Dual functions of iridium(III) 2-phenylpyridine complexes: Metastasis inhibition and lysosomal damage

Article abstract of DOI:10.1016/j.jinorgbio.2019.110983

Six N-phenylcarbazole/triphenylamine-appended half-sandwich iridium(III) 2-phenylpyridine complexes ([(η⁵-Cp*)Ir(C^N)Cl]) were prepared and characterized. Compared with cisplatin, these complexes exhibited potential antitumor activity against A549 and HeLa tumor cells, with IC₅₀ values (half-maximum inhibitory concentration) that changed from 2.8 ± 0.8 μM to 39.5 ± 2.7 μM, and could block the migration of tumor cells. These complexes also effectively bound to protein (binding constant: ~10⁴ M^{? 1}) and were transported through serum proteins, catalyzed the oxidation of coenzyme nicotinamide-adenine dinucleotide. Additionally, laser confocal microscopy and flow cytometry confirmed that these complexes possessed a non-energy-dependent cellular uptake mechanism, effectively accumulated in lysosomes (Pearson colocalization coefficient: ~0.74), damaged the integrity of acidic lysosomes, led to a change in the mitochondrial membrane potential, disrupted the cell cycle (G₀/G₁ phase), and eventually induced apoptosis. Above all, these complexes are potential antitumor agents with dual functions: metastasis inhibition and lysosomal damage.

Full text of DOI:10.1016/j.jinorgbio.2019.110983

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Dual functions of iridium(III) 2-phenylpyridine complexes:
Metastasis inhibition and lysosomal damage
Xicheng Liu, Shujiao Chen, Xingxing Ge, Ying Zhang, Yaoqi Xie,
Yingying Hao, Daiqun Wu, Jinmin Zhao, Xiang-Ai Yuan, Laijin
Tian, Zhe Liu
PII:
S0162-0134(19)30559-8
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110983
JIB 110983
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
23 August 2019
11 December 2019
26 December 2019
Please cite this article as: X. Liu, S. Chen, X. Ge, et al., Dual functions of iridium(III)
2-phenylpyridine complexes: Metastasis inhibition and lysosomal damage, Journal of
Inorganic Biochemistry (2018), https://doi.org/10.1016/j.jinorgbio.2019.110983
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Dual functions of Iridium(III) 2-phenylpyridine complexes: metastasis inhibition
and lysosomal damage
Xicheng Liu+*, Shujiao Chen+, Xingxing Ge, Ying Zhang, Yaoqi Xie, Yingying Hao, Daiqun Wu,
Jinmin Zhao, Xiang-Ai Yuan, Laijin Tian, Zhe Liu*
Institute of Anticancer Agents Development and Theranostic Application, The 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.
*Corresponding authors, Email: chemlxc@163.com (X. Liu); liuzheqd@163.com (Z. Liu)
+ X. Liu and S. Chen have equivalent contributions.
Abstract: Six N-phenylcarbazole/triphenylamine-appended half-sandwich iridium(III)
2-phenylpyridine complexes ([(η5-Cp*)Ir(C^N)Cl]) were prepared and characterized. Compared
with cisplatin, these complexes exhibited potential antitumor activity against A549 and HeLa
tumor cells, with IC50 values (half-maximum inhibitory concentration) that changed from 2.8 ± 0.8
μM to 39.5 ± 2.7 μM, and could block the migration of tumor cells. These complexes also
effectively bound to protein (binding constant: ~104 M-1) and were transported through serum
proteins, catalyzed the oxidation of coenzyme nicotinamide-adenine dinucleotide. Additionally,
laser confocal microscopy and flow cytometry confirmed that these complexes possessed a
non-energy-dependent cellular uptake mechanism, effectively accumulated in lysosomes (Pearson
colocalization coefficient: ~0.74), damaged the integrity of acidic lysosomes, led to a change in
the mitochondrial membrane potential, disrupted the cell cycle (G0/G1 phase), and eventually
induced apoptosis. Above all, these complexes are potential antitumor agents with dual functions:
metastasis inhibition and lysosomal damage.
Keywords: Iridium(III) complexes; Antitumor; Metastasis inhibition; Lysosomal damage
1. Introduction
Lysosomes, which are acidic intracellular organelles (pH: 4.5–5.5), are related to many
cellular processes, including apoptosis, cell migration, metabolism, plasma membrane repair and
the extracellular release of active enzymes [1,2]. In tumor cells, lysosomes are more numerous,
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less stable and show better cathepsin activity than that in normal cells, which can degrade almost
all kinds of biomacromolecules through the actions of more than 50 acid hydrolytic enzymes [3].
Interestingly, studies indicated that weakly basic drugs could effectively accumulate in lysosomes,
induce lysosomal swelling and rupture, and eventually, lead to lysosomal membrane
permeabilization (LMP) [4]. In general, LMP causes the release of cathepsins and other hydrolases
from the lysosomal lumen to the cytosol, thus inducing apoptosis or necrotic cell death [5]. Above
all, lysosomes are attractive targets for the selective killing of tumor cells [6-7]. Agents that can
interact with lysosomes, show promise for the development of novel and potent antitumor drugs.
Due to their unique antitumor mechanisms, including the inhibition of multiple protein
activities and involvement in cellular redox reactions, iridium(III) (IrIII) complexes have recently
emerged as promising alternatives to platinum-based metallodrugs (cisplatin, carboplatin and
oxaliplatin etc.) [8-11]. Among these, half-sandwich IrIII complexes have attracted particular
attention because of their favorable stability and antitumor activity [12-14]. The general formula
for half-sandwich IrIII complexes can be expressed as [(η5-Cp*)Ir(L^L)Cl], where Cp* represents
the electron-rich pentamethylcyclopentadienyl or its derivatives (e.g., phenyl, biphenyl), L^L is
various chelating ligands (e.g., N^N, C^N, N^O and C^O), and Cl is the leaving group. Due to the
easy modification of L^L ligands, a large number of weakly basic groups containing free electron
pairs, e.g., morpholine [15], benzimidazole [16, 17], rhodamine [18], β-carboline [19], and
imine-N-heterocyclic carbene [7], have been introduced into IrIII complexes and used as
lysosome-targeted and real-time tracking drugs, achieving ideal results.
Triphenylamine (TPA), N-phenylcarbazole (PhCz) and their derivatives, which are typical
hole-transporting and light-emitting materials, are widely used in organic light-emitting diodes
(OLEDs) [20], perovskite solar cells (PSCs) [21-23], electrostatic copying devices (OFETs) [24]
and electrostatic photocopying [25], etc. Additionally, these materials show promise for
applications in the biological probe and drug-targeting fields due to their favorable luminescent
properties, e.g., low excitation energy, high fluorescence quantum yield, suitable energy level,
favorable light and thermal stability. TPA-based spirobifluorene (SPF-TSA) enabled the
fluorescent imaging of both probe and mercury ions in cells by two-photon microscopy and
possessed excellent indicator properties within the acidic pH range [26, 27]. TPA-modified
cyclometalated IrIII phenanthroline complexes could effectively induce the production of singlet
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oxygen (1O2) after 730 nm laser irradiation and acted as an effective phototherapy drug against
HeLa cells [28]. In view of the favorable application prospects of triphenylamine in biological
probes and anticancer complexes, PhCz/TPA-modified half-sandwich IrIII phenylpyridine
complexes were synthesized and characterized (Figure 1). The antitumor activity of these
complexes against A549 (human lung cancer cells) and HeLa (human cervical cancer cells) cells
was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, and
the antitumor mechanism was studied through flow cytometry. Due to the favorable target
fluorescent properties, laser confocal technology was used to investigate the cellular uptake and
target sites in cells. Above all, PhCz/TPA-appended IrIII complexes are potential antitumor agents
for further evaluation.
Figure 1. Structures of C^N chelating ligands (L1-L3) and target complexes (1-6).
2. Results and discussion
The synthetic strategy for PhCz/TPA-modified phenylpyridine (C^N) chelating ligands (L1-L3)
and target IrIII complexes (1-6) is described in Scheme 1. Among these, L1-L3 were synthesized
by brominated PhCz/TPA (M1) and 4-(2-pyridyl)phenylboronic acid (M2) with good yields
(>80%) through the classical Suzuki reaction (palladium tetraphenylphosphine [Pd(PPh3)4] as
catalyst, anhydrous potassium carbonate (K2CO3) as acid binding agent) [29]. Target IrIII
complexes were obtained by the reaction of iridium dimers (Dimer 1 and 2) and C^N chelating
ligands (L1-L3), and sodium acetate (NaOAc) used as the acid extractor [30]. Structures and
1
compositions of chelating ligands and target complexes were confirmed by H NMR, elemental
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analyses, mass spectroscopy (MS) analyses, and 13C NMR. Chloroform-d (CDCl3, 7.26 ppm) was
used as a solvent for testing the 1H NMR spectrum of IrIII complexes. Hydrogen atoms of the five
1
methyl groups on cyclopentadienyl (Cp) were shown in the range of 1.59 to 1.90 ppm in the H
NMR spectrum, the methoxyl of TPA groups was present at 3.81 ppm, and hydrogen atoms on the
benzene and pyridine were in the range of 6.5 to 9.0 ppm. IrIII complexes used multiple solvents
such as dichloromethane (5.30 ppm) and acetic acid (2.10 ppm) in the synthesis and purification
1
processes, which showed certain amounts of residue in the H NMR spectrum. The MS data
agreed well with the results of the theoretical calculation (chloride ion loss).
Scheme 1. Design strategy of chelating ligands and target IrIII complexes.
Single crystals suitable for X-ray diffraction analysis were obtained by the slow diffusion of
n-hexane into a saturated tetrahydrofuran solution of [(η5-C5Me5)Ir(L2)Cl] (complex 3). The
X-ray crystal structure is illustrated in Figure 2, and the crystallographic data are listed in Tables
S1 and S2. Complex 3 shows the expected half-sandwich “three-leg piano-stool” geometry, and
the terminal TPA group exhibits a “propeller” shape with dihedral angles of 65.71, 74.41 and
75.29°. Additionally, the angle between P1 (benzene ring of phenylpyridine) and P2 (benzene ring
of TPA) is 34.87°. The smaller angle ensures the good coplanar property of the chelating ligand
and further improves its electron donor ability. The distance from the central iridium ion to the
cyclopentadiene ring is 1.832 Å, and the bond distances of Ir-N1, Ir-C7 and Ir-C1l are 2.095,
2.028 and 2.411 Å, respectively. The longer bond lengths correspond to the notion that the Ir-Cl
bond is the active center for half-sandwich IrIII complexes [12].
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It is worth noting that the stability of complexes in solution is closely related to their
biological activity [12]. Therefore, the stability of complexes 1-6 in a mixture of 20% MeOH/80%
phosphate buffered saline (PBS: pH ≈ 7.2, PBS is prepared from H2O) were evaluated by
ultraviolet-visible spectra at 25 °C. As shown in Figure S1, complexes did not show significant
changes within 8 h, which indicating that IrIII complexes are fairly stable and provide conditions
for subsequent biological assays.
Figure 2. Molecular structure of complex 3 (Hydrogen atoms have been omitted for clarity).
2.1. Cytotoxicity and antimetastatic assay
The antitumor activity of PhCz/TPA-appended half-sandwich IrIII phenylpyridine (PhPy)
complexes against A549 (human lung cancer) and HeLa (human cervical cancer) cells was
ascertained by the MTT assay after 24 h of treatment. The IC50 values (concentration when 50%
cell growth was inhibited) of complexes 1-6, cisplatin, and complexes 7 and 8 (Scheme S1,
half-sandwich IrIII 2-phenylpyridine complexes) are listed in Table 1. These complexes showed
better antitumor activity, the best of which (complex 1) was nearly eight times that of cisplatin
(widely used in the clinic) under the same conditions. Additionally, compared with the iridium
dimers (Dimer 1 and 2), chelating ligands (L1-L3) and complexes 7 and 8 , target complexes
exhibited better antitumor activity (Table S3), which confirms that the introduction of a PhCz/TPA
unit is beneficial for improving the antitumor activity of these complexes.
Table 1. IC50 values of complexes and cisplatin against A549, HeLa and BEAS-2B cells.
IC50 (μM)
Complex
BEAS-2B
1.9±0.1
7.0±1.5
1.6±0.5
A549
2.8±0.8
23.0±0.7
7.4±0.1
HeLa
[(η5-C5Me5)Ir(L1)Cl] (1)
[(η5-C5Me4C6H5)Ir(L1)Cl] (2)
[(η5-C5Me5)Ir(L2)Cl] (3)
1.6±0.2
7.3±1.6
2.5±0.1
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[(η5-C5Me4C6H5)Ir(L2)Cl] (4)
[(η5-C5Me5)Ir(L3)Cl] (5)
[(η5-C5Me4C6H5)Ir(L3)Cl] (6)
[(η5-C5Me5)Ir(PhPy)Cl] (7)
[(η5-C5Me4C6H5)Ir(PhPy)Cl] (8)
Cisplatin
39.5±2.7
3.5±0.1
40.6±2.8
1.3±0.1
8.6±0.6
35.6±1.6
11.0±0.1
7.5±0.2
45.7±1.4
1.7±0.2
13.0±0.5
27.6±0.6
8.5±1.4
11.8±2.4
17.1±4.9
11.9±0.7
38.4±2.8
21.3±1.7
Footnotes: The type of analysis is sample repeat; Exposure time is 24 h; A549 and HeLa cells are cancer cells, and
BEAS-2B: non-malignant human bronchus epithelial cell line; Each is repeated at least three times.
Additionally, complexes containing a pentamethylcyclopentadienyl (η5-C5Me5) ring
(complexes 1, 3 and 5) showed better antitumor activity than that of the corresponding phenyl
analogues (η5-C5Me4C6H5, complexes 2, 4 and 6). To understand these results, quantum chemical
computation was used for assessment after comprehensively considering the crystal structure of
complex 3. As shown in Figure S2, the natural population analysis (NPA) data for the central
iridium atom and the leaving group (Cl) were analyzed by density functional theory (DFT)
calculation at the B3LYP/6-31G(d, p) (C, H, N, Cl)/SDD (Ir) level [31, 32]. The NPA charge
population for Cl was almost the same, with values of -0.373 and -0.374; however, that of Ir was
0.078 and 0.147 in complexes 3 and 4, respectively. Obviously, the lower the charge of the central
iridium ion for complex 3, the more vulnerable it is to loss of the chloride ion, and the better the
anticancer activity [12]. Inductively coupled plasma mass spectrometry (ICP-MS) indicated that
the values of logP (partition coefficient in oil/water) were -0.19 and -0.75 for complexes 3 and 4,
respectively. The cumulative amount (equivalent concentration) of these complexes in A549 cells
after 12 h incubation was measured. The cell accumulation of complexes 3 and 4 were 85.6 and
39.8 (ppb/106 cells), respectively. All these indicate that improved lipid solubility is beneficial for
enhancing the antitumor activity of these complexes (1, 3 and 5 > 7); however, “going too far is as
bad as not going far enough”, and overlarge lipid solubility was unfavorable to the improvement
of antitumor activity (1>2; 3>4; 5>6; 2, 4 and 6＜8), which provides a basis for the design of
these complexes. The cytotoxicity of target complexes was further evaluated toward human lung
epithelial cells (BEAS-2B, Table 2). However, the results were unsatisfactory, and there was no
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significant selectivity between tumor cells and normal cells. Hence, more structural modification
is necessary in future work to decrease the cytotoxic action against normal cells without loss of
potential antitumor activity.
In general, tumor cell metastasis refers to the process of continuing to grow from the primary
site through lymphatic vessels, blood vessels or body cavities to other normal sites; therefore,
metastasis inhibition is an effective means of tumor treatment. Wound healing assays are an
effective approach to assess the antimetastatic capacity of target complexes [33]. As shown in
Figure 3, A549 cells treated with or without complexes 1 and 2 (0.25 × IC50) for 24 h, and the
middle area between two blue lines (“Wound”) is clear of tumor cells. In the absence of target
complexes (Control), the wound closure ratio (WCR = (R0-R1)/R0×100%) significantly improved
after 24 h (WCR: 25.00%), which confirmed the migration characteristics of tumor cells. However,
after the addition of complexes in this wound area, especially for 1, the WCR value (9.47%) was
relatively inhibited. This conclusion is consistent with the results of the MTT assay (1 showed
better antitumor activity than 2) and also confirms that these complexes have a potential effect on
the migration of cancer cells, which is helpful for tumor therapy.
Figure 3. Wound healing assay of A549 cells treated with or without complexes 1 and 2 (0.25 × IC50) for 24 h. (a)
Typical images were taken at 0 and 24 h. The widths of wounds are indicated with red lines. (b) Quantification of
wound closure ratio.
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2.2. Protein Binding assay
Serum albumin (SA), which is plentiful in blood plasma, plays an indispensable role in drug
transport and supersession. Interactions between drugs and SA are helpful for understanding the
intracellular situation [34]. Due to its structural homology with human serum albumin (HSA),
bovine serum albumin (BSA) offers a cost-effective model for protein binding; therefore, it was
utilized to study the binding characteristics with target complexes in this study.
As shown in Figures 4a and S3, the UV-vis absorption of BSA (10.0 μM) with or without target
IrIII complexes in Tris-HCl buffered solution (trishydroxymethylaminomethane- hydrochloric acid,
pH = 7.2) possesses two absorption peaks. With the increase in complexes (0-10.0 μM), the
maximum absorption at 228 nm (the absorption of BSA) decreased, which indicated that these
complexes can act on BSA and induce α-helical interference. Meanwhile, a significant red shift
(~6 nm) was found at 228 nm due to the influence of water (polar solvent). The absorption at 278
nm increased with the addition of complexes, which is related to the presence of three major
amino acid residues (tryptophan, tyrosine and phenylalanine) in BSA. Therefore, simultaneous
fluorescence spectra were utilized to ascertain the detailed changes of the microenvironment
around BSA under similar conditions. In this study, tyrosine and tryptophan residues of BSA were
detected through the wavelength intervals of Δλ = 15 nm and 60 nm by synchronous fluorescence,
respectively [35]. The weaker intensity of fluorescence at 291 nm and 285 nm (Δλ = 15 nm and 60
nm, respectively) for complexes 1-6 is shown in Figures 4c, 4d, S4 and S5. Interestingly, a minor
blue shift (~3 nm) is found at Δλ = 60 nm; however, almost no change occurs at the wavelength of
Δλ = 15 nm, which indicates that target complexes can affect the conformation of the tryptophan
microregion in BSA, in addition to tyrosine [36].
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Figure 4. (a) UV-vis spectrum of BSA (10.0 μM, 50.0 mM Tris-HCl, 50.0 mM NaCl, pH = 7.2) reacted with
complex 1 (0-10.0 μM). Inset: Wavelengths from 260 to 300 nm; (b) fluorescence spectra of BSA (10.0 μM; λex
=
280 nm; λem = 350 nm) in the absence and presence of complex 1 (0.0-10.0 μM); synchronous spectra of BSA
(10.0 μM) with the addition of complex 1 (0.0-10.0 µM) with wavelength differences of ∆λ = 15 nm (c) and ∆λ =
60 nm (d). The arrow shows the changes in intensity with increased complexes.
The interaction between target complexes and BSA was further confirmed by the
fluorescence quenching phenomenon of BSA [37]. As shown in Figures 4b and S6, with the
addition of target complexes, the fluorescence intensity of BSA rapidly quenched. Generally, the
quenching mechanism can be ascertained by the classical Stern-Volmer equation (Equation 1)
[38]:
⁄
[ ] [ ]
퐹0 퐹 = 1 + 퐾푠푣 푄 = 1 + 퐾푞휏0 푄
(1)
where Kq and Ksv represent the Stern-Volmer quenching rate constant and quenching constant
(obtained by the slope of the Stern-Volmer equation, Figure S7), F0 and F are the steady-state
fluorescence intensities without or with target complex, τ0 is the average life span of fluorophore
without complex (10-8 s) and [Q] is the concentration of complex. As shown in Table 2, the linear
fitting Stern−Volmer plots represent a single quenching mechanism. The values of Kq (changed
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from 0.76 × 1013 to 1.45 × 1013 M-1s-1) are far greater than the limit of the dynamic quenching
mechanism (2.0 × 1012 M-1s-1), which indicates that these IrIII complexes bind to BSA followed by
the static quenching mechanism [39]. Additionally, the binding constant (Kb) and the numbers of
binding sites (n) can be obtained through the Scatchard equation [40] (Equation 2):
[( [ ]
)⁄ ]
퐹0 퐹 퐹 = 퐾 + 푄
(2)
The binding constant Kb reflects the degree of interaction between target complexes and BSA,
and the values of n are the numbers of binding sites. As shown in Figure S8, Kb and n are the data
after the linear fitting of log((F0 - F)/F) vs log[Q]. As shown in Table 2, the values of n are almost
the same (~1) for all complexes, which is consistent with the results showing that the binding
Ⅲ
between BSA and Ir complexes can affect the conformation of the tryptophan microregion. In
addition, the binding constants (Kb) of complexes 1, 3 and 5 are a little bigger than those of
homologous phenyl-modified complexes (2, 4 and 6), respectively, which is also consistent with
the results of the cytotoxicity test. Above all, BSA may be an excellent carrier for the delivery of
these complexes in vivo.
Table 2. The values of Ksv, Kb, Kq and n for complexes 1-6 at 298 K.
Complex
Ksv (105 M-1)
1.20±0.20
1.11±0.21
1.45±0.15
0.76±0.10
1.55±0.19
1.16±0.16
Kq (1013 M-1 s-1) Kb (104 M-1)
n
1.20
1.11
1.45
0.76
1.55
1.16
10.16
7.99
9.42
5.68
9.44
8.75
1.05
1.08
1.13
1.10
1.18
1.09
1
2
3
4
5
6
2.3. Biocatalytic assay
The reduced state of nicotinamide adenine dinucleotide (NADH) is a control marker of the
energy production chain in mitochondria. Studies show that NADH can transfer hydrogen to
1
transition metal complexes and promote the production of reactive oxygen species (ROS, O2),
which manifests as an antitumor mechanism of oxidation [41, 42]. The interaction between target
complexes and NADH was determined by the UV-vis spectrum. As shown in Figures 5a and S9,
the maximum absorbance of NAD+ (oxidation state of NADH after hydrogen loss catalyzed by
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target complexes) and NADH can be determined at 259 and 339 nm; meanwhile, a significant
increase and decrease occur at 259 nm and 339 nm with the increase in target complexes, which
confirms the catalytic characteristics of these complexes with the change of NADH/NAD+.
Additionally, these changes can be effectively expressed by the turnover numbers (TONs, Figure
5b); the values of TONs for complexes 1, 3, and 5 (η5-C5Me5) are significantly larger than those of
phenyl-modified complexes 2, 4, and 6 (η5-C5Me4C6H5), which is consistent with the results of the
cytotoxicity test: the larger the TONs, the better the antitumor activity.
Figure 5. (a) UV-vis spectra of NADH (100.0 µM) with complex 1 (1.0 µM) in 20% MeOH/80% H2O (v/v) at 298
K for 8 h. The arrows show the changes in the absorbance spectra over time. (b) TONs of target complexes.
The levels of ROS in A549 cells, generated by the catalysis of these IrIII complexes after 24 h,
were identified by flow cytometry [43]. As shown in Figure S10, the levels of ROS are nearly 1.16
and 1.08 times that of the negative control for complexes 1 and 2 at the concentration of 0.50
×IC50, respectively. Compared with complex 2, the higher ROS levels induced by complex 1 are
consistent with the conclusion that complex 1 possesses better antitumor activity. However, the
minor TONs and ROS levels also confirmed the limited biocatalysis performance of these
compounds.
2.4. Apoptosis assay
To determine whether these IrIII complexes could induce apoptosis, A549 cells were treated with
complexes 1 and 2 at the concentrations of 0.5 × IC50, 1.0× IC50, 2.0× IC50 and 3.0× IC50 for 24 h
and then analyzed by flow cytometry [44]. As shown in Figure 6, complexes 1 and 2 can induce
apoptosis in a dose-dependent manner and chiefly affect late apoptosis. At the maximal incubated
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concentration (3.0 × IC50), the proportions of late apoptosis caused by complexes 1 and 2 were
65.85% and 66.71%, respectively (Tables S4 and S5). However, ~93% cells survived in the
control group under the same conditions. This conclusion further confirmed that target IrIII
complexes can induce the functional decline of tumor cells and lead to apoptosis. Additionally, IrIII
complexes can exert their antitumor effects and induce functional decline by disrupting the cell
cycle [45]. As shown (Figure S11, Tables S6 and S7), complexes 1 and 2 mainly lead to 63.89%
and 60.15% cell arrest in the G0/G1 phase, respectively, compared with the negative control, which
Ⅲ
increased by 7.87% and 3.29%. The results indicate that these Ir complexes may restrain the
proliferation of A549 cells by cell cycle arrest and, eventually, induce apoptosis.
Figure 6. Apoptosis analysis of A549 cells after exposure to complexes 1 and 2 for 24 h at 310 K and determined
by flow cytometry using Annexin VFITC/PI staining.
Mitochondria play an irreplaceable role in apoptosis due to their ability to provide biological
energy to cells. The decrease of the mitochondrial membrane potential (MMP) is a key indicator
of cell apoptosis [46, 47]. The effects of complexes 1 and 2 on MMP were assessed by JC-1
(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide), an ideal fluorescent
probe for detecting MMP with red and green fluorescence representing a high and low
mitochondrial membrane potential, respectively. As shown in Figure 7, the green fluorescence
increased, while the red fluorescence decreased with the addition of complexes 1 and 2. The
percentages of cells with mitochondrial membrane depolarization increased by 62.40% and 62.28%
for complexes 1 and 2, respectively, with concentrations changing from 0.5 × IC50 to 2.0 × IC50
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(Tables S8 and S9). The increase in the JC-1 green/red fluorescence intensity ratio further
confirmed that these IrIII complexes can induce a change in MMP, leading to apoptosis.
Figure 7. The loss of MMP induced by complexes 1 and 2 at the concentrations of 0.5 × IC50, 1.0 × IC50 and 2.0 ×
IC50 using JC-1 staining. The red aggregates and green monomers are gated.
2.5. Cellular uptake mechanism and cellular localization
Favorable cellular uptake mechanism is beneficial to improve the antitumor activity of drugs
[48]. The cellular uptake mechanism of complexes 1 and 2 in labeled A549 cells was assessed by
laser confocal microscopy. Cellular uptake mechanisms are mainly divided into two types:
energy-dependent mechanisms (including active transport and endocytosis) and
non-energy-dependent mechanisms (including passive transport and free diffusion) [49]. In this
study, A549 cancer cells were incubated with complexes 1 and 2 (10 μM) at 310 K and 277 K and
then treated with chlorocyanochlorophenyl (CCCP, metabolic inhibitor, 50 μM) and chloroquine
(endocytosis modulator, 50 μM) to ascertain the cell uptake mechanism. Compared with control
treatment, there were no significant changes (Figure S12), which indicated that these complexes
enter tumor cells, followed by a non-energy-dependent pathway [50].
Due to the favorable targeted luminescence properties of these complexes, laser confocal
microscopy was also utilized to determine the co-localization of complexes 1 and 2. In this study,
Lysol Tracker Deep Red (LTDR) and Mito Tracker Deep Red (MTDR) were used as the lysosome
and mitochondria fluorescent probes, respectively [51, 52]. As shown in Figure 8, complexes 1
and 2 effectively accumulated in lysosomes with Pearson Colocalization Coefficient (PCC) values
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of 0.81 and 0.70, respectively, after incubation for 1 h; however, the values for mitochondria were
almost negligible (-0.03 and -0.13, respectively). These results indicate that the introduction of a
weakly basic PhCz/TPA unit at the end of C^N chelating ligands (containing electron-rich
nitrogen atoms) is beneficial for targeting acidic lysosomes (pH = 4.5-5.5) for these complexes.
Interestingly, these complexes do not cause abnormal cell death immediately, which makes it easy
to track the changes in cells in real time.
Figure 8. The intercellular localization of complexes 1 and 2 was determined by confocal microscopy after 1 h
of incubation. Complexes were excited at 488 nm and collected at 520-580 nm. LTDR was excited at 594 nm
and collected at 600-660 nm. MTDR was excited at 543 nm and collected at 660-720 nm.
Lysosomal damage, a process known as lysosomal membrane permeabilization, can lead to
the release of cathepsins and other hydrolases from the lysosomal lumen to the cytosol, thus
inducing apoptosis [53]. In general, acridine orange (AO) is usually used to determine the integrity
of lysosomes [54-56]. Green fluorescence and red fluorescence represent the binding of AO to
RNA in the nucleus or cytoplasm and aggregation in the lysosome, respectively. As shown in
Figures 9 and S13, lysosomes showed obvious red fluorescence after A549 cells were treated with
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only AO (5.0 μM), which indicates the integrity of lysosomes. However, red fluorescence was
quickly quenched after exposure to complexes 1 and 2 at concentrations ranging from 1.0 × IC50
to 3.0 × IC50, which is the result of increased lysosomal damage. This result is mainly attributable
to the introduction of a PhCz/TPA unit, which increased the total alkalinity of target complexes,
leading to the accumulation and further damage to the integrity of acidic lysosomes while also
explaining the change of MMP. This result indicates that target complexes can induce apoptosis by
exerting lysosomal damage.
Figure 9. Lysosomal damage in A549 cells caused by complex 1 with AO (5.0 μM) staining.
3. Conclusion
Six PhCz/TPA-appended half-sandwich IrIII phenylpyridine complexes were prepared and
characterized. The introduction of the PhCz/TPA unit effectively increased the antitumor activity
of these complexes against A549 and HeLa tumor cells, the best of which had activity nearly eight
times that of clinical cisplatin and effectively blocked the migration of cancer cells. These
complexes can effectively bind to protein, undergo transport through serum proteins, and catalyze
the change of NADH to NAD+, leading to the increased ROS levels in cells. Additionally, flow
cytometry and laser confocal microscopy confirmed that these complexes possessed a
non-energy-dependent cellular uptake mechanism, effectively accumulated in lysosomes and
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damaged the integrity of acidic lysosomes, disrupted the cell cycle, induced a change in the
mitochondrial membrane potential, and eventually, induced apoptosis. Above all,
PhCz/TPA-appended half-sandwich IrIII phenylpyridine complexes are potential antitumor agents
with dual functions: metastasis inhibition and lysosomal damage.
4. Experimental
4.1.
General Information
IrCl3·3H2O, 1,2,3,4,5-pentamethyl-cyclopentadiene (95%), n-butyl lithium (1.6 M in hexane),
2,3,4,5-tetramethyl-2-cyclopentenone (95%), 2-(4-bromophenyl)pyridine, carbazole, Palladium
tetraphenylphosphine, triisopropyl borate, sodium acetate, N-bromosuccinimide, triphenylamine,
N,N′-di(4-methoxyphenyl)phenylamine, 1-bromo-4-iodobenzene were purchased from Ruiya
Biotechnology Co., Ltd. 4-bromotriphenylamine, 4-bromo-N,N′-di(4-methoxyphenyl)phenylamine,
N-(4-bromophenyl)carbazole (M1) were synthesized according to the literature [57]. 2-pyridyl
phenylboronic (M2) was synthesized through 2-(4-bromophenyl)pyridine and triisopropyl borate
according to the literature [58]. Fetal bovine serum, Nicotinamide adenine dinucleotide,
penicillin/streptomycin mixture, trypsin/EDTA, and phosphate-buffered saline (PBS) were
purchased from Sangon Biotech. A549 (lung cancer) and HeLa (human cervical cancer) cells were
obtained from Shanghai Institute of Biochemistry and Cell Biology (SIBCB). Dimers 1 and 2,
complexes 7 and 8 were synthesized according to the methods of literatures [12] (Scheme S1), the
data were shown in supporting information.
NMR spectra were obtained on Bruker DPX 500 spectrometers instrument. Mass spectrum
(ESI-MS) was measured on a LCQ Advantage MAX mass spectrometer. Elemental analysis was
performed on a VarioMICRO 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 5 nm slit width for both excitation and
emission. Induction of apoptosis, cell cycle and mitochondrial membrane potential (MMP)
determination were carried out by an ACEA Novocyte2040R flow cytometry. Viability assay
(MTT) was measured using a Perlong DNM-9606 microplate reader at an absorbance of 570 nm.
Cell uptake and cellular localization were carried out on a Carl Zeiss AG */LSM/880NLO two
photon laser Scanning microscope.
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The spectrograms of target complexes, the methods of biological performance testing are all
shown in Supporting Information.
4.2.
Synthesis
Synthesis of chelating ligands (L1-L3)
C^N chelating ligands were obtained by the classical Suzuki reaction (Scheme 1); the general
process is listed as follows: The corresponding brominated PhCz/TPA (4.0 mmol),
4-phenylpyridine-2-boronic acid (4.8 mmol, 0.96 g), palladium tetraphenylphosphine (0.08 mmol,
0.14 g), anhydrous potassium carbonate (8.0 mmol, 1.11 g), 60 mL of toluene and 12 mL of
deionized water were added to a 100 mL flask under N2 and stirred at 383 K for 12 h. The product
was extracted with dichloromethane (30 mL×3) and distilled under reduced pressure. Then, the
product was purified by chromatography on a silica gel column (petroleum ether: ethyl acetate
=15:1 as eluent), and the pure target chelating ligands were obtained. The 1H NMR spectrum, 13
C
NMR spectrum and ESI-MS of L1-L3 are presented in Figures S14, S16 and S18. The data were
as follows:
1
L1. Yield: 1.35g (86%). H NMR (500 MHz, CDCl3) δ 8.75 (d, J = 4.6 Hz, 1H), 8.17 (t, J = 7.0
Hz, 4H), 7.90 (d, J = 8.3 Hz, 2H), 7.82 (t, J = 7.2 Hz, 4H), 7.67 (d, J = 8.2 Hz, 2H), 7.50 (d, J =
8.2 Hz, 2H), 7.44 (t, J = 7.6 Hz, 2H), 7.31 (dd, J = 15.6, 8.3 Hz, 3H). 13C NMR (126 MHz, CDCl3)
δ 156.89, 149.79, 140.86, 140.75, 139.63, 138.62, 137.14, 136.90, 128.49, 127.52, 127.48, 127.40,
126.03, 123.49, 122.29, 120.54, 120.38, 120.06, 109.88. ESI-MS (m/z): calcd for C29H20N2, 396.2;
Found 397.2 [M+H]+.
1
L2. Yield: 1.29g (81%). H NMR (500 MHz, CDCl3) δ 8.72 (d, J = 4.8 Hz, 1H), 8.07 (d, J = 8.4
Hz, 2H), 7.78 (d, J = 4.0 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.7 Hz, 2H), 7.28 (dd, J =
8.4, 7.6 Hz, 5H), 7.17 – 7.11 (m, 6H), 7.04 (t, J = 7.3 Hz, 2H). 13C NMR (126 MHz, CDCl3) δ
157.07, 149.69, 147.64, 147.48, 141.19, 137.69, 136.81, 134.32, 129.33, 127.74, 127.30, 126.91,
124.53, 123.82, 123.05, 122.05, 120.40. ESI-MS (m/z): calcd for C29H22N2, 398.2; Found 398.2
[M].
L3. Yield: 1.45g (83%). 1H NMR (500 MHz, CDCl3) δ 8.76 (d, J = 4.3 Hz, 1H), 8.09 (d, J = 8.1
Hz, 2H), 7.85 (s, 2H), 7.70 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.34 (s, 1H), 7.10 (d, J =
8.8 Hz, 4H), 7.00 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.9 Hz, 4H), 3.81 (s, 6H). 13C NMR (126 MHz,
CDCl3) δ 156.03, 149.16, 142.01, 141.08, 140.75, 139.66, 138.38, 129.33, 127.52, 126.83, 126.76,
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124.58, 123.89, 123.10, 121.24, 120.51, 114.76, 55.52. ESI-MS (m/z): calcd for C31H26O2N2,
458.2; Found 459.2 [M+H]+.
Synthesis of [(η5-Cp*)Ir(C^N)Cl] (Complexes 1-6)
[(η5-Cpx)IrCl2]2 (dimer, 0.05 mmol, 1 equiv, ), chelating ligand L (0.1 mmol, 2 equiv) and
sodium acetate (0.6 mmol, 10 equiv) in methanol (40 mL) were stirred at ambient temperature
overnight. The solvent was removed under reduced pressure, and 20 mL of dichloromethane was
added, after which the precipitate (sodium acetate) was removed by filtration. Most of the solvent
was concentrated to 2.0 mL under vacuum and kept at 253 K for 12 h, filtered and washed with
cold methanol and diethyl ether. The 1H NMR, ESI-MS and 13C NMR spectrum of complexes 1-6
are presented in Figures S15, S17 and S19. The data were as follows:
1
[(η5-C5Me5)Ir(L1)Cl] (1). Yield: 77.3 mg (85%). H NMR (500 MHz, CDCl3) δ 8.74 (d, J = 5.2
Hz, 1H), 8.19 – 8.13 (m, 3H), 7.93 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.1 Hz, 1H), 7.80 (d, J = 8.0
Hz, 1H), 7.72 – 7.68 (m, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.2 Hz, 2H), 7.44 (t, J = 7.7 Hz,
2H), 7.39 (dd, J = 8.0, 1.7 Hz, 1H), 7.31 (t, J = 7.4 Hz, 2H), 7.12 (dd, J = 9.5, 3.5 Hz, 1H), 1.75 (s,
15H). 13C NMR (126 MHz, CDCl3) δ 166.90, 163.86, 151.45, 143.91, 142.00, 141.13, 140.96,
137.08, 136.62, 134.34, 128.68, 127.22, 125.97, 124.21, 123.41, 122.41, 121.44, 120.30, 119.91,
119.10, 109.98, 88.68, 9.08. Elemental Analysis: Found: C, 61.81; H, 4.60; N, 3.63%, calcd for C,
61.77; H, 4.52; N, 3.69%. ESI-MS (m/z): calcd for C39H34N2Ir: 723.2, Found: 723.3 [M-Cl]+.
[(η5-C5Me4C6H5)Ir(L1)Cl] (2). Yield: 80.7 mg (82%). 1H NMR (500 MHz, CDCl3) δ 8.56 (d, J =
5.2 Hz, 1H), 8.17 (d, J = 7.7 Hz, 2H), 7.98 (d, J = 1.6 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.80 (d, J
= 8.1 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.67 (td, J = 8.1, 1.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H),
7.52 – 7.38 (m, 9H), 7.36 (t, J = 7.3 Hz, 1H), 7.33 – 7.29 (m, 2H), 7.02 – 6.96 (m, 1H), 1.90 (s,
3H), 1.79 (d, J = 3.5 Hz, 6H), 1.67 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 166.89, 162.89, 151.59,
143.79, 141.62, 140.92, 140.63, 137.17, 136.58, 133.92, 132.37, 131.02, 128.74, 128.45, 127.33,
127.09, 125.96, 124.30, 123.40, 122.43, 121.35, 120.31, 119.91, 119.11, 109.96, 98.33, 97.95,
86.26, 85.19, 83.83, 14.14, 10.36, 9.92, 8.97. Elemental Analysis: Found: C, 64.51; H, 4.50; N,
3.40%, calcd for C, 64.41; H, 4.42; N, 3.41%. ESI-MS (m/z): calcd for C44H36N2Ir: 785.3, Found:
785.3 [M-Cl]+.
1
[(η5-C5Me5)Ir(L2)Cl] (3). Yield: 73.9 mg (81%). H NMR (500 MHz, CDCl3) δ 8.70 (d, J = 5.6
Hz, 1H), 8.04 (d, J = 1.7 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.67 – 7.64
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(m, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.29 (d, J = 8.2 Hz, 5H), 7.15 (dd, J = 8.1, 4.5 Hz, 6H), 7.06
(ddd, J = 18.4, 10.9, 4.3 Hz, 3H), 1.71 (s, 15H). 13C NMR (126 MHz, CDCl3) δ 167.03, 163.64,
151.36, 147.80, 147.02, 143.09, 142.37, 136.98, 135.93, 133.81, 129.28, 127.98, 124.43, 124.12,
123.91, 122.84, 122.11, 120.93, 118.92, 88.57, 9.04. Elemental Analysis: Found: C, 61.71; H, 4.90;
N, 3.63%, calcd for C, 61.60; H, 4.77; N, 3.68%. ESI-MS (m/z): calcd for C39H36N2Ir: 725.3,
Found: 725.3 [M-Cl]+.
[(η5-C5Me4C6H5)Ir(L2)Cl] (4). Yield: 81.9 mg (83%). 1H NMR (500 MHz, CDCl3) δ 8.49 (d, J =
5.6 Hz, 1H), 7.89 (d, J = 1.6 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.64 –
7.60 (m, 1H), 7.45 (t, J = 7.9 Hz, 4H), 7.39 – 7.32 (m, 3H), 7.28 (d, J = 8.2 Hz, 4H), 7.14 (d, J =
7.6 Hz, 5H), 7.08 (d, J = 8.6 Hz, 2H), 7.03 (t, J = 7.3 Hz, 2H), 6.95 – 6.91 (m, 1H), 1.83 (s, 3H),
1.77 (s, 6H), 1.61 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 167.01, 162.64, 151.51, 147.75, 142.14,
137.00, 133.34, 132.34, 131.17, 130.93, 129.89, 129.23, 128.67, 127.80, 127.28, 126.42, 124.37,
124.19, 123.81, 122.79, 122.08, 120.92, 118.87, 96.68, 94.72, 89.67, 87.80, 85.92, 10.23, 9.88,
8.96, 8.90. Elemental Analysis: Found: C, 64.29; H, 4.80; N, 3.43%, calcd for C, 64.26; H, 4.66;
N, 3.41%. ESI-MS (m/z): calcd for C44H38N2Ir: 787.3, Found: 787.4 [M-Cl]+.
[(η5-C5Me5)Ir(L3)Cl] (5). Yield: 80.7 mg (82%). 1H NMR (500 MHz, CDCl3) δ 8.69 (d, J = 5.7
Hz, 1H), 8.02 (d, J = 1.6 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.66 – 7.62
(m, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 1.7 Hz, 1H), 7.11 (d, J = 8.8 Hz, 4H), 7.07 – 7.04
(m, 1H), 7.00 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.9 Hz, 4H), 3.81 (s, 6H), 1.70 (s, 15H). 13C NMR
(126 MHz, CDCl3) δ 167.10, 163.58, 155.83, 151.32, 147.98, 142.76, 142.56, 141.04, 136.91,
133.84, 133.60, 127.74, 126.61, 124.08, 121.98, 120.76, 118.84, 115.82, 114.71, 88.53, 55.53,
9.02. Elemental Analysis: Found: C, 60.21; H, 5.10; N, 3.43%, calcd for C, 60.02; H, 4.91; N,
3.41%. ESI-MS (m/z): calcd for C41H40N2O2Ir: 785.3, Found: 785.4 [M-Cl]+.
[(η5-C5Me4C6H5)Ir(L3)Cl] (6). Yield: 88.9 mg (84%). 1H NMR (500 MHz, CDCl3) δ 8.48 (d, J =
5.3 Hz, 1H), 7.88 (s, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.60 (dd, J = 11.2, 4.3
Hz, 1H), 7.46 (d, J = 7.0 Hz, 2H), 7.42 – 7.28 (m, 6H), 7.09 (d, J = 7.3 Hz, 4H), 6.92 (dd, J = 15.6,
8.5 Hz, 3H), 6.85 (d, J = 8.6 Hz, 4H), 3.81 (s, 6H), 1.82 (s, 3H), 1.77 (d, J = 0.8 Hz, 6H), 1.59 (s,
3H). 13C NMR (126 MHz, CDCl3) δ 166.04, 162.67, 150.45, 146.55, 146.36, 146.20, 141.59,
139.99, 135.94, 132.09, 131.33, 129.90, 128.59, 127.64, 126.54, 126.24, 125.52, 123.15, 120.94,
119.75, 119.64, 117.78, 113.64, 98.50, 95.34, 85.09, 84.68, 82.10, 54.49, 13.10, 9.17, 8.85, 7.87.
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Elemental Analysis: Found: C, 62.71; H, 5.10; N, 3.23%, calcd for C, 62.61; H, 4.80; N, 3.17%.
ESI-MS (m/z): calcd for C46H42N2O2Ir: 847.3, Found: 847.4 [M-Cl]+.
Abbreviations
LMP: Lysosomal Membrane Permeabilization
Cp: pentamethylcyclopentadienyl
TPA: Triphenylamine
PhCz: N-phenylcarbazole
A549: human lung cancer cells
Hela: human cervical cancer cells
BEAS-2B: human lung epithelial cells
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
NPA: Natural Population Analysis
WCR: Wounds Closure Ratio
BSA: Bovine Serum Albumin
NADH: reduced state of Nicotinamide Adenine Dinucleotide
ROS: Reactive Oxygen Species
MMP: mitochondrial membrane potential
PCC: Pearson Colocalization Coefficient
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgments
We thank the University Research Development Program of Shandong Province (J18KA082), the
Student's Platform for Innovation and Entrepreneurship Training Program (2018A043), the
National Natural Science Foundation of China (Grant No. 21671118 and 21703118), the Taishan
Scholars Program and the High Performance Computing Center of Qufu Normal University for
support.
References
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Journal Pre-proof
[1] U. Repnik, B. Turk, Mitochondrion 10 (2010) 662-669.
[2] M.E. Guicciardi, M. Leist, G.J Gores, Oncogene 23 (2004) 2881-2890.
[3] J.P. Luzio, P.R. Pryor, N.A. Bright, Nat. Rev. Mol. Cell Biol. 8 (2007) 622-632.
[4] A. Serrano-Puebla, P. Boya, Ann. N. Y. Acad. Sci. 1371 (2016) 30-44.
[5] P. Saftig, J. Klumperman, Nat. Rev. Mol. Cell Biol. 10 (2009) 623-635.
[6] J.J. Li, X.C. Liu, H.F. Zhang, X.X. Ge, Y.H. Tang, Z.S. Xu, L.J. Tian, X.-A. Yuan, X.D. Mao,
Z. Liu, Inorg. Chem. 58 (2019) 1710-1718.
[7] B. Sun, J. Liu, Y. Gao, H.B. Zheng, L. Li, Q.W. Hu, H.Q. Yuan, H.X. Lou, Eur. J. Med. Chem.
136 (2017) 603-618.
[8] B. Rosenberg, L. Vancamp, J.E. Trosko, V.H. Mansour, Nature 222 (1969) 385-386.
[9] Y. Shi, S.A. Liu, D.J. Kerwood, J. Goodisman, J.C. Dabrowiak, J. Inorg. Biochem. 107 (2012)
6–14.
[10] S.J. Lucas, R.M. Lord, R.L. Wilson, R.M. Phillips, V. Sridharan, P.C. McGowan, Dalton
Trans. 41 (2012) 13800–13802.
[11] J. Ruiz, C. Vicente, C. de Haro, D. Bautista, Inorg. Chem. 52 (2013) 974−982.
[12] Z. Liu, P.J. Sadler, Acc. Chem. Res. 47 (2014) 1174-1185.
[13] K.K.W. Lo, K.Y. Zhang, RSC Adv. 2 (2012) 12069–12083.
[14] X.D. He, X.C. Liu, Y.H. Tang, J.Y. Du, M. Tian, Z.S. Xu, X.Y. Liu, Z. Liu, Dyes Pigments
160 (2019) 217–226.
[15] K.Q. Qiu, J.Q. Wang, C.L. Song, L.L. Wang, H.Y. Zhu, H.Y. Huang, J.J. Huang, H. Wang,
L.N. Ji, H. Chao, ACS Appl. Mater. Interfaces 8 (2016) 12702-12710.
[16] F.X. Wang, M.H. Chen, Y.N. Lin, H. Zhang, C.P. Tan, L.N. Ji, Z.W. Mao, ACS Appl. Mater.
Interfaces 9 (2017) 42471-42481.
[17] Y.L. Han, X.C. Liu, Z.Z. Tian, X.X. Ge, J.J. Li, M. Gao, Y.R. Li, Y. Liu, Z. Liu, Chem. Asian
J. 13 (2018) 3697-3705.
[18] W.L. Ma, Z.Z. Tian, S.M. Zhang, X.D. He, J.J. Li, X.R. Xia, X.B. Chen, Z. Liu, Inorg. Chem.
Front. 5 (2018) 2587–2597.
[19] L. He, Y. Li, C.P. Tan, R.R. Ye, M.H. Chen, J.J. Cao, L.N. Ji, Z.-W. Mao, Chem. Sci. 6 (2015)
5409-5418.
[20] X.C. Liu, J.F. Liang, J. You, L. Ying, Y. Xiao, S.R. Wang, X.G. Li, Dyes Pigments 131 (2016)
21

Journal Pre-proof
41-48.
[21] X.C. Liu, F. Zhang, Z. Liu, Y. Xiao, S.R. Wang, X.G. Li, J. Mater. Chem. C 5 (2017)
11429-11435.
[22] X.M. Zhao, F. Zhang, C.Y. Yi, D.Q. Bi, X.D. Bi, P. Wei, J.S. Luo, X.C. Liu, S.R. Wang, X.G.
Li, S.M. Zakeeruddin, M. Grätzel, J. Mater. Chem. A 4 (2016) 16330-16334.
[23] F. Zhang, X.M. Zhao, C.Y. Yi, D.Q. Bi, X.D. Bi, P. Wei, X.C. Liu, S.R. Wang , X.G. Li, S.M.
Zakeeruddin, M.Grätzel, Nano Energy 41 (2017) 469-475.
[24] Y. Shirota, H. Kageyama, Chem. Rev. 107 (2007) 953-1010.
[25] T.P. Bender, J.F. Graham, J.M. Duff, Chem. Mater. 13 (2001) 4105-4111.
[26] K.M. Omer, S.Y. Ku, J.Z. Cheng, S.H. Chou, K.T. Wong, A.J. Bard, J. Am. Chem. Soc. 133
(2011) 5492–5499.
[27] H.B. Xiao, Y.Z. Zhang, S.Z. Li, W. Zhang, Z.Y. Han, J.J. Tan, S.Y. Zhang, J.Y. Du, Sens.
Actuators, B 236 (2016) 233–240.
[28] F.F. Xue, Y. Lu, Z.G. Zhou, M. Shi, Y.P. Yan, H. Yang, S.P. Yang, Organometallics 34 (2015)
73-77.
[29] N.E. Leadbeater, M. Marco, Org. Lett. 4 (2002) 2973-2976.
[30] Z. Liu, I. Romero-Canelon, A. Habtemariam, G.J. Clarkson, P.J. Sadler, Organometallics 33
(2014) 5324-5333.
[31] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, et
al. Gaussian 09, revision D.01; Gaussian, Inc.: Wallingford, CT, 2013.
[32] X.A. Yuan, W.W. Zhang, L.H. Xie, J. Ma, W. Huang, W.J. Liu, J. Phys. Chem. B 119 (2015)
10316-10333.
[33] Q.J. He, J.L. Shi, Adv. Mater. 26 (2014) 391-411.
[34] O. Dömötör, C.G. Hartinger, A.K. Bytzek, T. Kiss, B.K. Keppler, E.A. Enyedy, J. Biol. Inorg.
Chem. 18 (2013) 9-17.
[35] M.E. Pacheco, L. Bruzzone, J. Lumin. 137 (2013) 138-142.
[36] D.S. Raja, G. Paramaguru, N. S. P. Bhuvanesh, J.H. Reibenspies, R. Renganathan, K.
Natarajan, Dalton Trans. 40 (2011) 4548-4559.
[37] Y.Z. Zhang, B. Zhou, Y.X. Liu, C.X. Zhou, X.L. Ding, Y. Liu, J. Fluoresc. 18 (2008) 109-118.
[38] M.R. Eftink, C.A.Ghiron, J. phys. Chem. 80 (1976) 486-493.
22

Journal Pre-proof
[39] A. Castiñeiras, N. Fernández-Hermidan, I. García-Santos, L. Gómez-Rodríguez, Dalton Trans.
41 (2012) 3486-3495.
[40] J.H. Tang, F. Luan, X.G. Chen, Biorg. Med. Chem. 14 (2006) 3210-3217.
[41] Z.J. Cheng, J. Lumin. 132 (2012) 2719-2729.
[42] S. Betanzos-Lara, Z. Liu, A. Habtemariam, A.M. Pizarro, B. Qamar, P.J. Sadler, Angew.
Chem. Int. Ed. 51 (2012) 3897-3900.
[43] Z. Liu, R.J. Deeth, J.S. Butler, A. Habtemariam, M.E. Newton, P.J.Sadler, Angew. Chem. Int.
Ed. 52 (2013) 4194-4197.
[44] I. Romero-Canelon, M. Mos, P.J. Sadler, J. Med. Chem. 58 (2015) 7874-7880.
[45] X. D. He, M. Tian, X.C. Liu, Y.H. Tang, C.F. Shao, P.W. Gong, J.F. Liu, S.M. Zhang, L.H.
Guo, Z. Liu, Chem. Asian J. 13 (2018) 1500 – 1509.
[46] V. Novohradsky, L. Zerzankova, J. Stepankova, A. Kisova, H. Kostrhunova, Z. Liu, P.J.
Sadler, J. Kasparkova, V. Brabec, Metallomics 6 (2014) 1491-1501.
[47] H. Antonicka, K. Choquet, Z.Y. Lin, A.C. Gingras, C.L. Kleinman, E.A. Shoubridge, Embo.
Rep. 18 (2017) 28-38.
[48] C.Qian, J. Wang, C. Song, L. Wang, L. Ji, H. Chao, Metallomics 5 (2013) 844-854.
[49] Y.Y. Jiang, G.H. Liu, X.R. Wang, J.M. Hu, G.Y. Zhang, S.Y. Liu, Macromolecules 48 (2015)
764-774.
[50] C. Jin, J. Liu, Y. Chen, G. Li, R. Guan, P. Zhang, L. Ji, H. Chao, Dalton Trans. 44 (2015)
7538–7547.
[51] C.Y. Li, M.X. Yu, Y. Sun, Y.Q. Wu, C.H. Huang, F.Y. Li, J. Am. Chem. Soc. 133 (2011)
11231-11239.
[52] S. Daum, V. Reshetnikov, M. Sisa, T. Dymych, M.D. Lootski, R. Bilyy, E. Bila, C. Janko, C.
Alexiou, M. Herrmann, L. Sellner, A. Mokhir, Angew. Chem. Int. Ed. 56 (2017) 15545–15549.
[53] Y. Zheng, L. He, D. Y. Zhang, C. P. Tan, L.-N Ji, Z. W. Mao, Dalton Trans. 46 (2017) 11395–
11407.
[54] J. Yang, J.X. Zhao, Q. Cao, L. Hao, D.X. Zhou, Z.J. Gan, L.N. Ji, Z.W. Mao, Acs Appl. Mater
Inter. 9 (2017) 13900-13912.
[55] X.X. Ge, S.J. Chen, X.C. Liu, Q.H. Wang, L.J. Gao, C.F. Zhao, L. Zhang, M.S. Shao, X.A.
Yuan, L.J. Tian, Z. Liu, Inorg. Chem. 58 (2019) 14175−14184.
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Journal Pre-proof
[56] H. Xu, K.Yin, W. Huang, Chem. Eur. J. 13 (2007) 10281-10293.
[57] S. Mabrouk, M.M. Zhang, Z.H. Wang, M. Liang, B. Bahrami, Y.G. Wu, J.H. Wu, Q.Q. Qiao,
S.F. Yang, J. Mater. Chem. A 6 (2018) 7950–7958.
[58] X.X Ge, X.C. Liu, Z.Z. Tian, S.J. Chen, X.Y. Liu, L.H. Guo, P.W. Gong, B.P. Ling, X.A.
Yuan, Z. Liu, Appl Organometal Chem. (2019) 5171.
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Graphical Abstract
Dual functions of Iridium(III) 2-phenylpyridine complexes: metastasis inhibition
and lysosomal damage
Xicheng Liu+*, Shujiao Chen+, Xingxing Ge, Ying Zhang, Yaoqi Xie, Yingying Hao, Daiqun Wu,
Jinmin Zhao, Xiang-Ai Yuan, Laijin Tian, Zhe Liu*
N-phenylcarbazole/triphenylamine-appended half-sandwich iridium(III) 2-phenylpyridine
complexes exhibited potential anticancer activity against A549 and HeLa cells. Complexes could
enter cells followed by non-energy-dependent cellular uptake mechanism, accumulate in
lysosomes, lead to lysosomal damage and eventually induce apoptosis.
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Highlights
1. Triphenylamine derivatives-appended iridium complexes exhibit antitumor activity.
2. Complexes inhibit the migration of tumor cells and induce lysosomal damage.
3. Complexes disrupt the cell cycle and induce apoptosis.
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