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New Organometallic Tetraphenylethylene⋅Iridium(III) Complexes with Antineoplastic Activity.
Accepted Article
Title: Synthesis and Antineoplastic Applications of TPE Appended
Organometallic Iridium(III) Complexes
Authors: Xicheng Liu, Xiangdong He, Xiaojing Zhang, Yongling Wang,
Jiaying Liu, Xiujuan Hao, Yue Zhang, Xiang-Ai Yuan, Laijin
Tian, and Zhe Liu
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To be cited as: ChemBioChem 10.1002/cbic.201900268
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10.1002/cbic.201900268
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Synthesis and Antineoplastic Applications of TPE Appended
Organometallic Iridium(III) Complexes
Xicheng Liu*[a], Xiangdong He[a], Xiaojing Zhang[a], Yongling Wang[a], Jiaying Liu[a], Xiujuan Hao[a], Yue
Zhang[a], Xiang-Ai Yuan[a], Laijin Tian[a], Zhe Liu[a]*
Abstract: IridiumIII (IrIII) complexes have attracted more and more
attention because of their potential antineoplastic activity for the past
few years. In this study, four IrIII complexes of the type [(η5Cpx)Ir(N^N)Cl]PF6 (1 and 2) and [Ir(Phpy)2(N^N)]PF6 (3 and 4) have
been synthesized and characterized. Complexes exhibit potential
antineoplastic activity towards A549 cells, especially for complex 1
(IC50: 3.56±0.5 µM), which was nearly six times of cis-platin
(21.31±1.7 µM). Additionally, these complexes show some
selectivity for cancer cells versus normal cells . Complexes could
be transported by serum albumin (binding constant changed from
0.37~81.71×105 M-1). IrIII complexes (1 and 2) could catalyze the
change of NADH to NAD+ (TONs: 43.2, 11.9) and induce the
accumulation of reactive oxygen species, which confirmed the
antineoplastic mechanism of oxidation, while cyclometalated
complexes (3 and 4) could target the lysosome (PCC: 0.73), lead to
lysosomal damage and induce apoptosis. Understanding the
mechanism of action would help further structure-activity optimization
on these novel IrIII complexes as emerging cancer therapeutics.
Introduction
The potential antineoplastic value of transition metal complexes
has been widely recognized since the discovery of cis-platin (cisdiamminedichloridoplatinum(II)) by Rosenberg etc. in 1965.[1]
Despite of numerous drawbacks, including strong resistance,
poor selectivity, unclear mechanism of action, and serious
adverse effects, platinum-based drugs are still considered as the
most active chemotherapeutic agents and widely used. [2] In fact,
treatment with platinum-based or platinum-related drugs accounts
for over 50% of all chemotherapeutic regimens. [3] To solve these
limitations, some alternative metals which showing different from
cis-platin in antineoplastic mechanism and target sites have been
developed and evaluated. In spite of the challenging, however,
[a]
Institute of Anticancer Agents Development and Theranostic
Application, The Key Laboratory of Life-Organic Analysis and Key
Laboratory of Phar maceutical Intermediates and Analysis of Natural
Medicine, School of Chemistry and Chemical Engineering, Qufu
Normal University, Qufu 273165, China.
Corresponding author (X. Liu) E-mail: chemlxc@163.com; Fax:
05374456301. (Z. Liu) E-mail: liuzheqd@163.com; Fax:
05374456301.
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
understanding the behavior of these complexes in tumor cells
would still benefit for the design and improvement. [4]
Very recently, organometallic iridiumIII (IrIII) complexes have
attracted much attention because of their potential anticancer
activity towards various tumor cells. [5] IrIII antitumor complexes
can be classified as two main types: half-sandwich structure and
cyclometalated structure complexes，and the general form can
be expressed as [(Cpx)Ir(L^L)Z]PF6 and [Ir(Phpy)2(L^L)]PF6,
respectively, where PF6 is the common counter ion, Z is an
chlorine anion or a neutral substituted pyridyl ligand, L^L is a
chelating bidentate ligand (such as N^N, C^N and N^O etc.), and
Cpx and Phpy represents the electron-rich cyclopentadienyl group
and 2-Phenylpyridine or its derivatives, respectively. Interestingly,
the nature of all the parts around central iridium atom have a
significant effect on the antineoplastic activity of these complexes,
among these, the type and the position of the substituents on the
L^L-chelating ligand were the most studied, and fine-regulating of
which could effectively modulate the mechanism of uptake, target
site of intracellular, and even the effects on the intracellular
tissues.[6] Moreover, the prominent targeted fluorescence
characteristics of organicmetallic IrIII complexes provided the
chance to investigate the microscopic mechanism of action.
Studies showed that organicmetallic Ir III complexes could enter
cells in an energy-dependent or nonenergy-dependent mode,
target lysosomes, mitochondria, endoplasmic reticulum or other
cellular tissues, and lead to lysosomal damage, change of
mitochondrial membrane potential or endoplasmic reticulum
stress, and eventually induce apoptosis. [7]
As a typical aggregation-induced emitting (AIE) fluorescence
group, tetraphenylethylene (TPE) has a propeller-shaped
structure and the rotatable peripheral benzene rings, and now
widely used in molecular design due to their distinctive
luminescence property and easy modification. [8] Recently, many
research achievements of TPE derivatives have been applied in
the fields of organic light-emitting diode (OLED)[9], chemical
sensing[10] and biological sensing[11]. However, the study of TPE
applied to organometallic antineoplastic field was rare. It was
established that the introduction of small molecular luminescent
group to organometallic IrIII complexes could effectively control
antineoplastic activity and the target position in organelle.[12]
Above all, in this study, four organometallic IrIII complexes of the
type [(Cpx)Ir(N^N)Cl]PF6 and [Ir(Phpy)2(N^N)]PF6 (Figure 1) have
been synthesized and characterized. Activity of target complexes
towards A549, BESA-2B and 16HBE cells was obtained by MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)
assay. Bovine serum albumin binding experiments were utilized
to verify the transport mechanism of these complexes. Compared
with cyclometalated IrIII TPE complexes (3 and 4), half-sandwich
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that TPE-appended IrIII complexes possess sufficient stability for
further biological assays.
Scheme 1. Design strategy of of organometallic IrIII TPE complexes.
Figure 1. Structures of as-synthesized organometallic IrIII TPE complexes.
Results and Discussion
Target IrIII TPE complexes were synthesized by dimer and
N^N-chelating ligands in good yields at ambient temperature, [13]
and the N^N ligands were synthesized by Suzuki reaction
between tetraphenylethylene boric acid and the corresponding
bromopyridine compounds under N2 (Scheme 1).[14] Multiple
attempts to grow single crystals failed because of the “propellershaped” structure of TPE molecule leading to the major steric
hindrance for target IrIII complexes. Instead, the N^N-chelating
ligands (L1 and L2) and target complexes (1-4) were characterized
by nuclear magnetic resonance spectrum (NMR) and mass
spectroscopy (MS), and all complexes were isolated as PF6 salts.
Deuterated reagent DMSO (2.50 ppm) and CHCl3 (7.26 ppm)
were used as the solvent for testing NMR of complexes.
Hydrogens and carbons of the five methyl groups on Cp ring is
shown in 1.66, 1.67 ppm and 8.58, 8.60 ppm for complex 1 and 2
in 1H and 13C NMR spectrum, and which on the benzene ring and
pyridine are shown in the range of 6.20~9.15 ppm and
119.48~167.86 ppm, respectively. Target complexes were
soluble in common organic solvents such as methanol, dimethyl
sulfoxide and chloroform, insoluble in the ether, hexane and
petroleum ether. Complexes 1 and 3, a specially selected
complexes, show the thermal decomposition temperatures (Td) of
323.8 and 451.5 °C (Figure S1), respectively, which indicate that
IrIII TPE complexes possess good thermal stability. Aqueous
stability of complexes 1 and 3 was also monitored in 50%
CH3OH/50% H2O (v/v) by ultraviolet-visible (UV-vis) absorption
spectrum at 298 K for 8 h, and no obvious changes for the
absorbance were observed (Figure S2). The studies confirmed
1 Cytotoxicity test
The cytotoxicity of target complexes (1-4), TPE-unattached
structural IrIII complexes 5 ([(η5-C5Me5)Ir(bipy)Cl]PF6) and 6
([Ir(Phpy)2(bipy)]PF6), cis-platin, Dimer (1 and 2) and chelating
ligands (L1 and L2) were determined by the MTT (3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay
after 24 h treatment towards A549 lung cancer cells (the leading
cause of death in both developed and developing countries). As
shown in Table 1, compared with dimer, chelating ligands and
TPE-unattached complexes, target IrIII TPE complexes (1-4)
exhibit potential antineoplastic activity, and the best of which
(complex 1) was nearly six times of cis-platin that widely used in
the clinic. This conclusion proves that the introduction of TPE
molecules is beneficial to enhance the antineoplastic activity of
organometallic iridium complex.
Table 1 Inhibition of growth against A549, BESA-2B and 16HBE cells by
complexes 1–6, cis-platin, dimer and chelating ligands after 24 h.
Complex
IC50 (µM)
A549
BEAS-2B
16HBE
1
3.56±0.5
6.31±0.9
6.61±0.2
2
3
17.27±0.1
32.73±0.5
4
>100
20.52±1.4
40.38±0.3
>100
35.10±0.7
21.04±0.5
>100
5
6
Cis-platin
Dimer 1
Dimer 2
L1
L2
>100
40.33 ± 0.8
21.31 ± 1.7
>100
>100
>100
>100
/
/
/
/
/
/
/
/
/
/
/
/
/
Half-sandwich IrIII complexes (1 and 2) exhibit better
antineoplastic activity than cyclometalated structure complexes (3
and 4) under the same conditions. Studies show the
antineoplastic activity of half-sandwich IrIII complex mainly
depends on the electronic properties of central iridium atom and
the leaving group (Cl).[4d] To further understand how the ancillary
ligands (TPE-appended bipyridine) influence the antineoplastic
activity, the optimal configurations, the frontier molecular orbitals
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IrIII TPE complexes (1 and 2) could induce the production of
reactive oxygen species, which confirmed the antitumor
mechanism of oxidation. Due to the favorable targeted
fluorescence property, complexes 3 and 4 could target the
lysosome in vivo, lead to lysosomal damage, arrest the cell cycle,
and eventually induce apoptosis. The results suggest that TPEappended IrIII complexes are of great significative for further
evaluation as antineoplastic drugs.
10.1002/cbic.201900268
ChemBioChem
and their electron cloud distribution were analyzed by density
functional theory (DFT) calculation at the B3LYP/6-31G(d) (C, H,
N, Cl)/lanl2dz (Ir) level.[15] As shown in Figure 2, the HOMOs of
complexes 1 and 2 are mainly localized on the bipyridine ligand
and the terminal TPE groups, while the LUMOs are mainly
localized on the central iridium atom, bipyridine ligand, phenyl of
TPE molecule connected with bipyridine and chlorine atom (the
leaving group), which indicate that the introduction of TPE
molecule improves the conjugate area and electron donor
capacity of bipyridine chelating ligand, [16] thus increasing the
electron cloud density of central iridium atoms, facilitating the
break of Ir-Cl bonds and leading to the favorable antineoplastic
activity. However, major dihedral angles between P1 (pyridine
ring) and P2 (benzene ring of TPE molecule) ring are 29.63°and
29.93°for complexes 1 and 2, respectively, which confirm that this
improvement is limited. This conclusion could further be
confirmed by the data of natural population analysis (NPA) and
Wiberg bond order of Ir-Cl bond at the B3LYP/6-31G(d, p) (C, H,
N, Cl)/SDD (Ir) level. The results of NPA charge population (Table
S1) for Ir and Cl in complexes 1 and 2 were almost the same with
the values of 0.19 and -0.33, respectively.[17] Ir-Cl bond levels
were also shown almost the same values for complex 1 (0.7553)
and 2 (0.7539). This conclusion is consistent with the hypothesis
of optimal configuration that the conjugation of chelating ligands
is finite because of the major dihedral angles between bipydine
and TPE molecule, which leads to the limited improvement of
electron cloud density for the central iridium atom, so there's not
much difference in Ir-Cl bond levels of complexes 1 and 2.
complexes can be effectively adjusted by reasonable design for
the type and number of ligands.[18]
In addition, the cytotoxicities of complexes 1-4 to human
bronchial epithelial cells (16HBE) and lung epithelial cells (BEAS2B) were further evaluated. As shown in Table 1, these
complexes show some selectivity for cancer cells versus normal
cells, especially for complexes 1 and 2, but it is not obvious.
Therefore, more structural modifcation is necessary to improve
the selectivity in future work.
2 Reaction with NADH
Being cofactor, NADH (the reduced state of nicotinamide
adenine dinucleotide) plays a major role in numerous biological
process such as cell death, antioxidation, oxidative stress and
energy metabolism.[19] In vivo, the transformation from NADH to
NAD+ could induce the accumulation of reactive oxygen species
(ROS) H2O2 and lead to apoptosis, which provided an
antineoplastic mechanism of oxidation. [20] As shown in Figures
3a and S3, there are significantly decreased absorbance at 339
nm (the maximum UV-vis absorbance of NADH) and increased
that at 259 nm (the maximum absorbance of NAD+) with the
addition of half-sandwich IrIII complexes (1 and 2), especially for
complex 1.[21] However, almost no changes occur for cycloiridium
complex (3 and 4). The turnover numbers (TONs) of complexes 1
(43.2), 2 (11.9) 3 (3.0) 4 (2.6) were calculated by measuring the
difference at 339 nm (Figure 3b), which was consistent with the
result that half-sandwich IrIII complexes possessed better
antineoplastic activity than cyclometalated IrIII complexes.
Fig. 3 (a) UV-vis spectrum of NADH (100 µM) dealed with complex 1 (1 µM) in
10% MeOH/90% H2O (v/v) at 298 K for 8 h; (b) The TONs of complexes.
Figure 2. Calculated optimal configurations, HOMO and LUMO diagrams of
complexes 1 and 2.
The partition coefficients (logP) of complexes 1-4 in oil/water
were determined by inductively coupled plasma mass
spectrometry and the values were -0.33, 0.12, 1.05 and 1.44,
respectively. These results indicate that the introduction of too
many TPE groups enhances the lipid solubility of the complexes,
but at the same time weakens their water solubility, thus affecting
the antineoplastic activity of complexes. [6d,7a] Studies have also
confirmed that the lipid solubility and water solubility of the
complexes can be improved, and the antineoplastic activity of
3 Study of BSA interactions
Being the most abundant protein in plasma, serum albumin
(SA) plays an major role in drug transport and metabolism, and
then the research on the interactions of target complexes and SA
is beneficial to investigate the transport mechanism of drugs. [22]
Because of the similar structure, easy availability and low price,
bovine serum albumin (BSA) could be a cost-efficent alternative
to human serum albumin (HSA). [23] Therefore, in this study, BSA
was used to determine the interaction between target complexes
and protein. As shown in Figures 4a and S4, with the increase of
IrIII TPE complexes, the maximum absorption of 218 nm (the
absorption of BSA) decreased, which indicated IrIII TPE
complexes could act on BSA and induce alphahelical interference.
Meanwhile, due to the influence of polar solvent (water), a
significant red shift was found at 218 nm. [24] Additionally, the
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of apoptotic cells after 24 h of treatment. The percentage of early
apoptosis and late apoptosis cells increased from 1.7% and 9.8%
to 8.1% and 65.9% when the concentration changed from 0.5 ×
IC50 to 3.0 × IC50 for complex 1. In addition, about 43.5% of A549
cells treated with complex 3 were undergoing apoptosis, 41.9% of
which were in late apoptosis. In comparison, 95.1% of A549 cells
survived for the control under the same conditions. This
conclusion was consistent with the results of the MTT assay, and
further confirmed IrIII TPE complexes could lead to functional
decline of tumor cells and induce apoptosis.
Figure 5. Apoptosis analysis of A549 cells after 24 h of exposure to complex 1
(a) and 3 (b) were determined by flow cytometry; Histogram of apoptosis
analysis after treated with complexes 1 (c) and 3 (d). Data are quoted as mean
± SD of three replicates.
Figure 4. (a) UV-vis spectrum of BSA (10.0 μM) in 5 mM Tris-HCl/50 mM
NaCl buffer solution (pH: 7.2) with the increase of complexe 2 (0-10.0 μM). (b)
Fluorescence spectra of BSA (10.0 μM; λex =280 nm; λem =343 nm) in the
absence and presence of complex 2 (0-10.0 μM).
Table 2. The values of Ksv, Kb and Kq for as-synthesized IrIII complexes.
Complex
Kq
(1013 M-1S-1)
Ksv
(105 M -1)
Kb
（105 M-1）
n
1
2
3
4
2.88
1.11
1.08
0.68
2.88±0.16
1.11±0.15
1.08±0.15
0.68±0.18
81.71
3.86
1.40
0.37
1.27
1.09
1.02
0.94
4 Apoptosis Assay
In order to clarify whether IrIII TPE complex could induce
apoptosis, A549 cells were treated with selected complexes 1 and
3 at the concentration of 0.5, 1.0, 2.0 and 3.0 × IC50. After staining
with Annexin V/Propidium Iodide, the data were obtained by flow
cytometry.[29] As shown in Figure 5 and Tables S2-S3,
complexes induce a dose-dependent increase in the percentage
5 Cell Cycle Analysis
The cell cycle was monitored by flow cytometry to determine
whether cell function decline was caused by the antiproliferative
activity of IrIII TPE complexes.[30] As shown in Figure 6 and
Tables S4-S5, A549 cells were exposed to complexes 1 and 3 at
the concentrations of 0.25, 0.5 and 1.0 × IC50 for 24 h. The
percentages of cells in the Sub-G1 phase increased from 61.3%
to 72.5% for complex 1 at the concentration of 1.0 × IC50. While
for complex 3, the Sub-G1 phase increased by only 1.6%. These
results confirm that IrIII TPE complexes could disturb the cell cycle
mainly in the Sub-G1 phase and eventually induce apoptosis,
especially for half-sandwiched IrIII complexes (1 and 2).
6 Induction of ROS
Reactive oxygen species (ROS) is a single electron
reduction product of a class of oxygen, and the accumulation in
vivo will lead to intrinsic apoptosis, which is used to explore the
function mechanism of anticancer agents.[4d] To further evaluate
the catalytic characteristic of IrIII TPE complexes, the levels of
ROS were determined by flow cytometry after exposure to
complexes 1-4 for 24 h at the concentrations of 0.25 × IC50 and
0.5 × IC50. As shown in Figure 7, compared with the positive
and negative, 82.01% and 89.08% of A549 cells were at high
ROS levels for complexes 1 and 2, respectively. This significant
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absorption peaks increased gradually at 278 nm, which
demonstrated that IrIII TPE complexes might interact with
Tryptophan, Tyrosine or Phenylalanine (three aromatic acid
residues in BSA) in the microenvironment.[25]
Binding properties between IrIII TPE complexes and BSA could
also be determined by fluorescence spectra (Figures 4b and S5),
and which were calibrated to correct the “inner filter” effect.[26] With
the increase of target complex, there is a significantly decrease at
343 nm (the flurescene emission peak of BSA) at 298 K. The
quenching rate constant (Kq) and Stern–Volmer quenching
constant (Ksv) were calculated using the classical Stern-Volmer
equation (Figure S6), and the binding constant (Kb) and binding
site number (n) of complexes were calculated by the Scatchard
equation (Figure S7).[27] As shown in Table 2, the Kq of all
complexes ranged from 0.68×1013 to 2.88×1013 M-1 s-1, which
were almost three orders of magnitude higher than that of a pure
dynamic quenching mechanism (2.0×1010 M-1 s-1).[28] This
conclusion indicate that static quenching mechanism plays a
decisive role in the process of IrIII TPE complexes acting on BSA.
In addition, the n and the Kb of complex 1 are the best among
these IrIII complexes, which indicate that the introduction of too
many TPE molecules will increase the steric hindrance between
the target complex and BSA. And also, this conclusion is
consistent with the result of cytotoxicity test that complex 1 show
the best antineoplastic activity than the other complexes.
10.1002/cbic.201900268
ChemBioChem
Figure 6. Cell cycle analysis of A549 cells after 24 h of exposure to complexes
1 (a) and 3 (b) by flow cytometry. The histogram of cell cycle distribution of
A549 cells for control and complexes 1 (c) and 3 (d). Data are quoted as mean
± SD of three replicates.
As the most popular luminous materials, cyclometalated IrIII
complexes have been widely used in the field of organic lightemitting diodes (OLEDs) not only because of the short excited
state lifetime, but also the high thermostability and easily
adjustable emission color.[31] Photophysical properties of
complexes 3 and 4 were determined by UV-vis and
photoluminescence (PL) spectrum. As shown in Figure 8,
complexes 3 and 4 show almost the same photophysical
properties, which have a strong intraligand absorption band (ππ*) at around 250-300 nm (ε >14000 M-1 cm-1) and a weaker
metal-to-ligand charge-transfer (MLCT) transition band at around
350-450 nm and exhibit a yellow intense emission located at ~572
nm. Electron cloud distribution of the frontier molecular orbitals
(Figure S8) shows that the HOMOs of 3 and 4 are mainly
localized on TPE molecules, and the LUMOs are mainly localized
on the Ir atom and dipyridine ligand. In addition, the distribution of
HOMO doesn't increase with the addition of TPE molecules for
complex 4. This conclusion indicates that TPE-appended ancillary
ligands have little effect on their electronic structures, and then,
which is in agreement with the result that complex 3 possess
almost the same UV-vis and PL spectrum with 4.
improvement of ROS level provides an effective basis for the
apoptosis of A549 cells, and further confirmed the antineoplastic
mechanism of oxidation for half-sandwich IrIII TPE complexes (1
and 2). However, for cyclometalated IrIII TPE complexes (3 and 4),
almost no A549 cells are in ROS level, which is consistent with
the result of NADH test, and also indicate that cyclometalated IrIII
TPE complexes possess different antineoplastic mechanism
compared with half-sandwiched IrIII TPE complexes.
Figure 8. UV-vis and PL of complexes 3 and 4 in methanol solution (1.0×10-5
M).
Figure 7. Effect of complexes 1 (a, c) and 3 (b, d) on intracellular ROS levels
in A549 lung cancer cells treated at the indicated concentrations for 24 h.
7 Evaluation of antineoplastic mechanism for cyclometalated
IrIII TPE complexes
Due to favorable luminescence property for cyclometalated
IrIII TPE complexes, laser confocal microscopy was used to
determine the subcellular localization in A549 cells. Lyso Tracker
Red DND-99 (LTRD) and Mito Tracker Deep Red (MTDR) were
utilized as lysosomes and mitochondria fluorescence probes,
respectively.[7b,32] As shown in Figure 9, complex 3 could
effectively accumulate in lysosomes with the Pearson’s colocalization coefficient (PCC) of 0.73 when incubated for 6 h.
However, the PCC of mitochondrion-targeted for complex 3 is
0.12. The results indicate that cyclometalated IrIII TPE complexes
could effectively target lysosomes in vivo. Additionally, target
complex did not cause abnormal cell death immediately, which
made it easy to track changes in lysosomal morphology timely.
Lysosomes are acidic intracellular organelles and play an
important role in many cellular processes, including posttranslational protein maturation, receptor degradation, apoptosis,
autophagy and the extracellular release of active enzymes. As the
organelles, lysosomes could break down proteins, nucleic acids,
polysaccharides and other biological macromolecules. When
damaged, they release hydrolases that digest the entire cell, and
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eventually promote apoptosis.[33] Acridine orange (AO), an
effective probe to determine the integrity of the acidic organelles
(which emitting red fluorescence in lysosomes and green
fluorescence in the cytosol and nuclei), was used as the probe to
study the lysosomal integrity of A549 cells. A549 cells were
incubated with complex 3 (10 μM) for 2 h and 6 h, and then stained
with AO (5 μM). As shown in Figure 10, there was a significant
decrease in red fluorescence when cells hatched in complex 3
(1.0 × IC50) for 2 h, and obvious lysosomal damage was found
after 6 h. The results confirmed that cyclometalated IrIII TPE
complexes could target lysosome, lead to lysosomal damage, and
eventually induce apoptosis.
In this paper, four organometallic iridiumIII complexes were
synthesized with simple synthetic procedures. The introduction of
TPE molecules could effectively adjust the lipid solubility of
complex drugs, and endowed IrIII complexes with potential
antineoplastic activity, the best of which (complex 1) was nearly
six times of cis-platin under the same conditions. Additionally,
these complexes show some selectivity for cancer cells versus
normal cells. Complexes could be transported through serum
albumin, disturbed the cell growth cycle and induced apoptosis.
Among these, half-sandwiched IrIII complexes (1 and 2) could
effectively catalyze the conversion of NADH to NAD+ and induce
the accumulation of ROS, which present the antitumor
mechanism of oxidation. Additionally, cycloiridium III complexes
could specifically accumulate in lysosomes, lead to the lysosomal
damage, and eventually induce apoptosis. Above all, TPEappended iridiumIII complexes could be a promising candidate for
further evaluation as antineoplastic drugs.
Experimental Section
Figure 9. Determination of intercellular localization for complex 3. A549 cells
were stained with LTRD and MTDR (200 nM) for 20 min and then incubated
with complexes 3 (the concentration is equivalent to IC50) for 6 h at 310 K. The
complexes were excited at 488 nm and the emission was collected at 493-630
nm. LTRD was excited at 594 nm and the emission was collected at 549-651
nm. MTDR was excited at 644 nm and the emission was collected at 660- 720
nm. Scale bar: 20 µm.
Fig. 10 (a) A549 cells were incubated with control (a) and complex 3 (10 μM)
for 2 h (b) and 6 h (c) and then stained with AO (5 μM). λex = 488 nm, λem =
510 ± 30 nm (green) and 625 ± 30 nm (red). Scale bar: 20 μm.
Conclusions
Materials
Iridium trichloride, 1,2,3,4,5-pentamethyl-cyclopentadiene(95%), 4,4'dibromo- 2,2'-bipyridine, 4-bromo-2,2'-bipyridine, tetratriphenylphosphine
palladium, anhydrous potassium carbonate, diphenylmethane, 4bromodiphenylketone, n-butyllithium, p-toluene sulphonic acid, triisopropyl
borate and phenylpyridine were purchased from Rhea biotechnology co.
LTD. For the biological experiments, DMEM medium, fetal bovine serum,
penicillin/streptomycin mixture and trypsin/EDTA were purchased from
Sangon Biotech. A549 lung cancer cells were obtained from Shanghai
Institute of Biochemistry and Cell Biology (SIBCB).
Syntheses and characterization
Synthesis of 4-(4-(1,2,2-triphenylvinyl)phenyl)-2,2'-bipyridine (L1):
Tetraphenylboric acid (0.56 g, 1.5 mmol), 4-bromo-2,2'-bipyridine
(0.24 g, 1.0 mmol), tetratriphenylphosphine palladium (0.12 g, 0.1 mmol),
anhydrous potassium carbonate (0.42 g, 3.0 mmol) were added into a 150
mL round-bottom flask under nitrogen. 60 mL toluene and 15 mL water
were deoxygenated and added, then the reaction mixture was refluxed for
30 h (monitored by thin-layer chromatography) and extracted with
dichloromethane. The organic layer was dried over anhydrous magnesium
sulfate. After solvent evaporation, the crude product was purified by silica
gel column chromatography (Petroleum ether: ethyl acetate =50:1 as
eluent) to give purified product (L1). Yield 0.27 g (55.4%). 1H NMR (500
MHz, CDCl3) δ 8.63 (t, J = 5.3 Hz, 2H), 8.58 (s, 1H), 8.41 (s, 1H), 7.78 (d,
J = 7.5 Hz, 1H), 7.47 (d, J = 8.2 Hz, 3H), 7.27 (s, 1H), 7.13 – 7.02 (m, 11H),
7.02 – 6.93 (m, 6H).
L2 was synthesized by tetraphenylboric acid and 4,4'-dibromo-2,2'bipyridine using the same method as L1. Yield 51.2%. 1H NMR (500 MHz,
CDCl3) δ 9.14 (s, 2H), 8.81 (d, J = 5.6 Hz, 2H), 7.80 (s, 2H), 7.74 (d, J =
8.1 Hz, 4H), 7.24 (s, 4H), 7.17 – 7.10 (m, 18H), 7.10 – 7.00 (m, 12H).
Synthesis of target complexes 1-4
Dimer (1 equiv) and chelating ligands (2 equiv) were stirred at ambient
temperature overnight in methanol solution. Then hexafluorophosphate (8
equiv) were added to above solution and reacted for 4 h. Most of the
solvent is concentrated in vacuum and kept at -20 °C for 12 h, filtered and
washed with cold methanol and diethyl ether. The 1H NMR and MADLITOF MS of complexes 1-4 are shown in Figures S9 and S10. The
concrete data are as follows:
[(η5-C5Me5)Ir(L1)Cl]PF6 (1): Yield 65.8%. 1H NMR (500 MHz, DMSO): δ
9.06 – 8.97 (m, 3H, H-Py), 8.91 (d, J = 6.1 Hz, 1H, H-Py), 8.35 (t, J = 7.2
Hz, 1H, H-Py), 8.08 (dd, J = 6.1, 1.9 Hz, 1H, H-Py), 7.92 (d, J = 8.5 Hz,
1H, H-Py), 7.90 – 7.83 (m, 2H, H-TPE), 7.28 – 7.10 (m, 11H, H-TPE), 7.11
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– 6.97 (m, 6H, H-TPE), 1.66 (s, 15H, H-CH3). 13C NMR (126 MHz, DMSO)
δ 155.91, 155.42, 152.54, 152.36, 150.32, 146.65, 143.35, 143.33, 143.22,
142.39, 142.34, 140.68, 140.06, 132.83, 132.17, 131.18, 131.16, 131.08,
129.45, 128.50, 128.35, 127.59, 127.43, 127.30, 125.81, 125.74, 124.99,
121.32, 89.58 (C- Cpx), 8.60 (CH3-Cpx); ESI-MS (m/z): Calcd for
C43H44ClIrN3 830.513 [M-PF6]+; Found: 830.863. Calcd for C43H44IrN3
795.063 [M-PF6-Cl]2+; Found: 794.926.
[(η5-C5Me5)Ir(L2)Cl]PF6 (2): Yield 60.2%. 1H NMR (500 MHz, DMSO): δ
9.15 (s, 2H, H-Py), 9.05 – 8.76 (m, 4H, H-Py), 8.08 (s, 2H, H-TPE), 7.93
(d, J = 7.6 Hz, 4H, H-TPE), 7.39 – 6.89 (m, 32H, H-TPE), 1.67 (s, 15H, HCH3). 13C NMR (126 MHz, DMSO) δ 155.53, 151.83, 150.89, 147.00,
143.24, 143.19, 143.15, 143.12, 143.07, 142.61, 142.52, 139.65, 132.90,
132.54, 131.32, 131.27, 127.98, 127.91, 127.71, 126.95, 126.91, 126.80,
126.04, 89.46 (C-Cpx), 8.55(CH3-Cpx); ESI-MS (m/z): Calcd for
C48H46ClIrN3 892.584 [M-PF6]+; Found: 892.884. Calcd for C48H46IrN3
857.134 [M-PF6-Cl] 2+; Found: 856.909.
[Ir(Phpy)2(L1)]PF6 (3): Yield 63.2%. 1H NMR (500 MHz, DMSO) δ 9.15 (d,
J = 8.2 Hz, 1H, H-Ar), 9.08 (s, 1H, H-Ar), 8.32 – 8.25 (m, 3H, H-Ar), 7.98
(dd, J = 5.9, 1.6 Hz, 1H, H-Ar), 7.96 – 7.85 (m, 7H, H-Ar), 7.80 (d, J = 5.9
Hz, 1H, H-Ar), 7.69 (dd, J = 11.7, 6.0 Hz, 2H, H-Ar), 7.63 (d, J = 5.4 Hz,
1H, H-Ar), 7.15 (ddd, J = 11.6, 10.5, 6.1 Hz, 12H, H-Ar/TPE), 7.06 – 6.98
(m, 8H, H-TPE), 6.91 (t, J = 7.4 Hz, 2H, H-TPE), 6.20 (dd, J = 7.1, 4.2 Hz,
3H, H-TPE). 13C NMR (126 MHz, DMSO) δ 167.86, 167.61, 156.24, 155.59,
151.10, 150.59, 150.25, 150.23, 150.12, 148.88, 148.80, 148.59, 146.85,
143.59, 143.47, 143.37, 143.32, 143.24, 143.09, 142.42, 139.96, 139.82,
138.12, 133.04, 132.58, 131.82, 131.74, 131.33, 131.28, 130.87, 130.74,
128.21, 127.97, 127.91, 127.87, 127.71, 126.85, 126.75, 126.71, 125.47,
125.05, 124.81, 124.72, 123.59, 123.32, 122.65, 122.61, 122.11, 119.64,
119.51; ESI-MS (m/z): Calcd for C54H50ClIrN3 968.682 [M-PF6]+; Found:
967.818.
[Ir(Phpy)2(L2)]PF6 (4): Yield 66.5%. 1H NMR (500 MHz, CDCl3): δ 8.59 (s,
2H, H-Ar), 7.89 (d, J = 6.9 Hz, 4H, H-Ar), 7.77 – 7.65 (m, 6H, H-Ar), 7.60
– 7.45 (m, 6H, H-Ar), 7.20 (s, 4H, H-Ar), 7.16 – 6.97 (m, 34H, H-TPE), 6.92
(t, J = 7.2 Hz, 2H, H-TPE), 6.32 (d, J = 7.4 Hz, 2H, H-TPE). 13C NMR (126
MHz, DMSO) δ 167.57, 156.08, 151.08, 150.60, 150.21, 149.00, 146.57,
143.61, 143.35, 143.29, 143.18, 142.26, 139.87, 138.12, 133.54, 132.49,
131.82, 131.38, 131.34, 131.30, 130.74, 127.93, 127.87, 127.71, 126.88,
126.83, 126.74, 126.71, 125.50, 124.68, 123.64, 122.55, 122.30, 119.48;
ESI-MS (m/z): Calcd for C48H46ClIrN3O2 1317.445 [M-PF6]+; Found: 1317.4.
Acknowledgements
[1]
[2]
[3]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
We thank the University Research Development Program of
Shandong Province (J18KA082) the Student's Platform for
Innovation and Entrepreneurship Training Program (2018A043),,
the National Natural Science Foundation of China (21671118 and
21703118), the Taishan Scholars Program, Shandong Provincial
Natural Science Foundation (ZR2017MB038) and High
Performance Computing Center of Qufu Normal University for
support.
Keywords:
Organometallic
•
Tetraphenylethylene • Anticancer
[4]
Iridium
complex
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
Tetraphenylethylene appended
organometallic IrIII complexes show
potential
antineoplastic
activity
towards A549 cells. Half-sandwich IrIII
complexes could lead to the
accumulation of reactive oxygen
species (ROS) and induce apoptosis.
Additionally, cycloiridiumIII complexes
could target lysosome, lead to
lysosomal damage, and eventually
induce apoptosis.
Xicheng Liu*, Xiangdong He, Xiaojing
Zhang, Yongling Wang, Jiaying Liu,
Xiujuan Hao, Yue Zhang, Xiang-Ai
Yuan, Laijin Tian, Zhe Liu*
Page No. – Page No.
Synthesis and Antineoplastic
Applications of TPE Appended
Organometallic Iridium(III)
Complexes
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