Half-sandwich Iridium(III) Benzimidazole-Appended Imidazolium-Based N-heterocyclic Carbene Complexes and Antitumor Application

Article abstract of DOI:10.1002/asia.201801323

A series of half-sandwich iridium(III) benzimidazole-appended imidazolium-based N-heterocyclic carbene (NHC) antitumor complexes [(η⁵-Cp^x)Ir(C^N)Cl]Cl, where Cp^x is pentamethylcyclopentadienyl (Cp*) or its biphenyl derivative (Cp^xbiph) and C^N is a NHC chelating ligand, were successfully synthesized and characterized. The Ir^III complexes showed potential antitumor activity against A549 cells, at most three times more potent than cis-platin under the same conditions. Complexes could bind to BSA by a static quenching mode, catalyzing the change of NADH to NAD⁺ and inducing the production of reactive oxygen species (maximum turnover number, 9.8), which play an important role in regulating cell apoptosis. Confocal microscopy showed that the complexes could specifically target lysosomes in cells with a Pearson's co-localization coefficient 0.76 and 0.72 after 1 h and 6 h, respectively, followed an energy-dependent cellular uptake mechanism and damaged the integrity of lysosomes. At the same time, complexes caused a marked loss of mitochondrial membrane potential.

Full text of DOI:10.1002/asia.201801323

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Half-sandwich Iridium(III) Benzimidazole-Appended Imidazolium-
Based N-heterocyclic Carbene Complexes and Antitumor
Application
Authors: Yali Han, Xicheng Liu, Zhenzhen Tian, Xingxing Ge, Juanjuan
Li, Min Gao, Yanru Li, Yi Liu, and Zhe Liu
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To be cited as: Chem. Asian J. 10.1002/asia.201801323
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10.1002/asia.201801323
Chemistry - An Asian Journal
Half-sandwich Iridium(III) Benzimidazole-Appended
Imidazolium-Based N-heterocyclic Carbene Complexes and
Antitumor Application
Yali Han, Xicheng Liu, Zhenzhen Tian, Xingxing Ge, Juanjuan Li, Min Gao, Yanru Li,
Yi Liu, Zhe Liu*
Institute of Antitumor 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 author (Email): liuzheqd@163.com
Abstract
Series of half-sandwich Iridium(III) benzimidazole-appended imidazolium-based
N-heterocyclic carbene (NHC) antitumor complexes [(η5-Cpx)Ir(C^N)Cl]Cl, where
Cpx is pentamethylcyclopentadienyl (Cp*) or its biphenyl derivatives (Cpxbiph) and
C^N is a NHC chelating ligand, were successfully synthesized and characterized. IrIII
complexes showed potential antitumor activity against A549 cells, at most three times
more potent than cis-platin under the same conditions. Complexes could bind to BSA
by a static quenching mode, catalyzing the change of NADH to NAD+ and inducing
the production of reactive oxygen species (maximum turnover number, 9.8), which
plays an important role in regulating cell apoptosis. Confocal microscopy showed that
the complexes could specific target lysosomes in cells with a Pearson’s
co-localization coefficient 0.76 and 0.72 after 1 h and 6 h, respectively, followed an
energy-dependent cellular uptake mechanism and damaged the integrity of lysosomes.
At the same time, complexes caused a marked loss of mitochondrial membrane
potential.
Key words: Half-sandwich iridium complex; N-heterocyclic carbene; Antitumor
1
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Introduction
The successful application of cis-platin in the clinic has promoted the
development of metal complexes antitumor drugs.[1-7] Platinum complexes are widely
used to treat various types of cancer, in fact, treatment with platinum-based or
platinum-related drugs accounts for over 50% of all chemotherapeutic regimens.[8-10]
Despite many benefits, however, these antitumor drugs also show several side effects,
such as strong resistance, poor selectivity, unclear mechanism of action, and serious
12]
adverse effects.[11,
Even so, the clinical application of platinum drugs has
stimulated to seek alternative antitumor drugs with greater efficiency, better targeting
ability, and novel antitumor mechanisms.[13] Among these, IrIII complexes have gained
particular attention because of their high stability and wide biological applications.[14]
These complexes exhibit high antiproliferative activity and a different mechanism of
action compared with those of conventional platinum-based drugs.[15]
Half-sandwich IrIII complexes, the general form of which is [(Cp)Ir(L^L)Z]X (Cp
represents electron-rich cyclopentadienyl group or its derivatives, Z is a leaving group,
L^L is a chelating ligand, and X is counter-ion), are fairly popular due to their high
antitumor activity. Among these, enhancing the electron donor capacity of Cp and
L^L could speed up the exchange of leaving group (Z), so as to improve the antitumor
activity of IrIII complexes,[16, 17] which can be reached by replacing Cp* with Cpxbiph (a
diphenyl derivative that having the stronger electron donor ability) or replacing the
bipyridine chelating ligand (N^N) with phenylpyridine ligand (C^N). In addition,
recent studies have found that the type and size of counter-ion (X) also have a greater
2
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impact on the antitumor activity of complexes. In general, the smaller volume of
counter-ion the better antitumor activity for the complexes was obtained.
NHC ligand applied in metal complexes as a widely used pharmacophore showed
significant effects in antitumor, anti-angiogenesis, anti-Alzheimer's disease and
anion-contagion studies.[18, 19] NHC ligand (C^N chelating ligand) can provide an
s-donor, which ensure high stability of the metal complex and reduces the probability
of decomposition of the compound in physiological media.[20-23] In addition, NHC
ligand can promote the reversible mechanism of coordination-dissociation, and affect
the catalytic activity of complex, which can interact at different stages of the cyclic
catalytic process in response the metal center.[24, 25] Above all, in this article, we report
the synthesis and characterization a serious of half-sandwich IrIII NHC complexes
([(η5-Cpx)Ir(C^N)Cl]Cl, Scheme 1). By adjusting the structure of cyclopentadiene
donor ligands (η5-Cpx), selecting the type of NHC chelating ligands (C^N) and using
the smaller chloride (Cl-) as counter-ion, IrIII NHC complexes show potential
antitumor activity against A549 human lung cancer cells by the MTT assay. The
antitumor mechanism of the complexes was also indentified by the binding to BSA,
catalyzing the change of NADH to NAD+, arresting the cell cycle, adjusting the
mitochondrial membrane potential, and lysosomal damage. Complexes could also
target the lysosome and mitochondria in cells and followed by an energy-dependent
cellular uptake mechanism. These results indicated that half-sandwich IrIII NHC
complexes have the potential to be new antitumor drugs.
Results and Discussion
3
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In this report, we use a simple and known method to create a new imidazole-rich
heterofunctional ligand. This is achieved by attaching an active (co-ordinative)
benzenimidazole function to the imidazolium-based NHC. Noteworthy, the carbene
ligand is a unusual protic NHC ligand of N-alkylated imidazole and the arene π orbital
27]
network with the NCN amidinylidene π orbitals,[26,
thus the resultant IrIII
complexes are chemically stable and catalytically active. Provided reaction times and
conditions are carefully adjusted depending on the starting material and the
appropriate dimer used, the reaction may be of considerable scope and readily
accessible. This structure provides direct access to a diversity of electronic properties
complexes,[28] which provide new prospects in targeted drugs.
The synthesis of the imidazolium ligand precursor of the C^N-chelated ligand was
carried out by direct N-alkylation of N-methylimidazole (ethyl, isopropyl and n-butyl)
with 2-chloromethyl-1-methyl-benzenimidazole (1), which was obtained as a hoar
solid in a one-step procedure from commercially available N-Methyl-1,2-phenyl-
enediamine with about 80% yield (Scheme 1).[29, 30] Adding 1-methylimidazole to a
solution of (1) in CH3CN at 90 °C for 48 h reacted to the formation of the
1-methyl-2-methyl-benzenimidazole-imidazolium salt L1, which was precipitated and
isolated as a gray solid in good yield (L2, L3 and L4 were synthesized using the same
method as L1). The formation of L1-L4 was confirmed by a characteristic downfield
1
signal in the H NMR spectrum at 9.41, 9.44, 9.51 and 9.43 ppm for the NCHN
proton. Half-sandwich IrIII NHC complexes [(η5-Cpx)Ir(C^N)Cl]Cl (1A-4B) were thus
obtained via a silver carbene intermediate, generated by adding silver oxide (Ag2O) to
4
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the NHC chelating ligands (L1-L4), remove excess silver oxide and add the
appropriate dimer [(η5-Cpx)IrCl2]2 in dichloromethane at ambient temperature. All
complexes were newly synthesized and achieved in good yields, purified to give a
yellow solid. The synthetic processes of ligands and complexes are shown in Scheme
1
1. The ligands were characterized by H NMR spectroscopy, electrospray ionization
mass spectrometry (ESI-MS) and elemental analysis. IrIII complexes were
1
characterized by H NMR, 13C NMR spectroscopy, mass spectrometry (MS),
elemental analysis and infrared spectroscopy (IR). And the characteristic peaks of
central imidazole carbon and other imidazole carbon of these complexes are 150.95 ~
13
154.07 ppm and 148.59 ~ 149.00 ppm in C NMR spectra (ESI Fig. S20-S27). The
characteristic peaks of these complexes are selectively labeled in IR spectra (ESI Fig.
S40-S47). Complexes were non-hygroscopic and highly soluble in common organic
solvents such as dichloromethane, chloroform, dimethyl sulfoxide, partially dissolved
in methanol, insoluble in ether, hexane and petroleum ether.
Scheme 1 Synthesis of IrIII NHC ligands (L1-L4) and complexes (1A-4B).
In Vitro Cytotoxicity
The antitumor activites of half-sandwich IrIII NHC complexes 1A-4B against A549
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human lung cancer cells were determined by the MTT assay. The IC50 value
(concentration when 50% cell growth was inhibited) after exposure to complexes and
cis-platin for 24 h are listed in Table 1. Except for complex 1A, other complexes
showed the potent antitumor activity against A549 lung cancer cells. In addition,
complex 4B have the best activity (IC50: 6.6 ± 1.7 μM), which was nearly three times
of cis-platin (IC50: 21.3 ± 1.7 μM) under the same conditions, and also better than the
ordinary Ir C^N (Phenylpyridine) complexes.
Table 1 IC50 values of complexes and cis-platin against A549 and BEAS-2B cells after 24 h
exposure.
IC50 (µM )
Complexes
A549
BEAS-2B
>100
[(η5-C5Me5)Ir(L1)Cl]Cl (1A)
[(η5-C5Me5)Ir(L2)Cl]Cl (2A)
>100
55.7 ± 3.3
42.8 ± 2.3
19.6 ± 1.3
51.0 ± 5.2
11.9 ± 0.8
7.9 ± 0.4
6.6 ± 1.7
21.3 ± 1.7
>100
[(η5-C5Me5)Ir(L3)Cl]Cl (3A)
>100
[(η5-C5Me5)Ir(L3)Cl]Cl (4A)
35.1 ± 2.1
32.6 ± 1.4
8.6 ± 0.1
5.7 ± 0.5
4.9 ± 0.1
/
[(η5-C5Me4C6H4C6H5)Ir(L1)Cl]Cl (1B)
[(η5-C5Me4C6H4C6H5)Ir(L2)Cl]Cl (2B)
[(η5-C5Me4C6H4C6H5)Ir(L3)Cl]Cl (3B)
[(η5-C5Me4C6H4C6H5)Ir(L4)Cl]Cl (4B)
Cis-platin
The cellular uptake capacity of metal-based antitumor complexes is usually related
to the size of lipophilicity and further affects the proliferation of tumor cells.[31]
Subject to the same NHC chelating ligand (C^N), Cpxbiph-based complexes show the
better activities than Cp*-based analogues (Fig. 1), which attribute to the stronger
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hydrophobicity for Cpxbiph ligand and help the complex to enter the tumor cells easier.
At the same time, IrIII complexes 1A-4A and 1B-4B also express the similar
cytotoxicity pattern of change: n-butyl > isopropyl > ethyl > methyl. The log P
(partition coefficient in oil/water) for complexes 1A, 4A and 4B were determined by
inductively coupled plasma mass spectrometry (ICP-MS), and the values were -2.01,
-1.07 and -0.29, respectively, which further confirmed this conclusion. These results
suggest that the antitumor activity of this type of complex can be tuned by varying
both the C^N-chelated ligand and the cyclopentadienyl ligand. In order to further
study the antitumor mechanism of IrIII NHC complexes, complex 4B with optimal
antitumor activity was selected as representative, and further studied.
Fig. 1 Histogram showed the IC50 values of complexes 1A-4B and cis-platin inhibited the growth
of A549 cells after 24 h.
In addition, the cytotoxicities of complexes 1A-4B were further evaluated against
the human bronchial epithelial normal cells BEAS-2B (Table 1). The results are not
satisfactory, no selectivity was observed for cancer cells versus normal cells with
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complexes 1B-4B. Although 1A-4A could selective for normal cells, ideal antitumor
activity and selectivity were not available at the same time, which may be related to
the structure of the complex. Hence, more structural modification is necessary in
future work to decrease the cytotoxic action toward normal cells without loss of the
selectivity between cancer and normal cells.
Hydrolysis Studies
The hydrolysis of M-Cl bonds to M-OH2 is considered to be a half-sandwich active
transition metal analogue, usually more reactive than the corresponding chloride
1
complex.[32] For the investigation of aqueous stability, studies were conducted by H
NMR and UV-Vis spectroscopy for complex 4B. Complex 4B was dissolved in 50%
CD3OD-d4/50% D2O (v/v) was monitored by 1H NMR spectroscopy, and the complex
showed no obvious change over 24 h (ESI Figure S1). It is possible that the aqua
1
complex was obtained before the H NMR spectra were acquired. Therefore, NaCl
(16 mol equiv) was then added to the solutions to further confirm the hydrolysis of
1
these complexes. There was also no change in the H NMR spectra. These results
suggest that the complexes remained stable under these conditions. Meanwhile, the
hydrolysis behaviors of complex 4B in 50% CH3OH/50% H2O (v/v) was also
monitored by UV-vis at 298 K. No absorbance changes were observed in UV-Vis
spectra (ESI Figure S2). As a result, the stability studies suggest that the
compounds have sufficient stability for the preparation of samples for biological
assays.
Reaction with NADH
Reduced coenzyme, nicotinamide adenine dinucleotide (NADH), plays a crucial
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role in many biological reactions.[33] It has been reported that NADH can provide
hydrides for half-sandwich IrIII complexes and promote the production of reactive
oxygen species (ROS) H2O2, thus providing an action mechanism of oxidants.[34] The
interaction of IrIII NHC complexes with NADH was detected by ultraviolet-visible
(UV-Vis) spectrum. The maximum absorbance of NADH and NAD+ can be
determined in 339 and 259 nm, respectively. As shown in Fig. 2, with the increase of
complex 4B, the absorption at 339 nm reduced, and an increase in 259 nm
significantly, which confirmed the catalytic activity of the IrIII NHC complex. And
also, the turnover number value of complex 4B was 9.8, which was calculated by
measuring the difference at 339 nm.
Fig. 2 Reaction of complex 4B (1.0 μM) and NADH (100 μM) in a 60% MeOH/40% H2O
(v/v) mixed solution was monitored by UV-Vis at 298 K over 7 h.
1
Complex 4B catalyze NADH to NAD+ was also monitored by H NMR at 298 K
with 7 h (Fig. 3). The addition of NADH (3.5 mM) to a solution of complex 4B (1.0
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mM) led to new peaks at 8.80, 9.17, and 9.35 ppm, assignable to the hydrogen atoms
at the C4, C6, and C2 positions of the nicotinamide ring of NAD+. In addition, a sharp
1
singlet at -12.85 ppm was observed in the H NMR spectrum, corresponding to Ir-H,
which confirm the conversion of NADH to its oxidized form NAD+.
Fig. 3 1H NMR spectra of the reaction between complex 4B (1.0 mM) and NADH (3.5 mM) in 60 %
CD3OD/40 % D2O (v/v) at 298 K: 10 min (bottom); 2 h (middle); 7 h (top). Left: Low-field region;
right: high-field region showing the Ir-H hydride peak (−12.85 ppm). Peaks labeled
correspond to chloride complex 4B and the formed Ir–H complex.
and
Protein (BSA) Binding Studies
Serum albumin (SA) is the most abundant protein in blood plasma, and playing an
important role in drug transport and metabolism.[35, 36] The research on the interaction
between antitumor agents and SA is conducive to understanding what happens in cells.
Bovine serum albumin (BSA) provides a cost-effective model because of its
resemblance to human serum albumin (HSA). Therefore, in this study, complex 4B
was selected to study the binding ability with BSA by UV-Vis absorption spectrum.
As shown in Fig. 4A, with the concentration of complex 4B increased, the maximum
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absorption peak at 227 nm decreases significantly, and the wavelength undergoes a
red shift, which is attributed to the interference between the α-helix of BSA and
complex. At 278 nm, red or blue shift wasn’t observed, indicating the complex 4B
changed the microenvironment of the Trp, Tyr and Phe aromatic acid residues in
BSA.[37]
Fig. 4 (A) UV-Vis spectroscopy of BSA in Tris-HCl / NaCI buffer solution (pH = 7.2), increasing
the concentration of complex 4B from 0 to 10μM. Inset: Wavelength range: 250-310 nm. (B)
Fluorescence spectrum of BSA (λex = 280 nm; λem = 343 nm) in the case where the concentration
of complex 4B was increased from 0 to 10 μM. Arrows: Direction of change in absorbance as the
concentration of the complex increases.
The fluorescence spectrum was determined and calibrated to correct the "inner
filter" effect. As shown in Fig. 4B, with the increase of complex 4B, the fluorescence
intensity of BSA quenched obviously and the maximum fluorescence intensity
wavelength has a small red shift, and the combination of the complex and BSA can be
further studied by a Stern-Volmer equation. The quenching rate constant Kq and
Stern-Volmer quenching constant Ksv were calculated via the classical Stern-Volmer
equation (ESI Fig. S3), and the binding constant Kb and binding site number n of all
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complexes were calculated by the Scatchard equation (ESI Fig. S4).[38] The Kq value
of complex 4B was calculated to be 1.52 × 1013 M-1 s-1, which is about three orders of
magnitude higher than the pure dynamic quenching mechanism (2.0 × 1010 M-1 s-1),
indicating that the IrIII NHC complex interacts with BSA by a static quenching
mechanism. The order of magnitude for Kq and Kb of complex 4B are 105 M-1s-1,
indicating a moderate combining level to BSA. Complex 4B showed the number of
binding site (n) with a value of 0.81 (~1), indicating about one binding site in BSA for
complex 4B.
Synchronous fluorescence spectroscopy plays an important role in obtaining
molecular environmental information near fluorescent molecules. As shown in ESI
Fig. S5 and S6, the synchronous fluorescence intensity decreases gradually with the
increase of the concentration of complex 4B, and the emission wavelength red shifts
to 3 nm at Δλ = 15 nm, but there is no obvious shift of emission wavelength at Δλ = 60
nm (Δλ: 15 nm and 60 nm, which are the characteristic of synchronous emission
spectrum for tyrosine and tryptophan residue, respectively).[39] The results indicate
IrIII NHC complex mainly changes the microenvironment of tyrosine for BSA.
Induction of apoptosis
To determine whether IrIII NHC complex could induce apoptosis, A549 cells were
treated with complex 4B at 0.5, 1.0, 2.0 and 3.0 equivalents IC50 for 24 h. After
staining with Annexin V/ Propidium Iodide, the data were obtained by flow
cytometry.[40] As shown in Fig. 5 and ESI Table S2, complex 4B induced apoptosis in
a dose-dependent manner, mainly manifested as late apoptosis and early apoptosis.
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The population of the late and early apoptosis had a significant increase with the value
changed from 3.3% and 0.3% to 30.4% and 4.3% after 24 h, respectively, when the
concentration changed from 0.5 × IC50 to 3.0 × IC50 for complex 4B. At the maximum
measured concentration (3.0 × IC50), complex 4B induced 34.7% apoptosis of A549
cells, while 97.3% of control cells are still alive under the same conditions. The
results indicated that IrIII NHC complex could reduce cell viability and induce
apoptosis.
Fig. 5 (A) Apoptosis analysis of A549 cancer cells after 24 h of exposure to complex 4B at 310 K
determined by flow cytometry using Annexin V-FITC vs PI staining. (B) Histogram showing
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populations for A549 cells in four stages treated by complex 4B. Data are quoted as mean ±
SD of three replicates.
ROS determination
The reactive oxygen species (ROS) mainly derived from mitochondria, and its
accumulation will lead to apoptosis, which usually used as one of the mechanisms to
explore the antitumor drugs.[41] Therefore, we detected the ROS level of A549 cells
treated with complex 4B by flow cytometry after 24 h. As shown in Fig. 6 and ESI
Table S3, compared with the control, the level of ROS increased by 1.3 and 2.1 times
for complex 4B in the concentration of 0.25 × IC50 and 0.5 × IC50, respectively. These
results indicate that IrIII NHC complexes could affect the mitochondria, induce the
production of ROS and lead to apoptosis.
Fig. 6 Flow cytometry analysis on ROS induction in A549 cancer cells treated with complex 4B at
concentration of 0.25 × IC50 and 0.5 × IC50.
Mitochondrial membrane potential
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Mitochondria provide biological energy to cells and play a vital role in triggering
intrinsic apoptotic pathway.[42] When the integrity of mitochondrial membrane was
destroyed, mitochondrial dysfunction happened and the signal of apoptosis would be
released.[43] The extent of mitochondrial dysfunction can be assessed by the loss of the
mitochondrial membrane potential (MMP). As shown in Fig. 7 and ESI Table S4, the
percentage of cells with mitochondrial membrane depolarization increased from 24.3%
to 35.0% when the concentration of complex 4B increased from 0.25 × IC50 to 2.0 ×
IC50. The increase of the ratio of JC-1 green/red fluorescence intensity further
confirmed IrIII NHC complexes could affect mitochondria and induce apoptosis.
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Fig. 7 Changes in mitochondrial membrane potential of A549 cancer cells induced by
complex 4B. (A) Flow cytometry histograms of the changes induced by the complex 4B at
concentrations of 0.25 × IC50, 0.5 × IC50, 1.0 × IC50 and 2.0 × IC50; (B) Histograms for the JC-1
monomers (Green)/JC-1 aggregates (Red) treated with different concentrations of complex 4B.
Data are quoted as mean ± SD of three replicates.
Cell imaging and cellular uptake
Laser confocal microscopy was used to study the co-localization because of the
intrinsic luminescence of complexes.[44] Lyso Tracker Red DND-99 (LTRD) and Mito
Tracker Deep Red (MTDR) were used as lysosomes and mitochondria fluorescence
probes, respectively.[45, 46] As shown in Fig. 8A, selected representative complex 4B
could effectively target lysosomes with the Pearson’s co-localization coefficient (PCC)
of 0.76 and 0.72 when incubated for 1 and 6 h, respectively. The value of PCC
indicated that IrIII NHC complex did not immediately cause A549 cells death, which
enabled to track the changes in lysosomal morphology timely. Lysosomes are acidic
intracellular organelles (pH: 4.5-5.5), which are involved in several cellular processes,
including receptor degradation, autophagy, apoptosis, post-translational protein
maturation, and the extracellular release of active enzymes. The NHC chelating
ligands of IrIII complexes contain a large number of nitrogen atoms that can provide
lone electron pair, which provide favorable conditions for the complexes to target
lysosomes.[47, 48] Additionally, the zeta potential of complex 4B (2.8 ± 0.3) were
positively charged, which could contribute to targeting with lysosomes after entering
the cytosol. Complex 4B showed much less targeting ability to mitochondria with low
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PCC of 0.21 and 0.15 after 1 h and 6 h, respectively, Fig. 8B.
Fig. 8 (A) Confocal microscopic images of A549 cells co-labeled with complex 4B (10 μM, 1 h, 6
h) and LTRD (75 nM, 1 h) for 1 h to 6 h, and collected at 549 - 651 nm (excitation: 594 nm, 1 h, 6
h), respectively. (B) Confocal microscopic images of A549 cells co-labeled with complex 4B (10
μM) and MTDR (500 nM, 30 min) for 1 h to 6 h, and collected at 610 - 756 nm (excitation: 644
nm). Complex 4B is excited at 488 nm. Scale bar: 20 μm.
The permeability of lysosomes depends on the integrity of lysosomes.[49, 50] Based
on co-localization experiments, complex 4B mainly localized the lysosomes of A549
cells. To investigate whether lysosomal damage induced by complex 4B is associated
with lysosome targeting specificity, lysosomal integrity of A549 cells were evaluated
by acridine orange (AO) staining after treatment with 4B for 6 h with the
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concentration of 1.0 and 2.0 × IC50. AO exhibits green fluorescence when binding to
RNA in nucleus or cytoplasm and red fluorescence when accumulated in
lysosomes.[51] As shown in Fig. 9, red fluorescence decreased significantly in
lysosomes when exposed to complex 4B (2.0 × IC50) after 6 h, and obvious lysosomal
damage was found, compared with control and 4B (1.0 × IC50). The results indicated
that IrIII NHC complexes could cause wreck to lysosomal integrity and induce
apoptosis.
Fig. 9 (a) A549 cells containing Acridine Orange (AO) as control group were not treated with
drugs. (b) After addition of complex 4B (1.0 × IC50) for 6 h, AO (5 μM) was added for 15 min
after laser confocal detection. (c) The concentration of the complex 4B is 2.0 × IC50, the rest of the
conditions are the same as (b). AO was excited at 488 nm and collected at a wavelength of
490-530 nm and 605-645 nm. Scale bar: 20 μm.
Small molecules can enter cells by different transport mechanisms, including
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energy-dependent mechanisms (such as endocytosis and active transport) and
energy-independent mechanisms (such as diffusion-promoting and passive
diffusion).[52] Complex 4B was selected to investigate the mode of cellular uptake
mechanism for IrIII NHC complexes. Incubation of A549 cells with complex 4B at
4 °C or pretreatment with carbonyl cyanide 3-chlorophenyl-hydrazone (CCCP), a
metabolic inhibitor, resulted in reduce cell uptake efficiency (Fig. 10). In addition, no
significant diversification of the uptake level of 4B was observed in A549 cells
pretreated with chloroquine, which usually regulated endocytosis by inhibiting
endoderm acidification.[53, 54] The results show that the cellular uptake complex 4B
mainly be controlled by the energy-dependent mechanism.
Fig. 10 Effect of incubation temperature (37 ºC and 4 ºC), metabolic inhibitor (CCCP, 50 μM)
and chloroquine (50 μM) on cellular uptake of 4B (10 μM, 30 min) measured by confocal
microscopy. Complex 4B was excited at 488 nm and emission was collected at 549-651 nm.
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Scale bar: 20 µm.
Conclusions
In this study, eight half-sandwich IrIII NHC complexes have been synthetized and
characterized. Complexes showed potential antitumor activity toward A549 cells, and
the complex 4B is even 3 times higher than the clinical antitumor drug cis-platin. The
antitumor activity can be regulated by changing the substituents of chelating ligands
and Cp* ligand, and following these rules: n-butyl > isopropyl > ethyl > methyl and
Cpxbiph > Cp*. Complexes can effectively bind with BSA by a static quenching
mechanism, catalyzing NADH to NAD+ and inducing ROS, which lead to apoptosis.
IrIII complexes can enter A549 cells by the energy-dependent mechanism, and mainly
targeted lysosomes and damaged the integrity of lysosomes. Therefore, half-sandwich
IrIII NHC complexes could be a promising candidate for further evaluation as
antitumor drugs.
Experimental Section
Materials
1-methylimidazole, 1-ethylimidazole, 1-isopropylimidazole, 1-n-butylimidazole,
N-Methyl-1,2-phenylenediamine, the appropriate dimer [(η5-Cpx)IrCl2]2 includes
[(η5-C5Me5)IrCl2]2 and [(η5-C5Me4C6H4C6H5)IrCl2]2 were prepared according to
literature procedures.[55] All other reagents are used as supplied by the commercial
supplier. Nitrogen was used as the drying and filling gas.
Synthesis of 2-(chloromethyl)-1-methylbenzimidazole
Monochloroacetic acid (8.0 g, 0.085 mol) and N-Methyl-1,2-phenylenediamine
(6.1 g, 0.05 mol) were refluxed in 50 mL of 4 N HC1 for 7.5 h with stirring. The
20
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reaction mixture was neutralized to neutral by K2CO3 solution. The precipitated
product was collected by vacuum filtration, and white solid was obtained after drying.
1
Yield: 4.9 g (80%). H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 7.9 Hz, 1H, Ar-H),
7.39 - 7.29 (m, 3H, Ar-H), 4.87 (s, 2H, NCH2C), 3.89 (s, 3H, N-CH3). Elemental
analysis: Found: C, 59.86; H, 5.01; N, 15.52%, calcd for C9H9ClN2: C, 59.84; H, 5.02;
N, 15.51%.
Synthesis of L1
To a solution of 2-chloromethyl-1-mehylbenzenimdazole (1.94 g, 10 mmol) in
CH3CN (100 mL) was added to 1-methylimidzole (1.64 g, 20 mmol). After reflux for
48 h, the solution was reduced to 10 mL by vacuum pump and Et2O was added to
precipitate the product. Then the resultant white powder was collected and washed
with ether (10 mL) three times. The product was dried in air and briefly purified.
1
Yield: 1.7 g (87%). H NMR (500 MHz, DMSO-d6) δ 9.41 (s, 1H, NCHN), 7.89 (s,
1H, imidazole-H), 7.79 (s, 1H, imidazole-H), 7.63 - 7.57 (m, 2H, Ar-H), 7.30 (t, J =
8.0 Hz, 1H, Ar-H), 7.22 (t, J = 7.6 Hz, 1H, Ar-H), 5.94 (s, 2H, NCH2C), 3.94 (s, 3H,
N-CH3), 3.88 (s, 3H, N-CH3). Elemental analysis: Found: C, 59.43; H, 5.75; N,
21.32%, calcd for C13H15ClN4: C, 59.41; H, 5.70; N, 21.36%. ESI-MS (m/z): calcd for
C13H15ClN4: 226.6 [C13H15N4]+; Found: 227.2.
L2, L3 and L4 were synthesized using the same method, the representation data is as
follows:
1
L2: Yield: 80%. H NMR (500 MHz, DMSO-d6) δ 9.44 (s, 1H, NCHN), 7.89 (d, J =
6.3 Hz, 2H, imidazole-H), 7.65 - 7.57 (m, 2H, Ar-H), 7.30 (t, J = 7.6 Hz, 1H, Ar-H),
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7.22 (t, J = 7.6 Hz, 1H, Ar-H), 5.89 (s, 2H, NCH2C), 4.28 (q, J = 7.3 Hz, 2H, N-CH2),
3.88 (s, 3H, N-CH3), 1.45 (t, J = 7.3 Hz, 3H, CH2CH3). Elemental analysis: Found: C,
60.76; H, 6.19; N, 20.24%, calcd for C14H17ClN4: C, 60.77; H, 6.17; N, 20.26%.
ESI-MS (m/z): calcd for C14H17ClN4: 240.6 [C14H17N4]+; Found: 241.2.
1
L3: Yield: 78%. H NMR (500 MHz, DMSO-d6) δ 9.51 (s, 1H, NCHN), 8.00 (t, J =
1.7 Hz, 1H, imidazole-H), 7.90 (t, J = 1.7 Hz, 1H, imidazole-H), 7.61 (t, J = 8.4 Hz,
2H, Ar-H), 7.32 - 7.28 (m, 1H, Ar-H), 7.25 - 7.20 (m, 1H, Ar-H), 5.87 (s, 2H,
NCH2C), 4.78 - 4.69 (m, 1H, N-iPr-CH), 3.88 (s, 3H, N-CH3), 1.51 (d, J = 6.7 Hz, 6H,
N-iPr-CH3). Elemental analysis: Found: C, 61.96; H, 6.59; N, 19.27%, calcd for
C15H19ClN4: C, 61.95; H, 6.57; N, 19.30%. ESI-MS (m/z): calcd for C15H19ClN4:
254.6 [C15H19N4]+; Found: 255.2.
1
L4: Yield: 83%. H NMR (500 MHz, DMSO-d6) δ 9.43 (s, 1H, NCHN), 7.88 (s, 2H,
imidazole-H), 7.60 (dd, J = 16.3, 8.0 Hz, 2H, Ar-H), 7.30 (t, J = 8.0 Hz, 1H, Ar-H),
7.22 (t, J = 8.0 Hz, 1H, Ar-H), 5.90 (s, 2H, NCH2C), 4.26 (t, J = 7.2 Hz, 2H,
N-nBu-CH2), 3.88 (s, 3H, N-CH3), 1.84 - 1.77 (m, 2H, N-nBu-CH2), 1.34 - 1.25 (m,
2H, N-nBu-CH2), 0.92 (t, J = 7.4 Hz, 3H, N-nBu-CH3). Elemental analysis: Found: C,
63.05; H, 6.94; N, 18.38%, calcd for C16H21ClN4: C, 63.02; H, 6.95; N, 18.37%.
ESI-MS (m/z): calcd for C16H21ClN4: 268.7 [C16H21N4]+; Found: 269.2.
Synthesis of IrIII NHC complexes (1A–4B)
General method: In a round bottom flask, silver oxide (2.4 eq) and (L1-L4) (2.0 eq)
were added to a solution of dichloromethane. After 8 h, the mixture was filtered
through celite and washed with CH2Cl2. The combined filtrates were added dropwise
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to a solution of CH2Cl2 containing Dimer ([Cp*IrCl2]2 and [CpxbiphIrCl2]2, 1.0 eq).
The mixture was stirred at room temperature for another 8 h and filtered through
celite. The solvent was removed in vacuum and the crude product was recrystallized
from CH2Cl2 / n-hexane to give a solid as a product.
[(η5-Cp*)Ir(L1)Cl]Cl (1A): Yield: 65%. 1H NMR (500 MHz, DMSO-d6) δ 7.79 (d, J =
8.1 Hz, 1H, imidazole-H), 7.65 (d, J = 1.9 Hz, 1H, imidazole-H), 7.57 (dd, J = 9.2,
5.0 Hz, 2H, Ar-H), 7.42 (dt, J = 23.2, 7.1 Hz, 2H, Ar-H), 5.98 (d, J = 16.7 Hz, 1H,
NCH2C), 5.09 (d, J = 16.7 Hz, 1H, NCH2C), 4.06 (s, 3H, N-CH3), 3.84 (s, 3H,
N-CH3), 1.68 (s, 15H, Cp*-H). 13C NMR (126 MHz, DMSO-d6) δ 153.88 (C-Ir),
148.67 (NCN), 138.58, 135.09, 124.74, 123.86 - 123.41, 119.22, 112.53, 90.49, 44.44,
37.29, 31.73, 9.44. Elemental analysis: Found: C, 44.13; H, 4.92; N, 8.94%, calcd for
C23H29Cl2IrN4: C, 44.16; H, 4.83; N, 8.96%. ESI-MS (m/z): calcd for C23H29ClIrN4:
589.17 [M-Cl]+; Found: 588.49. IR (KBr pellets, cm-1): 2973 (-CH3), 1636 (C=C),
1384(C-H), 1315 (C-N).
[(η5-Cp*)Ir(L2)Cl]Cl (2A): Yield: 72%. 1H NMR (500 MHz, DMSO-d6) δ 7.79 (d, J =
8.0 Hz, 1H, imidazole-H), 7.69 (s, 1H, imidazole-H), 7.67 (s, 1H, Ar-H), 7.61 (d, J =
8.0 Hz, 1H, Ar-H), 7.46 - 7.39 (m, 2H, Ar-H), 5.96 (d, J = 16.8 Hz, 1H, NCH2C), 5.09
(d, J = 16.7 Hz, 1H, NCH2C), 4.25 (dd, J = 13.1, 7.4 Hz, 1H, N-CH2), 4.13 - 4.08 (m,
1H, N-CH2), 4.06 (s, 3H, N-CH3), 1.66 (s, 15H, Cp*-H), 1.38 (t, J = 7.2 Hz, 3H,
N-CH2CH3). 13C NMR (126 MHz, DMSO-d6) δ 154.07 (C-Ir), 148.84 (NCN), 138.60,
135.09, 124.73, 124.05, 123.70, 121.22, 119.23, 112.52, 90.45, 44.90, 44.43, 31.76,
16.81, 9.40. Elemental analysis: Found: C, 44.97; H, 5.12; N, 8.75%, calcd for
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C24H31Cl2IrN4: C, 45.06; H, 5.04; N, 8.76%. ESI-MS (m/z): calcd for C24H31ClIrN4:
603.19 [M-Cl]+; Found: 602.56. IR (KBr pellets, cm-1): 2978 (-CH3), 2923 (-CH2),
1631 (C=C), 1382 (C-H), 1315 (C-N).
[(η5-Cp*)Ir(L3)Cl]Cl (3A): Yield: 67%. 1H NMR (500 MHz, DMSO-d6) δ 7.79 (d, J =
8.1 Hz, 1H, imidazole-H), 7.74 (d, J = 2.1 Hz, 1H, imidazole-H), 7.71 (d, J = 2.0 Hz,
1H, Ar-H), 7.64 (d, J = 8.2 Hz, 1H, Ar-H), 7.46 - 7.42 (m, 1H, Ar-H), 7.40 - 7.37 (m,
1H, Ar-H), 5.94 (d, J = 16.7 Hz, 1H, NCH2C), 5.05 (d, J = 16.7 Hz, 1H, NCH2C),
4.73 - 4.69 (m, 1H, N-iPr-CH), 4.06 (s, 3H, N-CH3), 1.66 (s, 15H, Cp*-H), 1.56 (d, J
= 6.8 Hz, 3H, N-iPr-CH3), 1.37 (d, J = 6.4 Hz, 3H, N-iPr-CH3). 13C NMR (126 MHz,
DMSO-d6) δ 152.99 (C-Ir), 149.00 (NCN), 138.39, 135.13, 124.71, 124.34, 123.65,
119.36, 119.12, 112.59, 90.51, 51.88, 44.50, 31.78, 25.57, 23.55, 9.59. Elemental
analysis: Found: C, 45.97; H, 5.12; N, 8.60 %, calcd for C25H33Cl2IrN4: C, 46.01; H,
5.10; N, 8.58 %. ESI-MS (m/z): calcd for C25H33ClIrN4: 616.97 [M-Cl]+; Found:
617.08. IR (KBr pellets, cm-1): 2982 (-CH3), 2908 (-CH), 1620 (C=C), 1382 (C-H),
1310 (C-N).
[(η5-Cp*)Ir(L4)Cl]Cl (4A): Yield: 77%. 1H NMR (500 MHz, DMSO-d6) δ 7.79 (d, J =
7.9 Hz, 1H, imidazole-H), 7.68 (d, J = 2.0 Hz, 1H, imidazole-H), 7.66 (d, J = 2.0 Hz,
1H, Ar-H), 7.60 (d, J = 7.9 Hz, 1H, Ar-H), 7.46 - 7.38 (m, 2H, Ar-H), 5.96 (d, J =
16.8 Hz, 1H, NCH2C), 5.09 (d, J = 16.7 Hz, 1H, NCH2C), 4.20 (dd, J = 12.0, 6.7 Hz,
1H, N-nBu-CH2), 4.06 (s, 3H, N-CH3), 4.00 (dd, J = 11.9, 6.9 Hz, 1H, N-nBu-CH2),
1.86 (dd, J = 14.2, 5.6 Hz, 1H, N-nBu-CH2), 1.66 (s, 15H, Cp*-H), 1.46 - 1.25 (m, 3H,
N-nBu-CH2), 0.92 (t, J = 7.4 Hz, 3H, N-nBu-CH3). 13C NMR (126 MHz, DMSO-d6) δ
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154.00 (C-Ir), 148.79 (NCN), 138.60, 135.09, 124.73, 123.95, 123.69, 121.48, 119.22,
112.51, 90.45, 49.81, 44.44, 33.03, 31.75, 20.03, 14.19, 9.39. Elemental analysis:
Found: C, 46.88; H, 5.31; N, 8.34 %, calcd for C26H35Cl2IrN4: C, 46.84; H, 5.29; N,
8.40 %. ESI-MS (m/z): calcd for C26H35ClIrN4: 630.98 [M-Cl]+; Found: 631.12. IR
(KBr pellets, cm-1): 2973 (-CH3), 2923 (-CH2), 1649 (C=C), 1387 (C-H), 1324 (C-N).
[(η5-Cpxbiph)Ir(L1)Cl]Cl (1B): Yield: 66%. 1H NMR (500 MHz, DMSO-d6) δ 7.79 (d,
J = 8.1 Hz, 1H, imidazole-H), 7.74 (d, J = 3.9 Hz, 1H, imidazole-H), 7.70 (ddd, J =
11.8, 8.0, 3.1 Hz, 5H, Ar-H), 7.62 (d, J = 8.4 Hz, 1H, Ar-H), 7.50 (ddd, J = 16.3, 6.8,
4.9 Hz, 4H, Ar-H), 7.40 (dt, J = 10.6, 7.3 Hz, 2H, Ar-H), 7.30 - 7.26 (m, 1H, Ar-H),
6.02 (d, J = 16.9 Hz, 1H, NCH2C), 5.16 (d, J = 16.7 Hz, 1H, NCH2C), 4.07 (s, 3H,
N-CH3), 3.56 (s, 3H, N-CH3), 1.82 (d, J = 15.5 Hz, 3H, Cpxbiph-H), 1.76 (d, J = 16.1
Hz, 6H, Cpxbiph-H), 1.71 (s, 3H, Cpxbiph-H). 13C NMR (126 MHz, DMSO-d6) δ 152.14
(C-Ir), 148.47 (NCN), 140.46, 139.96, 138.59, 135.09, 131.48, 130.79, 129.49,
128.18, 127.14, 124.82, 123.97, 123.66, 119.25, 112.58, 96.41, 92.46, 90.45, 87.93,
86.46, 31.80, 10.84, 10.34, 9.97, 9.76, 8.79. Elemental analysis: Found: C, 53.56; H,
4.32; N, 7.36 %, calcd for C34H35Cl2IrN4: C, 53.54; H, 4.63; N, 7.35 %. ESI-MS (m/z):
calcd for C34H35ClIrN4: 727.22 [M-Cl]+; Found: 726.95. IR (KBr pellets, cm-1): 2972
(-CH3), 1647 (C=C), 1382 (C-H), 1319 (C-N).
[(η5-Cpxbiph)Ir(L2)Cl]Cl (2B): Yield: 62%. 1H NMR (500 MHz, DMSO-d6) δ 7.80 (d,
J = 8.2 Hz, 1H, imidazole-H), 7.75 (s, 1H, imidazole-H), 7.75 - 7.70 (m, 4H, Ar-H),
7.63 (dd, J = 7.1, 5.2 Hz, 3H, Ar-H), 7.55 (d, J = 8.3 Hz, 1H, Ar-H), 7.50 (t, J = 7.7
Hz, 2H, Ar-H), 7.42 (dd, J = 7.7, 5.5 Hz, 2H, Ar-H), 7.30 (t, J = 7.4 Hz, 1H, Ar-H),
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6.01 (d, J = 16.8 Hz, 1H, NCH2C), 5.15 (d, J = 16.8 Hz, 1H, NCH2C), 4.08 (s, 3H,
N-CH3), 4.02 (dd, J = 13.2, 7.3 Hz, 1H, N-CH2), 3.79 (dd, J = 13.3, 7.0 Hz, 1H,
N-CH2), 1.85 (s, 3H, Cpxbiph-H), 1.78 (s, 3H, Cpxbiph-H), 1.73 (d, J = 11.3 Hz, 6H,
Cpxbiph-H), 1.12 (t, J = 7.2 Hz, 3H, Cpxbiph-H). 13C NMR (126 MHz, DMSO-d6) δ
152.29 (C-Ir), 148.63 (NCN), 140.22, 139.74, 138.51, 135.09, 130.80, 129.57, 128.31,
127.11, 124.80, 124.23, 123.66, 121.55, 119.32, 112.57, 96.69, 93.56, 91.74, 88.79,
87.07, 44.86, 44.48, 31.81, 16.19, 10.94, 10.19, 9.77. Elemental analysis: Found: C,
54.04; H, 4.82; N, 7.27 %, calcd for C35H37Cl2IrN4: C, 54.12; H, 4.80; N, 7.21 %.
ESI-MS (m/z): calcd for C35H37ClIrN4: 741.23 [M-Cl]+; Found: 741.83. IR (KBr
pellets, cm-1): 2973 (-CH3), 2927 (-CH2), 1645 (C=C), 1382 (C-H), 1324 (C-N).
[(η5-Cpxbiph)Ir(L3)Cl]Cl (3B): Yield: 69%. 1H NMR (500 MHz, DMSO-d6) δ 7.82 (d,
J = 8.2 Hz, 1H, imidazole-H), 7.75 (s, 1H, imidazole-H), 7.74 - 7.70 (m, 4H, Ar-H),
7.68 - 7.59 (m, 4H, Ar-H), 7.50 (t, J = 7.6 Hz, 2H, Ar-H), 7.47 - 7.37 (m, 3H, Ar-H),
6.02 (d, J = 16.8 Hz, 1H, NCH2C), 5.16 (d, J = 16.7 Hz, 1H, NCH2C), 4.53 (dd, J =
13.4, 6.9 Hz, 1H, N-iPr-CH), 4.10 (s, 3H, N-CH3), 1.98 (s, 3H, Cpxbiph-H), 1.77 (d, J
= 28.7 Hz, 6H, Cpxbiph-H), 1.64 (s, 3H, Cpxbiph-H), 1.24 (d, J = 6.5 Hz, 3H, N-iPr-CH3),
0.73 (d, J = 6.8 Hz, 3H, N-iPr-CH3). 13C NMR (126 MHz, DMSO-d6) δ 150.95 (C-Ir),
148.74 (NCN), 140.31, 139.81, 138.10, 135.16, 130.78, 129.58, 128.32, 127.11,
124.82, 124.48, 123.71, 119.57, 119.33, 112.71, 97.85, 96.74, 90.88, 87.79, 84.81,
51.76, 44.54, 31.86, 24.99, 21.93, 11.63, 10.05, 9.75. Elemental analysis: Found: C,
54.71; H, 4.93; N, 7.02 %, calcd for C36H39Cl2IrN4: C, 54.67; H, 4.97; N, 7.08 %.
ESI-MS (m/z): calcd for C36H39ClIrN4: 755.12 [M-Cl]+; Found: 755.25. IR (KBr
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pellets, cm-1): 2982 (-CH3), 2912 (-CH), 1635 (C=C), 1378 (C-H), 1315 (C-N).
[(η5-Cpxbiph)Ir(L4)Cl]Cl (4B): Yield: 64%. 1H NMR (500 MHz, DMSO-d6) δ 7.81 (d,
J = 8.3 Hz, 1H, imidazole-H), 7.75 (s, 1H, imidazole-H), 7.74 - 7.64 (m, 6H, Ar-H),
7.58 (dd, J = 13.4, 5.1 Hz, 2H, Ar-H), 7.51 (t, J = 7.7 Hz, 2H, Ar-H), 7.42 (q, J = 7.5
Hz, 2H, Ar-H), 7.33 (t, J = 7.7 Hz, 1H, Ar-H), 6.01 (d, J = 16.8 Hz, 1H, NCH2C),
5.17 (d, J = 16.8 Hz, 1H, NCH2C), 4.08 (s, 3H, N-CH3), 3.93 - 3.87 (m, 1H,
N-nBu-CH2), 3.63 (dd, J = 12.1, 7.2 Hz, 1H, N-nBu-CH2), 1.91 (s, 3H, Cpxbiph-H),
1.80 (s, 3H, Cpxbiph-H), 1.70 (d, J = 9.6 Hz, 6H, Cpxbiph-H), 1.60 - 1.55 (m, 1H,
N-nBu-CH2), 1.50 - 1.44 (m, 1H, N-nBu-CH2), 1.08 (dd, J = 14.9, 7.3 Hz, 1H,
N-nBu-CH2), 0.99 (dd, J = 14.7, 6.8 Hz, 1H, N-nBu-CH2), 0.71 (t, J = 7.4 Hz, 3H,
N-nBu-CH3). 13C NMR (126 MHz, DMSO-d6) δ 152.15 (C-Ir), 148.59 (NCN), 140.33,
139.87, 138.44, 135.13, 130.86, 129.56, 128.28, 127.17, 124.81, 124.09, 123.69,
121.70, 119.33, 112.60, 96.72, 95.68, 92.03, 86.97, 86.29, 49.60, 44.48, 32.68, 31.82,
19.74, 14.12, 11.12, 10.08, 9.79. Elemental analysis: Found: C, 55.13; H, 5.15; N,
6.95 %, calcd for C37H41Cl2IrN4: C, 55.21; H, 5.13; N, 6.96 %. ESI-MS (m/z): calcd
for C37H41ClIrN4: 769.26 [M-Cl]+; Found: 769.14. IR (KBr pellets, cm-1): 2973
(-CH3), 2932 (-CH2), 1636 (C=C), 1382 (C-H), 1315 (C-N).
Acknowledgments
We thank the University Research Development Program of Shandong Province
(J18KA082), the National Natural Science Foundation of China (Grant No. 21671118)
and the Taishan Scholars Program for support.
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