Triphenylamine-Appended Half-Sandwich Iridium(III) Complexes and Their Biological Applications

Article abstract of DOI:10.1002/asia.201800103

Organometallic half-sandwich Ir^III complexes of the type [(η⁵-Cp^x)Ir(N^N)Cl]PF₆ (Cp^x: Cp* or its phenyl Cp^xph or biphenyl Cp^xbiph derivatives; N^N: triphenylamine (TPA)-substitut

Full text of DOI:10.1002/asia.201800103

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Triphenylamine-appended half-sandwich iridiumIII complexes
and their biological applications
Authors: Zhe Liu, Xiangdong He, Meng Tian, Xicheng Liu, Yanhua
Tang, Changfang Shao, Peiwei Gong, Jinfeng Liu, Shumiao
Zhang, and Lihua Guo
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Chemistry - An Asian Journal
Triphenylamine-appended half-sandwich iridiumIII complexes and their
biological applications
Xiangdong He a,+, Meng Tian a,+, Xicheng Liu a*, Yanhua Tang a, Chang fang Shaoa, Peiwei Gong a,
Jinfeng Liu b, Shumiao Zhang a, Lihua Guo a, Zhe Liu a*
a 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, Department of Chemistry and Chemical Engineering, Qufu Normal University,
Qufu 273165, China.
b Qufu Normal University, School of Life Science, Qufu 273165, China.
*
Corresponding author: E-mail: chemlxc@163.com (Xicheng Liu); liuzheqd@163.com (Zhe
Liu).
+ X. D. He and M. Tian have equivalent contribution
Abstract: Organometallic half-sandwich IrIII complexes of type [(η5-Cpx)Ir(N^N)Cl]PF6 (Cpx:
Cp* or its phenyl Cpxph or biphenyl Cpxbiph derivatives, N^N: triphenylamine (TPA) substituted
bipyridyl ligand groups) have been synthesized and characterized. The complexes showed
excellent BSA and DNA binding properties, and oxidize NADH to NAD+ efficiently. Complexes
can induce apoptosis effectively and led to the emergence of reactive oxygen species (ROS) in
cells. All complexes showed potent cytotoxicity with IC50 values ranging from 1.5 to 7.1 µM
toward A549 human lung cancer cells after 24 h drug exposure, at most 14 times more potent than
cis-platin under the same conditions.
INTRODUCTION
The clinical success and drawbacks of Pt anticancer drugs have stimulated the exploration of
other metal-based anticancer complexes.[1] Recently, iridiumIII (IrIII) complexes have attracted
much attention because of their wide biological applications.[2-11] IrIII complexes are often viewed
as relatively inert, a common characteristic of low-spin d6 metal ions and especially third-row
transition metals.[12] However, IrIII complexes have shown potent anticancer activity,[2,13-15] and
displayed a different mechanism of action (MoA) compared with conventional platinum-based
drugs.[16,17]
Due to the advantages of high anticancer activity and unique MoA, half-sandwich
organometallic IrIII complexes have attracted extensive interest.[18-20] In a recent study, Sadler and
coworkers found that half-sandwich Cp* (pentamethylcyclopentadienyl) IrIII complexes with
N,N-bound ligand, such as 2,2′-bipyridine (bipy), 1,10-phenanthroline (phen) and ethylenediamine
(EDA), were all inactive against A2780 human ovarian cancer cells with IC50 values
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(concentrations at which 50% of the cell growth is inhibited) over 100 µM.[21,22] The increase of
benzene rings on the Cp ring has a great effect on the anticancer activity of the complexes. It is
noteworthy that the electronic and steric properties of the ligands have a major effect on the
chemical and biological activities to IrIII complexes.[23,24] Other work also proved that replacing
the chelating ligands has significant effect on changing the lipid solubility of the complexes and
can improve antitumor activity significantly.[25] This encouraged us to explore the activity of
complexes containing bipy derivatives in more details.
Triphenylamine (TPA) is a promising organic functional material in organic photoelectric field,
however, its biological application is rare. In this study, six half-sandwich complexes of the type
[(η5-Cpx)Ir(N^N)Cl]PF6 (Figure 1) have been synthesized and characterized, where (Cp*) is
phenyl derivatives (Cpxph), biphenyl derivatives (Cpxbiph), and the N^N-chelating ligands are bipy
linked with TPA groups through double bond, which can enhance the lipid solubility of IrIII
complexes, accordingly enhancing anticancer activity. The six IrIII complexes display higher
antitumor activity against A549 lung cancer cells (1.5 ~7.1 µM) than cis-platin (21.3 µM) under
the same conditions. Furthermore, all complexes can bind with BSA and DNA and oxidize NADH
to NAD+. IrIII complexes can effectively induce apoptosis and promoting the production of
reactive oxygen species (ROS) in cells. The results suggest that triphenylamine-appended
half-sandwich IrIII complexes are hopeful for development as new anticancer agents.
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Figure 1 The structure of as-synthesized half-sandwich organometallic IrIII complexes.
Results and Discussion
IrIII half-sandwich complexes of the type [(η5-Cpx)Ir(N^N)Cl]PF6 were synthesized in methanol
at ambient temperature, and the N^N-chelating ligands were synthesized by Wittig reaction and got
the trans structure by reflux with a catalytic amount of iodine. All complexes are new synthesized
complexes and obtained in good yields. Synthesis processes are shown in Scheme 1. IrIII
1
complexes and the intermediates were characterized by H NMR and mass spectroscopy. All IrIII
-
complexes were isolated as PF6 salts.
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Scheme 1 Synthesis process of IrIII complexes.
2.1 Cytotoxicity test
The antitumor activity of as-synthesized complexes against A549 human lung cancer cell
have been determined by MTT assay, the IC50 values after exposure to six complexes and
cis-platin for 24h are listed in Table 1. Notably, compared to cis-platin, as-synthesized six
complexes have better antitumor activity against A549 lung cancer cells with IC50 values range
from 1.5 to 7.1 µM. However, there is no better selectivity for the normal cell line 16HBE (human
bronchial epithelial cells) and BEAS-2B (human bronchial epithelial cells) for the complexes
(Table S1). As shown, complexes containing Cpxph ligand (complexes 2 and 5) showed higher
antiproliferative activity compare to their Cp* analogues (complexes 1 and 4), probably due to the
enhanced lipid solubility caused by the Cpxph ligand. However, introduction of the larger Cpxbiph
ligand in complexes 3 and 6 led to lower antiproliferative activity, probably due to the high
hydrophobicity of the chelating ligand. Therefore, the Cpxph ligand based complexes showed the
best anticancer activity. Sadler and coworkers have confirmed that Cp* IrIII complexes with
2,2′-bipyridine (bipy) are inactive against A2780 human ovarian cancer cells with IC50 values over
100 µM.[21,22] Therefore, the introduction of TPA group into bipy ligand played a crucial role to
switch on the anticancer activity of the complexes. However, variation of terminal groups (methyl
and methoxyl) on TPA group has less effect on the anticancer activity.
The cytotoxic activity of two ligands L1 and L2 was also tested, which showed no anticancer
activity, Table 1. In addition, the iridium dimers displayed inactivity or medium anti-cancer
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activity.[18] Interestingly, the complexes formed by coordination of iridium and TPA ligand
possessed high potency.
Table 1 Inhibition of growth toward A549 lung cancer cells by complexes 1–6 and comparison
with cis-platin recorded over a period of 24 h.
IC50 (µM)
Complex
A549
[(η5-C5Me5)Ir(L1)Cl]PF6 (1)
6.8 ± 0.5
[(η5-C5Me4C6H5)Ir(L1)Cl]PF6 (2)
1.5 ± 0.1
[(η5-C5Me4C6H4C6H5)Ir(L1)Cl]PF6 (3)
3.6 ± 0.5
[(η5-C5Me5)Ir(L2)Cl]PF6 (4)
4.8 ± 2.1
[(η5-C5Me4C6H5)Ir(L2)Cl]PF6 (5)
1.7 ± 0.2
[(η5-C5Me4C6H4C6H5)Ir(L2)Cl]PF6 (6)
7.1 ± 0.8
Cis-platin
21.3 ± 1.7
>100
L1
L2
>100
2.2 Reaction with NADH
In many biological processes, coenzyme NADH play a major role.[15] IrIII cyclopentadienyl
complexes can oxidize NADH to generate reactive oxygen species (ROS) H2O2, which provide a
pathway to a mechanism of oxidant.[26,27] The interaction of as-synthesized complexes with
NADH can be detected by measuring the maximum absorbance at 259 nm and 340 nm over time
with an ultraviolet-visible (UV-Vis) spectrophotometer (Figure 2 and Figure S1 in supporting
information). Among these, the peaks at short wavelength (~259 nm) can be assigned to the
absorption of NAD+ (The complexes capture hydrogen ions on NADH to produce NAD+), while
peaks at longer wavelength (~340 nm) are the absorption of NADH.[27] The turnover numbers
(TONs) of complexes 1 (42.4), 2 (27.5) 3 (2.1) 4 (31.3) 5 (14.4) and 6 (13.9) was calculated by
measuring the difference at 339 nm (Figure 2b). With the increase of the number of benzene
rings in Cp rings, the TONs of complexes decreased gradually. This phenomenon is attributed to
the increase of the benzene ring in the Cp ring, which increases the steric hindrance of the
complexes and decreases the ability of the complexes to oxidize NADH.
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Figure 2 (a) UV-Vis spectra of the reaction of NADH (100 µM) with complex 4 (1 µM) in 10%
MeOH/90% H2O (V:V) at 298 K for 8 h; (b) The TONs of as-synthesized complexes. The arrows
show changes in absorbance spectra over time.
2.3 Study of BSA interactions
Investigating the interaction between anticancer agents and protein is conductive to
understand what happened in cells.[28] Serum albumin (SA) is the main protein which play an
important role in drug transport and metabolism in blood plasma.[29] In this study, because of the
similar to human serum albumin (HSA) in structure and easily obtained, bovine serum albumin
(BSA) was used to test the binding to complexes by investigated the quenching of BSA. If the
quenching mechanism is a static quenching mechanism, changes in the absorption intensity
(278nm) are accompanied by significant wavelength shifts, while the dynamic quenching is
without any wavelength shifts.[12] From Figure 3a, we can see that there is no wavelength shift in
278 nm after adding complexes 1~6 to BSA solution, which indicate the interaction between BSA
and complexes is in the form of dynamic quenching.[30]
Figure 3 (a) UV-Vis spectrum of BSA (10 μM) in 5 mM Tris–HCl/50 mM NaCl buffer solution
(pH = 7.2) upon addition of the complexes 1-6 (5 μM). (b) Fluorescence spectra of BSA (10 μM;
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λex =280 nm; λem =343 nm) in the absence and presence of complex 3 (0–10 μM). The arrows
show changes in absorbance spectrum of BSA upon increasing the amounts of complexes 3.
Fluorescence quenching is another effective method to study the interaction between
complexes and proteins. As shown in Figure 3b (Figure S2 in supporting information), with the
increase of complex 3, the fluorescence intensity of BSA (10 μM) at 343 nm decreases
continuously.[31-35] To get a possible quenching mechanism, emission quenching data have been
analyzed by the classical Stern–Volmer equation and Scatchard equation.[12,36-39] As shown in
Table 2, the Stern–Volmer quenching constant Ksv and the quenching rate constant Kq and the
binding site number n of all complexes have no obvious change law, but the value of the binding
constant Kb will increase with the increase of the number of benzene rings on the Cp ring: Cp* >
CpXph > Cpxbiph. This result indicate that the introduction of benzene ring and diphenyl ring in Cp
have an adverse effect on the complexes binding to BSA. This is mainly due to the increasing
steric hindrance with the introduction of benzene ring and diphenyl ring on Cp, thereby affecting
the binding of complexes to BSA.
Table 2 The values of Ksv, Kb and Kq for as-synthesized complexes.
Kq
(1013 M-1S-1)
1.17
Ksv
Kb
（104 M-1）
22.56
6.08
Complex
n
(105 M -1)
1.17±0.16
0.77±0.15
1.24±0.15
1.05±0.18
1.11±0.16
1.39±0.14
1
2
3
4
5
6
1.05
0.98
0.87
1.00
0.96
0.91
0.77
1.24
2.33
1.05
10.08
6.51
1.11
1.39
3.71
Synchronous fluorescence spectra were obtained by a similar method to study the
microenvironment in the vicinity of the fluorophore.[40] The effect of the complexes 3 and 6 in the
BSA are shown in Figure 4 (Figure S5 and S6 in supporting information). With the increase of
complexes 3 and 6, both the synchronous fluorescence spectra for BSA markedly reduce in
fluorescence intensity at 283 and 275 nm (Δλ: 15 nm and 60 nm, which are the characteristic of
synchronous emission spectrum for tyrosine and tryptophan residue, respectively[41]). The
synchronous fluorescence spectrum has a displacement of 2-3 nm at Δλ = 15 nm, while no
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wavelength shift at Δλ = 60 nm, which indicating as-synthesized complexes mainly changes the
microenvironment of tyrosine for BSA.
Figure 4 Synchronous spectra of BSA (20 μM) with complexes 3 and 6, increasing (0−50 μM)
with a wavelength difference of [(a) 3, (b) 6] Δλ = 15 nm and [(c) 3, (d) 6 ] Δλ = 60 nm. The
arrows show changes in emission intensity of BSA upon increasing the amounts of complexes 3
and 6.
2.4 Interaction with CT-DNA
The interaction of base pairs of DNA with complexes usually involves intercalation,
interaction, non-intercalative, electrostatic interactions and damage of the DNA double helix.[42-45]
Change of the absorption wavelength of the as-synthesized complexes with increasing of
CT-DNA (DNA sodium from calf thymus) was achieved by UV-Vis titration, and the binding
constants (Kb) of the complexes were also calculated. The role of metal iridium in our complexes
probably is to provide a coordination center, then the metal to ligand (MLCT) or ligand to metal
charge transfer (LMCT) will appear, which can cause interaction between the complexes and
ct-DNA via electrostatic or covalent action.[12] As shown in Figure 5 (Figure S7 in supporting
information) and Table 3, subtractive and small red shift (Δλ = 6 nm) along with the increase of
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CT-DNA concentration indicate the interactions between complexes and DNA, which attributed to
the interaction of complexes with base pairs in the form of electrostatic binding or noncovalent
insertion.[46] The binding constants Kb were fitted by Benesi–Hildebrand equation.[12] As shown in
Table 3, Kb will increase with the increase of the number of benzene rings on the Cp ring: Cpxbiph.>
CpXph > Cp*. This result is in agreement with some previous studies.[47]
Figure 5 UV-Vis spectrum of complexes (a) 3 and (b) 6 dissolved in 10% DMSO 90% 5 mM
Tris–HCl/50 mM NaCl buffer solution, pH = 7.2 (V:V) ) upon addition of the CT-DNA (0-0.4 mM)
in 5 mM Tris–HCl/50 mM NaCl buffer solution (pH = 7.2). During the experiment, the
concentration of the complexes remained unchanged and the concentration of CT-DNA increased
continuously. The arrows show the direction of changes in absorbance upon increasing the
concentration of CT-DNA.
Table 3 Absorption spectroscopic properties of as-synthesized complexes on binding to CT-DNA.
Absorption λmax (nm)
Kb
Complex
∆λ
(103 mM-1)
Free
Bounda
1
2
3
4
5
6
474
466
466
486
478
475
479
475
472
490
484
479
5
9
6
4
6
4
1.70
2.94
3.04
0.37
3.03
3.06
a: [Complex]=20 μM at [DNA]/[Complex]=20
2.5 Ethidium Bromide (EtBr) Displacement Studies
Ethidium bromide (EtBr) as an effective intercalation agent has a strong effect on nucleic
acids and other biomolecules after dying, commonly used as fluorescent labeling of biological
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molecules. EtBr itself has weak fluorescence, but the fluorescence intensity will increase about 20
times when it is combined with DNA into EtBr-DNA.[31] As shown in Figure 6 (Figure S9 in
supporting information), fluorescence quenching was found when adding the complexes 3 and 6 to
a saturated EtBr-DNA solution. This phenomenon is attributed to the replacement of EtBr by IrIII
complexes from EtBr-DNA.[48-52] The quenching relative to the initial intensity was 46% (1), 35%
(2), 43% (3), 54% (4), 61% (5), and 65% (6), respectively. The quenching constants (Kq) are
calculated by the Stern-Volmer equation,[12] which were 1.13× 104 (1), 1.32 × 104 (2), 2.50 × 104
(3), 2.11 × 104 (4), 3.82 × 104 (5), and 4.27 × 104 (6), respectively, which is consistent with the
result of above CT-DNA binding test.
Figure 6 Fluorescence spectra of EtBr bound to DNA in the absence and presence of complexes
(a) 3 and (b) 6 (0-50 μM dissolved in DMSO) (EtBr, 10 μM; DNA, 10 μM; in 5 mM Tris–HCl/50
mM NaCl buffer solution (pH = 7.2)). The arrows show changes in emission intensity upon
increasing the amounts of complexes 3 and 6.
As shown, the values of the binding constants Kq have a little change, the complexes with
biphenyl rings on Cp ring have the strongest ability to replace EtBr compared to other complexes.
The complexes may bind DNA via intercalation obtained from electronic absorption studies and
the ability of the compound to bind to DNA increases with the increase of steric hindrance. From
Cp* to CpXph and Cpxbiph, the binding features to BSA decrease, while to DNA increase, which
could explain why complexes 2 an 4 showing the better anticancer activity.
2.6 Apoptosis Assay
In order to understand the function mechanism of complexes in cells, all complexes have
analyzed apoptotic populations against A549 lung cancer cells by flow cytometry when exposure
to as-synthesized complexes. As shown in Figure 7 (Table S2 and S3), flow cytometry analysis
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showed that treatment of A549 cells with complexes 3 and 6 led to a dose-dependent increase in
the percentage of apoptotic cells, respectively. After treatment for 24 h, when complex 3 at a
concentration of 1 × IC50, only 9.1% of A549 lung cancer cells were in late apoptotic phase. A
total of 86.6% (early apoptotic + late apoptotic) cells were undergoing apoptosis at 3 × IC50 for
complex 3, whereas untreated cells remained 95.1% viable, indicating obvious induction of
apoptosis at 3 equipotent concentrations of IC50. For complex 6, 66.3% A549 lung cancer cells
were in apoptotic phase at a concentration of 3 × IC50.
Figure 7 Apoptosis analysis of A549 lung cancer cells after 24 h of exposure to complexes (a) 3
and (b) 6 determined by flow cytometry using Annexin V-FITC vs PI staining. (c) 3 and (d) 6:
Populations for cells treated by complexes 3 and 6, respectively.
2.7 Cell Cycle Analysis
Cell cycle arrest analysis for complexes 3 and 6 against A549 lung cancer cells investigated
by flow cytometry was shown in Figure 8 (Tabel S4 and S5). The cell cycle progression was
analyzed at 0.25, 0.5 and 1 equipotent concentrations of IC50 of complexes for 24 h. At a
concentration of 1 × IC50, the percentages of cells in the S phase and G1 phase increased 4% and 2%
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when exposure to complex 3. For complex 6, at a concentration of 1 × IC50, the percentages of
cells in the G2/M phase of the cell cycle increased from 8.9% to 10.2% and the Sub-G1 phase
increased from 0.1% to 1.8%. Compared with the untreated control, complex 3 disturbs the cell
cycle at S phase, G1 phase and Sub-G1 phase, while complex 6 disturbing the cell cycle at S phase,
G2/M phase and Sub-G1 phase. The results indicate that the complexes can disturb the cell growth
cycle progression.
Figure 8 Cell cycle analysis of A549 lung cancer cells after 24 h of exposure to complexes (a) 3 and (b)
6 at the indicated concentrations by flow cytometry using PI staining. Populations in each cell cycle
phase for control and complexes (c) 3 and (d) 6.
2.8 Induction of ROS
Reactive oxygen species (ROS) plays an important role in regulating cell proliferation, death,
and signaling, which is also used to explore the function mechanism of anticancer agents.[15] The
level of ROS in A549 lung cancer cells induced by complexes 3 and 6 at concentrations of 0.25 ×
IC50 and 0.5 × IC50 investigated by flow cytometry analysis. As shown in Figure 9 (Tabel S6
and S7), treatment with complexes 3 and 6 after 24 h, the increasing of ROS levels at A549 lung
cancer cells were observed. The changes of ROS level have provided a basis for killing cancer
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cells.[16]
Figure 9 Effect of complexes (a) 3 and (b) 6 on intracellular ROS levels in A549 lung cancer cells
treated at the indicated concentrations for 24 h.
CONCLUSION
In this study, six new half-sandwich IrIII complexes were synthesized with simple synthetic
procedures. The introduction of TPA group to bipyridine ligand has effectively improved the
anticancer activity of complexes. Complex 2 showed the best activity towards A549 lung cancer
cells (IC50: 1.5 µM), which was more than ten times higher than the clinical anticancer drug
cis-platin. From Cp* to CpXph and Cpxbiph, the binding features to BSA decrease, while to DNA
increase, which further verifies why complexes 2 an 4 showing the better anticancer activity. And
also, IrIII complexes can induce ROS in cells, which can lead to apoptosis. The results of flow
cytometry further indicate that the complexes can disturb the cell growth cycle and lead to
apoptosis. Above all, triphenylamine-appended half-sandwich iridiumIII complexes could be a
promising candidate for further evaluation as anti-cancer drugs.
EXPERIMENTAL SCETION
Materials
IrCl3·nH2O, 4-(di-p-tolylamino)benzaldehyde, 4-(bis(4-methoxyphenyl)amino)benzaldehyde,
2,3,4,5-tetramethyl-2-cyclopentenone(95%), 1,2,3,4,5-pentamethyl-cyclopentadiene(95%), buty
llithium solution (1.6 M in hexane), 4'-methyl-[2,2'-bipyridine]-4-carbaldehyde, 4-(di-p-tolyla
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mino)benzaldehyde, 4-(bis(4-methoxyphenyl)amino)benzaldehyde and (λ2-bromanyl)triphenyl-λ
4-phosphane were purchased from Shanghai Pengteng Fine Chemical Co., Ltd. For the biol
ogical experiments, DMEM medium, fetal bovine serum, penicillin/streptomycin mixture an
d trypsin/EDTA were purchased from Sangon Biotech. A549 lung cancer cells were obtain
ed from Shanghai Institute of Biochemistry and Cell Biology (SIBCB).
Syntheses
Synthesis of Wittig reagents (F)
The intermediates (D) and Wittig reagents (F) were synthesized according to the literature.[53]
Synthesis of ligand (L).[54,55]
Wittig reagents F (3 mmol) and 4'-methyl-[2,2'-bipyridine]-4-carbaldehyde (1.5 mmol) were
added into a 100 mL round-bottom flask under N2. Anhydrous THF (40 mL) was added to above
flask, cooled down to 0 °C. The THF solution of t-BuOK (3 mmol,) was added dropwise to above
flask, stirred for 30 min at 0 °C, followed by stirring at room temperature until Wittig reagents F
was consumed completely (monitored by thin-layer chromatography). The reaction was
terminated with ice water. The crude product was refluxed for 8 h in THF with a catalytic amount
of iodine. Then the remaining iodine was removed by sodium hydroxide (NaOH) solution (Wt =
10%, 100 mL) with stirring for 2 h. After that, the product was purified by chromatographed on a
silica gel column (petroleum ether: ethyl acetate =15:1 as eluent) to give the title complexes as a
pure E stereoisomer.
(E)-4-methyl-N-(4-(2-(4'-methyl-[2,2'-bipyridin]-4-yl)vinyl)phenyl)-N-(p-tolyl)aniline
1
(L1): Yield: 2.9 g (51.6%). H NMR (500 MHz, CDCl3) δ 8.60 (dt, J = 15.7, 8.0 Hz, 2H), 7.42 –
7.35 (m, 4H), 7.14 – 7.08 (m, 4H), 7.04 – 6.79 (m, 10H), 3.83 (s, 6H), 2.49 (s, 3H).
(E)-4-methoxy-N-(4-methoxyphenyl)-N-(4-(2-(4'-methyl-[2,2'-bipyridin]-4-yl)vinyl)phen
1
yl)anilin (L2): Yield: 2.8 g (49.6%). H NMR (500 MHz, CDCl3) δ 8.62 – 8.55 (m, 2H), 8.48 (s,
1H), 8.26 (s, 1H), 7.43 – 7.32 (m, 4H), 7.14 (t, J = 6.3 Hz, 1H), 7.09 (d, J = 8.2 Hz, 4H), 7.04 –
6.94 (m, 7H), 2.45 (s, 3H), 2.33 (s, 6H).
Synthesis of the [(η5-C5Me5)IrCl2]2 (dimer1), [(η5-C5Me4C6H5)IrCl2]2 (dimer2) and
[(η5-C5Me4C6H4C6H5)IrCl2]2 (dimer3).
Dimers were prepared according to literature methods.[18]
[(η5-C5Me5)IrCl2]2 (dimer1): Yield: 0.397 g (64.6%). 1H NMR (500 MHz, CDCl3): δ 1.60 (s,
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J = 1.4 Hz, 15H).
1
[(η5-C5Me4C6H5)IrCl2]2 (dimer2): Yield: 0.37 g (58.5%). H NMR (500 MHz, CDCl3): δ
7.58 (m, 2H), 7.35 (m, 3H), 1.72 (s, 6H), 1.63 (s, 6H).
[(η5-C5Me4C6H4C6H5)IrCl2]2 (dimer3): Yield: 0.25 g (35.8%). 1H NMR (500 MHz, CDCl3):
δ 7.64 (m, 4H), 7.44 (m, 2H), 7.33 (m, 3H), 3.25 (m, 1H), 2.08 (s, 3H), 1.95 (s, 3H), 1.88 (s, 3H),
1.00 (d, 3H, J = 7.5 Hz).
Synthesis of the [(η5-Cpx)Ir(N^N)Cl]PF6.
A solution of [(η5-Cpx)IrCl2]2 (0.05 mmol) and complexes L (0.1 mmol) in methanol (40 mL)
resulting suspension was stirred at ambient temperature overnight. The mixture was then stirred
with addition of ammonium hexafluorophosphate (0.4mmol) for 4h. Most of the solvent is
concentrated in vacuum and kept at -20 °C for 12h ，filtered and washed with cold methanol and
1
diethyl ether. The H NMR (500 MHz, CD3Cl3) peak integrals of complexes 1-6 are shown in
(Figure S11 in supporting information). The high resolution mass spectrometry data of complexes
1-6 are shown in (Figure S12 in supporting information).
[(η5-Cpx)Ir(N^N)Cl]PF6 (1). Yield: 0.032 g (35.8%). 1H NMR (500 MHz, CDCl3): δ 8.54 (d,
J = 5.8 Hz, 1H), 8.50 (d, J = 6.0 Hz, 1H), 8.32 (d, J = 16.7 Hz, 2H), 7.58 (d, J = 5.9 Hz, 1H), 7.53
(d, J = 16.2 Hz, 1H), 7.44 (d, J = 8.8 Hz, 3H), 7.11 (d, J = 8.2 Hz, 4H), 7.04 (d, J = 8.3 Hz, 4H),
6.99 – 6.94 (t, 3H), 2.62 (s, 3H), 2.34 (s, 6H), 1.66 (s, 15H). ESI-MS (m/z): [M-PF6]+ Calcd for
C43H44ClIrN3, 830.513; Found 830.863. [M-PF6-Cl]+ Calcd for C43H44IrN3, 795.063; Found
794.926. Elemental analysis: Found: C, 52.89; H, 4.53; N, 4.32%, calcd for C43H44ClF6IrN3P: C,
52.95; H, 4.55; N, 4.31%.
1
[(η5-Cpx)Ir(N^N)Cl]PF6 (2). Yield: 0.025 g (30.2%). H NMR (500 MHz, CDCl3): δ 8.37
(dd, J = 16.5, 9.1 Hz, 3H), 8.30 (d, J = 6.1 Hz, 1H), 7.60 – 7.41 (m, 10H), 7.34 (d, J = 5.2 Hz, 1H),
7.10 (d, J = 8.3 Hz, 4H), 7.03 (d, J = 8.3 Hz, 4H), 6.95 (d, J = 8.6 Hz, 2H), 2.64 (s, 3H), 2.33 (s,
6H), 1.76 (d, J = 3.1 Hz, 6H), 1.72 (s, 6H). ESI-MS (m/z): [M-PF6]+ Calcd for C48H46ClIrN3,
892.584; Found 892.884. [M-PF6-Cl]+ Calcd for C48H46IrN3, 857.134; Found 856.909. Elemental
analysis: Found: C, 55.47; H, 4.43; N, 4.04%, calcd for C48H46ClF6IrN3P: C, 55.57; H, 4.47; N,
4.05%.
1
[(η5-Cpx)Ir(N^N)Cl]PF6 (3). Yield: 0.040 g (43.2%). H NMR (500 MHz, CDCl3): δ 8.40
(dd, J = 7.5, 4.3 Hz, 3H), 8.34 (d, J = 6.1 Hz, 1H), 7.74 (d, J = 8.3 Hz, 2H), 7.69 – 7.63 (m, 4H),
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7.55 (s, 1H), 7.53 – 7.47 (m, 3H), 7.47 – 7.42 (m, 3H), 7.37 (d, J = 5.7 Hz, 1H), 7.11 (d, J = 8.2
Hz, 3H), 7.07 (d, J = 8.6 Hz, 1H), 7.03 (d, J = 8.3 Hz, 3H), 7.00 (d, J = 4.8 Hz, 1H), 6.96 (d, J =
8.9 Hz, 2H), 6.89 (d, J = 8.6 Hz, 1H), 2.68 (s, 3H), 2.32 (d, J = 10.3 Hz, 6H), 1.78 (m, J = 12.2,
6.1 Hz, 12H). ESI-MS (m/z): [M-PF6]+ Calcd for C54H50ClIrN3, 968.682; Found 968.145.
[M-PF6-Cl]+ Calcd for C54H50IrN3, 933.282; Found 933.183. Elemental analysis: Found: C, 58.13;
H, 4.50; N, 3.78%, calcd for C54H50ClF6IrN3P: C, 58.24; H, 4.53; N, 3.77%.
[(η5-Cpx)Ir(N^N)Cl]PF6 (4). Yield: 0.033 g (36.5%). 1H NMR (500 MHz, CDCl3): 8.47 (d,
J = 5.8 Hz, 1H), 8.42 (d, J = 6.1 Hz, 1H), 8.25 (d, J = 12.5 Hz, 2H), 7.48 (dd, J = 22.5, 10.8 Hz,
2H), 7.40 – 7.30 (t, 3H), 7.01 (d, J = 13.3 Hz, 4H), 6.84 (dd, J = 35.5, 12.2 Hz, 7H), 3.75 (s, 6H),
2.55 (s, 3H), 1.59 (s, 15H). ESI-MS (m/z): [M-PF6]+ Calcd for C48H46ClIrN3O2, 862.275; Found
862.103 [M-PF6-Cl]+ Calcd for C48H46IrN3O2, 827.061; Found 827.141. Elemental analysis:
Elemental analysis: Found: C, 51.16; H, 4.42; N, 4.19%, calcd for C43H44ClF6IrN3O2P: C, 51.26;
H, 4.40; N, 4.17%.
1
[(η5-Cpx)Ir(N^N)Cl]PF6 (5). Yield: 0.036 g (38.3%). H NMR (500 MHz, CDCl3): δ 8.37
(dd, J = 14.1, 7.4 Hz, 3H), 8.29 (d, J = 6.1 Hz, 1H), 7.59 – 7.39 (m, 10H), 7.33 (d, J = 5.3 Hz, 1H),
7.09 (d, J = 8.8 Hz, 4H), 6.86 (d, J = 8.9 Hz, 6H), 3.81 (s, 6H), 2.63 (s, 3H), 1.76 (d, J = 3.5 Hz,
6H), 1.71 (s, 6H). ESI-MS (m/z): [M-PF6]+ Calcd for C43H44ClIrN3O2, 924.582; Found 924.705
[M-PF6-Cl]+ Calcd for C43H44IrN3O2, 889.132; Found 889.533. Elemental analysis: Found: C,
53.82; H, 4.32; N, 3.94%, calcd for C48H46ClF6IrN3O2P: C, 53.90; H, 4.34; N, 3.93%.
[(η5-Cpx)Ir(N^N)Cl]PF6 (6). Yield: 0.05 g (52.1%). 1H NMR (500 MHz, CDCl3) δ 8.42 –
8.38 (m, 3H), 8.32 (d, J = 6.1 Hz, 1H), 7.73 (d, J = 8.3 Hz, 2H), 7.66 (dd, J = 12.4, 8.3 Hz, 4H),
7.55 – 7.46 (m, 4H), 7.43 (t, J = 8.8 Hz, 3H), 7.36 (d, J = 6.1 Hz, 1H), 7.09 (d, J = 8.9 Hz, 4H),
6.96 (d, J = 16.1 Hz, 1H), 6.89 – 6.84 (m, 6H), 3.81 (s, 6H), 2.66 (s, 3H), 1.82 – 1.73 (m, 12H).
ESI-MS (m/z): [M-PF6]+ Calcd for C54H50ClIrN3O2, 1000.680; Found 1000.856. [M-PF6-Cl]+
Calcd for C54H50IrN3O2, 963.351; Found 963.902. Elemental analysis: Found: C, 56.52; H, 4.38;
N, 3.64%, calcd for C54H50ClF6IrN3O2P: C, 56.61; H, 4.40; N, 3.67%.
ACKNOWLEDGMENTS
We thank the National Natural Science Foundation of China (Grant No. 21671118) and the
Taishan Scholars Program for support.
Notes
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10.1002/asia.201800103
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The authors declare no competing financial interest.
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Table-of-Contents
Organometallic half-sandwich IrIII complexes of type [(η5-Cpx)Ir(N^N)Cl]PF6 (N^N:
triphenylamine substituted bipyridyl ligand) showed potent cytotoxicity and excellent BSA and
DNA binding properties, and catalytic conversion of NADH to NAD+. The complexes
significantly increased reactive oxygen species (ROS) in cells and induced apoptosis effectively.
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