Fluorescent half-sandwich phosphine-sulfonate iridium(III) and ruthenium(II) complexes as potential lysosome-targeted anticancer agents

Article abstract of DOI:10.1016/j.dyepig.2018.11.009

The synthesis, characterization and biological activity of neutral fluorescent Ir(III) and Ru(II) half-sandwich organometallic complexes containing phosphine-sulfonate ligands are reported. X-ray crystal structure of complexes 1–3, 10 and 11 exhibits the expected half-sandwich “three-legged piano-stool” pseudo-octahedral geometry. Spectroscopic properties study displays that these complexes show rich fluorescence properties. With the exception of 9, 10 and 11 toward A549 human lung cancer cells and 10 towards HeLa human cervical cancer cells, each complex shows promising cytotoxicity toward HeLa and A549 cells line with IC₅₀ values in the range of 3.6–53.1 μM, and 6.5–34.5 μM, respectively. Hydrolysis, DNA cleavage and depolarization of the mitochondrial membrane potential (MMP) appear not to be the main mechanism of action. However, these complexes are able to covert NADH to NAD⁺ via the transfer hydrogenation. Mechanism studies by flow cytometry display that the complexes exert their anticancer efficacy by inducing apoptosis, perturbing the cell cycle and increasing the intracellular ROS level. Furthermore, fluorescence property of these complexes provides a tool to investigate the microscopic mechanism by confocal microscopy. Notably, the typical Ir(III) complex 3 can specially localize to lysosome and damage it. In addition, complex 3 enters into HeLa cells mainly through energy-dependent pathway.

Full text of DOI:10.1016/j.dyepig.2018.11.009

Accepted Manuscript
Fluorescent half-sandwich phosphine-sulfonate iridium(III) and ruthenium(II)
complexes as potential lysosome-targeted anticancer agents
Qing Du, Yuliang Yang, Lihua Guo, Meng Tian, Xingxing Ge, Zhenzhen Tian, Liping
Zhao, Zhishan Xu, Juanjuan Li, Zhe Liu
PII:
S0143-7208(18)32140-5
https://doi.org/10.1016/j.dyepig.2018.11.009
DYPI 7153
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 26 September 2018
Revised Date: 4 November 2018
Accepted Date: 4 November 2018
Please cite this article as: Du Q, Yang Y, Guo L, Tian M, Ge X, Tian Z, Zhao L, Xu Z, Li J, Liu Z,
Fluorescent half-sandwich phosphine-sulfonate iridium(III) and ruthenium(II) complexes as potential
lysosome-targeted anticancer agents, Dyes and Pigments (2018), doi: https://doi.org/10.1016/
j.dyepig.2018.11.009.
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Graphical Abstract
A series of fluorescent phosphine-sulfonate iridium(III) and ruthenium(II)
half-sandwich complexes have been synthesized and characterized. Most of these
complexes display promising anticancer activities toward HeLa and A549 cancer cells.
The mechanism of actions (MoAs) of these complexes was explored by flow
cytometry and confocal microscopy imaging.

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Fluorescent Half-Sandwich Phosphine-Sulfonate
Iridium(III) and Ruthenium(II) Complexes as
Potential Lysosome-Targeted Anticancer Agents
Qing Du,a,† Yuliang Yang,a,† Lihua Guo,*a Meng Tian,a Xingxing Ge,a Zhenzhen Tian,a
Liping Zhao,a Zhishan Xu,a,b Juanjuan Li,a Zhe Liu*a
aInstitute of Anticancer Agents Development and Theranostic Application, The Key
Laboratory of Life-Organic Analysis and Key Laboratory of Pharmaceutical
Intermediates and Analysis of Natural Medicine, Department of Chemistry and
Chemical Engineering, Qufu Normal University, Qufu 273165, China.
bDepartment of Chemistry and Chemical Engineering, Shandong Normal University,
Jinan 250014, China.
*Corresponding Author: Email: guolihua@qfnu.edu.cn (L. H. Guo);
liuzheqd@163.com (Z. Liu).
†The first two authors are equal first authors.
‡Electronic supplementary information (ESI) available: Details of the Experimental
section, Figs. S1-S49, and Tables S1-S7. CIF and checkCIF files for 1-3, 10 and 11.
CCDC number of 1-3, 10 and 11 are 1813289, 1813290, 1813291, 1841617 and
1841618, respectively. These data can be obtained free of charge from the Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

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TOC:

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Abstract: The synthesis, characterization and biological activity of neutral
fluorescent Ir(III) and Ru(II) half-sandwich organometallic complexes containing
phosphine-sulfonate ligands are reported. X-ray crystal structure of complexes 1-3, 10
and 11 exhibits the expected half-sandwich “three-legged piano-stool”
pseudo-octahedral geometry. Spectroscopic properties study displays that these
complexes show rich fluorescence properties. With the exception of 9, 10 and 11
toward A549 human lung cancer cells and 10 towards HeLa human cervical cancer
cells, each complex shows promising cytotoxicity toward HeLa and A549 cells line
with IC50 values in the range of 3.6-53.1 µM, and 6.5-34.5 µM, respectively.
Hydrolysis, DNA cleavage and depolarization of the mitochondrial membrane
potential (MMP) appear not to be the main mechanism of action. However, these
complexes are able to covert NADH to NAD+ via the transfer hydrogenation.
Mechanism studies by flow cytometry display that the complexes exert their
anticancer efficacy by inducing apoptosis, perturbing the cell cycle and increasing the
intracellular ROS level. Furthermore, fluorescence property of these complexes
provides a tool to investigate the microscopic mechanism by confocal microscopy.
Notably, the typical Ir(III) complex 3 can specially localize to lysosome and damage it.
In addition, complex 3 enters into HeLa cells mainly through energy-dependent
pathway.
Keywords: half-sandwich; fluorescent; anticancer complexes; phosphine-sulfonate;
lysosome-targeted
1. Introduction

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A new area has been opened up in the field of inorganic drugs by Rosenberg who
discovered cisplatin’s biological activity [1,2]. The success of the platinum
chemotherapeutic drugs has prompted the investigation of other inorganic and
organometallic complexes (Ru, Ir, Cu, Rh, Fe, Au, Pd, Ni, Co, Ti, Ag, Ga, Zn, Sn, Os,
Mo, V, Re and lanthanide) to improve platinum anticancer complexes’ defects, such as
toxicity and resistance [3-8]. Among these compounds, organometallic
half-sandwich Ir(III) and Ru(II) complexes have attracted a lot of interest because of
their good anticancer activity and selectivity on various cancer cell cultures and the
different mechanism of actions (MoAs) from clinical platinum drugs [9-16]. In special,
half-sandwich Ir(III) and Ru(II) complexes bearing phosphorous ligands have
appeared as potencial anticancer drugs and even are pointing to clinical applications.
For instance, neutral Ru(II) arene complexes containing monodentate amphiphilic
phosphorous ligand (I, Scheme 1), have shown good antiangiogenic and
antimetastatic behavior and can inhibit the growth of primary tumors [17]. Recently,
we have studied a kind of cationic half-sandwich Ir(III) and Ru(II) complexes with
phosphine phosphonic amide (PPOA) as ligand and displayed that the remarkable
anticancer activities of the complexes was associated with the combinatorial action of
metal center and PPOA ligands (II, Scheme 1) [18]. On the other hand, the Sadler
group found that neutral half-sandwich Ir(III) complexes containing negatively
charged anionic C,N-chelating 2-phenylpyridine (phpy) ligand showed promising
biological activity while cationic half-sandwich Ir(III) complexes containing neutral
N,N-chelating ligand bipyridine (bpy) were inactive (III and IV, Scheme 1) [11].

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These results prompted us to prepare neutral half-sandwich Ir(III) and Ru(II)
complexes bearing anionic phosphorous bidentate ligands and investigate their
biological activities.
Scheme 1 The reported half-sandwich Ir(III) and Ru(II) anticancer complexes and our current work.
Moreover, confocal microscopy imaging has found wide application in the
biomedical field [19-22]. Fluorescent metal complexes are considered to be hopeful
anticancer agents that can combine imaging capabilities in multifunctional theranostic
platforms. So far, cyclometalated Ir or Ru anticancer complexes have been widely
used as probes and imaging agents due to their high photo stability, intense emission,
and large Stokes shifts [23-27]. However, the studies on the confocal microscopy
imaging of half-sandwich Ir(III) and Ru(II) anticancer complexes still remain scarce.
In this work, twelve neutral half-sandwich Ir(III) and Ru(II) complexes with
different phosphine-sulfonate ligands, have been synthesized and characterized. It
should be noted that phosphine-sulfonate ligands are currently attracting a lot of
interest for their easily tunable structures and have been applied in catalysis [28-31].
All of these synthesized complexes have been investigated for their spectroscopic

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properties, chemical reactivity and anticancer activity. On the basis of investigation
performed by flow cytometry and confocal microscopy imaging, we tried to
understand the MoAs of these complexes.
2. Results and discussion
2.1 Synthesis, characterization and spectroscopic properties.
Scheme 2. Synthesis of complexes 1-12.
[(η5-C5(CH3)5)IrCl2]2
(D1),
[(η5-C5(CH3)4C6H5)IrCl2]2
(D2),
[(η5-C5(CH3)4C6H4C6H5)IrCl2]2 (D3), [(η6-p-cymene)RuCl2]2) (D4) and the
phosphine-sulfonate ligands L1-L5 were synthesized according to reported methods
[32-36]. The complexes 1-12 were prepared in moderate to high yield (45%-88%)
from the reaction of the appropriate dimer with the corresponding
phosphine-sulfonate ligand and Na2CO3 in CH2Cl2 (Scheme 2 and Chart 1). These
1
31
13
complexes were characterized by H NMR, P NMR, C NMR, elemental analysis
and mass spectroscopy.

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Complex
Metal
Ir
Cpx/p-cym
Cp*
Cpxph
Cpxbiph
Cp*
P^O
L1
L1
L1
L2
L3
L4
L5
L1
L2
L3
L4
L5
1
2
Ir
3
Ir
4
Ir
5
Ir
Cp*
6
Ir
Cp*
7
Ir
Cp*
8
Ru
Ru
Ru
Ru
Ru
p-cym
p-cym
p-cym
p-cym
p-cym
9
10
11
12
Chart 1. Ir(III) and Ru(II) half-sandwich complexes 1-12 studied in this work. Cy = cyclohexyl.
The spectroscopic properties of complexes 1-6 and 8-12 were studied in CH2Cl2
(final DMSO concentration, 1% v/v) at 298 K. The UV-vis absorption spectra of Ir(III)
complexes 1-6 and Ru(II) complexes 8-12 in CH2Cl2 are shown in Fig. 1, a and b.
Ir(III) complexes 1-6 have strong absorption bands below 350 nm, while intense
absorption bands at approximately 250-450 nm was observed for Ru(II) complexes
8-12.

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2.0
1.5
1.0
0.5
0.0
1.5
1
2
3
4
5
6
8
9
10
11
12
1.0
0.5
0.0
250
300
350
400
450
500
550
250
300
350
400
450
500
550
Wavelength(nm)
Wavelength(nm)
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1
8
9
10
11
12
2
3
4
5
6
400
450
500
550
600
650
700
400
450
500
550
Wavelength(nm)
Wavelength(nm)
Fig. 1. (a) UV-vis spectra of iridium complexes 1-6 (20 µM) in CH2Cl2 at 298 K. (b) UV-vis spectra of ruthenium
complexes 8-12 (20 µM) in CH2Cl2 at 298 K. (c) Normalized emission spectra of iridium complexes 1-6 (20 µM)
in CH2Cl2 at 298 K (λex = 300 nm). (d) Normalized emission spectra of ruthenium complexes 8-12 (20 µM) in
CH2Cl2 at 298 K (λex = 365 nm).
The emission spectra of complexes 1-6 and 8-12 are obtained at 298 K. (Fig. 1, c
and d). Under ambient conditions, Ir(III) complexes 1-6 display purple emission with
a maximum wavelength of ca. 417 nm upon excitation at 300 nm. The emission
spectra show a strong resemblance for these Ir(III) complexes among each other.
Upon excitation at 365 nm, Ru(II) complexes 8-12 emit purple to blue (409-445 nm)
light. The ligand structure appears to have an influence on the emission wavelength of
these Ru(II) complexes. In contrast to these neutral complexes, our previously
reported cationic phosphine phosphonic amide (PPOA)-based half-sandwich Ir(III)
and Ru(II) complexes (II, Scheme 1) did not show emissions upon excitation at a

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wide wavelength range (280-500 nm). It should be noted that the fluorescent property
of the complexes provide useful information on the distribution, accumulation, and
uptake of the anticancer drugs in living cells. As a result, the rich fluorescent property
of these neutral half-sandwich P,O-chelating complexes represents a big advantage
and can provide an important research tool for investigating the MoAs of the
anticancer agents.
2.2 X-ray crystal structures.
Fig. 2. X-ray crystal structures with atom numbering schemes for (a) complex 1, (b) complex 2, (c) complex 3, (d)
complex 10 and (e) complex 11 with the thermal ellipsoids drawn at the 50% probability level. The hydrogen
atoms have been omitted for clarity. Selected bond lengths (Å): complex 1: Ir-C (centroid) = 1.8239, Ir-O =
2.134(6), Ir-P = 2.333(2), Ir-Cl = 2.403(3); complex 2: Ir-C (centroid) = 1.8172, Ir-O = 2.176(7), Ir-P = 2.330(3),
Ir-Cl = 2.384(3); complex 3: Ir-C (centroid) = 1.8167, Ir-O = 2.166(9), Ir-P = 2.345(3), Ir-Cl = 2.398(3); complex
10: Ru-C (centroid) = 1.6932, Ru-O = 2.088(5), Ru-P = 2.3744(10), Ru-Cl = 2.4010(11); complex 11: Ru-C
(centroid) = 1.7029, Ru-O = 2.113(4), Ru-P = 2.378(2), Ru-Cl = 2.4018(19).

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Single crystals of complexes 1-3, 10 and 11 were obtained from a slow diffusion of
Et2O to CH2Cl2 solutions of the corresponding complexes (Fig. 2). Crystallographic
data are listed in Table S1 and Table S2. Complexes 1-3, 10 and 11 adopt the expected
half-sandwich “three-legged piano-stool” pseudo-octahedral geometry. In general, the
Ir-O and Ru-O bonds (2.088-2.176 Å) are slightly shorter than that of Ir-P and Ru-P
bonds distance (2.330-2.378 Å). Intermolecular π-π stacking was not found in all of
the crystal structures.
2.3 In vitro cytotoxicity.
The anticancer activities of complexes 1-6 and 8-12 against HeLa (human cervical
cancer) and A549 cancer cell lines were studied by MTT assay with clinical cisplatin
as control (Table 1 and Fig. S39). The IC50 values for the complex 10 are >100 µM
against HeLa cells and A549 cells and thus deemed as inactive. Complexes 9 and 11
are only active in HeLa cells. However, other complexes show promising activity
toward the HeLa cancer cells and the A549 cancer cells with IC50 values of 3.6-53.1
µM, and 6.5-34.5 µM, respectively. No clear trend is observed for structure-activity
relationship of these complexes. Additionally, the introduction of the extended phenyl
substituents only results in little changes of the anticancer activity (e.g. iridium
complexes 1-3 with IC50 values of 3.6-7.9 µM), which is different from the reported
C,N and N,N-chelated half-sandwich Ir(III) complexes [10,37]. Notably, the
intercalative ability and hydrophobicity of phenyl substituents play important roles for
the biological activity of C,N and N,N-chelated half-sandwich Ir(III) complexes
[10,37]. In this work, the major role of the chelated phosphine-sulfonate ligands may

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have offset the advantages of the extended phenyl substituents. In particular, the
ruthenium(II) complexes in this system display a lower potency than iridium(III)
complexes, which can be attributed to the combinatorial action of metal and
phosphine-sulfonate ligand. It should be noted that most of the reported cytotoxic
organometallic ruthenium(II) compounds generally tend to be less potent than
cisplatin [38,39]. Furthermore, the investigated Ir(III) complexes have been shown a
slight depolarization of mitochondrial membranes (see “impact on mitochondrial
membrane potential (MMP)” section). As a result, the metabolization of MTT may
arise from the influence of complexes on the mitochondrial metabolism instead of the
viability of the cells. On the other hand, more than one assay should be used to
determine cell viability, as this would increase the reliability of the results obtained.
Therefore, the cytotoxicity of these complexes was also tested by colorimetric (neutral
red) assays (Table S3). No notable different results between this method with MTT
assay was observed, suggesting that the result of MTT assay is only slightly
influenced by change of MMP. The cytotoxicities of complexes 1-3, 8, 12 were
further evaluated against the human bronchial epithelial normal cells 16HBE (Table
1). Unfortunately, no selectivity was observed for cancer cells versus normal cells
with these complexes. Hence, more structural modification is necessary in future
work to decrease the cytotoxic action toward normal cells without loss of the
selectivity between cancer and normal cells.

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Table 1. IC50 values of complexes 1-12 tested toward cancer and normal cell lines and comparison with cisplatin.
IC50 (µM)
A549
Complex
HeLa
16HBE
(η5-C5Me5)Ir(L1)Cl (1)
7.9±0.2 23.2±1.4 26.7±0.6
5.9±0.3 6.5±0.4 11.9±0.9
(η5-C5Me4C6H5)Ir(L1)Cl (2)
(η5-C5Me4C6H4C6H5)Ir(L1)Cl (3) 3.6±0.3 20.8±2.5 7.0±0.3
(η5-C5Me5)Ir(L2)Cl (4)
(η5-C5Me5)Ir(L3)Cl (5)
(η5-C5Me5)Ir(L4)Cl (6)
(η5-C5Me5)Ir(L5)Cl (7)
(η6-p-cym)Ru(L1)Cl (8)
(η6-p-cym)Ru(L2)Cl (9)
(η6-p-cym)Ru(L3)Cl (10)
(η6-p-cym)Ru(L4)Cl (11)
(η6-p-cym)Ru(L5)Cl (12)
Cisplatin
17.8±0.6 34.4±0.3
10.9±0.1 12.5±0.5
32.4±0.2 25.8±0.2
a
-
-
53.1±1.1 34.5±4.3 39.9±3.0
27.9±1.0 ＞100
＞100
＞100
23.6±3.4 ＞100
24.4±0.1 22.7±4.2 35.0±0.6
7.5±0.2 21.3±1.7
a not determined due to its poor solubility
2.4 Aqueous stability.
M-Cl bonds in this type of complexes often undergo hydrolysis [11,40]. Thus, the
aqueous stability of complexes 3 and 9 was performed by UV-vis spectroscopy. The
time-dependent absorption spectra of complex 3 in 50% CH3OH/50% H2O (v/v)
solution and complex 9 in 40% CH3OH/60% H2O (v/v) solution were shown in Fig.
S40. The presence of methanol can enhance the solubility of the complexes. There are
no obvious changes in the absorption spectra of complexes 3 and 9 within 8 h.
Long-term stability monitoring was also performed by 1H NMR. After an incubation
for 24 h in 85% CD3OD-d4/15% D2O (v/v), no additional peaks were found in the 1H
NMR spectra for complexes 3 and 9. In addition, sodium chloride was added to the
solution to eliminate the existence of the fast equilibrium of 3 and 9 with aquo-species.
1
There was also no obvious change in the H NMR spectra (Figs S41 and S42).
Moreover, when AgNO3 (0.96 mol equiv) was mixed in the same solutions of

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complexes 3 and 9, the peaks of aqua compounds were observed after 16 h (Figs S43
and S44). All of the results suggested that the complexes were stable in aqueous
solution.
2.5 Interaction with nucleobases.
Targeting DNA can make complexes exhibit good anticancer activity [41-45]. Thus,
the binding of 9-ethylguanine (9-EtG) and 9-methyladenine (9-MeA) to complex 3
was examined. 1 mol equiv of 9-EtG or 9-MeA was added to an equilibrium solution
1
of 3 (0.5 mM) in 85% MeOD-d4/15% D2O (v/v) at 310 K. No additional H NMR
peaks over a period of 24 h were found (Figs S45 and S46), indicating that the
reaction between 9-MeA or 9-EtG and the complexes did not occur in this system.
Also, nucleobase adducts of these complexes was not monitored by mass
spectrometry. All of the results suggested that DNA is not the main target for the
phosphine-sulfonate half-sandwich complexes.
2.6 Plasmid DNA cleavage.
The DNA cleavage ability of complexes 3 and 9 (0-100 µM) was investigated by
agarose gel electrophoresis. However, no DNA cleavage was found for these
complexes even at a very high concentration (100 µM) (Fig. S47). This result further
confirmed that DNA was not the target for the complexes in this system.
2.7 Reaction with NADH.
It has been reported that neutral C,N-chelating half-sandwich Ir(III) complexes
were able to oxidize NADH by hydride transfer to the metal center and yield reactive
oxygen species in the form of H2O2 [46,47]. Thus, reactions between these complexes

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and NADH were investigated in 85% CD3OD-d4/15% D2O (v/v) solution by 1H NMR
spectrum (Fig. 3a). New peaks at 8.97, 9.34, and 9.57 ppm were assigned to the
hydrogen atoms at the C4, C6, and C2 positions of the nicotinamide ring of NAD+. In
addition, a sharp singlet at -11.39 ppm, which is corresponding to the Ir(III) hydride
complex 3, were found in the 1H NMR spectra after 5 min. In order to further evaluate
the catalytic activity, catalytic transfer hydrogenation reactions of complex 3 were
also studied in aqueous media by UV-vis spectroscopy (Figs 3b and S48). The
turnover numbers (TONs) of these complexes determined by the amount of UV
absorbance at 339 nm (Fig. 3c) showed the following trends in catalytic activity: 1
(11.6), 2 (11.4), 3 (19.6), 4 (12.4), 5 (15.6), 6 (12.2), 7 (15.8), 8 (8.5), 9 (12.7), 10
(16.1), 11 (11.9) and 12 (25.6). The changes of metal atoms led to little variation on
the catalytic activity of the complexes. However, the substituent effect of the chelating
ligands on catalytic activities of Ir(III) and Ru(II) complexes showed a similar trend
as follows: Ir: 7 (L5) > 5 (L3) > 4 (L2) > 6 (L4) > 1 (L1); Ru: 12 (L5) > 10 (L3) > 9
(L2) > 11 (L4) > 8 (L1). This fact leads to the assumption that the catalytic activity of
hydride transfer reaction in this system is mainly determined by the chelating ligands.

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1
Fig. 3. (a) H NMR spectra of the reaction between complex 3 (0.5 mM) and NADH (3.5 mol equiv) in 85%
MeOD-d4/15% D2O (v/v) at 310 K after 5 min, 2 h and 7 h. Left: Low-field region; right: high-field region
showing the Ir–H hydride peak(-11.39 ppm). Peaks labeled
and
correspond to the formed Ir–H complex; (b)
UV-vis spectra of the reaction of NADH (87 µM) with complex 12 (0.8 µM) in 5% MeOH/95% H2O (v/v) at 298
K for 8 h. (c) The turnover numbers (TONs) of complexes 1-12.

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2.8 Apoptosis assay.
100
80
60
40
20
0
Non-viable
Late
apoptosis
Early
apoptosis
Viable
control 0.5×IC
50
1×IC 2×IC 3×IC
50
50
50
Fig. 4. Apoptosis analysis of HeLa cells after 24 h of exposure to complex 3 at 310 K determined by flow
cytometry using annexin V-FITC vs. PI staining. (a) HeLa cells were left untreated (control) or treated with
different concentrations of complex 3 for 24 h. Lower left, living cells; lower right, early apoptotic cells; upper
right, late apoptotic cells; upper left, necrotic cells. (b) Histogram showing populations for HeLa cells in four
stages treated by complex 3. Data are quoted as mean ± SD of three replicates.
Cell apoptosis is regard as a programmed cell death process [48,49]. Hence, HeLa
cells were treated with complex 3 at 0.5, 1, 2 and 3 equipotent concentrations of IC50,
and analyzed by flow cytometry (Fig. 4 and Table S4). Obviously, treatment with
complex 3 enhanced the early and late apoptotic cell populations of the HeLa cells
compared with the control. When HeLa cells were incubated with complex 3 at 3 ×
IC50 concentration, a total of 50.6% of early apoptotic and late apoptotic cells were in
apoptosis phase, whereas untreated cells remained 90.3% viable. This suggested that

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cell death was induced through a high incidence of apoptosis.
2.9 Cell cycle analysis.
G
phase
80
70
60
50
40
30
20
10
0
1
S phase
G /M phase
2
0.25×IC
50
0.5×IC
control
50
Fig. 5. Cell cycle analysis of HeLa cells after 24 h of exposure to complex 3 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 the negative control (untreated cells) and complex 3 with 0.25 and 0.5 equipotent
concentrations of IC50. (b) Cell populations in each cell cycle phase for control and complex 3. Data are quoted as
mean ± SD of three replicates.
Cell cycle progression aberrant plays a critical role in cancer initiation and
progression [50]. Based on the encouraging results described above, we further
investigated complexes’ mechanism of action by recording changes on the cell cycle
phase distribution in HeLa cells using flow cytometry analysis. The cells were treated
with complex 3 at 0.25 × IC50 and 0.5 × IC50 concentrations, respectively, using
PI/RNase staining, and the observed FACS histogram statistics were shown in Fig. 5
and Table S5. The treatment of HeLa cells with complex 3 (0.5 × IC50) induced S
phase arrest and the proportions of cells increased from 21.6% to 32.5% compared to

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untreated cells, while no obvious changes were observed at the G2/M and G1 phase of
the cell cycle under the same test conditions. These results indicated that complex 3
mainly arrest the cell cycle at S phase with a concentration-dependent manner.
2.10 Induction of ROS in HeLa cancer cells.
Cells in Low ROS Levels
Cells in High ROS Levels
100
80
60
40
20
0
Negative
Control
Positive
Control
0.25×IC
50
Fig. 6. Flow cytometry analysis of ROS induction in HeLa cancer cells treated with complex
3 at concentration of
0.25 × IC50. (a) FL2 histogram for negative control (untreated cells), positive control and complex
3. (b)
Populations of cells in low and high level of ROS after treatment of complex
three replicates.
3. Data are quoted as mean ± SD of
Reactive oxygen species (ROS) can perturb the cellular antioxidant defense system
[51,52]. C,N-chelated half-sandwich Ir(III) anticancer complexes to generate the ROS
has been reported previously [47]. The ROS levels in HeLa cancer cells induced by
exposure to complex 3 for 24 h was determined at 0.25 × IC50 concentration by flow
cytometry. Significant increase of ROS levels in the cells was observed (Fig. 6 and
Table S6). Close to 90% of HeLa cells displayed a high ROS level, which was even

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comparable to positive control. The increase in ROS levels for complex 3 played an
important role in providing a basis for killing cancer cells. Thus, the production of
ROS was also considered to be the major MoA in this system.
2.11 Mitochondrial membrane potential assay.
There are a large number of evidences indicating that mitochondrial dysfunction
occurred in apoptotic cell death [52-54]. Analysis of the mitochondrial membrane
potential (MMP, ∆Ψm) in HeLa cells after the treatment with complex 3 at
concentrations of 0.25, 0.5, 1 and 2 × IC50 was performed by detecting the
fluorescence of JC-1. When the concentration of complex 3 increased, the MMP only
decreased slightly in HeLa cells (Fig. S49 and Table S7), suggesting that the
dysfunction of mitochondrial membrane potential was not the major mechanism of
action in this system.
2.12 Cellular localization and cell uptake.
Due to the rich fluorescent properties of the complexes in this system, the
subcellular localization of complex 3 was subsequently examined by confocal
microscopy. The HeLa cells were dual-stained with complex 3 and different
organelle-specific probes, respectively. Confocal microscopic imaging showed that
the complex 3 can effectively penetrate into HeLa cancer cells after 0.5 h incubation,
as suggested by the punctate green fluorescence in the cytoplasm (Fig. 7). Good
concordance between overlay images of the complex 3 (10 µM) and conventional dye
Lyso-Tracker Red DND-99 (LTDR, 75 nM) in HeLa cells was observed, suggesting
that complex 3 can specially localize to lysosome. Pearson's correlation coefficients

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for complex 3 and LTDR were 71% and 73% for 0.5 h and 3 h, respectively. However,
no co-localization between complex 3 (10 µM) and a mitochondrial specific dye
MitoTracker Deep Red FM (MTDR, 500 nM) in HeLa cells was observed. As a result,
the cytotoxic properties of these complexes in this system may originate from
lysosome-mediated cell death.
Fig. 7. Determination of intercellular localization of complex
3 by confocal microscopy. (a) Selective imaging of
HeLa cells incubated with complex
3 for 0.5 h and 3 h, then incubated with LTDR for 0.5h at 37 °C. The green and
red fluorescence represent complex
3 and lysosome, respectively. (b) Selective imaging of HeLa cells incubated
with complex
3 for 0.5 h and 3 h, then incubated with MTDR for 0.5 h at 37 °C. The green and red fluorescence
represent complex
3 and mitochondria, respectively. Complex 3 was excited at 488 nm and the emission was
collected at 530 ± 30 nm. LTDR was excited at 594 nm and the emission was collected at 630 ± 30 nm. MTDR
was excited at 644 nm and the emission was collected at 700 ± 30 nm. Scale bar: 20 µm.
Small molecules can pass through cells through energy independent or
energy-dependent pathways [55,56]. Incubation of HeLa cells with complex 3 (10 µM)

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at 4 oC or treatment with the metabolic inhibitor carbonyl cyanide
3-chloro-phenylhydrazone (CCCP, 50 uM) led to decreased cellular uptake levels
compared with control cells incubated at 37 °C. The decreased cellular uptake levels
of complex 3 (10 µM) were also observed in HeLa cells treated with chloroquine (50
µM) which can modulate endocytosis by inhibiting the acidification of endosomes
(Fig. 8). These observations indicated that complex 3 entered cells mainly through an
energy-dependent pathway.
Fig. 8. Cellular uptake mechanisms of complex 3. Confocal images of HeLa cells after incubation with complex
3
under different conditions. Cells were incubated with complex
3 at 37 °C for 1 h. This served as a control. Cells
were incubated with complex
3 at 4 °C for 1 h. Cells were pre-incubated with metabolic inhibitors CCCP at 37 °C
for 1 h and then incubated with complex
37 °C and then incubated with complex
3 at 37 °C for 1 h. Cells were pre-incubated with chloroquine for 1 h at
3
at 37 °C for 1 h. Complex 3 was excited at 488 nm and emission was
collected at 530 ± 30 nm. Scale bar: 20 µm
.

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2.13 Lysosomal damage.
Lysosomes often involved in the apoptotic pathways and lysosomal damage may
promote apoptosis [57]. The lysosomal integrity of the HeLa cells can be determined
by acridine orange (AO) since AO emits green fluorescence in cytosol or nuclei and
red fluorescence in lysosomes [58]. HeLa cancer cells treated with AO showed red
fluorescence in lysosomes (Fig. 9). Notably, the red fluorescence of AO decreased
with the increase of complex concentration, which indicated that lysosomal integrity
was jeopardized upon the treatment of complex 3. Thus, we conclude that complex 3
can induce apoptosis through lysosomal damage.
Fig. 9. Observation of lysosomal disruption in HeLa cells loaded with complex
3 (1, 2, 3, 4 and 5× IC50) for 12 h
at 37 ºC, then stained with AO (5 µM) at 37 °C for 15 min. Emission was collected at 510 ± 20 nm (green) and 625
± 20 nm (red) upon excitation at 488 nm. Scale bar: 20 µm.

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3. Conclusions
In conclusion, twelve neutral half-sandwich P,O-chelated Ir(III) and Ru(II)
complexes 1-12 have been synthesized and characterized. Their structures were
determined by X-ray crystallography. These complexes showed rich fluorescent
properties and can covert NADH to NAD+ via the transfer hydrogenation, which may
lead to the generation of ROS. Most of these complexes display promising anticancer
activities toward HeLa and A549 cancer cells, comparable to, and for some complexes
even higher than the clinical cisplatin. The mechanism of actions (MoAs) of the
complexes in this system was explored by flow cytometry and confocal microscopy
imaging. First, hydrolysis, DNA cleavage and loss of MMP were not the major
mechanism of action for this type of complexes. Next, the MoA of these complexes
was due to cell cycle perturbation, increase of ROS level and apoptosis induction.
Finally, these complexes can be quickly and effectively taken into HeLa cancer cells
through an energy-dependent pathway, and were mainly localized to lysosome. In
addition, lysosomal integrity can be jeopardized upon the treatment of these
complexes.
4. Experimental section
4.1. General materials and methods.
Dimers (D1-D4), phosphine-sulfonate ligands, complexes 4 and 9 were synthesized
according to previous reported procedures [32-36,59,60]. All other reagents and
solvents were purchased from commercial sources and used without purification
1
unless otherwise specified. H NMR spectra were acquired in 5 mm NMR tubes at

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298 K on Bruker DPX 500 (1H = 500.13 MHz). Mass spectra of complexes 1-12 were
recorded on a Thermo LTQ Orbitrap XL (ESI+) or a Atouflex Speed MALDI-TOF MS.
X-ray Diffraction data were obtained on a Bruker Smart CCD area detector with
graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). X-ray crystallographic
data for complexes 1-3, 10 and 11 have been deposited in the Cambridge
Crystallographic Data Centre under the accession numbers 1813289 (1), 1813290 (2),
1813291 (3), 1841617 (10) and 1841618 (11), respectively.
4.2 Synthesis of (Cpx/Arene)M(L)Cl.
General method: A solution of [(η5-Cpx)IrCl2]2 or [(η6-p-cym)RuCl2]2,
phosphine-sulfonate ligands (2 eq.) and Na2CO3 (4 eq.) in dichloromethane was
stirred for 36 h at ambient temperature and filtered. The volume of filtrate was slowly
reduced to a little on a rotary evaporator. Using solvent diffusion technique
(CH2Cl2/n-Hexane) gave the relatively stable yellow or red solid.
(η5-C5Me5)Ir(L1)Cl (1). [(η5-C5Me5)IrCl2]2 (47.8 mg, 0.06 mmol), L1 (42.5 mg,
1
0.12 mmol), Na2CO3 (25.4 mg, 0.24 mmol). Yield: 53.0 mg (62%). H NMR (500
MHz, CDCl3) δ 8.19 – 8.17 (m, 1H, Ar-H), 7.53 – 7.45 (m, 2H, Ar-H), 7.40 – 7.37 (m,
1H, Ar-H), 3.17 – 2.97 (m, 1H, Cy-H), 2.46 – 2.38 (m, 1H, Cy-H), 2.37 – 2.28 (m, 1H,
Cy-H), 2.23 – 1.57 (m, 19H, Cy-H (4H) + Cp*-H (15H)), 1.56 – 1.04 (m, 15H, Cy-H).
31P NMR (202 MHz, CDCl3) δ 0.88. 13C NMR (126 MHz, CDCl3) δ 130.83, 130.29,
129.18, 129.14, 91.66, 65.96, 36.50, 31.69, 29.18, 27.84, 27.77, 27.68, 27.58, 27.49,
27.43, 27.34, 26.21, 26.07, 15.39, 9.32, 0.11. ESI-MS (m/z): calcd for C28H41IrO3PS:
681.21, found: 681.21 [M-Cl]+. Elemental analysis: calcd (%) for C28H41ClIrO3PS: C,

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46.95; H, 5.77, found: C, 46.86; H, 5.81.
(η5-C5Me4C6H5)Ir(L1)Cl (2). [(η5-C5Me4C6H5)IrCl2]2 (46.0 mg, 0.05 mmol), L1
1
(35.4 mg, 0.10 mmol), Na2CO3 (21.2 mg, 0.20 mmol). Yield: 61.1 mg (79%). H
NMR (500 MHz, CDCl3) δ 8.19 – 8.17 (m, 1H, Ar-H), 7.62 – 7.60 (m, 2H, Ar-H),
7.55 – 7.52 (m, 1H, Ar-H), 7.47 – 7.43 (m, 1H, Ar-H), 7.39 – 7.35 (m, 4H, Ar-H), 3.00
– 2.80 (m, 1H, Cy-H), 2.39 – 2.30 (m, 1H, Cy-H), 2.27 – 2.18 (m, 1H, Cy-H), 1.88 –
31
1.72 (m, 10H, Cy-H), 1.58 (s, 12H, Cpxph-H), 1.33 – 0.94 (m, 9H, Cy-H). P NMR
13
(202 MHz, CDCl3) δ 4.81. C NMR (126 MHz, CDCl3) δ 131.14, 131.07, 130.54,
130.14, 129.01, 128.75, 128.48, 104.70, 104.66, 27.65, 27.56, 27.38, 27.34, 27.30,
27.25, 25.84, 25.77, 11.71, 9.64, 9.16, 0.11. ESI-MS (m/z): calcd for C33H43IrO3PS:
743.23, found: 743.23 [M-Cl]+. Elemental analysis: calcd (%) for C33H43ClIrO3PS: C,
50.92; H, 5.57, found: C, 50.83; H, 5.66.
(η5-C5Me4C6H4C6H5)Ir(L1)Cl (3). [(η5-C5Me4C6H4C6H5)IrCl2]2 (53.7 mg, 0.05
mmol), L1 (35.4 mg, 0.10 mmol), Na2CO3 (21.2 mg, 0.20 mmol). Yield: 39.2 mg
(46%). 1H NMR (500 MHz, CDCl3) δ 8.20 – 8.18 (m, 1H, Ar-H), 7.69 (d, J = 8.2 Hz,
2H, Ar-H), 7.63 – 7.56 (m, 4H, Ar-H), 7.56 – 7.53 (m, 1H, Ar-H), 7.47 – 7.43 (m, 3H,
Ar-H), 7.40 – 7.35 (m, 2H, Ar-H), 3.03 – 2.79 (m, 1H, Cy-H), 2.40 – 2.32 (m, 1H,
Cy-H), 2.27 – 2.19 (m, 1H, Cy-H), 1.98 – 1.89 (m, 2H, Cy-H), 1.88 – 1.69 (m, 12H,
31
Cpxbiph-H), 1.67 – 1.48 (m, 10H, Cy-H), 1.24 – 0.95 (m, 7H, Cy-H). P NMR (202
MHz, CDCl3) δ 4.87. 13C NMR (126 MHz, CDCl3) δ 141.22, 140.55, 131.08, 130.55,
130.20, 129.05, 127.76, 127.40, 127.09, 104.79, 104.75, 65.97, 53.57, 35.88, 27.36,
27.34, 28.98, 27.65, 27.56, 27.36, 27.34, 27.29, 27.25, 25.85, 25.73, 15.40, 11.79,

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11.76, 9.75, 9.18, 0.11. MALDI-TOF-MS (m/z): calcd for C39H46IrO3PS: 818.25,
found: 818.58 [M-Cl-H]+. Elemental analysis: calcd (%) for C39H47ClIrO3PS: C,
54.82; H, 5.54, found: C, 54.74; H, 5.59.
(η5-C5Me5)Ir(L3)Cl (5). [(η5-C5Me5)IrCl2]2 (47.8 mg, 0.06 mmol), L3 (57.4 mg,
0.12 mmol), Na2CO3 (25.4 mg, 0.24 mmol). Yield: 73.9 mg (73%). Two isomers are
1
obtained due to the chrial center of P atom. Isomer 1: H NMR (500 MHz, CDCl3) δ
8.54 – 8.49 (m, 1H, Ar-H), 8.03 – 8.01 (m, 1H, Ar-H), 7.60 – 7.16 (m, 7H, Ar-H), 7.01
– 6.93 (m, 4H, Ar-H), 6.61 – 6.58 (m, 1H, Ar-H), 6.18 – 6.17 (m, 1H, Ar-H), 5.77 –
5.76 (m, 1H, Ar-H), 3.27 (s, 3H, OMe), 2.84 (s, 3H, OMe), 1.37 (s, 15H, Cp*-H). 31P
NMR (202 MHz, CDCl3) δ 7.53. Isomer 2: 1H NMR (500 MHz, CDCl3) δ 8.63 – 8.59
(m, 1H, Ar-H), 8.06 – 8.04 (m, 1H, Ar-H), 7.60 – 7.16 (m, 7H, Ar-H), 7.10 – 7.04 (m,
4H, Ar-H), 6.86 – 6.83 (m, 1H, Ar-H), 6.31 – 6.33 (m, 1H, Ar-H), 5.84 – 5.82 (m, 1H,
31
Ar-H), 3.74 (s, 3H, OMe), 3.18 (s, 3H, OMe), 1.48 (s, 15H, Cp*-H). P NMR (202
MHz, CDCl3) δ 14.77. 13C NMR (126 MHz, CDCl3) δ 158.49, 156.50, 156.44, 149.30,
146.71, 146.60, 141.47, 141.27, 140.38, 139.83, 138.27, 138.10, 135.13, 135.10,
134.29, 134.24, 133.80, 133.75, 131.76, 131.75, 131.40, 131.19, 131.17, 131.02,
131.00, 130.96, 130.18, 130.08, 130.06, 129.72, 129.70, 129.42, 129.41, 129.29,
128.42, 128.36, 128.25, 128.19, 128.13, 128.02, 127.56, 127.49, 127.35, 127.24,
126.74, 126.34, 125.84, 125.80, 125.71, 125.34, 117.70, 117.69, 117.52, 103.63,
103.31, 103.13, 102.06, 91.82, 91.80, 90.93, 90.91, 54.69 (Isomer 1), 54.45 (Isomer
1), 8.85 (Isomer 1), 55.57 (Isomer 2), 54.28 (Isomer 2), 8.99 (Isomer 2).
MALDI-TOF-MS (m/z): calcd for C36H37IrO5PS: 805.17, found: 805.15 [M-Cl]+.

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Elemental analysis: calcd (%) for C36H37ClIrO5PS: C, 51.45; H, 4.44, found: C, 51.56;
H, 4.52.
(η5-C5Me5)Ir(L4)Cl (6). [(η5-C5Me5)IrCl2]2 (47.8 mg, 0.06 mmol), L4 (48.3 mg,
1
0.12 mmol), Na2CO3 (25.4 mg, 0.24 mmol). Yield: 44.9 mg (49%). H NMR (500
MHz, CDCl3) δ 8.33 – 8.01 (m, 2H, Ar-H), 7.77 – 7.59 (m, 1H, Ar-H), 7.43 – 7.38 (m,
4H, Ar-H), 7.10 – 6.90 (m, 3H, Ar-H), 6.93 – 6.85 (m, 1H, Ar-H), 6.79 –6.72(m, 1H,
Ar-H), 3.53 – 3.30 (m, 6H, OMe), 1.49 (s, 15H, Cp*-H). 31P NMR (202 MHz, CDCl3)
13
δ -6.92. C NMR (126 MHz, CDCl3) δ 130.59, 92.15, 58.61, 54.82, 53.57, 31.72,
29.83, 22.79, 18.58, 14.25, 8.88, 0.13. MALDI-TOF-MS (m/z): calcd for
C30H33IrO5PS: 729.14, found: 729.18 [M-Cl]+. Elemental analysis: calcd (%) for
C30H33ClIrO5PS: C, 47.15; H, 4.35, found: C, 47.06; H, 4.40.
(η5-C5Me5)Ir(L5)Cl (7). [(η5-C5Me5)IrCl2]2 (47.8 mg, 0.06 mmol), L5 (48.5 mg,
1
0.12 mmol), Na2CO3 (25.4 mg, 0.24 mmol). Yield: 51.3 mg (56%). H NMR (500
MHz, CDCl3) δ 9.14 (d, J = 8.7 Hz, 1H, Ar-H), 7.92 (d, J = 8.8 Hz, 1H, Ar-H), 7.80 (d,
J = 8.1 Hz, 1H, Ar-H), 7.63 – 7.59 (m, 1H, Ar-H), 7.56 – 7.53 (m, 1H, Ar-H), 7.48 –
7.43 (m, 1H, Ar-H), 3.20 – 3.17 (m, 1H, Cy-H), 2.45 – 2.34 (m, 2H, Cy-H), 2.13 –
2.06 (m, 2H, Cy-H), 1.98 – 1.87 (m, 2H, Cy-H), 1.83 – 1.65 (m, 7H, Cy-H), 1.62 –
1.50 (m, 15H, Cp*-H), 1.48 – 1.24 (m, 7H, Cy-H), 1.17 – 1.12 (m, 1H, Cy-H).
ESI-MS (m/z): calcd for C32H43IrO3PS: 731.23, found: 731.23 [M-Cl]+. Elemental
analysis: calcd (%) for C32H43ClIrO3PS: C, 50.15; H, 5.66, found: C, 50.09; H, 5.71.
(η6-p-cym)Ru(L1)Cl (8). [(η6-p-cym)RuCl2]2 (30.6 mg, 0.05 mmol), L1 (35.4 mg,
1
0.10 mmol), Na2CO3 (21.2 mg, 0.20 mmol). Yield: 29.8 mg (48%). H NMR (500

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MHz, CDCl3) δ 8.06 – 8.03 (m, 1H, Ar-H), 7.50 – 7.45 (m, 1H, Ar-H), 7.40 – 7.34 (m,
2H, Ar-H), 5.89 (d, J = 5.9 Hz, 1H, cym-H), 5.73 (d, J = 4.9 Hz, 1H, cym-H), 5.51 (d,
J = 5.9 Hz, 1H, cym-H), 5.41 (d, J = 5.8 Hz, 1H, cym-H), 2.96 (septet, J = 6.8 Hz, 1H,
cym-CH(CH3)2), 2.82 – 2.72 (m, 2H, Cy-H), 2.20 – 2.09 (m, 3H, Cy-H), 2.07 (s, 3H,
cym-CH3), 2.00 – 1.60 (m, 8H, Cy-H), 1.55 – 1.38 (m, 4H, Cy-H), 1.36 (d, J = 6.9 Hz,
3H, cym-CH(CH3)2), 1.32 (d, J = 6.9 Hz, 3H, cym-CH(CH3)2), 1.30 – 1.20 (m, 4H,
31
13
Cy-H), 1.19 – 1.12 (m, 1H, Cy-H). P NMR (202 MHz, CDCl3) δ 27.94. C NMR
(126 MHz, CDCl3) δ 148.94, 148.87, 131.01, 131.00, 130.50, 129.97, 129.92, 128.87,
128.83, 94.95, 85.19, 85.14, 85.08, 85.07, 80.88, 80.85, 40.23, 40.08, 36.82, 36.65,
30.84, 29.84, 29.40, 28.74, 27.80, 27.73, 27.67, 27.58, 27.50, 27.41, 26.45, 26.00,
22.31, 22.24, 18.40, 0.13. MALDI-TOF-MS (m/z): calcd for C28H40RuO3PS: 589.15,
found: 589.42 [M-Cl]+. Elemental analysis: calcd (%) for C28H40ClRuO3PS: C, 53.88;
H, 6.46, found: C, 53.84; H, 6.53.
(η6-p-cym)Ru(L3)Cl (10). [(η6-p-cym)RuCl2]2 (30.6 mg, 0.05 mmol), L3 (47.9 mg,
1
0.10 mmol), Na2CO3 (21.2 mg, 0.20 mmol). Yield: 60.0 mg (80%). H NMR (500
MHz, CDCl3) δ 8.62 – 8.57 (m, 1H, Ar-H), 8.00 – 7.97 (m, 1H, Ar-H), 7.54 – 7.47 (m,
2H, Ar-H), 7.40 – 7.35 (m, 1H, Ar-H), 7.28 – 7.25 (m, 2H, Ar-H), 7.21 – 7.17 (m, 1H,
Ar-H), 7.10 – 7.08 (m, 1H, Ar-H), 7.05 – 6.91 (m, 5H, Ar-H), 6.55 (d, J = 8.3 Hz, 1H,
Ar-H), 6.08 (d, J = 6.0 Hz, 1H, Ar-H), 5.85 – 5.81 (m, 1H, cym-H), 5.78 (d, J = 8.4
Hz, 1H, cym-H), 5.40 (d, J = 6.0 Hz, 1H, cym-H), 5.13 (d, J = 5.5 Hz, 1H, cym-H),
3.93 (s, 3H, OMe), 3.10 (s, 3H, OMe), 2.39 (septet, J = 6.8 Hz, 1H, cym-CH(CH3)2),
1.59 (s, 3H, cym-CH3), 1.21 (d, J = 6.9 Hz, 3H, cym-CH(CH3)2), 0.91 (d, J = 6.9 Hz,