“Half-sandwich” Schiff-base Ir(III) complexes as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2017.06.027

A series of “half-sandwich” Schiff-base Ir(III) complexes were synthesized and investigated for their in vitro activities against the leukemia K562 cell line. These compounds demonstrated antiproliferative activities against K562 cells with IC₅₀ values of 0.26–4.77 μM. In particular, compound 10c showed cytotoxicity against five cancer cell lines/sublines and stronger activities than cisplatin in K562, K562/A02, MCF-7, MCF-7/ADM, and A549 cells. Mechanism studies illustrated that compound 10c increased the level of reactive oxygen species and induced apoptosis of K562 cells. This compound effectively decreased the mitochondrial membrane potential and the protein level of Bcl-2. It also increased the protein levels of Bax, caspase-3, and caspase-9, and led to release of cytochrome c in K562 cells, indicating that the apoptosis induced by compound 10c was mediated by the intrinsic mitochondria apoptosis pathway.

Full text of DOI:10.1016/j.ejmech.2017.06.027

Accepted Manuscript
“Half-sandwich” Schiff-base Ir(III) complexes as anticancer agents
Ze-dong Mou, Ning Deng, Feng Zhang, Jiaying Zhang, Juan Cen, Xia Zhang
PII:
S0223-5234(17)30473-7
10.1016/j.ejmech.2017.06.027
EJMECH 9522
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 March 2017
Revised Date: 13 June 2017
Accepted Date: 14 June 2017
Please cite this article as: Z.-d. Mou, N. Deng, F. Zhang, J. Zhang, J. Cen, X. Zhang, “Half-sandwich”
Schiff-base Ir(III) complexes as anticancer agents, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.06.027.
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ACCEPTED MANUSCRIPT
Induce Apoptosis
Increase ROS
IC50 (K562) = 0.26 µM
IC50 (K562/A02) = 1.95 µM

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“Half-sandwich” Schiff-base Ir(III) complexes as anticancer agents
Ze-dong Moua, †, Ning Denga, †, Feng Zhang b, Jiaying Zhang a, Juan Cen b, ∗, and Xia Zhang a,
∗
a State Key Laboratory of Biotherapy and Department of Ophthalmology, West China Hospital, Sichuan
University and Collaborative Innovation Center, Chengdu 610041, China
b Key Laboratory of Natural Medicine and Immune Engineering, Henan University, Kaifeng 475004,
China
∗ Corresponding authors. E-mail addresses: cenjuan@henu.edu.cn (J. C.), zhang-xia@scu.edu.cn (X. Z.).
Tel./fax: +86 378 2864665(J. C.), +86 28 85503817 (X. Z.).
† These authors contributed equally.

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Abstract
A series of “half-sandwich” Schiff-base Ir(III) complexes were synthesized and
investigated for their in vitro activities against the leukemia K562 cell line. These compounds
demonstrated antiproliferative activities against K562 cells with IC50 values of 0.26 - 4.77 µM. In
particular, compound 10c showed cytotoxicity against five cancer cell lines/sublines and stronger
activities than cisplatin in K562, K562/A02, MCF-7, MCF-7/ADM, and A549 cells. Mechanism
studies illustrated that compound 10c increased the level of reactive oxygen species and induced
apoptosis of K562 cells. This compound effectively decreased the mitochondrial membrane
potential and the protein level of Bcl-2. It also increased the protein levels of Bax, caspase-3, and
caspase-9, and led to release of cytochrome c in K562 cells, indicating that the apoptosis induced
by compound 10c was mediated by the intrinsic mitochondria apoptosis pathway.
Key Words: Schiff-base Ir(III) complexes; antiproliferative activities; reactive oxygen species;
apoptosis.

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1. Introduction
Because of their variations in coordination numbers, geometries, and properties of the
metal ion and ligands, transition metals provide highly versatile scaffolds for drug design. [1-3]
Not surprisingly, the discovery of cisplatin and the other two platinum-based drugs, carboplatin
and oxaliplatin, represented the most successful examples in the field of medicinal inorganic
chemistry. Cisplatin and its analogues function as anticancer drugs mainly by forming platinum-
DNA adducts that interfere with DNA replication and further lead to cell death.[4, 5] Although
these platinum-based drugs are among the most widely used anticancer therapeutics, they
produce undesirable side effects and easily acquired drug resistance.[6, 7] The success and
limitations of these platinum-based drugs stimulated tremendous research efforts into the
discovery of other transition metal-based anticancer drugs. It is expected that transition metal-
based anticancer drugs with mechanisms of action different from those of the platinum-based
drugs would demonstrate diminished side effects, no easily developed drug resistance, and/or a
broader spectrum of anticancer activities.[8, 9]
Recently, in addition to other transition metal complexes including Ru-, Rh-, Re-, Os-,
and Au-based compounds, anticancer Ir(III) complexes received significant attentions.[1, 2, 9-11]
Interestingly, complexes of Ir(III), a third-row low-spin d6 metal ion, were initially considered to
be too inert to exhibit high anticancer activities.[12] This is probably because that hydrolysis is
usually an key step in the mechanism of action of metal-based anticancer agents, and yet the
process of exchanging an aqua ligand on [Ir(H2O)6]3+ takes hundreds of years.[13-15] In fact, an
Ir analogue of NAMI-A (a Ru-based antitumor metastasis agent on clinical trials), trans-
[IrCl4(DMSO)(Im)][ImH] (DMSO = dimethyl sulfoxide, ImH = imidazole), was kinetically inert
towards hydrolysis and was also biologically inactive.[16] However, Meggers and co-workers
took advantage of the kinetic inertness and structural opportunities provided by the octahedral
geometries of Ir(III), and successfully designed a selective Ir(III)-based inhibitor of the receptor
tyrosine kinase FLT4 (also known as VEGFR3) as anticancer agents (compound 1 in Figure
1).[17, 18] The Sadler group and the Sheldrick group found that the anticancer activities of Ir(III)
complexes strongly depended on their ligand sets.[19] For instance, the “half-sandwich”
pentamethylcyclopentadienyl (Cp*) iridium(III) complex 2 was biologically inactive, while by
xph
the incorporation of phenyl substituents as in phenyltetramethylcyclopentadienyl (Cp
xbiph
) and
biphenyltetramethylcyclopentadienyl (Cp
) ligands, iridium(III) complexes 3 and 4 both

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demonstrated activities against A2780 cancer cells.[14] The introduction of the C,N-chelating
ligands instead of the N,N-chelating ligands also improved the antiproliferative activities of the
iridium(III) complexes (see compounds 5 and 6 in Figure 1).[20] In addition, Ma and other
groups identified several series of cyclometalated Ir(III) complexes with favorable anticancer
activities.[21-25] However, to date, no Ir(III) complex has reached the market. Efforts are still
needed to discover novel Ir(III) complexes that will benefit the cancer patients.
Our group was previously inspired by the special characteristics of the iridium complexes
as catalysts and developed iridium-based asymmetric synthetic methodologies.[26, 27] The
recently reported anticancer activities of Ir(III)-based compounds intrigued our interest in
exploring the functions of Ir(III) complexes in biological systems. In order to identify novel
Ir(III)-based anticancer agents, we synthesized a series of “half-sandwich” Schiff-base Ir(III)
complexes. We report herein the anticancer activities, the structure-activity relationship analysis,
and the preliminary mechanism studies of these compounds.
2. Results and discussion
2.1 Chemistry
The general synthetic route for the targeted “half-sandwich” Schiff-base Ir(III) complexes
10a-o is outlined in Scheme 1.[28, 29] The easily accessible ketones 7 was treated with anilines
8 to obtain the ketimines 9. Then, the reaction of 1.0 equivalent of [Cp*IrCl2]2 with 2.0
equivalents of ketimine 9 in the presence of 10.0 equivalents of NaOAc in dichloromethane
(DCM) for 24 h afforded the Ir(III) complexes in moderate to high yields. All new complexes
were characterized by 1H NMR, 13C NMR, IR, and ESI-MS analysis.
2.2. Biological evaluation
2.2.1. In vitro antiproliferative activity and structure-activity relationship analysis
The in vitro antiproliferative activities of the compounds 10a-o were evaluated against
the leukemia K562 cell line and the corresponding IC50 (the concentration leading to 50%
inhibition of the cancer cell proliferation) values were listed in Table 1. Complexes 10a-o
demonstrated promising antiproliferative activities against K562 cells, and all showed higher
potency than cisplatin. Although the antiproliferative activities of these compounds fell within a
relatively narrow range (from 0.26 µM to 4.77 µM), the differences illustrated the effects of the

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Schiff-base ligands of these Ir(III) complexes on their activities in the K562 cells. In most cases,
compounds containing electron-donating groups showed better potency than their counterparts
without electron-donating groups. The complex 10k with a strong electron-withdrawing nitro
group showed the worst activity (IC50 = 4.77 µM), while compound 10c containing two electron-
donating methoxy groups demonstrated the highest potency (IC50 = 0.26 µM). Further analysis
revealed that electron-donating groups on the para-position of ring A favorably contributed to the
activity of the compounds and electron-withdrawing groups led to decrease of the activity of the
complexes (compounds 10j vs. 10e, 10i vs. 10h and 10g, and 10d vs. 10k, 10l, and 10n). This
probably is because hydrolysis is a key step in the mechanism of action as mentioned above and
the electron-donating groups facilitate the leaving of the chloride ions, accelerating the
hydrolysis. Substituents on ring B might affect the activity of the compounds through a
combination of electrostatic and other effects. For instance, in cases of compounds 10a, 10c, and
10d, compounds 10h and 10k, or compounds 10f and 10g, electrostatic effects dominantly
affected the activities of these compounds, where electron-donating groups on ring B resulted in
increase of the activities of the compounds and electron-withdrawing group led to decrease of the
activity of the complexes. In cases of compounds 10b and 10d, or compounds 10e and 10f, the
potency of complexes with no substituents on the phenyl ring was slightly lower than those with
electron-withdrawing chloro or trifluoromethyl substituents. This may indicate the specific
requirements of these complexes to interact with its targets in cells.
The activities of compound 10c (the most active in K562 cells) against a series of other
cancer cell lines/sublines including K562/A02, MCF-7, MCF-7/ADM, and A549 cells were
further investigated (Table 2). In all cancer cells tested, compound 10c displayed stronger
potency than cisplatin. Because of the strong activities of compound 10c in multiple cell lines,
the mechanisms of its antiproliferative effects were further investigated.
2.2.2. ROS assessment
The effect of compound 10c on the level of intracellular ROS (iROS) in K562 cells was
measured using the DCFH-DA method. The treatment with compound 10c as well as the
treatment with cisplatin both increased the levels of iROS compared with the untreated control
cells (Figure 2). In addition, the ROS scavenger N-acetyl-l-cysteine (NAC) attenuated the iROS
induced by compound 10c.

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2.2.3. Apoptosis assessment
The ability of compound 10c to provoke apoptosis in K562 cells was then evaluated. As
shown in Figure 3, the percentage of early apoptotic cells (Annexin V+/PI−) after the treatment
with compound 10c for 24 h increased from 0.9±0.5% (control) to 12.2±2.7%. NAC significantly
restored the percentage of early apoptosis cells in the presence of 1 µM compound 10c. This
effect was consistent with the result of the ROS assay, suggesting that compound 10c induced
apoptosis in a ROS-dependent manner. In addition, the percentage of cells in late apoptosis or
necrosis (annexin-V+/PI+) also increased after the treatment with compound 10c, but without
statistically significance compared with the effect caused by cisplatin.
2.2.4. The mitochondrial pathway of apoptosis induced by compound 10c
Our investigations then demonstrated that compound 10c triggered the mitochondrial
pathway of apoptosis. As shown in Figure 4, the intensity of green fluorescence (JC-1 monomers)
was substantially increased in the presence of compound 10c at 1 µM, indicating that compound
10c led to a decrease in the mitochondrial membrane potential of K562 cells. The decrease of the
mitochondrial membrane potential caused by compound 10c was significantly reversed by NAC
at 2 mM, demonstrating that the effect of compound 10c on mitochondrial membrane potential
was correlated with the ROS level in the cells. Figure 5A showed the concentration-dependent
release of cytochrome c (an apoptogenic factor) into the cytosol in K562 cells treated with
compound 10c, suggesting that the induced apoptosis was likely to be mediated by the intrinsic
mitochondria apoptosis pathway. Moreover, compound 10c down-regulated the expression of
Bcl-2 (an anti-apoptotic factor) and up-regulated Bax (a pro-apoptotic factor) in a dose-
dependent manner. The reduction of the ratio of Bcl-2/Bax also indicated the activation of
mitochondrial apoptotic pathway after the treatment with compound 10c. We then analyzed the
activity of caspase-9, a typical marker of mitochondrial apoptotic pathway. As shown in Figure
5B, compound 10c significantly elevated the activities of caspase-9 and caspase-3. Caspase-9
was 1.20, 1.80, 2.48, and 2.68 times as active as the control and caspase-3 was 1.16, 1.65, 2.14,
and 2.25 times as active as the control after the treatments with compound 10c at 0.1, 0.3, 1, and
3 µM, respectively.

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2.2.5. Cell cycle analysis
The effects of compound 10c on cell cycle distribution were examined using flow
cytometry. As shown in Figure 6, the treatment with 1 µM compound 10c for 48 h significantly
enhanced sub diploid cells and decreased cells in S phase and G0/G1 phase. The results also
demonstrated that cisplatin induced the accumulation of K562 cells in G0/G1 phase, indicating
that compound 10c had different mechanisms of action from cisplatin. The treatment with NAC
partially reversed the effects of compound 10c. In addition, the induction of cell death by the
treatment with compound 10c was also confirmed by the flow cytometric analysis of cell cycle.
3. Experimental Section
3.1. Synthesis
3.1.1. Materials and methods
1H NMR and 13C NMR spectra were recorded on a Bruker 400 MHz NMR spectrometer
(Bruker AMX 400, Fällanden, Switzerland) at 400 and 100 MHz, respectively. Chemical shifts
(δ) were reported in ppm with tetramethylsilane as the internal standard. IR spectra were
recorded on a Thermo Scientific Nicolet iS5 spectrometer (iD5 ATR, diamond). ESI-HRMS
spectra were recorded on a Waters SYNAPT G2. Melting points were recorded at SGW X-4
melting point instrument (Shanghai precision & scientific instrument Co., Ltd, Shanghai, China).
All reagents and solvents were purchased from Admas, Aldrich, Energy Chemical, J&K, and
Strem Chemicals, and used as received. Column chromatography was performed on silica gel
(300-400 mesh, Qingdao Marine Chemical Ltd, Qingdao, China).
3.1.2. General procedures for the preparation of [Cp*IrCl2]2
To a solution of IrCl3·3H2O (1.4950 g, 5.0mmol) in 10 mL methanol was added
pentamethylcyclopentadiene (1.0200 g, 7.5 mmol). The mixture was refluxed for 36 h. After the
mixture was cooled to room temperature, the product [Cp*IrCl2]2 was precipitated out, filtered,
washed with cold methanol, dried under vacuum, and used without further purification.
3.1.3. General procedures for the preparation of ketimines
Under a N2 atmosphere, dichloromethane (25 mL), Et3N (20 mmol), ketone (10 mmol),
and amine (12 mmol) were added into a round bottom flask. TiCl4 (5 mmol) in dichloromethane

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(10 mL) was added dropwise under N2 at 0 °C within 15 min. The reaction mixture was stirred at
0 °C for 0.5 h, then warmed up to 25 °C and stirred at 25 °C for 7-8 h. The reaction mixture was
quenched with a saturated solution of K2CO3 (30 mL), and the reaction mixture was filtered
through a Buchner funnel. The organic layer was separated from the filtrate, and the remaining
aqueous layer was extracted with dichloromethane (2×25 mL). The combined organic layer was
washed with brine (10 mL) and dried over anhydrous Na2SO4. The solvent was removed under
vacuum, and then the ketimine was recrystallized from ethanol.
3.1.4. General procedures for the preparation of the iridium complexes
[Cp*IrCl2]2 (1.0 equiv), ketimine (2.0 equiv), and NaOAc (10.0 equiv) were placed into a
Schlenk tube. The tube was then transferred into a nitrogen-filled glove-box. Dichloromethane
was then added and the resulting mixture was stirred at room temperature for 24 h. The reaction
mixture was filtered through celite, and dried over Na2SO4. After removal of the solvent under
vacuum the resulting solid was washed with diethyl ether/petroleum ether to afford the iridium
complexes.
3.1.5. Analytical and spectral characterization data
[(η5-Cp*)Ir((E) -4-((1-(4-methoxyphenyl)ethylidene)amino)benzonitrile)Cl] 10a:
Yellow solid (26.3 mg, 0.05 mmol, 86%); m.p. = 220 ~ 222 °C; 1H NMR (400 MHz, CDCl3, 298
K) δ (ppm): δ 7.93 (s, 1H), 7.74 (d, J = 7.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 1H), 7.35 (s, 1H), 6.96
(s, 1H), 6.61 (d, J = 8.4 Hz, 1H), 3.91 (s, 3H), 2.40 (s, 3H), 1.41 (s, 15H); 13C NMR (100 MHz,
CDCl3, 298 K) δ (ppm): 179.70, 170.89, 161.62, 153.47, 139.54, 129.88, 118.22, 117.49, 108.66,
107.41, 88.10, 54.08, 15.92, 7.54; IR (thin film, cm-1): 2960, 2357, 1534, 1456, 1342, 1257, 1013,
791, and 748; HRMS (ESI) for C26H28ClIrN2O [M-Cl]+ : m/z calc.: 577.1831, Found: 577.1855.
[(η5-Cp*)Ir((E) -1-(4-methoxyphenyl)-N-(4-(trifluoromethyl)phenyl)ethan-1-imine)Cl]10b:
Yellow solid (27.6 mg, 0.05 mmol, 84%); m.p. = 245 ~ 247 °C; 1H NMR (400 MHz, CDCl3, 298
K) δ (ppm): 8.19 – 7.81 (m, 1H), 7.70 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.6 Hz, 1H), 7.36 (d, J =
2.5 Hz, 1H), 7.19 – 6.91 (m, 1H), 6.61 (dd, J = 8.6, 2.5 Hz, 1H), 3.92 (s, 3H), 2.41 (s, 3H), 1.42
(s, 15H); 13C NMR (100 MHz, CDCl3, 298 K) δ (ppm): 180.51, 171.36, 162.40, 153.66, 140.71,
130.58, 128.08 (q, J = 23 Hz), 123.91 (q, J = 270 Hz), 119.18, 108.15, 89.01, 55.00, 16.83, 8.44;

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IR (thin film, cm-1): 2962, 2367, 1537, 1455, 1377, 1272, 1014, 795, and 749; HRMS (ESI) for
C26H28ClF3IrNO [M-Cl]+ : m/z calc.: 620.1752, Found: 620.1768.
[(η5-Cp*)Ir((E) -N,1-bis(4-methoxyphenyl)ethan-1-imine)Cl] 10c:
Yellow solid (purchased from Strem Chemicals); the characterization data are as follows and
consistent with the reported data.[30] 1H NMR (400 MHz, CDCl3, 253 K) δ (ppm): 7.79 (d, J =
8.8 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H), 7.02-6.95 (m, 2H), 6.91 (d, J =
8.1 Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 2.44 (s, 3H), 1.44 (s, 15H);
HRMS for C26H31ClIrNO2 [M-Cl]+ : m/z calc.: 582.1984, Found: 582.1980.
[(η5-Cp*)Ir((E)-1-(4-methoxyphenyl)-N-phenylethan-1-imine)Cl] 10d:
Orange solid (24.8 mg, 0.05 mmol, 84%); the characterization data are as follows and consistent
with the reported data.[31] m.p. = 228 ~ 230 °C; 1H NMR (300 MHz, CDCl3, 298 K) δ (ppm):
7.49- 7.35 (m, 4H), 7.23-7.20 (m, 3H), 6.59 (d, J = 7.9 Hz, 1H), 3.91 (s, 3H), 2.39 (s, 3H), 1.42(s,
15H); HRMS (ESI) for C25H29ClIrNO [M-Cl]+ : m/z calc.: 552.1878, Found: 552.1873.
[(η5-Cp*)Ir((E) -N-(4-chlorophenyl)-1-(p-tolyl)ethan-1-imine)Cl] 10e:
Yellow solid (27.5 mg, 0.05 mmol, 91%); m.p. = 287 ~ 289 °C; 1H NMR (400 MHz, CDCl3, 298
K) δ (ppm): 7.98 – 7.70 (m, 1H), 7.64 (s, 1H), 7.41 (t, J = 7.3 Hz, 3H), 6.85 (dd, J = 7.9, 1.1 Hz,
2H), 2.45 (s, 3H), 2.42 (s, 3H), 1.43 (s, 15H); 13C NMR (100 MHz, CDCl3, 298 K) δ (ppm):
181.53, 168.68, 149.31, 145.01, 142.50, 135.77, 131.58, 128.59, 122.73, 88.97, 53.40, 30.90,
21.98, 16.79, 8.52; IR (thin film, cm-1): 2963, 2359, 1575, 1455, 1375, 1261, 1013, 802, and 749;
HRMS (ESI) for C25H28Cl2IrN [M-Cl]+ : m/z calc.: 570.1540, Found: 570.1519.
[(η5-Cp*)Ir((E) -N-phenyl-1-(p-tolyl)ethan-1-imine)Cl] 10f:
Orange solid (25.4 mg, 0.05 mmol, 89%); the characterization data are as follows and consistent
with the reported data.[31] m.p. = 255 ~ 258 °C; 1H NMR (300 MHz, CDCl3, 298 K) δ (ppm):
7.64 (s, 1H), 7.41 (d, J = 7.4 Hz, 3H), 7.25-7.21 (m, 2H), 6.84 (d, J = 7.2 Hz, 2H), 2.45 (s, 3H),
2.41 (s, 3H), 1.41 (s, 15H); HRMS (ESI) for C25H29ClIrN [M-Cl]+ : m/z calc.: 536.1929, Found:
536.1925.

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[(η5-Cp*)Ir((E) -N,1-di-p-tolylethan-1-imine)Cl] 10g:
Yellow solid (27.2 mg, 0.05 mmol, 93%); m.p. = 238 ~ 240 °C; 1H NMR (400 MHz, CDCl3, 298
K) δ (ppm): 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 2H), 7.32 (d, J = 7.9 Hz, 1H), 7.13 (d, J = 7.3
Hz, 2H), 6.76 (dd, J = 7.9, 1.1 Hz, 2H), 2.37 (s, 3H), 2.32 (d, J = 2.5 Hz, 6H), 1.34 (s, 15H); 13C
NMR (100 MHz, CDCl3, 298 K) δ (ppm): 180.80, 168.11, 148.26, 145.33, 141.94, 135.63,
128.19, 122.45, 88.81, 21.89, 20.93, 16.68, 8.41; IR (thin film, cm-1): 2961, 2367, 1574, 1451,
1375, 1261, 1019, 800, and 749; HRMS (ESI) for C26H31ClIrN [M-Cl]+ : m/z calc.: 550.2086,
Found: 550.2081.
[(η5-Cp*)Ir((E) -1-(4-nitrophenyl)-N-(p-tolyl)ethan-1-imine)Cl] 10h:
Brown solid (27.7 mg, 0.05 mmol, 89%); m.p. = 275 ~ 277 °C; 1H NMR (400 MHz, CDCl3, 298
K) δ (ppm): 8.61 (d, J = 2.3 Hz, 1H), 7.86 (dd, J = 8.5, 2.3 Hz, 1H), 7.76 – 7.67 (m, 1H), 7.61 (d,
J = 8.5 Hz, 3H), 6.90 – 6.67 (m, 1H), 2.48 (s, 3H), 2.42 (s, 3H), 1.44 (s, 15H); 13C NMR (100
MHz, CDCl3, 298 K) δ (ppm): 180.23, 168.65, 153.38, 148.97, 147.86, 136.68, 129.16, 128.48,
116.79, 90.03, 21.03, 17.43, 8.50; IR (thin film, cm-1): 2961, 2357, 1504, 1446, 1332, 1260, 1019,
796, and 758; HRMS (ESI) for C25H28ClIrN2O2 [M-Cl]+ : m/z calc.: 581.1780, Found: 581.1812.
[(η5-Cp*)Ir((E) -1-phenyl-N-(p-tolyl)ethan-1-imine)Cl] 10i:
Yellow solid (22.5 mg, 0.05 mmol, 78%); m.p. = 202 ~ 204 °C; 1H NMR (400 MHz, CDCl3, 298
K) δ (ppm): 7.75 (d, J = 7.6 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.48 – 7.40 (m, 1H), 7.15 (dd, J =
7.2, 6.2 Hz, 3H), 6.94 (dd, J = 11.0, 4.0 Hz, 1H), 6.85 – 6.56 (m, 1H), 2.36 (s, 3H), 2.33 (s, 3H),
1.35 (s, 15H); 13C NMR (100 MHz, CDCl3, 298 K) δ (ppm): 181.28, 168.14, 148.25, 147.75,
135.78, 135.03, 131.91, 128.36, 121.30, 89.02, 20.97, 16.79, 8.44; IR (thin film, cm-1): 2982,
2364, 1579, 1448, 1374, 1261, 1025, 785, and 749; HRMS (ESI) for C25H29ClIrN [M-Cl]+ : m/z
calc.: 536.1929, Found: 536.1964.
[(η5-Cp*)Ir((E) -N-(4-chlorophenyl)-1-phenylethan-1-imine)Cl] 10j:
Orange solid (25.1 mg, 0.05 mmol, 85%); m.p. = 271 ~ 273 °C; 1H NMR (400 MHz, CDCl3, 298
K) δ (ppm): 7.76 (dd, J = 7.6, 0.7 Hz, 2H), 7.46 (dd, J = 7.8, 1.1 Hz, 1H), 7.34 (d, J = 8.0 Hz,
2H), 7.16 (dd, J = 7.5, 1.3 Hz, 1H), 6.97 (td, J = 7.7, 1.1 Hz, 1H), 6.77 (s, 1H), 2.38 (s, 3H), 1.37
(s, 15H); 13C NMR (100 MHz, CDCl3, 298 K) δ (ppm): 182.03, 168.67, 149.26, 147.40, 135.14,

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132.31, 131.74, 128.74, 121.53, 89.16, 30.91, 16.87, 8.53; IR (thin film, cm-1): 2961, 2354, 1539,
1448, 1374, 1260, 1012, 797, and 749; HRMS (ESI) for C24H26Cl2IrN [M-Cl]+ : m/z calc.:
556.1383, Found: 556.1357.
[(η5-Cp*)Ir((E) -1-(4-nitrophenyl)-N-phenylethan-1-imine)Cl] 10k:
Brown solid (25.9 mg, 0.05 mmol, 86%); m.p. = 275 ~ 277 °C; 1H NMR (400 MHz, CDCl3, 298
K) δ (ppm): 8.62 (d, J = 2.2 Hz, 1H), 7.88 (d, J = 2.3 Hz, 1H), 7.86 (d, J = 2.3 Hz, 1H), 7.63 (d, J
= 8.5 Hz, 1H), 7.48 (s, 2H), 7.29 (d, J = 7.5 Hz, 1H), 6.88 (s, 1H), 2.49 (s, 3H), 1.45 (s, 15H);
13C NMR (100 MHz, CDCl3, 298 K) δ (ppm): 180.30, 168.78, 153.25, 150.24, 148.94, 129.12,
128.58, 126.91, 116.76, 90.05, 17.42, 9.32, 8.43; IR (thin film, cm-1): 2961, 2367, 1510, 1448,
1378, 1258, 1011, 788, and 751; HRMS (ESI) for C24H26ClIrN2O2 [M-Cl]+ : m/z calc.: 567.1624,
Found: 567.1662.
[(η5-Cp*)Ir((E) -1-(4-chlorophenyl)-N-phenylethan-1-imine)Cl] 10l:
Yellow solid (22.7 mg, 0.05 mmol, 76%); m.p. = 231 ~ 233 °C; 1H NMR (400 MHz, CDCl3, 298
K ) δ (ppm): 7.77 (s, 1H), 7.68 (d, J = 2.0 Hz, 1H), 7.37 (d, J = 8.2 Hz, 2H), 7.16 (d, J = 7.4 Hz,
2H), 6.95 (dd, J = 8.3, 2.0 Hz, 1H), 6.80(s, 1H), 2.35 (s, 3H), 1.35 (s, 15H); 13C NMR (100 MHz,
CDCl3, 298 K) δ (ppm): 180.55, 169.84, 150.51, 146.18, 137.84, 134.43, 129.55, 126.44, 121.64,
89.36, 17.04, 8.42; IR (thin film, cm-1): 2961, 2371, 1530, 1448, 1373, 1259, 1014, 792, and 749;
HRMS (ESI) for C24H26Cl2IrN [M-Cl]+ : m/z calc.: 556.1383, Found: 556.1403.
[(η5-Cp*)Ir((E) -1-(2-chlorophenyl)-N-phenylethan-1-imine)Cl] 10m:
Yellow solid (23.3 mg, 0.05 mmol, 79%); m.p. = 232 ~ 234 °C; 1H NMR (400 MHz, CDCl3, 298
K) δ (ppm): 7.80 (s, 1H), 7.64 (d, J = 7.4 Hz, 1H), 7.38 (d, J = 32.7 Hz, 2H), 7.15 (t, J = 7.4 Hz,
1H), 7.01 (t, J = 7.6 Hz, 1H), 6.94 (d, J = 7.7 Hz, 1H), 6.77 (s, 1H), 2.63 (s, 3H), 1.32 (s, 15H);
13C NMR (100 MHz, CDCl3, 298 K) δ (ppm): 181.46, 171.91, 150.98, 143.34, 134.30, 132.39,
126.39, 124.74, 89.82, 23.13, 8.33; IR (thin film, cm-1): 2962, 2362, 1557, 1449, 1377, 1260,
1023, 790, and 748; HRMS (ESI) for C24H26Cl2IrN [M-Cl]+ : m/z calc.: 556.1383, Found:
556.1413.
[(η5-Cp*)Ir((E) -1-(4-fluorophenyl)-N-phenylethan-1-imine)Cl] 10n:

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Yellow solid (25.6 mg, 0.05 mmol, 89%); m.p. = 202 ~ 204 °C; 1H NMR (400 MHz, CDCl3, 298
K) δ (ppm): 7.84 (s, 1H), 7.52 (dd, J = 8.5, 5.6 Hz, 1H), 7.48 (dd, J = 9.4, 2.5 Hz, 1H), 7.44 (s,
2H), 7.23 (t, J = 7.4 Hz, 1H), 6.87 (s, 1H), 6.73 (td, J = 8.7, 2.5 Hz, 1H), 2.42 (s, 3H), 1.41 (s,
15H); 13C NMR (100 MHz, CDCl3, 298 K) δ (ppm): 180.07, 171.35, 166.08, 163.54, 150.56,
144.06, 130.44, 126.32, 120.81, 108.74, 89.25, 17.08, 8.39; IR (thin film, cm-1): 2960, 2369,
1544, 1448, 1373, 1259, 1014, 796, and 749; HRMS (ESI) for C24H26ClFIrN [M-Cl]+ : m/z calc.:
540.1679, Found: 540.1727.
[(η5-Cp*)Ir((E) -1-(naphthalen-2-yl)-N-phenylethan-1-imine)Cl] 10o:
Yellow solid (21.9 mg, 0.05 mmol, 72%); the characterization data are as follows and consistent
with the reported data.[30] m.p. = 247 ~ 249 °C; 1H NMR (400 MHz, CDCl3, 253 K) δ (ppm):
8.17 (s, 1H), 8.08 (s, 1H), 7.91 (d, J = 7.8 Hz, 1H), 7.81 (dd, J = 8.2, 2.8 Hz, 2H), 7.57-7.41 (m,
3H), 7.35-7.29 (m, 2H), 6.93 (d, J = 7.6 Hz, 1H), 2.58 (s, 3H), 1.45 (s, 15H); HRMS for
C28H29ClIrN [M]+: m/z calc.: 607.1605, Found: 607.1603.
3.2. Biological evaluation
3.2.1. Materials
Fetal bovine serum (FBS) was obtained from Hyclone (Thermo, South America). RPMI-
1640 medium was obtained from Gibco BRL (NY, USA.). N-acetyl-L-cysteine (NAC), 3-(4, 5-
dinethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), 7′-
dichlorodihydrofluorescein diacetate (DCFH-DA) were obtained from Sigma-Aldrich (St. Louis,
USA.). Antibodies for cytochrome c (sc-13156), COX4I2 (sc-100522), Bcl-2 (sc-7382), Bax (sc-
493), and β-Actin (sc-47778) were purchased from Santa Cruz Biotechnology (CA, USA).
Secondary anti-mouse (BA1050), anti-rabbit (BA1054) antibodies were obtained from Boster
Bio-Engineering Limited Company (Wuhan, China). The BCA protein kit, the Caspase-3
Activity Assay Kit, and the Caspase-9 Activity Assay Kit were products of the Beyontime
Institute of Biotechnology (Shanghai, China). The enhanced chemiluminescence (ECL) detection
Kit was obtained from Amersham Biosciences (Buckinghamshire, UK). Annexin V-FITC/PI
Apoptosis Detection Kit was purchased from Key GEN Biological Engineering Materials Co.
Ltd (Nanjing, China). Triton X-100, Bovine serum albumin (BSA), and 4’, 6-diamidino-2-

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phenylindole (DAPI) were purchased from Beijing Solarbio Science & Technology Co. Ltd
(Beijing, China).
3.2.2. Cell culture and treatments
Human leukemia cell line K562 and its adriamycin-selected multidrug resistant subline
K562/ADM were obtained from the Institute of Hematology, Chinese Academy of Medical
Sciences (Tianjin, China). Human breast cancer cell line MCF-7, adriamycin-selected multidrug
resistant subline MCF-7/ADM, and human non-small cell lung cancer cell line A549 were
purchased from Keygen Biotech (Nanjing, China). All the cell lines were grown in RPMI 1640
medium containing 10% bovine serum (FBS) and 100 U antibiotics (benzylpenicillin sodium and
gentamycin sulfate) at 37 °C in a humidified 5% CO2 incubator. To maintain MDR phenotype, 1
mg/L adriamycin was added to K562/ADM and MCF-7/ADM cultures and maintained in the
drug-free medium for at least two weeks before being used. Compounds were dissolved in
DMSO with a stock concentration of 0.1 M for in vitro assays.
3.2.3. The MTT assay
Cells were cultured at a density of 1×105/mL in a 96-well plate and allowed to attach
overnight. After the incubation with the testing chemicals for 48 h, the cells were treated with the
solution of MTT (5 mg/mL) at 37 °C for 4 h. The dark purple formazan crystals formed inside
the intact mitochondria were solubilized with DMSO, and the absorbance was measured at 570
nm using a microplate reader (Multiskan Spectrum, Thermo Scientific). The inhibition rates were
calculated on a plate-by-plate basis for the test wells. The IC50 values were calculated based on
the inhibitory rate curves using Bliss’ method.
3.2.4. The ROS assay
K562 cells were seeded on 6-well plates. After incubation with the testing chemicals,
cells from each group at a density of 1×106 were harvested and incubated with 10 µM DCFH-DA
at 37 °C for 30 min in the dark. Cells were then washed with PBS, and the fluorescence intensity
was measured in the channel at 515-545 nm (emission) for DCF (2′,7′-dichlorofluorescein) with
argon laser excitation at 488 nm using Flow Cytometry (BD FACSVerse). The intracellular DCF

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intensity was also confirmed by inverted fluorescence microscopy (Leica DMI4000) at a
magnification of 200-fold.
3.2.5. The analysis of mitochondrial membrane potential (MMP)
The membrane potential assay was based on the fluorescence intensity of JC-1 (a
lipophilic and cationic dye). JC-1 emitted green fluorescence when the MMP was relatively low
while JC-1 aggregates emitted red fluorescence when the mitochondrial membranes were
disrupted. Briefly, K562 cells were treated with the testing chemicals respectively for 24 h and
were incubated with 5 µg/mL JC-1 at 37 °C for 20 min in the dark. The cells were rinsed twice
with JC-1 staining buffer, and the fluorescence intensity was measured by flow cytometry (BD
FACSVerse) and was observed using a confocal microscopy (Leica TCS SP8).
3.2.6. Measurements of cytochrome c
After the treatment with compound 10c for 24 h, the cells were lysed in the preparation
buffer (250 mM sucrose, 20 mM HEPES-KOH at pH of 7.4, 10 mM NaCl, 1.5 mM MgCl2, 1
mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 2 µg/mL aprotinin, and 1 mM
phenylmethylsulfonyl fluoride) for 10 min on ice followed by centrifugation at 10,000 rpm at
4 °C for 20 min to separate the cytosolic and mitochondrial fractions. Protein concentrations
were measured using a BCA Protein Assay Kit (Beyotime). The contents of cytochrome c in the
cytosol and mitochondria were determined by western blot.
3.2.7. Extraction, fractionation, and detection of cytosolic and mitochondrial cytochrome c
K562 cells (2-5×107 per sample) were treated with the testing compounds for 24 h and
harvested by centrifugation at 800×g for 5 min. The extraction and fractionation of cytosolic and
mitochondrial cytochrome c were manipulated using mitochondia/cytosol isolation kit following
the manufacturer's instructions (Applygen Technologies Inc. Beijing, China). Briefly, the pellets
were washed once with ice-cold PBS, suspended with 1.5 ml pre-cold Mito-Cyto buffer in
grinder, and then grinded about 20 times. The cellular homogenate was centrifuged at 800×g at
4 °C for 5 min to obtain the supernatants. The supernatants were then centrifuged at 12000×g at
4 °C for 10 min. The obtained supernatants were used for the identification of cytosolic
cytochrome c and the pellets for mitochondrial cytochrome c were subjected to 15% SDS-PAGE

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and immunoblotted. Cytochrome c oxidase IV was used as the loading control for the
mitochondrial fraction.
3.2.8. Western blot
K562 cells from each group were treated with the testing compounds for 24 h and
suspended in lysis buffer for 30 min with shaking at 4 °C. After centrifugation (10000×g) for 10
min, the supernatants were collected. Cell lysate (50 µg) was resolved on 4-12 % SDS-PAGE
gels and then transferred onto the nitrocellulose membranes. The membranes were blocked with
Tris-buffered saline with 0.1% Tween 20 and 5% skim milk, and were incubated with the
primary antibodies overnight at 4 °C. The membranes were then washed with TBST for three
times and incubated with HRP-conjugated secondary antibody at room temperature for 1 h.
Following three-time wash with TBST, the membranes were developed by the ECL detection Kit.
The images of the western blot were captured and analyzed by the Bio-rad imaging system.
3.2.9. Measurements of the caspase activity
The activities of caspase-3 and -9 were measured using the Caspase-3 Activity Assay Kit
and the Caspase-9 Activity Assay Kit according to the kit manuals. K562 cells with or without
treatments of compounds were lysed using 100 µL lysis buffer, and subsequently centrifuged at
16000 g at 4 °C for 10 min. The supernatant was collected. Next, 70 µL of the assay buffer was
mixed with 20 µL of lysate in a 96-well plate, followed by the addition of 10 µL of 1 mM p-
nitroaniline (pNA). The plate was then incubated at 37 °C in the dark for 1 h, and the relative
fluorescence unit value for the emission at 405 nm was measured. The result was normalized to
the total protein measured using the Bradford assay.
3.2.10. Determination of the apoptotic rate
The apoptotic rate of K562 cells was detected by the Annexin V-FITC/PI double labeling
method. The cell suspension was centrifuged and re-suspended in 195 µL Annexin V-FITC
binding buffer and incubated with 5 µL Annexin V-FITC in the dark at ambient temperature for
10 min. Cells were centrifuged, and the pellets were re-suspended in 195 µL binding buffer.
Cells were then incubated with 10 µL PI solution on an ice bath in the dark for 10 min. The

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suspension of each group was analyzed by flow cytometry (BD FACSVerse). The fluorescence
of Annexin V-FITC and PI was also observed using a confocal microscopy (Leica TCS SP8).
3.2.11. The cell cycle assay
Following the incubation of the testing chemicals for 24 h, 1×106 cells were harvested
and fixed in 70% cold ethanol at 4 °C for 12 h. Then, cells were washed twice with PBS, re-
suspended in 150 µL of 100 ug/mL RNase solution (in 0.2 M citrate-phosphate buffer, pH 7.8),
and incubated at 37 °C for 30 min. Finally, cells were stained with 100 µg/mL PI solution at
room temperature in the dark. The fluorescence activity of the remaining DNA content was
analyzed by flow cytometry (BD FACSVerse).
3.2.12. Statistical analysis
All experiments were performed in triplicates. Significant differences among the groups
were determined by one-way ANOVA analysis followed by Dunnett’s multiple comparison tests.
P-values less than 0.05 were considered as statistically significant.
4. Concluding Remarks
In conclusion, we synthesized a series of “half-sandwich” Schiff-base Ir(III) complexes
and evaluated their activities against the K562 cell line. All compounds exhibited IC50 values of
less than 5 µM in K562 cells. The most potent compound 10c in K562 cells (IC50 = 0.26 µM)
also displayed cytotoxicity against a panel of other cancer cell lines/sublines including
K562/A02, MCF-7, MCF-7/ADM, and A549. Notably, compound 10c outperformed cisplatin in
all cells tested. The flow cytometric analysis with Annexin V-FITC/PI staining indicated that
compound 10c induced apoptosis of the cancer cells. Experiments also showed that compound
10c increased the iROS level and decreased MMP. Further investigation illustrated that the
treatment of 10c led to upregulation of Bax and caspase-9, downregulation of Bcl-2, and release
of cytochrome c in K562 cells, which suggested that compound 10c induce apoptosis through the
intrinsic mitochondria apoptosis pathway.
Acknowledgements
We appreciate the funding support from Sichuan University, the National Natural Science

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Foundation of China (Grant no. U1204830), and the Science and Technology Key Project of
Department of Education of Henan Province (Grants nos. 13A310064 and 15A360016).

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Figure 1. Representative Ir(III) complexes

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Figure 2. Compound 10c increased the generation of reactive oxygen species (ROS). (A) Flow
cytometric analysis of ROS generation. (B) Fluorescence microscopy analysis of ROS generation.
Cells were treated with 1 µM compound 10c, 1 µM compound 10c + 2 mM NAC, and 5 µM
cisplatin for 24 h, respectively, and then incubated with 10 µM DCFH-DA. Statistical data were
shown as mean ± SD (n =3). Significant differences relative to the control group were indicated
as * for P<0.05 and ** for P<0.01; significant differences relative to the group with 1 µM
compound 10c were indicated as # for P<0.05 and ## for P<0.01.

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Figure 3. Apoptosis induction of compound 10c in K562 cells were determined under light
microscopy (200×), Flow Cytometry and Confocal Microscopy. K562 cells were incubated with
1 µM compound 10c, 1 µM compound 10c + 2 mM NAC, and 5 µM cisplatin for 24 h,
respectively, and then incubated with Annexin V-FITC (green) and PI (red). Shown are the
representative results from three independent experiments.

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Figure 4. Compound 10c induced depolarization of mitochondria membrane potential in K562
cells. (A) Flow cytometric analysis of the depolarization of the mitochondria membrane potential.
(B) Confocal Microscopy analysis of the fluorescence of JC-1 aggregates (red) and JC-1
monomers (green). Cells were treated with 1 µM compound 10c, 1 µM compound 10c + 2 mM
NAC, and 5 µM cisplatin for 24 h, respectively, and then incubated with 5 µg/mL JC-1. Shown
are the representative results from three independent experiments.

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Figure 5. Compound 10c activated the mitochondrial apoptotic pathway in K562 cells. (A)
Concentration-dependent effects of compound 10c on cytochrome c release from mitochondria
to cytosol, and on the protein levels of Bcl-2 and Bax. The data were measured by western blot,
and the representative images from three independent experiments were shown. (B) Activation
of caspase-9 and caspase-3 induced by compound 10c. Enzymatic activities of caspases were
expressed as multiple proportions of the control. Statistical data were shown as mean ± SD (n
=3). Significant differences relative to the control group were indicated as * for P<0.05 and **
for P<0.01.

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Figure 6. Effects of compound 10c on the cell cycle of K562 cells treated for 48 h. Cells were
incubated with 1 µM compound 10c, 1 µM compound 10c + 2 mM NAC, and 5 µM cisplatin.
Cell cycle distributions of the control cells and the chemical-treated cells were determined by
propidium iodide (PI) staining and flow cytometric analysis. (A) Representative results from
three independent experiments. (B) Statistical data were shown as mean ± SD (n =3). Significant
differences relative to the control group were indicated as * for P<0.05 and ** for P<0.01;
significant differences relative to the group with 1 µM compound 10c were indicated as # for
P<0.05 and ## for P<0.01.

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Scheme 1. Synthetic route to Ir(III) complexes 10a-o and their in vitro antiproliferative activities
against K562 cells.
IC50 (µM)a,b
Entry
Compounds
R1
R2
1
2
3
4-OMe
4-OMe
4-OMe
4-CN
4-CF3
4-OMe
1.10
0.73
0.26
10a
10b
10c
4
5
4-OMe
4-Me
4-Me
4-Me
4-NO2
H
H
4-Cl
H
0.95
0.67
1.06
0.53
1.00
0.62
0.94
4.77
1.13
0.61
1.20
10d
10e
10f
6
7
4-Me
4-Me
4-Me
4-Cl
H
10g
10h
10i
8
9
10
11
12
13
14
H
10j
10k
10l
4-NO2
4-Cl
2-Cl
4-F
H
H
10m
10n
H
15
0.87
10o
aValues were the average of three determinations.
bThe IC50 of cisplatin against K562 is 5.86 µM.

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Table 1. Antiproliferative activities of compound 10c against K562/A02, MCF-7, MCF-7/ADM,
and A549 cells in vitro.
IC50
Compounds K562/A02 MCF-7 MCF-7/ADM A549
(µ
M)a
1.95
6.56
5.52
7.46
18.81
36.52
2.09
10c
16.03
cisplatin
aValues were the average of three determinations.

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Highlights:
1. Fifteen “half-sandwich” Schiff-base Ir(III) complexes were investigated.
2. Compound 10c displayed cytotoxicity against five cancer cell lines/sublines.
3. 10c increased ROS level and decreased the mitochondrial membrane potential of K562 cells.
4. 10c induced the apoptosis of K562 cells.