Ferrocene-appended iridium(III) Complexes: Configuration regulation, anticancer application, and mechanism research

Article abstract of DOI:10.1021/acs.inorgchem.9b02227

A series of ferrocene-appended half-sandwiched iridium(III) phenylpyridine complexes have been designed and synthesized. These complexes show better anticancer activity than cisplatin widely used in clinic under the same conditions. Meanwhile, complexes could effectively inhibit cell migration and colony formation. Complexes could interact with protein and transport through serum protein, effectively catalyzing the oxidation of nicotinamide-adenine dinucleotid and inducing the accumulation of reactive oxygen species (ROS, ¹O₂), which confirmed the anticancer mechanism of oxidation. Furthermore, laser scanning confocal detection indicates that these complexes can enter cells followed by a non-energy-dependent cellular uptake mechanism, effectively accumulating in the lysosome (Pearson's colocalization coefficient: ～0.90), leading to lysosome damage, and reducing the mitochondrial membrane potential (MMP). Taken together, ferrocene-appended iridium(III) complexes possess the prospect of becoming a new multifunctional therapeutic platform, including lysosome-targeted imaging and anticancer drugs.

Full text of DOI:10.1021/acs.inorgchem.9b02227

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Ferrocene-Appended Iridium(III) Complexes: Configuration
Regulation, Anticancer Application, and Mechanism Research
Xingxing Ge, Shujiao Chen, Xicheng Liu,* Qinghui Wang, Lijun Gao, Chengfeng Zhao, Lei Zhang,
Mingxiao Shao, Xiang-Ai Yuan, Laijin Tian, and Zhe Liu*
Institute of Anticancer Agents Development and Theranostic Application, Key Laboratory of Life-Organic Analysis and Key
Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, School of Chemistry and Chemical Engineering,
Qufu Normal University, Qufu 273165, China
S
* Supporting Information
ABSTRACT: A series of ferrocene-appended half-sandwiched
iridium(III) phenylpyridine complexes have been designed and
synthesized. These complexes show better anticancer activity than
cisplatin widely used in clinic under the same conditions.
Meanwhile, complexes could effectively inhibit cell migration
and colony formation. Complexes could interact with protein and
transport through serum protein, effectively catalyzing the
oxidation of nicotinamide−adenine dinucleotid and inducing the
accumulation of reactive oxygen species (ROS, 1O2), which
confirmed the anticancer mechanism of oxidation. Furthermore,
laser scanning confocal detection indicates that these complexes
can enter cells followed by a non-energy-dependent cellular uptake
mechanism, effectively accumulating in the lysosome (Pearson’s
colocalization coefficient: ∼0.90), leading to lysosome damage,
and reducing the mitochondrial membrane potential (MMP). Taken together, ferrocene-appended iridium(III) complexes
possess the prospect of becoming a new multifunctional therapeutic platform, including lysosome-targeted imaging and
anticancer drugs.
Additionally, the last one has entered clinical trials.8 Compared
with other metal complexes, the anticancer potential of simple
1. INTRODUCTION
Globally, cancer is responsible for one-sixth of the deaths,
ferrocene derivatives was generally low; however, the
including 9.6 million in 2018.1 Although most small organic
incorporation of a ferrocene group into other transition-
molecules and biologically derived compounds have been
metal complexes can effectively improve the anticancer activity
approved for medical treatment and dominate the pharma-
ceutical market, metallodrugs still attract much attention
of ferrocene.9 Von Poelhsitz et al. attempted to combine
nitrosylruthenium complexes with ferrocene and indicated that
because of their unique biological and chemical character-
istics.2−4 Cisplatin was the first metalloanticancer therapeutic
agent for clinical application and initiated a new field in the
application of transition-metal complexes for therapeutic
design. However, some undesirable side effects, including
the ferrocene ligand plays a crucial role on the anticancer
activity of these complexes.10,11 Rajput et al. prepared a series
of ferrocenyl compounds containing pyridine ligands and
further studied their coordination behavior with platinum,
palladium, rhodium, and iridium.12 In our previous research, a
high systemic toxicity, drug resistance, low selectivity for the
target DNA, and inefficient bioavailability,4 have greatly
ferrocene molecule was introduced into triphenyltin com-
pounds.13 All of these studies indicated that, compared with
limited its application and development, fueling tremendous
research effort into the discovery of other transition-metal
complexes with therapeutic properties.
single metal complexes (ferrocene or other transition-metal
complexes), heteronuclear metal complexes all displayed more
excellent cytotoxic activity against the cancer cell line.
Ferrocene or its derivatives, typical organometallic com-
Iridium(III) (IrIII) complexes have recently burgeoned as
pounds possessing the advantages of mild reversible redox
promising alternatives to platinum-based organometallic
properties, high stability, lipophilicity, and low toxicity, are
anticancer drugs because of their unique anticancer mecha-
highly active against several diseases, especially cancer. Studies
nisms, e.g., involvement in cellular redox reactions (catalyze
showed that the oxidation state of the ferrocenyl moiety plays a
the oxidation of nicotinamide−adenine dinucleotid and induce
crucial role in their cytotoxic effects and put forward an
oxidation-related mechanism.5 Ferrocenium could produce
1
reactive oxygen species (ROS, O2), which, in turn, induce
Received: July 24, 2019
DNA damage and finally exhibit their cytotoxic activities.5−7
© XXXX American Chemical Society
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the accumulation of ROS) and inhibitor of various proteins
[vascular endothelial growth factor receptor 3 (VEGF R3), B-
cell lymphoma-2 (Bcl-2), etc.].14,15 Additionally, the rational
design of the ligands around iridium has achieved success in
the development of anticancer complexes, including the
introduction of C^N-chelating ligands instead of N^N-
chelating ligands,15,16 the replacement of smaller counter-
anions with larger ones,17 etc. Most importantly, integration of
the anticancer potencies and the favorable targeted fluo-
rescence characteristics of IrIII complexes supplies us with the
possibility to construct novel theranostic platforms.18
Different from traditional single metal complexes, multi-
nuclear or heteronuclear metal complexes have confirmed
potential advantages in the field of anticancer, including better
biological activity and different anticancer mechanisms.19
Given the great potential of ferrocene and IrIII complexes in
the field of anticancer, in this paper, ferrocene-modified
phenylpyridine was prepared by the classical Wittig reaction,
further reacted with the dimer of IrIII, and constructed the
iridium−iron heteronuclear anticancer complexes (complexes
1−4, Figure 1). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-
iridium(III) phenylpyridine (5 and 6) complexes, especially for
trans-configuration complexes (1 and 3). In addition, we
investigated the mechanism of action and evaluated the
imaging capabilities of iridium−iron heteronuclear complexes.
2. RESULTS AND DISCUSSION
Ferrocene-appended half-sandwiched IrIII complexes ([(η5-
Cp*)Ir(C^N)Cl]) were obtained by the reaction of [(η5-
C5Me5)IrCl2]2 (dimer 1) and [(η5-C5Me4C6H5)IrCl2]2 (dimer
2) with ferrocene-modified phenylpyridine (C^N) ligands (L1
and L2, Scheme 1). C^N-chelating ligands (L1 and L2) were
obtained by the classical Wittig reaction, and pure products
were obtained by column chromatography.20 The cis and trans
1
structures were certified by H NMR spectroscopy (Figure
S1). Hydrogen atoms of the double bonds are shown in the
range of 6.0−7.0 ppm with coupling constants of 11.9 and 16.1
Hz, respectively.21 Complexes 1−6 were newly synthesized
and obtained in good yields (>80%; Figure 1) and fully
1
characterized by H NMR spectroscopy, mass spectrometry
(MS), and elemental analysis (Figures S2−S10). Deuterated
reagents CDCl3 (7.26 ppm) and (CD3)2SO (2.50 ppm) were
1
used as solvents to test the H NMR spectra of 1−6. The
hydrogen atoms of methylcyclopentadiene (η5-C5Me5) are
shown in the range of 1.5−2.0 ppm, and the hydrogen atoms
on ferrocene are shown in the range of 4.0−4.5 ppm.
Additionally, iridium−iron complexes used multiple solvents
in the process of synthesis and purification, including methanol
(MeOH; 3.32 ppm) and dichloromethane (5.30 ppm), which
1
showed some residues in the H NMR spectra.
Single crystals of complexes 1 and 2 suitable for X-ray
diffraction analysis were obtained by the slow diffusion of n-
hexane into a saturated dichloromethane solution of
complexes. The crystal structures of 1 and 2 are depicted in
Figure 2A, and crystallographic data are listed in Table S1.
Complexes show a “three-leg piano-stool” geometry; interest-
ingly, one crystal unit cell contains two molecules for complex
2. Obviously, complexes possess the expected trans/cis-olefin
configuration, and ferrocene exhibits the superimposed
configuration. The selected bond lengths and angles are
shown in Table 1; around the central iridium ion, the distance
of Ir−Cl is the longest. This conclusion is consistent with the
result that the Ir−Cl bond is the active center of half-
sandwiched iridium complexes.22
Figure 1. Structures of the designed complexes (1−6).
trazolium bromide (MTT) assay indicates that iridium−iron
complexes show better anticancer activity than ferrocene-
modified phenylpyridine (L1 and L2) and half-sandwiched
Scheme 1. Synthesis Processes of Ferrocene-Modified Chelating Ligands and Iridium−Iron Complexes
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subjected to two consecutive scans from 0 to 1.3 V at a scan
rate of 100 mV s−1. As shown in Figure 3, similar chemical
redox behavior, and two reversible redox processes were found
to give very strong cathodic peaks at +0.583 and +1.066 V and
anodic peaks at +0.259 and +0.754 V for 1 and cathodic peaks
at +0.639 and +1.189 V and anodic peaks at +0.242 and
+0.804 V for complex 2. The reversible redox peaks of a1 and
a2 correspond to the redox reaction of the ferrocene group,
while for the metal iridium, this reaction occurs at b1 and b2.
The electrochemical data show that complexes with the
analogous structures have similar CV behaviors. A comparison
of the cyclic voltammograms of complexes 1 and 2 and
ferrocene indicates that ferrocene-modified ligands have no
significant effect on the electronic properties of these
complexes.
2.2. Cell Cytotoxicity Assay. The anticancer activity of
these iron−iridium heteronuclear complexes toward three
human cancer cell lines [A549 (lung cancer cells), HeLa
(cervical cancer cells), and HepG2 (hepatoma cells)] and the
normal cell BEAS-2B (human bronchial epithelioid cells) was
determined by MTT assay, and cisplatin was included as a
control. The cancer cells were cultivated with different
concentrations of complexes for 24 h to obtain the IC50
values (the concentration of 50% cell growth is inhibited);
unfortunately, no selectivity was observed for cancer cells
versus normal cells, and the results are listed in Table 2.
Figure 2. (A) Molecular structures for complexes 1 and 2. Hydrogen
atoms have been omitted for clarity. (B) Color-coded electrostatic
potential calculated on the van der Waals surfaces of complexes 1 and
2.
Table 1. Distances around the Central Iridium and Selected
Angles for the Crystals of 1 and 2
distance (Å)
θ (deg)
complex
Ir−Cp1
Ir−N1
Ir−C7
Ir−Cl1
P1−Cp2
1
2
1.833
1.830
2.101
2.108
2.056
2.042
2.406
2.417
9.39
55.35
Table 2. IC50 Values of Complexes 1−6, Ferrocene-
Modified C^N Ligands (L1 and L2), and Cisplatin against
A549, Hela, HepG2, and BEAS-2B Cells Determined by
MTT Assay after 24 h
2.1. Stability and Electrochemical Properties. The
stabilities of complexes 1 and 2 were investigated by 1H NMR
spectroscopy at 310 K. Complexes 1 and 2 were dissolved in
80% dimethyl sulfoxide (DMSO)-d6/20% D2O (v/v). As
shown in Figures S12 and S13, no additional peaks were found
IC50 (μM)
1
in the H NMR spectra after 24 h, indicating the stability of
complex
A549
Hela
HepG2
BEAS-2B
iridium−iron complexes under these conditions. In addition,
the stability of complexes 1 and 2 was also monitored by UV−
vis spectroscopy in a 20% DMSO/80% H2O (v/v) solution at
298 K for 8 h, and no obvious changes were monitored for the
absorbance (Figure S11). Overall, stability studies suggested
that these complexes possessed sufficient stability for further
studies of the chemical reactivity and biological activity.23
Cyclic voltammetry (CV) was performed to further investigate
the electrochemical properties of complexes 1 and 2 and
ferrocene using dichloromethane as the solvent and
tetrabutylammonium hexafluorophosphate as the supporting
electrolyte (Figures 3 and S21). Complexes 1 and 2 were
1
2
3
4
5
6
L1
L2
3.1 0.1
5.3 0.1
3.2 0.2
7.6 0.1
27.6 0.6
8.5 1.4
91.9 0.3
72.5 1.6
21.3 1.7
2.4 0.01
3.9 0.7
3.7 0.4
5.3 0.7
35.6 1.6
11.0 0.1
10.0 2.0
>100
2.2 0.01
2.4 0.1
1.8 0.6
3.5 0.3
32.3 0.8
13.6 1.2
>100
3.4 0.4
3.6 0.1
3.3 0.1
4.1 0.2
>100
22.7 1.1
cisplatin
7.5 0.2
Figure 3. Evaluation of the cyclic voltammograms of 1 and 2 (1 mM) in dichloromethane solutions. Scan rate = 100 mV s−1. Data are quoted as
mean SD of three replicates.
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Figure 4. (A) Wound healing assay against A549 cells incubated with complexes 1 and 2 (1.0 × IC50) for 24 h. Scale bar: 200 μM. (B) Histograms
of wound healing assay after 24 h. Data are quoted as mean SD of three replicates.
Figure 5. (A) UV−vis spectrum of BSA in a 5 mM Tris-HCl/10 mM NaCl buffer solution (pH = 7.2) with an increase of complex 1 (0−10 μM).
The arrows show the direction of changes in absorbance. Inset: Wavelength from 260 to 290 nm. (B) Fluorescence spectra of BSA (5 μM; λex
280 nm; λem = 350 nm) in the absence and presence of 1 (0−10 μM).
=
Obviously, these complexes all exhibit consistent sensitivity
to A549, Hela, and HepG2 cancer cells. As shown in Table 2,
compared with ferrocene-modified C^N ligands (L1 and L2)
and ferrocene-unattached structural IrIII complexes 5 ([(η5-
C5Me5)Ir(bipy)Cl]PF6) and 6 ([(η5-C5Me4C6H5)Ir(bipy)Cl]-
PF6) (Scheme S1), iridium−iron heteronuclear complexes
possess better antineoplastic activity under the same
conditions and exhibit a certain broad-spectrum characteristic
(Figure S22), the best of which (complex 3) was almost 13
times that of cisplatin (widely used in the clinic) toward
HepG2 cells. The results indicate that these heteronuclear
complexes possess better anticancer potential after being
constructed by the combination of iridium complexes (5 and
6) and ferrocene (L1 and L2), which individually show the
weaker anticancer activity. Interestingly, the activities of trans-
configurational complexes (1 and 3) are higher than those of
cis-configurational complexes (2 and 4), as evidenced by the
lower IC50 values. To further understand these, quantum
chemical computation was used for assessment after
comprehensive consideration of the crystal structures of
complexes 1 and 2. The data of natural population analysis
(NPA) for the central iridium atom and the leaving group
(chlorine) were analyzed by density functional theory
calculation at the B3LYP/6-31G(d,p) (C, H, N, Cl)/SDD
(Ir) level (Figure 2B). The results of NPA charge population
for chlorine in complexes 1 and 2 were almost the same, with
values of −0.376 and −0.375; however, those for iridium were
0.078 and 0.093, respectively. This is mainly attributed to the
minor dihedral angle (θ) between the P1 and Cp2 rings of
complex 1 (9.39°), leading to better coplanar property and
electron donor capacity for the chelating ligand (L1; Table 1).
Therefore, the lower the charge of the central iridium ion for
complex 1, the more vulnerable it is to loss of the chlorine ion
and the better the anticancer activity.18,22
2.3. Inhibition of Migration and Colony Formation.
The metastasis of cancer cells is a complicated process,
including digestion of the extracellular matrix, growth of
tumors and angiogenesis at new sites, and cell migration to and
invasion of the lymphnodes or circulation system.24−27
Metastasis inhibition is an effective means for cancer
treatment. Herein, wound healing assay was performed toward
A549 cells to confirm whether iridium−iron complexes might
inhibit the migration and invasive properties of tumor cells. As
shown in Figure 4, after 24 h of incubation, complexes 1 and 2
(1.0 × IC50) might inhibit cell migration at wound closure
ratios [WCR = (R0 − R1)/R0 × 100%] of 2.01% and 5.31%,
respectively. Compared with the vehicle control (19.03%), the
smaller WCR indicates that complexes 1 and 2 might inhibit
the migration of A549 cells in vitro, and also 1 performs better
than 2, which was consistent with the results of anticancer
activity in vitro.
Metastatic cancer cells have to proliferate at new sites to
form secondary tumors. Accordingly, for malignant cancer
cells, especially for cancer stem cells, colony formation is a
crucial feature.28−30 Complexes 1 and 2 at different
concentrations were cultivated to ascertain the inhibition of
colony formation. As shown in Figure S23, colony formation
can obviously be inhibited after incubation with complexes 1
and 2 at the same concentration (375 and 750 nM). These
results showed that complexes could effectively inhibit the
growth of cancer cells during a significant period of time.
2.4. Protein Binding Assay. Human serum albumin
(HSA), which can bind a variety of substrates, including metal
cations, hormones, and most therapeutic drugs, is the most
abundant carrier protein in plasma. Because of the advantages
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of abundance, ease of purification, stability, medical signifi-
cance, ligand binding properties, and resemblance to HSA,
bovine serum albumin (BSA) was used as a favorable model to
study the mechanism of binding.23,31 In this work, UV−vis
absorption and fluorescence spectra were used to investigate
the binding of complexes with BSA. As shown in Figures 5A
and S14, with an increase of the complexes, a significant
reduction of absorption was observed at 227 nm, which is
related to the interference between the α-helix of BSA and the
complexes. Because of the influence of water (polar solvent), a
significant red shift was also found at 218 nm. In addition, a
dramatically increase, without any shift at 278 nm, suggested
that complexes can interact with BSA and closely relate to the
resut that the microenvironment of tryptophan, tyrosine, or
phenylalanine (three aromatic acid residues in BSA) was alter.
Therefore, to further explore the microenvironment changes in
the bonding process, synchronous fluorescence spectra with
various concentrations of 1−4 were also recorded at the
wavelength intervals of Δλ = 15 and 60 nm, respectively. As
shown in Figures S16 and S17, the fluorescence intensities for
BSA at 291 and 285 nm (Δλ = 15 and 60 nm, respectively)
were weakened with an increase of the complexes. Addition-
ally, a minor red shift (1−3 nm) occurs at 285 nm (Δλ = 60
nm) at the investigated concentration range; however, almost
no change occurred at the wavelength of Δλ = 15 nm. The
results indicated that the conformation of BSA was changed
and the polarity around the tryptophan residues was
increased.32
In summary, the stable combination with BSA suggested that
BSA is expected to become an excellent carrier for the delivery
of anticancer agents in vivo.
2.5. Catalytic Assay. Being a cofactor, NADH (the
reduced state of nicotinamide adenine dinucleotide) plays an
important role in regulating energy production in mitochon-
dria. Ruthenium(II), rhodium(III), and iridium(III) complexes
have been reported that could catalyze the change of NADH to
NAD+ and induce the production of ROS (1O2), followed by
an anticancer mechanism of oxidation.35 The interaction
between NADH (100 μM) and iridium−iron complexes (1.0
μM) was monitored by UV−vis spectroscopy in 10% MeOH/
90% H2O (v/v) at 298 K after various time intervals. The
maximum absorbances of NADH and NAD+ were monitored
at 339 and 259 nm, respectively (Figures 6 and S20). It is
evident that the intensity of the absorbance at 339 nm
decreased and increased at 259 nm significantly over time.
Furthermore, the turnover numbers (TONs), change ratio at
339 nm, were calculated to assess the catalytic efficiency of
each complex. The TONs changed from 8.68 to 50.32,
especially for complex 1, indicating the favorable catalytic
behavior toward the oxidation of NADH. Additionally, TONs
follow the trend 1, 2 > 3, 4 and 1 > 2; 3 > 4, which contribute
to the introduction of a cis-configuration double bond and
higher numbers of phenyl, leading to a bigger steric hindrance
between the iridium−iron complexes and NADH. Meanwhile,
these results were consistent with the conclusions of cellular
cytotoxicity and BSA binding.
In this study, fluorescence spectra were also used to find the
nature of complex binding to BSA. As shown in Figures 5B and
S15, a significant quenching at 343 nm (the fluorescent
emission peak of BSA) was found with an increase of the
iridium−iron complexes. The quenching rate constant (Kq)
and Stern−Volmer quenching constant (KSV) can be obtained
through the classical Stern−Volmer equation, and the binding
constant (Kb) and binding site number (n) were calculated
through the Scatchard equation (Figures S18 and S19).
Fluorescence quenching is usually divided into two different
mechanisms: dynamic and static.33 As shown in Table 3, the
Excessive ROS accumulation was reported as a major factor
causing cell apoptosis.36,37 In order to investigate the effect of
iridium−iron complexes on the production of ROS in cells, the
intracellular ROS levels of A549 cells were measured by flow
cytometry using an oxidant-sensitive fluorescent probe,
dichlorofluorescein−diacetate (DCFH-DA), after treatment
with complexes 1 and 2 for 24 h. As shown in Figure S24, the
ROS levels showed a dose-dependent increase after treatment
with iridium−iron complexes from 0.25 to 0.5 × IC50.
Compared with the negative, 2.0 and 1.7 times A549 cells
were at high ROS levels for complexes 1 and 2 at the
concentration of 0.5 × IC50, respectively. These data were
consistent with the catalytic effect of complexes, and excessive
production of ROS appears to play a critical role in apoptosis.
2.6. Apoptosis Assay. Apoptosis is a programmed cell
death; many transition-metal-based anticancer drugs can
inhibit cell growth by activating apoptosis.38 In this study,
cells undergoing apoptosis were identified by flow cytometry
with annexin V-FITC/propidium iodide (PI) dual staining
after exposure to complexes. As shown in Figure 7 and Tables
S4 and S5, complexes induced a dose-dependent increase for
apoptotic cells after 24 h. The percentages of late apoptotic
cells increased by 78.8% and 53.2% when the concentration
changed from 1.0 to 3.0 × IC50 for complexes 1 and 2,
respectively. In comparison, 93.7% of the A549 cells survived
for the control. This result confirmed that iridium−iron
complexes could lead to cell death through a high incidence of
apoptosis, which was consistent with the conclusion of MTT
assay.
Table 3. Values of KSV, Kb, Kq, and n for Complexes 1 and 4
at 298 K
complex KSV (×105 M−1
)
Kq (×1013 M−1 s−1
)
Kb (×104 M−1
)
n
1
2
3
4
1.49 0.11
2.16 0.07
1.25 0.10
1.43 0.10
1.49
2.16
1.25
1.43
8.75
8.20
7.29
1.89
1.18
1.26
1.18
0.87
values of Kq ranged from 1.25 × 1013 to 2.16 × 1013 M−1 s−1,
which were almost 1 order of magnitude higher than that of a
pure dynamic quenching mechanism (>2.0 × 1012 M−1 s−1)
and confirmed that iridium−iron complexes could interact
with BSA following a static quenching mechanism.34 Although
the values of n (≈1) were almost similar, the Kb values of
complexes 1 and 2 are higher than those of complexes 3 and 4,
which indicated that the introduction of too many phenyl
groups could increase the steric hindrance between complexes
and BSA. For the same reason, complex 1 (trans configuration)
shows favorable Kb compared with 2 (cis configuration);
similarly, complex 3 > complex 4. Meanwhile, this conclusion
corresponds to the cytotoxicity test in which complexes 1 and
3 show better anticancer activity than the other two complexes.
Generally, IrIII complexes exert their anticancer effects and
induce function decline through disruption of the cell cycle. To
investigate the effect of these complexes on cell cycle arrest,
flow cytometry was used to analyze the DNA content of A549
cells stained by PI. The results are shown in Figure 8 and
Tables S2 and S3, which were obtained after A549 cells were
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Figure 6. Reaction of complex 1 (1.0 μM) and NADH (100 μM) in a 60% MeOH/40% H2O (v/v) mixed solution monitored by UV−vis at 298 K
over 8 h. (B) TONs of complexes 1−4.
Figure 7. (A) Apoptosis of A549 cells analyzed by flow cytometry after treatment with complexes 1 and 2 (concentrations = 1.0, 2.0, and 3.0 ×
IC50) and annexin V-FITC/PI stained for 24 h at 310 K. (B) Histograms of four stages for A549 cells induced by complexes 1 and 2. Data were
averaged for three replicate experiments SD. p values were calculated after a t test against the negative control data: *, p < 0.05; **, p < 0.01.
exposed to complexes 1 and 2 for 24 h at the concentrations of
0.25, 0.5, 1.0, and 2.0 × IC50. As shown, complexes 1 and 2
might induce cell cycle disorder and lead to a dose-dependent
increase in the Sub-G1 phase, the population of which
increased by 10.03% and 14.85% for complexes 1 and 2 at
the concentration of 2.0 × IC50, respectively. The obviously
dose-dependent-increased Sub-G1 peak usually correlated with
apoptosis,39 and this conclusion indicated that iridium−iron
complexes might induce cell death through regulation of the
cell cycle.40
Apoptosis is often closely related to the regulation
mechanism of mitochondria and accompanied by changes of
the mitochondrial membrane potential (MMP); meanwhile,
the decrease of MMP is an important signal in the early
apoptotic process.41 To clarify whether complex-induced
apoptosis occurred through the destruction of mitochondrial
homeostasis, flow cytometry was used to detect the
depolarization of MMP with JC-1 (an ideal fluorescent probe
for detecting MMP) staining. JC-1-treated cells demonstrate
green and red channels on the flow cytometers, and red
fluorescence (JC-1 aggregate) can shift to green fluorescence
(JC-1 monomer) when MMP declines. As shown in Figure
S25 and Tables S6 and S7, a significant dose-dependent
decline of MMP occurs in A549 cells induced by complexes 1
and 2 for 24 h, and the population of mitochondrial-
membrane-depolarized cells increased by 62.4% and 52.6%
for complexes 1 and 2 with a concentration change from 0.25
× IC50 to 2.0 × IC50, respectively. The decline of MMP further
confirmed that iridium−iron complexes could influence the
mitochondria and contribute to apoptosis.
2.7. Cellular Uptake and Localization Assay. A
favorable intracellular uptake mechanism is beneficial to
improvement of the anticancer activity for drugs.42 The
cellular uptake mechanism is mainly divided into two types:
energy-dependent (including active transport and endocytosis)
and non-energy-dependent (including passive transport and
free diffusion). In this study, A549 cells were treated with 1
and 2 at 277 and 310 K for 1 h and then treated with
chlorocyanochlorophenyl (metabolic inhibitor, 50 μM) and
chloroquine (endocytosis modulator, 50 μM) to determine the
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Figure 8. (A) Altered A549 cell cycle analyzed by flow cytometry after being induced by complexes 1 and 2 (concentrations = 0.25, 0.5, 1.0, and
2.0 × IC50) for 24 h at 310 K. (B) Histograms of cell cycle distributions. Data are quoted as mean SD of three replicates. p values were calculated
after a t test against the negative control data: *, p < 0.05; **, p < 0.01.
cell uptake mechanisms. As shown in Figures S26 and S27,
there were almost no changes compared with the control,
which confirmed that the manner in which iridium−iron
complexes enter the cells is independent of the endocytosis
and energy, therefore, corresponding to cellular uptake of the
non-energy-dependent mechanism.43,44
Because of the favorable targeted luminescence property of
iridium−iron complexes, Lysol Tracker Deep Red (LTDR;
lysosome fluorescent probe) and Mito Tracker Deep Red
(MTDR; mitochondria fluorescent probe) were used to
ascertain the subcellular localization in A549 cells through
laser confocal microscopy. As shown in Figure 9, complexes 1
and 2 could effectively accumulate in lysosomes with high
Pearson correlation coefficients (PCCs) of 0.90 and 0.91;
however, the PCCs of the mitochondria were 0.21 and 0.19,
respectively. The results indicated that iridium−iron complexes
can enter the cells and accumulate abundantly in lysosomes.
Lysosomes are acidic intracellular organelles (pH = 4.5−5.5)
and possess the ability to destroy polysaccharides, proteins,
nucleic acids, and other biological macromolecules.44,45 Once
damaged, they release hydrolases, digest the entire cell, and
eventually promote apoptosis. Acridine orange (AO), an
Figure 9. Determination of the intercellular localization of complexes
effective probe (which emits red/green fluorescence in
1 and 2 in A549 cells by confocal microscopy. A549 cells were labeled
lysosomes/cytosol and nuclei), was utilized to ascertain the
with MTDR (A) and LTDR (B) and then exposed to 1 and 2 (1.0 ×
integrity of the acidic lysosomes of A549 cells. A549 cells were
exposed to complexes 1 and 2 (1.0 × IC50 and 3.0 × IC50) for
6 h and then stained with AO (5 μM). As shown in Figure 10,
there was a obvious decrease of red fluorescence in the
presence of the complexes (1.0 × IC50), and obvious lysosomal
damage was found when the concentration of the complexes
increased to 3.0 × IC50. The results confirmed that iridium−
iron complexes might target lysosomes, lead to lysosomal
damage, and eventually induce apoptosis.
IC50). Complexes 1 and 2 were excited at 488 nm, and the emission
was collected at 550 30 nm. MTDR was excited at 644 nm, and the
emission was collected at 690 30 nm. LTDR was excited at 594 nm,
and the emission was collected at 630 30 nm. Scale bar: 20 μm.
3. CONCLUSION
In this work, four iridium−iron heteronuclear complexes have
been designed and synthesized. Iridium−iron complexes show
potent anticancer activities toward A549, Hela, and HepG2
G
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Inorganic Chemistry
Article
localization were carried out on a Carl Zeiss AG */LSM/880NLO
two-photon laser scanning microscope.
4.2. Synthesis of Ferrocene-Modified Ligands (L1 and L2).
L1 and L2 were obtained by the classical Wittig reaction:20,46
Ferrocene Wittig reagents (0.81 g, 1.5 mmol) and 2-(4-
phenylformaldehyde)pyridine (0.18 g, 1.0 mmol) were added to a
100 mL round-bottomed flask under nitrogen. Anhydrous THF (40
mL) was added to the above flask and cooled to 0 °C. The THF
solution of potassium tert-butoxide (0.17 g, 1.5 mmol) was added
dropwise to the above flask and stirred for 30 min at 0 °C, followed by
stirring at room temperature until 2-(4-phenylformaldehyde)pyridine
was consumed completely (monitored by thin-layer chromatogra-
phy). The reaction was terminated with ice water (80 mL). The
resulting precipitate was extracted with THF (30 mL × 3), dried with
anhydrous magnesium sulfate, and distilled under reduced pressure.
The final product was purified by chromatography on a silica gel
column (petroleum ether:ethyl acetate = 15:1 as the eluent), and the
data are as follows.
Figure 10. Observation of lysosomal disruption in A549 cells caused
by complexes 1 and 2 (1.0 and 3.0 × IC50) with AO (5 μM) staining
at 37 °C for 15 min. Emissions were collected at 510 20 nm (green)
and 625 20 nm (red) upon excitation at 488 nm. Scale bar: 20 μm.
1
L1. Yield: 0.194 g (53.2%). H NMR (500 MHz, CDCl3): δ 8.70
(d, J = 4.2 Hz, 1H), 7.99 (d, J = 8.2 Hz, 2H), 7.76 (s, 2H), 7.54 (d, J =
8.1 Hz, 2H), 7.26 (s, 4H), 6.97 (d, J = 16.1 Hz, 1H), 6.74 (d, J = 16.1
Hz, 1H), 4.49 (s, 2H), 4.31 (s, 2H), 4.16 (s, 5H).
1
L2. Yield: 0.189 g (51.8%). H NMR (500 MHz, CDCl3): δ 8.70
cells compared with iridium complexes and ferrocene
monomers while inhibiting the migration and colony
formation of cancer cells. Moreover, these complexes can
bind with BSA effectively, show favorable catalytic effects upon
a change of the coenzyme NADH to NAD+, and induce the
production of ROS. Laser confocal monitoring indicates that
complexes enter the cells in a non-energy-dependent manner,
accumulate abundantly in lysosomes, induce damage of the
lysosomes, disturb the cell circle, and eventually induce
apoptosis. Above all, the combination of ferrocene with
iridium(III) complexes may be a promising strategy for the
construction of a potential multifunctional heteronuclear metal
complex anticancer therapeutic platform.
(d, J = 4.7 Hz, 1H), 7.93 (d, J = 8.3 Hz, 2H), 7.75 (s, 2H), 7.46 (d, J =
8.2 Hz, 2H), 7.24 (d, J = 13.8 Hz, 2H), 6.46 (d, J = 11.9 Hz, 1H),
6.36 (d, J = 11.9 Hz, 1H), 4.22 (s, 2H), 4.16 (s, 2H), 4.11 (s, 5H).
4.3. Synthesis of Iridium−Iron Complexes (1−4). The general
synthesis method is as follows: A solution of dimers of iridium (dimer
1/dimer 2; 39.8 mg/46 mg, 0.05 mmol), sodium acetate (32.8 mg, 0.4
mmol), and ferrocene-modified phenylpyridine ligands L (36.5 mg,
0.1 mmol) in MeOH (40 mL) was stirred at ambient temperature
overnight. Most of the solvents were concentrated in vacuum, kept at
253 K for 12 h, filtered, and washed with cold MeOH and diethyl
ether. 1H NMR (500 MHz, DMSO-d6/CDCl3), ESI-MS, matrix-
assisted laser desorption/ionization (MALDI)-TOF-MS, and ele-
mental analysis of complexes 1−4 are shown in Figures S2, S3, and
S6−S9. The data are as follows.
Complex 1. Yield: 0.063 g (86.5%). 1H NMR (500 MHz, CDCl3):
δ 8.67 (d, J = 5.4 Hz, 1H), 7.82−7.76 (m, 2H), 7.63 (dd, J = 11.2 and
7.6 Hz, 2H), 7.18 (d, J = 7.2 Hz, 1H), 7.05 (t, J = 6.3 Hz, 1H), 6.93
(d, J = 16.0 Hz, 1H), 6.76 (d, J = 15.9 Hz, 1H), 4.52 (d, J = 24.5 Hz,
2H), 4.31 (s, 2H), 4.18 (s, 5H), 1.71 (s, 15H). MALDI-TOF-MS.
Calcd for C33H33NFeIr ([M − Cl]+): m/z 691.704. Found: m/z
691.994. Elem anal. Calcd for C33H33NIrFeCl: C, 54.51; H, 4.57; N,
1.93. Found: C, 54.78.; H, 4.61; N, 1.90.
4. EXPERIMENTAL SECTION
4.1. General Information. IrCl3·3H2O, phenylpyridine, ferroce-
necarboxaldehyde, sodium borohydride, triphenylphosphine hydro-
bromate, 2-(4-phenylformaldehyde)pyridine, sodium acetate, 2,3,4,5-
tetramethyl-2-cyclopentenone (95%), 1,2,3,4,5-pentamethylcyclopen-
tadiene (95%), a n-butyllithium solution (1.6 M in hexane), and all
kinds of organic solvents [methanol (MeOH), tetrahydrofuran
(THF), methylbenzene, etc.] were purchased from Rhea Biotechnol-
ogy Co. Ltd. For the biological experiments, Dulbecco’s modified
Eagle medium, fetal bovine serum, a penicillin/streptomycin mixture,
and trypsin/ethylenediaminetetraacetic acid were purchased from
Sangon Biotech. A549 (lung cancer cells), Hela (cervical cancer cells),
and HepG2 (hepatic carcinoma cells) were obtained from Shanghai
Institute of Biochemistry and Cell Biology. The appropriate dimers of
iridium ([(η5-Cpx)IrCl2]2) were prepared according to literature
procedures.20
NMR spectra were obtained on Bruker DPX 500 spectrometers,
with the chemical shifts reported in parts per million using
tetramethylsilane as the internal standard. MS spectra were measured
on LCQ Advantage MAX and Bruker MicroTOF QII electrospray
ionization (ESI)-quadrupole-time-of-flight (TOF) mass spectrome-
ters. Elemental analysis was performed on a Vario MICRO CHNOS
elemental analyzer. UV−vis spectroscopy was performed on a
PERSEE TU-1901 UV spectrometer. Fluorescence spectra were
collected by a Hitachi F-4600 fluorescence spectrophotometer, with a
400 V voltage and a 5 nm slit width for both excitation and emission.
Cyclic voltammogram experiments were carried out using a CH
Instrument model 600D electrochemical analyzer/workstation.
Induction of apoptosis, cell cycling, and MMP determination were
carried out by an ACEA Novocyte 2040R flow cytometry. Viability
assay (MTT) was measured using a Perlong DNM-9606 microplate
reader at an absorbance of 570 nm. Cell uptake and cellular
Complex 2. Yield: 0.060 g (82.1%). 1H NMR (500 MHz, CDCl3):
δ 8.68 (d, J = 5.4 Hz, 1H), 7.77 (d, J = 7.8 Hz, 2H), 7.67−7.55 (m,
2H), 7.09−7.02 (m, 2H), 6.50 (d, J = 11.8 Hz, 1H), 6.32 (d, J = 11.9
Hz, 1H), 4.31 (d, J = 58.0 Hz, 2H), 4.16 (d, J = 19.7 Hz, 2H), 4.11 (s,
5H), 1.65 (d, J = 12.8 Hz, 15H). ESI-MS. Calcd for C33H33NFeIr ([M
− Cl]+): m/z 691.7. Found: m/z 692.3. Elem anal. Calcd for
C33H33NIrFeCl: C, 54.51; H, 4.57; N, 1.93. Found: C, 54.75.; H,
4.63; N, 1.86.
Complex 3. Yield: 0.070 g (88.6%). 1H NMR (500 MHz, CDCl3):
δ 8.52 (d, J = 5.4 Hz, 1H), 7.78 (d, J = 7.2 Hz, 1H), 7.68−7.58 (m,
3H), 7.56−7.49 (m, 2H), 7.44 (d, J = 6.2 Hz, 3H), 7.05 (d, J = 7.0
Hz, 1H), 6.94 (t, J = 6.2 Hz, 1H), 6.60 (d, J = 6.2 Hz, 2H), 4.48 (s,
2H), 4.32 (s, 2H), 4.17 (s, 5H), 1.88 (s, 3H), 1.72 (d, J = 16.1 Hz,
6H), 1.64 (s, 3H). MALDI-TOF-MS. Calcd for C38H35NFeIr ([M −
Cl]+): m/z 753.770. Found: m/z 754.044. Elem anal. Calcd for
C38H35NIrFeCl: C, 57.83; H, 4.47; N, 1.77. Found: C, 58.10.; H,
4.51; N, 1.73.
Complex 4. Yield: 0.068 g (86.9%). 1H NMR (500 MHz, DMSO):
δ 8.46 (d, J = 5.5 Hz, 1H), 8.08 (d, J = 8.1 Hz, 1H), 7.85−7.78 (m,
2H), 7.48 (s, 1H), 7.32 (d, J = 2.8 Hz, 5H), 7.17 (t, J = 6.6 Hz, 1H),
6.99 (d, J = 8.0 Hz, 1H), 6.38 (d, J = 12.0 Hz, 1H), 6.32 (d, J = 12.0
Hz, 1H), 4.23 (d, J = 32.5 Hz, 2H), 4.17 (s, 2H), 4.10 (s, 5H), 1.71
(s, 3H), 1.61 (d, J = 14.6 Hz, 6H), 1.48 (s, 3H). ESI-MS. Calcd for
C38H35NFeIr ([M − Cl]+): m/z 753.8. Found: m/z 754.3. Elem anal.
H
DOI: 10.1021/acs.inorgchem.9b02227
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Inorganic Chemistry
Article
Calcd for C38H35NIrFeCl: C, 57.83; H, 4.47; N, 1.77. Found: C,
58.04; H, 4.52; N, 1.72.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b02227.
■
S
Details of the experimental section, Scheme S1, Figures
S1−S27, and Tables S1−S7 (PDF)
Accession Codes
CCDC 1940401 and 1940402 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: chemlxc@163.com (X.L.). Fax: 0086-537-4455228.
*E-mail: liuzheqd@163.com (Z.L.). Fax: 0086-537-4455228.
■
ORCID
Xicheng Liu: 0000-0002-5932-7206
Zhe Liu: 0000-0001-5796-4335
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the University Research Development Program of
Shandong Province (J18KA082), the National Natural Science
Foundation of China (Grants 21671118 and 21703118), the
Taishan Scholars Program, Shandong Provincial Natural
Science Foundation (Grant ZR2017MB038), and the High
Performance Computing Center of Qufu Normal University
for support.
(18) Hao, H. L.; Liu, X. C.; Ge, X. X.; Zhao, Y.; Tian, X.; Ren, T.;
Wang, Y.; Zhao, C. F.; Liu, Z. Half-sandwich iridium(III) complexes
with α-picolinic acid frameworks and antitumor applications. J. Inorg.
Biochem. 2019, 192, 52−61.
(19) Anderson, C. M.; Jain, S. S.; Silber, L.; Chen, K.; Guha, S.;
Zhang, W.; McLaughlin, E. C.; Hu, Y.; Tanski, J. M. Synthesis and
characterization of water-soluble, heteronuclear ruthenium(III)/
ferrocene complexes and their interactions with biomolecules. J.
Inorg. Biochem. 2015, 145, 41−50.
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