Novel and Versatile Imine-N-Heterocyclic Carbene Half-Sandwich Iridium(III) Complexes as Lysosome-Targeted Anticancer Agents

Article abstract of DOI:10.1021/acs.inorgchem.8b01656

We, herein, report the synthesis, characterization, luminescence properties, anticancer, and antibacterial activities of a family of novel half-sandwich iridium(III) complexes of the general formula [(n⁵-Cp^x)Ir(C^N)Cl]PF₆^- [Cp^x = pentamethylcyclopentadienyl (Cp) or tetramethyl(biphenyl)-cyclopentadienyl (Cp^xbiph)] bearing versatile imine-N-heterocyclic carbene ligands. In this complex framework, substituents on four positions could be modulated, which distinguishes this class of complex and provides a large amount of flexibility and opportunity to tune the cytotoxicity of complexes. The X-ray crystal structures of complexes 4 and 10 exhibit the expected "piano-stool" geometry. With the exception of 1, 2, and 11, each complex shows potent cytotoxicity, with IC₅₀ (half-maximum inhibitory concentration) values ranging from 1.99 to 25.86 μM toward A549 human lung cancer cells. First, the effect of four positions bearing different substituents in the complex framework on the anticancer activity, that is, structure-activity relationship, was systematically studied. Complex 8 (IC₅₀ = 1.99 μM) displays the highest anticancer activities, whose cytotoxicity is more than 10-fold higher than that of the clinical platinum drug cisplatin against A549 cancer cells. Second, their chemical reactivity including nucleobases binding, catalytic activity in converting coenzyme NADH to NAD⁺, reaction with glutathione (GSH), and bovine serum albumin (BSA) binding is investigated. No reaction with nucleobase is observed. However, these iridium(III) complexes bind rapidly to GSH and can catalyze oxidation of NADH to NAD⁺. In addition, they show moderate binding affinity to BSA and the fluorescence quenching of BSA by the iridium (III) complexes is due to the static quenching. Third, the mode of cell death was also explored through flow cytometry experiments, including cell cycle, apoptosis induction, reactive oxygen species (ROS) and mitochondrial membrane potential. It seems that cell cycle perturbation, apoptosis induction, increase of ROS level and loss of mitochondrial membrane potential together contribute to the anticancer potency of these complexes. Last, the use of confocal microscopy provides insights into the microscopic mechanism that the typical and most active complex 8 enters A549 lung cancer cells mainly through energy-dependent pathway and is located in lysosome. Furthermore, lysosome damage and nuclear morphology were detected by confocal microscopy. Nuclear condensation and apoptotic bodies may finally induce cells apoptosis. Interestingly, complex 8 also shows antibacterial activity against Gram-positive Staphylococcus aureus. This work may provide an alternative and effective strategy to smart design of potent organometallic half-sandwich iridium(III) anticancer drugs.

Full text of DOI:10.1021/acs.inorgchem.8b01656

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Novel and Versatile Imine-N-Heterocyclic Carbene Half-Sandwich
Iridium(III) Complexes as Lysosome-Targeted Anticancer Agents
Yuliang Yang,† Lihua Guo,*,† Zhenzhen Tian,† Yuteng Gong,† Hongmei Zheng,† Shumiao Zhang,†
Zhishan Xu,†,‡ Xingxing Ge,† and Zhe Liu*,†
†Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of Life-Organic Analysis and Key
Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, Department of Chemistry and Chemical
Engineering, Qufu Normal University, Qufu 273165, China
‡Department of Chemistry and Chemical Engineering, Shandong Normal University, Jinan 250014, China
S
* Supporting Information
ABSTRACT: We, herein, report the synthesis, characterization,
luminescence properties, anticancer, and antibacterial activities of a
family of novel half-sandwich iridium(III) complexes of the general
−
formula [(η5-Cpx)Ir(C^N)Cl]PF6 [Cpx = pentamethylcyclopenta-
dienyl (Cp*) or tetramethyl(biphenyl)-cyclopentadienyl (Cpxbiph)]
bearing versatile imine-N-heterocyclic carbene ligands. In this complex
framework, substituents on four positions could be modulated, which
distinguishes this class of complex and provides a large amount of
flexibility and opportunity to tune the cytotoxicity of complexes. The
X-ray crystal structures of complexes 4 and 10 exhibit the expected
“piano-stool” geometry. With the exception of 1, 2, and 11, each
complex shows potent cytotoxicity, with IC50 (half-maximum
inhibitory concentration) values ranging from 1.99 to 25.86 μM
toward A549 human lung cancer cells. First, the effect of four positions bearing different substituents in the complex framework
on the anticancer activity, that is, structure−activity relationship, was systematically studied. Complex 8 (IC50 = 1.99 μM)
displays the highest anticancer activities, whose cytotoxicity is more than 10-fold higher than that of the clinical platinum drug
cisplatin against A549 cancer cells. Second, their chemical reactivity including nucleobases binding, catalytic activity in
converting coenzyme NADH to NAD+, reaction with glutathione (GSH), and bovine serum albumin (BSA) binding is
investigated. No reaction with nucleobase is observed. However, these iridium(III) complexes bind rapidly to GSH and can
catalyze oxidation of NADH to NAD+. In addition, they show moderate binding affinity to BSA and the fluorescence quenching
of BSA by the iridium (III) complexes is due to the static quenching. Third, the mode of cell death was also explored through
flow cytometry experiments, including cell cycle, apoptosis induction, reactive oxygen species (ROS) and mitochondrial
membrane potential. It seems that cell cycle perturbation, apoptosis induction, increase of ROS level and loss of mitochondrial
membrane potential together contribute to the anticancer potency of these complexes. Last, the use of confocal microscopy
provides insights into the microscopic mechanism that the typical and most active complex 8 enters A549 lung cancer cells
mainly through energy-dependent pathway and is located in lysosome. Furthermore, lysosome damage and nuclear morphology
were detected by confocal microscopy. Nuclear condensation and apoptotic bodies may finally induce cells apoptosis.
Interestingly, complex 8 also shows antibacterial activity against Gram-positive Staphylococcus aureus. This work may provide an
alternative and effective strategy to smart design of potent organometallic half-sandwich iridium(III) anticancer drugs.
combine a hard-donor imine group with the soft-donor
properties of an NHC.4
INTRODUCTION
■
The N-heterocyclic carbenes (NHCs) have been proved to be
the efficient ancillary ligands because of their strong
coordination ability and easily tunable structure by the steric
and electronic substituents on the metal center.1 Most of the
transition metals from group 7 to group 11 in the periodic
table can be coordinated by the NHC ligands.1h,2 Metal
complexes bearing different NHC ligands have been prepared
and employed in various fields, particularly as catalysts and
anticancer agents.1h,3 Among these ligands, imine-N-hetero-
cyclic carbene is an attractive class of chelating ligands that
The successful application of platinum-based anticancer
agents in the clinical has stimulated the research for other
metal-based diagnostic drugs and chemotherapeutic drugs,
which may be able to diminish severe toxic side effects,
overcome platinum resistance, and broaden the spectrum of
sensitive tumors.5 For metal-based chemotherapeutic drugs,
both metal ions and the nature of chelating ligands play key
Received: June 15, 2018
© XXXX American Chemical Society
A
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roles in the therapeutic efficiency of anticancer agents.6
Recently, a series of highly potent half-sandwich organo-
metallic anticancer agents bearing different chelating ligands,
such as bis-NHC ligands, imine-pyridyl ligands, and pyridyl-
NHC ligands (Scheme 1), have been reported.6,7 Our group
substituents, have been prepared and fully characterized. In
this C^N-ligand framework, three positions of the ligand could
be modulated, respectively, which distinguishes this class of
ligand and provides a large amount of flexibility and
opportunities to tune the cytotoxicity of such complexes.
These complexes have been systematically investigated for
their chemical and biological reactivity, cancer cell toxicity
against A549 cancer cells, antibacterial activity against
Staphylococcus aureus (S. aureus) and molecular imaging in
live cells. On the basis of these studies, we tried to understand
the mechanism of actions of these iridium(III) complexes. The
results demonstrated that this type of iridium(III) complexes
has a great potential in cancer chemotherapy.
Scheme 1. Reported Half-Sandwich Anticancer Complexes
and Our Current Work
RESULTS AND DISCUSSION
■
Synthesis, Characterization, and Spectroscopic Prop-
erties. The synthetic routes to imine-N-heterocyclic carbene
ligands L1−L11 and novel half-sandwich iridium(III)
complexes 1−12 are depicted in Scheme 2. A series of
Scheme 2. Synthetic Routes for Imine-N-Heterocyclic
Carbene Ligands L1−L11 and [(η5-Cpx)Ir(C^N)Cl]PF6
Complexes 1−12
have developed a series of half-sandwich iridium(III)
complexes containing bis-NHC ligands (I, Scheme 1).7a
These complexes exhibit potent cytotoxicity toward HeLa
human cervical cancer cells, and the anticancer activity of these
complexes can be tuned by varying substitutions at two
different interior positions. Our group has also synthesized a
class of imine-pyridyl half-sandwich iridium(III) and
ruthenium(II) complexes (II, Scheme 1).6,7b The cytotoxicity
and cancer cell selectivity of such complexes can be governed
via alternative metal ions and chelating ligands around the
metal. Recently, Hartinger et al. designed some pyridyl-NHC
organoruthenium anticancer complexes (III, Scheme 1) and
studied their antiproliferative properties and reactions with
biomolecules.7c In this system, introduction of different
substituents gave complexes with a wide variety of properties.
These results encouraged us to prepare a series of half-
sandwich iridium(III) complexes bearing imine-N-heterocyclic
carbene chelating ligands and explore their anticancer activity
and reactivity toward biomolecules.
Moreover, confocal microscopy imaging has achieved a great
success in the field of biology and medicine.8 It has a lot of
advantages, such as higher resolution than conventional optical
microscopes, enabling continuous nondestructive optical
sectioning of samples, removing the influence of stray light,
and increasing the clarity of the image. The development of
fluorescent anticancer complexes can help researchers to real-
time track drug intracellular transport and distribution in cells,
and to monitor the interactions between the drug and the
biological target molecule, thus providing an important tool for
exploring the mechanism of actions (MoAs) of the anticancer
drugs. Cyclometalated iridium(III) or ruthenium(II) anti-
cancer complexes have been widely used as imaging agents and
probes by virtue of their intense emission, long emission
lifetimes, large Stokes shifts, and high photo stability.9
However, half-sandwich anticancer iridium(III) complexes
used for confocal microscopy imaging have been rarely
reported.
imidazolium salts (C^imine·HCl) (L1−L11) containing
different substituents were synthesized in good yields by a
coupling reaction of the corresponding imidoyl chlorides with
imidazole bearing the different substituents. Novel half-
sandwich iridium(III) complexes 1−12 were synthesized in
high yields (75−97%) via the classical transmetalation method.
All the synthesized iridium(III) complexes were isolated as
−
1
PF6 salts and fully characterized by H NMR spectroscopy
(Figures S1−S12), CHN elemental analysis, ESI-MS (Figures
S13−S33), and X-ray crystallography.
Due to the similarity in the structure of these complexes, the
complex 8 was selected as a representative complex to monitor
spectroscopic properties. First, the UV−vis absorption of the
complex 8 in CH3CN solution was determined utilizing UV
spectrophotometer. As seen from Figure 1a, a weak absorption
peak at ∼320 nm was detected. Next, as illustrated in Figure
1b, the maximum emission wavelengths of the complex 8 were
conducted by fluorescence spectroscopy, which were at 503
and 565 nm when excited at 322 nm. The emission spectrum
of complex 8 located at 565 nm was dominated.
In this contribution, 12 novel half-sandwich iridium(III)
complexes of the form [(η5-Cpx)Ir(C^N)Cl]PF6, where C^N
are imine N-heterocyclic carbene ligands with different
X-ray Crystal Structures. The X-ray crystal structures of
−
complexes [(η5-C5Me5)Ir(L4)Cl]PF6 (4) and [(η5-C5Me5)-
B
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Figure 1. UV−vis absorption spectra (a) and fluorescence spectra of (b) of complex 8 in CH3CN solution (20 μM).
Ir(L10)Cl]PF6− (10) were unambiguously confirmed and their
Table 1. Inhibition of the Growth of A549 Cancer Cells by
Complexes 1−12 and Cisplatin
a
molecular structures are shown in Figure 2. X-ray crystallo-
complex
IC50 (μM)
>100
>100
25.86 1.2
14.05 0.1
9.15 0.2
3.04 0.5
2.21 0.2
1.99 0.1
3.94 0.3
3.64 0.3
[(η5-Cp*)Ir(L1)Cl]PF6 (1)
[(η5-Cp*)Ir(L2)Cl]PF6 (2)
[(η5-Cp*)Ir(L3)Cl]PF6 (3)
[(η5-Cp*)Ir(L4)Cl]PF6 (4)
[(η5-Cp*)Ir(L5)Cl]PF6 (5)
[(η5-Cp*)Ir(L6)Cl]PF6 (6)
[(η5-Cp*)Ir(L7)Cl]PF6 (7)
[(η5-Cp*)Ir(L8)Cl]PF6 (8)
[(η5-Cp*)Ir(L9)Cl]PF6 (9)
[(η5-Cp*)Ir(L10)Cl]PF6 (10)
[(η5-Cp*)Ir(L11)Cl]PF6 (11)
Figure 2. X-ray crystal structures of compounds of (A) [(η5-
C5Me5)Ir(L4)Cl]PF6− (4) and (B) [(η5-C5Me5)Ir(L10)Cl]PF6− (10)
with the thermal ellipsoids drawn at the 50% probability level. The
>100
b
[(η5- Cpxbiph)Ir(L1)Cl]PF6 (12)
7.44 0.3
−
cisplatin
21.30 1.7
hydrogen atoms and PF6 counterions have been omitted for clarity.
a
Selected bond lengths (Å) and angles (deg): complex 4 Ir−
C(centroid) = 1.8372, Ir−Ccarbene = 2.006(4), Ir−N = 2.131(4), Ir−
Cl = 2.4036(13), Ccarbene−Ir−N = 75.53(16), Ccarbene−Ir−Cl =
82.39(14), N−Ir−Cl = 89.58(11); complex 10 Ir−C(centroid) =
1.8392, Ir−Ccarbene = 2.009(5), Ir−N = 2.135(5), Ir−Cl = 2.4009(15),
Ccarbene−Ir−N = 76.2(2), Ccarbene−Ir−Cl = 80.97(17), N−Ir−Cl =
88.65(13).
IC50 (half-maximum inhibitory concentration) values are drug
concentrations necessary for 50% inhibition of cell viability. Data
are presented as means
standard deviations and cell viability is
b
assessed after 24 h of incubation. Cpxbiph = tetramethyl(biphenyl)-
cyclopentadienyl.
graphic data are listed in Table S2, and selected bond lengths
and angles are summarized in Table S3. Complexes 4 and 10
are arranged in monoclinic crystal systems with the C2/c and
P2(1)/c space group, respectively. Each complex adopts the
expected half-sandwich distorted-octahedral “three-legged
piano-stool” geometry. The distance between the iridium(III)
center and the centroid of Cp* (pentamethylcyclopentadienyl)
ring is 1.8372 and 1.8392 Å for 4 and 10, respectively.
Study of the Structure−Activity Relationship. The in
vitro cytotoxicity of complexes 1−12 and cisplatin toward
A549 cancer cells was determined after a 24 h exposure period
using the MTT assay.10 Although these iridium(III) complexes
possess very similar structures, they have different anticancer
activities. Subtle changes in the framework of such complexes
result in a significant change on their biological behaviors. As
depicted in Table 1 and Figure 3, the resulting 50% growth
inhibitory concentration (IC50) values for the complexes 1, 2,
and 11 were >100 μM and thus deemed as inactive. However,
other nine complexes display promising activity toward A549
cancer cells comparable to or even higher than cisplatin.
Notably, an especially high anticancer activity is showed for
complexes 7 and 8. They give a ∼10-fold increase in cytotoxic
potency than that of cisplatin against A549 cells. On the whole,
the in vitro anticancer activities of these complexes revealed
Figure 3. Inhibition of the growth of A549 cells by complexes 1−12
and cisplatin.
the following trends: 8 ≈ 7 ≈ 6 > 12 > 10 > 9 > 5 > 4 >
cisplatin > 3.
First, the steric hindrance of ortho-substituents in the aniline
shows a significant effect on the cytotoxicity of complexes.
Replacement of the aniline substituents from 2,6-dimethyl to
2,6-diisopropyl results in an excellent increase in anticancer
activity. For example, complexes 5, 6, 7, and 8, whose IC50
values are 9.15, 3.04, 2.21, and 1.99 μM, respectively, exhibit in
C
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1
−
Figure 4. (a) H NMR spectra of the reaction between complex [(η5-C5Me5)Ir(L8)Cl]PF6 (8) (1 mM) and NADH (3.5 mol equiv) in 50%
CD3OD-d4/50% D2O (v/v) at 310 K after 5 min, 2 h, and 7 h. Left: Low-field region. Right: High-field region showing the Ir−H hydride peak
(−16.05 ppm). Peaks labeled with a green circle and red box correspond to the formed Ir−H complex. (b) UV−vis spectra of the reaction of
NADH (100 μM) with complex [(η5-C5Me5)Ir(L8)Cl]PF6− (8) (1 μM) in 5% MeOH/95% H2O (v/v)at 298 K for 8 h. (c) The turnover numbers
(TONs) of complexes 1, 4, 8, and 10.
vitro anticancer activity significantly superior to complexes 1
(>100 μM), 2 (>100 μM), 3 (25.86 μM), and 4 (14.05 μM)
containing the same substituents (R1 and R2) against A549
cancer cell lines. In addition, when ortho-substituents in the
aniline is changed into the hydrogen atom, complex 11
becomes inactive (>100 μM). Next, maintaining the aniline
ring substituent unchanged and increasing tether length on the
imidazole ring gradually, the cytotoxicity follows the order of
butyl- > isopropyl- > ethyl- > methyl-substituted NHCs. As a
result, the tether length perturbations also exhibit variation on
the anticancer activities of these complexes.
cells lines, respectively. This result suggests that anticancer
activity of these complexes could be tuned through electronic
changes of the substituents on the imine carbon.
In this system, minor structural changes on four positions of
the complex have a pronounced effect on their biological
behaviors. As a result, this class of iridium(III) complexes
represents a versatile and potent platform for development of
organometallic anticancer metal containing drugs.
Reaction with NADH. In a sea of biocatalyzed processes,
the coenzyme NADH and NAD+ are a significant redox pair.
Previous work has shown that half-sandwich iridium(III)
anticancer complexes can accept a hydride from NADH and
promote the production of ROS H2O2, thus providing a
pathway to an oxidant mechanism of action.12 Reactions
between the iridium(III) complexes and NADH in 50%
Pervious work has shown that the anticancer activity of half-
sandwich iridium(III) complexes containing classical bipyr-
idine chelating ligands increases by the incorporation of phenyl
substituents on Cp*.11 In this work, the introduction of
biphenyl substituents onto the tetramethylcyclopentadienyl
ring to obtain the complex [(η5-C5Me4C6H4C6H5)Ir(L1)Cl]-
1
CD3OD-d4/50% D2O (v/v) were monitored by H NMR
spectroscopy (complex 1, Figure S34; complex 8, Figure 4a).
When NADH (3.5 mol equiv) was added to a 1 mM
solution of complex 8, NADH was converted into its oxidized
form NAD+. A new set of low-field peaks at 8.92, 9.28, and
9.44 ppm assignable to the protons at the C4, C6, and C2
positions of the nicotinamide ring of NAD+ and a sharp singlet
at −16.05 ppm corresponding to the IrIII hydride complex
−
PF6 (12) (IC50 = 7.44 μM) also results in enhanced
cytotoxicity as compared to its parent Cp* complex [(η5-
−
C5Me5)Ir(L1)Cl]PF6 (1) (IC50 > 100 μM). Finally, the
influence of the substituent on the imine carbon on anticancer
activity of these complexes was further investigated. Replace-
ment of the methyl group on the imine carbon by a more
electron-poor phenyl ring leads to complexes 9 (IC50 = 3.94
μM) and 10 (IC50 = 3.64 μM), which display approximately
6.6 and 3.9 times higher anticancer efficacy than complexes 3
(IC50 = 25.86 μM) and 10 (IC50 = 14.05 μM) against A549
[(η5-Cp*)Ir(L8)H]PF6 (Figure 4a) were observed after 5
−
min. The reaction of complex 1 with NADH also had the
similar results (Figure S34). In a solution of complex 8 (0.5
mM) with NADH (3 mol equiv) in 50% MeOH/50% H2O (v/
D
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Figure 5. Dependence on time of the interaction of complex 4 (1 mM in CD3OD-d4/D2O (1/1 v/v)) with GSH (25 mM, in D2O, pH* adjusted to
1
7.2 0.1), monitored by H NMR (500 MHz) at 310 K.
Figure 6. UV−vis spectrum of BSA (10 μM) in 5 mM Tris-HCl/10 mM NaCl buffer solution (pH 7.2) upon addition of the complex 4 (A) or 8
(B) (0−10 μM). Inset: Wavelength from 260 to 290 nm. Fluorescence spectra of BSA (10 μM; λex = 280 nm; λem = 343 nm) in the absence and
presence of the complex 4 (C) or 8 (D) (0−10 μM). The arrow shows the intensity changes in increasing concentration of the iridium(III)
complex.
v) at 298 K, the ROS hydrogen peroxide was detected by the
appearance of a blue color on hydrogen peroxide-test paper. In
order to investigate the effect of four positions bearing different
substituents in this complex framework on the catalytic ability,
the reactions of complexes 1, 4, 8, and 10 (∼1 μM) with
NADH (100 μM) in 5% MeOH/95% H2O (v/v) were also
investigated by UV−vis at 298 K (Figures 4b and S35). The
turnover numbers (TONs) of complexes 1 (7.3), 4 (6.6), 8
(6.6), and 10 (8.0) were calculated by detecting the changes in
absorbance at 339 nm (Figure 4c). These results suggest that
the steric hindrance of ortho-substituents on the aniline
moiety, the length of the alkyl substitutions on the imidazole
ring and the electronic perturbations on the imine carbon
appear to exhibit little variation on the catalytic activity of
these complexes.
Interaction with Nucleobases. Reactions of complexes 4
and 8 with model nucleobase 9-ethylguanine (9-EtG) or 9-
1
methyladenine (9-MeA) were monitored using the H NMR
spectroscopy from 5 min to 24 h. Solutions of 4 and 8 (∼1
mM) and 2.0 molar equiv of 9-EtG or 9-MeA in 50% CD3OD-
1
d4/50% D2O (v/v) were prepared, respectively, and H NMR
spectra were recorded at different time intervals at 310 K. The
1
NMR data do not show additional H NMR peaks over a
period of 24 h (Figures S36−S39), suggesting that no reaction
with model nucleobase occurred for complexes 4 and 8. Also,
the formation of nucleobase adducts was not detected by mass
E
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Table 2. Quenching Parameters and Binding Parameters for the Interaction of the Complexes 4 and 8 with BSA
complex
T (K)
Ksv (104)
Kq (×1012 M−1 S−1
)
Kb (×104 M−1
)
n
4
8
298
298
2.68 0.28
3.66 0.15
2.68
3.66
1.73
12.8
1.181
0.304
spectrometry. So DNA may not be the main target for this type
of iridium(III) complexes.
understand the nature of binding complexes with BSA. In
this work, the fluorescence measured was calibrated to correct
the “inner filter” effect.18 As shown in Figure 6, C and D, the
fluorescence intensity of BSA at ∼343 nm gradually decreased
with the increase of concentration of complexes, suggesting
that the complexes 4 and 8 can interact with BSA through
static quenching mode. The possible quenching mechanism
can be interpreted using the Stern−Volmer equation (eq 1)19
Reaction with GSH. Glutathione (GSH), the most
plentiful nonprotein molecule in the cell, is involved in the
detoxification of a lot anticancer agents.13 Previous work has
shown that GSH can reaction with iridium(III) and
ruthenium(II) complexes through the coordination of thiol
group to metal center.14 The time dependence of the reaction
of complexes 4 and 8 with tripeptide glutathione (GSH) were
monitored by 1H NMR under the same conditions: complex 4
or 8 (1 mM) in CD3OD-d4/D2O (1/1 v/v, pH* adjusted to
F /F = 1 + Ksv[Q] = 1 + Kqτ0[Q]
0
(1)
where F0 and F are the steady-state fluorescence intensities in
the absence and presence of the quenching agent, respectively,
[Q] represents the total concentration of the quenching agent,
Kq is the quenching rate constant, and τ0 is the average lifetime
of protein in the absence of quencher, and its value is 10−8 s.20
Ksv is Stern−Volmer quenching constant which can be
obtained from the ratio of the slope to the intercept of the
plot of F0/F versus the concentration of the tested complex
(Figure S41). The corresponding Stern−Volmer quenching
constants Ksv and quenching rate constants kq are given in
Table 2.
7.2
0.1) with GSH (25 mM) tested by 1H NMR
spectroscopy from 5 min to 7 h at 310 K. As shown in
Figures 5 and S40, the low-field ligand-phenyl and imidazole
ring peaks of Ir−Cl complex decreased gradually, and a new set
of low-field resonances appeared when the excess GSH (25
mol equiv) was added. After 7 h, the Ir-Cl complex has been
converted into the glutathione adducts (Ir−SG) completely.
On the other hand, the five methyl groups in the Cp* ring of
complexes 4 and 8 give rise to one singlet, but split into one
doublet with an intensity ratio of 1:1 for Ir-SG. Complexes 4
and 8, which contain an unsymmetric chelating ligand, are
chiral. Thus, two diastereomeric glutathione adducts are
expected.14b These results suggest that complexes 4 and 8
can reaction with GSH and Ir-SG adduct formed rapidly. This
high glutathione affinity may mean that these iridium(III)
complexes bind rapidly to GSH on entering cells. Thus, the
binding of these iridium(III) complexes to DNA was blocked.
This may explain why DNA is not a major target in this system.
Protein Binding Studies. The reactions of metal-base
anticancer agents with proteins have attracted much attention
since these interactions might feature processes that are
significant for the toxicity, the biodistribution, and even the
mechanism of action of anticancer agents.15 Serum albumin,
which possesses significant binding properties and highest
content in blood plasma, plays an essential role in the drug
transport system.16 In this work, BSA (bovine serum albumin),
owing to its structural similarity with human serum albumin
(HSA), is chosen to study the binding of complexes to serum
albumin.
The calculated value of Kq for the complexes 4 and 8 are
2.68 × 1012 M−1 s−1 and 3.66 × 1012 M−1 s−1, respectively,
which are about 2 orders of magnitude higher than that of
purely dynamic quenching mechanism (2.0 × 1010
M
−1 s−1).21
The value of Kq indicates that a static quenching mechanism
dominates in the interaction between iridium(III) complexes
and BSA. The binding constant Kb and number of complex
bound to BSA (n) are calculated (Figure S42), using the
following formula (eq 2):22
log[(F − F)/F] = log Kb + n log[Q]
(2)
0
By comparison of the values of Kq and Kb, it is inferred that
complex 8 interacts with BSA more strongly than the complex
4. Thus, when the bulkiness of ortho-substituents in the aniline
increases, the binding constant value (K) also increases. The
difference of this binding ability between complexes 4 and 8
may cause differences in the anticancer activity of these
complexes.
Synchronous fluorescence spectrometry is a very useful
technique for obtaining information about molecular environ-
ment in the vicinity of the fluorophore molecules at low
concentrations under analogous conditions.23 The use of Δλ =
15 nm and Δλ = 60 nm gives the spectral property of tyrosine
residues and tryptophan residues, respectively.24 It can be seen
from Figure S43, that the synchronous fluorescence intensity
for BSA with increasing concentrations of complex 4 displayed
a decrease at 292 nm (Δλ = 15 nm) with an inappreciable blue
shift of 1 or 2 nm. At the same time, the emission wavelength
of tryptophan residues did not undergo significant changes
during the binding process. These results indicated that the
reaction of complexes with BSA can influence the con-
formation of the tyrosine microregion. In addition, the slight
blue shift is mainly due to the fact that the active site in protein
is buried in a hydrophobic environment.25 Therefore, the
hydrophobicity around tyrosine residues is reinforced in this
system. The valid binding of the complex with the BSA was
The UV−vis absorption spectra of BSA were measured
before and after the addition of complexes 4 and 8. The results
are shown in Figure 6, A and B. Upon addition of the
complexes, the absorption peak at 228 nm decreased
dramatically and shifted toward longer wavelength. The
decrease of absorbance is due to the induced perturbation of
the α-helix of BSA by the transition metal-based complexes.17
The obvious red-shift in the absorption spectra is associated
with the polarity of their surroundings. With the addition of
iridium(III) complexes to BSA, a progressive increase without
any shift was found in the absorption peak of BSA at 278 nm
for these complexes, suggesting that the anticancer metal-
lodrugs can interact with the BSA molecule and the
microenvironments of the three aromatic acid residues in
BSA (Trp, Tyr, and Phe) are altered.
Quenching of the emission intensity of BSA has been
performed in the presence of the complexes to further
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Figure 7. Flow cytometry data for cell cycle distribution of A549 cancer cells exposed to complexes 4 (A) and 8 (B) for 24 h. Concentrations used
were 1 and 2 equipotent concentrations of IC50. Cell staining for flow cytometry was carried out using PI/RNase. Data are quoted as mean SD of
three replicates. P-Values were calculated after a t test against the negative control data, *p < 0.05.
Figure 8. Apoptosis analysis of A549 cells after 24 h of exposure to complexes 4 and 8 at 310 K determined by flow cytometry using annexin V-
FITC vs PI staining. Populations for cells in four stages treated by complexes 4 and 8. Data are quoted as mean SD of three replicates. p-Values
were calculated after a t test against the negative control data, *p < 0.05 and **p < 0.01.
confirmed via the hydrophobicity observed in fluorescence and
synchronous experiments. Hence, the strong interaction of
these complexes with BSA suggested that BSA can be
considered as an excellent carrier for delivery of anticancer
agents in vivo.
Cell Cycle Arrest. Generally, most anticancer metallodrugs
exert their anticancer efficacy via genomic DNA damage and
cell cycle perturbation.26 To understand the impact of the new
complexes on cell growth, complexes 4 and 8 effect on the cell
cycle by flow cytometry analysis in A549 cancer cells was
evaluated. A549 cells were treated with different concen-
trations (1 × IC50 and 2 × IC50) of complexes 4 and 8 for 24 h
(Figures 7 and S44 and Tables S4−S5), respectively. In
comparison to negative control populations, both complexes 4
and 8 arouse an increased 10.2% of cells in the G2/M phase at
a concentration of 2 × IC50, suggesting that complexes 4 and 8
arrest the cell cycle at the G2/M phase in a concentration-
dependent manner.
Apoptosis Assay. To quantify the amount of cells in
different apoptosis stages, A549 cells were treated with
complexes 4 and 8 at 0.5, 1, 2, and 3 equipotent concentrations
of IC50 for 24 h, and then analyzed by flow cytometry. The
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results are shown in Figure 8 and Tables S6−S7. When
complexes 4 and 8 is at 3 equipotent concentrations of IC50, a
total of 78.1% and 80.1% of cells were undergoing apoptosis,
respectively. As shown in the histogram (Figure 8), the most
apoptotic cells are in late apoptosis stage. This indicated that
cell death can be induced through a high incidence of
apoptosis. In addition, complex 8 showed slightly enhanced
ability to induce apoptosis of A549 cells, which is consistent
with its higher anticancer activity compared to complex 4.
ROS Determination. Mitochondria are the main sites of
cellular ROS production, and mitochondrial dysfunction may
lead to ROS accumulation during the process of apoptosis.27
The levels of ROS in A549 cells were estimated by flow
cytometry analysis. Treatment of A549 cells with complex 8 for
24 h results in a dose-dependent increase in the production of
ROS (Figure 9). This result indicates that complex 8 may lead
to ROS accumulation. Elevated levels of reactive oxygen
species may be responsible for the cell toxicity of these
complexes.
dide (JC-1) using flow cytometry. As shown in Figure 10
and Tables S8−S9, increasing the concentration of complexes
4 and 8 result in an obvious red-to-green color shift, which
marks the loss of MMP. The percentage of cells with
mitochondrial membrane depolarization increases from
10.72% to 61.15% and 7.67% to 50.10% for 4 and 8 at a
concentration of 2 × IC50, respectively. Additionally, as shown
in Figure S45 and Table S10, the JC-1 red/green fluorescence
ratios reduced from 8.34 0.42 to 0.64 0.01 and 12.14
1.67 to 1.00 0.04 for 4 and 8 at a concentration of 2 × IC50,
respectively. As a result, the dysfunction of MMP may
contribute to the anticancer activity of these iridium(III)
complexes.
Cellular Localization. Because of luminescence properties
of these iridium(III) complexes, we subsequently examined the
cellular localization of these complexes using confocal
microscopy. Organelle-specific probes are powerful tools that
can real-time monitor morphological changes of organelle and
intracellular dynamic processes.28 To evaluate which organelles
would be targeted by these iridium(III) complexes, the A549
cells were dual-stained with complex 8 and different organelle-
specific probes, respectively. Confocal microscopic imaging
indicates that the complex 8 can effectively enter A549 cells
after 1 h incubation, as indicated by the intense and punctate
green fluorescence in the cytoplasm (Figure 11). Co-
localization analysis with the organelle-specific stain for
lysosome shows excellent concordance between overlay images
of the complex 8 (10 μM) and conventional LTDR (75 nM)
in A549 cells. Pearson’s correlation coefficients for complex 8
and LTDR is 82%. Clearly, complex 8 is selectively localized
within lysosome and the cytotoxic properties may originate
from lysosome mediated cell death.
Lysosomal Damage. The dysfunction of lysosomes of
A549 cells was evaluated via acridine orange (AO) staining.
AO is a useful probe employed to assess the lysosomal
functional state at subcellular level, because it emits a
concentration-dependent red/green fluorescence.29 As shown
in Figure 12, A549 cells treated with only acridine orange(AO)
(5 μM) showed distinct red fluorescence in lysosomes,
suggesting that lysosomes under such conditions were intact.
However, the red fluorescence of AO significantly decreased
with the increase of drug concentration, which suggested that
lysosomal integrity was jeopardized upon complex 8 treat-
ments. These results indicated that complex 8 can induce
apoptosis through lysosomal damage.
Figure 9. Analysis of ROS levels by flow cytometry after A549 cells
were treated with complex 8 at the 0.25 and 0.5 equipotent
concentrations of IC50 for 24 h and stained with H2DCFDA. p-Values
were calculated after a t test against the negative control data, *p <
0.05.
Impact on Mitochondrial Membrane Potential
(MMP). Mitochondrial plays a central role in apoptosis
because they can generate a large of the ATP in cells. The
decrease of mitochondrial membrane potential is an important
indicator for the detection of apoptosis. The effects of
complexes 4 and 8 on MMP were determined by 5,5′,6,6′-
tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine io-
Figure 10. Changes in mitochondrial membrane potential of A549 cancer cells induced by complexes 4 (a) and 8 (b).
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Figure 11. Determination of intercellular localization of complex 8 by confocal microscopy. A549 cells were incubated with complex 8 (10 μM) for
1 h at 37 °C, then coincubated with MTDR (500 nM), LTDR (75 nM) and DAPI (1 μg/mL) for 20 min, respectively. Complex 8 was excited at
488 nm and the emission was collected at 520 20 nm. MTDR was excited at 543 nm and the emission was collected at 690 30 nm; LTDR was
excited at 594 nm and the emission was collected at 630 30 nm. DAPI was excited at 405 nm and the emission was collected at 460 30 nm.
Scale bar: 20 μm.
Figure 12. Observation of lysosomal disruption in A549 cells loaded with complex 8 for 6 h at 37 °C, then stained with acridine orange (AO) (5
μM) at 37 °C for 15 min. Emission was collected at 510 20 nm (green) and 625 20 nm (red) upon excitation at 488 nm. Scale bar: 20 μm.
The A549 cells were treated with (a) only acridine orange (AO); (b) acridine orange (AO) and complex 8 (1× IC50); (c) acridine orange (AO)
and complex 8 (3× IC50).
Cellular Uptake Mechanisms. We also further inves-
tigated the cellular uptake mechanisms of complex 8 in A549
cells. The ways in which small molecules enter cells include
energy-dependent or energy-independent pathways.30 As
shown in Figure 13, A549 cells incubated with complex 8 at
4 °C or pretreated with carbonyl cyanide m-chlorophenyl
hydrazone (CCCP) resulted in a reduced intracellular
luminescence intensity compared to untreated control group
(37 °C). However, intracellular luminescence intensity was not
significantly influenced by the endocytosis inhibitor chlor-
oquine. These results suggest that the cellular uptake of
complex 8 is mainly through a well-known energy-dependent
mechanism, such as active transport. Endocytosis was not
responsible for the uptake of complex 8.
Observation of Nuclear Morphology. The investigation
of nuclear morphology of the A549 cells was also performed by
confocal microscopy using nucleus-staining dye 4′,6-diamidi-
no-2-phenylindole (DAPI). Confocal microscopic images are
shown in Figure S46. As time goes on, the volume of nucleus
gradually decreases and the surface of the nucleus becomes
nonsmooth. In addition, nuclear condensation occurs, and
then the nucleus is broken into fragments of different sizes.
Further, the presence of apoptotic bodies was also detected.
These results suggested that complex 8 can change nuclear
morphology effectively and finally induce cells apoptosis.
Antibacterial Studies. To further investigate the
potentiality of such complexes as antibacterial agents,
complexes 4 and 8 were chosen and preliminary studied
against two bacterial strains, the Gram-positive Staphylococcus
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This result is consistent with their cytotoxicity. Additionally, as
shown in Figure 14b, complexes 4 and 8 showed no activity
against P. vulgaris. It seems that complex 8 exhibited selective
antibacterial activity toward S. aureus.
CONCLUSIONS
■
To conclude, a series of novel and versatile imine-N-
heterocyclic carbene-based half-sandwich iridium(III) anti-
cancer complexes of the type [(η5-Cpx)Ir(C^N)Cl]PF6 have
−
been designed and prepared. Spectroscopic properties study
displays that complex 8 has a detectable fluorescence. This
type of imine-N-heterocyclic carbene half-sandwich iridium-
(III) complex appears to be the first time used as anticancer
agents. In this complex framework, substituents on four
positions could be modulated, which provides a large amount
of flexibility and opportunities to tune the cytotoxicity of
complexes. The structure−activity relationships are very
significant. With increasing the size of the substituents on
aniline moiety and imidazole ring, the anticancer activity
increased. In addition, the complexes containing the electron-
poor groups seem to have the higher anticancer activity than
that containing the electron-rich groups.
No nucleobase binding was observed for these complexes,
indicating that DNA is not a possible target. This type of
complexes can react with GSH and catalyze oxidation of
NADH to NAD+. Further, they showed moderate binding
affinity to BSA and the fluorescence quenching of BSA by the
metal complexes is due to the static quenching. As a result,
binding of complexes 4 and 8 with BSA can reach the
designated target through blood transport. Additionally, the
complexes 4 and 8 aroused a decrease in mitochondrial
membrane potential, disturbed the cell cycle at G2/M phase
and induced obvious cell apoptosis in A549 cancer cells.
Complex 8 can also induce high increase in the level of ROS in
A549 cancer cells. It seems that cell cycle, apoptosis induction,
ROS level and mitochondrial membrane potential together
contribute to the anticancer potency of these iridium(III)
complexes.
Figure 13. Confocal images of A549 cells after incubation with
complex 8 (10 μM) under different conditions. (A) Cells were
incubated with complex 8 (10 μM) at 37 °C for 1 h. (B) Cells were
incubated with complex 8 (10 μM) at 4 °C for 1 h. (C) Cells were
preincubated with CCCP (50 μM) for 1 h at 37 °C and then
incubated with complex 8 (10 μM) at 37 °C for 1 h. (D) Cells were
preincubated with chloroquine (50 μM) for 1 h at 37 °C and then
incubated with complex 8 (10 μM) at 37 °C for 1 h. Complex 8 was
excited at 488 nm and emission was collected at 520 20 nm. Scale
bar: 20 μm.
aureus (S. aureus) and the Gram-negative Proteus vulgaris (P.
vulgaris). As illustrated in Figure 14, the S. aureus and P.
Confocal imaging studies indicated that the typical and most
active complex 8 entered A549 lung cancer cells mainly
through energy-dependent pathway and was located in
lysosome. The cytotoxic properties may originate from
lysosome mediated cell death. Furthermore, lysosomal damage
and nuclear morphology were detected in A549 cancer cells by
confocal microscopy. Nuclear condensation and apoptotic
bodies may finally induce cells apoptosis. Interestingly,
complex 8 also showed antibacterial activity against Gram-
positive S. aureus. All of these preliminary results suggest that
this type of iridium(III) complexes containing versatile imine-
N-heterocyclic carbene ligands could be a promising candidate
for future cancer therapy.
Figure 14. Antibacterial activity of complexes 4 and 8 (1, 5, 25, and
125 μM) were determined by the Oxford Cup method. (a) The anti-
S. aureus activity of complexes 4 and 8. (b) The anti-P. vulgaris activity
of complexes 4 and 8. Ampicillin (AMP) was used as the positive
control (100 μg mL−1). Scale bar: 10 mm.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b01656.
vulgaris diameter of inhibition zone are 19 mm and 33 mm,
respectively. Interestingly, an obviously inhibition zone was
observed when the S. aureus was treated with maximum
concentration of complex 8 (the diameter of inhibition zone is
11 mm), and no inhibition zone was observed when the S.
aureus was treated with the same concentration of complex 4
(Figure 14a), suggesting that complex 8 exhibited higher
efficiency than complex 4 in the growth inhibition of S. aureus.
Details of the experimental section, Figures S1−S46, and
Tables S1−S10 (PDF)
Accession Codes
CCDC 1843724 and 1843726 contain the supplementary
crystallographic data for this paper. These data can be obtained
J
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Inorganic Chemistry
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free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: guolihua@qfnu.edu.cn.
*E-mail: liuzheqd@163.com.
■
ORCID
Lihua Guo: 0000-0002-0842-9958
Zhe Liu: 0000-0001-5796-4335
Notes
Preishuber-Pflugl, P.; Bildstein, B. Heteroditopic Imino N-Hetero-
̈
cyclic Carbenes and Their Sulfur, Selenium, and Tungsten
The authors declare no competing financial interest.
Tetracarbonyl Derivatives. Eur. J. Inorg. Chem. 2005, 2005, 1325−
ACKNOWLEDGMENTS
■
̈
1333. (e) Frøseth, M.; Netland, K. A.; Tornroos, K. W.; Dhindsa, A.;
We thank Shandong Provincial Natural Science Foundation
(ZR2018MB023), the National Natural Science Foundation of
China (Grant No. 21671118), the Taishan Scholars Program,
the Key Laboratory of Polymeric Composite & Functional
Materials of Ministry of Education (PCFM-2017-01), and the
excellent experiment project of Qufu Normal University
(jp201705) for support.
Tilset, M. Synthesis and characterization of novel Pd(II) complexes
with chelating and non-chelating heterocyclic iminocarbene ligands.
Dalton Trans. 2005, 249, 1664−1674. (f) Frøseth, M.; Netland, K. A.;
Rømming, C.; Tilset, M. Synthesis and characterization of novel
Pd(II) and Pt(II) complexes with 5-ring chelating iminoylcarbene
ligands. J. Organomet. Chem. 2005, 690, 6125−6132. (g) Coleman, K.
S.; Dastgir, S.; Barnett, G.; Alvite, M. J. P.; Cowley, A. R.; Green, M.
L. H. A nonenolizable imino-N-heterocyclic carbene ligand and
corresponding silver(I) metal complex. J. Organomet. Chem. 2005,
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DOI: 10.1021/acs.inorgchem.8b01656
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