Half-sandwich iridium(III) complexes with α-picolinic acid frameworks and antitumor applications

Article abstract of DOI:10.1016/j.jinorgbio.2018.12.012

Eight half-sandwich iridium^III (Ir^III) complexes of the general formula [(η⁵-Cp^xbiph)Ir(O^N)Cl] (Cp^xbiph is tetramethyl(biphenyl)cyclopentadienyl, and the O^N is α-picolinic acid chelating ligand and

Full text of DOI:10.1016/j.jinorgbio.2018.12.012

JournalofInorganicBiochemistry192(2019)52–61
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Half-sandwich iridium(III) complexes with α-picolinic acid frameworks and
antitumor applications
⁎
Hailong Haoa, Xicheng Liua, , Xingxing Gea, Yao Zhaob, Xue Tiana, Ting Rena, Yan Wanga,
⁎
Chengfeng Zhaoa, Zhe Liua,
a Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of Life-Organic Analysis, Key Laboratory of Pharmaceutical Intermediates
and Analysis of Natural Medicine, Department of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China
b CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Eight half-sandwich iridiumIII (IrIII) complexes of the general formula [(η5-Cpxbiph)Ir(O^N)Cl] (Cpxbiph is tetra-
methyl(biphenyl)cyclopentadienyl, and the O^N is α-picolinic acid chelating ligand and its derivatives) were
synthesized and characterized. Compared with cis-platin widely used in clinic, target IrIII complexes showed at
most five times more potent antitumor activity against A549 cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay. IrIII complexes could be transported by serum albumin, bind with DNA,
catalyze the oxidation of nicotinamide-adenine dinucleotid (NADH) and induce the production of reactive
oxygen species, which confirmed the antitumor mechanism of oxidation. IrIII complexes could enter A549 cells
followed by an energy-dependent cellular uptake mechanism, meanwhile, target the mitochondria and lyso-
somes with the Pearson's colocalization coefficient of 0.33 and 0.74, respectively, lead to the lysosomal de-
struction and the change of mitochondrial membrane potential (ΔΨm), and eventually induce apoptosis.
Half-sandwich structure
Iridium complexes
α-Picolinic acid
Antitumor
1. Introduction
cyclopentadiene (Cp), tetramethyl(biphenyl)cyclopentadienyl (Cpxbiph),
the diphenyl derivatives of Cp and showing the stronger electron donor
Platinum-based antitumor drugs have proved to be indispensable in
the treatment of cancer, and accounting for almost 50% cancer ther-
apeutic drugs on a world scale [1,2]. However, widely used it is today,
platinum antitumor drugs [3] also show some adverse factors, such as
unclear mechanism of action, poor selectivity, strong resistance and
serious side effects, which prompted to seek the alternative antitumor
drugs with more efficient and better targeted [4,5]. IrIII complexes have
recently emerged as the potential alternatives, compared with the
conventional platinum-based drugs, which have shown favorable anti-
proliferative activity and a diverse antitumor mechanism [1,6].
characteristics, could further enhance the rate of ligand exchange and
hydrolyze rapidly for complexes after introduction, therefore improving
the overall antitumor activity [1,10]. In addition, the favorable targeted
fluorescence characteristics of half-sandwich IrIII complexes provided
an insight into microscopic mechanism, after which enter the tumor cell
organelles and target mitochondria, lysosomes and other tissues [8,11].
Due to the strong complexation ability of nitrogen atom of pyridine
and the oxygen atoms of carboxylate to various transition metal ions, α-
picolinic acid (α-PA) has attracted extensive attention as a potential
chelating ligand [12–15]. The study show complex containing α-PA
chelating ligand could effectively improve the catalytic property for
hydrolysis, reduction and oxidation [16,17]. And also, these types of
complexes showed good biological applications, such as carcinostatic,
antitumor, antiviral and antibacterial activity [18]. Above all, as the
type of O^N chelating ligands, α-PA and its derivatives were used to
construct the half-sandwich structure IrIII antitumor complexes (Fig. 1
and Table 1). Compared with cis-platin (21.3 μM) widely used in clinic,
the introduction of α-PA could effectively adjust the hydrophilicity and
lipophilicity of target complexes, and leading to the potential antitumor
IrIII antitumor complexes included two main types: half-sandwich
iridium complexes and cyclometalated iridium. Because of the excellent
antitumor activity, half-sandwich structure IrIII complexes have at-
tracted much attention [7]. The general formula of half-sandwich IrIII
complexes is [(Cp*)Ir(L^L)Z]PF6, where Cp* represents the electron-rich
cyclopentadienyl group or its derivatives, and Z is the leaving group,
L^L is various chelating ligands [8]. The study show the rational design
of ligands around Ir center may change the hydrophobic and hydro-
philic of complexes, and therefore change biological targets, and even
the performance in cells [9]. And also, compared with simple
activity (IC50 value of the best was 4.41
0.43 μM) [9,19]. In
⁎ Corresponding authors.
E-mail addresses: chemlxc@163.com (X. Liu), liuzheqd@163.com (Z. Liu).
https://doi.org/10.1016/j.jinorgbio.2018.12.012
Received 15 October 2018; Received in revised form 2 December 2018; Accepted 21 December 2018
Availableonline24December2018
0162-0134/©2018ElsevierInc.Allrightsreserved.

H. Hao et al.
JournalofInorganicBiochemistry192(2019)52–61
Fig. 1. Synthesis process of half-sandwich IrIII α-PA complexes.
2.1. Cytotoxicity test
Table 1
IC50 values of IrIII α-PA complexes, cis-platin and Dimer for 24 h.
A549 cells were hatched in as-synthesized IrIII complexes, and
subsequently tested by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide) assay to evaluate the cytotoxicity. The IC50
values (the concentration of 50% cell growth is inhibited) are listed in
Table 1 after hatched in IrIII complexes and cis-platin for 24 h. Except
for complex 4, other complexes displayed higher potency against A549
lung tumor cells than the dimer. In addition, complex 6 showed the best
Complexes
N^O
IC50(μM)
A549 cells
[(η5-Cpxbiph)Ir(L1)Cl] (1)
[(η5-Cpxbiph)Ir(L2)Cl] (2)
[(η5-Cpxbiph)Ir(L3)Cl] (3)
[(η5-Cpxbiph)Ir(L4)Cl] (4)
[(η5-Cpxbiph)Ir(L5)Cl] (5)
[(η5-Cpxbiph)Ir(L6)Cl] (6)
[(η5-Cpxbiph)Ir(L7)Cl] (7)
[(η5-Cpxbiph)Ir(L8)Cl] (8)
cis-Platin
R1 = H, R2 = H, R3 = H
31.3
5.6
0.9
R1 = H, R2 = H, R3 = Br
R1 = H, R2 = Cl, R3 = H
R1 = H, R2 = H, R3 = COOH
R1 = CH3, R2 = H, R3 = H
R1 = H, R2 = H, R3 = CF3
R1 = OH, R2 = H, R3 = H
R1 = H, R2 = H, R3 = OH
/
0.4
0.4
4. 9
> 100
15.7
4.4
1.0
0.4
0.1
4.9
1.7
0.8
activity (IC50: 4.41 0.43 μM), which was nearly 5 times of cis-platin
1.7 μM) under the same conditions. The logP (partition
15.4
43.8
21.3
57.7
(IC50: 21.3
coefficient in oil/water) values of complexes 1, 4 and 6 were −0.15,
−2.73 and 1.05, respectively, which were determined by inductively
coupled plasma mass spectrometry (ICP-MS). Notably, compared with
complex 1, complex 4 improve lipophilicity by introducing hydrophilic
carboxyl substituents to α-picolinic acid chelating ligand, which in-
dicating that the stronger of hydrophobicity for complex 6 caused by
trifluoromethyl (hydrophobic group), the greater of cytotoxicity.
Dimer
/
addition, complexes showed excellent bovine serum albumin (BSA)
binding properties, and catalyzing the change of nicotinamide-adenine
dinucleotid (NADH) to NAD+. Due to the favorable targeted fluores-
cence, these complexes could also target mitochondria and lysosomes in
A549 cells by the confocal microscopy imaging [20–22], and leading to
the damage of lysosomes and the change of mitochondrial membrane
potential. And also, energy-dependent cellular uptake mechanism is the
main way for complexes to enter A549 tumor cells. The results suggest
that α-PA-appended half-sandwich IrIII complexes are hopeful for de-
veloping as new potential antitumor agents.
2.2. Reaction with BSA
Serum albumin (SA), the most abundant protein in plasma, plays a
crucial role in drug transport and metabolism [23,24]. The research on
the interaction between IrIII complexes and SA is conducive to study the
antitumor mechanism of drugs. Bovine serum albumin (BSA) could be a
cost-effective alternative model for human serum albumin (HSA) be-
cause of their similar structure, easy availability, and low price [24,25].
Therefore, in this study, complexes 1 and 6 were selected to study the
binding ability with BSA by ultraviolet-visible (UV–Vis) absorption
spectrum. As shown in Fig. 2a and ESI Fig. S4a, the maximum ab-
sorption at 218 nm decreased when increasing IrIII complexes, which
attributed to the interaction between IrIII complex and BSA. Meanwhile,
a significant red shift was found at 218 nm because of the influence of
polar solvent (water) [26,27]. The increase in absorption at 278 nm is
accompanied by the addition of complex, which indicate that the IrIII α-
PA complexes induces the changes of BSA in three aromatic acid re-
sidues (Tryptophan, tyrosine, and phenylalanine) [28–31].
2. Results and discussion
Half-sandwich IrIII α-PA complexes of the general formula [(η5-
Cpxbiph)Ir(O^N)Cl] were synthesized at ambient temperature and ob-
tained in good yields (61.0–79.0%) (Fig. 1 and Table 1). Target IrIII
complexes and the dimer of iridium were characterized by hydrogen
nuclear magnetic resonance (1H NMR) spectrum, mass spectrum (MS)
and elemental analysis, and all the analytical data are consistent with
the proposed structures (ESI Figs. S1–S2). Deuterated reagent DMSO
(2.50 ppm) was used as a solvent for testing 1H NMR of target com-
plexes. The hydrogen on the five methyl groups on the Cp ring is shown
in the range of 1.60 to 1.80 ppm in the 1H NMR spectrum, while on the
benzene ring and pyridine are shown in the range of 7.0 to 9.0 ppm.
Other than that, target complexes used multiple solvents such as di-
chloromethane (5.76 ppm), methanol (3.16, 4.01 ppm), H2O
(3.33 ppm), ether (1.09, 3.38 ppm) or n-hexane (0.86, 1.25 ppm) in the
synthesis and purification process, which showed a certain amount of
residue in the 1H NMR spectrum. Single crystal of complex 6 suitable
for X-ray diffraction analysis was obtained by the slow diffusion of
hexane into a saturated dichloromethane solution of complex. The X-
ray crystal structures are shown in ESI Fig. S3, the crystallographic data
and the selected bond lengths, bond angles are listed in ESI Tables S1
and S2, respectively. Complex 6 has the expected half-sandwich “three-
leg piano-stool” geometry. The distances of iridium atom to η5-cyclo-
pentadienyl ring centroid were 1.747 Å. The IreCl, IreO and IreN bond
distances were 2.374(7), 2.153(19) and 2.050(2) Å, respectively. The
larger bond length of IreCl determines the weaker bond energy, which
is convenient for the hydrolysis of half-sandwich IrIII α-PA complex and
provides the basis for better anticancer activity.
Information on tyrosine or tryptophan residued in BSA could be
determined by a synchronous fluorescence spectrophotometer at a
wavelength interval of Δλ = 15 nm and Δλ = 60 nm, respectively. The
weaked of fluorescence intensity at 291 nm and 285 nm (Δλ = 15 nm
and 60 nm, respectively) for complexes 1 and 6 were shown in ESI Figs.
S5–S6. As shown, there is barely change at the wavelength of
Δλ = 60 nm, but a shift of 2–3 nm occurs at Δλ = 15 nm, which in-
dicate that tyrosine residued in BSA is more involved in the binding
reaction than tryptophan.
The binding properties between IrIII α-PA complexes and BSA could
also be reflected by fluorescence emission spectroscopy. As shown in
Fig. 2b (ESI Fig. S4b), with the increase of complexes 1 and 6 at 298 K, a
rapid weaken of the fluorescence intensity for BSA was occurred. The
Stern–Volmer quenching constant Ksv, quenching rate constant Kq, the
binding constant Kb and binding site number n were calculated by
classical Stern-Volmer equation (ESI Fig. S7) and Scatchard equation
(ESI Fig. S8). Among these, the values of Kq were 2.87 × 1013 and
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H. Hao et al.
JournalofInorganicBiochemistry192(2019)52–61
Fig. 2. (a) Absorbance of BSA (10 μM) was detected by UV–Vis spectrum after reaction with complex 6 (0–10 μM) in Tris-HCl/NaCl buffer solution (pH = 7.2) at
298 K. The arrows showed the change of absorbance with the increase of complex. Inset: Wavelength absorbance changes from 250 to 300 nm. (b) The fluorescence
intensity of BSA (10 μM) was detected by fluorescence spectra after reaction with complex 6 (0–10 μM) in Tris-HCl/NaCl buffer solution (pH = 7.2) at 298 K. The
arrows showed the change fluorescence intensity with the increase of complex.
of DNA [35]. The extent of the hypochromism commonly parallels the
intercalative binding strength [36]. As shown in Table 3, with the in-
crease of DNA, the spectral perturbation follows the order: complex
1 > 6. Although IrIII α-PA complexes could interact with ctDNA
probably via noncovalent binding modes, DNA could not be the main
target due to the absence of formation of nucleobase adducts and
plasmid DNA cleavage. These findings suggested that DNA-complex
interaction was a possible cause of antitumor activity.
Table 2
The values of Ksv, Kb, Kq and n for complexes 1 and 6 at 298 K.
Complexes
Ksv (105 M−1
)
Kq (1013 M−1 S−1
)
Kb (M−1
)
n
1
6
2.87
4.95
0.18
0.14
2.87
4.95
4.23 × 104
0.85
1.20
3.14 × 105
4.95 × 1013 M−1 s−1 for complexes 1 and 6, respectively, which were
almost three orders of magnitude higher than that of a pure dynamic
quenching mechanism (2.0 × 1010 M−1 s−1) in Table 2 [32]. The re-
sults indicate that static quenching mechanism is the main way for IrIII
α-PA complexes acting on BSA. And also, complex 6 had a larger
binding constant (Kb) value and binding site number (n) than complex
1, which was consistent with that complex 6 showing the better anti-
tumor activity.
2.4. Reaction of complexes with NADH
The coenzyme, nicotinamide adenine dinucleotide (NADH), plays a
vital role in the biocatalysis process [10]. Our previous study indicated
that half-sandwich IrIII complexes were able to catalyze the change of
NADH to NAD+ and induce the production of reactive oxygen species
(ROS), which provide a pathway for the mechanism of oxidation
[37,38]. The interaction between NADH and IrIII α-PA complexes was
obtained by UV–Vis spectrum in Fig. 3 (ESI Fig. S10). A slight decrease
2.3. Interaction with ctDNA
in 339 nm and increase in 259 nm (the absorbance of NADH and NAD+
,
To understand whether the selected complexes 1 and 6 have a
possible binding to DNA, the interaction of the complex with ctDNA
(DNA sodium from the calf thymus) was investigated by UV–Vis titra-
tion, meanwhile, the binding constant (Kb) was calculated [33]. As
shown in Table 3 and ESI Fig. S9, with the increase of ctDNA, the de-
crease of absorption value and small red shift (Δλ) at 262 nm were
found, which concluded that the base pair interacted with IrIII complex
was informed by electrostatic-binding or non-covalent insertion [34].
The binding constants Kb were obtained by Benesi–Hildebrand equation
[26]. As shown, compared with complex 1, the introduction of tri-
fluoromethyl substituent induces the greater Kb for complex 6, which
was consistent with the change of antitumor activity. Antitumor me-
tallodrugs binding with DNA usually leads to hypochromism and
bathchromism, due to the intercalative mode involving a strong
stacking interaction between aromatic chromophore and the base pairs
respectively [38]) were obtained with the increase of complexes. The
turnover numbers (TONs) of complexes 1 and 6 were shown in Fig. 3b.
Complex 6 show the bigger TON value (7.09) than 1 (5.95), which was
correspond with the result of MTT assay.
2.5. Apoptosis assay
Flow cytometry was used to determine whether cell function decline
was associated with apoptosis by means of A549 cells treated with IrIII
complexes after stained with annexin V-FITC and Propidium iodide
[39,40]. As shown in Fig. 4 and ESI Tables S3–S4, the values of the
early and late apoptotic phase had a significant increase when the
concentration of complex 6 changed from 1.0 × IC50 to 3.0 × IC50 after
24 h (from 2.6% and 12.6% to 3.4% and 80.2%, respectively). In
comparison, approximately 30.6% of A549 cells treated with complex 1
were undergoing apoptosis, of which 29.1% were in late apoptosis,
however, 92.8% survived for control under the same conditions. The
results were consistent with the results of MTT assay, and further
confirmed IrIII α-PA complexes could lead to function decline of tumor
cells and induce apoptosis.
Table 3
Absorption spectroscopic properties of the IrIII complexes with ctDNA.
Complexes Absorption λmax (nm)
Δλ Hypochromicity (%) Kb (104 mM−1
)
Free
Bound
1
6
262
263
263
265
1
2
30.19
20.08
3.74
9.79
2.6. Cell cycle analysis
[Complex] = 20 μM at [DNA]/[Complex] = 20.
The cell cycle was monitored to determine whether cell cycle arrest
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H. Hao et al.
JournalofInorganicBiochemistry192(2019)52–61
Fig. 3. (a) Absorbance of NADH (100 μM) was detected by UV–Vis spectrum after reaction with complex 6 (1 μM) in methanol solution (MeOH/H2O = 20%/80%
(V:V)) at 298 K for 8 h. Inset: Wavelength from 220 to 260 nm. The arrows showed absorbance changed over time. (b) The histogram of TONs for complexes 1 and 6.
was caused by the antiproliferative activity of IrIII complexes [41,42].
As shown in Fig. 5 (ESI Tables S5–S6), the results of cell cycle arrest for
A549 cells were determined by flow cytometry after 24 h with different
concentration (0.25, 0.5, 1.0 and 2.0 × IC50 for complexes 1 and 6). As
shown, the percentages of cells in G0/G1 + G2/M phase increased by
only 1.1% at the concentration of 2.0 × IC50 for complex 1. However,
for complex 6, which increased by 16.9%. Compared with the control,
IrIII α-PA complexes can interfere with the cell growth cycle progression
of G0/G1 + G2/M phase, and achieving the purpose of apoptosis.
2.7. Induction of ROS
The ROS levels of A549 tumor cells hatched in complexes 1 and 6
(0.25 and 0.5 × IC50) were detected by flow cytometry. As shown in
Fig. 6 (ESI Tables S7–S8), compared with the control, there is hardly no
change for the ROS level. These results were consistent with the con-
clusion of the NADH test, the oxidation is not the main mechanism for
the antitumor activity of IrIII α-PA complexes.
Fig. 4. (a) Apoptosis of A549 cells was analyzed by flow cytometry after treated with complexes 1 and 6 (concentrations = 0.5, 1.0, 2.0 × IC50, 3.0 × IC50) and
annexin V-FITC and PI stained for 24 h at 310 K. (b) The histogram of four stages for A549 cells induced by complexes 1 and 6. Data was averaged for three replicate
experiments
SD.
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H. Hao et al.
JournalofInorganicBiochemistry192(2019)52–61
Fig. 5. (a) The altered A549 cell cycle was analyzed by flow cytometry after induced by complexes 1 and 6 (concentrations = 0.25, 0.5,1.0 and 2.0 × IC50) for 24 h at
310 K. (b) The histogram of cell cycle distributions. Data are quoted as mean
SD of three replicates.
7
7
1
6
6
6
5
4
3
2
1
0
5
4
3
2
1
0
Negative
control
0.25×IC50
0.5×IC50
Positive
control
Negative
control
0.25×IC50
0.5×IC50
Positive
control
Fig. 6. The histogram of ROS level to A549 tumor cells hatched by complexes 1 and 6 (concentrations = 0.25, 0.5, 1.0 and 2.0 × IC50). Data was averaged for three
replicate experiments SD.
2.8. Mitochondrial membrane potential (ΔΨm)
concentrations of 0.25, 0.5, 1.0 and 2.0 × IC50) and subsequently
stained. As shown in Fig. 7 and ESI Tables S9–S10, a significant con-
centration-dependent increase was found at the indicated concentra-
tions, with a remarkable loss from 6.73% and 5.46% (control) to
29.71% and 82.08% (2.0 × IC50) for complexes 1 and 6, respectively.
This conclusion is reflected more intuitively by the ratios of JC-1
Monomers (Green)/JC-1 Aggregates (Red) in Fig. 7b, which further
confirmed IrIII α-PA complexes could act on target mitochondria and
induced apoptosis.
Mitochondria provided energy for the life activities of cells and were
the main sites for the formation of adenosine triphosphate (ATP) in cells
[42]. Mitochondrial dysfunction could lead to cell death, and the
change of mitochondrial membrane potential (ΔΨm) was usually used to
assess [43]. The values of ΔΨm were determined through evaluating the
change of JC-1 (a frequently-used fluorescent probe to assess ΔΨm)
when A549 cells were hatched in complexes
1 and 6 (the
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H. Hao et al.
JournalofInorganicBiochemistry192(2019)52–61
Fig. 7. (a) Changes in mitochondrial membrane potential of A549 tumor cells induced by complexes 1 and 6 (concentrations = 0.25, 0.5, 1.0 and 2.0 × IC50). (b)
Histograms for the JC-1 Monomers/JC-1 Aggregates (the ratios of green to red fluorescence) treated with complexes 1 and 6. Data are quoted as mean
SD of three
replicates.
2.9. Cell imaging and cellular uptake mechanism
probes were Mito Tracker Deep Red (MTDR) and Lyso Tracker Red
DND-99 (LTRD) [45,45], respectively. As shown in Fig. 8a, complex 6
could effectively target lysosomes with a high Pearson's colocalization
coefficient (PCC) of 0.74. On the other hand, complex 6 could also
target mitochondria with the PCC value of 0.33. Interestingly, IrIII α-PA
Laser confocal microscopy was used to determine the subcellular
localization in A549 cells due to the inherent luminescence property for
IrIII α-PA complex [43]. The mitochondrial and lysosomal fluorescent
Fig. 8. (a) Confocal laser images of A549 cells hatched in complex 6 (15 μM, 1 h) and LTRD (100 nM, 30 min). (b) Confocal laser images of A549 cells hatched in
complex 6 (15 μM, 1 h) and MTDR (50 nM, 30 min). Scale bars: 20 μm.
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H. Hao et al.
JournalofInorganicBiochemistry192(2019)52–61
Fig. 9. Laser confocal microscopy images of A549
cells after hatched in complex 6 and stained by AO.
(a) A549 cells were stained by AO (5 μM). (b) A549
cells were hatched in complex 6 (10 μM) for 1 h and
then stained by AO (5 μM). (c) A549 cells were hat-
ched in complex 6 (10 μM) for 6 h and then stained
by AO (5 μM). λex = 488 nm, λem = 510
30 nm
30 nm (red). Scale bar: 20 μm.
(green) and 625
complex did not cause abnormal cell death immediately, which made it
easy to track the changes in cells in real time.
were synthesized and charactered by 1H NMR, MS and element ana-
lysis. Target complexes showed the potential antitumor activity towards
A549 tumor cells, and the best of which (complex 6) was nearly five
times cis-platin under the same conditions. Complexes could be trans-
Compared with normal cells, most of the lysosomal changes are
caused by tumor suppressor genes in tumor cells [46]. Therefore, re-
searchers have shown a high interest in studying the effects of lyso-
somal destruction on tumor cell death and tumor cell apoptosis me-
chanisms [47,48]. Drug-treated A549 cells were stained by acridine
orange (AO, an effective probe to research lysosomal integrity due to its
characteristics with red fluorescence in the lysosomes and green
fluorescence in the cytoplasm and nucleus.) to test the lysosomal in-
tegrity. As shown in Fig. 9, there was a significant decrease for red
fluorescence in lysosomes when A549 cells hatched in complex 6
(1.0 × IC50) for 1 h, and obvious lysosomal damage was found after 6 h.
The results confirmed that IrIII α-PA complexes could target lysosomes,
induce lysosomal damage, and eventually lead to apoptosis.
ported by serum albumin, catalyze the conversion of NADH to NAD+
.
Complexes could enter tumor cells by the means of energy-dependent
mechanisms, specifically targeted lysosomes and mitochondria, even-
tually leaded to lysosomal damage and the change of mitochondrial
membrane potential, disturbed the cell growth cycle and induced
apoptosis. Above all, half-sandwich IrIII α-PA complexes could be
hopeful for a potential candidate for antitumor drugs.
4. Experimental section
4.1. Materials
Energy demand mechanisms (divided into active transport and en-
docytosis) and non-energy demand mechanisms (divided into passive
transport and free diffusion) were two major cellular uptake mechan-
isms for drugs [48]. Chloroquine and carbonyl cyanide m-chlor-
ophenylhydrazine (CCCP) were selected as the endocytosis inhibitor
and the energy inhibitor to study the uptake mechanism of complexes,
respectively [49]. Cells should be incubated for 2 h at 277 K and 310 K
before pre-treating cells with chloroquine and CCCP. The ingestion ef-
ficiency of A549 tumor cells was lowered at 277 K compared to the cells
at a temperature of 310 K, from which it was seen that the complex 6
was taken up by cells to follow the energy demand mechanism as shown
in Fig. 10.
IrCl3·3H2O, butyllithium solution (1.6 M in hexane), 2,3,4,5-tetra-
methyl-2-cyclopentenone (95%), 4-bromobiphenyl, trimethyl(2,3,4,5-
tetramethyl-2,4-cyclopentadien-1-yl)silane (97%) and different pyr-
idine acid ligands (L1–L8) were purchased from Ruiya Technology
Company Limited. DMEM medium, fetal bovine serum, penicillin/
streptomycin mixture, trypsin/EDTA, and phosphate-buffered saline
(PBS) were purchased from Sangon Biotech. IrIII α-PA complexes was
dissolved in DMSO and diluted with the tissue culture medium before
use.
4.2. Synthesis of [(η5-C5Me4C6H4C6H5)IrCl2]2 (dimer)
3. Conclusions
Dimer was prepared according to literature method [9]. A solution
of Cpxbiph (6.83 g, 25 mmol) and IrCl3·nH2O (7.33 g, 25 mmol) in MeOH
(60 mL) was heated to reflux under N2 atmosphere for 48 h. The dark
In this paper, eight half-sandwich structure IrIII α-PA complexes
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H. Hao et al.
JournalofInorganicBiochemistry192(2019)52–61
Fig. 10. Confocal images of A549 cells after hatched in complex 6 (10 μM) under different conditions. (a) A549 Cells were hatched in complex 6 (10 μM) for 10 min at
277 K. (b) A549 cells were hatched in complex 6 (10 μM) for 10 min at 310 K. (c) A549 cells were hatched in CCCP (10 μM) at 310 K for 1 h and then complex 6
(10 μM) at 310 K for 10 min. (d) A549 cells were hatched in chloroquine (50 μM) for 1 h at 310 K and then complex 6 (10 μM) for 10 min at 310 K (λex = 405 nm,
λem = 590
30 nm). Scale bars: 20 μm.
red product was concentrated to 8 mL on a rotary evaporator. An or-
ange crystal was precipitated at −5 °C. The product was collected by
filtration, washed with dichloromethane and ether and dried in vacuo.
Yield 52.2%. 1H NMR (500 MHz, DMSO) δ 7.70 (qd, J = 8.6, 1.8 Hz,
6H), 7.48 (t, J = 7.7 Hz, 2H), 7.40 (d, J = 7.3 Hz, 1H), 1.72 (d,
J = 14.5 Hz, 12H).
complexes 1–8 are shown in ESI Fig. S2.
[(η5-Cpxbiph)Ir(L1)Cl] (1). Yield: 72.2%. 1H NMR (500 MHz, DMSO)
δ 8.69 (d, J = 5.2 Hz, 1H), 8.17 (td, J = 7.7, 1.3 Hz, 1H), 7.96 (d,
J = 7.7 Hz, 1H), 7.78 (ddd, J = 7.4, 5.5, 1.5 Hz, 1H), 7.73 (d,
J = 8.4 Hz, 4H), 7.63 (d, J = 8.3 Hz, 2H), 7.49 (t, J = 7.7 Hz, 2H), 7.40
(t, J = 7.4 Hz, 1H), 1.75 (d, J = 8.8 Hz, 6H), 1.68 (d, J = 17.7 Hz, 6H).
ESI-MS (m/z): [M-Cl]+ Calcd for C27H25IrNO2, 587.72; Found 587.98.
Elemental Analysis: found: C, 51.99; H, 4.01; N, 2.28%, calcd for
5. Synthesis of complexes 1–8
C
27H25ClIrNO2: C, 52.04; H, 4.04; N, 2.25%.
[(η5-Cpxbiph)Ir(L2)Cl] (2). Yield: 73.3%. 1H NMR (500 MHz, DMSO)
General procedure: Under N2 atmosphere, [(η5-C5Me4C6H4C6H5)
IrCl2]2 (Dimer) (0.05 mmol) and α-PA chelating ligands (L1–L8)
(0.10 mmol) are dissolved in methanol (40 mL) to obtain a mixed so-
lution, which was stirred for 12 h at 37 °C (monitored by thin-layer
chromatography). The solvent was removed on a rotary evaporator and
recrystallized from methanol and ether. The crystalline pellets were
washed with ether and dried. The 1H NMR (500 MHz, DMSO) peak
integrals of complexes 1–8 are shown in ESI Fig. S1. The MS of
δ 8.64 (d, J = 1.9 Hz, 1H), 8.45 (dd, J = 8.3, 2.0 Hz, 1H), 7.90 (d,
J = 8.3 Hz, 1H), 7.74 (t, J = 7.7 Hz, 4H), 7.62 (d, J = 8.3 Hz, 2H), 7.49
(t, J = 7.7 Hz, 2H), 7.40 (t, J = 7.4 Hz, 1H), 1.77 (d, J = 1.7 Hz, 6H),
1.69 (s, 6H). ESI-MS (m/z): [M-Cl]+ Calcd for C27H24BrIrNO2, 665.58;
Found 665.96. Elemental Analysis: found: C, 46.12; H, 3.41; N, 2.07%,
calcd for C27H24BrClIrNO2: C, 46.19; H, 3.45; N, 2.00%.
[(η5-Cpxbiph)Ir(L3)Cl] (3). Yield: 68.2%. 1H NMR (500 MHz, DMSO)
59

H. Hao et al.
JournalofInorganicBiochemistry192(2019)52–61
δ 8.64 (d, J = 6.0 Hz, 1H), 8.00 (d, J = 2.4 Hz, 1H), 7.92 (dd, J = 6.0,
2.5 Hz, 1H), 7.76–7.71 (m, 4H), 7.64 (d, J = 8.3 Hz, 2H), 7.49 (t,
J = 7.7 Hz, 2H), 7.40 (t, J = 7.4 Hz, 1H), 1.75 (d, J = 4.0 Hz, 6H), 1.68
(d, J = 17.7 Hz, 6H). ESI-MS (m/z): [M-Cl]+ Calcd for C27H24ClIrNO2,
622.16; Found 621.98. Elemental Analysis: found: C, 49.35; H, 3.64; N,
2.14%, calcd for C27H24Cl2IrNO2: C, 49.31; H, 3.68; N, 2.13%.
[(η5-Cpxbiph)Ir(L4)Cl] (4). Yield: 61.0%. 1H NMR (500 MHz, DMSO)
δ 14.15 (s, 1H), 8.94 (d, J = 1.2 Hz, 1H), 8.57 (dd, J = 8.0, 1.6 Hz, 1H),
8.06 (d, J = 8.0 Hz, 1H), 7.74 (t, J = 7.4 Hz, 4H), 7.67 (d, J = 8.3 Hz,
2H), 7.49 (t, J = 7.6 Hz, 2H), 7.40 (t, J = 7.4 Hz, 1H), 1.75 (d,
J = 1.2 Hz, 6H), 1.69 (d, J = 2.6 Hz, 6H). ESI-MS (m/z): [M-Cl]+ Calcd
for C28H25IrNO4, 631.73; Found 631.96. Elemental Analysis: found: C,
50.38; H, 3.78; N, 2.12%, calcd for C28H25ClIrNO4: C, 50.41; H, 3.78; N,
2.10%.
Acknowledgments
We thank the National Natural Science Foundation of China (Grant
No. 21671118), the Taishan Scholars Program, the University Research
Development Program of Shandong Province (J18KA082), and the
Student's Platform for Innovation and Entrepreneurship Training
Program of Shandong Province (201710446042) for support.
Notes
The authors declare no competing financial interest.
Appendix A. Supplementary data
[(η5-Cpxbiph)Ir(L5)Cl] (5). Yield: 78.8%. 1H NMR (500 MHz, DMSO)
δ 8.55 (s, 1H), 7.96 (s, 1H), 7.72 (s, 4H), 7.65 (s, 3H), 7.49 (s, 2H), 7.40
(s, 1H), 2.63 (s, 3H), 1.73 (d, J = 7.8 Hz, 6H), 1.66 (d, J = 18.6 Hz,
6H). ESI-MS (m/z): [M-Cl]+ Calcd for C28H27IrNO2, 601.75; Found
602.03. Elemental Analysis: found: C, 52.76; H, 4.24; N, 2.25%, calcd
for C28H27ClIrNO2: C, 52.78; H, 4.27; N, 2.20%.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jinorgbio.2018.12.012.
References
[1] S. Mukhopadhyay, R.K. Gupta, R.P. Paitandi, N.K. Rana, G. Sharma, B. Koch,
L.K. Rana, M.S. Hundal, D.S. Pandey, Organometallics 34 (2015) 100–107.
[2] R.R. Barefoot, J. Chromatogr. B Biomed. Sci. Appl. 751 (2001) 205–211.
[3] E. Wong, C.M. Giandomenico, Chem. Rev. 99 (1999) 2451–2466.
[4] M. Callari, J.R. Aldrich-Wright, P.L.D. Souza, M.H. Stenzel, Prog. Polym. Sci. 39
(2014) 1614–1643.
[(η5-Cpxbiph)Ir(L6)Cl] (6). Yield: 72.5%. 1H NMR (500 MHz, DMSO)
δ 8.70 (s, 1H), 8.65 (dd, J = 8.2, 1.5 Hz, 1H), 8.18 (d, J = 8.1 Hz, 1H),
7.77–7.71 (m, 4H), 7.65 (d, J = 8.3 Hz, 2H), 7.50 (t, J = 7.6 Hz, 2H),
7.41 (t, J = 7.3 Hz, 1H), 1.78 (s, 6H), 1.71 (d, J = 14.4 Hz, 6H). ESI-MS
(m/z): [M-Cl]+ Calcd for C28H24F3IrNO2, 655.72; Found 656.02.
Elemental Analysis: found: C, 48.72; H, 3.48; N, 2.01%, calcd for
C28H24ClF3IrNO2: C, 48.66; H, 3.50; N, 2.03%.
[5] X.C. Liu, Y.H. Tang, X.D. He, X.X. Ge, J. Liu, X.Y. Meng, M.X. Shao, Y.M. Jin,
L.J. Tian, Z. Liu, J. Inorg. Biochem. 191 (2019) 194–202.
[6] V. Novohradsky, L. Zerzankova, J. Stepankova, A. Kisova, H. Kostrhunova, Z. Liu,
P.J. Sadler, J. Kasparkova, V. Brabec, Metallomics 6 (2014) 1491–1501.
[7] X. He, M. Tian, X. Liu, Y. Tang, C. Shao, P. Gong, J. Liu, S. Zhang, L. Guo, Z. Liu,
Chem. Asian J. 13 (2018) 1500–1509.
[(η5-Cpxbiph)Ir(L7)Cl] (7). Yield: 77.3%. 1H NMR (500 MHz, DMSO)
δ 12.83 (s, 1H), 8.22 (dd, J = 3.6, 2.7 Hz, 1H), 7.75 (dd, J = 8.4,
6.7 Hz, 4H), 7.69–7.63 (m, 4H), 7.49 (t, J = 7.6 Hz, 2H), 7.40 (t,
J = 6.8 Hz, 1H), 1.74 (d, J = 2.1 Hz, 6H), 1.68 (d, J = 15.6 Hz, 6H).
ESI-MS (m/z): [M-Cl]+ Calcd for C27H25IrNO3, 603.72; Found 603.99.
Elemental Analysis: found: C, 50.72; H, 3.91; N, 2.23%, calcd for
[8] X. He, X. Liu, Y. Tang, J. Du, M. Tian, Z. Xu, X. Liu, Z. Liu, Dyes Pigments 160
(2019) 217–226.
[9] Z. Liu, A. Habtemariam, A.M. Pizarro, S.A. Fletcher, A. Kisova, O. Vrana, L. Salassa,
P.C. Bruijnincx, G.J. Clarkson, V. Brabec, J. Med. Chem. 54 (2011) 3011–3026.
[10] Z. Liu, I. Romerocanelón, B. Qamar, J.M. Hearn, A. Habtemariam, N.P. Barry,
A.M. Pizarro, G.J. Clarkson, Angew. Chem. Int. Ed. 53 (2014) 3941–3946.
[11] Z. Xu, D. Kong, X. He, L. Guo, X. Ge, X. Liu, H. Zhang, J. Li, Y. Yang, Z. Liu, Inorg.
Chem. Front. 5 (2018) 2100–2105.
C
27H25ClIrNO3: C, 50.74; H, 3.94; N, 2.19%.
[(η5-Cpxbiph)Ir(L8)Cl] (8). Yield: 66.4%. 1H NMR (500 MHz, DMSO)
[12] H. Imam, B. Kumar, Shafayat, Orient. J. Chem. 27 (2011) 287–291.
[13] S.M. Shohayeb, R.G. Mohamed, H. Moustafa, S.M. El-Medani, J. Mol. Struct. 1119
(2016) 442–450.
δ 11.30 (s, 1H), 8.14 (d, J = 2.5 Hz, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.74
(d, J = 8.4 Hz, 4H), 7.64 (d, J = 8.3 Hz, 2H), 7.48 (dd, J = 12.7,
4.8 Hz, 3H), 7.40 (t, J = 7.4 Hz, 1H), 1.73 (s, 6H), 1.66 (d, J = 15.3 Hz,
6H). ESI-MS (m/z): [M-Cl]+ Calcd for C28H29IrNO3, 603.72; Found
604.00. Elemental Analysis: found: C, 50.71; H, 3.94; N, 2.20%, calcd
for C27H25ClIrNO3: C, 50.74; H, 3.94; N, 2.19%.
[14] M.A. Yurovskaya, O.D. Mit'Kin, F.V. Zaitseva, Review, Chem. Heterocycl. Compd.
34 (1998) 871–899.
[15] A. Salvini, P. Frediani, E. Rivalta, Inorg. Chim. Acta 351 (2003) 225–234.
[16] R.I. Kureshy, N.H. Khan, S.H.R. Abdi, S.T. Patel, P. Iyer, J. Mol. Catal. A Chem. 150
(1999) 163–173.
[17] R. Ramesh, Inorg. Chem. Commun. 7 (2004) 274–276.
[18] P. Viswanathamurthi, K. Natarajan, Transit. Met. Chem. 24 (1999) 638–641.
[19] D. Xu, Y. Xu, N. Cheng, X. Zhou, Y. Shi, Q. He, J. Coord. Chem. 63 (2010)
2360–2369.
Abbreviations
[20] Y. Li, B. Liu, X.R. Lu, M.F. Li, L.N. Ji, Z.W. Mao, Dalton Trans. 46 (2017)
11363–11371.
IrIII
iridiumIII
[21] R.R. Ye, C.P. Tan, M.H. Chen, L. Hao, L.N. Ji, Z.W. Mao, Chem. Eur. J. 22 (2016)
7800–7809.
α-PA
HSA
BSA
Ksv
α-picolinic acid
human serum albumin
bovine serum albumin
Stern–Volmer quenching constant
quenching rate constant
binding constant
[22] J. Adler, I. Parmryd, Cytometry A 77 (2010) 733–742.
[23] V. Thamilarasan, P. Karunakaran, N. Kavitha, C. Selvaraju, N. Sengottuvelan,
Polyhedron 118 (2016) 12–24.
[24] B.P. Espósito, R. Najjar, Coord. Chem. Rev. 232 (2002) 137–149.
[25] T. Keleş, B. Barut, Z. Biyiklioglu, A. Özel, Dyes Pigments 139 (2017) 575–586.
[26] J. Li, L. Guo, Z. Tian, M. Tian, S. Zhang, K. Xu, Y. Qian, Z. Liu, Dalton Trans. 46
(2017) 15520–15534.
Kq
Kb
NADH
nicotinamide adenine dinucleotide
[27] R. Esteghamat-Panah, H. Hadadzadeh, H. Farrokhpour, J. Simpson, A. Abdolmaleki,
F. Abyar, Eur. J. Med. Chem. 127 (2016) 958–971.
ICP-MS inductively coupled plasma mass spectrometry
logP
partition coefficient in oil/water
[28] D. Li, M. Zhu, C. Xu, B. Ji, Eur. J. Med. Chem. 46 (2011) 588–599.
[29] S. Tabassum, R. Singh, M. Zaki, M. Ahmad, M. Afzal, RSC Adv. 5 (2015)
35843–35851.
UV–Vis ultraviolet-visible spectrum
TONs
MTT
turn over numbers
[30] X.F. Zhao, Y. Ouyang, Y.Z. Liu, Q.J. Su, H. Tian, C.Z. Xie, J.Y. Xu, New J. Chem. 38
(2014) 955–965.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
mide
bro-
[31] T.S. Kamatchi, P. Kalaivani, P. Poornima, V.V. Padma, F.R. Fronczek, K. Natarajan,
RSC Adv. 4 (2013) 2004–2022.
ROS
reactive oxygen species
[32] N.K. Das, L. Pawar, N. Kumar, S. Mukherjee, Chem. Phys. Lett. 635 (2015) 50–55.
[33] T. Mukherjee, M. Mukherjee, B. Sen, S. Banerjee, G. Hundal, P. Chattopadhyay, J.
Coord. Chem. 67 (2014) 2643–2660.
ΔΨm
PCC
mitochondrial membrane potential
Pearson's colocalization coefficient
Mito Tracker Deep Red
[34] S.A.M. Sukri, L.Y. Heng, N.H.A. Karim, J. Fluoresc. 27 (2017) 1009–1023.
[35] P.K. CUmadevi, H. alaivani, S. Puschmann, P.S. Murugan, R. Mohan,
J. Prabhakaran, Photochem. Photobiol. B 167 (2017) 45–57.
[36] Q.L. Zhang, J.G. Liu, H. Chao, G.Q. Xue, L.N. Ji, J. Inorg. Biochem. 83 (2001)
49–55.
MTDR
LTRD
CCCP
AO
Lyso Tracker Red DND - 99
m-chlorophenylhydrazone
acridine orange
[37] Z. Liu, R.J. Deeth, J.S. Butler, A. Habtemariam, M.E. Newton, P.J. Sadler, Angew.
60

H. Hao et al.
JournalofInorganicBiochemistry192(2019)52–61
Chem. Int. Ed. 125 (2013) 4288–4291.
[44] J. Li, L. Guo, Z. Tian, S. Zhang, Z. Xu, Y. Han, R. Li, Y. Li, Z. Liu, Inorg. Chem. 57
[38] B.L. Soledad, L. Zhe, H. Abraha, A.M. Pizarro, Q. Bushra, P.J. Sadler, Angew. Chem.
Int. Ed. 124 (2012) 3963–3966.
(2018) 13552–13563.
[45] Y. Han, Z. Tian, S. Zhang, X. Liu, J. Li, Y. Li, Y. Liu, M. Gao, Z. Liu, J. Inorg.
[39] W. Ma, X. Ge, L. Guo, S. Zhang, J. Li, X. He, Z. Liu, Dyes Pigments 162 (2019)
385–393.
Biochem. 189 (2018) 163–171.
[46] J. Wang, Y. Yu, K. Lu, M. Yang, Y. Li, X. Zhou, Z. Sun, Int. J. Nanomedicine 12
[40] J. Li, Z. Tian, Z. Xu, S. Zhang, Y. Feng, L. Zhang, Z. Liu, Dalton Trans. 47 (2018)
15772–15782.
(2017) 809–825.
[47] Y. Zheng, L. He, D. Zhang, C. Tan, L. Ji, Z. Mao, Dalton Trans. 46 (2017)
[41] M. Tian, J. Li, S. Zhang, L. Guo, X. He, D. Kong, H. Zhang, Z. Liu, Chem. Commun.
53 (2017) 12810–12813.
11395–11407.
[48] C. Jin, J. Liu, Y. Chen, R. Guan, O. Cheng, Y. Zhu, L. Ji, H. Chao, Sci. Rep. 6 (2016)
[42] A. Karabatsiakis, C. Böck, J. Salinas-Manrique, S. Kolassa, E. Calzia, D.E. Dietrich,
I.T. Kolassa, Transl. Psychiatry 4 (2014) 397.
22039.
[49] Y. Han, X. Liu, Z. Tian, X. Ge, J. Li, M. Gao, Y. Li, Y. Liu, Z. Liu, Chem. Asian J 13
(2018) 3697–3705.
[43] Z. Tian, J. Li, S. Zhang, Y. Yang, Z. Xu, D. Kong, H. Zhang, X. Ge, J. Zhang, Z. Liu,
Inorg. Chem. 57 (2018) 10498–10502.
61