Activation of C-H bonds in nitrones leads to iridium hydrides with antitumor activity

Article abstract of DOI:10.1021/jm4004973

We report the design and synthesis of a series of new cyclometalated iridium hydrides derived from the C-H bond activation of aromatic nitrones and the biological evaluation of these iridium hydrides as antitumor agents. The nitrone ligands are based on the structure of a popular antioxidant, α-phenyl-N-tert-butylnitrone (PBN). Compared to cisplatin, the iridium hydrides exhibit excellent antitumor activity on HepG2 cells. The metal-coordinated compound with the most potent anticancer activity, 2f, was selected for further analysis because of its ability to induce apoptosis and interact with DNA. During in vitro studies and in vivo efficacy analysis with tumor xenograft models in Institute of Cancer Research (ICR) mice, complex 2f exhibited antitumor activity that was markedly superior to that of cisplatin. Our results suggest, for the first time, that metal hydrides could be a new type of metal-based antitumor agent.

Full text of DOI:10.1021/jm4004973

Brief Article
pubs.acs.org/jmc
Activation of C−H Bonds in Nitrones Leads to Iridium Hydrides with
Antitumor Activity
Xiaoda Song,†,∥ Yong Qian,†,∥ Rong Ben,† Xiang Lu,† Hai-Liang Zhu,*,† Hui Chao,*,§ and Jing Zhao*,†,‡
†Institute of Chemistry and BioMedical Sciences, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences,
Nanjing University, Nanjing 210093, China
‡Shenzhen Key Lab of Nano-Micro Material Research, School of Chemical Biology and Biotechnology, Shenzhen Graduate School of
Peking University, Shenzhen 518055, China
§School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China
S
* Supporting Information
ABSTRACT: We report the design and synthesis of a series of new cyclometalated iridium hydrides derived from the C−H
bond activation of aromatic nitrones and the biological evaluation of these iridium hydrides as antitumor agents. The nitrone
ligands are based on the structure of a popular antioxidant, α-phenyl-N-tert-butylnitrone (PBN). Compared to cisplatin, the
iridium hydrides exhibit excellent antitumor activity on HepG2 cells. The metal-coordinated compound with the most potent
anticancer activity, 2f, was selected for further analysis because of its ability to induce apoptosis and interact with DNA. During in
vitro studies and in vivo efficacy analysis with tumor xenograft models in Institute of Cancer Research (ICR) mice, complex 2f
exhibited antitumor activity that was markedly superior to that of cisplatin. Our results suggest, for the first time, that metal
hydrides could be a new type of metal-based antitumor agent.
in inorganic chemistry, and we will use this term here).5 More
INTRODUCTION
Metal-based chemotherapy has become an important treatment
■
recently, iridium complexes have shown potential as anticancer
agents.6 Meggers and co-workers demonstrated an octahedrally
option for suppressing tumor growth and inhibiting metastasis
progression.1 The design of metallodrugs is distinct from the
coordinated iridium(III) complex as a nanomolar-level selective
inhibitor of the receptor protein kinase VEGFR3 (Flt4).7 An
design of traditional organic pharmacophores in that a different,
sometimes unique, chemical space is accessible, with multiple
iridium(III) complex with one of the strongest activity at
inhibiting tumor necrosis factor-α (TNF-α) has been reported
coordination numbers, coordination geometries, and a large
by Ma et al.8 In addition to these two rationally designed
iridium(III) complexes, other iridium-containing complexes
with anticancer activity were designed to contain bulky ligands,
causing significant distortions to the DNA structure.9a,b Several
complexes containing iridium−halide bonds have been
reported,2,6c and these covalently bind to guanine and adenine
bases through N7 sites, similarly to platinum-based complex-
es.9c−e
selection of ligands.2 Considerable efforts have been focused on
platinum compounds since the discovery of cisplatin, which is
still one of the best-selling anticancer drugs after its approval by
Food and Drug Administration in 1978 (Scheme 1).3 However,
Scheme 1. Classes of Metal-Based Drugs
In addition to the traditional coordination complexes and
organometallic complexes featuring metal−carbon bonds, we
report in this manuscript the design, synthesis, and biological
evaluation of a series of new iridium(III) complexes with a
metal−hydrogen bond. The aromatic C−H bonds in the
bidentatenitrone ligands were activated to yield the iridium
hydrides. One of the products was fully characterized using
NMR spectroscopic methods and X-ray analysis. The in vitro
studies revealed that the complexes have strong antiproliferative
effects on HepG2 cells, a human hepatocellular liver carcinoma
cell line. Complex 2f, which had the highest activity in cell
culture, exhibited greater activity than cisplatin in our in vivo
tumor model system. Mechanistic experiments indicated that
complex 2f has a strong interaction with DNA and induces
cancer cells to generate reactive oxygen species (ROS). These
severe side effects and drug resistance limit its applications.3
Thus, drug design strategies, such as varying the metal center
and ligands, have been employed to improve the properties of
metal-based anticancer agents.4 For example, the ruthenium
complexes KP1019 and NAMI-A are currently in clinical trials
(ligand-coordinated compounds are referred to as “complexes”
Received: April 6, 2013
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
Brief Article
results suggest a new strategy to expand our capacity for
developing new anticancer drugs.
RESULTS AND DISCUSSION
■
Organometallic Hydride Design and Synthesis. We
selected bidentate arylnitrone ligands based on the structure of
α-phenyl-N-tert-butylnitrone (PBN), which has been shown to
be an effective radical scavenger and a nitric oxide donor, as
well as to decrease oxidative stress.10 PBN derivatives have also
been shown to exhibit anticancer activity.11 The synthesis of the
nitrone ligands and their complexes is outlined in Scheme 2.
Scheme 2. Synthesis of the Octahedral Iridium Complexes
Used in This Study
Figure 1. ORTEP representation (50% thermal ellipsoids) of the
crystal structure of complex 2a acetonitrile analogue.
Acetone ligand in complexes 2 is easily replaced by other
strong-binding ligands such as acetonitrile and carbon
monoxide (CO). When the acetone ligand is replaced with a
carbon monoxide (CO) ligand, the CO ligand will be more
likely to stay on iridium. As a comparison, iridium carbonyl
complexes 3a−k were prepared by bubbling CO (1 atm, 10
min) through a CH2Cl2 solution of complexes 2a−k, resulting
1
in the substitution of the acetone ligand (Scheme 2). The H,
13C, and 31P NMR spectroscopic analyses were performed to
confirm the formation of these complexes.
In Vitro Biological Evaluation. With the nitrone ligands
(1a−k), the iridium acetone complexes (2a−k), and the
iridium CO complexes (3a−k) in hand, their antiproliferative
activity was evaluated by using HepG2 cells. The IC50 values
(the drug concentration at which 50% of the cells were viable
relative to the control) were calculated from the dose−survival
curves, which were obtained by using the 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after
48 h of drug treatment. The results are summarized in Table 1.
The nitrones (1a−k) are proposed to provide astronger
donating oxygen than alcohols. A series of nitrones were
prepared through one-pot reactions of aldehydes and N-tert-
butyl hydroxylamine hydrochloride in CH2Cl2 with excellent
yields. [IrH2(Me2CO)2(PPh3)2]+ has been shown to be
effective in many catalytic reactions, including C−H
activation,12 and appears to be a reasonable choice as a starting
material. The [IrH2(Me2CO)2(PPh3)2]SbF6 and the corre-
sponding nitrone ligand were refluxed in acetone in the
presence of 3,3-dimethylbut-1-ene as a hydrogen acceptor.
After simple purification, the expected iridium complexes (2a−
k) as the only products with good yields (Scheme 2). These
products are formed via C−H bond activation and are stable
toward air and moisture in both their solid and solution forms.
The complex 2f was generally stable in human microsome
metabolic tests (Supporting Information (SI), pp S10−S11).
The new iridium complexes (2a−k) were fully characterized
Table 1. IC50 Values of the Iridium Complexes 2a−k and
3a−k, Nitrone Ligands, and Cisplatin (IC50 SD(μM)) on
the HepG2 Cell Line
nitrone ligand
(1)
iridium acetone complex
(2)
iridiumCO complex
(3)
a
b
c
d
e
f
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
4.76 0.4
1.85 0.22
1.92 0.18
1.46 0.12
1.76 0.14
0.82 0.08
1.46 0.15
1.75 0.16
2.27 0.24
5.03 0.48
4.98 0.52
>20
1.26 0.12
1.19 0.15
1.72 0.2
1.15 0.08
3.07 0.41
1.63 0.17
2.78 0.31
3.15 0.36
1.12 0.15
3.79 0.37
3.58 0.32
using H, 13C, and 31P NMR spectroscopy. In the H NMR
spectra (acetone-d6), complexes 2a−k presented a high-field
triplet resonance at δ −20 to −30, indicative of the hydrides
due to their coupling to two phosphines. A single crystal of
complex 2a acetonitrile analogue suitable for structure
determination by X-ray crystallography was obtained via the
diffusion method (Figure 1). A crystallographic study of 2a
acetonitrile analogue revealed that the iridium complex has an
octahedral geometry. The oxygen of the nitrone moieties was
coordinated trans to the hydride and acted as a directing group
to assist the activation of the ortho C−H bond via
cyclometalation. The two phosphines are in a trans arrange-
ment with a P(2)−Ir−P(1)angle of 176.46(6)°. The Ir−
C(1)bond distance is 2.007(6) Å, and the Ir−O(1) bond
distance is 2.169(5) Å.
1
1
g
h
i
j
k
[IrH2(Me2CO)2(PP-
h3)2]SbF6
cisplatin13
21.55 1.45
Complex 2f exhibited the strongest inhibitory activity, with an
IC50 value that is approximately 26-fold lower than that of
cisplatin. The nitrone ligands alone showed no significant
antiproliferative activity (IC50 values all greater than 20 μM),
indicating that the iridium−ligand complexation is essential for
inhibiting HepG2 cell growth. Complex 2f was further
B
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Journal of Medicinal Chemistry
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investigated on NCI-60 cell lines by National Cancer Institute
Developmental Therapeutics Program, and the in vitro testing
results were included in the SI (Figure S3).
negative CD band at 245 nm was observed, suggesting that a
strong interaction occurs between the DNA and complex 2f
and that DNA undergoes conformational changes due to this
interaction (SI Figure S4).
To investigate the cell-killing mechanism, HepG2 cells
treated with a range of concentrations of 2f were stained
with DAPI, a blue nucleolar DNA staining dye. The treated
cells exhibited apoptotic features, including chromatin
aggregation and nucleolus fragmentation in a dose-dependent
manner (SI Figure S1). To further confirm that complex 2f
induces HepG2 cells to undergo apoptosis, HepG2 cells treated
with 2f were tested using an Annexin V-FITC/propidium
iodide (PI) assay. This assay is based on the translocation of
phosphatidylserine from the inner leaflet of the plasma
membrane to the cell surface in cells during early apoptosis.
The cells in early apoptosis are in the lower right quadrant
(annexin V+PI−), and the cells in late apoptosis are in the upper
right quadrant (annexin V+PI+) (SI Figure S2). The culture of
HepG2 cells growing in medium with the DMSO treatment
contained a low number of apoptotic cells (SI Figure S2A). A
significant induction of apoptosis was observed upon treatment
with complex 2f at 0.5, 1, and 1.5 μM for 48 h. The proportions
of cells in late apoptosis and early apoptosis were 5.90% and
14.25%, 6.52% and 18.36%, and 7.21% and 25.17%, respectively
(SI Figure S2B−D).
Mechanistic Experiments. A cell cycle analysis was
performed to elucidate the mechanism of growth inhibitory
activity of complex 2f (SI Figure S8). HepG2 cells were treated
with complex 2f at a concentration of 5 μM for 48 h using the
same concentration of cisplatin as the control. The cells were
subsequently fixed and stained with PI, and the DNA content
was analyzed by flow cytometry. Relative to the DMSO-treated
control, complex 2f caused an increase in the number of cells
undergoing apoptosis (SubG1-peak), with a concomitant
decrease in the number of cells in the G1, S, and G2/M
phases. Furthermore, complex 2f was more effective than
cisplatin at inducing cell apoptosis (SI Figure S8). This cell
cycle analysis clearly indicates that complex 2f causes apoptosis
to reduce the cell numbers.
1
Taking advantage of their unique shifts in H NMR and 31P
NMR spectra, the Ir−H and Ir−P resonances could provide
insights for the interaction between complex 2f and DNA. We
set out to mix complex 2f with a segment of oligoDNA and five
nucleosides (uridine, thymidine, guanosine, cytidine, and
adenosine). First, we selected a synthetic oligonucleotide
containing a 25-mer (5′-ATGATTTAGGTGACACTATAG-
CAGT-3′), a dinucleotide (dGpG) that has been used for the
cisplatin−DNA binding test.15 After incubation of complex 2f
and the complementary double-stranded oligoDNA at 310 K
for 6 h in 86% DMSO-d6−14%D2O, the resonance of the high-
field triplet Ir−H was found to shift from −22.6 ppm to
−23.94, −24.01, and −24.34 ppm. This pattern was similar to
the combination of complex 2f with adenosine (−24.07,
−24.30 ppm) and cytidine (−24.04 ppm) (SI Figure S7a). In
contrast, uridine and thymidine did not appear to interact with
1
complex 2f based on the H NMR spectroscopy measurement
(SI Figure S7a).
In addition, because there are significant amounts of iridium
accumulated in the membrane (∼17%), the cell membrane
could be a potential target for iridium complex 2f. To
investigate this possibility, we utilized the membrane-permeable
JC-1 dye to monitor the effect of complex 2f on the
mitochondrial membrane. The JC-1 dye exhibits potential-
dependent accumulation in mitochondria, indicated by a
fluorescence emission shift from green (∼529 nm) to red
(∼590 nm). We did not observe a significant fluorescence
change after treatment with complex 2f (SI Figure S5),
suggesting that complex 2f did not induce a decrease in the
mitochondrial membrane potential.
Inspired by the recent findings that the antitumor activity of
organometallic complexes may involve the redox signaling
pathways in cancer cells,16 the fluorescent probe 7-dichlor-
odihydrofluoresceindiacetate (DCFH-DA) was used to explore
the possible generation of cellular ROS with the introduction of
an iridium complex. The green fluorescence was detected over
a period of 4 h after treating the cells with complex 2f (5 μM)
(SI Figure S6). The ROS generation capacity of complex 2f was
found to be stronger than that of 12 μM H2O2.
The cell accumulation, cell fraction distribution, and DNA
binding of the iridium complex 2f in HepG2 cells were
determined. The iridium contents in the whole cell, nucleus,
cytosol, membrane, cytoskeleton fractions, and genome DNA
were quantified (via ICP-MS, Table 2) after 24 h of exposure to
In Vivo Biological Evaluation. On the basis of these in
vitro studies, complex 2f was used to examine its effect on in
vivo xenograft models in ICR mice. The tumor xenograft
nodules were formed by subcutaneous injection of H22 cells.
The mice were then treated with complex 2f at a dose of 3 mg/
kg by intraperitoneal injection, and cisplatin was used as a
control for comparison with the antitumor efficiency in the
xenograft model. Figure 2a shows the change in tumor volume
by intraperitoneal administration in H22 cells via the xenograft
tumor in mice. Both cisplatin and complex 2f effectively
inhibited tumor growth. The tumor volumes in the control
group receiving saline increased rapidly from day 6, with their
mean tumor volumes reaching more than 4000 mm3 on day 24.
In comparison, groups receiving either cisplatin or complex 2f
showed retardation in tumor growth, with the mean tumor
volumes near 1000 mm3 on day 24. Importantly, complex 2f
showed at least the same level of efficacy in inhibiting tumor
growth compared to that of cisplatin. Statistical analysis
revealed that the cohort receiving the treatment with complex
Table 2. Iridium Accumulation and Cell Fraction
Distribution in HepG2 Cells (ng Ir/106 cell)
mean
SD
cell
31.9
0.5
1.9
0.06
3.0
0.4
1.6
0.2
genome DNA
membrane
cytoskeleton
cytosol
16.6
3.5
3.9
nucleus
1.2
complex 2f. The amount of iridium located within genome
DNA (1.8%) was similar to that of cisplatin (1%),14 suggesting
that DNA might be a potential target for these cytotoxic iridium
complexes.
CD spectroscopy was performed to investigate the
interaction of complex 2f with plasmid pMD18 DNA. After
incubation of DNA with complex 2f, a significant decrease in
the intensity of the positive CD band at 275 nm and the
C
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Journal of Medicinal Chemistry
Brief Article
EXPERIMENTAL SECTION
■
General Methods for Synthesis. Unless otherwise mentioned, all
manipulations were performed using standard Schlenk techniques; all
reagents were purchased from commercial suppliers without any
further purification, and all solvents were distilled before use unless
otherwise noted. Nuclear magnetic resonance (NMR) spectra were
acquired on Bruker DRX-500/300 operating at 125 and 75 MHz for
1H NMR and 13C NMR, respectively. All 1H NMR spectra are
reported in δ units, parts per million (ppm) downfield from
tetramethylsilane as the internal standard, and the coupling constants
are indicated in hertz (Hz). The following abbreviations are used for
spin multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, and br = broad. ESI-MS spectra were recorded on a Mariner
System 5304 mass spectrometer. TLC was performed on glass-backed
silica gel sheets (silica gel HG/T2354-92 GF254) and visualized under
UV light (254 nm). Column chromatography was performed using
silica gel (200−300 mesh), eluting with ethyl acetate and petroleum
ether. The purity of all compounds was >95% prior to biological
testing and was determined by ultrahigh pressure liquid chromatog-
raphy (Waters ACQUITY UHPLC).
General Method for the Synthesis of 1a−k. A mixture of
aldehydes (1.59 mmol), N-(tert-butyl) hydroxylamine hydrochloride
(200 mg, 1.59 mmol), Na2SO4 (678 mg, 4.77 mmol), and NaHCO3
(401 mg, 4.77 mmol) were added to CH2Cl2 (10 mL), and the
reaction mixture was stirred and refluxed for approximately 48 h. The
resulting mixture was filtered, and the solvent was then removed under
vacuum. The target products (1a−k) were purified by column
chromatography (silica gel, petroleum ether/ethyl acetate 3/1−1/1).
General Method for the Synthesis of Iridium Acetone Complexes
2a−k. Under an argon atmosphere, a 25 mL Schlenk tube was charged
with N-tert-butyl-α-phenylnitrone (0.31 mmol, 1.1 equiv), 3,3-
dimethylbut-1-ene (1.5 mL), IrH2(PPh3)2(C3H6O)2SbF6 (300 mg,
0.28 mmol), and dry acetone (6 mL). The mixture was stirred under
reflux for 12 h. The solvent was removed, and the residue was washed
with diethyl ether or pentane to obtain complexes 2a−k.
General Method for the Synthesis of Iridium CO Complexes 3a−
k. An iridium acetone complex (100 mg) was dissolved into CH2Cl2
(6 mL) in a Schlenk tube then bubbled into carbon monoxide gas for
approximately 10 min. The solvent was subsequently removed, and the
residue was washed with diethyl ether or pentane to obtain complexes
3a−k.
Figure 2. In vivo efficacy of complex 2f against H22 tumor cells in ICR
mice. (a) Tumor volume established in ICR mice during therapy
under different treatments. (b) Body weight change of ICR mice
receiving different treatments during therapy. Data are presented as
the mean body weight SD (n = 6).
2f had similar tumor volumes compared to the cisplatin-treated
group from day 9 to day 24.
To determine the adverse effects of these therapeutic
treatments, we analyzed the variations in body weights during
these experiments. The complex 2f-treated group showed
favorable results without any obvious loss of body weight,
whereas treatment with cisplatin led to severe weight loss in the
mice (Figure 2b). The in vivo efficacy results were consistent
and correlated well with the data obtained from in vitro cell
culture analyses, indicating that complex 2f exhibits an
anticancer activity that is better than that of cisplatin. When
healthy mice were treated with complex 2f, no noticeable
weight loss was observed (SI Figure S8).
ASSOCIATED CONTENT
■
S
* Supporting Information
Morphological analysis, annexin V/propidium iodide staining,
MTT assay, NCI-60 cell lines test, CD, ICP-MS, UHPLC
CONCLUSION
■
1
diagram crystal structure, and spectral data of MS, H NMR,
In conclusion, we report the synthesis of iridium hydrido
complexes (2a−k) and iridium hydrido CO complexes (3a−k)
with aromatic nitrones (PBN) as ligands (1a−k). Biological
evaluation of the antitumor activity of these ligands (1a−k) and
their iridium complexes (2a−k, 3a−k) indicated that these
iridium hydrides exhibited excellent antitumor activity on
tumor cells. The complex with the most potent antitumor
activity, 2f, was selected for further screening using NCI-60 cell
lines and exhibited promising activity. Further analysis
confirmed its ability to induce apoptosis. Preliminary
mechanistic studies revealed a strong interaction between the
iridium complex 2f and DNA. Finally, complex 2f was shown to
be effective in vivo using tumor xenograft models in ICR mice,
exhibiting less toxicity than that of cisplatin. Previous research
on metal hydrides has mainly focused on their catalytic roles,
especially in the area of C−H bond activation. Numerous metal
hydrides have been obtained from intensive research on C−H
bond activation. We expect that our findings could open doors
to further investigation of the biological activities of metal
hydrides.
13C NMR, and 31P NMR. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +86 0755 2603 3151. Fax: +86 0755 2603 3174. E-
mail: jingzhao@nju.edu.cn.
Author Contributions
∥X.S. and Y.Q. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work is financially supported by grants of the National
Basic Research Program of China (2010CB923303 to J.Z.) and
the Jiangsu Natural Science Foundation (BK2011547 to J.Z.).
J.Z. thanks the Shenzhen Government (SW201110018,
JC201104210113A, KQC201105310016A) for support. We
D
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Journal of Medicinal Chemistry
Brief Article
(7) Wilbuer, A.; Vlecken, D. H.; Schmitz, D. J.; Kraling, K.; Harms,
K.; Bagowski, C. P.; Meggers, E. Iridium complex with antiangiogenic
properties. Angew. Chem., Int. Ed. Engl. 2010, 49, 3839−3842.
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thank the National Cancer Institute Developmental Therapeu-
tics Program for the in vitro testing results of complex 2f.
(8) Leung, C. H.; Zhong, H. J.; Yang, H.; Cheng, Z.; Chan, D. S.; Ma,
V. P.; Wong, C. Y.; Ma, D. L. A metal-based inhibitor of tumor
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binding properties of half-sandwich organometallic intercalators:
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ABBREVIATIONS USED
■
PBN, α-phenyl-N-tert-butylnitrone; ICR mice, Institute of
Cancer Research mice; VEGFR, vascular endothelial growth
factors receptor; (TNF-α), tumor necrosis factor-α; HepG2,
hepatocellular carcinoma cell line; ROS, reactive oxygen
species; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide; DAPI, 4′,6-diamidino-2-phenylindole; PI,
propidium iodide; FITC, fluorescein isothiocyanate; JC-1,
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine io-
dide; DCFH-DA, dichlorodihydrofluorescein diacetate
(b) Schmitt, F.; Govindaswamy, P.; Suss-Fink, G.; Ang, W. H.;
̈
Dyson, P. J.; Jeanneret, L. J.; Therrien, B. Ruthenium porphyrin
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1842−1844. (e) Wang, D.; Lippard, S. J. Cellular processing of
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