We report the synthesis and characterization of eight half-sandwich cyclopentadienyl IrIII pyridine complexes of the type [(η5-Cpxph)Ir(phpy)Z]PF6, in which Cpxph = C5Me4C6H5 (tetramethyl(phenyl)cyclopentadienyl), phpy = 2-phenylpyridine as C∧N-chelating ligand, and Z = pyridine (py) or a pyridine derivative. Three X-ray crystal structures have been determined. The monodentate py ligands blocked hydrolysis; however, antiproliferative studies showed that all the Ir compounds are highly active toward A2780, A549, and MCF-7 human cancer cells. In general the introduction of an electron-donating group (e.g., Me, NMe2) at specific positions on the pyridine ring resulted in increased antiproliferative activity, whereas electron-withdrawing groups (e.g., COMe, COOMe, CONEt2) decreased anticancer activity. Complex 5 displayed the highest anticancer activity, exhibiting submicromolar potency toward a range of cancer cell lines in the National Cancer Institute NCI-60 screen, ca. 5 times more potent than the clinical platinum(II) drug cisplatin. DNA binding appears not to be the major mechanism of action. Although complexes [(η5-Cpxph)Ir(phpy)(py)]+ (1) and [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]+ (5) did not cause cell apoptosis or cell cycle arrest after 24 h drug exposure in A2780 human ovarian cancer cells at IC50 concentrations, they increased the level of reactive oxygen species (ROS) dramatically and led to a loss of mitochondrial membrane potential (ΔΨm), which appears to contribute to the anticancer activity. This class of organometallic Ir complexes has unusual features worthy of further exploration in the design of novel anticancer drugs.
We report the synthesis and characterization of eight half-sandwich cyclopentadienyl IrIII pyridine complexes of the type [(η5-Cpxph)Ir(phpy)Z]PF6, in which Cpxph = C5Me4C6H5 (tetramethyl(phenyl)cyclopentadienyl), phpy = 2-phenylpyridine as C∧N-chelating ligand, and Z = pyridine (py) or a pyridine derivative. Three X-ray crystal structures have been determined. The monodentate py ligands blocked hydrolysis; however, antiproliferative studies showed that all the Ir compounds are highly active toward A2780, A549, and MCF-7 human cancer cells. In general the introduction of an electron-donating group (e.g., Me, NMe2) at specific positions on the pyridine ring resulted in increased antiproliferative activity, whereas electron-withdrawing groups (e.g., COMe, COOMe, CONEt2) decreased anticancer activity. Complex 5 displayed the highest anticancer activity, exhibiting submicromolar potency toward a range of cancer cell lines in the National Cancer Institute NCI-60 screen, ca. 5 times more potent than the clinical platinum(II) drug cisplatin. DNA binding appears not to be the major mechanism of action. Although complexes [(η5-Cpxph)Ir(phpy)(py)]+ (1) and [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]+ (5) did not cause cell apoptosis or cell cycle arrest after 24 h drug exposure in A2780 human ovarian cancer cells at IC50 concentrations, they increased the level of reactive oxygen species (ROS) dramatically and led to a loss of mitochondrial membrane potential (ΔΨm), which appears to contribute to the anticancer activity. This class of organometallic Ir complexes has unusual features worthy of further exploration in the design of novel anticancer drugs.
The clinical use of
platinum anticancer drugs has stimulated the
search for other transition metal anticancer complexes with improved
features.[1] In particular other platinum
complexes[2] and some group 8 metal complexes
containing iron[3] and ruthenium[4] centers show promising anticancer activity both in vitro and in vivo.Very recently,
possible biological applications of iridium compounds
have attracted attention.[5] Half-sandwich
organometallic IrIII compounds in particular display high
versatility and show promising anticancer activity.[6] For example, Sheldrick et al. have designed monoiridium
and di-iridium polypyridyl intercalators that target DNA in cancer
cells.[7] We have studied a series of half-sandwich
IrIII anticancer agents of formula [(Cpx)Ir(L∧L′)Z]0/, where
Cpx = Cp*, Cpxph (tetramethyl(phenyl)cyclopentadienyl),
or Cpxbiph (tetramethyl(biphenyl)cyclopentadienyl), L∧L′ = bidentate ligand with nitrogen, oxygen,
and/or carbon donor atoms, and Z = Cl, H2O, or pyridine
(py).[5a,6a] We found that potent activity can be achieved
by modification of ligands around the iridium center and that small
changes in structure can have a major effect on biological activity.
For example, antiproliferative activity as measured by IC50 values (concentration at which 50% of cell growth is inhibited)
decreased dramatically from inactive (>100 μM) to highly
potent
(submicromolar) when phenyl or biphenyl was introduced in place of
a methyl group on the Cp* ring. We also reported that anticancer activity
can be improved significantly by replacement of neutral N∧N-chelating ligands with negatively charged C∧N-chelating
ligands, leading to increased cellular uptake and nucleobase binding.[6c] The monodentate ligand Z (which in most of these
IrIII half-sandwich compounds is Cl) is often readily substituted
by water in aqueous solution (hydrolysis), followed by interaction
with biological molecules. A relationship between hydrolysis and anticancer
activity has been established for RuII arene compounds,
where readily hydrolyzed compounds are cytotoxic and those that do
not hydrolyze are inactive or weakly active toward cancer cells.[8] For cyclopentadienyl Ir C∧N
compounds, we found that decreasing hydrolysis by substitution of
Cl by pyridine (py) does not result in loss of anticancer activity.
In fact, the py complex is highly potent, ca. 10 times and 6 times
more active than the clinically used platinum drug cisplatin (CDDP)
and the chloride analogue, respectively.[6a] These results encouraged us to explore in more detail the activity
of complexes containing py derivatives.In this study, the complexes
contain Cpxph and C∧N-bound 2-phenylpyridine
(phpy) as the cyclopentadienyl
and chelating ligands, respectively, and various pyridine derivatives
as the monodentate ligand Z. Thus, eight half-sandwich IrIII compounds of the type [(η5-Cpxph)Ir(phpy)Z]PF6, where Z = pyridine or its derivatives, were synthesized
and characterized. Their chemical behavior and antiproliferative activity
toward cancer cells have been investigated.
Experimental
Section
Materials
2-Phenylpyridine, 4-pyridinemethanol, 4-dimethylaminopyridine,
methylnicotinate, N,N-diethylnicotinamide, 3-picoline,
4-picoline, 3-acetylpyridine, 9-ethylguanine, and 9-methyladenine
were purchased from Sigma-Aldrich. For the biological experiments,
RPMI-1640 medium, fetal bovine serum, l-glutamine, penicillin/streptomycin
mixture, trypsin/EDTA, and phosphate-buffered saline (PBS) were purchased
from PAA Laboratories GmbH. Cisplatin CDDP (≥99.9%), trichloroacetic
acid (≥99%), sulforhodamine B (75%), sodium phosphate monobasic
monohydrate (≥99%), sodium phosphate dibasic heptahydrate (≥99%),
acetic acid (≥99%), staurosporine, propidium iodide (>94%),
and RNase A were obtained from Sigma-Aldrich. Complex [(η5-Cpxph)Ir(phpy)Cl] was prepared according to reported
methods.[6d]
Syntheses
Compounds 1–8 were prepared by the same general
method: A solution of the chlorido
complex [(η5-Cpxph)Ir(phpy)Cl] and AgNO3 (1 mol equiv) in MeOH and water (1:1, v/v) was heated under
reflux in an N2 atmosphere for 3 h. The precipitate (AgCl)
was removed by filtration through Celite, and pyridine derivative
(10 molar equiv) was added to the filtrate. The reaction mixture was
stirred at ambient temperature for 12 h. NH4PF6 (10 mol equiv) was then added to the solution. The yellow precipitate
was dissolved in acetone. The solution was evaporated slowly at ambient
temperature, and the crystalline product was collected by filtration,
washed with diethyl ether, and recrystallized from methanol/diethyl
ether.
Suitable crystals
of compounds 2, 5, and 8 were
selected and mounted
on a glass fiber with Fromblin oil on an Oxford Diffraction Gemini
Xcalibur diffractometer with a Ruby CCD area detector. The crystals
were kept at 100(2) or 150(2) K during data collection. Using Olex2,[9] the structures of 2, 5, and 8 were solved with the XS[10] structure solution program using direct methods and refined with
the XL[10] refinement package using least
squares minimization.X-ray crystallographic data for compounds 2, 5, and 8 have been deposited
in the Cambridge Crystallographic Data Centre under the accession
numbers CCDC 1007223, 1007225, and 1007224, respectively.
NMR Spectroscopy
1H NMR
spectra were acquired
in 5 mm NMR tubes at 298 or 310 K on either a Bruker DPX 400 (1H = 400.03 MHz) or an AVA 600 (1H = 600.13 MHz)
spectrometer. 1H NMR chemical shifts were internally referenced
to CHD2OD (3.33 ppm) for methanol-d4 or CHCl3 (7.26
ppm) for chloroform-d1. MeOD-d4 was used to aid solubility. All data processing was
carried out using MestReC or TOPSPIN version 2.0 (Bruker U.K. Ltd.).
Mass Spectrometry
Electrospray ionization mass spectra
(ESI-MS) were obtained by preparing the samples in 50% CH3CN and 50% H2O (v/v) or using NMR samples for infusion
into the mass spectrometer (Bruker Esquire 2000). The mass spectra
were recorded with a scan range of m/z 400–1000 for positive ions.
Elemental Analysis
CHN elemental analyses were carried
out on a CE-440 elemental analyzer by Warwick Analytical (UK) Ltd.
pH Measurement
pH or pH* values (pH meter reading without
correction for effect of deuterium on glass electrode) of NMR samples
in H2O or D2O were measured at ca. 298 K directly
in the NMR tube, before and after recording NMR spectra, using a Corning
240 pH meter equipped with a micro combination electrode calibrated
with Aldrich buffer solutions of pH 4, 7, and 10.
Inductively
Coupled Plasma Mass Spectrometry (ICP-MS)
All ICP-MS analyses
were carried out on an Agilent Technologies 7500
series ICP-MS instrument. The water used for ICP-MS analysis was doubly
deionized (DDW) using a Millipore Milli-Q water purification system
and a USF Elga UHQ water deionizer. The iridium Specpure plasma standard
(Alfa Aesar, 1000 ppm in 10% HCl) was diluted with 5% HNO3 DDW to prepare freshly calibrants at concentrations of 50 000,
10 000, 5000, 1000, 500, 200, 50, 10, and 5 ppt. The
ICP-MS instrument was set to detect 193Ir with typical
detection limits of ca. 2 ppt using no gas mode.
Hydrolysis
Solutions of complexes 1–8 with
final concentrations of 150 μM in 10% MeOD-d4/90% D2O (v/v) were prepared by
dissolution of the complex in MeOD-d4 followed
by rapid dilution with D2O. 1H NMR spectra were
recorded after various time intervals at 310 K.
Interactions
with Nucleobases
The reaction of complexes 1–8 (1 mM) with nucleobases 9-EtG or 9-MeA
typically involved addition of 1 mol equiv of nucleobase to an equilibrium
solution of complexes 1–8 in 20%
MeOD-d4/80% D2O (v/v). 1H NMR spectra of these solutions were recorded at 310 K after
various time intervals.
NCI-60 Screening
Compounds 2 and 5 were evaluated by the National Cancer
Institute Developmental
Therapeutics Program (NCI/DTP, USA) for in vitro cytotoxicity
toward ca. 60 human cancer cell lines. The cells were treated with
iridium compounds for 48 h at five concentrations ranging from 0.01
to 100 μM. Every compound was tested twice, and data are the
average of the two experiments. Data for cisplatin and oxaliplatin
are from NCI/DTP screening performed in October 2009 and 2010, respectively.
The protocol for the determination of cytotoxicity toward the 60-cell-line
panel can be found at http://dtp.nci.nih.gov/branches/btb/ivclsp.html. The DTP homepage can be accessed at http://dtp.cancer.gov/.
Cell Culture
A2780 ovarian carcinoma, A549 lung and
MCF7 breast human adenocarcinoma cells were obtained from the European
Collection of Cell Cultures (ECACC) and were grown in Roswell Park
Memorial Institute medium (RPMI-1640) or Dubelco's Modified Eagle
Medium (DMEM). All media were supplemented with 10%(v/v) fetal calf
serum, 1%(v/v) 2 mM glutamine, and 1% (v/v, 10k units/mL) penicillin/streptomycin.
All cells were grown as adherent monolayers at 310 K in a 5% CO2 humidified atmosphere and passaged regularly at ca. 80% confluence.
In Vitro Growth Inhibition Assay
Briefly,
5000 cells were seeded per well in 96-well plates. The cells were
preincubated in drug-free media at 310 K for 48 h before adding different
concentrations of the compounds to be tested. In order to prepare
the stock solution of the drug, the solid complex was dissolved first
in 5% DMSO and then diluted in a 50:50 v/v mixture of RPMI-1640/saline.
This stock was further diluted using cell culture medium until working
concentrations were achieved. The drug exposure period was 24 h. After
this, supernatants were removed by suction, and each well was washed
with PBS. A further 72 h was allowed for the cells to recover in drug-free
medium at 310 K. The SRB assay[11] was used
to determine cell viability. Absorbance measurements of the solubilized
dye (on a BioRad iMark microplate reader using a 470 nm filter) allowed
the determination of viable treated cells compared to untreated controls.
IC50 values (concentration of drug resulting in a 50% cell
growth inhibition) were determined as duplicates of triplicates in
two independent sets of experiments, and their standard deviations
were calculated.
Metal Accumulation in Cancer Cells
Iridium accumulation
studies for complexes 1 and 5 were conducted
on A2780 ovarian cells. Briefly, 1.5 × 106 cells were
seeded on a six-well plate. After 24 h of preincubation time in drug-free
medium at 310 K, the complexes were added to give final concentrations
equal to IC50, and a further 24 h of drug exposure was
allowed. After this time, excess drugs were removed by suction, and
cells were washed with PBS and then treated with trypsin-EDTA. A suspension
of single cells was counted, and cell pellets were collected. Each
pellet was digested overnight in concentrated nitric acid (73%) at
353 K; the resulting solutions were diluted with double-distilled
water to a final concentration of 5% HNO3, and the amount
of Ir taken up by the cells was determined by ICP-MS. These experiments
did not include any cell recovery time in drug-free media; they were
carried out in triplicate, and the standard deviations were calculated.
Cell Cycle Analysis
A2780 cells at 1.5 × 106 per well were seeded in a six-well plate. Cells were preincubated
in drug-free media at 310 K for 24 h, after which drugs were added
at equipotent concentrations equal to IC50. After 24 h
of drug exposure, supernatants were removed by suction and cells were
washed with PBS. Finally, cells were harvested using trypsin-EDTA
and fixed for 24 h using cold 70% ethanol. DNA staining was achieved
by resuspending the cell pellets in PBS containing propidium iodide
(PI) and RNAse. Cell pellets were washed and resuspended in PBS before
being analyzed in a Becton Dickinson FACScan flow cytometer using
excitation of DNA-bound PI at 536 nm, with emission at 617 nm. Data
were processed using Flowjo software.
Induction of Apoptosis
Flow cytometry analysis of apoptotic
populations of A2780 cells caused by exposure to complexes 1 and 5 was carried out using the annexin V-FITC apoptosis
detection kit (Sigma-Aldrich) according to the supplier’s instructions.
Briefly, 1.5 × 106 A2780 cells per well were seeded
in a six-well plate. Cells were preincubated in drug-free media at
310 K for 24 h, after which drugs were added at equipotent concentrations
equal to IC50. After 24 h of drug exposure, supernatants
were removed by suction, and cells were washed with PBS. Finally,
cells were harvested using trypsin-EDTA. Sample staining was achieved
by resuspending the cell pellets in buffer containing annexin V-FITC
and PI. For positive-apoptosis controls, A2780 cells were exposed
to staurosporine (1 μg/mL) for 2 h. Cells for apoptosis studies
were used with no previous fixing procedure as to avoid nonspecific
binding of the annexin V-FITC conjugate.
ROS Determination
Flow cytometry analysis of ROS/superoxide
generation in A2780 cells caused by exposure to complexes 1 and 5 was carried out using the Total ROS/Superoxide
detection kit (Enzo-Life Sciences) according to the supplier’s
instructions. Briefly, 1.5 × 106 A2780 cells per well
were seeded in a six-well plate. Cells were preincubated in drug-free
media at 310 K for 24 h in a 5% CO2 humidified atmosphere,
and then drugs were added to triplicates at concentrations of IC50 and 2 × IC50. After 1 h of drug exposure,
supernatants were removed by suction and cells were washed and harvested.
Staining was achieved by resuspending the cell pellets in buffer containing
the orange/green fluorescent reagents. Cells were analyzed in a Becton
Dickinson FACScan flow cytometer using FL1 channel Ex/Em: 490/525
nm for the oxidative stress and FL2 channel Ex/Em: 550/620 nm for
superoxide detection. Data were processed using Flowjo software. At
all times, samples were kept under dark conditions to avoid light-induced
ROS production.
Mitochondrial Membrane Assay
Analysis
of the changes
of mitochondrial potential in A2780 cells after exposure to complexes 1 and 5 was carried out using the Abcam, JC-10
mitochondrial membrane potential assay kit according to the manufacturer’s
instructions. Briefly, 1.5 × 106 cells were seeded
in six-well plates left to incubate for 24 h in drug-free medium at
310 K in a humidified atmosphere. Drug solutions, at equipotent concentrations
equal to IC50 and 2 × IC50, were added
in triplicate, and the cells were left to incubate for a further 24
h under similar conditions. Supernatants were removed by suction,
and each well was washed with PBS before detaching the cells using
trypsin-EDTA. Staining of the samples was done in flow cytometry tubes
protected from light, incubating for 30 min at ambient temperature.
Samples were immediately analyzed on a Beckton Dickinson FACScan,
reading the reduction of fluorescence in the FL2 channel. For positive
controls, A2780 cells were exposed to carbonyl cyanide 3-chlorophenylhydrazone,
CCCP (5 μM), for 15 min. Data were processed using Flowjo software.
Results
Novel Ir compounds 1–8 were synthesized
in moderate yields from the chlorido analogue [(η5-Cpxph)Ir(phpy)Cl][6d] by substitution
of chloride by pyridine or its derivatives in the presence of silver
nitrate, Scheme 1. All the synthesized complexes
were isolated as PF6– salts and were
fully characterized by 1H NMR spectroscopy, CHN elemental
analysis, and ESI-MS. The complexes studied in this work are shown
in Scheme 1.
Scheme 1
Synthesis of Ir Compounds
Studied in This Work
X-ray Crystal Structures
The X-ray crystal structures
of [(η5-Cpxph)Ir(phpy)(4-Me-py)]PF6 (2), [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]PF6 (5), and [(η5-Cpxph)Ir(phpy)(3-CONEt2-py)]PF6 (8) were determined. The complexes adopt the expected
half-sandwich pseudo-octahedral “three-leg piano-stool”
geometry with the Ir bound to a η5-cyclopentadienyl
ligand occupying three coordination sites, the nitrogen atom of the
py derivative (2.099–2.118 Å), and a 2-phenylpyridine
C∧N-chelating ligand. Their structures are shown
in Figure 1. Crystallographic data are shown
in Table S1, and selected bond lengths
and angles are listed in Table 1.
Figure 1
X-ray crystal structures for (A) [(η5-Cpxph)Ir(phpy)(4-Me-py)]PF6 (2), (B) [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]PF6 (5), and (C) [(η5-Cpxph)Ir(phpy)(3-CONEt2-py)]PF6 (8),
with thermal ellipsoids drawn at 50% probability. The hydrogen atoms
and counterions have been omitted for clarity.
Table 1
Selected Bond Lengths (Å) and
Angles (deg) for [(η5-Cpxph)Ir(phpy)(4-Me-py)]PF6 (2), [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]PF6 (5), and [(η5-Cpxph)Ir(phpy)(3-CONEt2-py)]PF6 (8)
2
5
8
Ir–C
2.170(3)
2.1676(19)
2.163(3)
(cyclopentadienyl)
2.173(2)
2.1727(18)
2.168(3)
2.202(3)
2.1766(19)
2.168(3)
2.223(3)
2.2316(18)
2.245(3)
2.236(3)
2.2442(18)
2.263(3)
Ir–C(centroid)
1.827
1.825
1.832
Ir–C(phpy)
2.065(2)
2.0505(17)
2.053(3)
Ir–N*a
2.073(2)
2.0811(16)
2.093(3)
Ir–N#b
2.106(2)
2.0994(15)
2.118(2)
C–Ir–N*
78.34(9)
78.40(6)
78.15(12)
C–Ir–N#
85.94(8)
84.66(6)
86.67(10)
N*–Ir– N#
88.27(9)
87.01(6)
80.87(9)
N* is the
nitrogen atom in the 2-phenylpyridine
chelating ligand.
N# is the nitrogen atom
in the monodentate ligand.
The
crystal structures reported here are the second examples of crystal
structures containing the Cpxph ligand.[6b] The phenyl ring of Cpxph is twisted by about
45° relative to the cyclopentadienyl ring in 2 and 5 and 83° in 8. The Ir–cyclopentadienyl
(centroid) bond distances for compounds 2, 5, and 8 ranged from 1.825 to 1.832 Å, longer than
that of [(η5-Cpxph)Ir(bpy)Cl]PF6 (bpy = 2,2′-bipyridine)[6b] (1.789
Å), probably due to the negatively charged phpy ligand. The change
in monodentate ligands in 2, 5, and 8 does not give rise to much difference in bond lengths between
Ir and coordinated atoms; however, a smaller N–Ir–N
angle of 80.87(9)° for 8 is observed compared to
88.27(9)° and 87.01(6)° for 2 and 5, respectively. There is weak π–π intermolecular
ring stacking between the neighboring phenylpyridine rings in the
unit cell of compound 2, Figure S1. The two interacting π systems are parallel, with a centroid–centroid
distance of 4.291 Å.X-ray crystal structures for (A) [(η5-Cpxph)Ir(phpy)(4-Me-py)]PF6 (2), (B) [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]PF6 (5), and (C) [(η5-Cpxph)Ir(phpy)(3-CONEt2-py)]PF6 (8),
with thermal ellipsoids drawn at 50% probability. The hydrogen atoms
and counterions have been omitted for clarity.N* is the
nitrogen atom in the 2-phenylpyridine
chelating ligand.N# is the nitrogen atom
in the monodentate ligand.
Hydrolysis
The hydrolysis of complexes 1–8 (150 μM) in 10% MeOD-d4/90% D2O (v/v) was studied by 1H NMR spectroscopy at 310
K. The presence of methanol ensured sufficient
solubility of the complex. The 1H NMR spectra showed no
obvious change over 24 h, indicating that these Ir compounds remained
stable under these conditions.
Antiproliferative Activity
The activity of complexes 1–8 toward
A2780 human ovarian, A549 lung,
and MCF-7 breast cancer cells was investigated, Table S2 and Figure 2. The IC50 values (concentration at which 50% of the cell growth is inhibited)
for all IrIII complexes are comparable to or lower than
that of cisplatin, suggesting that all these compounds are highly
active. Complex 5, [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]+, containing 4-dimethylaminopyridine,
displayed the highest anticancer activity, with an IC50 value of 0.20 ± 0.04 μM toward MCF-7 cells, ca. 36 times
more potent than cisplatin. Complex 8, containing N,N-diethylnicotinamide, showed the lowest anticancer activity
toward all three cancer cell lines.
Figure 2
Inhibition of growth of (A) A2780 human
ovarian cancer; (B) A549
lung cancer; and (C) MCF-7 breast cancer cells by compounds 1–8 and comparison with cisplatin (CDDP).
Inhibition of growth of (A) A2780 human
ovarian cancer; (B) A549
lung cancer; and (C) MCF-7 breast cancer cells by compounds 1–8 and comparison with cisplatin (CDDP).With regard to the effects of
substitutions on the pyridine ring
on anticancer activity, overall, complexes containing electron-withdrawing
groups on the pyridine ring show less activity compared to those complexes
with an electron-donating group.NCI-60 GI50 mean graphs for
complexes 2 (right) and 5 (left). The midpoint
(log10 GI50) is −6.14 (2) and −6.46
(5). Bars to the right of the mean indicate lower activity
relative to the mean, and those to the left, higher activity.The antiproliferative activity
of compounds 2 and 5 was further evaluated
in the National Cancer Institute NCI-60
human cancer cell screen, consisting of nine tumor subtypes.[12] Three end points were determined: GI50 (the concentration that causes 50% cell growth inhibition), TGI
(the concentration that causes 100% cell growth inhibition), and LC50 (the concentration that decreases the original cell number
by 50%). The GI50 mean graph for 2 and 5 is shown in Figure 3. The midpoint
(log10 GI50) of 2 and 5 is −6.14 (GI50 = 724 nM) and −6.46 (GI50 = 347 nM), respectively. Bars extending to the left in the
mean graph represent higher activity than the mean of all tested cell
lines. Bars extending to the right correspondingly imply activity
less than the mean. Complex 5 shows high potency in a
wide range of cancer cell lines (Figure 3),
with particular selectivity toward MDA-MB-468 (breast), A498 (renal),
and COLO-205 (colon), with GI50 values of <170 nM. Notably,
complex 2 displayed potency toward the A498 (renal) cell
line with a GI50 of 19 nM. Complex 5 showed
good selectivity toward leukemia, CNS cancer, colon cancer, and breast
cancer. In comparison with cisplatin (CDDP), Ir complexes displayed
higher activity against NCI-60 cancer cell lines, especially Ir complex 5, which is 4–5 times more active than cisplatin, Figure 4.
Figure 3
NCI-60 GI50 mean graphs for
complexes 2 (right) and 5 (left). The midpoint
(log10 GI50) is −6.14 (2) and −6.46
(5). Bars to the right of the mean indicate lower activity
relative to the mean, and those to the left, higher activity.
Figure 4
GI50, TGI, and LC50 values (μM)
of 2, 5, and CDDP in the NCI-60 screen.
GI50, TGI, and LC50 values (μM)
of 2, 5, and CDDP in the NCI-60 screen.
Interactions with Nucleobases
Reactions of complexes 1–8 with
nucleobase derivatives 9-ethylguanine
(9-EtG) and 9-methyladenine (9-MeA) were investigated. Solutions of 1–8 (ca. 1 mM) and 1 molar equiv of 9-EtG
or 9-MeA in 20% MeOD-d4/80% D2O (v/v) were prepared, and 1H NMR spectra were recorded
at different time intervals at 310 K.No reaction with 9-MeA
was observed for all complexes, as addition of nucleobase model to
a solution of 1–8 resulted in no
additional 1H NMR peaks over 24 h. In contrast, all the
complexes reacted with 9-EtG. For example, in the 1H NMR
spectrum of a solution containing 8 and 1 molar equiv
of 9-EtG, one set of new peaks assignable to the 9-EtG adduct 8G appeared, showing that 32% of 8 had reacted
after 24 h (Figure 5). A significant change
in chemical shift of the CH=N (phpy ligand) proton of complex 8 from 8.88 to 9.29 ppm for 8G was observed.
A new 9-EtG H8 peak appeared at 7.43 ppm (singlet), shifted by 0.39
ppm to high field relative to that of free 9-EtG. The ESI-MS of an
equilibrium solution contained a major peak at m/z 723.2, confirming the formation of the 9-EtG adduct 8G, [(η5-C5Me4C6H5)Ir(phpy)(9-EtG)]+ (calcd m/z 722.9). The percentages of nucleobase
adducts formed by all the complexes after 24 h reaction, based on 1H NMR peak integrals, are shown in Table
S3 and Figure 6.
Figure 5
Low-field region of the 1H NMR spectra for reaction
of [(η5-Cpxph)Ir(phpy)(3-CONEt2-py)]PF6 (8) with 9-EtG: (A) 10 min after
addition of 1 mol equiv of 9-EtG to an equilibrium solution of complex 8 (1.0 mM) in 20% MeOD-d4/80%
D2O (v/v) at 310 K; (B) after 24 h reaction. Peak assignments:
(red squares) 8; (blue squares) guanine adduct 8G. After 24 h, 32% of 8 had reacted.
Figure 6
Bar chart showing the extent of binding of complexes 1–8 (ca. 1 mM in 20% MeOD-d4/80% D2O) to the nucleobase 9-EtG over 24
h at
310 K.
Low-field region of the 1H NMR spectra for reaction
of [(η5-Cpxph)Ir(phpy)(3-CONEt2-py)]PF6 (8) with 9-EtG: (A) 10 min after
addition of 1 mol equiv of 9-EtG to an equilibrium solution of complex 8 (1.0 mM) in 20% MeOD-d4/80%
D2O (v/v) at 310 K; (B) after 24 h reaction. Peak assignments:
(red squares) 8; (blue squares) guanine adduct 8G. After 24 h, 32% of 8 had reacted.Bar chart showing the extent of binding of complexes 1–8 (ca. 1 mM in 20% MeOD-d4/80% D2O) to the nucleobase 9-EtG over 24
h at
310 K.
Cellular Ir Accumulation
Complex 5, which
displayed the highest anticancer activity, and complex 1, containing a nonsubstituted py ligand, were selected for further
studies. First we investigated the cellular accumulation of Ir from
complexes 1 and 5 in A2780 ovarian cancer
cells. After 24 h of drug exposure at equipotent concentrations corresponding
to IC50 values, 3.5 times more Ir, as determined by ICP-MS,
from the pyridine complex 1 was accumulated in the cells
compared to the py-NMe2 analogue 5 (7.8 ±
0.5 ng of Ir vs 2.2 ± 0.3 ng of Ir per 106 cells).
Apoptosis Assay
In order to investigate whether the
reduction in cell viability observed by the SRB assay is based on
apoptosis, A2780 cells were treated with complexes 1 and 5 at equipotent concentrations of IC50 for 24 h,
then stained with annexin V/propidium iodide and analyzed by flow
cytometry. This allowed determination of cell populations as viable
(unstained, only self-fluorescence), early apoptosis (stained by annexin
V only, green fluorescence), late apoptosis (stained by annexin V
and PI, green and red fluorescence), and nonviable (stained by PI
only, red fluorescence). Dot plots (Figure 7 and Table S4) showed that around 95%
of A2780 cells remained in the viable phase after 24 h of exposure
to 1 and 5, indicating no obvious induction
of apoptosis at equipotent concentrations of IC50.
Figure 7
Apoptosis analysis
of A2780 human ovarian cells after 24 h of exposure
to complexes 1 and 5 at 310 K determined
by flow cytometry using annexin V-FITC vs PI staining. (A) FL1 vs
FL2 histogram for cells exposed to complexes 1 and 5 at equipotent concentrations of IC50. (B) Populations
for cells treated by 1 and 5. p-Values were calculated after a t test against the
negative control data, *p < 0.05, **p < 0.01, ap > 0.05.
Apoptosis analysis
of A2780 human ovarian cells after 24 h of exposure
to complexes 1 and 5 at 310 K determined
by flow cytometry using annexin V-FITC vs PI staining. (A) FL1 vs
FL2 histogram for cells exposed to complexes 1 and 5 at equipotent concentrations of IC50. (B) Populations
for cells treated by 1 and 5. p-Values were calculated after a t test against the
negative control data, *p < 0.05, **p < 0.01, ap > 0.05.
Cell Cycle Studies
Next we performed
cell cycle arrest
analysis for complexes 1 and 5 toward A2780
cells by flow cytometry to determine whether the induced cell growth
inhibition was the result of cell cycle arrest. In comparison to the
control population, the cell cycle data (Figure 8 and Table S5) clearly show no significant
population change, indicating that Ir compounds 1 and 5 did not cause cell cycle arrest at equipotent concentrations
of IC50.
Figure 8
Cell cycle analysis of A2780 human ovarian cancer cells
after 24
h of exposure to complexes 1 and 5 at 310
K. Concentrations used were equipotent at IC50. Cell staining
for flow cytometry was carried out using PI/RNase. (A) FL2 histogram
for negative control (cells untreated) and complexes 1 and 5. (B) Cell populations in each cell cycle phase
for control and complexes 1 and 5. p-Values were calculated after a t test
against the negative control data, *p < 0.05,
**p < 0.01, ap >
0.05.
Cell cycle analysis of A2780 human ovarian cancer cells
after 24
h of exposure to complexes 1 and 5 at 310
K. Concentrations used were equipotent at IC50. Cell staining
for flow cytometry was carried out using PI/RNase. (A) FL2 histogram
for negative control (cells untreated) and complexes 1 and 5. (B) Cell populations in each cell cycle phase
for control and complexes 1 and 5. p-Values were calculated after a t test
against the negative control data, *p < 0.05,
**p < 0.01, ap >
0.05.
Induction of ROS in A2780
Cancer Cells
We determined
the level of reactive oxygen species (ROS) in A2780 human ovarian
cancer cells induced by complexes 1 and 5 at concentrations of IC50 and 2 × IC50 by flow cytometry fluorescence analysis (Figure 9 and Table S6). This allowed the
determination of the total level of oxidative stress (combined levels
of H2O2, peroxy and hydroxyl radicals, peroxynitrite,
and NO), while also monitoring superoxide production. After only 1
h of drug exposure, we observed a dramatic increase in both total
ROS levels and superoxide levels in cells treated with complexes 1 and 5 compared to untreated cells. ROS were
detected in more than 97% of A2780 cells. A concentration dependence
of ROS induction was observed for both Ir complexes: the population
of cells that shows high fluorescence in both FL-1 and FL-2 channels
(indicating high total oxidative stress as well as high superoxide
levels) increased from 48 ± 2% at IC50 to 64 ±
3 at 2 × IC50 for complex 1 and increased
from 40 ± 3% at IC50 to 47 ± 2 at 2 × IC50 for complex 5 (Figure 9 and Table S6).
Figure 9
ROS induction in A2780
cancer cells treated with complexes 1 and 5. FL1 channel detects total oxidative
stress, and FL2 channel detects superoxide production. (A) Comparison
between the four different populations caused by IC50 and
2 × IC50 of 1. (B) Comparison between
the four different populations caused by IC50 and 2 ×
IC50 of 5. p-Values were
calculated after a t test against the negative control
data, *p < 0.05, **p < 0.01, ap > 0.05.
ROS induction in A2780
cancer cells treated with complexes 1 and 5. FL1 channel detects total oxidative
stress, and FL2 channel detects superoxide production. (A) Comparison
between the four different populations caused by IC50 and
2 × IC50 of 1. (B) Comparison between
the four different populations caused by IC50 and 2 ×
IC50 of 5. p-Values were
calculated after a t test against the negative control
data, *p < 0.05, **p < 0.01, ap > 0.05.
Polarization of the Membrane Potential
Analysis of
the changes of mitochondrial membrane potential (ΔΨm)
in A2780 cells after exposure to complexes 1 and 5 was carried out by observing the fluorescence of JC-10,
a cationic lipophilic dye, using flow cytometry. JC-10 aggregates
inside mitochondria and emits red fluorescence; however, upon membrane
polarization, JC-10 is disaggregated, reducing the red emission. The
level of membrane polarization after cells were exposed to complexes 1 and 5 at concentrations of IC50 and
2 × IC50 for 24 h is shown in Figure 10 and Table S7. Both Ir complexes
have significant effects on cell membrane polarization; around 70%
of cells lost ΔΨm. The impairment induced by 1 and 5 (which is reflected in ΔΨm) is clearly
concentration-dependent (Figure 10).
Figure 10
Changes in
mitochondrial membrane potential of A2780 human ovarian
cancer cells induced by complexes 1 and 5. (A) Flow cytometry histograms of the changes induced by the complexes
at concentrations of IC50 and 2 × IC50.
(B) Populations of cells that exhibit a reduction in the FL2 fluorescence,
indicative of changes in the mitochondrial membrane potential. p-Values were calculated after a t test
against the negative control data, **p < 0.01.
Changes in
mitochondrial membrane potential of A2780 human ovarian
cancer cells induced by complexes 1 and 5. (A) Flow cytometry histograms of the changes induced by the complexes
at concentrations of IC50 and 2 × IC50.
(B) Populations of cells that exhibit a reduction in the FL2 fluorescence,
indicative of changes in the mitochondrial membrane potential. p-Values were calculated after a t test
against the negative control data, **p < 0.01.
Discussion
IrIII complexes are often considered to be relatively
inert, a common characteristic of low-spin d6 metal ions
and especially third-row transition metals.[13] Compared to platinum- or ruthenium-based anticancer agents, iridium
anticancer complexes are still in their infancy.With regard
to half-sandwich IrIII complexes [(Cpx)Ir(L∧L′)Z]0/, we found
that both the cyclopentadienyl Cpxph or Cpxbiph ligand and chelating ligand C∧N– can dramatically
influence anticancer activity.[6b−6d] In addition, we have shown that
complexes containing pyridine as
the monodentate ligand exhibit 6 times higher anticancer activity
compared to the chlorido analogue.[6a] Therefore,
we have investigated a series of IrIII complexes of type
[(Cpxph)Ir(phpy)Z]+ containing phenyl-substituted
Cp*, C∧N-bound 2-phenylpyridine, and pyridine or
its derivatives (Scheme 1) in this work. Novel
compounds 1–8 have been synthesized
and are reported for the first time.Encouragingly, all eight
compounds exhibit high potency against
human ovarian A2780 cancer, A549 lung cancer, and MCF-7 breast cancer
cells, at least comparable with cisplatin, Figure 2. In general an electron-donating substituent on the py ring
confers higher activity in comparison with electron-withdrawing groups.
This may arise from strengthening the Ir–N(py) bond, thus reducing
side reactions on the way to target sites. In addition, lipophilicity
might as well influence the potency of these complexes.[14] Complex 5, [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]+, showed
the highest anticancer activity, ca. 3–9 times more active
than unmodified py complex 1. In addition, complex 5 shows submicromolar activity toward a wide range of cancer
cell lines in the NCI-60 screen, with selectivity for leukemia, CNS
cancer, colon cancer, and breast cancer cell lines, being 4–5
times more potent than CDDP (Figures 3 and 4). Cpxph py complex 1 displayed
ca. 8 times less anticancer potency than the Cpxbiph analogue,
which is consistent with the general finding we reported previously
that the anticancer efficiency increases with phenyl substitution
on the Cp* ring.[6a,6b,6d]Hydrolysis often presents an activation step for transition
metal
anticancer complexes.[15] However, no significant
hydrolysis was observed for complexes 1–8 in aqueous solution. DNA is usually a potential target for
transition metal anticancer drugs.[16] Although 1–8 are inert to hydrolysis, they can
react with nucleobase 9-EtG to various extents from 11% to 50% (Figure 6), depending on the electronic effect of the substituent
on the py ring. Electron-withdrawing groups (such as acetyl and ester
groups) facilitate ligand substitution of the py derivative by 9-EtG,
whereas electron donor groups (such as methyl and dimethylamino groups)
hamper formation of the Ir-EtG adduct. No reaction of 1–8 with 9-MeA was observed, consistent with our
previous studies that guanine binds stronger to IrIII than
adenine.[6a,6b,6d] The extent
of nucleobase binding does not correlate with antiproliferative activity.
Compared to complexes 1–5, complexes 6–8 bind to 9-EtG to a greater extent;
however, they show lower activity toward cancer cells. Therefore,
although DNA is a potential target for these iridium compounds, DNA
binding may not be the major mechanism of action.Apoptosis
is a process of cell death in a programmed fashion. A
large number of transition metal-based anticancer agents have been
reported to inhibit cell growth by activation of apoptosis.[17] Induction of apoptosis is usually dependent
on the concentration of administered compounds[17f,18] and on exposure time.[19] No apoptosis
was observed when A2780 cells were exposed to complexes 1 and 5 at their IC50 concentrations for 24
h. Also IC50 concentrations of complexes 1 and 5 did not cause significant cell cycle arrest after
24 h of drug exposure. Lack of accumulation of cells in the sub-G1
phase in cell cycle experiments is consistent with the absence of
apoptotic cell death.[20]Reactive
oxygen species play important roles in regulating cell
proliferation, death, and signaling. They can also play significant
roles in the mechanism of action of anticancer agents.[21] In fact, dinuclear Cp*Ir(III) complexes containing
bridging dipyridyl ligands have been reported to generate ROS and
induce apoptosis in Jurkat leukemia cells.[17f] We suggested previously that the antiproliferative mechanism for
the iridium pyridine complex in this series is related to ROS generation.[6a] Consequently, we also determined the ROS levels
in A2780 ovarian cancer cells induced by 1 and 5. Both complexes increased ROS levels significantly even
at IC50 concentration after 1 h drug exposure (Figure 9), which led to the majority of cancer cells (>97%)
being affected by generation of ROS. These increases in ROS levels
may provide a basis for killing cancer cells.Mitochondria are
involved in a number of important tasks in living
cells, such as energy production and generation of ROS. Mitochondrial
dysfunction can participate in the induction of cell death and was
assessed by measuring changes in the mitochondrial membrane potential.
Intriguingly, both complexes 1 and 5 (IC50 concentration) induced significant changes in mitochondrial
membrane potential (Figure 10); more than 70%
of A2780 cells lose ΔΨm after exposure to Ir compounds
for 24 h. This may contribute to the anticancer activities of these
Ir compounds.
Conclusions
In this work, we have
prepared eight new organometallic IrIII cyclopentadienyl
complexes [(η5-Cpxph)Ir(phpy)Z]PF6 to explore the effect of a monodentate
pyridine-based ligand on their chemical and anticancer activity. The
X-ray crystal structures of [(η5-Cpxph)Ir(phpy)(4-Me-py)]PF6 (2), [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]PF6 (5), and [(η5-Cpxph)Ir(phpy)(3-CONEt2-py)]PF6 (8) were determined.All the complexes display high potency toward A2780, A549, and
MCF-7 human cancer cells, comparable to, and for some complexes even
higher than, the clinical anticancer drug cisplatin. The anticancer
activity can be fine-tuned by varying the pyridine-based ligand; the
presence of an electron-donating group confers higher anticancer activity.
The most active complex, 5, contains a 4-dimethylamine
substituent on pyridine. The results of the NCI 60 cancer cell line
screening show that complex 5 is 4–5 times more
potent than cisplatin and exhibits submicromolar activity in a wide
range of cancer cell lines, especially against leukemia, CNS cancer,
colon cancer, and breast cancer. Nanomolar activity (GI50 19 nM) was obtained for complex 2 toward the renal
A498 cancer cell line.No distinct hydrolysis was observed for
this type of complex in
aqueous solution; however, all complexes display weak nucleobase binding
to 9-ethylguanine, suggesting that DNA could be a possible target,
although other targets appear to be more important. Additionally,
no obvious apoptosis and cell cycle arrest were induced when A2780
cancer cells were treated with IC50 concentrations of complexes 1 and 5. However, the iridium complexes 1 and 5 induce a dramatic increase in the level
of ROS in ovarian cancer cells within 1 h and caused mitochondrial
dysfunction by loss of the mitochondrial membrane potential. Our work
suggests that this type of iridium complex could be a promising candidate
for further evaluation as chemotherapeutic agents for human cancers.
Authors: Riccardo Rubbiani; Suzan Can; Igor Kitanovic; Hamed Alborzinia; Maria Stefanopoulou; Malte Kokoschka; Susann Mönchgesang; William S Sheldrick; Stefan Wölfl; Ingo Ott Journal: J Med Chem Date: 2011-11-17 Impact factor: 7.446
Authors: V Venkatesh; Raul Berrocal-Martin; Christopher J Wedge; Isolda Romero-Canelón; Carlos Sanchez-Cano; Ji-Inn Song; James P C Coverdale; Pingyu Zhang; Guy J Clarkson; Abraha Habtemariam; Steven W Magennis; Robert J Deeth; Peter J Sadler Journal: Chem Sci Date: 2017-10-20 Impact factor: 9.825
Authors: Wen-Ying Zhang; Samya Banerjee; George M Hughes; Hannah E Bridgewater; Ji-Inn Song; Ben G Breeze; Guy J Clarkson; James P C Coverdale; Carlos Sanchez-Cano; Fortuna Ponte; Emilia Sicilia; Peter J Sadler Journal: Chem Sci Date: 2020-05-15 Impact factor: 9.825
Scheme 1
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10