Structure-activity relationships for highly potent half-sandwich organoiridium(III) anticancer complexes with C^N-chelated ligands

Article abstract of DOI:10.1016/j.jinorgbio.2018.11.007

We herein report the synthesis, characterization, catalytic ability in converting coenzyme NADH to NAD⁺ and anticancer activity of half-sandwich iridium(III) complexes, [(η⁵-Cp^xbiph)Ir(C^N)Cl]PF₆ ^?, where Cp^xbiph = tetramethyl(biphenyl)cyclopentadienyl, C^N = varying imine-N-heterocyclic carbene ligands. The molecular structure of [(η⁵-Cp^xbiph)Ir(L6)Cl]PF₆ (complex Ir6), exhibiting the familiar “piano-stool” geometry, has been authenticated by X-ray crystallography. The anticancer activities of these complexes can be governed via substituent effects of three tunable domains and the ligand substituted variants offer an effective chelate ligand set that distinguishes anticancer activity and catalytic ability. Notably, complex Ir6 displays the greatest cytotoxic activities (IC₅₀ = 0.85 μM), whose anticancer activity is more approximately 25-fold higher than that of cisplatin. The initial cell death mechanistic insight displays that this group of iridium(III) complexes exerts anticancer effects via cell cycle arrest, apoptosis induction and loss of the mitochondrial membrane potential. In addition, the confocal microscopy imaging shows that the complex Ir6 can damage lysosome. Overall, preliminary structure–activity relationships study and understanding of the cell death mechanism perhaps provide a rational strategy for enhancing anticancer activity of this family of complexes.

Full text of DOI:10.1016/j.jinorgbio.2018.11.007

Accepted Manuscript
Structure-activity relationships for highly potent half-sandwich
organoiridium(III) anticancer complexes with C^N-chelated
ligands
Yuliang Yang, Lihua Guo, Xingxing Ge, Shaopeng Shi, Yuteng
Gong, Zhishan Xu, Xiaofeng Zheng, Zhe Liu
PII:
S0162-0134(18)30515-4
DOI:
Reference:
https://doi.org/10.1016/j.jinorgbio.2018.11.007
JIB 10594
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
1 September 2018
16 October 2018
8 November 2018
Please cite this article as: Yuliang Yang, Lihua Guo, Xingxing Ge, Shaopeng Shi, Yuteng
Gong, Zhishan Xu, Xiaofeng Zheng, Zhe Liu , Structure-activity relationships for highly
potent half-sandwich organoiridium(III) anticancer complexes with C^N-chelated ligands.
Jib (2018), https://doi.org/10.1016/j.jinorgbio.2018.11.007
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
Structure-Activity
Relationships
for
Highly
Potent
Half-Sandwich Organoiridium(III) Anticancer Complexes with
C^N-Chelated Ligands
Yuliang Yanga, Lihua Guo*a, Xingxing Gea, Shaopeng Shia, Yuteng Gonga, Zhishan
Xua,b, Xiaofeng Zhenga, 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).
Graphical abstract:
Highly potent half-sandwich iridium(III) anticancer complexes with C^N-chelated
ligands were prepared. The substituent effects of three adjustable domains on
cytotoxic potency were significant. Mechanistic studies reveal that these complexes
exert anticancer activity via cell cycle arrest, apoptosis induction, loss of the
mitochondrial membrane potential and lysosomal damage.
1

ACCEPTED MANUSCRIPT
ABSTRACT: We herein report the synthesis, characterization, catalytic ability in
converting coenzyme NADH to NAD+ and anticancer activity of half-sandwich
−
iridium(III)
complexes,
[(η5-Cpxbiph)Ir(C^N)Cl]PF6 ,
where
Cpxbiph
=
tetramethyl(biphenyl)cyclopentadienyl, C^N = varying imine-N-heterocyclic carbene
ligands. The molecular structure of complex Ir6, exhibiting the familiar “piano-stool”
geometry, has been authenticated by X-ray crystallography. The anticancer activities
of these complexes can be governed via substituent effects of three tunable domains
and the ligand substituted variants offer an effective chelate ligand set that
distinguishes anticancer activity and catalytic ability. Notably, complex Ir6 displays
the greatest cytotoxic activities (IC50 = 0.85 µM), whose anticancer activity is more
approximately 25-fold higher than that of cisplatin. The initial cell death mechanistic
insight displays that this group of iridium(III) complexes exerts anticancer effects via
cell cycle arrest, apoptosis induction and loss of the mitochondrial membrane
potential. In addition, the confocal microscopy imaging shows that the complex Ir6
can damage lysosome. Overall, preliminary structure–activity relationships study and
understanding of the cell death mechanism perhaps provide a rational strategy for
enhancing anticancer activity of this family of complexes.
Key words: imine-N-heterocyclic carbene; half-sandwich; iridium(III) complexes;
structure-activity relationships; anticancer.
1. Introduction
Since cancer has an increasing impact on mortality worldwide, a growing number
of researchers are being devoted to the exploration of emerging cancer therapy
strategies. Among these, cytotoxic chemotherapeutic agents play a vital role in
fighting cancer. Platinum-based complexes are represented by cisplatin, which are
extensively employed for the treatment of a wide range of tumors [1-3]. At present,
more than 50% of chemotherapy treatments involve the use of platinum drugs.
However, patients receiving these agents have suffered severe adverse effects,
including nephrotoxicity, myelosuppression, peripheral neuropathy, ototoxicity, and
2

ACCEPTED MANUSCRIPT
nausea, which limits the dose that can be administered [4-7]. Therefore, the
investigation of other metal-based anticancer agents as promising candidates and
alternative drugs to cisplatin and its derivatives containing highly anticancer efficacy,
low-toxicity and low side effects in normal tissues has received a significant amount
of attention [8, 9].
Scheme 1. Reported N-heterocyclic carbene anticancer complexes and our current work.
Organometallic compounds, which provide a potentially unexplored field for
biological and medicinal application, recently attract increasing interest in the field of
bioorganometallic chemistry [10-24]. In particular, metal complexes ligated by
N-heterocyclic carbenes (NHCs) have found wide applications in various fields,
particularly as catalysts and anticancer agents [25-33]. At present, various transition
metal complexes, including Pt, Os, Pd, Ag, Au, Ru, Rh and Ir have been explored as
novel cancer treatment options [34-39]. For example, Che et al. reported a panel of
cyclometalated palladium(II) N-heterocyclic carbene compounds (I, Scheme 1) and
explored their reaction with biomolecules glutathione (GSH), ascorbic acid and
human serum albumin (HSA) [34]. The mechanisms of action (MoAs) of this class of
complexes were studied via different analysis assays, including mitochondrial
membrane depolarization and inhibition of epidermal growth factor receptor (EGFR)
signaling pathway in association with apoptotic cell death of the cancer cells.
3

ACCEPTED MANUSCRIPT
Similarly, Mao’s group developed a family of mitochondria-targeted Ag(I)- and
Au(I)-NHC anticancer agents bearing imidazolium-linked cyclophanes, which
showed potent cytotoxicity toward cancer cell lines (II, Scheme 1) [35]. The
selectivity, stable in aqueous solutions and cell death mechanism of these complexes
can be remarkably distinguished via alternative metal ions. In addition, a new series
of iridium(I)-NHC complex has been prepared by Metzler-Nolte et al (III, Scheme 1)
[36]. This complex showed high cytotoxicity against three different cancer cell lines,
which is attributed to an unusual oxidative mechanism. Numerous metal-NHC
complexes have emerged as promising antitumor agents, but only a few iridium-NHC
anticancer agents have been limitedly developed. These investigations motivated us to
design new type of half-sandwich iridium(III) complexes bearing
imine-N-heterocyclic carbene ligands and explore their possible anticancer
mechanisms.
In this study, a series of structurally analogous half-sandwich iridium(III)
−
complexes with similar structure [(η5-Cpxbiph)Ir(C^N)Cl]PF6 were successfully
synthesized and systematically studied for chemical and biological activities, mainly
including catalytic potential in oxidize NADH to NAD+, in vitro cytotoxic activity
toward A549 cancer cells and their possible anticancer mechanisms. Specially, the
structural–activity relationships (SARs) of anticancer activity and catalytic potential
in converting coenzyme NADH to NAD+ of the complexes were adequately studied.
The cell death mechanism, including arresting cell cycle, inducing apoptosis,
dropping mitochondrial membrane potential (MMP) and the increase of lysosomal
permeabilization, is also studied.
2. Results and discussion
2.1. Synthesis and characterization
The imidazolium salts (C^imine·HCl) (L1–L6) were prepared by reaction of
corresponding imidoyl chlorides with N-substituted imidazole (Scheme 2). The
synthetic route of the iridium(III) complexes in this work is depicted in Scheme 3.
Complexes Ir1–Ir6 with various imine-N-heterocyclic carbene ligands were obtained
in moderate to high yields (55–88%) by corresponding silver NHC complexes as
4

ACCEPTED MANUSCRIPT
−
effective transmetalating agents. Complexes Ir1–Ir6 were isolated as PF6 salts,
1
purified by recrystallization and characterized by H NMR (Fig. S1–S6), elemental
analysis and ESI-MS (Fig. S7–S17). Moreover, the molecular structure of complex
Ir6 was also authenticated by X-ray crystallography (Fig. 1 and Tables S1-S2). These
results suggested that the complexes were in good agreement with the expected
structures. All complexes are scarcely soluble in water. However, they readily
dissolve in CH3CN, CH2Cl2, CHCl3 and DMSO.
Scheme 2. Synthetic scheme of the imine-N-heterocyclic carbene ligands L1–L6.
−
Scheme 3. Synthetic scheme of [(η5-Cpxbiph)Ir(C^N)Cl]PF6 complexes Ir1–Ir6.
2.2. X-ray crystal structure.
Crystals of complex Ir6 were obtained by slow diffusion of hexane into a saturated
solution of complex Ir6 in CH2Cl2/CHCl3 (1:1, v:v). The molecular structure for
complex Ir6 with atomic numbering scheme is shown in Fig. 1. X-ray
crystallographic data and selected bond lengths and angles are summarized in Tables
S1 and S2, respectively. The complex crystallized in the triclinic system with the P-1
space group. Complex Ir6 adopt the expected half-sandwich pseudo-octahedral
“three-legged piano-stool” geometry. The bond distance between the iridium center to
the centroid of Cpxbiph ring is 1.8445 Å and the Ir–Cl bond distance is 2.397(3) Å. The
5

ACCEPTED MANUSCRIPT
ortho-metalated Ir–C bond distance [Ir–C = 2.014(8) Å] is slightly shorter than Ir–N
bond distance [Ir–N = 2.121(7) Å]. Further, no intermolecular π-π stacking in the unit
cell of complex Ir6 is observed.
−
Fig. 1. X-ray crystal structure for complex Ir6. The hydrogen atoms and PF6 counterions have
been omitted for clarity. Selected bond lengths (Å) and angles (deg): Ir–C(centroid) = 1.8445, Ir–
Ccarbene = 2.014(8), Ir–N = 2.121(7), Ir–Cl = 2.397(3), Ccarbene–Ir–N = 76.1(3), Ccarbene–Ir–Cl =
81.0(3), N–Ir–Cl=88.2(2).
2.3. Study of the Structure–Activity Relationship
Table 1. Test data of IC50 values for ligands L1-L6, complexes Ir1–Ir6 and cisplatina towards
A549 lung cancer cells and BEAS-2B human bronchial epithelial normal cells.
IC50 (µM)
IC50 (µM)
A549
Ligand
Complex
A549
BEAS-2B
>100
BEAS-2B
3.58 ± 0.3
2.03 ± 0.2
0.60 ± 0.3
3.43 ± 0.1
1.21 ± 0.3
4.60 ± 0.5
42.0 ± 2.3
L1
L2
L3
L4
L5
L6
>100
>100
>100
>100
>100
>100
[(η5-Cpxbiph)Ir(L1)Cl]PF6 (Ir1)
[(η5-Cpxbiph)Ir(L2)Cl]PF6 (Ir2)
[(η5-Cpxbiph)Ir(L3)Cl]PF6 (Ir3)
[(η5-Cpxbiph)Ir(L4)Cl]PF6 (Ir4)
[(η5-Cpxbiph)Ir(L5)Cl]PF6 (Ir5)
[(η5-Cpxbiph)Ir(L6)Cl]PF6 (Ir6)
Cisplatin
4.21 ± 0.3
3.25 ± 0.2
1.84 ± 0.1
4.39 ± 0.1
2.67 ± 0.1
0.85 ± 0.2
21.3 ± 1.7
>100
>100
>100
>100
>100
aIC50 values are drug concentrations necessary for 50% inhibition of cell viability. Data are presented as means ±
standard deviations and cell viability is assessed after 24 h of incubation.
To assess whether the present six imine-N-heterocyclic carbene ligands and their
corresponding iridium(III) complexes possess cytotoxicity and potential structure–
activity relationships (SARs), the in vitro cytotoxicity of ligands L1-L6 and
complexes Ir1-Ir6 toward BEAS-2B human bronchial epithelial normal cells and
6

ACCEPTED MANUSCRIPT
A549 lung cancer cells were investigated after treatment for 24 h using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay, with the
well-known cisplatin as a positive control. The resulting 50% growth inhibitory
concentration (IC50) values are listed in Fig. 2 and Table 1. All of the
imine-N-heterocyclic carbene ligands displayed exceedingly low cytotoxicity toward
BEAS-2B cells and A549 cells (>100 µM), they were thus deemed as inactive.
Excitingly, this new class of complexes is highly potent against A549 cancer cells line
with IC50 values of 0.85–4.39 µM and display more high potency than the widely
used clinical platinum drug cisplatin (21.3 µM). The cytotoxicity of these complexes
may be derived from the combinatorial action of metal and imine-N-heterocyclic
carbene ligands. Notably, complex Ir6 was ca. 25 times as active as cisplatin in the
A549 cell line (0.85 μM vs 21.3 μM), demonstrating great potential in cancer
25
20
15
10
5
0
Cisplatin Ir1
Ir6
Ir2
Ir5
Ir3
ComplexIr4
Fig. 2. Histogram showing the comparison of IC50 values of complexes Ir1–Ir6 and cisplatin
towards A549 cancer cells after incubation for 24 h.
chemotherapy. Overall, the cytotoxic activities of complexes and cisplatin against
A549 cancer cells are in the following order: Ir6 > Ir3 > Ir5 > Ir2 > Ir1 > Ir4 >
cisplatin. In addition, to understand if the synthesized complexes are selective only
against cancer cells, the human normal bronchial epithelial cells BEAS-2B was
chosen to expound on the cytotoxicity of this family of complexes. The IC50 values
for complexes Ir1–Ir6 toward BEAS-2B have been indicated in Table 1.
Unfortunately, their selectivity towards cancer cells and normal cells was not
observed. Therefore, more structural modification is necessary to reduce the cytotoxic
7

ACCEPTED MANUSCRIPT
activity towards normal cells without loss of the selectivity between cancer cells and
normal cells in the future work.
The toxicological properties of this family of iridium(III) complexes can be
governed via substitution effects of three tunable domains. First, when the length of
the alkyl substitutions on the imidazole ring increased from methyl- to isopropyl-
group, the complex display enhanced cytotoxicity (Ir1: 4.21 vs Ir2: 3.25 vs Ir3: 1.84
µM, Ir3 > Ir2 > Ir1). The increased tether length on the imidazole ring may increase
the lipophilicity of this family of complexes and thus result in the enhanced
cytotoxicity. Next, the anticancer activity can also be enhanced by increasing the size
of ortho-substituents in aniline. For instance, under the same conditions as the other
substituents (R1 and R2), complexes Ir4 (4.39 µM) and Ir5 (2.67 µM) exhibit in vitro
anticancer activity dramatically surpass complexes Ir2 (3.25 µM) and Ir3 (1.84 µM),
respectively. Ultimately, the effect of the substituent on the imine carbon on
cytotoxicity of such complexes was further studied. When the substituent on imine
carbon was changed from methyl to phenyl, the increased cytotoxic potency was
observed (complex Ir5: IC50 = 2.67 μM vs complex Ir6: IC50 = 0.85 μM). Clearly, the
anticancer properties of this class of complexes can be governed by subtle structure
changing in cheating ligands. Previous studies have demonstrated that the cytotoxicity
and lipophilicity of structurally similar half-sandwich iridium(III) anticancer
complexes could be enhanced through the introduction of phenyl substituents on the
Cp* ligand [40]. Within this class, the potency of these anticancer agents could be
modulated by altering the lipophilicity of the substituents on three positions of the
chelated ligands. These results build a structure–activity relationship for this type of
half-sandwich iridium(III) complexes, which may be a rational strategy to design
novel iridium(III) anticancer agents.
2.4. Reaction with NADH
In the human body, coenzyme NADH/NAD+ play an essential role and participate
in numerous metabolic reactions [41]. It has been reported that the similar
half-sandwich iridium(III) anticancer agents as an efficient biocatalysts can oxidize
NADH by hydride transfer to O2 to yield reactive oxygen species (ROS) H2O2, which
8

ACCEPTED MANUSCRIPT
offered a pathway to an oxidant mechanism of action [42-44]. In order to explore the
influence of three tunable domains on the catalytic ability in this class of new
complexes, the time-dependent reactions of complexes Ir1-Ir6 (ca. 1 µM) with
NADH (100 µM) in 5% MeOH/95% H2O (v/v) were performed using
ultraviolet-visible (UV-vis) spectrophotometer at 298 K (Fig. 3a and Fig. S18). The
turnover numbers (TONs) of complexes Ir1 (80.0), Ir2 (85.0), Ir3 (65.0), Ir4 (70.0)
Ir5 (53.7) and Ir6 (33.7) were computed by monitoring the absorbance change at 339
nm (Fig. 3, b). Although no clear trends were observed, it appears that the substituent
effects of three modifiable domains on the catalytic activity were pronounced. In
particular, complex Ir2 show ca. 8 times higher catalytic ability in the oxidation of
NADH than the reported similarly half-sandwich iridium(III) complexes containing
bis-NHC-based ligands under the same test conditions [45]. Thus, the strong catalytic
ability of this family of complexes in converting NADH to NAD+ may promote
production of ROS and increase the anticancer potency in an oxidant mechanism of
action.
Fig. 3. (a) UV-vis spectra of the reaction of NADH (100 µM) with complex Ir2 (1 µM) in 5%
MeOH/95% H2O (v:v) at 298 K for 8 h; (b) The turnover numbers (TONs) of complexes Ir1–Ir6.
2.5. Interaction with model nucleobase
Complex Ir6 was chosen as a representative to react with the DNA model
9-ethylguanine (9-EtG) and 9-methyladenine (9-MeA). The time-dependent reactions
of complex Ir6 with model nucleobase 9-EtG or 9-MeA were performed utilizing 1H
NMR spectroscopy under the same conditions: complex Ir6 (1 mM) in
9

ACCEPTED MANUSCRIPT
CD3OD-d4/D2O (8/2, v/v) with model nucleobase 9-EtG or 9-MeA (2 mM) tested
1
from 5 min to 24 h at 310 K. As illustrated in Figs. S19 and S20, no new H NMR
peaks were observed even after 24 h. Also, the formation of nucleobase adducts by
complex Ir6 were not monitored using mass spectrometry. These results revealed that
DNA may not be the target for this class of iridium(III) complexes.
2.6. Cell cycle arrest.
Fig. 4. Flow cytometry data for cell cycle distribution of A549 cancer cells exposed to complex
Ir6 for 24 h. Concentrations used were 0.25, 0.5, 1 and 2 equipotent concentrations of IC50. Cell
staining for flow cytometry was carried out using PI/RNase. Data are quoted as mean ± SD of
three replicates.
Usually, most of the metal anticancer drugs inhibit the growth of cancer cells by
disrupting the regulation of cell cycle distribution. To understand whether this class of
complexes inhibited cell viability of A549 cells is on account of cell cycle arrest, cell
cycle distribution of A549 cells was determined via quantitation of the DNA content.
A549 cancer cells were treated with complex Ir6 at concentrations of 0.25×IC50,
0.5×IC50, 1×IC50 and 2×IC50 for 24 h, then stained with propidium iodide (PI) and
further analyzed by flow cytometry. As expected, the cell cycle distribution in A549
cells significantly changed, and the DNA content in G0/G1 phase increased from 51.16
10

ACCEPTED MANUSCRIPT
± 0.3% to 55.71 ± 0.6%, 57.27 ± 1.0%, 59.10 ± 1.0%, 61.45 ± 0.4% in a
concentration-dependent manner after treatments with 0.25×IC50, 0.5×IC50, 1×IC50
and 2×IC50 of complex Ir6, respectively (Fig. 4, Fig. S21 and Table S3). These results
indicated that complex Ir6 strongly induced A549 cells to undergo G0/G1 phase arrest,
which may be one of the possible mechanisms of cell growth inhibition.
2.7. Apoptosis Assay.
Fig. 5. Apoptosis analysis of A549 cells after 24 h of exposure to complex Ir6 at 310 K
determined by flow cytometry using annexin V-FITC vs PI staining. Populations for cells in four
stages treated by complex Ir6. Data are quoted as mean ± SD of three replicates.
Apoptosis and necrosis are the two main types of cell death [46]. Previous studies
have suggested that most anticancer drugs currently used in clinical trigger apoptosis
in susceptible cells [47]. To understand the pathway potentially induced by complex
Ir6, the Annexin V/propidium (PI) staining was employed to monitor the apoptosis of
A549 cells. As illustrated in Fig. 5, Fig. S22 and Table S4, treatment of A549 cancer
cells with complex Ir6 at concentrations of 0.5×IC50 and 1×IC50 for 24 h, a
concentrate-dependent apoptotic effect was observed. At the 1×IC50 tested
concentration, complex Ir6 caused early and late apoptosis in 13.48% and 20.85% of
11

ACCEPTED MANUSCRIPT
A549 cells, respectively, suggesting that the induction of cell death can occur through
the apoptotic pathway.
2.8. Impact on mitochondrial membrane potential (MMP)
The loss of mitochondrial membrane potential (MMP, ΔΨm), an early event during
apoptosis, is a significant indicator for the evaluation of apoptosis. The impact of
complex Ir6 on MMP was measured by staining with lipophilic cationic JC-1 dye. As
shown in Fig. 6 and Table S5, with the concentration increases, a dose-dependent
red-to-green color shift was observed, suggesting the decrease of MMP. The
percentage of cells with mitochondrial membrane depolarization increases from 6.05%
to 19.58% at a concentration of 1 × IC50 compared to untreated cells. Hence, the
dysfunction of mitochondria may contribute to the cytotoxic activity of this class of
complexes.
Fig. 6. Complex Ir6 induced mitochondrial membrane potential changes in A549 cells.
2.9. Lysosomal damage
Lysosomes, an organelle in eukaryotic cells, control cell apoptosis or death at
several levels, since they are involved in various aspects of cancer cell
immortalization [48]. The lysosomal integrity is a crucial indicator for assess cell
apoptosis or death. Acridine orange (AO) is a concentration-dependent fluorescent
dye utilized to monitor the lysosomal integrity of A549 cells [49-51]. As shown in Fig.
7, a distinct decrease in red fluorescence intensity was observed as the concentration
12

ACCEPTED MANUSCRIPT
of complex Ir6 increased, suggesting that the integrity of lysosomes was jeopardized,
i.e., the increase of lysosomal permeabilization. Thus, we conclude that the cell
apoptosis or death may depend on the disruption of lysosomes.
Fig. 7. Observation of lysosomal disruption in A549 cells loaded with complex Ir6 for 6 h at 37
ºC, then stained with acridine orange (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. The A549 cells were
treated with (a) only acridine orange (AO); (b) acridine orange (AO) and complex Ir6 (1× IC50);
(c) acridine orange (AO) and complex Ir6 (3× IC50). Scale bar: 20 µm.
3. Conclusions
In conclusion, a set of half-sandwich iridium(III) complexes containing
imine-N-heterocyclic carbene ligands were synthesized and characterized. These
iridium(III) complexes exhibited highly potent cytotoxicity (up to 25-fold more
effective than cisplatin) against A549 cancer cells. The substituent effects of three
adjustable domains on cytotoxic potency were significant. The larger size of
substituents on aniline and imidazole ring results in the higher anticancer efficiency.
Additionally, introducing a more lipophilic phenyl ring also enhances the anticancer
activity of the complexes. These complexes have strong catalytic ability in the
oxidation of NADH. However, no clear trend was observed for the substituent effects
of three adjustable domains on the catalytic ability. No binding to 9-EtG or 9-MeA
was observed for this type of complexes. Flow cytometric analysis showed that
complex Ir6 can stall cell in the G0/G1 phase of the cell cycle, lead to loss of the
MMP and trigger early- and late-stage apoptosis in A549 cells. Interestingly,
13

ACCEPTED MANUSCRIPT
lysosomal damage was detected in A549 cancer cells utilizing confocal microscopy.
These initial studies point out that the half-sandwich iridium(III) complexes in this
system possess some interesting biological effects and worthy of further investigation.
4. Experimental Section
4.1. General information
2,3,4,5-tetramethyl-2-cyclopentenone (95%), butyllithium solution (1.6 M in
hexane), 4-bromo-biphenyl, IrCl3·nH2O, 2,6-dimethylaniline, 2,6-diisopropylaniline,
acetyl chloride, benzoyl chloride, triphosgene, thionyl chloride, 1-methylimidazole,
1-ethylimidazole, 1-isopropylimidazole, Ag2O, 9-ethylguanine and 9-methyladenine
were purchased from commercial source. [(η5-C5Me4C6H4C6H5)IrCl2]2 was
synthesized according to previously reported methods [40]. The intermediate
compounds were prepared according to previously reported procedure [52-57]. The
ligands L1-L5 [52], L6 [53-57] were prepared according to slightly modified
literature procedure, respectively.
1H NMR spectra were acquired at 298 K on Bruker DPX 500 spectrometers using
TMS as an internal standard and CDCl3 as solvent. Mass spectra of the ligands were
recorded on a Thermo LTQ Orbitrap XL (ESI+). Mass spectra of the complexes were
recorded on 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).
4.2 Synthesis of [(η5-Cpxbiph)Ir(C^N)Cl]PF6
A round-bottomed flask was charged with a mixture of ligands (0.10 mmol) and
Ag2O (1.2 eq) in CH2Cl2 (20 mL) were stirred at room temperature for 6 h. The
mixture was filtered through Celite. The filtrate was charged with
[(η5-Cpxbiph)IrCl2]Cl2 (0.05 mmol). The mixture was then stirred at room temperature
for 12 h. Subsequently, KPF6 (6 eq) was added and further stir for 30 min. The
suspension was filtered and the solvent was removed using vacuum system. The
orange solid was obtained by recrystallizing from CH2Cl2 and hexane solution.
14

ACCEPTED MANUSCRIPT
Ir1 Yield: 77.1 mg (83%). 1H NMR (500 MHz, CDCl3) δ 7.68 (d, J = 2.3 Hz, 1H),
7.65 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 7.4 Hz, 2H), 7.46 (t, J = 7.6 Hz, 2H), 7.38 (t, J =
7.4 Hz, 1H), 7.35 (s, 1H), 7.34 – 7.29 (m, 4H), 7.17 (dd, J = 6.2, 3.0 Hz, 1H), 3.73 (s,
3H), 3.61 – 3.52 (m, 1H), 2.53 (s, 3H), 2.38 – 2.29 (m, 1H), 1.87 (s, 3H), 1.64 (s, 3H),
1.51 (s, 3H), 1.35 (s, 3H), 1.19 (d, J = 6.6 Hz, 3H), 0.97 (d, J = 6.7 Hz, 6H), 0.68 (d, J
= 6.5 Hz, 3H). ESI-MS (m/z): calcd for C39H46IrClN3: 784.301, found: 784.295,
[(η5-C5Me4C6H4C6H5)Ir(L1)Cl]+. Elemental analysis: calcd (%) for C39H46N3IrClPF6:
C, 50.40; H, 4.99; N, 4.52; found: C, 50.56; H, 4.94; N, 4.69.
Ir2 Yield: 83.3 mg (88%). 1H NMR (500 MHz, CDCl3) δ 7.73 (d, J = 2.2 Hz, 1H),
7.67 (d, J = 8.1 Hz, 2H), 7.62 (d, J = 7.3 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.39 (d, J =
7.4 Hz, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.34 – 7.29 (m, 3H), 7.20 (dd, J = 6.5, 2.7 Hz,
1H), 4.00 (dq, J = 14.4, 7.2 Hz, 1H), 3.90 (dq, J = 14.6, 7.4 Hz, 1H), 3.63 –3.55 (m,
1H), 2.55 (s, 3H), 2.45 – 2.36 (m, 1H), 1.83 (s, 3H), 1.62 (s, 3H), 1.47 (s, 3H), 1.41 (d,
J = 2.4 Hz, 3H), 1.39 (d, J = 7.3 Hz, 3H), 1.20 (d, J = 6.6 Hz, 3H), 1.01 (d, J = 6.6 Hz,
3H), 0.98 (d, J = 6.8 Hz, 3H), 0.78 (d, J = 6.4 Hz, 3H). ESI-MS (m/z): calcd for
C40H48IrClN3: 798.3166, found: 798.2949, [(η5-C5Me4C6H4C6H5)Ir(L2)Cl]+.
Elemental analysis: calcd (%) for C40H48N3IrClPF6: C, 50.92; H, 5.13; N, 4.45; found:
C, 50.78; H, 5.30; N, 4.58.
Ir3 Yield: 67.2 mg (70%). 1H NMR (500 MHz, CDCl3) δ 7.79 (s, 1H), 7.69 (d, J = 7.8
Hz, 2H), 7.61 (d, J = 7.6 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.40 (dd, J = 13.0, 7.1 Hz,
3H), 7.36 – 7.31 (m, 2H), 7.30 (s, 1H), 7.22 (dd, J = 6.8, 2.4 Hz, 1H), 4.33 –4.22 (m,
1H), 3.64 – 3.55 (m, 1H), 2.56 (s, 3H), 2.52 – 2.41 (m, 1H), 1.82 (s, 3H), 1.58 (s, 3H),
1.45 (d, J = 12.0 Hz, 6H), 1.38 (dd, J = 6.4, 4.4 Hz, 6H), 1.20 (d, J = 6.6 Hz, 3H),
1.04 (d, J = 6.3 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.2 Hz, 3H). ESI-MS
(m/z):
calcd
for
C41H50IrClN3:
812.332,
found:
812.317,
[(η5-C5Me4C6H4C6H5)Ir(L3)Cl]+. Elemental analysis: calcd (%) for C41H50N3IrClPF6:
C, 51.43; H, 5.26; N, 4.39; found: C, 51.62; H, 5.33; N, 4.61.
Ir4 Yield: 59.4 mg (67%). 1H NMR (500 MHz, CDCl3) δ 7.69 (d, J = 8.2 Hz, 3H),
7.66 – 7.62 (m, 2H), 7.53 (d, J = 8.1 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.39 (t, J = 7.4
Hz, 1H), 7.26 –7.23 (m, 1H), 7.21 (t, J = 4.1 Hz, 2H), 7.18 (d, J = 2.1 Hz, 1H), 3.73 –
15

ACCEPTED MANUSCRIPT
3.62 (m, 2H), 2.57 (s, 3H), 2.29 (s, 3H), 2.06 (s, 3H), 1.83 (s, 3H), 1.73 (s, 3H), 1.35
(s, 3H), 1.21 (t, J = 7.3 Hz, 3H), 1.18 (s, 3H). ESI-MS (m/z): calcd for C36H40IrClN3:
742.2540, found: 742.2304, [(η5-C5Me4C6H4C6H5)Ir(L4)Cl]+. Elemental analysis:
calcd (%) for C36H40N3IrClPF6: C, 48.73; H, 4.54; N, 4.74; found: C, 48.90; H, 4.60;
N, 4.78.
Ir5 Yield: 70.1 mg (78%). 1H NMR (500 MHz, CDCl3) δ 7.80 (s, 1H), 7.69 (d, J = 8.2
Hz, 2H), 7.61 (d, J = 7.5 Hz, 2H), 7.56 (d, J = 8.1 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H),
7.39 (t, J = 7.3 Hz, 1H), 7.25 – 7.19 (m, 3H), 7.17 (d, J = 2.3 Hz, 1H), 4.07 – 3.99 (m,
1H), 2.58 (s, 3H), 2.28 (s, 3H), 2.06 (s, 3H), 1.83 (s, 3H), 1.76 (s, 3H), 1.29 (s, 3H),
1.21 (d, J = 6.7 Hz, 3H), 1.19 (s, 3H), 1.15 (d, J = 6.7 Hz, 3H). ESI-MS (m/z): calcd
for C37H42IrClN3: 756.2697, found: 756.2418, [(η5-C5Me4C6H4C6H5)Ir(L5)Cl]+.
Elemental analysis: calcd (%) for C37H42N3IrClPF6: C, 49.30; H, 4.70; N, 4.66; found:
C, 49.48; H, 4.90; N, 4.77.
Ir6 Yield: 52.6 mg (55%). 1H NMR (500 MHz, CDCl3) δ 8.11 (s, 1H), 7.76 (d, J = 8.1
Hz, 2H), 7.71 (d, J = 8.1 Hz, 2H), 7.65 (d, J = 7.7 Hz, 2H), 7.60 (s, 1H), 7.52 – 7.44
(m, 3H), 7.39 (t, J = 7.3 Hz, 1H), 7.27 (d, J = 3.9 Hz, 1H), 7.20 (s, 1H), 7.15 (d, J =
7.5 Hz, 1H), 7.11 (d, J = 1.8 Hz, 1H), 7.03 (dd, J = 13.8, 6.2 Hz, 2H), 6.93 (d, J = 7.5
Hz, 1H), 4.17 – 4.05 (m, 1H), 2.55 (s, 3H), 1.92 (s, 6H), 1.88 (s, 3H), 1.32 – 1.15 (m,
12H). ESI-MS (m/z): calcd for C42H44IrClN3: 818.285, found: 818.323,
[(η5-C5Me4C6H4C6H5)Ir(L6)Cl]+. Elemental analysis: calcd (%) for C42H44N3IrClPF6:
C, 52.36; H, 4.60; N, 4.36; found: C, 52.59; H, 4.83; N, 4.49. Single crystals were
obtained by slow diffusion of hexane into a mixture solution of CH2Cl2 and CHCl3
(v:v, 1:1).
16

ACCEPTED MANUSCRIPT
Abbreviations
Cpxbiph
NHCs
tetramethyl(biphenyl)cyclopentadienyl
N-heterocyclic carbenes
human serum albumin
mechanisms of action
HSA
MoAs
EGFR
9-EtG
9-MeA
IC50
epidermal growth factor receptor
9-ethylguanine
9-methyladenine
50% growth inhibitory concentration
bovine serum albumin
glutathione
BSA
GSH
SARs
ROS
structural-activity relationships
reactive oxygen species
acridine orange
AO
MMP
UV-vis
mitochondrial membrane potential
ultraviolet-visible
Acknowledgements
We are grateful for the supports of the Shandong Provincial Natural Science
Foundation (ZR2018MB023), the National Natural Science Foundation of China
(Grant No. 21671118) and the Taishan Scholars Program, the Key Laboratory of
Polymeric Composite & Functional Materials of Ministry of Education
(PCFM-2017-01), excellent experiment project of Qufu Normal University
(jp201705).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. xxx.
References
[1] E.R. Jamieson, S.J. Lippard, Chem. Rev. 99 (1999) 2467–2498.
[2] G. Chu, J. Biol. Chem. 269 (1994) 787–790.
17

ACCEPTED MANUSCRIPT
[3] V. Cepeda, M.A. Fuertes, J. Castilla, C. Alonso, C. Quevedo, J.M. Pérez,
Anti-Cancer Agents Med. Chem. 7 (2007) 3–18.
[4] L. Kelland, Nat. Rev. Cancer 7 (2007) 573–584.
[5] R.P. Miller, R.K. Tadagavadi, G. Ramesh, W.B. Reeves, Toxins 2 (2010) 2490–
2518.
[6] Y.R. Zheng, K. Suntharalingam, T.C. Johnstone, H. Yoo, W. Lin, J.G. Brooks,
S.J. Lippard, J. Am. Chem. Soc. 136 (2014) 8790–8798.
[7] A.M. Florea, D. Büsselberg, Cancers 3 (2011) 1351–1371.
[8] Z.Z. Tian, J.J. Li, S.M. Zhang, Z.S. Xu, Y.L. Yang, D.L. Kong, H.R. Zhang, X.X.
Ge, J.M. Zhang, Z. Liu, Inorg. Chem. 57 (2018) 10498–10502.
[9] M. Tian, J.J. Li, S.M. Zhang, L.H. Guo, X.D. He, D.L. Kong, H.R. Zhang, Z.
Liu, Chem. Commun. 53 (2017) 12810–12813.
[10] Q. Du, L.H. Guo, M. Tian, X.X. Ge, Y.L. Yang, X.Y. Jian, Z.S. Xu, Z.Z. Tian, Z.
Liu, Organometallics 37 (2018) 2880–2889.
[11] L.L. Zeng, P. Gupta, Y.L. Chen, E.J. Wang, L.N. Ji, H. Chao, Z.S. Chen, Chem.
Soc. Rev. 46 (2017) 5771–5804.
[12] T.T. Zou, C.T. Lum, C.N. Lok, J.J. Zhang, C.M. Che, Chem. Soc. Rev. 44 (2015)
8786–8801.
[13] M. Li, L.H. Lai, Z.N. Zhao, T.F. Chen, Chem. Asian J. 11 (2016) 310–320.
[14] Z.Q. Zhang, X.Y. Wang, C. Luo, C.C. Zhu, K. Wang, C.L. Zhang, Z.J. Guo,
Chem. Asian J. 12 (2017) 1659–1667.
[15] L.J. Liu, W.H. Wang, S.Y. Huang, Y.J. Hong, G.D. Li, S. Lin, J.L. Tian, Z.W.
Cai, H.M.D. Wang, D.L. Ma, C.H. Leung, Chem. Sci. 8 (2017) 4756–4763.
[16] J. Arshad, M. Hanif, S. Movassaghi, M. Kubanik, A. Waseem, T. Söhnel, S.M.F.
Jamieson, C.G. Hartinger, J Inorg. Biochem. 177 (2017) 395–401.
[17] G.S. Yellol, A. Donaire, J.G. Yellol, V. Vasylyeva, C. Janiak, J. Ruiz, Chem.
Commun. 49 (2013) 11533–11535.
[18] L. Biancalana, L.K. Batchelor, T. Funaioli, S. Zacchini, M. Bortoluzzi, G.
Pampaloni, P.J. Dyson, F. Marchetti, Inorg. Chem. 57 (2018) 6669–6685.
18

ACCEPTED MANUSCRIPT
[19] Q. Wu, L.Y. Liu, S.L. Li, F.X. Wang, J. Li, Y. Qian, Z. Su, Z.W. Mao, P.J. Sadler,
H.K. Liu, J Inorg. Biochem. 189 (2018) 30–39.
[20] W.L. Ma, Z.Z. Tian, S.M. Zhang, X.D. He, J.J. Li, X.R. Xia, X.B. Chen, Z. Liu,
Inorg. Chem. Front. 5 (2018) 2587–2597.
[21] W.Y. Hua, J. Zhao, W.W. Hu, S.H. Gou, J Inorg. Biochem. 186 (2018) 17–23.
[22] K.G. Samper, S.C. Marker, P. Bayón, S.N. MacMillan, I. Keresztes, Ò. Palacios,
J.J. Wilson, J Inorg. Biochem. 177 (2017) 335–343.
[23] L.H. Guo, H.R. Zhang, M. Tian, Z.Z. Tian, Y.J. Xu, Y.L. Yang, H.W. Peng, P.
Liu, Z. Liu, New J. Chem. 42 (2018) 16183−16192.
[24] M. Ouyang, L.L. Zeng, H.Y. Huang, C.Z. Jin, J.P. Liu, Y. Chen, L.N. Ji, H.
Chao, Dalton Trans. 46 (2017) 6734–6744.
[25] Y. Li, B. Liu, X. R. Lu, M. F. Li, L.N. Ji, Z.W. Mao, Dalton Trans. 46 (2017)
11363−11371.
[26] W.A. Herrmann, Angew. Chem. Int. Ed. 41 (2002) 1290−1309.
[27] L.H. Guo, S.Y. Dai, X.L. Sui, C.L. Chen, ACS Catal. 6 (2016) 428−441.
[28] Y. Li, C.P. Tan, W. Zhang, L. He, L.N. Ji, Z.W. Mao, Biomaterials 39 (2015)
95−104.
[29] Y.L. Yang, L.H. Guo, Z.Z. Tian, X.C. Liu, Y.T. Gong, H.M. Zheng, X.X. Ge, Z.
Liu, Chem. Asian J. 13 (2018) 2923−2933.
[30] L.H. Guo, C.L. Chen, Sci. China: Chem. 58 (2015) 1663−1673.
[31] S. Díez-González, N. Marion, S.P. Nolan, Chem. Rev. 109 (2009) 3612−3676;
[32] L.H. Guo, W.J. Liu, C.L. Chen, Mater. Chem. Front. 1 (2017) 2487−2494.
[33] Y.L. Yang, L.H. Guo, X.X. Ge, Z.Z. Tian, Y.T. Gong, H.M. Zheng, Q. Du, X.F.
Zheng, Z. Liu, Dyes and Pigments. 161 (2019) 119−129.
[34] T.T. Fong, C.N. Lok, C.Y. Chung, Y.E. Fung, P.K. Chow, P.K. Wan, C.M. Che,
Angew. Chem. Int. Ed. 55 (2016) 11935–11939.
[35] Y. Li, G.F. Liu, C.P. Tan, L.N. Ji, Z.W. Mao, Metallomics 6 (2014) 1460–1468;
[36] Y. Gothe, T. Marzo, L. Messori, N. Metzler-Nolte, Chem. Commun. 51 (2015)
3151–3153.
19

ACCEPTED MANUSCRIPT
[37] W. Streciwilk, A. Terenzi, X. Cheng, L. Hager, Y. Dabiri, P. Prochnow, J.E.
Bandow, S. Wölfl, B. Keppler, I. Ott, Eur. J Med. Chem. 156 (2018) 148–161.
[38] R.W. Sun, L.F Chow, X.H. Li, J.J. Yan, S.Y Chui, C.M. Che, Chem. Sci. 2 (2011)
728–736.
[39] D. Jantke, M. Cokoja, A. Pöthig, W.A. Herrmann, F.E. Kühn, Organometallics
32 (2013) 741–744.
[40] 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.
[41] F.S. Omar, N. Duraisamy, K. Ramesh, S. Ramesh, Biosens. Bioelectron. 79
(2016) 763–775.
[42] 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. 126 (2014) 4022–
4027.
[43] H.R. Zhang, L.H. Guo, Z.Z. Tian, M. Tian, S.M. Zhang, Z.S. Xu, P.W. Gong,
X.F. Zheng, J. Zhao, Z. Liu, Chem. Commun. 54 (2018) 4421–4424.
[44] Z. Liu, P.J. Sadler, Acc. Chem. Res. 47 (2014) 1174–1185.
[45] C.L. Wang, J.F. Liu, Z.Z. Tian, M. Tian, L.J. Tian, W.Q. Zhao, Z. Liu, Dalton
Trans. 46 (2017) 6870−6883.
[46] I. Vermes, C. Haanen, Adv. Clin. Chem. 31 (1994) 177–246.
[47] N.A. Thornberry, Y. Lazebnik, Science 281 (1998) 1312–1316.
[48] M.T. Gyparaki, A.G. Papavassiliou, Trends Mol. Med. 20 (2014) 239–241.
[49] X.Z. Zhao, M.L. Li, W. Sun, J.L. Fan, J.J. Du, X.J. Peng, Chem. Commun. 54
(2018) 7038–7041.
[50] L. He, Y. Li, C.P. Tan, R.R. Ye, M.H. Chen, J.J. Cao, L.N. Ji, Z.W. Mao, Chem.
Sci. 6 (2015) 5409–5418.
[51] P. Boya, G. Kroemer, Oncogene 27 (2008) 6434–6451.
[52] J.A. Deng, H.Y. Gao, F.M. Zhu, Q. Wu, Organometallics 32 (2013) 4507−4515.
[53] M. Frøseth, K.A. Netland, C. Rømming, M. Tilset, J. Organomet. Chem. 690
(2005) 6125–6132.
20

ACCEPTED MANUSCRIPT
[54] M.L. Rosenberg, A. Krivokapic, M. Tilset, Org. Lett. 11 (2009) 547–550.
[55] M.L. Rosenberg, E. Langseth, A. Krivokapic, N.S. Gupta, M. Tilset, New J.
Chem. 35 (2011) 2306–2313.
[56] A. Krajete, G. Steiner, H. Kopacka, K.H. Ongania, K. Wurst, M.O. Kristen, P.
Preishuber-Pflügl, B. Bildstein, Eur. J. Inorg. Chem. 8 (2004) 1740−1752.
[57] S. Dastgir, K.S. Coleman, A.R. Cowley, M.L.H. Green, Organometallics 25
(2006) 300−306.
21

ACCEPTED MANUSCRIPT
Highlights
 Six new half-sandwich iridium(III) anticancer complexes have been synthesized
and characterized.
 These complexes exhibit highly potent cytotoxicity toward A549 cancer cells.
 The substituent effects of three adjustable domains on cytotoxic potency were
significant.
22