Preparation and the anticancer mechanism of configuration-controlled Fe(ii)-Ir(iii) heteronuclear metal complexes

Article abstract of DOI:10.1039/d0dt02408b

A series of configuration-controlled Fe(ii)-Ir(iii) heteronuclear metal complexes, including ferrocene and half-sandwich like iridium(iii) complex units, have been designed and prepared. These complexes show better anticancer activity than cisplatin under the same conditions, especiallycis-configurational ones. Laser confocal microscopy analysis confirms that the complexes follow a non-energy-dependent cellular uptake mechanism, accumulate in lysosomes (pearson co-localization coefficient: ～0.7), lead to lysosomal damage, and eventually induce apoptosis. These complexes can reduce the mitochondrial membrane potential, disturb the cell circle, catalyze the oxidation of nicotinamide-adenine dinucleotide (NADH) and increase the levels of intracellular reactive oxygen species (ROS), following an anticancer mechanism of oxidation. In addition, the complexes could bind to serum protein, and transport through it. Above all, the Fe(ii)-Ir(iii) heteronuclear metal complexes hold promise as potential anticancer agents for further study.

Full text of DOI:10.1039/d0dt02408b

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Preparation and anticancer mechanism of configuration-controlled Fe(II)-Ir(III)
heteronuclear metal complexes
Mingxiao Shao, Xicheng Liu*, Yiwei Sun, Shuaihua Dou, Qi Chen, Xiang-Ai Yuan, Laijin Tian,
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, School of
Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China.
*Corresponding author, (X. Liu) E-mail: chemlxc@163.com; Fax: 0086-537-4456301; (Z. Liu) E-mail:
liuzheqd@163.com, Fax: 0086-537-4456301.
Abstract
Series of configuration-controlled Fe(II)-Ir(III) heteronuclear metal complexes, including
ferrocene and half-sandwich iridium(III) complex units, have been designed and prepared. These
complexes show better anticancer activity than cisplatin under the same conditions, especially for
cis-configurational one. Laser confocal verifies complexes follow a non-energy-dependent cellular
uptake mechanism, accumulate in lysosomes (Pearson co-localization coefficient: ~0.7), lead to
lysosomal damage, and eventually induce apoptosis. These complexes can decrease the
mitochondrial membrane potential, disturb the cell circle, catalyze the oxidation of
nicotinamide-adenine dinucleotide (NADH) and increase the levels of intracellular reactive
oxygen species (ROS), follow an anticancer mechanism of oxidation. Additionally, complexes
could bind to serum protein, and transport through it. Above all, Fe(II)-Ir(III) heteronuclear metal
complexes hold promise as potential anticancer agents for further study.
Keywords: Ferrocene; Iridium(III) complex; Lysosome-targeted; Anticancer
1 Introduction
Recently, organometallic compounds, which have at least one metal-carbon bond, have
aroused wide concern due to their unique chemical structure and favourable biological activities
[1]. Among them, ferrocene and iridium(III) complexes, which have a sandwich structure
(η5-(C5H5)2Fe) and half-sandwich structure ([(η5-Cp*)Ir(L^L)Cl]PF6), have shown extensive
applications in in various fields, including bio-organometallic chemistry, polymer chemistry,
organic synthesis, materials science, crystal engineering and electrochemistry, especially in the
anticancer drug design and development [2-4].
Cancer is the second leading cause of death in developed nations according to health
organizations. It has been estimated that approximately 1,806,590 cancer cases will be diagnosed
in the United States in 2020, which is equivalent to approximately 4,950 new cases each day of
the year [5]. Although various organic small molecules and biological derivatives have been
widely used for anticancer treatment, platinum-based metallodrugs have attracted attention
because of their unique biological and chemical properties [6-8]. Ferrocene and its derivatives,
typical organometallic complexes with advantages such as reversible redox properties, low
toxicity, low cost and inherent stability in air, are currently being evaluated as anticancer agents
[9-12]. Studies have shown that the oxidation state of the ferrocene moiety plays a crucial role in
its cytotoxic actions and is involved in inducing the accumulation of reactive oxygen species (1O2)
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via an oxidation-related mechanism [13-15]. Additionally, some derivatives of ferrocene havVeiew Article Online
DOI: 10.1039/D0DT02408B
been used as ligands to form many types of heteronuclear metal complexes, which have exhibited
an important influence on the coordination behaviour of these ligands, and further affect their
biological activity in vitro [16]. In general, the anticancer potential of ferrocene is lower than that
of other metallodrugs but can be improved by combining it with other transition metal complexes
[17]. Ferrocenylpyridines, ferrocenylphenylpyridines and 1,1′-di(2-pyridyl)ferrocene ligands and
their platinum, palladium, rhodium, and iridium complexes were screened for activity against two
human cancer cell lines [18]. At least two of the complexes displayed growth inhibition similar to
that of the widely used chemotherapeutic agent, cisplatin. In our previous study,
ferrocene-appended organotin compounds were used to catalyse the oxidation of
nicotinamide-adenine dinucleotide (NADH), leading to the accumulation of reactive oxygen
species (ROS) and inducing A549 tumour cell apoptosis and oncosis [19]. These results indicate
that ferrocene-based heteronuclear metal complexes display more excellent cytotoxic activity
against the cancer cell lines compared with the ferrocene monomer, providing guidance regarding
the design of metal anticancer drugs.
Iridium(III) (IrIII) complexes have been rapidly developed as the effective substitutes for
platinum anticancer drugs because of their special anticancer mechanisms, including favourable
biocatalysis performance (Catalyze the oxidation of NADH, leading to production of ROS) and as
various proteins inhibitors [20-22]. Among them, half-sandwich IrIII complexes, generally
described as [(η5-Cp*)Ir(L^L)X]Z, have attracted more attention because of their favourable
anticancer activity [23,24]. Interestingly, each part has a certain effect on the anticancer activity of
these complexes, and the rational design has achieved success in the development of these
complexes [3]. Cyclopentadienyl or its phenyl and biphenyl derivatives (Cp*) occupy three
coordination sites at the octahedral metal center and provide to the metal a lipophilic protecting
face. The dentate ligand (L^L) and the leaving group (X, usually chlorine) occupy the remaining
three coordination sites, which endow the whole complex with hydrophilic properties. Therefore,
precise regulation between the lipophilic cyclopentadienyl derivative and the other coordinated
ligands can effectively control the solubility, the ligand-exchange kinetics and the stability of
these complexes [25]. Of course, the size and coordination ability of the counter ions (Z, usually
-
PF6 ) also has a significant influence on the chemical reactivity and anticancer activity of these
complexes [26].
Multinuclear or heteronuclear metal complexes differ from the traditional metal complex
monomers and have shown good application potential for treatment [27]. Considering the
advantage of ferrocene derivatives and half-sandwich iridium(III) complexes in the field of
anticancer, Fe(II)-Ir(III) heteronuclear metal complexes (Fe-Ir1~Fe-Ir4, Scheme 1) were prepared
and characterised in this paper. An MTT (3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium
bromide) assay indicated that these complexes had better anticancer activity than the monomers of
ferrocene-bipyridine pro-ligands (L1 and L2, Scheme 2) and half-sandwich IrIII bipyridine
complexes (5 and 6, Scheme S1), particularly those with a cis-configurational ones (2 and 4). We
also investigated the intracellular tissue targeting and the anticancer mechanism of these
complexes. The results indicated that Fe(II)-Ir(III) heteronuclear metal complexes had potential as
anticancer agents for further evaluation.
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Scheme
1
Structures of designed Fe(II)-Ir(III) heteronuclear metal complexes,
non-energy-dependent cellular uptake mechanism, targeted and damaged lysosome, led to the
decrease of mitochondrial membrane potential (MMP) and the increase of reactive oxygen species
(ROS), eventually induced apoptosis.
2 Results and discussion
2.1 Synthesis and general aspects
Fe(II)-Ir(III) heteronuclear metal complexes were obtained by the reaction of
[(η5-C5Me5)IrCl2]2 (Dimer 1) or [(η5-C5Me4C6H5)IrCl2]2 (Dimer 2) with the corresponding
ferrocene-modified bipyridine (N^N) bidentate ligands (L1 and L2), Scheme 2. N^N-bidentate
ligands (L1 and L2) were obtained by the interaction of 4-formyl-4'-methyl-2,2'-bipyridine
(biPy-CHO) and ferrocene methyl triphenylphosphine hydrobromate through the classical Wittig
reaction and purified by column chromatography [28]. The trans- and cis-spatial configurations
were confirmed by the hydrogen nuclear magnetic resonance (1H NMR) spectra (Fig. S1),
hydrogen atoms of the olefin were shown in the range of 6.4 ppm to 6.8 ppm with the coupling
constant of 16.1 Hz and 12.0 Hz, respectively [29]. Complexes were synthesized and isolated as
hexafluorophosphate in good yields (>85%), and fully characterised by NMR spectra (Figs. S3
and S4), electrospray ionization mass spectra (ESI-MS, Fig. S5) and elemental analysis. Hydrogen
atoms on the methyl of cyclopentadiene were shown in the range of 1.6 ppm to 1.8 ppm, and that
of bipyridine changed from 2.6 ppm to 3.4 ppm. Hydrogen atoms on ferrocene were shown in the
range of 4.2 ppm to 4.9 ppm, and others on pyridine and benzene appear in the range of 6.3 ppm
to 8.9 ppm. The methyl carbon atoms of bipyridine were shown in 21.2~21.5 ppm, and which on
Cp* were at 8.4~9.8 ppm in 13C NMR spectra. The carbon atoms of ferrocene units listed in the
range of 68.5~82.2 ppm. The results of ESI-MS were consistent with the theoretically calculated
values (loss of PF6− or Cl−).
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N
N
PF6
PPh3Br
Cl
R
L1
1) t-BuOK, THF, 273 K 2 h
2) biPy-CHO, r.t. 6 h
1) Dimer, CH3OH, r.t. 24 h
2) NH4PF6, r.t. 6 h
Fe
Ir
Fe
N
N
N
N
Wittig reagent
Fe
R=CH3; C6H5
L2
Cl
CHO
Cl
Cl
Cl
Cl
Ir
Ir
Ir
Ir
Cl
Cl
Cl
N
N
biPy-CHO
Dimer 1
Dimer 2
Scheme 2 Synthesis process of Fe(II)-Ir(III) heteronuclear metal complexes.
Single crystals of Fe-Ir1 suitable for X-ray diffraction analysis were obtained by slow
diffusion of n-hexane into a saturated solution of dichloromethane. Crystal structure was shown in
Fig. 1, crystallographic data and selected bond length and angles were listed in Tables S1 and S2.
Obviously, Fe-Ir1 shows expected trans-configuration. In addition, metal iridium cell can be
described as a “three-leg piano-stool” geometry [3], and ferrocene unit exhibit a superimposed
configuration. Around the central iridium ion, the distance of Ir-Cl is longest, which is consistent
with the conclusion that Ir-Cl bond is the active center for half-sandwich structural iridium(III)
complexes [30].
Fig. 1 X-ray crystal structure of Fe-Ir1. The thermal ellipsoids drawn at the 50% probability level
(H atoms were omitted for clarity).
2.2 Cell cytotoxicity assay
The anticancer activity of Fe(II)-Ir(III) heteronuclear metal complexes against A549 (lung
cancer cells) and HeLa (cervical cancer cells) cell lines was determined using the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after 24 h treatment.
Cisplatin, widely used clinically, was included as a control. The IC50 values (concentration at
which 50% of the cell growth is inhibited) are listed in Table 1. As shown, compared with the
basic ferrocene-appended bipyridine pro-ligands (L1 and L2) and simple half-sandwich IrIII
bipyridine complexes (Ir5 and Ir6, Scheme S1), Fe(II)-Ir(III) heteronuclear metal complexes
exhibited better antitumor activity under the same conditions, the best of which (Fe-Ir4) showing
almost 5-fold higher activity than cisplatin against A549 cells. These results indicate that
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combining the ferrocene monomer and metal iridium(III) complex monomer can effectively
improve the overall anticancer activity, particularly when they are connected through the
cis-configuration olefin. However, the cytotoxicity of these complexes towards human lung
epithelial cells (BEAS-2B, human normal cells) was also high under the same conditions, and no
significant selectivity existed between normal cells and tumour cells. However, this favourable
antitumor activity provided a structural foundation for the further design and optimization of such
compounds.
Table 1 IC50 values of target complexes, ferrocene-modified bipyridine pro-ligands (L1, L2) and
cisplatin against A549, HeLa and BEAS-2B cell lines determined by MTT assay after 24 h.
IC50 (μM)
Complex
A549
30.34±4.88
19.90±0.66
6.70±0.40
4.81±0.27
＞100
Hela
21.42±0.81
13.90±0.79
7.42±0.41
5.51±0.10
＞100
BEAS-2B
33.48±0.41
23.66±0.72
8.06±0.03
7.32±0.42
＞100
Fe-Ir1
Fe-Ir2
Fe-Ir3
Fe-Ir4
Ir5
Ir6
＞100
＞100
＞100
L1
92.91±1.32
＞100
＞100
＞100
L2
＞100
＞100
Cisplatin
21.32±1.71
7.52±0.21
38.40±2.83
Obviously, the anticancer activity of cis-configurational complexes is higher than that of the
corresponding trans-configurational one (Fe-Ir2 > Fe-Ir1; Fe-Ir4 > Fe-Ir3) with the evidence of
the lower IC50 values, respectively. To understand these, quantum chemical computation was used
to evaluate after adequately considering the crystal structure of Fe-Ir1. The data of natural
population analysis (NPA) for central atom (Ir) and the leaving group (Cl) and Wiberg bond order
of Ir-Cl bond in Fe-Ir1 and Fe-Ir2 were analysed by density functional theory (DFT) calculation
at the B3LYP/6-31G(d, p) (C, H, N, Cl)/SDD (Ir) level. Fe-Ir1 exhibit a little larger Wiberg bond
order (0.6217) than Fe-Ir2 (0.6187). Meanwhile, the values of NPA charge population for Cl in
Fe-Ir1 and Fe-Ir2 are -0.343 and -0.327, however, which for Ir are almost the same (0.173 and
0.172, respectively). Although the N^N bidentate pro-ligand (L1) containing trans-configurational
alkene has the smaller bond angles (Table S3), the electron cloud configuration of front orbitals,
HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular
Orbital), shows the combination of ferrocene and bipyridine does not effectively improve the
conjugation of the whole pro-ligands (HOMOs are mainly localized on ferrocene and olefin;
LUMOs are mainly localized on Ir, Cl, bipyridine unit and olefin; Fig. S6), which have almost the
similar electron-donating abilities and contribute to the similar charges in the central iridium ions.
Therefore, the lower the charge of Cl−, the stronger the Ir-Cl bond, and the worse the anticancer
activity of Fe-Ir1 [30]. Additionally, dimer 2-based complexes (Fe-Ir3 and Fe-Ir4) showed better
antitumor activity than the corresponding dimer 1-based one. The oil-water distribution
coefficients (logPo/w) were determined by UV-visible (UV-vis) spectra, and the values were 0.38
and 0.61 for Fe-Ir1 and Fe-Ir3, respectively. This conclusion indicates the increase of lipid
solubility is beneficial to the improvement of anticancer activity for these complexes. All these
provide the basic premise for further design and optimization of such complexes.
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Hydrolysis of M–Cl bonds can represent an activation step for half-sandwich transition metal
anticancer complexes. M–OH2 aqua complexes are usually more reactive than the corresponding
chloride complexes [3,30]. The hydrolysis characteristic of Fe-Ir3 and Fe-Ir4 in 20%
CH3OH/80% H2O (v/v) solution at 298 K over 8 h was monitored by UV-vis absorption spectra
(Fig. S7). The presence of methanol ensured the solubility of these complexes. Time dependence
of the formation of the aqua adducts of complexes was fitted to pseudo first-order kinetics (Fig.
S8). The values of hydrolysis rate constants are 0.00128 min-1 and 0.00305 min-1, and the
half-lives of hydrolysis are 541.5 min and 227.3 min for Fe-Ir3 and Fe-Ir4, respectively.
Fe(II)-Ir(III) heteronuclear metal complexes undergo a much slower hydrolysis, and the hydrolytic
half-life of Fe-Ir4 is about 2.5 times faster than that of Fe-Ir3, which helps Fe-Ir4 to exhibit the
better anticancer activity.
2.3 Cellular uptake and localization assay
Owning to the favourable anticancer activity against the A549 cell line, the subcellular
localisation in A549 cells of these complexes was determined by using laser confocal microscopy.
Lyso Tracker Red DND-99 (LTRD) and Mito Tracker Deep Red (MTDR) were employed as
fluorescent probes for the lysosomes and mitochondria, respectively [31]. At an appropriate
fluorescence emission (no conflict with the fluorescent probe of Lyso Tracker Red DND-99 and
Mito Tracker Deep Red, Fig. S9), Fe-Ir3 and Fe-Ir4 were shown to effectively accumulate in
lysosomal tissues with the Pearson’s colocalization coefficients of 0.72 and 0.76 over 2 h (Fig. 2).
However, the Pearson’s colocalisation coefficients for mitochondria targeting were 0.11 and 0.17,
respectively. This result indicates that Fe(II)-Ir(III) heteronuclear metal complexes are mainly
target lysosomes in vivo. Additionally, these complexes do not cause abnormal cell death
immediately, making them convenient for tracking real-time changes in lysosomal morphology.
Fig. 2 Determination of intracellular localization of Fe-Ir3 and Fe-Ir4 in A549 cells by laser
confocal microscopy. A549 cells were incubated with LTRD and MTDR, and then exposed to
Fe-Ir3 and Fe-Ir4 (1.0 × IC50). LTRD was excited at 594 nm and collected at 600-660 nm.
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MTDR was excited at 644 nm and collected at 660-720 nm. Complexes were excited at 405 nm
and the emission was collected at 420-500 nm. Scale bar: 20 µm.
Lysosomes, which are acidic intracellular organelles (pH=3.5-5.5), can destroy biological
macromolecules [32]. Widespread disruption of lysosomal integrity can lead to the release of
cathepsins and other hydrolases from the lysosomal lumen into the cytosol, thus initiating
apoptosis [33]. Acridine orange (AO), with red/green fluorescence in the lysosomes/cytosol, is
typically utilised as a proble to determine the integrity of lysosomes. A549 cells were exposed to
Fe-Ir3 and Fe-Ir4 (1.0×IC50 and 3.0×IC50) for 2 h, and then stained with AO (5 μM), Figs. 3 and
S10. Compared with the control, an obviously decrease in red fluorescence was observed in the
presence of these complexes (1.0×IC50), and obvious lysosomal damage was found at a
concentration of 3.0×IC50. This suggests that Fe(II)-Ir(III) heteronuclear metal complexes may
accumulate in lysosomes, followed by disruption of lysosomal integrity and eventually induction
of cell death.
Fig. 3 Lysosomal damage in A549 cells caused by Fe-Ir4 with AO (5.0 μM) staining 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.
A good intracellular uptake mechanism is beneficial for improving drug activity. After
treatment with Fe-Ir3 and Fe-Ir4 at 277 K and 310 K and preincubation with
chlorocyanochlorophenyl (CCCP, metabolic inhibitor, 10 μM) and chloroquine (endocytosis
modulator, 50 μM) for 2 h, A549 cells were evaluated to determine the cellular uptake
mechanisms (Fig. S11). Energy-dependent mechanisms (including active transport and
endocytosis) and non-energy-dependent mechanisms (including passive transport and free
diffusion) are the two main cellular uptake mechanisms of organometallic drugs [34]. As shown,
there was almost no change compared with the control, indicating that Fe(II)-Ir(III) heteronuclear
metal complex entering cell follows a non-energy-dependent pathway [35].
2.4 Anticancer mechanism assay
Apoptosis refers to the autonomous and orderly death of cells controlled by genes in order to
maintain internal environment stability, and most transition metal anticancer drugs can clear
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tumour cells by inducing apoptosis [36]. In order to investigate whether FeD(IOI)I:-1I0r.(1I0I3I9)/D0DT02408B
heteronuclear metal complexes could induce apoptosis, A549 cells were stained with annexin
V-FITC/PI (propidium iodide) and then hatched with Fe-Ir3 and Fe-Ir4 at the concentrations of
1.0×IC50, 2.0×IC50 and 3.0×IC50 for 24 h, followed by flow cytometry analysis (Fig. 4 and Tables
S4-S5). Fe-Ir3 and Fe-Ir4 induced the apoptosis of A549 cell in a dose-dependent manner,
mainly in the late apoptosis phase. The percentage of late apoptosis cells increased from 15.6%
and 21.3% to 25.5% and 30.1% when the concentrations of Fe-Ir3 and Fe-Ir4 were changed from
1.0×IC50 to 3.0×IC50, respectively. Additionally, >94% of A549 cells survived in the control
groups under the same conditions. This confirms that Fe(II)-Ir(III) heteronuclear metal complexes
can lead to tumour cell death via apoptosis.
Fig. 4 Flow cytometric quantification of annexin V-FITC and propidium iodide (PI)
double-labeled A549 cells treated with Fe-Ir3 and Fe-Ir4 for 24 h.
Generally, organometallic anticancer complexes could cause a functional decline by
disrupting the cell cycle and exert their effects. [37] To investigate the effect of Fe(II)-Ir(III)
heteronuclear metal complexes on tumour cell cycle arrest, A549 cells, stained by PI, were
exposed to Fe-Ir3 and Fe-Ir4 at the concentration of 0.5×IC50 1.0×IC50 and 2.0×IC50 for 24 h and
analysed by flow cytometry. As shown in Fig. 5 and Tables S6-S7, the percentages of cells in the
G2/M phase increased from 12.9% to 22.2% when the concentration changed from 0.5×IC50 to
2.0×IC50 for Fe-Ir4, however, which for Fe-Ir3 only increased by 2.0%. More important, though
less visible, is the contribution to blocking the cell cycle.
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Fig. 5 Distribution of A549 cells labeled with PI after incubation with Fe-Ir4 (0.5×IC50 1.0×IC50
and 2.0×IC50) for 24 h. The marked numbers were the percentages of cells in the G2/M phase.
Additionally, mitochondria, the source of energy or adenosine triphosphate (ATP) in cells,
play a unique role in the process of apoptosis. [38] The decrease of mitochondrial membrane
potential (MMP) is
a
vital signal in the early apoptotic process. [39] JC-1
(5,5,6,6′-tetrachloro-1,1′,3,3′ tetraethyl benzimidazolyl carbocyanine iodide), an ideal fluorescent
probe for detecting MMP (the high and low MMP corresponding to the red and green fluorescence
in the aggregate and monomer state), was utilised to clarify whether apoptosis induced through
destroying mitochondrial homeostasis and estimated by flow cytometry [40], Fig. S12. As shown,
a dose-dependent decline of MMP occurs in A549 cells induced by Fe-Ir3 and Fe-Ir4. Compared
with the negative control, the population of mitochondrial membrane depolarized cells increase by
8.1% and 21.8% for Fe-Ir3 and Fe-Ir4 at the concentration of 2.0×IC50, respectively. It's not hard
to see that although these complexes do not target mitochondria, widespread lysosomal damage
verified by laser confocal testing results in the release of hydrolases from the lysosomal lumen to
the cytosol, which directly leads to the mitochondrial dysfunction and eventually apoptosis.
Reactive oxygen species (ROS) are mainly produced and stored in the mitochondria, and
increased intracellular ROS levels may induce apoptosis [41,42]. Oxidant-sensitive DCFH-DA
(2,7-dichlorodi-hydrofluorescein diacetate), a fluorescent probe, was used to monitor the
intracellular ROS levels in A549 cells using flow cytometry, Fig. S13. The ROS levels in A549
cells show a dose-dependent increase as the concentrations of Fe-Ir3 and Fe-Ir4 were increased
from 0.25×IC50 to 0.5×IC50 after 24 h exposure. Compared with the negative control, intracellular
ROS levels showed 1.23/1.44-fold increases for Fe-Ir3/Fe-Ir4 when the concentration was
0.5×IC50, respectively. Additionally, NADH (the reduced state of nicotinamide adenine
dinucleotide) plays a key role in regulating energy production in mitochondria.45. Transition metal
complexes can catalyse the oxidation of NADH and lead to the accumulation of ROS (1O2),
exerting an antitumor mechanism of oxidation [43]. The UV-vis spectrum was utilised to
investigate the interaction between complexes and NADH in 10% CH3OH/90% H2O (v/v) at 298
K at different time points, Fig. 6. The intensity of absorbance at 339 nm (the maximum
absorbance of NADH) and 259 nm (the maximum absorbance of NAD+, oxidation state of
NADH, loss of hydrogen catalyzed by organometallic compounds) significantly decreased and
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increased over time, respectively [44]. These changes were well-reflected by the turnover numberVsiew Article Online
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(TONs, the change ratio at 339 nm). Fe-Ir4 showed a higher TON than Fe-Ir3, indicating that
cis-configurational complexes possess the better biocatalytic performance relative to
trans-configurational complexes. Overall, widespread lysosomal damage leads to apoptosis, which
can be revealed by a decrease in mitochondrial membrane potential, increase in intracellular
reactive oxygen species (Scheme 1), catalytic acceleration of NADH oxidation, and an antitumor
mechanism of oxidation.
Fig. 6 UV-vis spectra of NADH (100 μM) with Fe-Ir3 (a) and Fe-Ir4 (b) in 10% CH3OH/90%
H2O (v:v) over 8 h at 298 K. The arrows show the changes over time.
2.5 Transport mechanism assay
Human serum albumin (HSA) is the most abundant carrier protein in the human plasma,
which can bind a variety of substrates, including hormones, metal cations and most therapeutic
drugs. Bovine serum albumin (BSA) is usually utilised as a model to investigate the transport
mechanism of organometallic anticancer drugs due to the advantages of resemblance to HSA,
abundance, stability, ease of purification, etc. [45] Therefore, in this study, UV-vis spectra and
fluorescence spectra were used to determine the binding mechanism between these complexes and
serum albumin. As shown in Figs. 7a and S14a, a significant reduction is found at 227 nm (the
UV-vis absorption of BSA) along with a red shift (~7 nm, induced by the polar solvent) with the
increase of Fe-Ir3 and Fe-Ir4, which indicates that Fe(II)-Ir(III) heteronuclear metal complexes
can change the conformation of BSA, reduce the content of the α-helix structure and loosen the
conformation of the protein [46]. In addition, a slight change (increase and decrease for Fe-Ir3 and
Fe-Ir4, respectively) without any shift at 278 nm suggest that complexes can interact with BSA
and closely relate to the microenvironment of aromatic acid residues in BSA. Among them,
tyrosine and tryptophan, two main aromatic acid residues, can be reflected by the wavelength
interval of Δλ = 15 nm and 60 nm through synchronous fluorescence, respectively [47]. As shown
in Figs. 7C, 7d, S14c and S14d, the fluorescence intensity for BSA at 291 nm and 285 nm (Δλ =
15 nm and 60 nm, respectively) weaken quickly with the increase of complexes. Additionally, a
minor red shift (~3 nm) occurs at 285 nm (Δλ = 60 nm) at the investigated concentration range,
however, almost no change occurred at the wavelength of Δλ = 15 nm. The results indicated that
the conformation of BSA was changed, and the polarity around the tryptophan residues increased
[48].
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Fig. 7 (a) UV-vis spectra of BSA in 5 mM Tris-HCl/10 mM NaCl buffer solution (pH = 7.2) with
the increase of Fe-Ir3 (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
Fe-Ir3 (0-10 μM); Synchronous spectra of BSA (5 μM) in the presence of increasing amounts of
Fe-Ir3(0-10 μM) with a wavelength of Δλ = 15 nm (c) and Δλ = 60 nm (d).
The interaction mechanism between Fe-Ir3/Fe-Ir4 and BSA was further investigated by the
fluorescence quenching phenomenon of BSA [49], Figs. 7b and S14b. A significant quenching at
343 nm (the fluorescent emission peak of BSA) was found with the increase of Fe-Ir3 and Fe-Ir4.
The Stern–Volmer quenching constant (Ksv) and quenching rate constant (Kq) can be obtained
through the classical Stern–Volmer equation (Figs. S15a and S15b). Dynamic mechanism and
static mechanism are two main mechanism of fluorescence quenching [50]. As shown in Table 2,
the values of Kq were 8.6×1012 M−1 s−1 and 1.24×1013 M−1 s−1 for Fe-Ir3 and Fe-Ir4, respectively,
which were almost one order of magnitude higher than that of a pure dynamic quenching
mechanism (<2.0×1012 M-1 s-1) [51]. This conclusion indicated that Fe(II)-Ir(III) heteronuclear
metal complexes could interact with BSA abiding by a static quenching mechanism. Additionally,
binding site number (n) and the binding constant (Kb) were further calculated through the
Scatchard equation (Figs. S15c and S15d). Although the values of n (≈1) were almost the similar,
Fe-Ir4 exhibited the higher binding constant (Kb) than that of Fe-Ir3, which indicated that the
introduction of cis-configurational olefin could decrease the steric hindrance between complex
and BSA. This conclusion is consistent with that of toxicity test that Fe-Ir2 and Fe-Ir4 show the
better anticancer activity than the corresponding trans-configurational ones. In summary, the
stable combination with BSA suggested that BSA is expected to become an excellent carrier for
delivery of anticancer agents in plasma.
Table 2 The values of Ksv, Kb, Kq and n for complexes 1 and 4 at 298 K.
Ksv
(105 M−1)
Kq
(1013 M−1 s−1)
11
Kb
(104 M−1)
n

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0.86±0.05
1.24±0.13
0.86
1.24
5.64
6.50
1.15
1.23
Fe-Ir3
Fe-Ir4
Conclusion
In this paper, four configuration-controlled Fe(II)-Ir(III) heteronuclear metal complexes were
prepared and characterized. The combination of ferrocene monomer and half-sandwich IrIII
complex monomer effectively improved the anticancer activity of target complexes, especially for
the cis-configurational one. Fe-Ir4, the best of these complexes, were almost five times of
cisplatin towards A549 cells under the same conditions. Laser confocal confirms complexes enter
cells followed a non-energy-dependent mechanism, accumulate in lysosomes and induce the
lysosomal damage, and lead to apoptosis. Complexes could decrease the mitochondrial membrane
potential, disturb the cell circle at G2/M phase, catalyze the oxidation of NADH and improve the
levels of intracellular ROS, provide an anticancer mechanism of oxidation. Above all,
Fe(II)-Ir(III) heteronuclear metal complexes may be a promising strategy for constructing
potential organometallic anticancer therapeutic platform.
3 Experimental section
3.1 General information
Iridium trichloride hydrated, 1,2,3,4,5-pentamethyl-cyclopentadiene (95%), Phenyl
magnesium bromide (1.0
M
in THF), 2,3,4,5-tetramethyl-2-cyclopentenone (95%),
ferrocenecarboxaldehyde, 4,4'-dimethyl-2,2'-bipyridine, selenium dioxide, sodium borohydride,
triphenylphosphine hydrobromate, potassium tert-butoxide, ammonium hexafluorophosphate and
all kinds of organic solvents (methylene dichloride, methanol, tetrahydrofuran and 1,4-dioxane
etc.) were purchased from Rhea biotechnology co. LTD. For the biological experiments, DMEM
medium, fetal bovine serum, penicillin/streptomycin mixture and trypsin/EDTA were purchased
from Sangon Biotech. A549 (lung cancer cells) and Hela (cervical cancer cell) were obtained from
Shanghai Institute of Biochemistry and Cell Biology (SIBCB). The appropriate dimer of iridium
(Dmier 1 and Dimer 2) were prepared according to literature procedures [50].
NMR spectra were obtained on Bruker DPX 500 spectrometers instrument, with the chemical
shifts reported in ppm using tetramethylsilane (TMS) as an internal standard. Mass spectrum (MS)
was measured on a Waters Q-TOF MicroTM mass spectrometer. Elemental analysis was performed
on a VarioMICRO CHNOS elemental analyzer. UV-vis spectra and fluorescence spectra were
collected on a PERSEE TU-1901 UV spectrometer and Hitachi F-4600 fluorescence
spectrophotometer, repectively. Induction of apoptosis, cell cycle and mitochondrial membrane
potential (MMP) determination were carried out by an ACEA Novocyte2040R flow cytometry.
Viability assay (MTT) was measured using a Perlong DNM-9606 microplate reader at an
absorbance of 570 nm. Cell uptake and cellular localization were carried out on a Carl Zeiss AG
*/LSM/880NLO two photon laser Scanning microscope.
3.2 Synthesis of ferrocene-modified bipyridine pro-ligands (L1-L2)
L1 and L2 were synthesized by the classical Wittig reaction [28]: Ferrocene methyl
triphenylphosphine hydrobromate (0.98 g, 1.82 mmol) was added into a 100 mL Schlenk flask
under nitrogen. Anhydrous tetrahydrofuran (20 mL) was added to above flask and cooled down to
273 K. Tetrahydrofuran solution (10 mL) of potassium tert-butoxide (0.68 g, 6.05 mmol) was
added dropwise to above flask and stirred for 30 min at 273 K. Then, tetrahydrofuran solution (10
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mL) of 4-formyl-4'-methyl-2,2'-bipyridine (0.30 g, 1.51 mmol) was added and stirred at room
temperature until 4-formyl-4'-methyl-2,2'-bipyridine was consumed completely (monitored by
thin-layer chromatography). The reaction was terminated by ice water (50 mL). Extracted with
ethyl acetate (30 mL×3), dried with anhydrous magnesium sulfate, and distilled under reduced
pressure. The final product was purified by chromatograph on a silica gel column (petroleum
ether: ethyl acetate =30:1 as eluent). The 1H NMR, and ESI-MS of L1 and L2 were shown in Figs.
S1-S2. The data were listed as follows:
L1: Yield: 0.26 g (45.1%). 1H NMR (500 MHz, CDCl3) δ 8.59 (t, J = 4.7 Hz, 2H), 8.43 (d, J
= 20.9 Hz, 1H), 8.29 (s, 1H), 7.30 (dd, J = 8.5, 7.3 Hz, 2H), 7.17 (d, J = 4.6 Hz, 1H), 6.72 (d, J =
16.1 Hz, 1H), 4.55 – 4.50 (m, 2H), 4.39 – 4.34 (m, 2H), 4.16 (s, 5H), 2.46 (s, 3H). ESI-MS (m/z):
calcd for C23H20N2Fe, 380.1, found 381.2 [M+H]+.
L2: Yield: 0.21g (36.5%). 1H NMR (500 MHz, CDCl3) δ 8.56 (dd, J = 4.9, 2.3 Hz, 2H), 8.40
(s, 1H), 8.30 (s, 1H), 7.36 (s, 1H), 7.18 (d, J = 4.5 Hz, 1H), 6.55 (d, J = 11.9 Hz, 1H), 6.41 (d, J =
12.0 Hz, 1H), 4.23 (s, 4H), 4.14 (s, 5H), 2.47 (s, 3H). ESI-MS (m/z): calcd for C23H20N2Fe, 380.1,
found 381.2 [M+H]+.
3.3 Synthesis of Fe(II)-Ir(III) heteronuclear metal complexes (Fe-Ir1 ~ Fe-Ir4)
The general synthesis method is as follows: Dimer of iridium (Dimer 1/Dimer 2, 39.8 mg/46.0
mg, 0.050 mmol) and ferrocene-modified bipyridine (L1 and L2, 38.0 mg, 0.10 mmol) were
added into a 100 mL Schlenk flask under nitrogen, and added methanol (30 mL) and stirred at
ambient temperature overnight. Ammonium hexafluorophosphate (65.2 mg, 0.40 mmol) was
added into the mixture and stirred for 6 h. The solvent was removed in vacuum and recrystallized
from dichloromethane and n-hexane. The 1H NMR, 13C NMR, ESI-MS and elementary analysis of
complexes are shown in Figs. S3-S5. The data were listed as follows:
Fe-Ir1: Yield: 81.8 mg (92.1%). 1H NMR (500 MHz, CDCl3) δ 8.60 – 8.44 (m, 2H), 8.35 (d, J =
20.7 Hz, 2H), 7.53 (d, J = 15.2 Hz, 1H), 7.45 (d, J = 13.0 Hz, 2H), 6.62 (d, J = 14.0 Hz, 1H), 4.83
(s, 2H), 4.61 (s, 2H), 4.34 (s, 5H), 2.66 (s, 3H), 1.67 (s, 15H). 13C NMR (126 MHz, d6-DMSO) δ
155.41, 155.21, 152.77, 152.18, 151.65, 149.03, 139.10, 129.84, 125.38, 125.05, 121.24, 119.32,
89.21, 81.17, 71.21, 69.97, 68.58, 21.30, 8.63. ESI-MS (m/z): calcd for C33H35N2ClFeIr, [M-PF6]+,
743.147, found 743.097; calcd for C33H35N2FeIr, [M-PF6-Cl]2+, 708.178, found 708.125.
Elemental analysis: calcd (%) for C33H35N2ClF6PFeIr: C, 44.63; H, 3.97; N, 3.15; found: C,
44.78.; H, 4.04; N, 3.11.
Fe-Ir2: Yield: 81.0 mg (91.2%). 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J = 4.5 Hz, 1H), 8.50 (d,
J = 5.5 Hz, 1H), 8.26 (s, 1H), 7.97 (s, 1H), 7.67 (d, J = 4.9 Hz, 1H), 7.50 (d, J = 4.2 Hz, 1H), 6.73
(d, J = 10.3 Hz, 1H), 6.43 (d, J = 11.5 Hz, 1H), 4.42 (dd, J = 29.5, 15.1 Hz, 4H), 4.25 (s, 5H), 2.63
(s, 3H), 1.68 (s, 15H). 13C NMR (126 MHz, d6-DMSO) δ 155.40, 155.19, 152.77, 152.15, 151.62,
149.02, 139.10, 129.83, 125.34, 125.04, 121.19, 119.34, 89.20, 81.17, 71.21, 69.96, 68.57, 21.29,
8.62. ESI-MS (m/z): calcd for C33H35N2ClFeIr, [M-PF6]+, 743.147, found 743.076; calcd for
C33H35N2FeIr, [M-PF6-Cl]2+, 708.178, found 708.096. Elemental analysis: calcd (%) for
C33H35N2ClF6PFeIr: C, 44.63; H, 3.97; N, 3.15; found: C, 44.81.; H, 4.03; N, 3.13.
1
Fe-Ir3: Yield: 83.8 mg (88.2%). H NMR (500 MHz, DMSO) δ 8.84 (s, 1H), 8.79 (s, 1H), 8.54
(d, J = 5.6 Hz, 1H), 8.46 (d, J = 5.7 Hz, 1H), 7.80 (d, J = 16.1 Hz, 1H), 7.73 (s, 1H), 7.65 (s, 1H),
7.50 (s, 5H), 6.93 (d, J = 15.9 Hz, 1H), 4.70 (s, 2H), 4.55 (s, 2H), 4.22 (s, 5H), 2.65 (s, 3H), 1.78
(d, J = 9.0 Hz, 6H), 1.69 (s, 6H). 13C NMR (126 MHz, d6-DMSO) δ 155.58, 155.34, 153.06,
151.32, 150.93, 150.91, 149.26, 139.49, 130.47, 129.91, 129.75, 129.43, 125.31, 125.20, 121.03,
13

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119.78, 98.82, 87.32, 86.68, 82.15, 71.27, 69.98, 68.56, 21.30, 9.73, 8.55. ESI-MS (m/z): calcd for
C38H37N2ClFeIr, [M-PF6]+, 805.162, found 806.240; calcd for C38H37N2FeIr, [M-PF6-Cl]2+,
770.194, found 771.304. Elemental analysis: calcd (%) for C38H37N2ClF6PFeIr: C, 48.03; H, 3.93;
N, 2.95; found: C, 48.32; H, 4.05; N, 2.91.
1
Fe-Ir4: Yield: 81.3 mg (85.6%). H NMR (500 MHz, CDCl3) δ 8.36 (d, J = 5.8 Hz, 1H), 8.30 –
8.26 (m, 2H), 7.99 (s, 1H), 7.58 (dd, J = 7.5, 1.9 Hz, 2H), 7.50 (t, J = 6.2 Hz, 4H), 7.36 (d, J = 5.8
Hz, 1H), 6.71 (d, J = 11.5 Hz, 1H), 6.40 (d, J = 11.7 Hz, 1H), 4.46 (s, 1H), 4.41 (s, 2H), 4.34 (s,
1H), 4.24 (s, 5H), 2.60 (s, 3H), 1.77 (d, J = 6.2 Hz, 6H), 1.73 (d, J = 1.3 Hz, 6H). 13C NMR (126
MHz, CDCl3) δ 155.32, 155.15, 153.07, 150.10, 149.91, 149.68, 137.63, 130.32, 129.60, 129.44,
129.29, 128.60, 127.29, 124.97, 123.59, 121.69, 99.56, 99.41, 99.33, 86.77, 85.94, 81.81, 71.27,
69.98, 68.56, 21.47, 9.78, 9.76, 8.52, 8.48. ESI-MS (m/z): calcd for C38H37N2ClFeIr, [M-PF6]+,
805.162, found 805.167. Elemental analysis: calcd (%) for C38H37N2ClF6PFeIr: C, 48.03; H,
3.93; N, 2.95; found: C, 48.31; H, 4.07; N, 2.92.
Acknowledgments
We thank the National Natural Science Foundation of China (21671118 and 21703118), the
Taishan Scholars Program, the Student’s Platform for Innovation and Entrepreneurship Training
Program (2018A043) and High Performance Computing Center of Qufu Normal University for
support.
Supporting Information
Details of the experimental section, Scheme S1, Figures S1−S15, and Tables S1−S7.
Accession Codes
CCDC 1996137 contain the supplementary crystallographic data for this paper. These data can be
obtained 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: +441223 336033.
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DOI: 10.1039/D0DT02408B
Graphical Abstract
Configuration-modified Fe(II)-Ir(III) heteronuclear metal complexes show potential
anticancer activity, especially for cis-configurational one.
1