← Back
Valproic Acid-Functionalized Cyclometalated Iridium(III) Complexes as Mitochondria-Targeting Anticancer Agents.
DOI: 10.1002/chem.201703157
Full Paper
& Antitumor Agents
Valproic Acid-Functionalized Cyclometalated Iridium(III)
Complexes as Mitochondria-Targeting Anticancer Agents
Rui-Rong Ye, Jian-Jun Cao, Cai-Ping Tan, Liang-Nian Ji, and Zong-Wan Mao*[a]
Abstract: Valproic acid (VPA) is a short-chain, fatty acid type
histone deacetylase inhibitor (HDACi), which can cause
growth arrest and induce differentiation of transformed
cells. Phosphorescent cyclometalated IrIII complexes have
emerged as potential anticancer agents. By conjugation of
VPA to IrIII complexes through an ester bond, VPA-functionalized cyclometalated iridium(III) complexes 1 a–3 a were designed and synthesized. These complexes display excellent
two-photon properties, which are favorable for live-cell
imaging. The ester bonds in 1 a–3 a can be hydrolyzed
Introduction
Iridium complexes have recently emerged as promising alternatives to platinum-based metallo-anticancer drugs.[1] Phosphorescent cyclometalated IrIII complexes are regarded as excellent probes for bioimaging and biosensing, due to their outstanding photophysical properties, including relatively high
quantum yields, long emission lifetimes, large Stokes shifts,
two-photon absorption (TPA) and high photobleaching resistance.[2] On the other hand, cyclometalated IrIII complexes are
also considered to be potent anticancer candidates, as they
can target subcellular organelles,[3] inhibit protein activities,[4]
and act as photodynamic therapeutic agents.[5] We endeavored
to develop cyclometalated IrIII complexes as multifunctional
theranostic agents integrating anticancer properties and imaging capabilities.[3f, 4c, 5a, 6]
Mitochondria are known as the power houses of a cell, and
they also play important roles in many important cellular processes, for example, apoptosis regulation and intracellular signaling.[7] Defects in mitochondrial function are directly related
to aging, cancer, and neurodegenerative disorders.[8] With a diverse range of mitochondria-targeted drugs currently in clinical
trials, targeting mitochondria as a cancer therapy strategy has
had great success in recent years.[9]
[a] Dr. R.-R. Ye, J.-J. Cao, Dr. C.-P. Tan, Prof. L.-N. Ji, Prof. Z.-W. Mao
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry
School of Chemistry and Chemical Engineering
Sun Yat-Sen University, Guangzhou 510275 (P. R. China)
E-mail: cesmzw@mail.sysu.edu.cn
Supporting information and the ORCID identification number for the
author of this article can be found under https://doi.org/10.1002/
chem.201703157.
Chem. Eur. J. 2017, 23, 15166 – 15176
quickly by esterase and display similar inhibition of HDAC
activity to VPA. Notably, 1 a–3 a can overcome cisplatin resistance effectively and are about 54.5–89.7 times more cytotoxic than cisplatin against cisplatin-resistant human lung
carcinoma (A549R) cells. Mechanistic studies indicate that
1 a–3 a can penetrate into human cervical carcinoma (HeLa)
cells quickly and efficiently, accumulate in mitochondria, and
induce a series of cell-death-related events mediated by mitochondria. This study gives insights into the design and anticancer mechanisms of multifunctional anticancer agents.
Histone deacetylase inhibitors (HDACis) are emerging as a
new class of epigenetic anticancer drugs that can promote the
acetylation of histones and non-histone proteins and induce
transcriptional events involved in growth arrest, differentiation,
and apoptotic cell death.[10] With suberoylanilide hydroxamic
acid (SAHA) approved by the FDA in 2006 for the treatment of
the rare cancer cutaneous T-cell lymphoma, more and more organic-molecule HDACis have been explored for cancer therapy,
and several HDACis are now in phase I and II clinical trials.[11]
By equipping SAHA with phosphorescent compounds of
metals such as ruthenium(II), iridium(III), and rhenium(I), we
have developed a series of metal-based HDACis.[4c, 12]
Valproic acid (VPA), a clinically used antiepileptic and anticonvulsant drug,[13] has recently been demonstrated as a shortchain, fatty acid type HDACi.[14] Similar to more widely studied
HDACis, VPA can cause growth arrest and induce differentiation of transformed cells in culture.[15] Combining VPA with
platinum, Shen,[16] Brabec,[18] Gibson[19] and their respective coworkers reported a series of PtIV-VPA prodrugs, and these complexes exhibited strong synergistic cytotoxicity.
Esterification is an efficient and convenient optimization
method for carboxylic acid-containing compounds, which can
markedly improve their cellular uptake efficacy.[20] Free VPA
hardly enters cells, so that, starting from millimolar concentrations in medium, only low micromolar concentrations were observed in cells.[18] Herein, by conjugating VPA with cyclometalated IrIII complexes through an ester bond, VPA-functionalized
IrIII complexes 1 a–3 a (Scheme 1) were designed and synthesized. Complexes 1 b–3 b, which lack the VPA group, were used
as references. Fluorescence spectroscopy, time-resolved emission spectroscopy, and ESI-MS studies were performed to investigate the hydrolytic process of the ester bonds in 1 a–3 a.
The TPA properties, HDAC inhibitory activities, in vitro
15166
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper
Scheme 1. Chemical structures of ligand L, HMbpy, cyclometalated IrIII complexes 1 a–3 a, and their corresponding hydrolysis products 1 b–3 b and VPA.
antiproliferative activity, and anticancer mechanisms including
subcellular localization, impact on mitochondrial integrity, elevation of reactive oxygen species (ROS), cell-cycle arrest, and
induction of apoptosis, were investigated in detail.
Results and Discussion
Synthesis, characterization, and photophysical properties
Ligand L was obtained by the reaction of 4-hydroxymethyl-4’methyl-2,2’-bipyridyl (HMbpy) with 2-propylvaleryl chloride for
12 h at room temperature. Complexes 1 a–3 a and 1 b–3 b were
synthesized by heating two equivalents of L or HMbpy with
the corresponding IrIII chlorido-bridged dimer in refluxing
CH2Cl2/CH3OH under nitrogen for 4 h. The synthetic routes to
ligand L, 1 a–3 a, and 1 b–3 b are shown in Schemes S1–S3 of
the Supporting Information. Complexes 1 a–3 a and 1 b–3 b
were characterized by ESI-MS, 1H and13C NMR spectroscopy
(Figures S1–S12 of the Supporting Information), and elemental
analysis. The structures of 2 a and 2 b were also determined by
X-ray diffraction (Figure 1). The crystal data and selected bond
lengths and angles are listed in Tables S1 and S2 of the Supporting Information.
The photophysical properties of the IrIII complexes were investigated in phosphate buffered saline (PBS), CH2Cl2, and
CH3CN. They all show intense absorption bands at 250–450 nm
(Figure S13A of the Supporting Information), which can be assigned to mixed ligand-centered (LC) transition, ligand-toligand charge transfer, and singlet and triplex metal-to-ligand
charge transfer (1MLCT and 3MLCT). On excitation at 405 nm,
1 a–3 a and 1 b–3 b exhibit green to red phosphorescent emissions (Figure S13B of the Supporting Information). Quantum
yields of the IrIII complexes range from 0.002 to 0.29, and phosphorescence lifetimes lie between 18.60 and 951.56 ns in different solvents at room temperature (Table S3 of the Supporting Information). Compared with reference complexes 1 b–3 b,
conjugation of VPA to IrIII complexes can increase the emission
lifetimes of conjugates 1 a–3 a in PBS. As shown in Table S3 of
Chem. Eur. J. 2017, 23, 15166 – 15176
www.chemeurj.org
Figure 1. X-ray crystal structures of 2 a and 2 b. The hydrogen atoms and
counterions are omitted for clarity.
the Supporting Information, the emission lifetimes of 1 a–3 a in
PBS are increased approximately 6.7-, 2.5-, and 17.6-fold compared with those of 1 b–3 b, respectively. Moreover, the emission spectra of 1 a–3 a also show redshifts, due to the electrondonating OCH(CH2CH2CH3)2 group of VPA. For complexes with
_
the same CN ligand, the energy gap between the HOMO and
the LUMO can be decreased by increasing the conjugated
_
length of NN ligands, which elicits a redshift of the emission
wavelength and an increase in emission lifetime.[21]
Two-photon absorption (TPA) cross sections
Cyclometalated iridium(III) complexes are good candidates for
two-photon phosphorescent imaging.[2b, 3f] Their two-photon
excitation has several advantages over conventional
15167
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper
one-photon excitation, such as decreased photobleaching effects, minimal photodamage to cellular structures, low background interference, and deep penetration.[22] The TPA properties of 1 a–3 a and 1 b–3 b were investigated by using a twophoton-induced fluorescence method with rhodamine B as a
reference in CH3OH, and the cross sections were determined in
the wavelength range of 720–890 nm. Over the measured
range, the maximum TPA cross sections dmax of 1 a–3 a and
1 b–3 b ranged from 38 to 129 GM (1 GM = 10@50 cm4 photon@1)
at l = 740 nm (Figure S14 and Table S3 of the Supporting Information). These values are moderate compared with those of
other IrIII complexes measured by the same method.[5b,c, 23]
From the log–log plot of the emission intensity against incident power, the linear regression slopes of 1 a–3 a and 1 b–3 b
are 1.98 : 0.01, 2.00 : 0.03, 2.05 : 0.01, 1.95 : 0.02, 2.05 : 0.01
and 1.94 : 0.01, respectively, which indicate nonlinear TPA of
the IrIII complexes (Figure S15 of the Supporting Information).
Hydrolysis by esterase in vitro
Ester-modified compounds have been widely used as prodrugs. They can be hydrolyzed selectively or nonselectively by
a variety of esterases to release active pharmacophores.[24] The
responses of 1 a–3 a to esterase were monitored by fluorescence spectroscopy, time-resolved emission spectroscopy,[25]
and ESI-MS.[26] Herein, porcine liver esterase (PLE) was used as
a model to investigate the hydrolytic process of the ester
bonds in 1 a–3 a. Time-dependent emission studies showed
that the 3MLCT emissions of 1 a–3 a show a decrease in intensity on treatment with PLE for 1 h (Figure 2 A). The emission intensities decreased by a factor of about 3.4, 2.9, and 11.4 for
1 a–3 a, respectively. The hydrolytic half-lives of 1 a–3 a are
14.5, 12.7, and 7.3 min, respectively. No significant changes in
the emission of 1 b–3 b were found on treatment with PLE for
the same time (Figure S16 of the Supporting Information),
which indicates that the observed emission decrease of 1 a–3 a
is a result of the release of VPA after hydrolysis by esterase.
Time-resolved emission decay was also used to investigate
the hydrolytic process of the ester bonds in 1 a–3 a. As shown
in Figure 2 B, the emission decay curves of 1 a–3 a treated with
PLE for different time intervals gradually approach that of reference complexes 1 b–3 b, respectively. The luminescence lifetimes are 326.75 and 48.61 ns for 1 a and 1 b, respectively. On
treatment of 1 a with PLE for 10, 30, and 60 min, the luminescence lifetimes of the system were 218.38, 177.50, and
74.95 ns, respectively (Figure 2 B a). The decreased lifetimes are
probably due to hydrolysis of the ester linkage. Similar results
were also obtained after incubation of 2 a or 3 a with PLE for
different time intervals (Figure 2 B b and c).
Equal amounts of 1 a–3 a were incubated with PLE in PBS for
different time intervals. The mass spectra of 1 a–3 a showed
peaks assigned to intact complexes as well as peaks
corresponding to reference complexes (Figure S17–S19 of the
Supporting Information). The results indicate that 1 a–3 a can
undergo hydrolysis in the presence of esterase.
HDAC enzyme inhibition assay
The HDAC inhibitory activities of VPA, 1 a–3 a, and 1 b–3 b
were measured by using a commercially available HDAC assay
kit, and the results are summarized in Figure 3. In the present
study, we found that 1 mm VPA could inhibit 59 % of the
Figure 2. A) Time-dependent changes in emission spectra (2 V 10@5 m, lex = 405 nm) of 1 a (a), 2 a (b), and 3 a (c) with PLE at 298 K; insets: plots of relative
emission intensities at 584 nm (1 a), 527 nm (2 a), and 631 nm (3 a) versus time of esterase treatment. B) Time-resolved emission decay curves of 1 a (a), 2 a
(b), and 3 a (c) on treatment with PLE at 298 K for different time intervals, which were compared with those of 1 b, 2 b and 3 b, respectively.
Chem. Eur. J. 2017, 23, 15166 – 15176
www.chemeurj.org
15168
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper
Table 1. IC50 values of tested compounds towards different cell lines.[a]
Compound
Figure 3. HDAC inhibition activity of VPA, 1 a–3 a, 1 b–3 b, PLE, and mixtures
of 1 a–3 a with PLE. Data are shown as mean : SD of three independent experiments.
HDAC activity. Complexes 1 a–3 a and 1 b–3 b at 1 mm VPAequivalent dose showed 23, 17, 19, 20, 16, and 14 % inhibition
of the HDAC activity, respectively. When 1 a–3 a were pretreated with PLE at 298 K for 10 h, their HDAC inhibitory activity
dramatically increased to 56, 52, and 59 %, respectively. This indicates that once the ester bonds in 1 a–3 a are hydrolyzed,
free VPA is released, the VPA effectively elicits HDAC inhibition
activity, and the produced 1 b–3 b do not interfere with HDAC
inhibition by VPA.
Lipophilicity and cellular uptake efficacy of IrIII complexes
Lipophilicity lg P can strongly influence the cellular uptake, localization, and cytotoxicity of a compound.[27] The lipophilicity
of 1 a–3 a and 1 b–3 b was determined by the flask-shaking
method. The lg P values obtained for the compounds are in
the
following order: 2 a (2.9) > 1 a (2.1) > 3 a (2.0) > 2 b (1.8) > 1 b
(1.0) > 3 b (0.9) (Table S4 of the Supporting Information). The
result indicates that the conjugation of VPA to IrIII complexes
can enhance the lipophilicity of 1 a–3 a.
As iridium is an exogenous element, the quantitative measurement of the cellular uptake levels of 1 a–3 a and 1 b–3 b
was determined by inductively coupled plasma mass spectrometry (ICP-MS). On incubation of 5 mm complexes with
human cervical carcinoma (HeLa) cells for 30 min, the intracellular iridium contents of compounds were in the following
order (Table S4 of the Supporting Information): 2 a (1174.7(:
100.5) ng per 106 cells) > 1 a (610.0(: 59.2) ng per 106 cells) >
3 a (578.4(: 46.8) ng per 106 cells) > 2 b (564.2(: 50.0) ng per
106 cells) > 1 b (434.2(: 48.2) ng per 106 cells) > 3 b (361.0(:
35.1) ng per 106 cells). The cellular uptake efficiency of 1 a–3 a
and 1 b–3 b is well correlated with their lipophilicity.
In vitro cytotoxicity
The cytotoxicities of VPA, 1 a–3 a, 1 b–3 b, and the mixtures of
1 b–3 b with VPA (1 b + VPA, 2 b + VPA, and 3 b + VPA) were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against HeLa, human pulmonary carcinoma (A549), cisplatin-resistant A549 (A549R), human hepatocellular liver carcinoma (HepG2), and human normal liver
(LO2) cells. On the basis of the IC50 values (Table 1), the in vitro
Chem. Eur. J. 2017, 23, 15166 – 15176
www.chemeurj.org
1a
2a
3a
1b
2b
3b
1 b + VPA
2 b + VPA
3 b + VPA
VPA
cisplatin
HeLa
A549
IC50 [mm]
A549R
HepG2
LO2
0.54 : 0.05
2.0 : 0.2
1.0 : 0.1
0.62 : 0.06
4.1 : 0.4
0.48 : 0.04
1.9 : 0.2 0.79 : 0.06 0.75 : 0.05
3.3 : 0.3
0.64 : 0.05
1.7 : 0.1
1.3 : 0.1
0.92 : 0.08
3.8 : 0.3
2.1 : 0.2
13.8 : 1.1
8.2 : 0.7
3.6 : 0.3
17.8 : 1.7
1.5 : 0.1
10.4 : 1.0
5.6 : 0.5
2.2 : 0.2
13.1 : 1.3
2.2 : 0.2
14.7 : 1.0 10.9 : 1.0
3.5 : 0.3
16.2 : 1.5
2.6 : 0.2
12.5 : 1.2
8.5 : 0.8
3.4 : 0.4
18.0 : 1.8
1.4 : 0.1
9.8 : 1.0
6.0 : 0.6
2.8 : 0.2
13.5 : 1.3
2.8 : 0.2
15.2 : 1.5 10.0 : 1.0
3.5 : 0.4
17.6 : 1.7
> 100
> 100
> 100
> 100
> 100
23.5 : 2.3
20.6 : 2.1 70.9 : 7.1
20.5 : 2.1
30.8 : 2.9
[a] Data are presented as mean : SD, and cell viability was assessed after 48 h
of incubation.
antiproliferative efficacies of the compounds are in the following order: 1 a, 2 a, 3 a > 1 b, 2 b, 3 b & 1 b + VPA, 2 b + VPA, 3 b +
VPA > cisplatin > VPA. VPA (IC50 > 100 mm) is inactive against all
the cell lines tested. This is in agreement with the above HDAC
inhibition results and reports in literature that VPA needs millimolar doses to elicit HDAC inhibition and cytotoxicity.[15, 28]
Conjugates 1 a–3 a, which have IC50 values ranging from
0.48–2.0 mm, show higher cytotoxicity than the other compounds screened against all of the human cancer cells. Specifically, 1 a (IC50 = 1.0 mm), 2 a (IC50 = 0.79 mm), and 3 a (IC50 =
1.3 mm) are approximately 70.9, 89.7, and 54.5 times more
potent than cisplatin (IC50 = 70.9 mm) against A549R cells, respectively, and thus these complexes can overcome cisplatin
resistance. Furthermore, they exhibit a certain selectivity
toward human cancer cells over noncancerous cells, as 1 a–3 a
show 6.6, 4.4, and 4.1 times lower cytotoxicity against LO2
cells than against HepG2 cells, respectively. Reference complexes 1 b–3 b show much higher cytotoxicity than VPA and
cisplatin, with IC50 values ranging from 1.5 to 14.7 mm. The mixtures of 1 b–3 b with free VPA (1 b + VPA, 2 b + VPA, and 3 b +
VPA) show comparable cytotoxicity to 1 b–3 b, that is, simply
mixing them does not improve the antiproliferative efficacy.
Moreover, conjugation of the VPA to the IrIII complexes can enhance their anticancer potency; 1 a–3 a are about 2.9–8.9 times
more cytotoxic than 1 b–3 b or their simple mixtures with VPA
against all of the human cancer cells.
Cellular localization and uptake mechanisms of IrIII complexes
Studies on cellular localization of phosphorescent metal complexes can provide further clues for the investigations of anticancer mechanisms.[27] By taking advantage of the rich photophysical properties of cyclometalated IrIII complexes, the intracellular distribution of 1 a–3 a was investigated by one-photon
microscopy (OPM) and two-photon microscopy (TPM) confocal
luminescence imaging. As shown in Figure 4, all IrIII complexes
can be effectively taken up by HeLa cells and exhibit obvious
organelle accumulation after 30 min incubation. Co-localization
experiments on 1 a–3 a with commercial MitoTracker Deep Red
15169
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper
Figure 4. OPM and TPM images of HeLa cells co-labeled with IrIII complexes
(5 mm, 30 min) and MTDR (150 nm, 30 min). Complexes 1 a–3 a were excited
at 405 nm (OPM) or 740 nm (TPM). MTDR was excited at 633 nm. The phosphorescence/fluorescence was collected at 600(: 20) nm for 1 a,
530(: 20) nm for 2 a, 630(: 20) nm for 3 a, and 665(: 20) nm for MTDR.
Overlay 1: overlay of the 2nd and 3rd columns. Overlay 2: overlay of the 2nd
and 4th columns. Scale bar: 20 mm.
FM (MTDR) under one- and two-photon excitation
demonstrate specific mitochondrial staining of the complexes.
Under two-photon excitation, the Pearson’s correlation coefficients of 1 a–3 a with MTDR are all greater than 0.85. Meanwhile, negligible co-localization for 1 a–3 a with LysoTracker
Deep Red FM (LTDR) was observed (Figure S20 of the Supporting Information), which indicates that 1 a–3 a can specifically
label mitochondria.
We further investigated the cellular uptake mechanisms of
1 a–3 a. Incubation of HeLa cells with 1 a–3 a at 4 8C or on pretreatment with the metabolic inhibitor carbonyl cyanide 3chloro-phenylhydrazone (CCCP) leads to reduced cellular
uptake efficiency (Figure S21–S23 of the Supporting Information). However, no obvious alteration of the uptake level of
1 a–3 a is observed in cells pretreated with chloroquine, which
modulates endocytosis by inhibiting the acidification of endosomes. The results indicate that the cellular uptake of 1 a–3 a
was mainly through an energy-dependent mechanism, which
is similar to other cyclometalated IrIII complexes previously reported.[3e, 5a, 6b]
Figure 5. Representative JC-1 red/green ratio obtained from three independent experiments. Data are shown as mean : SD of three independent experiments.
1 a: 1.6 : 0.1; 2 a: 1.8 : 0.1; 3 a: 1.2 : 0.1). The results indicate
that 1 a–3 a can impair mitochondrial integrity.
Elevation of intracellular ROS levels
Mitochondria are major sites of cellular ROS production, and
the loss of MMP may lead to an increase in cellular ROS.[30] The
effect of IrIII complexes on intracellular ROS levels was detected
by flow cytometry with 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) staining. The nonfluorescent H2DCFDA can be
oxidized to the highly bright 2’,7’-dichlorofluorescein (DCF) by
cellular ROS.[31] As shown in Figure 6, treatment of HeLa cells
with 1 a–3 a increases the DCF fluorescence signals in a dosedependent manner. Compared with the control, for cells treated with 1 a–3 a (6 mm) at concentrations of approximately ten
times the IC50 values for 6 h, the mean fluorescence intensity
(MFI) increases about 5.6-, 4.5-, 5.1-fold for 1 a, 2 a, and 3 a, respectively. The results indicate that 1 a–3 a can significantly
induce elevation of intracellular ROS levels. Additionally, pretreatment of cells with N-acetylcysteine (NAC, an ROS scavenger) remarkably reduces the cytotoxicity of 1 a–3 a (Figure S25
of the Supporting Information). These results suggest that ROS
play a vital role in IrIII-induced cell death.
Impact on mitochondrial membrane potential (MMP)
As 1 a–3 a could localized to mitochondria, their impact on mitochondrial integrity was monitored by flow cytometry. The
changes in MMP were detected by 5,5’,6,6’-tetrachloro-1,1’3,3’-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) staining. Mitochondrial depolarization is indicated by a decrease in
the red/green fluorescence intensity ratio.[29] As shown in Figure S24 of the Supporting Information, compared with the vehicle-treated cells, cells treated with 1 a–3 a cause a marked
red-to-green color shift, indicating the loss of MMP. Representative JC-1 red/green ratio signals are shown in Figure 5; cells
treated with 1 a–3 a for 6 h show a concentration-dependent
decrease in JC-1 red/green fluorescence ratios. Notably, treatment of HeLa cells with 1 a–3 a (6 mm) at concentrations of approximately 10 times the IC50 values significantly decreased the
JC-1 red/green fluorescence ratios (control: 12.2 : 1.0;
Chem. Eur. J. 2017, 23, 15166 – 15176
www.chemeurj.org
Cell-cycle arrest
Cell cycle is tightly correlated with the proliferation and development of cancer cells. The cytotoxicity of many anticancer
drugs is often associated with genomic DNA damage and cellcycle perturbation.[32] It has been reported that VPA mainly induces G0/G1 cell-cycle arrest in many tumor types.[33] The effects of 1 a–3 a on cell-cycle progression in HeLa cells were investigated. As shown in Figure S26 of the Supporting Information, on treatment of HeLa cells with 1 a–3 a at different concentrations for 24 h, cell population in the G2/M phase was
significantly increased, along with a concomitant decrease in
the fraction of G0/G1 and S cells (Table S5 of the Supporting
Information). The cell cycle is regulated by highly complicated
molecular machinery.[34] The perturbations of cell-cycle progression induced by 1 a–3 a and VPA are different, which indicates
15170
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper
Figure 6. Effects of 1 a–3 a on ROS generation. HeLa cells were treated with
1 a–3 a at the indicated concentrations for 6 h, after which they were labeled
with H2DCFDA and analyzed by flow cytometry (reflected by MFI of DCF; excitation at 488 nm and emission at 525(: 20) nm).
the impact of VPA on cell-cycle distribution of the conjugates
is negligible.
Induction of apoptosis
Apoptosis is one of the extensively studied types of cell death,
and the contribution of apoptosis to the pathogenesis of
cancer has been well documented.[35] It has been reported that
both iridium(III) complexes[36] and VPA[28] can induce apoptotic
cell death. To investigate the mechanisms of 1 a–3 a-induced
cell death, the morphological changes of HeLa cells caused by
1 a–3 a were examined by 2’-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-1 H,3’H-2,5’-bibenzimidazole (Hoechst 33342) staining. As shown in Figure 7 A, vehicle-treated control cells show
a normal overall morphology and a homogeneous nuclear
staining pattern. After being treated with 1 a–3 a (1 mm) at concentrations of approximately twice the IC50 values for 24 h,
cells display morphological changes typical of apoptosis, including cell shrinkage, membrane blebbing, bright staining,
chromatin condensation, nuclei fragmentation, and the presence of apoptotic bodies.[37]
Annexin V/propidium iodide (PI) dual staining can differentiate early apoptotic (annexin V-positive and PI-negative), late
apoptotic and necrotic (annexin V-positive and PI-positive), and
viable (annexin V-negative and PI-negative) cells. Flow-cytoChem. Eur. J. 2017, 23, 15166 – 15176
www.chemeurj.org
metric analysis showed that treatment of HeLa cells with 1 a–
3 a leads to a dose-dependent increase in the percentage of
apoptotic cells. As shown in Figure 7 B, after HeLa cells are
treated with 1 a–3 a (2 mm) at concentrations of approximately
four times the IC50 values for 24 h, the percentages of cells in
apoptotic phase (annexin V-positive) are 2.5, 78, 70.9, and
73.5 % for control, 1 a, 2 a, and 3 a, respectively. The apoptosisinducing capabilities of 1 b–3 b under the same conditions
were also investigated (Figure S27 of the Supporting Information). After HeLa cells were treated with 1 a–3 a and 1 b–3 b at
concentrations on the same magnitude as the IC50 value, the
percentages of 1 a–3 a-treated cells in apoptotic phase were
much higher than those of 1 b–3 b-treated cells. This result implies that the released VPA may be involved in 1 a–3 a-induced
cell apoptosis.
To further clarify the mechanism by which the complexes
induce apoptosis, the activity of caspase-3/7 was examined by
using the Caspase-GloS assay kit in HeLa cells after 6 h exposure to different concentrations of 1 a–3 a. This caspase has
been identified as a key executor of apoptosis under various
stimuli.[38] As shown in Figure 7 C, treatment of 1 a–3 a markedly stimulates activation of caspase-3/7 in a dose-dependent
manner. Moreover, cells pretreated with the pan caspase inhibitor z-VAD-FMK (50 mm) show a marked increase in cell viability
compared with cells treated with IrIII complexes alone (Figure 7 D). These results collectively suggest that cell death induced by 1 a–3 a mainly occurs through the caspase-dependent apoptotic pathway.
Conclusions
Three VPA-functionalized cyclometalated IrIII complexes 1 a–3 a
were synthesized and characterized. All of them display excellent two-photon properties. Fluorescent spectroscopy and ESIMS studies demonstrate that 1 a–3 a can be quickly hydrolyzed
on treatment with esterase. In vitro HDAC activity assay
showed that conjugates 1 a–3 a show similar inhibition of
HDAC activity to VPA once hydrolyzed by esterase. Conjugation of the VPA to the IrIII complexes can improve the lipophilicity and cellular uptake efficacy of conjugates 1 a–3 a, and further enhances their anticancer potency. Complexes 1 a–3 a
show much higher antiproliferative activities than cisplatin
against various cancer cells, especially cisplatin-resistant A549
cells, which indicates that they can overcome cisplatin resistance. Owing to their high lipophilicity and electropositivity,
1 a–3 a can be effectively taken into HeLa cells and specifically
localized to mitochondria. Further anticancer mechanistic studies indicate that these complexes can induce a series of events
associated with mitochondrial damage in HeLa cells including
MMP depolarization, ROS production, cell-cycle arrest, caspase
activation, and apoptosis. Our study demonstrates that multifunctional anticancer drug may be achieved by conjugating
metal complexes with other organic drugs through ester linkages, and the resulting conjugates provide a new strategy to
build potential anticancer agents for future cancer therapy.
15171
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper
Figure 7. A) Hoechst 33342-stained HeLa cells after incubation with 1 a–3 a for 24 h. B) Flow-cytometric quantification of annexin V and PI double-labeled
HeLa cells after treatment with 1 a–3 a for 24 h at the indicated concentrations. C) Activation of caspase-3/7 in HeLa cells treated with 1 a–3 a at the indicated
concentrations for 6 h. D) The impact of z-VAD-FMK on the cytotoxicity of 1 a–3 a. HeLa cells were treated with 1 a–3 a for 48 h at the indicated concentrations
in the absence or presence of z-VAD-FMK. Cell viability was measured by MTT assay. Data are represented as mean : SD of three independent experiments.
**p < 0.005, compared with the cell viability of IrIII treatment alone.
Experimental Section
General instrumentation
General materials and methods
ESI-MS, microanalysis (C, H, N), and 1H NMR and 13C NMR measurements were performed with an LCQ DECA XP spectrometer (USA),
an Elementar Vario EL CHNS analyzer (Germany), and a Bruker
Avance 400 spectrometer (Germany), respectively. The XRD pattern
was collected with a Bruker D8 Advance diffractometer. UV/Vis
spectra emission spectra and time-resolved emission data were recorded with a Varian Cary 300 spectrophotometer (USA) and an
FLS 920 combined fluorescence lifetime and steady-state spectrometer (Japan), respectively. The two-photon fluorescence data
were acquired with an OpoletteTM 355II (pulse width , 100 fs,
80 MHz repetition rate, tuning range 720–890 nm, Spectra Physics
Inc., USA). Ir content was measured with a Thermo X Series 2 ICPMS (USA). The cell viability and HDAC inhibition activity were determined with a TECAN Infinite M200 Pro microplate reader (Switzerland). Confocal microscopy images were obtained with a LSM
710 confocal laser scanning fluorescence microscope (Carl Zeiss,
Germany). Flow-cytometric analysis was performed with a BD FACS
CaliburTM flow cytometer (Becton Dickinson, USA).
Iridium chloride hydrate, 2-phenylpyridine (ppy), 2-(2,4-difluorophenyl)pyridine (dfppy), 2-(2-thienyl)pyridine (thpy), HMbpy, 2-propylvaleryl chloride, and NH4PF6 were purchased from Alfa Aesar. Cisplatin, DMSO, PLE [(NH4)2SO4 suspension, + 150 units mg@1 protein],
VPA, CCCP, chloroquine, MTT, JC-1, H2DCFDA, NAC, PI, Annexin VFITC apoptosis detection kit, Hoechst 33342, and z-VAD-fmk were
purchased from Sigma-Aldrich. The fluorescent HDAC activity assay
kit was purchased from Enzo Life Sciences (USA). MTDR and LTDR
were purchased from Life Technologies (USA). Caspase-3/7 activity
assay kit was purchased from Promega (USA).
The cyclometalated IrIII chlorido-bridged dimers [{Ir(ppy)2Cl}2],[39]
[{Ir(dfppy)2Cl}2][40] and [{Ir(thpy)2Cl}2][41] were prepared according to
literature methods. The purity of the synthesized compounds was
analyzed by combustion analysis and they were found to be
+ 95 % pure. All the tested compounds were dissolved in DMSO
just before the experiments, and the concentration of DMSO was
1 vol % in PBS. The solutions of 1 a–3 a and 1 b–3 b in PBS proved
to be stable for at least 48 h at room temperature, as monitored
by UV/Vis spectroscopy.
Chem. Eur. J. 2017, 23, 15166 – 15176
www.chemeurj.org
General synthetic procedures for ligand L and iridium(III)
complexes
Ligand L: As shown in Scheme S1 of the Supporting Information,
HMbpy (2.5 mmol) and Et3N (15 mmol) were dissolved in dry DMF
15172
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper
(45 mL), and 2-propylvaleryl chloride (2.5 mmol) in dry DMF was
then added dropwise. The mixture was stirred under nitrogen for
12 h at room temperature, and then concentrated to give yellow
oily liquid, which was used directly for the next step without further purification.
Complexes 1 a–3 a. As shown in Scheme S2 of the Supporting Information, 1 a–3 a were synthesized by heating ligand L
(0.46 mmol, 2 equiv) and the appropriate iridium(III) chloridobridged dimer (0.23 mmol, 1 equiv) in refluxing CH2Cl2/CH3OH (2:1
v/v), followed by anion exchange with saturated NH4PF6 solution
and purification by recrystallization from CH2Cl2/diethyl ether.
Complexes 1 b–3 b: Complexes 1 b–3 b were synthesized according to the synthetic procedure for 1 a–3 a, with slight modification,
by using HMbpy instead of ligand L.
[Ir(ppy)2(L)](PF6) (1 a): Complex 1 a was synthesized according to
the synthetic procedure described above, which gave the product
as an orange powder. Yield: 0.353 g (79 %). 1H NMR (400 MHz,
[D6]DMSO): d = 8.84 (d, J = 4.0 Hz, 1 H), 8.72 (d, J = 4.1 Hz, 1 H), 8.28
(d, J = 7.1 Hz, 2 H), 7.93 (d, J = 6.5 Hz, 4 H), 7.88 (d, J = 5.6 Hz, 1 H),
7.73–7.61 (m, 4 H), 7.56 (d, J = 5.2 Hz, 1 H), 7.20–7.13 (m, 2 H), 7.06–
6.98 (m, 2 H), 6.95–6.87 (m, 2 H), 6.27–6.16 (m, 2 H), 5.33 (d, J =
4.9 Hz, 2 H), 2.55 (d, J = 5.3 Hz, 3 H), 1.49 (m, 4 H), 1.26–1.15 (m, 4 H),
1.09 (d, J = 6.9 Hz, 1 H), 0.86–0.76 ppm (m, 6 H); 13C NMR (100 MHz,
[D6]DMSO): d = 175.49, 167.35, 155.93, 155.16, 152.05, 150.90,
150.43, 149.66, 149.41, 144.23, 139.20, 131.57, 130.69, 129.89,
126.91, 126.08, 125.52, 124.33, 123.48, 122.70, 120.48, 63.70, 44.66,
34.30, 21.40, 20.42, 14.24 ppm; ESI-MS (CH2Cl2): m/z 827.55
[M@PF6] + ; elemental analysis calcd (%) for C42H42F6IrN4O2P·3 H2O: C
49.16, H 4.72, N 5.46; found: C 49.39, H 4.39, N 5.77.
[Ir(dfppy)2(L)](PF6) (2 a): Complex 2 a was synthesized according to
the synthetic procedure described above, which gave the product
as a bright yellow powder. Yield: 0.393 g (82 %). 1H NMR (400 MHz,
[D6]DMSO): d = 8.85 (s, 1 H), 8.74 (s, 1 H), 8.30 (d, J = 8.3 Hz, 2 H),
8.04 (t, J = 7.7 Hz, 2 H), 7.94 (d, J = 5.7 Hz, 1 H), 7.78–7.66 (m, 4 H),
7.57 (d, J = 5.5 Hz, 1 H), 7.28–7.22 (m, 2 H), 6.97 (t, J = 10.9 Hz, 2 H),
5.63 (t, J = 6.9 Hz, 2 H), 5.35 (s, 2 H), 2.57 (s, 3 H), 1.61–1.38 (m, 4 H),
1.21 (dd, J = 14.6, 7.3 Hz, 4 H), 1.09 (t, J = 7.0 Hz, 1 H), 0.82 ppm (td,
J = 7.2, 4.0 Hz, 6 H); 13C NMR (100 MHz, [D6]DMSO): d = 175.47,
163.30, 155.33, 152.69, 150.93, 150.24, 149.97, 140.47, 130.27,
128.06, 127.13, 126.31, 124.93, 123.72, 113.65, 99.46, 63.66, 44.69,
34.30, 21.44, 20.43, 14.23 ppm; ESI-MS (CH2Cl2): m/z 899.65 [MPF6] + ; elemental analysis calcd (%) for C42H38F10IrN4O2P·H2O: C
47.50, H 3.80, N 5.28; found: C 47.25, H 3.63, N 5.47.
[Ir(thpy)2(L)](PF6) (3 a): Complex 3 a was synthesized according to
the synthetic procedure described above, which gave the product
as a brown powder. Yield: 0.384 g (85 %). 1H NMR (400 MHz,
[D6]DMSO): d = 8.82 (s, 1 H), 8.71 (s, 1 H), 7.80 (m, 5 H), 7.71–7.64 (m,
4 H), 7.59 (d, J = 5.5 Hz, 1 H), 7.53 (t, J = 4.9 Hz, 2 H), 6.96 (dd, J = 9.1,
5.6 Hz, 2 H), 6.18 (t, J = 4.9 Hz, 2 H), 5.34 (s, 2 H), 2.56 (s, 3 H), 1.60–
1.40 (m, 4 H), 1.21 (dd, J = 14.7, 7.3 Hz, 4 H), 1.10 (t, J = 7.0 Hz, 1 H),
0.82 ppm (td, J = 7.2, 3.6 Hz, 6 H); 13C NMR (100 MHz, [D6]DMSO):
d = 175.49, 163.44, 156.07, 155.29, 152.83, 152.19, 151.05, 150.17,
149.74, 139.77, 136.64, 131.34, 130.76, 130.02, 127.02, 126.02,
123.40, 121.38, 118.73, 63.69, 44.67, 34.30, 21.40, 20.43, 14.24 ppm;
ESI-MS (CH2Cl2): m/z 839.40 [M@PF6] + ; elemental analysis calcd (%)
for C38H38F6IrN4O2PS2·H2O: C 45.55, H 4.02, N 5.59; found: C 45.41,
H 3.90, N 5.62.
[Ir(ppy)2(HMbpy)](PF6) (1 b): Complex 1 b was synthesized according to the synthetic procedure described above, which gave the
product as an orange powder. Yield: 0.330 g (85 %). 1H NMR
(400 MHz, [D6]DMSO): d = 8.76 (d, J = 7.1 Hz, 2 H), 8.27 (d, J = 8.1 Hz,
2 H), 7.93 (t, J = 8.7 Hz, 4 H), 7.80 (d, J = 5.6 Hz, 1 H), 7.70 (d, J =
Chem. Eur. J. 2017, 23, 15166 – 15176
www.chemeurj.org
5.6 Hz, 1 H), 7.66–7.60 (m, 3 H), 7.52 (d, J = 5.4 Hz, 1 H), 7.17 (t, J =
6.5 Hz, 2 H), 7.02 (t, J = 7.4 Hz, 2 H), 6.90 (t, J = 7.3 Hz, 2 H), 6.21 (d,
J = 7.1 Hz, 2 H), 5.76 (s, 1 H), 4.75 (s, 2 H), 2.54 ppm (s, 3 H); 13C NMR
(100 MHz, [D6]DMSO): d = 167.36, 156.33, 155.52, 152.06, 151.15,
149.82, 149.48, 149.19, 144.28, 139.13, 131.57, 130.66, 129.69,
126.04, 125.50, 124.31, 122.62, 122.22, 120.44, 61.83, 21.31 ppm;
ESI-MS (CH2Cl2): m/z 701.25 [M@PF6] + ; elemental analysis calcd (%)
for C34H28F6IrN4OP·0.4(C2H5)2O: C 48.84, H 3.68, N 6.40; found: C
48.55, H 3.88, N 6.25.
[Ir(dfppy)2(HMbpy)](PF6) (2 b): Complex 2 b was synthesized according to the synthetic procedure described above, which gave
the product as a bright yellow powder. Yield: 0.337 g (80 %).
1
H NMR (400 MHz, [D6]DMSO): d = 8.78 (d, J = 11.0 Hz, 2 H), 8.30 (d,
J = 8.4 Hz, 2 H), 8.04 (t, J = 7.8 Hz, 2 H), 7.86 (d, J = 5.6 Hz, 1 H), 7.76
(d, J = 5.6 Hz, 1 H), 7.71 (t, J = 5.0 Hz, 2 H), 7.65 (d, J = 5.5 Hz, 1 H),
7.55 (d, J = 5.4 Hz, 1 H), 7.26 (t, J = 6.6 Hz, 2 H), 6.97 (t, J = 10.6 Hz,
2 H), 5.76 (s, 1 H), 5.64 (d, J = 7.9 Hz, 2 H), 4.77 (d, J = 5.3 Hz, 2 H),
2.56 ppm (s, 3 H); 13C NMR (100 MHz, [D6]DMSO): d = 163.26,
156.94, 155.32, 152.71, 150.27, 149.91, 140.41, 130.07, 128.05,
126.35, 124.93, 123.79, 122.42, 113.71, 61.80, 21.35 ppm; ESI-MS
(CH2Cl2): m/z 773.35 [M@PF6] + ; elemental analysis calcd (%) for
C34H24F10IrN4OP·2 H2O: C 42.82, H 2.96, N 5.87; found: C 42.68, H
2.87, N 5.76.
[Ir(thpy)2(HMbpy)](PF6) (3 b): Complex 3 b was synthesized according to the synthetic procedure described above, which gave the
product as a brown powder. Yield: 0.319 g (81 %). 1H NMR
(400 MHz, [D6]DMSO): d = 8.74 (d, J = 5.9 Hz, 2 H), 7.81 (t, J = 7.7 Hz,
2 H), 7.76 (t, J = 6.6 Hz, 3 H), 7.65 (dd, J = 9.0, 5.1 Hz, 4 H), 7.57–7.51
(m, 3 H), 6.96 (t, J = 6.5 Hz, 2 H), 6.19 (dd, J = 4.7, 1.9 Hz, 2 H), 5.75 (s,
1 H), 4.76 (s, 2 H), 2.55 ppm (s, 3 H); 13C NMR (100 MHz, [D6]DMSO):
d = 163.49, 156.46, 155.66, 153.11, 152.21, 150.42, 150.06, 149.70,
139.70, 136.57, 131.29, 130.82, 129.82, 126.22, 126.08, 122.15,
121.39, 118.69, 61.81, 21.31 ppm; ESI-MS (CH2Cl2): m/z 713.25
[M@PF6] + ; elemental analysis calcd (%) for C30H24F6IrN4OPS2·1.5 H2O:
C 40.72, H 3.08, N 6.33; found: C 40.99, H 3.05, N 6.07.
Crystallographic structure determination
Crystals of 2 a and 2 b suitable for X-ray analysis were obtained by
slow diffusion of diethyl ether in to CH2Cl2 solutions. The crystal
structures of 2 a and 2 b were solved by direct methods with
SHELXS and refined by the full-matrix least-squares technique with
SHELXL.[42] Crystal data and selected bond lengths and angles are
listed in Tables S1 and S2 of the Supporting Information.
Determination of TPA cross sections
The TPA spectra were determined at 720–890 nm by the typical
two-photon laser-induced fluorescence method.[43] Two-photon
fluorescence measurements were performed in fluorometric quartz
cuvettes with IrIII complexes (5 V 10@4 m) in CH3OH at 298 K by
using rhodamine B (5 V 10@5 m) as reference. The TPA cross section
was calculated according to Equation (1):[44]
ds ¼ dr
Fr cr Is ns
Fs cs Ir nr
ð1Þ
where d is the TPA cross section, F the quantum yield, c the concentration, I the integrated fluorescence intensity, and n the refractive index. Subscript r stands for reference, and s for sample.
15173
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper
Hydrolysis of 1 a–3 a by PLE in vitro
@5
Time-dependent emission spectra: Mixtures of 1 a–3 a (2 V 10 m)
with degassed PBS were freshly prepared in quartz cuvettes (3 mL),
and then a suspension of PLE (1 mL) in (NH4)2SO4 was added. Timedependent emission spectra were recorded after reaction for 5 min
at 298 K.
@5
Luminescence decay signals: Mixtures of 1 a–3 a (2 V 10 m) with
degassed PBS were freshly prepared in quartz cuvettes (3 mL), and
then a suspension of PLE (1 mL) in (NH4)2SO4 was added. After the
mixtures were incubated at 298 K for the indicated time intervals,
luminescence decay curves were recorded.
ESI-MS: Complexes 1 a–3 a (2 V 10@5 m) were dissolved in freshly
prepared PBS buffer (3 mL), and then a suspension of PLE (1 mL) in
(NH4)2SO4 was added. After the mixtures were incubated at 298 K
for the indicated time intervals, acetone (400 mL, 253 K) was added
to quench the enzymatic hydrolysis. The samples were centrifuged
(15 000 g, 10 min). The supernatant was collected and analyzed by
ESI-MS.
HDAC enzyme inhibition assay
The ability of the compounds to inhibit HDACs was investigated in
triplicate by following the experimental procedure supplied with
the assay kit. First, the master mixture was prepared from the
HDAC substrate solution (25 mL/well) and HDAC assay buffer
(25 mL/well). The master mixture was pipetted to all wells (50 mL/
well). After that, 1 mm solutions of tested inhibitors (VPA, 1 a–3 a,
and 1 b–3 b), which were prepared in water from their stock solutions in DMSO, were added to the plate (10 mL/well). For the inhibition activity in the presence of the esterase, 1 a–3 a (1 mm) were
pretreated with PLE (1 mL) at 298 K for 10 h, and then directly
added to the corresponding wells (10 mL/well). The plates were incubated at 37 8C for 30 min. HDAC developer (50 mL/well) was pipetted into all wells and the plates were incubated at room temperature for an additional 30 min. The fluorescence was detected
at excitation/emission wavelengths of 340 and 460 nm by using a
fluorescence microplate reader. IC50 values (the drug concentration
required to inhibit HDAC activity by 50 %) were calculated according to a regression analysis of the concentration/inhibition data.
Determination of lipophilicity
The lg P values were determined by the flask-shaking method.
Briefly, equal amounts of aqueous sodium chloride (0.9 wt %) and
n-octanol were mutually saturated for one week, and then the mixture was separated to obtain oil and aqueous phases. Complexes
1 a–3 a and 1 b–3 b were dissolved in the aqueous phase, and an
equal volume of the saturated n-octanol was added. The solutions
were shaken in the oscillator for 24 h, and then centrifuged at
1500 g for 10 min. The IrIII content of the n-octanol and aqueous
phases was determined by UV/Vis spectroscopy, and lg P was calculated as the logarithmic ratio of IrIII concentration in n-octanol to
that in the aqueous phases.
ICP-MS measurement
Cellular accumulation of IrIII complexes was determined in HeLa
cells. Cells were seeded in 10 cm tissue culture dishes and incubated for 24 h. The medium was removed and replaced with fresh
medium containing the tested IrIII complexes (5 mm). After 30 min
incubation, the cells were collected, counted, and then digested
with HNO3 (65 %, 0.2 mL) at room temperature for at least 24 h.
Chem. Eur. J. 2017, 23, 15166 – 15176
www.chemeurj.org
The quantity of Ir taken up by HeLa cells was determined by ICPMS.
Cytotoxicity assay
The growth inhibitory effect of the tested compounds towards
HeLa, A549, A549R, HepG2 and LO2 cells lines was evaluated by
MTT assay as previously described.[27] For the cytotoxicity assay in
the presence of the inhibitors (z-VAD-FMK or NAC), HeLa cells were
preincubated with inhibitors at the indicated concentrations for
1 h before the complexes were added.
One- and two-photon cellular imaging
HeLa cells were co-incubated with IrIII complexes (5 mm) and MTDR
(150 nm) or LTDR (50 nm) at 37 8C for 30 min. Cells were washed
three times with PBS and visualized by confocal microscopy immediately; lex = 405 (OPM) or 740 nm (TPM) for IrIII complexes and
633 nm for MTDR and LTDR; lem = 600(: 20) nm for 1 a, 530(:
20) nm for 2 a, 630(: 20) nm for 3 a, 665(: 20) nm for MTDR, and
668(: 20) nm for LTDR, respectively.
Cellular uptake mechanism studies
To investigate the impact of temperature and metabolic or endocytic inhibitors on cellular uptake of 1 a–3 a, HeLa cells were
seeded in 35 mm dishes and cultured for 24 h. For temperature
tests, cells were incubated with IrIII complexes (5 mm) at 4 and 37 8C
for 30 min; for inhibitor tests, cells were pretreated with 30 mm
CCCP or 50 mm chloroquine for 1 h at 37 8C and then incubated
with IrIII complexes (5 mm) at 37 8C for a further 30 min. In each
case, cells were washed three times with PBS and visualized by
confocal microscopy; lex = 405 nm (OPM) or 740 nm (TPM); lem =
600(: 20) nm for 1 a, 530(: 20) nm for 2 a, and 630(: 20) nm for
3 a.
Analysis of MMP
The impact of 1 a–3 a on MMP was determined as previously described.[27] Briefly, HeLa cells were treated with 1 a–3 a at the indicated concentrations for 6 h, collected, and stained with 5 mg mL@1
JC-1. The fluorescence intensity of the cells was measured immediately by flow cytometry with excitation at 488 nm and dual emission at 530 (green) and 590 nm (red). MFI was analyzed with
FlowJo 7.6 software.
Measurement of intracellular ROS
The impact of 1 a–3 a on ROS levels was determined as previously
described.[27] Briefly, HeLa cells were treated with 1 a–3 a at the indicated concentrations for 6 h, collected, and incubated with
10 mm H2DCFDA in serum-free Dulbecco’s modified Eagle’s
medium (DMEM) for 15 min at 37 8C. The fluorescence intensity of
the cells was measured by flow cytometry with excitation at
488 nm and emission at 530 nm. Green MFI was analyzed with
FlowJo 7.6 software.
Cell-cycle analysis
The impact of the tested complexes on cell-cycle distribution was
analyzed by flow cytometry and PI staining as previously described.[12a]
15174
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper
Detection of apoptosis
Hoechst staining: HeLa cells were seeded into 35 mm dishes and
treated with 1 a–3 a for 24 h. The cells were then washed once
with PBS and fixed with 4 % paraformaldehyde at room temperature for 10 min. Then, cells were labeled with Hoechst 33342
(5 mg mL@1 in PBS) for 5 min. The cells were analyzed immediately
with a confocal microscope.
Annexin V/PI assay: For apoptosis determination assays, HeLa
cells were cultured in six-well plates and incubated with the indicated concentrations of IrIII complexes for 24 h. After treatment,
cells were harvested and stained with 5 mL annexin V and 10 mL PI
at room temperature for 10 min in the dark, and analyzed immediately by flow cytometry with excitation at 488 nm. Data were analyzed with FlowJo 7.6 software.
Caspase-3/7 activity assay: Activation of Caspase-3/7 by 1 a–3 a
was measured by using the Caspase-GloS Assay kit according to
the manufacturer’s instructions with a slight modification. Briefly,
HeLa cells were cultured in 48-well plates and treated with different concentration of IrIII complexes for 6 h. Cells were washed
three times with PBS and then lysed. 50 mL cell lysate was added
to each well, followed by the addition of 50 mL Caspase-GloS 3/7
reagent. The mixture was incubated at room temperature for
30 min and then the luminescence was measured with a TECAN Infinite M200 station. Cells treated with vehicle control DMSO (1 %,
v/v) were used as the reference group.
Statistical analysis
All biological experiments were performed at least twice in triplicate in each experiment. Representative results are depicted in this
report and data were presented as mean : SD.
Acknowledgements
This study was supported by the National Natural Science
Foundation of China (Nos. 21231007, 21572282 and 21571196),
the 973 program (Nos. 2014CB845604 and 2015CB856301), the
Guangdong Natural Science Foundation (2015A030306023),
China Postdoctoral Science Foundation (2016M590832) and
the Fundamental Research Funds for the Central Universities.
Conflict of interest
The authors declare no conflict of interest.
Keywords: antitumor agents · apoptosis · imaging agents ·
iridium · N ligands
[1] a) Z. Liu, P. J. Sadler, Acc. Chem. Res. 2014, 47, 1174 – 1185; b) A. Wilbuer,
D. H. Vlecken, D. J. Schmitz, K. Kraling, K. Harms, C. P. Bagowski, E. Meggers, Angew. Chem. Int. Ed. 2010, 49, 3839 – 3842; Angew. Chem. 2010,
122, 3928 – 3932.
[2] a) D.-L. Ma, S. Lin, W. Wang, C. Yang, C.-H. Leung, Chem. Sci. 2017, 8,
878 – 889; b) Y. Chen, R. L. Guan, C. Zhang, J. J. Huang, L. N. Ji, H. Chao,
Coord. Chem. Rev. 2016, 310, 16 – 40.
[3] a) M. H. Chen, F. X. Wang, J. J. Cao, C. P. Tan, L. N. Ji, Z. W. Mao, ACS Appl.
Mater. Interfaces 2017, 9, 13304 – 13314; b) K. Qiu, H. Huang, B. Liu, Y.
Liu, Z. Huang, Y. Chen, L. Ji, H. Chao, ACS Appl. Mater. Interfaces 2016, 8,
12702 – 12710; c) J. S. Nam, M.-G. Kang, J. Kang, S.-Y. Park, S. J. C. Lee,
H.-T. Kim, J. K. Seo, O.-H. Kwon, M. H. Lim, H.-W. Rhee, T.-H. Kwon, J. Am.
Chem. Eur. J. 2017, 23, 15166 – 15176
www.chemeurj.org
Chem. Soc. 2016, 138, 10968 – 10977; d) W. Lv, Z. Zhang, K. Y. Zhang, H.
Yang, S. Liu, A. Xu, S. Guo, Q. Zhao, W. Huang, Angew. Chem. Int. Ed.
2016, 55, 9947 – 9951; Angew. Chem. 2016, 128, 10101 – 10105; e) R. R.
Ye, C. P. Tan, L. N. Ji, Z. W. Mao, Dalton Trans. 2016, 45, 13042 – 13051;
f) L. He, C.-P. Tan, R.-R. Ye, Y.-Z. Zhao, Y.-H. Liu, Q. Zhao, L.-N. Ji, Z.-W.
Mao, Angew. Chem. Int. Ed. 2014, 53, 12137 – 12141; Angew. Chem.
2014, 126, 12333 – 12337.
[4] a) L.-J. Liu, L. Lu, H.-J. Zhong, B. He, D. W. J. Kwong, D.-L. Ma, C.-H.
Leung, J. Med. Chem. 2015, 58, 6697 – 6703; b) C.-H. Leung, H.-J. Zhong,
H. Yang, Z. Cheng, D. S.-H. Chan, V. P.-Y. Ma, R. Abagyan, C.-Y. Wong, D.L. Ma, Angew. Chem. Int. Ed. 2012, 51, 9010 – 9014; Angew. Chem. 2012,
124, 9144 – 9148; c) R.-R. Ye, C.-P. Tan, L. He, M.-H. Chen, L.-N. Ji, Z.-W.
Mao, Chem. Commun. 2014, 50, 10945 – 10948.
[5] a) L. He, Y. Li, C.-P. Tan, R.-R. Ye, M.-H. Chen, J.-J. Cao, L.-N. Ji, Z.-W. Mao,
Chem. Sci. 2015, 6, 5409 – 5418; b) X. Tian, Y. Zhu, M. Zhang, L. Luo, J.
Wu, H. Zhou, L. Guan, G. Battaglia, Y. Tian, Chem. Commun. 2017, 53,
3303 – 3306; c) L. K. McKenzie, I. V. Sazanovich, E. Baggaley, M. Bonneau,
V. Guerchais, J. A. G. Williams, J. A. Weinstein, H. E. Bryant, Chem. Eur. J.
2017, 23, 234 – 238.
[6] a) J. J. Cao, C. P. Tan, M. H. Chen, N. Wu, D. Y. Yao, X. G. Liu, L. N. Ji, Z. W.
Mao, Chem. Sci. 2017, 8, 631 – 640; b) Y. Li, C.-P. Tan, W. Zhang, L. He, L.N. Ji, Z.-W. Mao, Biomaterials 2015, 39, 95 – 104.
[7] S. Fulda, L. Galluzzi, G. Kroemer, Nat. Rev. Drug Discovery 2010, 9, 447 –
464.
[8] A. H. V. Schapira, Lancet 2012, 379, 1825 – 1834.
[9] S. E. Weinberg, N. S. Chandel, Nat. Chem. Biol. 2015, 11, 9 – 15.
[10] J. E. Bolden, M. J. Peart, R. W. Johnstone, Nat. Rev. Drug Discovery 2006,
5, 769 – 784.
[11] B. S. Mann, J. R. Johnson, M. H. Cohen, R. Justice, R. Pazdur, Oncologist
2007, 12, 1247 – 1252.
[12] a) R.-R. Ye, Z.-F. Ke, C.-P. Tan, L. He, L.-N. Ji, Z.-W. Mao, Chem. Eur. J. 2013,
19, 10160 – 10169; b) R.-R. Ye, C.-P. Tan, Y.-N. Lin, L.-N. Ji, Z.-W. Mao,
Chem. Commun. 2015, 51, 8353 – 8356.
[13] W. Lçscher, CNS Drugs 2002, 16, 669 – 694.
[14] D. C. Drummond, C. O. Noble, D. B. Kirpotin, Z. X. Guo, G. K. Scott, C. C.
Benz, Annu. Rev. Pharmacol. Toxicol. 2005, 45, 495 – 528.
[15] N. Gurvich, O. M. Tsygankova, J. L. Meinkoth, P. S. Kein, Cancer Res.
2004, 64, 1079 – 1086.
[16] J. Yang, X. R. Sun, W. W. Mao, M. H. Sui, J. B. Tang, Y. Q. Shen, Mol.
Pharm. 2012, 9, 2793 – 2800.
[17] M. Alessio, I. Zanellato, I. Bonarrigo, E. Gabano, M. Ravera, D. Osella, J.
Inorg. Biochem. 2013, 129, 52 – 57.
[18] V. Novohradsky, L. Zerzankova, J. Stepankova, O. Vrana, R. Raveendran,
D. Gibson, J. Kasparkova, V. Brabec, Biochem. Pharmacol. 2015, 95, 133 –
144.
[19] R. Raveendran, J. P. Braude, E. Wexselblatt, V. Novohradsky, O. Stuchlikova, V. Brabec, V. Gandin, D. Gibson, Chem. Sci. 2016, 7, 2381 – 2391.
[20] R. Humerickhouse, K. Lohrbach, L. Li, W. F. Bosron, M. E. Dolan, Cancer
Res. 2000, 60, 1189 – 1192.
[21] a) Q. Zhao, S. Liu, M. Shi, C. Wang, M. Yu, L. Li, F. Li, T. Yi, C. Huang,
Inorg. Chem. 2006, 45, 6152 – 6160; b) R. Liu, N. Dandu, J. Chen, Y. Li, Z.
Li, S. Liu, C. Wang, S. Kilina, B. Kohler, W. Sun, J. Phys. Chem. C 2014,
118, 23233 – 23246.
[22] W. R. Zipfel, R. M. Williams, W. W. Webb, Nat. Biotechnol. 2003, 21,
1369 – 1377.
[23] G. Li, Q. Lin, L. Sun, C. Feng, P. Zhang, B. Yu, Y. Chen, Y. Wen, H. Wang, L.
Ji, H. Chao, Biomaterials 2015, 53, 285 – 295.
[24] T. Imai, Drug Metab. Pharmacokinet. 2006, 21, 173 – 185.
[25] F. C. M. Leung, A. Y. Y. Tam, V. K. M. Au, M. J. Li, V. W. W. Yam, Acs Appl.
Mater. Interfaces 2014, 6, 6644 – 6653.
[26] Q. Fu, J. Tang, M. Cui, Z. Zheng, Z. Q. Liu, S. Y. Liu, J. Chromatogr. B
2015, 990, 169 – 173.
[27] R.-R. Ye, C.-P. Tan, M.-H. Chen, L. Hao, L.-N. Ji, Z.-W. Mao, Chem. Eur. J.
2016, 22, 7800 – 7809.
[28] N. Takai, N. Kawamata, D. Gui, J. W. Said, I. Miyakawa, H. P. Koeffler,
Cancer 2004, 101, 2760 – 2770.
[29] S. T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T. W. Smith,
G. D. Steele, Jr., L. B. Chen, Proc. Natl. Acad. Sci. USA 1991, 88, 3671 –
3675.
[30] P. T. Schumacker, Cancer Cell 2006, 10, 175 – 176.
[31] R. Cathcart, E. Schwiers, B. N. Ames, Anal. Biochem. 1983, 134, 111 – 116.
15175
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Full Paper
[32] G. I. Shapiro, J. W. Harper, J. Clin. Invest. 1999, 104, 1645 – 1653.
[33] Y. W. Chou, N. K. Chaturvedi, S. G. Ouyang, F. F. Lin, D. Kaushik, J. Wan, I.
Kim, M. F. Lin, Cancer Lett. 2011, 311, 177 – 186.
[34] A. W. Murray, Cell 2004, 116, 221 – 234.
[35] C. P. Tan, Y. Y. Lu, L. N. Ji, Z. W. Mao, Metallomics 2014, 6, 978 – 995.
[36] a) J. M. Hearn, I. Romero-Caneljn, B. Qamar, Z. Liu, I. Hands-Portman,
P. J. Sadler, ACS Chem. Biol. 2013, 8, 1335 – 1343; b) A. Kastl, A. Wilbuer,
A. L. Merkel, L. Feng, P. Di Fazio, M. Ocker, E. Meggers, Chem. Commun.
2012, 48, 1863 – 1865.
[37] R. S. Hotchkiss, A. Strasser, J. E. McDunn, P. E. Swanson, N. Engl. J. Med.
2009, 361, 1570 – 1583.
[38] N. A. Thornberry, Y. Lazebnik, Science 1998, 281, 1312 – 1316.
[39] K. A. King, R. J. Watts, J. Am. Chem. Soc. 1987, 109, 1589 – 1590.
Chem. Eur. J. 2017, 23, 15166 – 15176
www.chemeurj.org
[40] Y. M. You, S. Y. Park, J. Am. Chem. Soc. 2005, 127, 12438 – 12439.
[41] C. Li, M. Yu, Y. Sun, Y. Wu, C. Huang, F. Li, J. Am. Chem. Soc. 2011, 133,
11231 – 11239.
[42] G. M. Sheldrick, Acta Crystallogr. Sect. A 2008, 64, 112 – 122.
[43] N. S. Makarov, M. Drobizhev, A. Rebane, Opt. Express 2008, 16, 4029 –
4047.
[44] C. Xu, W. W. Webb, J. Opt. Soc. Am. B 1996, 13, 481 – 491.
Manuscript received: July 8, 2017
Revised manuscript received: August 14, 2017
Accepted manuscript online: August 22, 2017
Version of record online: October 9, 2017
15176
T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim