Electronic effects on reactivity and anticancer activity by half-sandwich N,N-chelated iridium(iii) complexes

Article abstract of DOI:10.1039/C8NJ03360A

The synthesis and characterization of a series of organometallic half-sandwich N,N-chelated iridium(iii) complexes bearing a range of electron-donating and withdrawing substituents were described. The X-ray crystal structures of complexes 1, 3 and 5 have been determined. This work demonstrated how the aqueous chemistry, catalytic activity in converting coenzyme NADH to NAD⁺ and anticancer activity can be controlled and fine-tuned by the modification of the ligand electronic perturbations. In general, the introduction of an electron-withdrawing group (-Cl and-NO₂) on the bipyridine ring resulted in increased anticancer activity, whereas an electron-donating group (-NH₂,-OH and-OCH₃) decreased the anticancer activity. Complex 6 bearing a strongly electron-withdrawing NO₂ group displayed the highest anticancer activity (7.3 ± 1.2 μM), ca. three times as active as cisplatin in the A549 cell line. Notably, selective cytotoxicity for cancer cells over normal cells was observed for complexes 1 and 6. DNA binding does not seem to be the primary mechanism for cancer fighting. However, the aqueous chemistry, cell apoptosis and cell cycle, which show similar dependence on the ligand electronic perturbations as the anticancer activity, appear to together contribute to the anticancer potency of theses complexes. This work may provide an alternative strategy to enhance anticancer activity for these N,N-chelated organometallic half-sandwich iridium(iii) complexes.

Full text of DOI:10.1039/C8NJ03360A

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

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Electronic Effects on Reactivity and Anticancer Activity by
HalfꢀSandwich N, NꢀChelated Iridium(III) Complexes
Lihua Guo, Hairong Zhang, Meng Tian, Zhenzhen Tian, Yanjian Xu, Yuliang
Yang, Hongwei Peng, Peng Liu, Zhe Liu*
The Key laboratory of Life-Organic Analysis and key Laboratory of Pharmaceutical
Intermediates and Analysis of Natural Medicine, Institute of Anticancer Agents
Development and Theranostic Application, Department of Chemistry and Chemical
Engineering, Qufu Normal University, Qufu 273165, China.
*Corresponding author. Email: liuzheqd@163.com.
ABSTRACT: The synthesis and characterization of a series of organometallic
halfꢀsandwich N, Nꢀchelated iridium(III) complexes bearing a range of electronꢀdonating
and withdrawing substituents were described. The Xꢀray crystal structures of complexes
1, 3 and 5 have been determined. This work demonstrated how the aqueous chemistry,
catalytic activity in converting coenzyme NADH to NAD+ and anticancer activity can be
controlled and fineꢀtuned by the modification of the ligand electronic perturbations. In
general, the introduction of an electronꢀwithdrawing group (ꢀCl and ꢀNO2) on the
bipyridine ring resulted in increased anticancer activity, whereas electronꢀdonating group
(ꢀNH2, ꢀOH and ꢀOCH3) decreased anticancer activity. Complex 6 bearing strongly
electronꢀwithdrawing NO2 group displayed the highest anticancer activity (7.3 ± 1.2 ꢁM),
ca. three times as active as cisplatin in the A549 cell line. Notably, a selective cytotoxicity
for cancer cells over normal cells was observed for complexes 1 and 6. DNA binding
does not seem to be the primary mechanism for cancer fighting. However, the aqueous
chemistry, cell apoptosis and cell cycle, which show the similar dependence on the ligand
electronic perturbations as the anticancer activity, appear to together contribute to the
anticancer potency of theses complexes. This work may provide an alternative strategy to
enhance anticancer activity for these N, Nꢀchelated organometallic halfꢀsandwich
iridium(III) complexes.
INTRODUCTION
In a search for less toxic and more potent alternatives to cisplatin,1ꢀ3 organometallic

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complexes have recently been shown to be promising anticancer agents due to their
versatile structures, potential redox features, and wide range of ligand substitution
rates.4ꢀ18 Among these compounds, halfꢀsandwich iridium(III) complexes bearing
bidentate N, N or C, N ligands received great attention for their redoxꢀmediated
mechanism of actions (MoAs) different from platinumꢀbased drugs.19ꢀ33
The Sadler
groups
have developed
two
kinds
of halfꢀsandwich
pentamethylcyclopentadienyl (Cp*) iridium(III) complexes containing neutral N,
Nꢀchelated ligands and anionic C, Nꢀchelated ligands respectively (Scheme 1, I and
II).29ꢀ31 On the one hand, the biological activity of these complexes increases by the
incorporation of phenyl substituents on Cp*. The hydrophobicity and intercalative ability
of extended cyclopentadienyl systems make the major contribution to the anticancer
activity of their iridium(III) complexes.31 On the other hand, the electronic and steric
properties of the bidentate ligands can also have a large effect on the chemical and
biological activities.30, 32, 33 For example, Sadler et al. have investigated the effects of
electronꢀdonating and electronꢀwithdrawing substituents on anionic C, Nꢀchelated ligands
on the physiochemical and biological activity of their complexes (II).30 The anticancer
activity also can be fineꢀtuned by varying the monodentate pyridine derivatives (III). The
introduction of an electronꢀdonating group on the pyridine ring resulted in increased
anticancer activity.33 These studies have shown that the anticancer activity of these
complexes is strongly dependent on the type of substituent and its position on the ligand,
thus providing a new strategy that could be beneficial for the development of these C,
Nꢀchelated ligandꢀbased complexes. However, the effect of incorporating functionality on
the N, Nꢀchelated ligand in halfꢀsandwich iridium(III) anticancer complexes has not been
widely explored. Because of halfꢀsandwich Cp* iridium(III) complexes’ wide applications
in catalysis,34ꢀ36 previous studies have investigated the electronic effects on the catalytic
disproportionation of formic acid to methanol by halfꢀsandwich Cp* iridium(III)
complexes.36 These results encouraged us to systematically explore the ligand electronic
effects on reactivity and cancer cell cytotoxicity of halfꢀsandwich iridium(III) anticancer
complexes containing neutral N, Nꢀchelated ligands of 4, 4′ꢀdiꢀRꢀ2, 2′ꢀbipyridine (R =
electronꢀdonating and electronꢀwithdrawing groups). In this work, six halfꢀsandwich N,
Nꢀchelated iridium(III) complexes bearing a range of electronꢀdonating and withdrawing

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substituents (ꢀNH2, ꢀOH, ꢀOCH3, ꢀH, ꢀCl or ꢀNO2) were synthesized and characterized.
These complexes have been investigated for their aqueous chemistry, electrostatic
potential surfaces (EPS), nucleobase binding and cancer cell toxicity against A549 cancer
cells. The work also explores the MoA of these iridium(III) complexes by catalytic
hydride transfer analysis, cell cycle, apoptosis and ROS (reactive oxygen species).
Scheme 1. Halfꢀsandwich Iridium(III) Complexes and Their Substituents Electronic
Effect on Anticancer Activity
RESULTS AND DISCUSSIONS
Scheme 2. Synthesis of Iridium(III) Complexes Studied in This Work
Syntheses. The synthetic routes for the complexes 1ꢀ6 are shown in Scheme 2. [Cpbiph
IrCl2]2 (Cpbiph =η5ꢀC5Me4C6H4C6H5) were synthesized by microwave heating of
IrCl3·nH2O and relative cyclopentadienyl ligand.37 These complexes were synthesized in
moderate yields by reaction of bipyridine ligands (bpy) with the dinuclear iridium

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ꢀ
precursors in methanol at ambient temperature. All complexes were isolated as PF6 salts.
The introduction of electronꢀdonating groups (ꢀNH2, ꢀOH and ꢀOCH3) and
electronꢀwithdrawing groups (ꢀCl and ꢀNO2) in complexes 1ꢀ3, 5 and 6 is the first to be
reported in complexes of the type [CpbiphIr(N^N)Cl]PF6. All of the synthesized complexes
were fully characterized by 1HNMR (Figure S2ꢀS6 in the Supporting Information), mass
spectroscopy (Figure S7ꢀS11 in the Supporting Information), FTIR (Figure S12ꢀS16 in
the Supporting Information) and CHN elemental analysis. Single crystals of complexes 1,
3 and 5 suitable for Xꢀray diffraction were also obtained by slow evaporation of a
methanol/diethyl ether solution at ambient temperature.
Figure 1. Xꢀray crystal structures with atom numbering schemes for complexes 1, 3 and
5 with thermal ellipsoids drawn at 50% probability. The hydrogen atoms and
ꢀ
counteranion PF6 have been omitted for clarity.
Xꢀray Crystal Structures. The molecular structures of [CpbiphIr(bpyꢀNH2)Cl]PF6 (1),
[CpbiphIr(bpyꢀOCH3)Cl]PF6 (3) and [CpbiphIr(bpyꢀCl)Cl]PF6 (5) were determined by
Xꢀray diffraction and are shown in Figure 1. CCDC numbers of 1, 3 and 5 are 1569686,
1569665 and 1569685, respectively. The selected bond lengths and angles are shown in
Table 1 and crystallographic data are listed in Table S1. Complexes 1, 3 and 5 exhibit the
expected halfꢀsandwich pseudoꢀoctahedral “threeꢀleg pianoꢀstool” geometry, similar to
reported complex 4.29 The distances of iridium bound to η5ꢀcyclopentadienyl ring
centroid for complexes 1, 3 and 5 are 1.781, 1.814 and 1.795 Å, respectively. The
complex 1 has the longest IrꢀCl bond length of these three Xꢀray structures. The IrꢀN1
(chelating ligand) bond lengths in 1, 3 and 5 are 2.098 (5), 2.095 (8) and 2.115 (5) Å,
respectively and the IrꢀN2 bond lengths are 2.103 (5), 2.082 (8) and 2.095 (5) Å,
respectively. These results suggest that the introduction of electronꢀdonating and

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electronꢀwithdrawing groups on the bipyridine can have a certain degree of influence on
the structure.
Table 1. Selected Bond Lengths (Å) and Angles (deg) for Complexes 1, 3 and 5.
1
3
5
Ir−C(cyclopentadienyl) 2.147(6)
2.189(12)
2.187(11)
2.177(10)
2.184(10)
2.179(11)
2.155(6)
2.169(6)
2.170(6)
2.178(7)
2.201(7)
2.152(6)
2.159(7)
2.173(6)
2.186(7)
Ir−C(centroid)
Ir−N1
1.7813
1.8135
1.7948
2.098(5)
2.103(5)
2.4103(18)
76.62(19)
84.77(16)
84.54(15)
2.095(8)
2.082(8)
2.244(10)
76.7(3)
83.0(3)
84.4(4)
2.115(5)
2.095(5)
2.3856(18)
76.1(2)
Ir−N2
Ir−Cl
N1−Ir−N2
N1−Ir−Cl
N2−Ir−Cl
85.65(15)
87.30(16)
Electrostatic potential surfaces. The electrostatic potentials surfaces (EPSs) of
complexes 1ꢀ6 were calculated performing in the framework of density functional theory
(DFT) by using of the hybrid B3LYP function implemented in the Gaussian 09 program
package.38 We select the Los Alamos LANL2DZ effective nuclear pseudopotentials
(ECPs) and valence bisꢀzeta group for the iridium atom and the 6ꢀ31+G (d, p) basis group
for the carbon, nitrogen, chlorine, oxygen and hydrogen atoms.39, 40 The structures of the
complexes were fully optimized without any symmetry constraints. Charge delocalization
was performed using natural bond orbital (NBO) analysis.41 The resulting EPS of each
complex is shown in Figure 2. There are no major differences in the charge distribution at
the iridium center, Cpbiph ring and chloride ligands among the complexes 1ꢀ6. The
electronꢀdonating and electronꢀwithdrawing substituent on the chelating ligand only
causes a localized effect. The EPS of the chelating ligand are affected by enhancing or
diminishing the electronic charge density on the bipyridine ring to which the substituent
is bound. Complexes 5 and 6 bearing the electronꢀwithdrawing groups provide a more
positive EPS within the ring, while complexes 1ꢀ3 containing the electronꢀdonating
groups generate a more negative EPS within the ring. Weak electrostatic forces are often

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important for binding and recognition interactions with biomolecules such as peptides,
proteins, and enzymes.42 As a result, the nature of the substituent may play a key role in
the interaction of the complexes with target sites.
Figure 2. EPSs of the complexes 1ꢀ6. EPSs surfaces are shown in both space (with
positive and negative regions in blue and red, respectively) and mapped on electron
density (isovalue 0.004) of the molecules. The electrostatic potential is represented with a
color scale going from red (ꢀ0.15 au) to blue (+0.15 au).
Scheme 3. Behaviour of Complexes 1ꢀ6 in Methanol/Water Solutions.
2+
2+
+
Cpbiph
Cpbiph
Cpbiph
[XꢀH2O]2+
or
Solv 2
Solv 1
Ir H2O
N
Ir Cl
N
Ir H2O
N N
ESIꢀMS
ꢀ H2O or H3O+
N
N
[XꢀH2OꢀH]+
×
detected by
ESIꢀMS
R
R
R
R
R
R
X=CpbiphIr(N^N)H2O
Solv 1=CH3OH:H2O 1:9 v/v
Solv 2=CH3OH:H2O 5:5 v/v
Hydrolysis Studies. Since MꢀOH2 complexes often show higher activity than the
corresponding chlorido complexes.43, 44 Hydrolysis of MꢀCl bonds is considered to be an
activation step for anticancer complexes.45 Due to limited solubility in water, the
hydrolysis studies on complexes 1ꢀ6 were carried out in 50% CD3OD /50% D2O (v/v)
solutions by 1HNMR at 298 K. No hydrolysis was observed by 1HNMR spectroscopy. In
1
addition, no obvious changes in the HNMR spectra of complexes 1ꢀ6 can be observed

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1
over 24 h. It is possible that the aqua complex was obtained before the HNMR spectra
were acquired. Therefore, NaCl (20 mol equiv) was then added to the solutions to further
1
confirm the hydrolysis of these complexes. There was also no change in the HNMR
spectra (Figure S17ꢀS23 in the Supporting Information). In addition, when AgNO3 (0.95
mol equiv) was added to the same solution of complex 3, the peaks of aqua complex was
observed after 10 h. These results indicated that the complexes were stable towards
hydrolysis under these conditions (Figure S20 in the Supporting Information). Previous
studies have shown that the hydrolysis of the similar complexes can be detected by
UVꢀVis spectroscopy.29 As a result, the hydrolysis of complexes 1ꢀ6 in 10% MeOH/90%
H2O (v/v) solution was also monitored by ESIꢀMS and UVꢀVis (Figure S24ꢀS35 in the
Supporting Information). As shown in Scheme 3, metal species [XꢀH2O]2+ or [XꢀH2OꢀH]
+(X = CpbiphIr(N^N)H2O), which arise from the Clꢀ/H2O exchange, were detected by
ESIꢀMS. The aqua adducts [CpbiphIr(N^N)H2O]2+ may lose a H2O or H3O+ in the testing
process.46 Furthermore, the NMR analysis mentioned above showed that the complexes
in waterꢀmethanol solution were still Cpbiphꢀ and bpyꢀbound Ir(III) species and did not
suffer from decomposition or ligand dissociation. These results suggested that the
hydrolysis of complexes 1ꢀ6 occurred in 10% MeOH/90% H2O (v/v) solution. Thus, the
absorbance changes in UVꢀVis spectra can be ascribed to the production of aqua adducts.
However, when 50% MeOH/50% H2O (v/v) solution was employed, no absorbance
changes were observed in UVꢀVis spectra (Figures S24(B)ꢀS29(B) in the Supporting
Information), which indicated that no hydrolysis occurred under these conditions. This
result was consistent with NMR analysis. It seems reasonable that, in accordance with the
behavior of some previously reported halfꢀsandwich metal complexes,47ꢀ49 complexes 1ꢀ6
may undergo Clꢀ/H2O exchange more easily in diluted solutions with a higher relative
content of water, as is the case of cell culture. Hydrolysis rate constants and halfꢀlives can
be calculated (Table 2) by fitting the UVꢀVis absorption difference to pseudo firstꢀorder
kinetics. To a certain extent，the hydrolysis of IrꢀCl bonds is influenced by the electronic
perturbation. Basically, the halfꢀlives of hydrolysis were observed to be lower for the
complexes with electronꢀwithdrawing substituents. Complex 3 containing the
electronꢀdonating OCH3 group showed the longest halfꢀlife (78.0 ± 6.0 min, Table 2),
which is almost ca. 13 times as that of complex 6 containing the electronꢀwithdrawing

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NO2 group. This result may attribute to the shortest IrꢀCl bond of complex 3 (2.244 Å),
which facilitate chloride loss.
Table 2. Hydrolysis Data for Complexes 1ꢀ6 at 298K Monitored by UVꢀVis.
Complex
k ( minꢀ1 )
0.023
t1/2 ( min )
31.1 ± 3.2
54.0 ± 1.0
78.0 ± 6.0
70.2 ± 5.6
29.0 ± 2.8
6.0 ± 0.8
1
2
3
4
5
6
0.013
0.009
0.010
0.024
0.117
Cytotoxicity. The cytotoxicity of complexes 1ꢀ6 in A549 human lung cancer cells was
shown in the Table 3. The IC50 value (concentration at which 50% of the cell growth is
inhibited) covered a wide range from 7.3 to 35.2 ꢁM in A549 cells, indicating that the
electronic perturbations exhibit some variation on the cytotoxicity of the complexes. The
IC50 value revealed the following trends in activity: NO2 > Cl > H > OCH3 > NH2 > OH.
It is worth noting that the presence of electronꢀwithdrawing groups in complexes
increases the cytotoxicity, which is opposite to the reported C, Nꢀchelated iridium
complexes33 (Scheme 1, III) with a substituent on the monodentate pyridine ring.
Complex 6 bearing electronꢀwithdrawing NO2 group displays the most potent anticancer
activity (7.3 ± 1.2 ꢁM) compared to other complexes 1ꢀ5. In particular, IC50 values of
complex 6 are ca. three times as active as cisplatin in the A549 cell line (7.3 ± 1.2 ꢁM Vs
21.3 ± 1.7 ꢁM), demonstrating great potential in cancer chemotherapy. Previously studies
have shown that iridium bipydineꢀchelated half sandwich complexes can hydrolyze
rapidly and may have a major contribution to anticancer activity.29 In our system,
although the structure activity relationships are not very significant, the complexes
containing the electronꢀwithdrawing groups seem to have the faster hydrolysis rate and
the higher anticancer activity than that containing the electronꢀdonating groups,
indicating that activation by aquation is crucial to the MoA of these iridium(III)
complexes.
The cytotoxicity of complexes 1ꢀ6 were further studied against two human bronchial
epithelial normal cells BEASꢀ2B and 16HBE, in the Table 3. Interestingly, complexes 1
and 6 both exhibited much less cytotoxicity towards normal cells than cancer cells.
Notably, complex 6 achieved good selectivity with IC50 ratio of 3.0 and 5.9 of BEASꢀ2B

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and 16HBE normal cells to A549 cancer cells. However, complexes 1 and 6 are still
active against human bronchial epithelial cells BEASꢀ2B and 16HBE normal cells with
IC50< 75 µM. Hence, more structural modification is necessary to decrease the cytotoxic
action towards normal cells without loss the selectivity between cancer and normal cells
in the future work.
Table 3. Inhibition of Growth of A549 Human Lung Cancer Cells by Complexes 1–6 and
Comparison with Cisplatin; Complexes 1, 4, 6 and Cisplatin Comparison with BEASꢀ2B
and 16HBE.a
IC50 (ꢁM)
A549
IC50 (ꢁM)
BEASꢀ2B
IC50 (ꢁM)
16HBE
Complex
R1
R2
30.7 ± 1.4
35.2 ± 1.4
14.9 ± 0.3
14.3 ± 1.2
11.3 ± 0.9
7.3 ± 1.2
70.3 ± 1.9
28.1 ± 3.1
14.7 ± 1.3
14.3 ± 1.3
5.0 ± 0.4
74.6 ± 0.6
19.6 ± 1.4
26.6 ± 1.2
16.7 ± 1.7
3.9 ± 0.1
2.3
0.7
0.9
1.0
0.4
3.0
2.0
2.4
0.5
1.8
1.2
0.3
5.9
0.9
1
2
3
4
5
6
21.7 ± 2.3
42.0 ± 2.3
43.0 ± 3.7
18.6 ± 2.0
Cisplatin
21.3 ± 1.7
a
Cells were treated with various concentrations of tested compounds for 24 h. Cell
viability was determined by the MTT assay and IC50 values were calculated as
described in the Experimental Section. Each value represents the mean SD of three
independent experiments. R1 represents the ratio of IC50 of BEASꢀ2B normal cells to
A549 cancer cells. R2 represents the ratio of IC50 of 16HBE normal cells to A549 cancer
cells.
Interaction with Nucleobases. DNA often represents a potential target site for many
transition metal anticancer complexes,50, 51 complexes 1ꢀ6 were evaluated for their ability
to bind to 9ꢀethylguanine (9ꢀEtG) and 9ꢀMethyladenine (9ꢀMeA). 9ꢀMeA or 9ꢀEtG was
added to an equilibrium solution of complexes 1ꢀ6 (1.0 mM) in 50% CD3OD /50% D2O
1
(v/v) at 310 K. No new peaks were detected in the HNMR over a period of 24 h (Figure
S36ꢀS38 in the Supporting Information), indicating that complexes 1ꢀ6 did not show any
binding to model nucleobases 9ꢀMeA under these conditions (Table S3 in the Supporting
Information). Also, the formation of nucleobase adducts by these iridium(III) complexes
was not detected by mass spectrometry. So DNA may not be the main target for this type
of iridium(III) complexes and the regulation of the electronic effect also does not affect

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their ability to bind to DNA. Nevertheless, considering the effect of water content on
hydrolysis, it is highly possible that interaction with nucleobases can occur in diluted
solutions with a higher relative content of water.
Cleavage plasmid DNA. We also studied the DNA binding ability of complexes 1, 6
through agarose gel electrophoresis of supercoiled pBR322 plasmid DNA in buffer.
However, no DNA cleavage can be observed for these iridium(III) complexes even at a
high concentration of 150 µM. Likewise, DNA may not be the major target of these
complexes. (Figure S39ꢀS40 in the Supporting Information).
Reaction with NADH. Coenzyme NADH and NAD+ plays a crucial role in a series of
biocatalyzed processes. Previously, we have demonstrated that NADH can donate a
hydride to aqua C, Nꢀchelated iridium(III) complexes and produce ROS in the form of
H2O2 , thus providing a catalytic behavior to an oxidant mechanism of action.52, 53 As a
result, reactions between iridium(III) complexes 1ꢀ6 and NADH were studied (Figure 3).
The reaction of a 0.9 mM solution of complex 1 with NADH (3.6 mol equiv.) in 50%
1
CD3OD /50% D2O (v/v) at 298 K was monitored by HNMR spectroscopy over a 7 h
period. After 2 h, a sharp singlet peak at ꢀ10.91 ppm, which is corresponding to IrꢀH
1
species, was observed. After 7 h, this peak was still visible. A new set of peaks in the H
NMR spectra corresponding to the formation of NAD+ was also observed. In addition,
the reaction of complexes 1ꢀ6 with NADH in 1: (ca. 90) ratio in 10% MeOH/90% H2O
(v/v) was also monitored by UVꢀVis at 298 K in order to examine catalytic ability (Figure
S41 in the Supporting Information). The conversion of NADH to NAD+ can be measured
by the amount of UV absorption at 339 nm as NADH has an absorption at 339 nm while
NAD+ not. The turnover numbers (TONs) of complexes calculated by the amount of UV
absorption at 339 nm (Figure 3) revealed the following trends in catalytic activity: 1
(35.3), 2 (1.4), 3 (4.4), 4 (1.0), 5 (5.3) and 6 (36.3), NO2 > NH2 > Cl > OCH3 > OH > H.
Despite no clear trend was observed, it is apparent that ligand electronic effects were
pronounced. Complex 6 exhibits the best catalytic activity, which is 8 times as much as
that of complex 3. The presence of NH2 and NO2 group in complexes 1 and 6 seems to
enhance the catalytic activity. Very good catalytic activity of the oxidation of NADH was
achieved, with TONs 35.3 for complex 1 and 36.3 for complex 6. As these complexes can
covert NADH to NAD+, the good catalytic performance may provide a potential pathway

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to induce of ROS and enhance the killing of cancer cells in an oxidant mechanism of
action.52 In a solution of 6 (0.5 mM) with NADH (3 mol equiv) in 50% MeOH/ 50%H2O
(v/v) at 298 K, the ROS hydrogen peroxide was detected by the appearance of a blue
color on hydrogen peroxideꢀtest paper. It is found that the concentration of H2O2 is about
0.20 mM, and its level may be limited by the oxygen solubility (ca. 0.23 mM at 288 K).52
However, complexes 1 and 6, which had the opposite electronic contribution, behaved the
similar catalytic activity toward NADH reaction. This result suggested the formation of
IrꢀH did not play a vital role in killing cancer cells in this system. It is expected that the
anticancer activity of these complexes is more dependent on other pathways such as the
cell cycle arrest.
Figure 3. (A) 1HNMR spectra of the reaction between complex [Cpbiph
Ir(bpyꢀNH2)Cl]PF6 (1) (0.9 mM) and NADH (3.6 mol equiv.) in 50% CD3OD /50% D2O
(v/v) at 298 K, 5 min; 2.0 h; 7.0 h. Left: lowꢀfield region; right: highꢀfield region showing
the IrꢀH hydride peak (ꢀ10.91 ppm). (B) The normalized UVꢀVis spectra of the reaction of
NADH (ca. 90 ꢁM) with complex [CpbiphIr(bpyꢀNH2)Cl]PF6 (1), [Cpbiph Ir (bpyꢀNO2)ꢀ
ꢀCl]PF6 (6) (1 ꢁM) respectively in 10% MeOH / 90% H2O (v/v) at 298 K for 8 h. (C) The
turnover numbers (TONs) of complexes 1ꢀ6.

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Apoptosis Assay. Complex 1 containing electronꢀdonating NH2 group and complex 6
containing electronꢀwithdrawing NO2 group were selected for more investigation on their
mechanism of actions. A lot of metalꢀbased anticancer drugs have been reported to inhibit
cell growth by inducing apoptosis, which is regard as a programmed cell death
process.54ꢀ62 Hence, complexes 1 and 6 at 0.5, 1 and 2 equipotent concentrations of IC50
were used for the treatment of A549 human lung cancer cells for 24 h, which were then
stained with Annexin V/Propidium Iodide and analyzed by flow cytometry. Cell
populations as viable, early apoptosis, late apoptosis and nonviable can be determined
through this experiment. The results were shown in Figure 4 and Table S4 in the
supporting information. In general, cells in apoptosis increased with the concentrations of
iridium(III) complexes. The plots showed that around 16.8% A549 cells were in early
apoptotic phase and 28.9% cells were in the late apoptotic phase after 24 h of exposure to
complex 1 at a concentration of IC50. When the complex 1 is at 2 × IC50 concentrations,
52.3% A549 cells were in apoptosis phase (the total proportion of early and late apoptotic
cells), whereas untreated cells remained 90.4% viable, indicating obvious induction of
apoptosis at 2 × IC50 concentrations. Complex 6 showed the similar mode of action. The
proportion of early and late apoptotic cells was 22.9% and 37.6%, respectively at a
concentration of IC50 after 24 h. At 2 × IC50 concentrations, a total of 83.1% (the total
proportion of early and late apoptotic cells) cells were undergoing apoptosis. Therefore,
the complex 6 containing the electronꢀwithdrawing NO2 groups has the stronger ability to
induce apoptosis of A549 cells, which is consistent with its higher anticancer activity
compared to complex 1 containing the electronꢀdonating NH2 groups.
Figure 4. Apoptosis analysis of A549 cells after 24 h of exposure to complexes 1 and 6 at
310 K determined by flow cytometry using Annexin VꢀFITC vs PI staining. (A) A549

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cells were control and treated with different concentrations of complexes 1 and 6 for 24 h;
(B) Populations for cells treated by complexes 1 and 6.
Cell Cycle Analysis. Through the cell cycle analysis, we can determine whether the
induced cell growth inhibition was the result of cell cycle arrest.63 Hence, cell cycle arrest
analysis for complexes 1 and 6 toward A549 human lung cancer cells was performed by
flow cytometry (Table 5 and Table S5ꢀS6 in the Supporting Information). Treating of
A549 cells with complex 1 at a concentration of 0.25 × IC50 and 0.5 × IC50 led to G1
phase arrest, where the percentages of cells increased 1.5% and 5.9% respectively
compared to untreated cells. This small change was deemed as a very weak cell cycle
arrest. In comparison with complex 1, complex 6 displayed stronger cell cycle arrest
ability and showed significant increases in the proportions of cells at G2/M phase, about
5.9% and 10.8% respectively at a concentration of 0.25 × IC50 and 0.5 × IC50. In addition,
a small subꢀdiploid peak corresponds to subꢀG1 phase change was observed for complex
6, indicating that cells are undergoing apoptosis. In consistent with apoptosis assay
analysis, introducing electronꢀwithdrawing groups to the complexes can enhance the cell
cycle arrest.
Figure 5. Cell cycle analysis of A549 cells after 24 h of exposure to complexes 1 and 6 at
310 K. Concentrations used were 0.25 and 0.5 equipotent concentrations of IC50. Cell
staining for flow cytometry was carried out using PI/RNase. (A) FL2 histogram for
negative control (cells untreated) and complexes 1 and 6. (B) Cell populations in each
cell cycle phase for control and complexes 1 and 6.
Induction of ROS in A549 Human Lung Cancer Cells. High concentration of

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reactive oxygen species (ROS) often result in oxidative stress and damages to cells.64 The
level of reactive oxygen species (ROS) in A549 human lung cancer cells induced by
complexes 1 and 6 at concentrations of 0.25 × IC50 and 0.5 × IC50 are also determined by
flow cytometry analysis (Figure 6 and Table S7ꢀS8 in the Supporting Information).
Compared to untreated cells, a dramatic increase in the ROS levels was observed for
complexes 1 and 6 even at low concentration (0.25 × IC50) after 24 h of drug exposure.
The generation of ROS by the complexes may provide a basis for killing cancer cells. It
should be noted that the increased ROS levels in cancer cells by these iridium(III)
complexes may be attributed to the catalytic reaction of NADH to NAD+. On the one
hand, the possible chemical mechanism of induction of ROS induced by iridium(III)
complexes has been reported previously.52 On the other hand, complexes 1 and 6,
which displayed the similar catalytic activity toward the reaction of NADH to NAD+,
also generated the similar amounts of ROS. However, considering the different anticancer
activity of complexes 1 and 6, producing ROS also seems to not be a major contributor to
the cytotoxicity in this system.
Figure 6. Flow cytometry analysis on ROS induction in A549 human lung cancer cells
treated with complex 1 and 6.
CONCLUSION
In summary, a series of N, Nꢀchelated iridium complexes bearing a series of

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electronꢀdonating and ꢀwithdrawing substituents were synthesized. The aim is to
systematically investigate the ligand electronic effects on reactivity and cancer cell
cytotoxicity in this class of halfꢀsandwich iridium(III) complexes. The Xꢀray crystal
structures of complexes 1, 3 and 5 were obtained. DFT calculations showed that
substituents caused only localized effects on the electrostatic potential surface of the
complexes. The more rapid hydrolysis rate of IrꢀCl bonds was obtained with complexes
bearing more strongly electronꢀwithdrawing groups. No nucleobase binding was
observed for this type of complexes under the conditions mentioned in the experiment.
All complexes showed promising activity toward the A549 human lung cancer cell,
comparable to, and for some complexes even higher than the clinical anticancer drug
cisplatin. The ligand electronic structure of the complexes had some influence on the
anticancer activity. Complex 6 bearing strongly electronꢀwithdrawing NO2 group
displayed the highest anticancer activity, ca. three times as active as cisplatin in the A549
cell line, but even more importantly, complexes 1 and 6 displayed the anticancer
selectivity. The work also explores the mechanism of actions (MoAs) of these iridium(III)
complexes by cell apoptosis and cycle. Both two experiments also exhibited the same
trend that the electronꢀwithdrawing groups on the bipyriding ring led to better effect than
the electronꢀdonating groups. In addition, these iridium complexes can also induce a
dramatic increase in the level of ROS in A549 cancer cells.
Pervious work has shown that the biological activity of these complexes increases by
the incorporation of phenyl substituents on Cp*.29, 31 This study shows that the electronic
changes in the bipyridine in halfꢀsandwich iridium complexes can also have an effect on
the chemical and biological activity of these complexes. This may provide an alternative
strategy to enhance anticancer activity for these N, Nꢀchelated organometallic
halfꢀsandwich iridium anticancer complexes.
EXPERIMENTAL SECTION
Materials.
IrCl3·nH2O,
2,3,4,5ꢀtetramethylꢀ2ꢀcyclopentenone
(95%),
4ꢀbromoꢀbiphenyl, butyllithium solution (1.6 M in hexane), 4, 4′ꢀdimethoxyꢀ2,
2′ꢀbipyridine, 2, 2′ꢀbipyridine, concentrated sulfuric acid, fuming nitric acid, 10% Pd/C,
hydrazine hydrate, ammonium hexafluorophosphate and NaCl, anhydrous MgSO4, HCl

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(36%), HBr solution (41 %), hexane, diethyl ether, ethanol, dry THF, 9ꢀethylguanine and
9ꢀmethyladenine were purchased from SigmaꢀAldrich. CpxbiphH, [CpbiphIrCl2]2 and
complex 4 were synthesized according to the reported methods.29, 37 For the biological
experiments, phosphateꢀbuffered saline (PBS), penicillin/streptomycin mixture, fetal
bovine serum, trypsin/EDTA and DMEM medium were purchased from Sangon Biotech..
Testing compounds was dissolved in DMSO and diluted with the tissue culture medium
before use.
[CpbiphIr(bpyꢀNH2)Cl]PF6 (1). A solution of [CpbiphIrCl2]2 (108 mg, 0.1 mmol) and 4,
4′ꢀdiaminoꢀ2, 2′ꢀbipyridine (47.4 mg, 0.25 mmol) in MeOH was heated to reflux in an N2
atmosphere for 16 hours, cooled and filtered. The volume of the solution was slowly
reduced to a small amount on a rotary evaporator, NH4PF6 (135 mg, 0.8 mmol) was
added and after stirring at 277 K for 0.5 h, a precipitate formed in the solution. It was
collected by filtration, washed with ether and recrystallized from methanol/ether. Yield:
52 mg (32%). A single crystal was obtained by slow evaporation of a methanol/diethyl
ether solution at room temperature. 1H NMR (500 MHz, DMSOꢀd6): δ 8.00 (d, J = 6.6 Hz,
ArꢀH, 2H), 7.75 (t, J = 7.1 Hz, ArꢀH, 4H), 7.53ꢀ7.47 (m, ArꢀH, 4H), 7.41 (t, J = 7.4 Hz,
ArꢀH, 1H), 7.27 (s, NH2, 4H), 7.23 (d, J = 2.5 Hz, ArꢀH, 2H), 6.71 (dd, J = 6.6, 2.5 Hz,
ArꢀH, 2H), 1.73 (s, CpbiphꢀH, 6H), 1.66 (s, CpbiphꢀH, 6H). Anal. Calcd for
[CpbiphIr(bpyꢀNH2)Cl]PF6 (832.25): C, 44.74; H, 3.75; N, 6.73. Found: C, 44.62; H, 3.71;
N, 6.76. MS: m/z 687.02 [CpbiphIr(bpyꢀNH2)Cl]+. IR (KBr pellets, cmꢀ1): 3461 (NH2),
3361 (NH), 1640 (NH), 1318 (CꢀN).
[CpbiphIr(bpyꢀOH)Cl]PF6 (2). The synthesis was performed as for 1 using
[CpbiphIrCl2]2 (72 mg, 0.07 mmol) and 4, 4′ꢀdihydroxyꢀ2, 2′ꢀbipyridine (30.7 mg, 0.16
mmol). Yield: 63 mg (56%). 1HNMR (500 MHz, DMSOꢀd6): δ 7.99 (s, OH, 2H), 7.74 (s,
ArꢀH, 4H), 7.53 (d, J = 8.0 Hz, ArꢀH, 3H), 7.48 (d, J = 7.2 Hz, ArꢀH, 2H), 7.40 (s, ArꢀH,
2H), 7.28 (s, ArꢀH, 2H), 6.60 (s, ArꢀH, 2H), 1.71 (s, CpbiphꢀH, 6H), 1.64 (s, CpbiphꢀH, 6H).
Anal. Calcd for [CpbiphIr(bpyꢀOH)Cl]PF6 (834.21): C, 44.63; H, 3.50; N, 3.36. Found: C,
44.51; H, 3.46; N, 3.41. MS: m/z 689.00 [CpbiphIr(bpyꢀOH)Cl]+. IR (KBr pellets, cmꢀ1):
3445 (ꢀOH), 1036 (CꢀO).
[CpbiphIr(bpyꢀOCH3)Cl]PF6 (3). The synthesis was performed as for 1 using
[CpbiphIrCl2]2 (109 mg, 0.1 mmol) and 4, 4′ꢀdimethoxyꢀ2, 2′ꢀbipyridine (52.5 mg, 0.24

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mmol). Yield: 127 mg (72%). A single crystal was obtained by slow evaporation of a
1
methanol/diethyl ether solution at room temperature. H NMR (500 MHz, DMSOꢀd6): δ
8.48 (d, J = 6.6 Hz, ArꢀH, 2H), 8.42 (d, J = 2.7 Hz, ArꢀH, 2H), 7.82 (d, J = 8.3 Hz, ArꢀH,
2H), 7.77 (d, J = 7.4 Hz, ArꢀH, 2H), 7.61 (d, J = 8.2 Hz, ArꢀH, 2H), 7.51 (t, J = 7.7 Hz,
ArꢀH, 2H), 7.44ꢀ7.39 (m, ArꢀH, 3H), 4.07 (s, OCH3, 6H), 1.76 (s, CpbiphꢀH, 6H), 1.71 (s,
CpbiphꢀH, 6H). Anal. Calcd for [CpbiphIr(bpyꢀOCH3)Cl]PF6 (862.27): C, 45.97; H, 3.86; N,
3.25. Found: C, 45.85; H, 3.81; N, 3.28. MS: m/z 717.02 [CpbiphIr(bpyꢀOCH3)Cl]+. IR
(KBr pellets, cmꢀ1): 1384 (CꢀH), 1047 (CꢀO).
[CpbiphIr(bpyꢀCl)Cl]PF6 (5). The synthesis was performed as for 1 using
[CpbiphIrCl2]2 (109 mg, 0.1 mmol) and 4, 4′ꢀdichloroꢀ2, 2′ꢀbipyridine (55.0 mg, 0.24
mmol). Yield: 144 mg (81%). A single crystal was obtained by slow evaporation of a
1
methanol/diethyl ether solution at room temperature. H NMR (500 MHz, DMSOꢀd6): δ
9.19 (d, J = 2.2 Hz, ArꢀH, 2H), 8.66 (d, J = 6.2 Hz, ArꢀH, 2H), 8.00 (dd, J = 6.2, 2.2 Hz,
ArꢀH, 2H), 7.82 (d, J = 8.4 Hz, ArꢀH, 2H), 7.79ꢀ7.76 (m, ArꢀH, 2H), 7.62 (d, J = 8.3 Hz,
ArꢀH, 2H), 7.51 (t, J = 7.6 Hz, ArꢀH, 2H), 7.43 (t, J = 7.4 Hz, ArꢀH, 1H), 1.78 (s, CpbiphꢀH,
6H), 1.72 (s, CpbiphꢀH, 6H). Anal. Calcd for [CpbiphIr(bpyꢀCl)Cl]PF6 (871.09): C, 42.74;
H, 3.12; N, 3.22. Found: C, 42.63; H, 3.09; N, 3.24. MS: m/z 724.90
[CpbiphIr(bpyꢀCl)Cl]+. IR (KBr pellets, cmꢀ1): 558 (CꢀCl).
[CpbiphIr(bpyꢀNO2)Cl]PF6 (6). The synthesis was performed as for 1 using
[CpbiphIrCl2]2 (108 mg, 0.1 mmol) and 4, 4′ꢀdinitroꢀ2, 2′ꢀbipyridine (60.2 mg, 0.24 mmol).
Yield: 62 mg (35%). A single crystal was obtained by slow evaporation of a
1
methanol/diethyl ether solution at room temperature. H NMR (500 MHz, DMSOꢀd6): δ
8.48 (d, J = 6.5 Hz, ArꢀH, 2H), 8.42 (d, J = 2.5 Hz, ArꢀH, 1H), 7.82 (d, J = 8.1 Hz, ArꢀH,
2H), 7.77 (d, J = 7.6 Hz, ArꢀH, 2H), 7.60 (d, J = 8.1 Hz, ArꢀH, 2H), 7.51 (t, J = 7.5 Hz,
ArꢀH, 3H), 7.45ꢀ7.37 (m, ArꢀH, 3H), 1.80–1.75 (m, CpbiphꢀH, 6H), 1.74ꢀ1.69 (m, CpbiphꢀH,
6H). Anal. Calcd for [CpbiphIr(bpyꢀNO2)Cl]PF6 (892.21): C, 41.73; H, 3.05; N, 6.28.
Found: C, 41.64; H, 3.02; N, 6.31. MS: m/z 747.10 [CpbiphIr(bpyꢀNO2)Cl]+. IR (KBr
pellets, cmꢀ1): 1619 (NO2), 1384 (NO2), 1315 (CꢀN), 1256 (NꢀO).
ASSOCIATED CONTENT
Supplementary Materials

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Full experimental details for the synthesis of ligands, NMR, IR and MS spectra of all
complexes, crystallographic data for 1, 3, and 5, the data for nucleobase studies, aqueous
chemistry, cancer cell studies, DNA reaction and NADH reaction kinetics.
CIF and checkCIF files for 1, 3 and 5. CCDC numbers of 1, 3 and 5 are 1569686,
1569665, and 1569685, respectively. These data can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
AUTHOR INFORMATION
Corresponding Author
*Email: liuzheqd@163.com.
ORCID
Lihua Guo: 0000ꢀ0002ꢀ0842ꢀ9958.
Zhe Liu: 0000ꢀ0001ꢀ5796ꢀ4335.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
We thank the National Natural Science Foundation of China (Grant No. 21671118),
Shandong Provincial Natural Science Foundation (ZR2018MB023) and the Taishan
Scholars Program for support.
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