Half-sandwich IridiumIII N-heterocyclic carbene antitumor complexes and biological applications

Article abstract of DOI:10.1016/j.jinorgbio.2018.09.009

Series of half-sandwich Ir^III N-heterocyclic carbene (NHC) antitumor complexes [(η⁵-Cp*)Ir(C^C)Cl] have been synthesized and characterized (Cp* is pentamethyl cyclopentadienyl, and C^C are four NHC chelating ligands containing phenyl

Full text of DOI:10.1016/j.jinorgbio.2018.09.009

Accepted Manuscript
Half-sandwich IridiumIII N-heterocyclic carbene antitumor
complexes and biological applications
Yali Han, Zhenzhen Tian, Shumiao Zhang, Xicheng Liu, Juanjuan
Li, Yanru Li, Yi Liu, Min Gao, Zhe Liu
PII:
DOI:
Reference:
S0162-0134(18)30458-6
doi:10.1016/j.jinorgbio.2018.09.009
JIB 10564
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
31 July 2018
10 September 2018
11 September 2018
Please cite this article as: Yali Han, Zhenzhen Tian, Shumiao Zhang, Xicheng Liu,
Juanjuan Li, Yanru Li, Yi Liu, Min Gao, Zhe Liu , Half-sandwich IridiumIII N-
heterocyclic carbene antitumor complexes and biological applications. Jib (2018),
doi:10.1016/j.jinorgbio.2018.09.009
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Half-sandwich IridiumIII N-heterocyclic Carbene Antitumor
Complexes and Biological Applications
Yali Han, Zhenzhen Tian, Shumiao Zhang, Xicheng Liu*, Juanjuan Li, Yanru Li, Yi Liu,
Min Gao, Zhe Liu*
Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of
Life-Organic Analysis and Key Laboratory of Pharmaceutical Intermediates and Analysis of
Natural Medicine, School of Chemistry and Chemical Engineering, Qufu Normal University, Qufu
273165, China.
*Corresponding author (Email): chemlxc@163.com (X.C. Liu); liuzheqd@163.com (Z. Liu)
Abstract
Series of half-sandwich IrIII N-heterocyclic carbene (NHC) antitumor complexes
[(η5-Cp*)Ir(C^C)Cl] have been synthesized and characterized (Cp* is pentamethyl
cyclopentadienyl, and C^C are four NHC chelating ligands containing phenyl rings at
different positions). IrIII complexes showed potent antitumor activity with IC50 values
ranged from 3.9 to 11.8 µM against A549 cells by the MTT assay. Complexes can
catalyze the conversion of the coenzyme NADH to NAD+ and induce the production
of reactive oxygen species (ROS), and bonding to BSA by static quenching mode.
Complexes can arrest the cell cycle in G1 or S phase and reduce the mitochondrial
membrane potential. Confocal microscopy test show complexes could target the
lysosome and mitochondria in cells with the Pearson’s colocalization coefficient of
0.82 and 0.21 after 12 h, respectively, and followed by an energy-dependent cellular
uptake mechanism.
Key words: Half-sandwich; IridiumIII complex; N-heterocyclic carbene; Antitumor
1. Introduction
Cancer is threatening people's health, and the research on anticancer drugs has
caused widespread concern. Anticancer drugs mainly include natural drugs and
chemical synthetic drugs [1, 2], among these, metal-centered platinum antitumor
drugs are most widely used in clinical practice. Although cisplatin and its derivatives
have achieved great success in clinical applications, the development of which is
hindered by many drawbacks, e.g., serious side-effects and easily acquired drug
1

ACCEPTED MANUSCRIPT
resistance [3-6]. Even so, the successful clinical application of platinum drugs has
promoted the research process of other metal antitumor drugs [7]. Recently,
non-platinum-based compounds such as iridium complexes are attracting more and
more attention [8, 9], and which have significant potential to become alternatives to
platinum-based metal anticancer agents [10, 11].
Iridium antitumor complexes mainly include two types: half-sandwich and
cyclometalated iridium complexes. Due to the higher antitumor activity,
half-sandwich IrIII complexes have attracted considerable attention. The general form
of half-sandwich IrIII complexes can be expressed as [(Cpx)Ir(L^L)Z], Cpx represents
the electron-rich cyclopentadienyl group and its derivatives, Z is the leaving group,
and L^L is chelating ligand. Previous study showed the introduction of electron-rich
Cp* in IrIII complexes can increase the stability of ligand binding to metal iridium
[12-14]. And also, the type and the size of chelating ligands could obviously influence
the targeted sites, the lipid solubility and even antitumor activity for half-sandwich
IrIII complexes.
N-heterocyclic carbene (NHC) metal complexes have been extensively used in
various fields, especially as potential anticancer agents [15-17]. Previous study
showed cyclic double carbene ligands IrIII complexes directly activate the
mitochondrial energy production system of cancer cells, generating reactive oxygen
species (ROS) and activating mitochondria-dependent cell death signaling pathways
[18-20]. However, the study of half-sandwich IrIII NHC complex applied to antitumor
field was rare [21, 22]. In this study, four half-sandwich IrIII NHC complexes of the
type [(Cp*)Ir(C^C)Cl] (Complexes 1-4, Scheme 1) were synthesized and
characterized. Cp* is pentamethylcyclopentadienyl, C^C are four diverse NHC
chelating ligands containing phenyl rings at different positions [23]. As-synthesized
IrIII complexes displayed a favorable antitumor activity against A549 lung cancer cells
(IC50: 3.9-11.8 μm) than cisplatin (21.3 μm) under the same conditions. Complexes
were able to bind with bovine serum albumin (BSA) and oxidize NADH
(nicotinamide adenine dinucleotide) to NAD+, which inducing the production of
reactive oxygen species (ROS) [24, 25]. Complexes can arrest the cell cycle and
2

ACCEPTED MANUSCRIPT
induce apoptosis. Confocal microscopy test show complexes could target the
lysosome and mitochondria in cells with the Pearson’s colocalization coefficient of
0.82 and 0.21 after 12 h, respectively, and followed by an energy-dependent cellular
uptake mechanism [26-28]. The results suggest that half-sandwich IrIII NHC
complexes are hopeful for development as new antitumor agents.
Scheme 1. The selcted NHC chelating ligands (L1-L4) and the synthetic process
of IrIII NHC complexes (1-4).
2. Results and Discussion
2.1 Synthesis and Characterization
Half-sandwich IrIII NHC complexes of the type [(Cp*)Ir(C^C)Cl] were synthesized
in dichloromethane at ambient temperature using silver oxide (Ag2O) catalyzed
reaction. NHC chelating ligands were obtained by the reaction of the corresponding
imidazole and iodine hydrocarbon. All of IrIII NHC complexes were newly
synthesized complexes and achieved in good yields. The synthetic processes are
shown in Scheme 1. IrIII NHC complexes and the intermediates were marked by
nuclear magnetic resonance spectrum (1H NMR), mass spectroscopy (MS) and
elemental analysis. Complexes were non-hygroscopic and highly soluble in common
organic solvents such as dichloromethane, chloroform, dimethyl sulfoxide, partially
dissolved in methanol, insoluble in ether, hexane and petroleum ether.
3

ACCEPTED MANUSCRIPT
2.2 Cytotoxicity Test
The antitumor activity of IrIII NHC complexes against A549 human lung cancer
cells was determined by the MTT assay [29-31]. As shown in Table 1, all complexes
showed better antitumor activity than cisplatin (widely used in clinical practice), with
the IC50 values ranged from 3.9 to 11.8 μM. Compared with complex 1, complexes
2-4 have the better antitumor activity, which is mainly due to the enhanced lipid
solubility caused by the introduction of more phenyl to NHC chelating ligands. The
log P (partition coefficient in oil/water) for complexes 1 and 4 were determined by
inductively coupled plasma mass spectrometry (ICP-MS), the values were -1.12 and
-0.57, respectively. The data show that the increase of the benzene ring to NHC
chelating ligands can effectively increase the lipid solubility of the complex. However,
the position and number of benzene rings have little change in the activity of
complexes 2-4. In order to further study the antitumor mechanism of IrIII NHC
complexes, complexes 2 and 3 were selected as representatives, and further studied.
Table 1. IC50 values of complexes 1-4 and cisplatin against A549 cells determined by MTT
assay after 24 h.
Complex
IC50 (µM)
11.8±1.2
5.9±0.4
4.6±0.2
3.9±0.7
[(η5-C5Me5)Ir(L1)Cl] (1)
[(η5-C5Me5)Ir(L2)Cl] (2)
[(η5-C5Me5)Ir(L3)Cl] (3)
[(η5-C5Me5)Ir(L4)Cl] (4)
Cisplatin
21.3±1.7
2.3 Reaction with NADH
NADH and NAD+ are indispensable substances in various bio-catalytic reactions.
[32]. NADH can contribute hydride to transition metal-based complex and promote
the production of ROS (H2O2), thus serving as a pathway for the oxidation mechanism
[33]. Interaction between IrIII NHC complexes and NADH was determined by
ultraviolet-visible (UV-Vis) spectrum. The maximum absorbance of NADH and
NAD+ can be determined in 339 and 259 nm, respectively. As shown in Fig. 1A (Fig.
4

ACCEPTED MANUSCRIPT
S1, ESI), with the increase of complexes, the absorption of 339 nm showed a
significantly reduced and an increase in 259 nm which further confirmed the catalytic
activity of IrIII NHC complexes [33]. The turn over numbers (TONs) values of
complexes 2 and 3 were shown in Fig. 2B. As shown, complex 3 had the bigger TONs
value, which was correspond with the result of MTT assay. The bigger of the TONs
value, the better of antitumor activity.
Fig. 1 (A) Reaction of complex 3 (1.0 μM) and NADH (100 μM) in a 60% MeOH/40% H2O (v/v)
mixed solution was monitored by UV-Vis at 298 K over 8 h. (B) The TONs of complexes 2 and 3.
2.4 Protein interaction
Serum albumin (SA) has the highest abundance in plasma and excellent binding
properties, so it is typically used in the delivery of drugs in the blood system [34-37].
As a kind of transport protein, bovine serum albumin (BSA) has the advantages of
cheap, stable, easy to purify and similar to human serum albumin (HSA) [38], which
was chose as a model to study the interaction between complexes and proteins.
The UV-Vis absorption spectrum of BSA in the presence of complexes 2 and 3 are
shown in Fig. 2 and Fig. S2, ESI. The internal filtration effect can be eliminated by
UV-Vis and aim at further fluorescence intensity studies. With the increase of
complexes, the maximum absorption at 228 nm decreased, which is mainly due to the
induced perturbation of BSA because of IrIII compounds. [39-41]. Obvious red-shift
was found at 228 nm attribute to the effect of the polar solvent (water). However,
there is no obvious shift in 278 nm, which indicated the microenvironment of the
three aromatic acid residues in BSA (Trp, Tyr and Phe) was changed because of IrIII
5

ACCEPTED MANUSCRIPT
NHC complexes [42].
Fig. 2 (A) Complex 3 was increased from 0 μM to 10 μM, UV-Vis spectra of BSA in
Tris-HCl/NaCl buffer solution (pH=7.2). Arrows indicate the direction of change in absorbance as
the concentration of the complex increases. Inset: Wavelength absorbance changes from 200 to
320 nm. (B) The complex 3 was increased from 0 μM to 10 μM, and the fluorescence spectrum of
BSA was changed (0.5 μM, λex=280 nm, λem=343 nm).
The fluorescence emission spectra of BSA with different concentrations of
complexes 2 and 3 at 298 K are presented in Fig. 2B and Fig. S2B, ESI. As shown,
fluorescence intensity of BSA quenched obviously with the increase of complexes.
The quenching rate constant Kq and Stern–Volmer quenching constant Ksv were
calculated by classical Stern-Volmer equation (Fig. S3, ESI), and the binding constant
Kb and binding site number n of complexes were obtianed by the Scatchard equation
(Fig. S4, ESI) [43]. As shown in Table 3, the values of Kq for complexes 2 and 3 were
3.92×1012 and 3.21×1012 M-1 s-1, which are about two orders of magnitude higher than
that of a pure dynamic quenching mechanism (2.0×1010 M-1 s-1) [44]. These indicated
that the interaction between IrIII NHC complexes and BSA have been followed by a
static quenching mechanism dominates. The binding site number (n) was almost the
same (~1), but complex 3 having a slightly larger binding constant (Kb) values than
complex 2, which was consistent with the conclusion that complex 3 had the higher
antitumor activity.
6

ACCEPTED MANUSCRIPT
Table 3. Quenching parameters and binding parameters for the interaction of the complex 2 and
complex 3 with BSA.
Complex Ksv (104M-1)
Kq (1012 M-1s-1) Kb (104 M-1)
n
3.92±0.73
3.21±0.29
3.92
3.21
2.06
2.55
1.29
1.14
2
3
Information on tyrosine residues and tryptophan residues in the BSA
microenvironment can be displayed at the same fluorescence spectra Δλ = 15 nm and
Δλ = 60 nm [45]. As shown in Figs. S5-S6, ESI, the fluorescence intensity at 291 nm
and 285 nm (Δλ = 15 nm and 60 nm), which are the characteristic of the synchronous
emission spectra for tyrosine and tryptophan residues of BSA, reduced with the
increase of compounds 2 and 3. At Δλ = 15 nm, a slight blue shift of 2 nm was
observed in the complex 3 (Fig. S6), and a slight red shift of 3 nm was observed for
complex 2 at Δλ = 60 nm. The above results clearly shows that IrIII NHC complexes
could act on BSA by affecting its microenvironment.
2.5 Cell cycle analysis
As shown in Fig. 3 (Fig S7, Table S1 and S2, ESI), the cell cycle arrest for A549
cells exposure to complexes 2 and 3 with the concentrations of 0.25, 0.5 and 1.0×IC50
for 24 h have been determined by flow cytometry. At a concentration of 2.0×IC50, the
percentages of cells in the Sub-G1 phase increased from 58.1% to 64.7% when
exposure to complex 2, which indicating cells failed to synthesize RNA and protein
normally, or affecting the energy supply to the next stage of the cell [31, 35]. For
complex 3, the percentages of cells in the S phase increased 7.8%, which indicating
complex 3 may block the synthesis of DNA and histone, or some DNA
replication-related enzymes [35]. Compared with the untreated control, the cell cycle
was disturbed in sub-G1 and S phase for complexes 2 and 3, respectively. The results
indicate that IrIII NHC complexes can disturb the cell growth cycle progression, and
achieving the objective of apoptosis.
7

ACCEPTED MANUSCRIPT
Fig. 3 Histogram data of cell cycle distribution of A549 cancer cells of complexes 2 and 3 after 24
h. Tests were performed using 0.25, 0.5, 1.0, and 2.0 times of the IC50 equivalent concentration.
The data is taken as mean ± SD of three measurements. Cell staining for flow cytometry was
carried out using PI/RNase.
2.6 Induction of apoptosis
To determine whether cell function decline was associated with apoptosis, A549
cells were treated with complexes 2 and 3 at 1.0, 2.0, and 3.0 equivalents IC50 for 24
hours. After staining with Annexin, data were measured by flow cytometry [46]. As
shown in Fig. 4 and Tables S3- S4, ESI, the population of the early and late apoptotic
phase had a significant increase with the value ranged from 1.0% and 4.3% to 2.1%
and 29.8% after 24 h, respectively, when the concentration changed from 1.0×IC50 to
3.0×IC50 for complex 2. For complex 3 (Fig. 4), about 39.0% of the A549 cells were
undergoing apoptosis, including 32.0% of cells in the late apoptosis, while 95.8% of
untreated cells are still alive under the same conditions.
8

ACCEPTED MANUSCRIPT
Fig. 4 (A) Apoptosis analysis of A549 cancer cells after 24 h of exposure to complexes 2 and 3 at
310 K determined by flow cytometry with Annexin V-FITC vs PI staining. (B) Histogram showing
populations for A549 cells in four stages treated by complexes 2 and 3. Data are quoted as mean ±
SD of three replicates.
2.7 Induction of ROS
The accumulation of ROS produced in mitochondrial will lead to apoptosis, which
can be used to explore the function mechanism of antitumor agents. By means of flow
cytometry, the level of ROS was determined in A549 cells for complexes 2 and 3 after
24 h. As shown in Fig. 5 and Tables S5-S6, ESI, compared with the control, the level
of ROS increased by 1.3 and 1.4 times for complex 2 in the concentration of
0.25×IC50 and 0.50×IC50, respectively. For complex 3, the level of ROS only
increased by 1.1 times with the concentration of 0.5×IC50. The results indicate that IrIII
NHC complexes could induce the production of ROS, which provide a basis that
complexes could affect the mitochondria and lead to apoptosis.
9

ACCEPTED MANUSCRIPT
Fig. 5 A549 cancer cells were incubated with complexes 2 (A) and 3 (B) at concentrations of
0.25×IC50 and 0.5×IC50 to induce ROS production. Taking the ROS level of negative control as
the standard to make the histogram.
2.8 Mitochondrial membrane potential
Mitochondrial dysfunction may be involved in the cause of cell death [47-49]. The
extent of mitochondrial dysfunction can be assessed by the loss of the mitochondrial
membrane potential (MMP). As shown in Fig. 6, Fig. S8 and Tables S7-S8, ESI, the
loss of MMP induced by complexes 2 and 3 was assessed by detecting the JC-1 dye
(the decrease in red fluorescence and increased green fluorescence) using flow
cytometry. At the indicated concentrations, a significant concentration-dependent
increase was found with a remarkable loss of MMP, e.g. from 34.3% and 7.8%
(control) to 60.1% and 30.6% (2.0× IC50) for complexes 2 and 3, respectively, which
proving IrIII NHC complexes could act on mitochondria and induce apoptosis.
10

ACCEPTED MANUSCRIPT
Fig. 6 (A) At concentration of 0.25×IC50, 0.5×IC50, 1.0×IC50 and 2.0×IC50, the loss of MMP
induced by complex 2 using JC-1 dye. The red aggregates and green monomers are gated. (B)
Histograms for the MMP treated with different concentrations of complex 2. The above data
is the average ± SD of three measurements repeated.
2.9 Cell imaging and cellular uptake
Subcellular localization of complexes 2 and 3 can be easily determined by laser
confocal microscopy in A549 cells on account of their intrinsic luminescence. The
Lyso Tracker Red DND-99 (LTRD) and Mito Tracker Deep Red (MTDR) were used
as lysosomes and mitochondria fluorescence probes, respectively. As shown in Fig. 7
11

ACCEPTED MANUSCRIPT
(Fig. S9, ESI), selected representative complex 3 could effectively target lysosomes
with the Pearson coefficients of 0.75, 0.88, and 0.82 when incubated for 1, 6 and 12 h,
respectively. In addition, complex 3 could also target mitochondria with the Pearson
coefficients of 0.21, 0.17, and 0.23 after 1, 6 and 12 h, respectively, although it was
not apparent. And also, the complex did not immediately lead to abnormal cell death,
enabling us to track changes in lysosome morphology in real time [6].
Fig. 7 Confocal microscopic images of A549 cells co-labeled with complex 3 (10 μM, 1h, 6h, 12h)
and LTRD (75 nM, 1h). Complex 3 and LTRD were excited at 488 nm and 594 nm, respectively.
Complex 3 collects fluorescence in 549-651nm and LTRD collects fluorescence in 493-630nm.
Scale bar: 20 μm.
Lysosomal permeability is often triggered by the disruption of the lysosomal
integrity [50]. To investigate whether lysosomal damage induced by complex 3 was
accompanied by lysosome targeting specificity. Lysosomal integrity of A549 cells
were evaluated by acridine orange (AO) staining after treatment with 3 for 6 h with
the concentration of 1.0 × IC50 and 3.0 × IC50 [51, 52]. AO exhibits red fluorescence
when accumulated in lysosome and green fluorescence when bounding to RNAs in
12

ACCEPTED MANUSCRIPT
the nuclei or cytosol. As shown in Fig. 8, compared with control, red fluorescence
basically disappears in lysosome when exposed to complex 3 (1.0 × IC50) after 6 h,
and obvious lysosomal damage was found at 3.0 × IC50.
Fig. 8 AO-loaded A549 cells (a) as a control group, no drug was added to the cells. (b) After
addition of complex 3 (1.0 × IC50) for 6 h, AO (5 μM) was added for 15 min after laser confocal
detection. (c) After addition of complex 3 (3.0 × IC50) for 6 h, AO (5 μM) was added for 15 min
after laser confocal detection. Complex 3 was excited at 488 nm and collected at a wavelength of
493-630 nm. Scale bar: 20 μm.
Drug molecules can enter cells with different transport mechanisms, including
energy-dependent mechanisms (such as endocytosis and active transport) and
energy-independent mechanisms (such as diffusion-promoting and passive diffusion)
[53]. Therefore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and chloroquine
were used as energy inhibitor and endocytic inhibitor to study the mechanism of
cellular uptake, respectively. The cells incubated at 277 K and 310 K for 2 h and then
pretreatment with CCCP and chloroquine. As shown in Fig. 9, the uptake of cells
13

ACCEPTED MANUSCRIPT
reduced efficiency after incubated at 273 K, which suggested that cellular uptake of
complex 3 was followed by an energy-dependent mechanism.
Fig. 9 Effect of incubation temperature (37 ºC and 4 ºC), metabolic inhibitor (CCCP, 50 μM) and
chloroquine (50 μM) on cellular uptake of 3 (10 μM, 30 min) measured by confocal microscopy.
Complex 3 was excited at 488 nm and emission was collected at 493-630 nm. Scale bar: 20 µm.
3. Conclusions
In this study, four new half-sandwich IrIII NHC complexes were synthesized with
simple synthetic procedures. All complexes showed favorable antiproliferative
activity. Complex 4 showed the best activity towards A549 lung cancer cells, which
were five times higher than the clinical antitumor drug cisplatin. Complexes can
effectively bind with BSA, catalyzing NADH to NAD+ and inducing ROS, which will
disturb the cell growth cycle and lead to apoptosis. Complexes can specifically target
lysosomes and mitochondria, entering cells through energy-dependent mechanisms,
14

ACCEPTED MANUSCRIPT
and destroying the integrity of lysosomes. Above all, half-sandwich IrIII NHC
complexes could be a promising candidate for further evaluation as antitumor drugs.
4. Experimental section
1H-imidazole, 1H-benzimidazole, 1-benzyl-3-methylimidazoliumiodide (L1),
1-benzylbenzimidazole,
1-diphenylmethylimidazole,
1-(diphenylmethyl)
-1H-benzimidazole and the dimer [Cp*IrCl2]2 were prepared according to literature
procedures [54]. All other reagents are used as supplied by the commercial supplier.
Nitrogen was used as the drying and filling gas.
Synthesis of L2-L4.
1-benzylbenzimidazole (2.5 g, 12 mmol) was dissolved in 10 mL of acetonitrile,
and then iodomethane (1.1 mL, 18 mmol) were added. The mixture was refluxed
overnight. After rotary evaporation, yellow oil product was washed with diethyl ether,
1
pure yellow salt was obtained. Yield: 2.2 g (86%). H NMR (500 MHz, CDCl3) δ
11.23 (s, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.65 (s, 1H), 7.60 (s, 2H), 7.53 (d, J = 6.4 Hz,
2H), 7.39 (d, J = 7.3 Hz, 3H), 5.83 (s, 2H), 4.28 (s, 3H).
L3 and L4 were synthesized using the same method, the representation data is as
follows:
L3: Yield: 79%. 1H NMR (500 MHz, CDCl3) δ 9.67 (s, 1H), 7.43 (s, 2H), 7.41 (s, 5H),
7.33 – 7.30 (m, 5H), 7.08 (s, 1H), 4.08 (s, 3H).
L4: Yield: 74%. 1H NMR (500 MHz, CDCl3) δ 10.21 (s, 1H), 7.71 (d, J = 8.3 Hz, 1H),
7.64 (d, J = 7.6 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.45 (s, 10H), 7.29 (s, 1H), 7.20 (s,
1H), 4.31 (s, 3H).
Synthesis of the complexes 1–4.
In a round bottom flask, silver oxide (2.4 eq) and chelating ligands (L1-L4) (2.0 eq)
were added to a solution of dichloromethane (CH2Cl2). After 8 h, the mixture was
filtered through Celite and washed with CH2Cl2. The combined filtrates were added
dropwise to a solution of CH2Cl2 containing [Cp*IrCl2]2 (1.0 eq). The mixture was
stirred at room temperature for another 8h and filtered through Celite. The solvent
was removed in vacuum， and the product was recrystallized from CH2Cl2 / n-hexane
to give a pure yellow product. Detailed characterization data of complexes 1–4 were
15

ACCEPTED MANUSCRIPT
as follows:
1
[(η5-C5Me5)Ir(L1)Cl] (1): Yield: 79%. H NMR (500 MHz, CDCl3) δ 7.62 (d, J = 7.5
Hz, 1H), 6.97 (dd, J = 12.9, 7.0 Hz, 2H), 6.94 (d, J = 2.0 Hz, 1H), 6.90 (d, J = 1.9 Hz,
1H), 6.81 (t, J = 7.3 Hz, 1H), 4.86 (d, J = 13.8 Hz, 1H), 4.65 (d, J = 13.9 Hz, 1H),
3.93 (s, 3H), 1.68 (s, 15H). Elemental analysis: Found: C, 50.49; H, 5.21; N, 5.64%,
calcd for C21H26ClIrN2: C, 50.58; H, 5.26; N, 5.62%. ESI-MS (m/z): calcd for
C21H26IrN2: 498.67 [M-Cl]+; found 499.42.
1
[(η5-C5Me5)Ir(L2)Cl] (2): Yield: 72%. H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 7.6
Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.34 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 6.7 Hz, 1H),
7.23 (d, J = 7.4 Hz, 1H), 7.08 – 7.05 (m, 1H), 6.92 (t, J = 7.4 Hz, 1H), 6.81 (t, J = 7.0
Hz, 1H), 5.16 (d, J = 14.2 Hz, 1H), 5.03 (d, J = 14.1 Hz, 1H), 4.04 (s, 3H), 1.82 (s,
15H). Elemental analysis: Found: C, 50.78; H, 5.10; N, 5.09%, calcd for C25H28ClIrN2:
C, 50.72; H, 5.14; N, 5.11%. ESI-MS (m/z): calcd for C25H28IrN2: 548.73 [M-Cl]+;
found 549.33.
[(η5-C5Me5)Ir(L3)Cl] (3): Yield: 74%. 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J = 7.6
Hz, 1H), 7.51 (d, J = 6.7 Hz, 3H), 7.44 (s, 2H), 6.84 (t, J = 7.1 Hz, 1H), 6.80 (s, 1H),
6.61 (t, J = 7.4 Hz, 1H), 6.38 (d, J = 8.7 Hz, 2H), 5.98 (s, 1H), 3.90 (s, 3H), 1.81 (s,
15H). Elemental analysis: Found: C, 52.49; H, 5.23; N, 4.35%, calcd for C27H30ClIrN2:
C, 53.06; H, 5.11; N, 4.28%. ESI-MS (m/z): calcd for C27H30IrN2: 574.77 [M-Cl]+;
found 575.33.
[(η5-C5Me5)Ir(L4)Cl] (4): Yield: 68%. 1H NMR (500 MHz, CDCl3) δ 7.95 (d, J = 7.7
Hz, 1H), 7.53 (s, 1H), 7.46 (d, J = 6.5 Hz, 4H), 7.09 – 7.04 (m, 2H), 6.84 (t, J = 7.5
Hz, 1H), 6.70 (t, J = 7.4 Hz, 1H), 6.61 (t, J = 7.5 Hz, 1H), 6.46 (d, J = 7.7 Hz, 2H),
5.56 (d, J = 8.6 Hz, 1H), 4.11 (s, 3H), 1.77 (s, 15H). Elemental analysis: Found: C,
59.51; H, 5.18; N, 4.45%, calcd for C31H32ClIrN2: C, 59.59; H, 5.16; N, 4.48%.
ESI-MS (m/z): calcd for C31H32IrN2: 624.57 [M-Cl]+; found 625.33.
Acknowledgments
We thank the University Research Development Program of Shandong Province
(J18KA082), the National Natural Science Foundation of China (Grant No. 21671118)
and the Taishan Scholars Program for support.
16

ACCEPTED MANUSCRIPT
References
[1] L. Galluzzi, L. Senovilla, I. Vitale, J. Michels, I. Martins, O. Kepp, M. Castedo, G. Kroemer,
Oncogene 31 (2011)1869-1873.
[2] C.-H. Leung, H.-J. Zhong, D.S.-H. Chan, D.-L. Ma, Coord. Chem. Rev. 257 (2013) 1764-1776.
[3] F.-X. Wang, M.-H. Chen, X.-Y. Hu, R.-R. Ye, C.-P. Tan, L.-N. Ji, Z.-W. Mao, Sci. Rep. 6 (2016)
38954.
[4] H. Zhang, L. Guo, Z. Tian, M. Tian, S. Zhang, Z. Xu, P. Gong, X. Zheng, J. Zhao, Z. Liu, Chem.
Commun. 54 (2018) 4421-4424.
[5] A. Wilbuer, D.H. Vlecken, D.J. Schmitz, K. Kräling, K. Harms, C.P. Bagowski, E. Meggers, Angew.
Chem. Intl. Ed. 49 (2010) 3839-3842.
[6] Y. Li, C.-P. Tan, W. Zhang, L. He, L.-N. Ji, Z.-W. Mao, Biomaterials 39 (2015) 95-104.
[7] L. Zeng, P. Gupta, Y. Chen, E. Wang, L. Ji, H. Chao, Z.-S. Chen, Chem. Soc. Rev. 46 (2017)
5771-5804.
[8] Z. Liu, P.J. Sadler, Acc. Chem. Res. 47 (2014) 1174-1185.
[9] N. Muhammad, Z. Guo, Curr. Opin. Chem. Biol. 19 (2014) 144-153.
[10] L. Feng, Y. Geisselbrecht, S. Blanck, A. Wilbuer, G.E. Atilla-Gokcumen, P. Filippakopoulos, K.
Kräling, M.A. Celik, K. Harms, J. Maksimoska, R. Marmorstein, G. Frenking, S. Knapp, L.-O.
Essen, E. Meggers, J. Am. Chem. Soc. 133 (2011) 5976-5986.
[11] Z. Liu, I. Romero-Canelón, B. Qamar, J.M. Hearn, A. Habtemariam, N.P.E. Barry, A.M. Pizarro,
G.J. Clarkson, P.J. Sadler, Angew. Chem. Intl. Ed. 126 (2014) 4022-4027.
[12] M. Böge, C. Fowelin, P. Bednarski, J. Heck, Organometallics 34 (2015) 1507-1521.
[13] L.C. Sudding, R. Payne, P. Govender, F. Edafe, C.M. Clavel, P.J. Dyson, B. Therrien, G.S. Smith,
J. Org. Chem. 774 (2014) 79-85.
[14] G.S. Yellol, A. Donaire, J.G. Yellol, V. Vasylyeva, C. Janiak, J. Ruiz, Chem. Commun. 49 (2013)
11533-11535.
[15] S. Díez-González, N. Marion, S.P. Nolan, Chem. Rev. 109 (2009) 3612-3676.
[16] B.K. Paul, N. Guchhait, Photochem. Photobiol. Sci. 10 (2011) 980-991.
[17] F. Schmitt, K. Donnelly, J.K. Muenzner, T. Rehm, V. Novohradsky, V. Brabec, J. Kasparkova, M.
Albrecht, R. Schobert, T. Mueller, J. Inorg. Biochem. 163 (2016) 221-228.
[18] Z. Deng, L. Yu, W. Cao, W. Zheng, T. Chen, Chem. Commun. 51 (2015) 2637-2640.
[19] Y. Li, X. li, W. Zheng, C. Fan, Y. Zhang, T. Chen, J. Mater. Chem. 1 (2013) 6365-6372.
[20] J. Liu, Y. Chen, G. Li, P. Zhang, C. Jin, L. Zeng, L. Ji, H. Chao, Biomaterials. 56 (2015) 140-153.
[21] S. Fulda, L. Galluzzi, G. Kroemer, Nat. Rev. Drug Discovery 9 (2010) 447.
[22] R.A.J. Smith, R.C. Hartley, M.P. Murphy, Antioxid. Redox Signaling 15 (2011) 3021-3038.
[23] J. Zhu, L. Wu, Q. Zhang, X. Chen, X. Liu, Spectrochim. Acta Part A. 95 (2012) 252-257.
[24] Z. Xu, D. Kong, X. He, L. Guo, X. Ge, X. Liu, H. Zhang, J. Li, Y. Yang, Z. Liu, Inorg. Chem.
Front. 2018.
[25] J.J. Soldevila-Barreda, A. Habtemariam, I. Romero-Canelón, P.J. Sadler, J. Inorg. Biochem. 153
(2015) 322-333.
[26] W. Zhang, C.-H. Tung, Chem. Eur. J. 24 (2018) 2089-2093
[27] H. Huang, L. Yang, P. Zhang, K. Qiu, J. Huang, Y. Chen, J. Diao, J. Liu, L. Ji, J. Long, H. Chao,
Biomaterials. 83 (2016) 321-331.
[28] L. He, Y. Huang, H. Zhu, G. Pang, W. Zheng, Y.-S. Wong, T. Chen, Adv. Funct. Mater. 24 (2014)
2754-2763.
17

ACCEPTED MANUSCRIPT
[29] E. Schuh, C. Pflüger, A. Citta, A. Folda, M.P. Rigobello, A. Bindoli, A. Casini, F. Mohr, J. Med.
Chem. 55 (2012) 5518-5528.
[30] C. Wang, J. Liu, Z. Tian, M. Tian, L. Tian, W. Zhao, Z. Liu, Dalton. Trans. 46 (2017) 6870-6883.
[31] A. Pflug, M. Lukarska, P. Resa-Infante, S. Reich, S. Cusack, Virus. Research. 234 (2017) 103-117.
[32] Z. Liu, R.J. Deeth, J.S. Butler, A. Habtemariam, M.E. Newton, P.J. Sadler, Angew. Chem. Int. Ed.
52 (2013) 4194-4197.
[33] S. Betanzos-Lara, Z. Liu, A. Habtemariam, A.M. Pizarro, B. Qamar, P.J. Sadler, Angew. Chem. Int.
Ed. 51 (2012) 3897-3900.
[34] S. Chatterjee, T.K. Mukherjee, Phys. Chem. Chem. Phys. 16 (2014) 8400-8408.
[35] E. Ramachandran, D. Senthil Raja, N.S.P. Bhuvanesh, K. Natarajan, Dalton. Trans. 41 (2012)
13308-13323.
[36] D.S. Raja, N.S.P. Bhuvanesh, K. Natarajan, Dalton. Trans. 41 (2012) 4365-4377.
[37] Z. Chen, J. Zhang, C. Liu, BioMetals. 26 (2013) 827-838.
[38] J. Fernández-Gallardo, B.T. Elie, F.J. Sulzmaier, M. Sanaú, J.W. Ramos, M. Contel,
Organometallics 33 (2014) 6669-6681.
[39] H. Naz, P. Khan, M. Tarique, S. Rahman, A. Meena, S. Ahamad, S. Luqman, A. Islam, F. Ahmad,
M.I. Hassan, Int. J. Biol. Macromol. 96 (2017) 161-170.
[40] N. Selvakumaran, N.S.P. Bhuvanesh, A. Endo, R. Karvembu, Polyhedron 75 (2014) 95-109.
[41] B. Buscher, S. Laakso, H. Mascher, K. Pusecker, M. Doig, L. Dillen, W. Wagner-Redeker, T.
Pfeifer, P. Delrat, P. Timmerman, Bioanalysis 6 (2014) 673-682.
[42] X. He, M. Tian, X. Liu, Y. Tang, C.F. Shao, P. Gong, J. Liu, S. Zhang, L. Guo, Z. Liu, Chem Asian
J. 13 (2018) 1500-1509.
[43] A. Baral, L. Satish, D.P. Das, H. Sahoo, M.K. Ghosh, New. J. Chem. 41 (2017) 8130-8139.
[44] P. Zhang, S. Zhuo, L. Sun, P. Zhang, C. Zhu, R. Soc. Chem. 39 (2015) 4551-4555.
[45] M. Polson, S. Fracasso, V. Bertolasi, M. Ravaglia, F. Scandola, Inorg. Chem. 43 (2004)
1950-1956.
[46] R. Rubbiani, S. Can, I. Kitanovic, H. Alborzinia, M. Stefanopoulou, M. Kokoschka, S.
Mönchgesang, W.S. Sheldrick, S. Wölfl, I. Ott, J. Med. Chem. 54 (2011) 8646-8657.
[47] Y. Jiang, G. Liu, X. Wang, J. Hu, G. Zhang, S. Liu, Macromolecules 48 (2015) 764-774.
[48] M. Nichi, T. Rijsselaere, J.D.A. Losano, D.S.R. Angrimani, G.K.V. Kawai, I.G.F. Goovaerts, A.
Van Soom, V.H. Barnabe, J.B.P. De Clercq, P.E.J. Bols, Reprod. Domest. Anim. 52 (2017) 257-263.
[49] H. Antonicka, K. Choquet, Z.Y. Lin, A.C. Gingras, C.L. Kleinman, E.A. Shoubridge, EMBO Rep.
18 (2017) 28-38.
[50] X. Wang, M. Zhu, F. Gao, W. Wei, Y. Qian, H.-K. Liu, J. Zhao, J. Inorg. Biochem. 180 (2018)
179-185.
[51] S. Daum, M.S.V. Reshetnikov, M. Sisa, T. Dumych, M.D. Lootsik, R. Bilyy, E. Bila, C. Janko, C.
Alexiou, M. Herrmann, L. Sellner, A. Mokhir, Angew. Chem. Int. Ed. 56 (2017) 15545-15549.
[52] L. He, C.P. Tan, R.R. Ye, Y.Z. Zhao, Y.H. Liu, Q. Zhao, L.N. Ji, Z.W. Mao, Angew. Chem. Int. Ed.
53 (2014) 12137-12141.
[53] C. Li, M. Yu, Y. Sun, Y. Wu, C. Huang, F. Li, J. Am. Chem. Soc. 133 (2011) 11231-11239.
[54] Z. Liu, A. Habtemariam, A.M. Pizarro, S.A. Fletcher, A. Kisova, O. Vrana, L. Salassa, P.C.A.
Bruijnincx, G.J. Clarkson, V. Brabec, P.J. Sadler, J.Med. Chem. 54 (2011) 3011-3026.
18

ACCEPTED MANUSCRIPT
Graphical Abstracts
Ⅲ
Ir N-heterocyclic carbene complexes can catalyze nicotinamide
adenine dinucleotide (NADH) to NAD+, induce reactive oxygen species
(ROS), bonding to bovine serum albumin (BSA) and reduce the
mitochondrial membrane potential (MMP). Complexes could target
lysosome, and through energy-dependent cellular uptake mechanism.
19

ACCEPTED MANUSCRIPT
Highlights
1. IrIII complexes showed potent antitumor activity against A549 cells
than cisplatin.
2. IrIII complexes could target the lysosome and mitochondria in tumor
cells.
3. IrIII complexes enter cells followed by an energy-dependent cellular
uptake mechanism.
20