1 μM
compound 3c, with significant inhibition after treatment with
0.25 and 0.5 μM concentrations. In contrast, cisplatin
treatment resulted in minimal effects on the ISV formation
(E and E′) even at higher concentration (1 μM). The positive
control displayed significant inhibition at all of the tested
concentrations with complete growth inhibition at 0.5 μM
axitinib. Overall, these results indicate that gold(I) alkyne 3c
displayed antiangiogenic properties in a transgenic zebrafish
model.
Inhibition of Angiogenesis in a Transgenic Zebrafish
Model. It has been reported that TrxR inhibition could also
contribute to angiogenesis inhibition apart from the inhibition
of tumor growth and the induction of cancer cell death.35
Further, gold(I) alkynyl compounds containing napthalimide
ligands have a significant effect on angiogenesis inhibition.36
Therefore, we investigated the angiogenesis-inhibiting proper-
ties of 3c using a transgenic Tg(fli1a:EGFP) zebrafish model.
The latter line expresses enhanced green fluorescent protein
(EGFP) in endothelial cells and provides a rapid in situ biotest
to visualize the development of characteristic patterns of
intersegment vessels (ISVs). The biotest principles are based
on the induction of dose-dependent inhibition of ISV
formation, which can be microscopically visualized with the
reduction of the fluorescence signal and quantified to provide
dose response analysis.37 In this assay, Tg(fli1a:EGFP)
embryos at the 24 h postfertilization (hpf) stage, before
sprouting of ISVs had begun, were exposed to concentrations
of 3c and cisplatin that did not induce any discernible
phenotypic effects or embryo mortality. The embryos treated
with axitinib, a pyrazole compound that displays strong kinase
inhibition properties, were used as the positive controls.
CONCLUSIONS
■
In summary, we have synthesized a new series of gold(I)
alkynyl complexes containing methoxy-substituted cinnamide
ligands and investigated their cytotoxic properties on 2D and
3D cancer models. The results showed that hybridization of
tertiary phosphine gold(I) chloride with methoxy cinnamide
alkynes led to higher cytotoxicity profiles on different cancer
cells than their individual counterparts. Trimethoxy substitu-
tion on the cinnamide moiety appeared to be particularly
advantageous (3c and 4c) over the mono- and dimethoxy
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Figure 11. Nuclear morphological changes in the D24 cells after treatment with different concentrations of complex 3c as determined by Hoechst
33242 staining.
or external 85% H3PO4 (31P), and coupling constants (J) are given in
hertz. Mass spectra were acquired on a PerkinElmer Axion 2 time-of-
ligands. Replacement of triphenylphosphine by PTA resulted
in a reduction of the activity. The most potent compound in
flight electrospray ionization (ESI) spectrometer. Elemental analyses
the series 3c displayed antiproliferative activities in the range of
were carried out by the Chemical Analysis Facility in the Department
1.53−6.02 μM, comparable to, or better than, cisplatin (0.63−
23.5 μM). The cytotoxicity of 3c was 20-fold higher than that
of Molecular Sciences, Macquarie University, New South Wales,
Australia.
of cisplatin toward D24 melanoma cells. Moreover, 5-fold
X-ray Crystallography. Crystals of complex 3a suitable for single-
higher selectivity toward melanoma cells than normal
Hek293T cells was observed. The more physiologically
crystal X-ray diffraction were obtained from dichloromethane/hexane.
A crystal was mounted on a nylon loop using a drop of inert oil
relevant 3D tumor spheroidal assays on D24 melanoma cells
revealed the cytotoxic potential of 3c. Anticancer effects in
D24 cells were triggered by 3c through TrxR enzyme
inhibition and mitochondrial-mediated apoptosis. Compound
3c also has significant antiangiogenic effects in zebrafish
embryos, in contrast to cisplatin, which is almost inactive at the
tested concentrations. In summary, the newly synthesized
alkynyl gold complexes of the type (triphenylphosphine)gold-
(I) alkyne showed promising potential as future cancer
therapeutics.
(Nujol) before being transferred into a stream of cold nitrogen. The
reflections were collected on a D8 Bruker diffractometer equipped
with an APEX-II area detector using graphite-monochromated Mo Kα
radiation (λ = 0.71073 Å) from a 1 μS microsource. For data
collection and data processing, SMART40 and SAINT41 were used,
respectively, and absorption corrections were performed using
SADABS.42 The structure was solved using direct methods and
refined with full-matrix least-squares methods on F2 using the SHELX-
TL package.43,44 The CCDC number for complex 3a is 1873465.
Synthesis. General Procedure for the Synthesis of N-(Prop-2-yn-
1-yl)cinnamamides (2a−2c). To a solution of substituted cinnamic
acid (1a−1c; 1.0 mmol) and EDC (1.2 mmol) in anhydrous
dimethylformamide were added DIPEA (1.2 mmol) and HOBT (1.2
mmol) at 0 °C under a nitrogen atmosphere. After the reaction
mixture was stirred for 10 min, propargylamine (1.0 mmol) was added
and stirring continued at room temperature for 12 h. The reaction
mixture was poured into ice-cold water (25 mL) and extracted with
ethyl acetate (3 × 15 mL). The organic layer was separated, washed
with a NaCl solution, and dried over anhydrous Na2SO4, and the
EXPERIMENTAL SECTION
■
Chemistry. [AuCl(PPh3)]38 and [AuCl(PTA)]39 were prepared
following literature methods. The other chemicals were purchased
and used without further purification. 1H (300 MHz), 31P (121
MHz), and 13C (75 MHz) NMR spectra were obtained as CDCl3
solutions on a Bruker Avance 300 spectrometer at room temperature.
Chemical shifts are referenced to residual solvent signals (1H and 13C)
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Figure 12. Angiogenesis assay in transgenic Tg(fli1a:EGFP) zebrafish embryos. Embryos were treated with different concentrations of compound
3c, with cisplatin and axitinib as positive controls, at 24 hpf. The images of the zebrafish larvae at 4 dpf were captured using a fluorescence
microscope in a green channel: (A and A′) vehicle-treated control embryos; (B−D and B′−D′) compound-3c-treated embryos at 0.1, 0.25, and 0.5
μM; (E and E′) cisplatin-treated embryos (1 μM); (F−H and F′−H′) axitinib-treated embryos at 0.1, 0.2, and 0.5 μM concentrations. ISVs are
shown by white arrows. Inhibition of ISV sprouting is marked with an asterisk. The magnified portions of images A−H are shown in A′−H′,
respectively.
solvent was evaporated. The crude products were purified by column
NMR: δ 165.85, 161.11, 141.63, 129.49, 127.34, 117.30, 114.33,
79.61, 71.75, 55.40, 29.45. ESI-MS: m/z 216.2 ([M + H]+).
(E)-3-(3,4-Dimethoxyphenyl)-N-(prop-2-yn-1-yl)acrylamide (2b).
1H NMR: δ 7.63 (d, J = 15.6 Hz, 1H), 7.12 (dd, J = 8.3 and 1.8 Hz,
1H), 7.05 (d, J = 1.8 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.31 (d, J =
15.6 Hz, 1H), 5.80 (br s, 1H), 4.23 (dd, J = 5.2 and 2.5 Hz, 2H), 3.94
(s, 6H), 2.29 (t, J = 2.5 Hz, 1H). 13C NMR: δ 165.73, 150.83, 149.20,
chromatography using ethyl acetate/hexane (30−50%) as the eluent
to obtain the desired cinnamide alkynes (2a−2c) in 75−90% yield.
(E)-3-(4-Methoxyphenyl)-N-(prop-2-yn-1-yl)acrylamide (2a). 1H
NMR: δ 7.65 (d, J = 15.6 Hz, 1H), 7.48 (d, J = 8.7 Hz, 2H), 6.92 (d, J
= 8.7 Hz, 2H), 6.30 (d, J = 15.6 Hz, 1H), 5.80 (br s, 1H), 4.22 (dd, J
= 5.1 and 2.5 Hz, 2H), 3.87 (s, 3H), 2.29 (t, J = 2.5 Hz, 1H). 13C
I
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Inorganic Chemistry
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141.85, 127.60, 122.16, 117.57, 111.14, 109.74, 79.57, 71.80, 56.00,
55.92, 29.48. ESI-MS: m/z 246.2 ([M + H]+).
(100 μL well−1). 1% DMSO in a complete growth medium was used
as the negative control. After 72 h of incubation, the growth medium
containing the compounds was replaced with 100 μL of a 0.5 mg
mL−1 MTT solution. Incubation was continued for 4 h at 37 °C in the
dark. Unreacted MTT was removed, and 100 μL of DMSO was added
to each well to dissolve the formazan crystals. The absorbances of the
solutions were recorded at 570 nm using a microplate reader
(SpectraMax). The absorbance is proportional to the number of living
cells, and the IC50 values were calculated using Probit software.
Cell Viability on 3D Cellular Spheroids. D24 cells (4 × 103
well−1) were cultured in a blackwell plate and grown in a RPMI
medium supplemented with 10% FBS for 24 h to allow the formation
of 3D cellular spheroids. The formed spheroids were treated with
different concentrations of complex 3c for 72 h, and the effect on
spheroidal growth was examined by phase-contrast light microscopy.
For live dead staining, the drug-treated spheroids were stained with
calcein (2 μM) and propidium iodide (4 μM) for 30 min at room
temperature and the images were captured using a fluorescence
microscope (Bio-Rad).
DNA Binding Studies. The UV−vis absorption studies were
primarily employed to analyze the binding modes of the gold
complexes to CT-DNA. In this process, the gold complexes (3a−3c
and 4a−4c) were dissolved in DMSO and then diluted to the desired
concentration with Tris-buffered saline (25 μM). The quartz cell was
used to carry out spectroscopic titrations by adding increasing
concentrations of CT-DNA to a solution of the complex (25 μM),
and the spectrum was recorded after each addition using an Agilent
Cary 500 spectrophotometer.
(E)-N-(Prop-2-yn-1-yl)-3-(3,4,5-trimethoxyphenyl)acrylamide
(2c). 1H NMR: δ 7.60 (d, J = 15.5 Hz, 1H), 6.76 (s, 2H), 6.35 (d, J =
15.5 Hz, 1H), 5.87 (br s, 1H), 4.23 (dd, J = 5.0 and 2.5 Hz, 2H), 3.91
(s, 9H), 2.30 (t, J = 2.5 Hz, 1H). 13C NMR: δ 165.45, 153.47, 141.95,
139.83, 130.17, 119.07, 105.08, 79.45, 71.88, 61.00, 56.18, 29.51. ESI-
MS: m/z 276.2 ([M + H]+).
General Procedure for the Synthesis of Gold(I) Alkynes (3a−3c
and 4a−4c). To a suspension of [AuCl(PPh3)] or [AuCl(PTA)] (1.0
mmol) in methanol were added cinnamide alkynes (2a−2c, 1.0
mmol) and NaOH (1.1 mmol), and the mixture was stirred for 8 h at
room temperature. The precipitated solid was isolated by filtration,
washed with methanol and diethyl ether, and dried in vacuo to give
the title compounds in good yield (90−95%).
3a. 1H NMR: δ 7.34−7.43 (m, 18H), 6.92 (d, J = 8.6 Hz, 2H), 6.26
(d, J = 15.6 Hz, 1H), 5.78 (br s, 1H), 4.37 (d, J = 4.6 Hz, 2H), 3.86
(s, 3H). 31P NMR: δ 42.12. ESI-MS: m/z 674.3 ([M + H]+). Elem
anal. Calcd for C31H27AuNO2P (673.14): C, 55.28; H, 4.04; N, 2.08.
Found: C, 54.66; H, 3.92; N, 1.99.
3b. 1H NMR: δ 7.60−7.44 (m, 15H), 7.10 (dd, J = 8.3 and 1.8 Hz,
1H), 7.04 (d, J = 1.8 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H), 6.28 (d, J =
15.6 Hz, 1H), 5.83 (br s, 1H), 4.37 (d, J = 4.7 Hz, 1H), 3.93 (s, 6H).
13C NMR: δ 165.49, 150.55, 149.14, 140.79, 134.30, 134.21, 131.66,
129.23, 129.15, 127.93, 121.88, 118.49, 111.12, 109.76, 55.95, 55.91,
31.02. 31P NMR: δ 42.10. ESI-MS: m/z 704.3 ([M + H]+). Elem anal.
Calcd for C32H29AuNO3P (703.15): C, 54.63; H, 4.15; N, 1.99.
Found: C, 54.78; H, 3.95; N, 1.97.
3c. 1H NMR: δ 7.60−7.44 (m, 15H), 6.76 (d, J = 5.8 Hz, 2H), 6.31
(d, J = 15.6 Hz, 1H), 5.86 (br s, 1H), 4.37 (d, J = 4.7 Hz, 2H), 3.92
(s, 6H), 3.89 (s, 3H). 13C NMR: δ 165.19, 153.41, 140.94, 134.39,
134.21, 131.67, 130.51, 130.41, 129.97, 129.31, 129.16, 119.94,
104.93, 77.25, 61.00, 56.17, 31.06. 31P NMR: δ 42.06. ESI-MS: m/z
734.3 ([M + H]+). Elem anal. Calcd for C33H31AuNO4P (733.16): C,
54.03; H, 4.26; N, 1.91. Found: C, 54.31; H, 4.10; N, 1.89.
4a. 1H NMR: δ 7.67−7.51 (m, 1H), 7.47 (d, J = 8.6 Hz, 2H), 6.91
(d, J = 8.7 Hz, 2H), 6.29 (d, J = 15.6 Hz, 1H), 5.99 (br s, 1H), 4.64−
4.45 (m, 6H), 4.33−4.25 (m, 8H), 3.86 (s, 3H). 13C NMR: δ 165.65,
160.92, 140.61, 129.38, 127.61, 118.17, 114.30, 73.32, 73.22, 55.39,
52.54, 52.27, 30.88. 31P NMR: δ −51.18. ESI-MS: m/z 569.2 ([M +
H]+).
DNA Cleavage Studies. The interactions of supercoiled pBR322
plasmid DNA and the complexes were determined by using an
agarose gel electrophoresis experiment. In this method, supercoiled
pBR322 plasmid DNA (300 ng) in a Tris−HCl buffer (5 mM) with
50 mM NaCl (pH 7.2) was treated with different concentrations of
metal complexes (10, 5, and 1 μM), followed by dilution with a Tris−
HCl buffer to a total volume of 20 μL with 10% gel loading buffer, i.e.,
Blue Juice (2 μL). The samples were then incubated at 37 °C for 1 h
and loaded onto 1.5% agarose gel containing 1.0 mg mL−1 SYBR safe
(Thermofischer). Electrophoresis was carried out at 70 V for 60 min
in a TBE buffer (Tris-borate EDTA). UV light was used for
visualizing the obtained bands, and the images were captured by a Gel
Documentation System (Bio-Rad).
Molecular Docking. A docking study was performed using
Molegro Virtual Docker software version 6. ChemDraw 2D Ultra was
used for the formation of 2D structures of the compounds. Cleanup of
the compounds was done, and the energy was minimized using the
MM2 and MOPAC tools of ChemDraw 3D to achieve the local energy
minima. Human mitochondrial enzyme TrxR (PDB ID: 2J3N) was
extracted from the Protein Data Bank (https://www.rcsb.org/) based
on the criteria that they had reasonable resolution (≤2.8 Å). The pdb
had three small molecules, namely, flavin adenine dinucleotide
(FAD), nicotinamide adenine dinucleotide phosphate (NADP), and
(4s)-2-methyl-2,4-pentanediol (MPD). From the three, MPD was
selected as the template for locating the active sites available on chains
A and B, and the rest of the structures were removed. The grid
resolution was set to 0.30 Å. The cavity was created using the
following dimensions while keeping the constraint radius at 14: X,
−95.32 Å; Y, 99.04 Å; Z, 34.77 Å. Along with the other assessment
parameters used in the default settings, ligand evaluation was done
using the Lennard-Jones potential, sp2−sp2 torsions, internal hydro-
gen bond, and internal entropy. The synthesized ligands were
imported, and the docking was performed using the template ligand
MPD. Because of the stochastic nature of the ligand−protein docking
search algorithm, 10 runs were conducted and 10 docking solutions
(pose) were retained for each ligand. From the 10 poses, only one
pose was extracted based on the higher moldock score, rerank score,
and lowest root-mean-square deviation.
1
4b. H NMR: δ 7.56 (d, J = 15.6 Hz, 1H), 7.14−7.03 (m, 2H),
6.90−6.86 (m, 1H), 6.29 (d, J = 15.6 Hz, 1H), 5.88 (br s, 1H), 4.66−
4.48 (m, 6H), 4.34−4.24 (m, 8H), 3.93 (s, 6H). 31P NMR: δ −51.09.
ESI-MS: m/z 599.2 ([M + H]+).
4c. 1H NMR: δ 7.59 (d, J = 15.6 Hz, 1H), 6.79 (s, 2H), 6.28 (d, J =
15.6 Hz, 1H), 5.88 (br s, 1H), 4.65−4.47 (m, 6H), 4.44−4.33 (m,
8H), 3.93 (s, 6H), 3.89 (s, 3H). 31P NMR: δ −50.62. ESI-MS: m/z
629.2 ([M + H]+).
Cell Culture. A549 (lung) and D24 (melanoma) cells were
maintained in a humidified atmosphere of 5% CO2 at 37 °C in a
RPMI medium, and HT1080 (fibrosarcoma) and Hek293T (normal
kidney) cells were grown in Dulbecco’s modified Eagle’s medium
(Gibco, Invitrogen). Both media were supplemented with 10% fetal
bovine serum (FBS), 1% penicillin, and streptomycin (1000 U mL−1).
All of the cell lines were passaged with 0.25% trypsin/1 mM
ethylenediaminetetraacetic acid (EDTA) after reaching 80% con-
fluence and used for the experiments. For cell culture experiments,
stock concentrations of the compounds (10 mM) were made in
DMSO, and the final working concentrations were prepared in a
complete cell growth medium.
MTT Assay. The antiproliferative ability of compounds 2a−2c,
3a−3c, and 4a−4c was investigated using MTT assay. The assay is
dependent on the ability of live cells to reduce MTT (Invitrogen) to a
purple formazan product, which can be measured spectrophotometri-
cally. In this assay, A549 (lung), D24 (melanoma), HT1080
(fibrosarcoma), and Hek293T cells were plated in 96-well plates at
a density of 5000 cells well−1 and incubated overnight. For
determination of the IC50 values, the cells were incubated with
different concentrations of compounds in the range of 100−0.001 μM
Analysis of Intracellular ROS Production. D24 cells were
incubated with the control and different concentrations of the gold
complex 3c (1, 2, and 4 μM) for 48 h. After treatment, the cells were
washed with PBS, collected by trypsinization, and further incubated
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with 10 μM carboxy-H2DCFDA (Invitrogen) for 30 min at 37 °C.
Again, the cells were washed with PBS and immediately subjected to
flow-cytometry analysis.
Assessment of Mitochondrial Membrane Potential (DΨm).
Briefly, D24 cells were untreated or treated with complex 3c (1, 2, and
4 μM) for 48 h. After treatment, the cells were trypsinized, collected
by centrifugation, and then incubated with JC-1 at 37 °C for 20 min.
The stained cells were washed with PBS twice and measured by flow
cytometry.
ACKNOWLEDGMENTS
■
The authors acknowledge the Micro Nano Research Facility,
RMIT University, for providing the facilities to carry out cell
culture experiments.
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AUTHOR INFORMATION
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Corresponding Authors
*E-mail: srinivasareddy.telukutla@rmit.edu.au (T.S.R.).
*E-mail: nedaossadat.mirzadeh@rmit.edu.au (N.M.).
*E-mail: suresh.bhargava@rmit.edu.au (S.B.).
(17) Reddy, V. G.; Bonam, S. R.; Reddy, T. S.; Akunuri, R.; Naidu,
V. G. M.; Nayak, V. L.; Bhargava, S. K.; Kumar, H. M. S.; Srihari, P.;
Kamal, A. 4β-amidotriazole linked podophyllotoxin congeners: DNA
topoisomerase-IIα inhibition and potential anticancer agents for
prostate cancer. Eur. J. Med. Chem. 2018, 144, 595−611.
ORCID
T. Srinivasa Reddy: 0000-0002-2806-4300
Steven H. Priver: 0000-0001-5521-9884
Donald Wlodkowic: 0000-0002-0780-3362
Suresh Bhargava: 0000-0002-3127-8166
Notes
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Anticancer and Antibacterial Activity Studies of Gold(I)-Alkynyl
Chromones. Molecules 2015, 20, 19699−19718.
The authors declare no competing financial interest.
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DOI: 10.1021/acs.inorgchem.9b00281
Inorg. Chem. XXXX, XXX, XXX−XXX