Preparation and Bioactivity of Iridium(III) Phenanthroline Complexes with Halide Ions and Pyridine Leaving Groups

Article abstract of DOI:10.1002/cbic.202000511

A series of half-sandwich structural iridium(III) phenanthroline (Phen) complexes with halide ions (Cl^?, Br^?, I^?) and pyridine leaving groups ([(η⁵-Cp^X)Ir(Phen)Z](PF₆)_n, Cp^x: electron-rich cyclopentadienyl group, Z: leaving group) have been prepared. Target complexes, especially the Cp^xbiph (biphenyl-substituted cyclopentadienyl)-based one, showed favourable anticancer activity against human lung cancer (A549) cells; the best one (Ir8) was almost five times that of cisplatin under the same conditions. Compared with complexes involving halide ion leaving groups, the pyridine-based one did not display hydrolysis but effectively caused lysosomal damage, leading to accumulation in the cytosol, inducing an increase in the level of intracellular reactive oxygen species and apoptosis; this indicated an anticancer mechanism of oxidation. Additionally, these complexes could bind to serum albumin through a static quenching mechanism. The data highlight the potential value of half-sandwich iridium(III) phenanthroline complexes as anticancer drugs.

Full text of DOI:10.1002/cbic.202000511

Combining Chemistry and Biology
Accepted Article
Title: Preparation and bioactivity of iridium(III) phenanthroline
complexes with halide ions and pyridine leaving groups
Authors: Xicheng Liu, Mingxiao Shao, Congcong Liang, Jinghang Guo,
Guangxuan Wang, Xiang-Ai Yuan, Zhihong Jing, Laijin Tian,
and Zhe Liu
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202000511
Link to VoR: https://doi.org/10.1002/cbic.202000511
01/2020

10.1002/cbic.202000511
ChemBioChem
FULL PAPER
Preparation and bioactivity of iridium(III) phenanthroline
complexes with halide ions and pyridine leaving groups
Dr. Xicheng Liu*[a], Mingxiao Shao[a], Congcong Liang[a], Jinghang Guo[a], Guangxuan Wang[a], Xiang-Ai
Yuan[a], Zhihong Jing[a], Laijin Tian[a], Prof. Dr. Zhe Liu*[a]
[a]
Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of Life-Organic Analysis and Key Laboratory of Phar
maceutical Intermediates and Analysis of Natural Medicine, School of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China.
Corresponding author (X. Liu) E-mail: chemlxc@163.com; Fax: 05374456301. (Z. Liu) E-mail: liuzheqd@163.com; Fax: 05374456301.
Supporting information for this article is given via a link at the end of the document.
Abstract:
A
series of half-sandwich structural iridium(III)
bidentate ligand played a key role in the activity of these
complexes. Bidentate ligands with different coordination modes,
including N^N,[7] C^N,[8] C^C,[9] N^O,[10] N^S,[11] S^S,[12] O^O[13]
etc., had been introduced into such complexes, which could
induce a much higher intracellular accumulation of Ir and
contribute to the cytotoxicity of these complexes. Meanwhile, the
phenanthroline (Phen) complexes with halide ions (Cl−, Br−, I−) and
pyridine leaving groups ([(η5-CpX)Ir(Phen)Z](PF6)n, Cpx: electron-rich
cyclopentadienyl group, Z: leaving group) were prepared. Target
complexes, especially Cpxbiph (biphenyl-substituted cyclopentadienyl)-
based one, showed favourable anticancer activity against human lung
cancer (A549) cells, and the best of which (Ir8) was almost five times
that of cisplatin under the same conditions. Compared with complexes
involving halide ion leaving groups, the pyridine-based one did not
display hydrolysis but effectively caused lysosomal damage, leading
to accumulation in the cytosol, inducing an increase in the level of
intracellular reactive oxygen species and apoptosis, indicating an
anticancer mechanism of oxidation. Additionally, these complexes
could bind to serum albumin via a static quenching mechanism. The
data highlight the potential value of half-sandwich iridium(III)
phenanthroline complexes as anticancer drugs.
mode of connection between the substituent and the bidentate
[14]
ligand (trans- and cis-double bonds)
or the position of the
substituents [15] could effectively affect the anticancer activity of
these complexes. Additionally, recent studies also revealed that
the type and size of counter-ion (X) displayed a greater effect on
the anticancer activity of these complexes.[16]
Hydrolysis is often considered as an important activation step
for transition metal complexes.[17] In general, half-sandwich IrIII
complexes with chloride ion (Cl−) leaving group can rapidly
hydrolyse in an aqueous solution to form Ir−OH2 complexes that
may have pronounced reactivity. However, the rapid hydrolysis
and strong binding to biomolecules may account for the low
cytotoxicity of half-sandwich IrIII complexes.[6] Therefore, the
effects of different leaving groups on the hydrolysis and toxicity of
half-sandwich IrIII complexes are significant. Hence, in this study,
a series of half-sandwich IrIII phenanthroline complexes with
halide ions (Cl−, Br−, I−) and pyridine leaving groups were
prepared (Figure 1). The bioactivity towards A549 human lung
cancer cells was established using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) based assay. These
complexes displayed favourable anticancer activity. The
relationship between the anticancer activity and the hydrolysis of
the complexes was analysed via ultraviolet-visible (UV-vis)
spectroscopy and quantitative calculation. The anticancer
mechanism was further analysed using flow cytometry. The data
provide a foundation for its further evaluations.
Introduction
In-depth research on the mechanism of tumour formation has
revealed the potentially valuable role of chemical drugs in tumour
therapy.[1] The rapid development of platinum-based drugs has
exemplified the medicinal value and anticancer potential of metal
complexes.[2] Despite their excellent benefits, these anticancer
drugs have severe side effects, such as strong resistance, serious
adverse effects, and poor selectivity. These detriments have
prompted searches for alternative anticancer drugs with greater
efficiency and different anticancer mechanisms.[3] Half-sandwich
iridium(III) (IrIII) complexes have drawn particular attention owing
to their particular anticancer mechanisms, which include
catalysing the intracellular reduction–oxidation (REDOX) reaction
and inhibition of proteins (VEGF R3 and Bcl-2, etc.), which can
lead to the accumulation of intracellular reactive oxygen species
(ROS, 1O2).[4]
The general formula of half-sandwich IrIII complexes is
usually expressed as [(η5-Cpx)Ir(L^L)Z]X, where Cpx represents
electron-rich cyclopentadienyl group, L^L is bidentate ligand, Z is
leaving group, and X is counter-ion (usually hexafluorophosphate).
Interestingly, the chemical characteristics and bioactivity of these
complexes could be effectively regulated and controled through
fine-tuning the steric and electronic properties of ligands.[5]
Among them, the introduction of phenyl or biphenyl units on Cp to
replace methyl group, could effectively change the hydrolysis rate
and extent of IrIII complexes.[6] It was also established that the L^L
Figure 1. Structures of half-sandwich IrIII phenanthroline complexes.
1
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10.1002/cbic.202000511
ChemBioChem
FULL PAPER
Results and Discussion
in lipid solubility owing to the introduction of biphenyl and pyridine
was beneficial for improving the antitcancer activity of these
complexes.[18] In addition, the cytotoxicity of Ir5 and Ir8 toward
human lung epithelial cells (BEAS-2B) was further assessed
using the MTT assay, and the IC50 values were 21.1±0.6 and
6.3±0.8 μM, respectively. These results were not optimistic, and
no significant selectivity for tumour cells was evident. However,
the evidence of favourable anticancer activity should prompt
further design refinements and optimisation of such complexes.
Half-sandwich IrIII phenanthroline (Phen) complexes (Ir1 and Ir5;
Cl− is the leaving group) were synthesised by the reaction of [(η5-
Cp*)IrCl2]2 (Dimer 1) or [(η5-Cpxbiph)IrCl2]2 (Dimer 2) with
phenanthroline (N^N bidentate ligand) at the ambient temperature.
The complexes were further reacted with sodium bromide/sodium
iodide/pyridine (Py) to obtain other complexes with Br−, I−, and Py
leaving groups, Scheme 1. Ir1–Ir8 were isolated as
hexafluorophosphates. The composition and structures of the
complexes were characterised via nuclear magnetic resonance
spectroscopy (1H NMR and 13C NMR, Figures S1 and S2),
electrospray ionization mass spectrometry (ESI-MS, Figure S3),
and elemental analysis. Hydrogen atoms of the Cpx ring ranged
from 1.6 to 2.0 ppm, and those on the benzene and
phenanthroline units were shown in the range of 7.0 to 8.0 ppm
and 8.0 to 10.0 ppm, respectively, in 1H NMR spectra. The carbon
atoms of the methyl and Cpx groups ranged from 8.1 to 10.9 ppm
and from 78.5 to 98.8 ppm, respectively, in 13C NMR spectra. The
carbon atoms of phenanthroline and pyridine ranged from 124.5
to 153.4 ppm. The ESI-MS results were consistent with the
theoretically calculated values (loss of PF6− or Py).
Table 1. IC50 values of [(η5-Cpx)Ir(Phen)Z](PF6)n, cisplatin and [(η5-Cpx)IrCl2]2
(Dimer) for 24 h.
IC50 (μM)
Complexes
Cpx
X
n
A549 cell
＞100
BEAS-2B cell
Ir1
Ir2
Ir3
Cp*
Cp*
Cp*
Cl
Br
I
1
1
1
/
/
/
＞100
＞100
Ir4
Ir5
Cp*
Py
Cl
Br
I
2
1
1
1
2
/
＞100
/
Cpxbiph
Cpxbiph
Cpxbiph
Cpxbiph
/
11.1 ± 1.2
16.9 ± 0.2
25.3 ± 0.1
4.4 ± 1.2
21.3 ± 1.7
＞100
21.1 ± 0.6
Ir6
/
Ir7
/
Ir8
Py
/
6.3 ± 0.8
Cisplatin
38.4 ± 2.8
Cp*
/
/
Dimer
/
/
Cpxbiph
57.7 ± 0.8
2. Hydrolysis assay
Hydrolysis of Ir–Z (Z is the leaving group in half-sandwich IrIII
complexes) bond represents an activation step for half-sandwich
organometallic anticancer complexes. M–OH2 aqua complexes
are often more reactive than the corresponding complexes.[19] The
hydrolysis characteristics of Ir1, Ir5 and Ir8 in 20% DMSO/80%
H2O (v/v) solution at 298 K over 8 h were measured by UV-vis
absorption spectra (Figures 2 and S6), and the presence of
DMSO ensured the solubility of these complexes. As shown, the
UV-vis spectroscopy of Ir8 displayed almost no change over time,
which indicated the stability of this complex. Time dependence of
the formation of the aqua adducts of Ir1 and Ir5 was fitted to the
pseudo first-order kinetics (Figure S7). The values of hydrolysis
rate constants were 0.00284 min-1 and 0.00186 min-1, and the
half-lives of hydrolysis were 244.07 min and 372.66 min for Ir1
and Ir5, respectively, the hydrolytic half-life of Ir1 was slightly
faster than that of Ir5.
Scheme 1. Synthesis process of IrIII phenanthroline complexes with different
leaving groups.
1. Bioactivity assay
In comparison with well-developed catalytic molecules, Ir-based
pharmaceuticals are still in their infancy. So far, organometallic IrIII
complexes have shown the potential for use in the field of
anticancer therapy[5]. In this study, the anticancer activities of
these complexes were evaluated using the MTT assay in A549
human lung cancer cells (a leading cause of death both in
developing and developed countries), and the dose response
curves of Ir1–Ir8 were shown in Figure S4. Cisplatin (widely used
in clinical practice) was used as a control, and the IC50 values (the
concentration at which 50% cell growth was inhibited) were listed
in Table 1 after incubated in IrIII Phen complexes or cisplatin for
24 h. As shown, compared with the corresponding dimer of iridium
(Dimer 1, Scheme 1), the anticancer activity of the Cp*-based
To further understand the differences in hydrolysis between
Ir1 and Ir5, the Wiberg bond order of Ir–Cl bond was ascertained
by density functional theory (DFT) calculation at the B3LYP-
D3(BJ)/6-31G(d,p) (C, H, N, Cl)/LANL2DZ (SMD) (Ir) level with
the values of 0.6385 and 0.6632 for Ir1 and Ir5, respectively.[21]
Addionally, the values of natural population analysis (NPA)
charge population for the central iridium ions of Ir1 and Ir5 were
0.308 and 0.314. Obviously, the bigger angles between
cyclopentadienyl and diphenyl (the inserts of Figure S8) damaged
the conjugation of η5-Cpxbiph tridentate ligand and decreased the
electron donor capacity, then increased the formal charge of the
central iridium ion and Wiberg bond order of Ir–Cl bond to some
extent. The higher charge of central iridum ion and the stronger
bond order of Ir–Cl (Ir5) contributed to the weaker hydrolysis than
complexes (Ir1-Ir4) was not improved. However, Ir5−Ir8 (Cpxbiph
-
based one) displayed the better anticancer activity than Dimer 2
(Figure S5). The best of these complexes (Ir8) was almost 5 times
that of cisplatin under the same conditions, which was even better
than the common half-sandwich IrIII bipyridine[4d] and
phenylpyridine[14] complexes. The values of logP (the oil/water
partition coefficient) were −0.68, 0.97 and 1.32 for Ir1, Ir5 and Ir8,
respectively. The findings indicated that an appropriate increase
2
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10.1002/cbic.202000511
ChemBioChem
FULL PAPER
that of Ir1. However, Ir5, η5-Cpxbiph-based complex, displayed the
better anticancer activity than Ir1, which indicated that hydrolysis
was not the right decisive factor affecting the anticancer activity
for half-sandwich IrIII Phen complexes.[6]
Figure 2. UV-vis spectra of Ir1 and Ir5 in 20% DMSO/80% H2O (v/v) solution at
298 K over 8 h. The arrows show the change over time.
Figure 3. Apoptosis assay of A549 cells after 24 h exposure to Ir8 determined
by flow cytometry after Annexin V-FITC/PI staining and histograms of late
apoptosis for A549 cells induced by Ir8. Data were averaged for three replicate
experiments ± SD. p values were calculated after a t test against the control
data: *, p < 0.05; **, p < 0.01.
3. Anticancer mechanism assay
Apoptosis, a major form of cell death controlled by genes,
plays a crucial role in the growth and development of healthy and
cancerous cells. Organometallic anticancer complexes, e.g.
Ru(II), Re(I), Os(II), Fe(II) etc., can clear cancer cells through
inducing apoptosis.[21] In this study, A549 cells (stained by
annexin V-FITC/PI) were incubated with Ir5 and Ir8 at the
concentration of 0.5×IC50, 1.0×IC50, 2.0×IC50 and 3.0×IC50 for 24
h. The samples were analysed using flow cytometry to determine
whether IrIII Phen complexes could induce apoptosis. As shown in
Figures 3 and S9, the percentage of apoptotic cells showed a
dose-dependent increase after the 24 h incubation. When the
concentration changed from 0.5×IC50 to 3.0×IC50, the percentage
of the apoptotic phase (including early apoptosis and late
apoptosis) increased by 28.25% and 25.85% for Ir5 and Ir8,
respectively. In comparison, the survival exceeded 95% in the
control. This findings indicated that these complexes could induce
cell death through apoptosis, especially late apoptosis. Generally,
IrIII complexes can induce a functional decline through disrupting
the cell cycle, lead to apoptosis and display their anticancer
effects.[22] To investigate the impact of these complexes on cell
cycle arrest, A549 cells (stained by PI) were incubated with Ir5
and Ir8 for 24 h at the concentration of 0.25×IC50, 0.5×IC50,
1.0×IC50, 2.0×IC50 and determined by flow cytometry (Figure S10
and Tables S1-S2). As shown, there was almost no change
happened to A549 cells at G0/G1 phase, S phase and G2/M phase
after exposed to Ir5 and Ir8 with the concentration changed from
0.25×IC50 to 2.0×IC50. This finding indicated that half-sandwich IrIII
Phen complexes did not disturb the cell growth cycle progression
and obtain the purpose of apoptosis.
usually results from an increase in intracellular ROS levels[24], was
determined by assessing the change in JC-1 (a frequently-used
fluorescent probe to assess ΔΨm) after the A549 cells were
incubated with Ir5 and Ir8 and subsequently stained. As shown in
Figures 4 and S12, an obvious dose-dependent increase from
7.82% (control) to 60.81% for Ir5 and 71.50% (2.0 × IC50) for Ir8
was observed at the specified concentrations. These findings
further confirmed that half-sandwich IrIII phenanthroline
complexes could induce a decrease in ΔΨm, which contributed to
the increase in the intracellular ROS and led to apoptosis.
Reactive oxygen species (ROS, 1O2), mainly originating from
mitochondria, are well known activators of apoptosis.[23]
Intracellular ROS levels of A549 cells were monitored by flow
cytometry utilizing oxidant-sensitive DCFH-DA (2,7-dichlorodi-
hydrofluorescein diacetate) as a fluorescent probe at the
concentration of 0.25×IC50 and 0.5×IC50 (Ir5 and Ir8) after 24 h
incubation. As shown in Figure S11, the intracellular ROS levels
display a dose-dependent increase with an increase in the
concentrations of Ir5 and Ir8. Compared with the negative control,
the ROS levels increased by 1.45- and 1.67-fold for Ir5 and Ir8,
respectively, at the concentration of 0.5×IC50. Additionally, the
change in the mitochondrial membrane potential (ΔΨm), which
Figure 4. Changes of mitochondrial membrane potential (ΔΨm) for A549 cells
induced by Ir5 and Ir8 (concentrations: 0.25×IC50, 0.5×IC50, 1.0×IC50 and
2.0×IC50).
The reduced state of nicotinamide adenine dinucleotide
(NADH), existing extensively in the cytoplasm, is a control marker
in the energy production chain of organisms.[25] The oxidation of
NADH induced by organometallic complexes, including Ir(III),
Ru(II), Rh(III) and Os(II) complexes, can lead to the accumulation
of intracellular ROS and provide an oxidation-based anticancer
mechanism.[26] In this study, UV-vis spectra were utilized to
3
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10.1002/cbic.202000511
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ascertain the interaction between these complexes and NADH in
10% CH3OH/90% H2O (v/v) at 298 K over time, Figure 5. As
shown, the absorbance at 339 nm (which is the maximum
absorbance of NADH) was significantly reduced with time after
incubation with Ir5 and Ir8, and this reduction was accompanied
by increased absorption at 259 nm (the maximum absorbance of
NAD+, oxidation state of NADH).[19] These changes can be well
reflected through the turnover numbers (TONs). Ir8 displayed
slightly higher TON than Ir5. This result indicated that Ir8 had a
better biocatalytic function. The finding was consistent with the
results of the MTT assay, in which the IrIII complexes with greater
TONs displayed more effective anticancer activity. Above all, the
widespread apoptosis induced by the IrIII Phen complexes was
further demonstrated by the increase in intracellular ROS levels
due to the decrease in ΔΨm and the catalytic acceleration to the
oxidation of NADH, which displayed an oxidation-based
anticancer mechanism.
Figure 6. Laser confocal microscopy images of A549 cells after hatched in Ir8
(0.5 × IC50) for 1 h and 6 h, and then stained by AO. λex = 488 nm, λem = 510 ±
30 nm (green) and 625 ± 30 nm (red). Scale bar: 20 μm.
5. Protein binding assay
Serum albumin (SA), the main protein component of serum total
protein, plays an important role in maintaining the blood osmotic
pressure, transporting metabolic substances in body, and
carrying out drug delivery in body fluids.[30] The interaction of
drugs and SA may influence the biodistribution of drugs in the
body, then affect the toxicity of drugs. Because of numerous
advantages, including low cost, abundance, similar structure and
properties to human serum albumin (HSA), bovine serum albumin
(BSA) was generally utilized as a model to study the mechanism
of protein binding.[31] The UV-vis spectroscopy of BSA, before and
after the addition of Ir5 and Ir8 in trishydroxymethyl
aminomethane-hydrochloric acid buffered solution (Tris-HCl, pH
= 7.2), were shown in the Figures 7a and S13a. With the increase
of Ir5 and Ir8, a significant reduction was found at 227 nm (the
UV-vis absorption of BSA) along with a red shift phenomenon (~5
nm, induced by the polar solvent), which indicated that these
compounds could change the conformation of BSA, reduce the
content of the α-helix structure and loosen the conformation of the
protein.[32] Additionally, the absorption at 278 nm displayed minor
increase, which reflected the presence of three major amino acid
residues (tryptophan, tyrosine, and phenylalanine) in BSA.[33]
Among these, tyrosine and tryptophan residues can be reflected
by the wavelength interval of Δλ = 15 nm and 60 nm through
synchronous fluorescence, respectively.[34] As shown in Figures
7c, 7d, S13c and S13d, the fluorescence intensity decreased
significantly with the increase of Ir5 and Ir8, a minor red shift (2
nm and 3 nm for Ir5 and Ir8, respectively) was found at Δλ = 15
nm, however, no changes occured at the wavelength of Δλ = 60
nm. These findings indicated that the changes of the conformation
of BSA, and the polarity around the tyrosine residues is
increased.[35]
Figure 5. UV-vis spectra of NADH (100 µM) were determined after incubated
with Ir5 and Ir8 (1 µM) in CH3OH/H2O= 10%/90% (v:v) at 298 K for 8 h. The
arrows showed the absorbance change over time.
4. Cellular Localization assay
The intracellular Ir8 contents (5 μM) in the cytosol, nucleus,
nuclear chromatin and cytoskeleton fractions isolated from A549
cells were determined by using inductively coupled plasma mass
spectrometry (ICP-MS) after 24 h of exposure to determine the
cellular localisation. Ir8 accumulated mainly in the cytosol
(0.38±0.11 ng/106 cells), and the values for nucleus, nuclear
chromatin and cytoskeleton fractions were 0.10±0.09, 0.05±0.07
and 0.04±0.05 ng/106 cells, respectively. Lysosomes (pH:
4.5~5.5), containing more than 60 acid hydrolases, could
decompose biological macromolecules and involved in many
important physiological processes, including cell metabolism,
migration and apoptosis etc.[27] Research showed that IrIII
complexes could effectively accumulate in lysosome and lead to
partial lysosomal membrane permeabilization (LMP)[28]. And
widespread LMP would lead to the hydrolase in lysosome
entering cytoplasm and induce cell apoptosis. Acridine orange
The interaction between these complexes and BSA was
further investigated by the fluorescence quenching of BSA. As
shown in Figures 7b and S13b, a significant quenching at 351 nm
(the fluorescent emission peak of BSA) was found with the
increase of Ir5 and Ir8. The binding constant (Kb), the binding
number (n), the Stern–Volmer quenching constant (Ksv) and the
quenching rate constant (Kq) were calculated by the classical
Scatchard equation[36] and Stern-Volmer equation[37], Figure S14.
As shown in Table 2, the values of Kq were 1.426×1014 M-1 s-1 and
5.335×1013 M-1 s-1 for Ir5 and Ir8, respectively. The values were
obviously higher than that of a pure dynamic quenching
mechanism (<2.0×1012 M-1 s-1),[38] which indicated that static
quenching mechanism was the main way for the IrIII Phen
complexes binding to BSA. The values of n were almost the same
(~1), which was consistent with the results that the binding
(AO),
a
probe with red/green fluorescence in the
lysosome/cytoplasm or nucleus, was used to ascertain the
lysosomal integrity of tumor cells.[29] A549 cells were incubated
with Ir8 over 1 h and 6 h, then stained with AO (5 μM, 15 min). As
shown in Figure 6, a significant decrease in red fluorescence was
observed after the A549 cells were incubated with Ir8 (0.5×IC50)
for 6 h. This finding indicated that the IrIII Phen complexes could
cause lysosomal damage, leading to accumulation in the cytosol,
action on NADH and mitochondria, increased intracellular ROS,
and eventually the induction of apoptosis.
4
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10.1002/cbic.202000511
ChemBioChem
FULL PAPER
between BSA and Ir1-Ir6 can affect the microregion of tyrosine in
BSA. Meanwhile, Ir5 displayed a larger binding constant (Kb)
value and binding site number (n) than Ir8. This finding indicated
that half-sandwich IrIII Phen complexes displayed more excellent
hydrolysis properties and possessed superior serum protein
binding properties, which would be more readily transported
through plasma.
ions and pyridine leaving groups provide the foundation for the
further design of organometallic anticancer drugs.
Experimental Section
1. General information
Iridium trichloride hydrated, 1,2,3,4,5-pentamethylcyclopentadiene (95%),
2,3,4,5-tetramethyl-2-cyclopentenone
(95%),
4-bromobiphenyl,
ammonium hexafluorophosphate, phenanthroline, sodium chloride,
sodium bromide, sodium iodide, silver nitrate, pyridine and all kinds of
organic solvents (methanol, dichloromethane, DMSO etc.) were
purchased from Rhea biotechnology co. LTD. Trypsin/EDTA, bovine
serum albumin, DMEM medium, streptomycin/penicillin mixture, and
phosphate-buffered saline (PBS) were provided by Sangon Biotech. A549
(lung cancer cells) were obtained from Shanghai Institute of Biochemistry
and Cell Biology (SIBCB). Testing complexes were dissolved in DMSO
and diluted with the tissue culture medium before use. Stock solutions of
cisplatin (10 mM) and complexes (10 mM) were prepared in DMSO. All
stock solutions were stored at -20 °C, thawed and diluted with culture
medium prior to each experiment.
NMR spectra were ascertained through Bruker DPX 500
spectrometers instrument, with the chemical shifts reported in ppm and
tetramethylsilane (TMS) as an internal standard. Mass spectra were
determined by a Waters Q-TOF MicroTM mass spectrometer. Elemental
analysis was obtained through a VarioMICRO CHNOS elemental analyzer.
UV-vis spectra and fluorescence spectra were dertermined through a
PERSEE TU-1901 UV spectrometer and Hitachi F-4600 fluorescence
spectrophotometer, respectively. Induction of apoptosis, cell cycle and
mitochondrial membrane potential were carried out on an ACEA Novocyte
2040R flow cytometry. Viability assay (MTT) was measured using a
Perlong DNM-9606 microplate reader at an absorbance of 570 nm. The
cellular localization was determined by Carl Zeiss AG*/LSM/880NLO two-
photon laser scanning microscope and VG Elemental inductively coupled
plasma mass spectrometer (ICP-MS).
Figure 7. (a) UV-vis spectra of BSA in 5 mM Tris-HCl/10 mM NaCl buffer
solution (pH = 7.2) with the increase of Ir8 (0-10 μM). The arrows show the
direction of changes in absorbance; (b) Fluorescence spectra of BSA (5 μM; λex
= 280 nm; λem = 350 nm) in the absence and presence of Ir8 (0-10 μM);
Synchronous spectra of BSA (5 μM) in the presence of increasing amounts of
Ir8 (0-10 μM) with a wavelength of Δλ = 15 nm (c) and Δλ = 60 nm (d).
2. Synthesis of [(η5-C5Me5)IrCl2]2 (Dimer1) and [(η5-C5Me4C6H5)IrCl2]2
(Dimer2)
Table 2. The values of Kq, Ksv, Kb and n for Ir5 and Ir8 at 298 K.
Kq
Ksv
Kb
Dimer of iridium (Dimer 1 and Dmier 2) were prepared according to
literature procedure,[39] the synthesis process is as follows: Iridium
trichloride hydrate (1.00 g, 2.8 mmol) was dissolved in methanol (30 mL)
in a microwave vial, pentamethyl cyclopentadiene (1.38 g, 10.0 mmol) was
added and reacted at 127 °C , 400 psi for 30 min in a microwave instrument.
The reaction mixture was cooled to ambient temperature, filtered and then
concentrated the filtrate to about 10 mL on a rotary evaporator. Cool, filter,
and get an orange precipitate. The product was washed with methanol and
diethyl ether, dried in the air, and pure Dimer 1 was obtained. Yield: 0.79
g (66%). 1H NMR (500 MHz, CDCl3): δ 1.63 (s, 30H).
Dimer 2 was synthesized using biphenyltetramethylcyclopentadiene
(2.74 g, 10.0 mmol) and Iridium trichloride hydrate (1.0 g, 2.8 mmol)
according to the method of Dimer 1. Yield: 0.83 g (53%). 1H NMR (500
MHz, CDCl3): δ 7.64 (m, 8H), 7.44 (m, 4H), 7.33 (m, 6H), 2.08 (s, 6H), 1.95
(s, 6H), 1.88 (s, 6H), 1.00 (d, J = 7.5 Hz, 6H).
Complex
n
(1013 M-1S-1)
(105 M -1
)
（105 M-1）
Ir5
Ir8
14.263
5.335
14.263±0.18
5.335± 0.14
15.666
4.630
1.049
0.934
Conclusion
In summary, eight half-sandwich IrIII phenanthroline complexes
with halide ions and pyridine leaving groups were synthesised and
characterised. The IrIII complexes showed potential anticancer
activity against A549 cells, especially for Cpxbiph-based one. The
logP values indicated that an appropriate increase in lipid
solubility was beneficial for improving the anticancer activity of the
complexes. These complexes with halide ion and pyridine leaving
groups exhibited different anticancer mechanisms. Among them,
the pyridine-based complexes, although no hydrolysis, could
effectively induce the production of intracellular ROS. Halide ion-
based catalysts exhibit favourable hydrolytic properties. However,
hydrolysis is not the decisive factor affecting the anticancer
activity of these complexes. Instead, an increase in the
intracellular ROS contributes more towards the apoptosis induced
by these complexes. Additionally, these complexes caused
lysosomal damage and led to the accumulation in the cytosol,
binding to serum albumin, and transport through plasma. The data
of the half-sandwich IrIII phenanthroline complexes with halide
3. Synthesis of IrIII phenanthroline complexes (Ir1 and Ir5)
The general method is as follows: Dimer of iridium (Dimer 1/Dimer 2, 79.6
mg/107.2 mg, 0.10 mmol) and phenanthroline (36.0 mg, 0.20 mmol) were
added into a 100 mL Schlenk flask under nitrogen, added methanol (30
mL) as solvent and stirred at 298 K overnight (monitored by thin-layer
chromatography). Ammonium hexafluorophosphate (130.4 mg, 0.80
mmol) was added into the mixture and stirred for another 6 h. The solvent
was removed in vacuum and recrystallized from dichloromethane and n-
hexane. The 1H NMR (500 MHz, d6-DMSO), 13C{1H} NMR (126 MHz, d6-
DMSO), ESI-MS and elementary analysis of Ir1 and Ir5 are shown in
Figures S1-S3. The specific NMR data of target complexes were shown in
Figure 8. The data were listed as follows:
5
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10.1002/cbic.202000511
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FULL PAPER
[(η5-Cp*)Ir(Phen)bpy](PF6)2 (Ir4). Yield: 66%. 1H NMR (500 MHz, DMSO)
δ 9.69 (d, 3J = 6.1 Hz, 2H, H9-Py), 9.07 (d, 3J = 8.2 Hz, 2H, H1-Phen), 8.47
3
3
(d, J = 5.2 Hz, 2H, H3-Phen), 8.40 (dd, J = 8.2, 5.2 Hz, 2H, H2-Phen),
8.36 (s, 2H, H5-Phen), 7.91 (t, 3J = 7.7 Hz, 1H, H11-Py), 7.46 – 7.40 (m, 2H,
H10-Py), 1.62 (s, 15H, H8-CH3). 13C{1H} NMR (126 MHz, DMSO) δ 153.28
(C1-Phen), 151.77 (C9-Py), 146.98 (C6-Phen), 141.32, 141.02, 131.33,
128.81, 128.80, 128.46, 91.66 (C7-Cp*), 8.13 (C8-Cp*). ESI-MS (m/z):
Calcd for C27H28N3F12P2Ir, 877.12; Found 507.33 [M-bpy-2PF6]2+
.
Elemental Analysis: Calcd for C27H28N3F12P2Ir: C, 36.99; H, 3.22; N, 4.79%.
Found: C, 36.96; H, 3.27; N, 4.72%.
Figure 8. Attribution of NMR for the target complexes.
[(η5-Cpxbiph)Ir(Phen)Br]PF6 (Ir6). Yield: 68%. 1H NMR (500 MHz, DMSO) δ
9.13 (d, 3J = 5.3 Hz, 2H, H1-Phen), 8.97 (d, 3J = 8.2 Hz, 2H, H3-Phen), 8.39
(s, 2H, H5-Phen), 8.18 (dd, 3J = 8.2, 5.3 Hz, 2H, H2-Phen), 7.81 (d, 3J = 8.3
Hz, 2H, H15-Cpxbiph), 7.77 (d, 3J = 7.3 Hz, 2H, H14-Cpxbiph), 7.63 (d, 3J = 8.2
Hz, 2H, H16-Cpxbiph), 7.51 (t, 3J = 7.6 Hz, 2H, H13-Cpxbiph), 7.42 (t, 3J = 7.4
Hz, 1H, H12-Cpxbiph), 1.88 (s, 6H, H19-CH3), 1.82 (s, 6H, H8-CH3). 13C{1H}
NMR (126 MHz, DMSO) δ 152.48 (C1-Phen), 146.66 (C6-Phen), 141.01,
139.88, 139.55, 131.16, 129.57, 128.58, 128.44, 128.26, 128.03, 127.95,
127.19, 98.75 (C17-Cpxbiph), 88.09 (C7-Cpxbiph), 83.23 (C18-Cpxbiph), 10.23
(C8-Cpxbiph), 8.81 (C19-Cpxbiph). ESI-MS (m/z): Calcd for C33H29N2BrF6PIr,
870.1; Found 725.2 [M-PF6]+. Elemental Analysis: Calcd for
C33H29N2BrF6PIr: C, 45.52; H, 3.36; N, 3.22%. Found: C, 45.77; H, 3.42;
N, 3.13%.
[(η5-Cp*)Ir(Phen)Cl]PF6 (Ir1). Yield: 82%. 1H NMR (500 MHz, DMSO) δ
9.40 (d, 3J = 5.3 Hz, 2H, H1-Phen), 8.97 (d, 3J = 8.2 Hz, 2H, H3-Phen), 8.37
(d, J = 3.1 Hz, 2H, H5-Phen), 8.22 (dd, J = 8.2, 5.3 Hz, 2H, H2-Phen),
1.73 (s, 15H, H8-CH3). 13C{1H} NMR (126 MHz, DMSO) δ 152.60 (C1-
Phen), 146.60 (C6-Phen), 139.66 (C3-Phen), 130.95 (C4-Phen), 128.42
(C5-Phen), 127.94 (C2-Phen), 89.59 (C7-Cp*), 8.70 (C8-Cp*). ESI-MS
(m/z): Calcd for C22H23N2ClF6PIr, 688.07; Found 543.33 [M-PF6]+.
Elemental Analysis: Calcd for C22H23N2ClF6PIr: C, 38.40; H, 3.37; N,
4.07%. Found: C, 38.24; H, 3.53; N, 4.00%.
4
3
[(η5-Cpxbiph)Ir(Phen)Cl]PF6 (Ir5). Yield: 79%. 1H NMR (500 MHz, DMSO)
1H NMR (500 MHz, DMSO) δ 9.13 (d, 3J = 5.2 Hz, 2H, H1-Phen), 8.99 (d,
1
[(η5-Cpxbiph)Ir(Phen)I]PF6 (Ir7). Yield: 65%. H NMR (500 MHz, DMSO) δ
3
3J = 8.2 Hz, 2H, H3-Phen), 8.39 (s, 2H, H5-Phen), 8.19 (dd, J = 8.2, 5.3
9.16 (d, 3J = 5.2 Hz, 2H, H1-Phen), 8.96 (d, 3J = 8.2 Hz, 2H, H3-Phen), 8.40
(s, 2H, H5-Phen), 8.16 (dd, 3J = 8.2, 5.3 Hz, 2H, H2-Phen), 7.76 (dd, 3J =
12.9, 7.9 Hz, 4H, H14/H15-Cpxbiph), 7.56 (d, 3J = 8.2 Hz, 2H, H16-Cpxbiph),
7.50 (t, 3J = 7.6 Hz, 2H, H13-Cpxbiph), 7.41 (t, 3J = 7.3 Hz, 1H, H12-Cpxbiph),
1.95 (s, 6H, H19-CH3), 1.91 (s, 6H, H8-CH3). 13C{1H} NMR (126 MHz,
DMSO) δ 153.17 (C1-Phen), 146.45 (C6-Phen), 141.04, 139.80, 139.48,
131.27, 131.22, 129.56, 128.63, 128.45, 127.94, 127.18, 98.57 (C17-
Cpxbiph), 88.97 (C7-Cpxbiph), 85.30 (C18-Cpxbiph), 10.90 (C8-Cpxbiph), 9.37
(C19-Cpxbiph). ESI-MS (m/z): Calcd for C33H29N2IF6PIr, 918.1; Found 773.2
[M-PF6]+. Elemental Analysis: Calcd for C33H29N2IF6PIr: C, 43.19; H, 3.19;
N, 3.05%. Found: C, 43.33; H, 3.26; N, 2.97%.
Hz, 2H, H2-Phen), 7.83 (d, 3J = 8.2 Hz, 2H, H15-Cpxbiph), 7.78 (d, 3J = 7.6
Hz, 2H, H14-Cpxbiph), 7.66 (d, 3J = 8.2 Hz, 2H, H16-Cpxbiph), 7.51 (t, 3J = 7.6
Hz, 2H, H13-Cpxbiph), 7.43 (t, 3J = 7.3 Hz, 1H, H12-Cpxbiph), 1.85 (s, 6H, H19-
CH3), 1.78 (s, 6H, H8-CH3). 13C{1H} NMR (126 MHz, DMSO) δ 152.10 (C1-
Phen), 146.75 (C6-Phen), 140.98, 139.94, 139.60, 131.13, 131.12, 129.57,
128.56, 128.48, 128.43, 128.06, 127.94, 127.20, 98.78 (C17-Cpxbiph), 87.55
(C7-Cpxbiph), 82.28 (C18-Cpxbiph), 9.86 (C8-Cpxbiph), 8.65 (C19-Cpxbiph). ESI-
MS (m/z): Calcd for C33H29N2BrF6PIr, 826.2; Found 681.2 [M-PF6]+.
Elemental Analysis: Calcd for C33H29N2BrF6PIr: C, 47.97; H, 3.54; N,
3.39%. Found: C, 48.21; H, 3.66; N, 3.16%.
[(η5-Cpxbiph)Ir(Phen)bpy](PF6)2 (Ir8). Yield: 59%.1H NMR (500 MHz,
4. Synthesis of other IrIII phenanthroline complexes
3
3
DMSO) δ 9.56 (d, J = 5.1 Hz, 2H, H9-Py), 9.09 (d, J = 8.2 Hz, 2H, H1-
Phen), 8.51 (d, 3J = 5.3 Hz, 2H, H3-Phen), 8.40 (s, 2H, H5-Phen), 8.35 (dd,
3J = 8.2, 5.3 Hz, 2H, H2-Phen), 7.95 (t, 3J = 7.6 Hz, 1H, H11-Py), 7.61 (d,
3J = 7.3 Hz, 2H, H15-Cpxbiph), 7.53 (d, 3J = 8.4 Hz, 2H, H14-Cpxbiph), 7.46 (m,
4H, H10-Py/H16-Cpxbiph), 7.40 (d, 3J = 8.2 Hz, 1H, H12-Cpxbiph), 7.02 (d, 3J =
8.2 Hz, 2H, H13-Cpxbiph), 1.81 (s, 6H, H19-CH3), 1.72 (s, 6H, H8-CH3).
13C{1H} NMR (126 MHz, DMSO) δ 153.36 (C1-Phen), 151.89 (C9-Py),
150.46 (C6-Phen), 146.85, 141.61, 141.40, 141.28, 139.19, 137.18,
131.62, 130.59, 129.56, 128.99, 128.68, 127.59, 127.07, 124.69, 94.42
(C17-Cpxbiph), 91.98 (C7-Cpxbiph), 78.59 (C18-Cpxbiph), 9.42 (C8-Cpxbiph), 8.24
(C19-Cpxbiph). ESI-MS (m/z): Calcd for C38H34N3F12P2Ir, 1015.2; Found
645.8 [M-bpy-2PF6]2+. Elemental Analysis: calcd for C38H34N3F12P2Ir: C,
44.97; H, 3.38; N, 4.14%. Found: C, 45.21; H, 3.46; N, 4.01%.
The general method is as follows: Ir1 or Ir5 (50.0 mg/60.0 mg, 0.073 mmol)
were added to a 100 mL Schlenk flask under nitrogen. The mixture of
methanol (15 mL) and water (15 mL) was added and stirred until Ir1 or Ir5
dissolved. Silver nitrate (12.0 mg, 0.073 mmol) was added and reacted for
5 h with light shielded. Remove silver chloride by filtration, and sodium
bromide/sodium iodide/pyridine (0.58 mmol) was added and reacted
overnight. Ammonium hexafluorophosphate (95.2 mg, 0.58 mmol) was
added into the mixture and stirred for another 6 h. The solvent was
removed in vacuum and recrystallized from dichloromethane and n-
hexane. The 1H NMR (500 MHz, d6-DMSO), 13C{1H} NMR (126 MHz, d6-
DMSO), ESI-MS and elementary analysis of complexes are shown in
Figures S1-S3. The specific NMR data of target complexes were shown in
Figure 8. The data were listed as follows:
[(η5-Cp*)Ir(Phen)Br]PF6 (Ir2). Yield: 60%. 1H NMR (500 MHz, DMSO) δ
9.40 (d, 3J = 5.3 Hz, 2H, H1-Phen), 8.96 (d, 3J = 8.2 Hz, 2H, H3-Phen), 8.37
3
(s, 2H, H5-Phen), 8.21 (dd, J = 8.2, 5.3 Hz, 2H, H2-Phen), 1.76 (s, 15H,
Statistical analysis
H8-CH3). 13C{1H} NMR (126 MHz, DMSO) δ 153.04 (C1-Phen), 146.49 (C6-
Phen), 139.61 (C3-Phen), 130.97 (C4-Phen), 128.43 (C5-Phen), 127.90
(C2-Phen), 90.04 (C7-Cp*), 8.95 (C8-Cp*). ESI-MS (m/z): Calcd for
C22H23N2BrF6PIr, 732.03; Found 587.42 [M-PF6]+. Elemental Analysis:
Calcd for C22H23N2BrF6PIr: C, 36.07; H, 3.16; N, 3.82%. Found: C, 36.11;
H, 3.09; N, 3.86%.
Analysis of variance was performed for all data analysis and values were
presented as means ± standard deviation. Differences were considered
statistically significant at p < 0.05 (*) and p < 0.01 (**). P values were
calculated after a t test.
[(η5-Cp*)Ir(Phen)I]PF6 (Ir3). Yield: 67%. 1H NMR (500 MHz, DMSO) δ 9.41
(d, 3J = 6.0 Hz, 2H, H1-Phen), 8.95 (d, 3J = 8.2 Hz, 2H, H3-Phen), 8.38 (s,
2H, H5-Phen), 8.20 (dd, 3J = 8.2, 5.3 Hz, 2H, H2-Phen), 1.83 (s, 15H, H8-
CH3). 13C{1H} NMR (126 MHz, DMSO) δ 152.61 (C1-Phen), 146.60 (C6-
Phen), 139.67 (C3-Phen), 130.96 (C4-Phen), 128.43 (C5-Phen), 127.95
(C2-Phen), 89.60 (C7-Cp*), 8.71 (C8-Cp*). ESI-MS (m/z): Calcd for
C22H23N2IF6PIr, 780.02; Found 635.25 [M-PF6]+. Elemental Analysis:
Calcd for C22H23IF6IrN2P: C, 33.90; H, 2.97; N, 3.59%. Found: C, 33.95; H,
2.97; N, 3.52%.
Acknowledgements
We thank the University Research Development Program of
Shandong Province (J18KA082), the Student’s Platform for
Innovation and Entrepreneurship Training Program (2019A003),
the National Natural Science Foundation of China (21671118,
6
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000511
ChemBioChem
FULL PAPER
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10.1002/cbic.202000511
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Iridium(III) phenanthroline anticancer complexes with different leaving groups (Z) could lead to lysosomal damage, catalyzing the
change of NADH/NAD+, decreasing the mitochondrial membrane potential (ΔΨm), increasing the level of reactive oxygen species (ROS),
and eventually leading to apoptosis.
8
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