Contrasting Anticancer Activity of Half-Sandwich Iridium(III) Complexes Bearing Functionally Diverse 2-Phenylpyridine Ligands

Article abstract of DOI:10.1021/acs.organomet.5b00097

We report the synthesis, characterization, and antiproliferative activity of 15 iridium(III) half-sandwich complexes of the type [(η⁵-Cp?)Ir(2-(R′-phenyl)-R-pyridine)Cl] bearing either an electron-donating (-OH, -CH₂OH, -CH₃) or electron-withdrawing (-F, -CHO, -NO₂) group at various positions on the 2-phenylpyridine (2-PhPy) chelating ligand giving rise to six sets of structural isomers. The X-ray crystal structures of [(η⁵-Cp?)Ir(2-(2′-fluorophenyl)pyridine)Cl] (1) and [(η⁵-Cp?)Ir(2-(4′-fluorophenyl)pyridine)Cl] (2) exhibit the expected "piano-stool" configuration. DFT calculations showed that substituents caused only localized effects on the electrostatic potential surface of the chelating 2-PhPy ligand of the complexes. Hydrolysis of all complexes is rapid, but readily reversed by addition of NaCl. The complexes show preferential binding to 9-ethylguanine over 9-methyladenine and are active catalysts for the oxidation of NADH to NAD⁺. Antiproliferative activity experiments in A2780 ovarian, MCF-7 breast, A549 lung, and HCT116 colon cancer cell lines showed IC₅₀ values ranging from 1 to 89 μM, with the most potent complex, [(η⁵-Cp?)Ir(2-(2′-methylphenyl)pyridine)Cl] (13) (A2780 IC₅₀ = 1.18 μM), being 10× more active than the parent, [(η⁵-Cp?)Ir(2-phenylpyridine)Cl], and 2× more active than [(η⁵-Cp^xPh)Ir(2-phenylpyridine)Cl]. Intriguingly, contrasting biological activities are observed between structural isomers despite exhibiting similar chemical reactivity. For pairs of structural isomers both the nature and position of the functional group can affect the hydrophobicity of the complex. An increase in hydrophobicity resulted in enhanced cellular-iridium accumulation in A2780 ovarian cells, which generally gave rise to an increase in potency. The structural isomers [(η⁵-Cp?)Ir(2-(4′-fluorophenyl)pyridine)Cl] (2) and [(η⁵-Cp?)Ir(2-phenyl-5-fluoropyridine)Cl] (4) preferentially localized in the cytosol > membrane and particulate > nucleus > cytoskeleton. This work highlights the strong dependence of biological behavior on the nature and position of the substituent on the chelating ligand and shows how this class of organometallic anticancer complexes can be fine-tuned to increase their potency without using extended cyclopentadienyl systems. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.organomet.5b00097

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Article
pubs.acs.org/Organometallics
Contrasting Anticancer Activity of Half-Sandwich Iridium(III)
Complexes Bearing Functionally Diverse 2‑Phenylpyridine Ligands
Adam J. Millett, Abraha Habtemariam, Isolda Romero-Canelon, Guy J. Clarkson, and Peter J. Sadler*
́
Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry, CV4 7AL, U.K.
*
S Supporting Information
ABSTRACT: We report the synthesis, characterization, and antiproliferative
5
activity of 15 iridium(III) half-sandwich complexes of the type [(η -Cp*)Ir(2-
(
R′-phenyl)-R-pyridine)Cl] bearing either an electron-donating (−OH,
−
CH OH, −CH ) or electron-withdrawing (−F, −CHO, −NO ) group at
2
3
2
various positions on the 2-phenylpyridine (2-PhPy) chelating ligand giving
5
rise to six sets of structural isomers. The X-ray crystal structures of [(η -
Cp*)Ir(2-(2′-fluorophenyl)pyridine)Cl] (1) and [(η -Cp*)Ir(2-(4′-
5
fluorophenyl)pyridine)Cl] (2) exhibit the expected “piano-stool” config-
uration. DFT calculations showed that substituents caused only localized
effects on the electrostatic potential surface of the chelating 2-PhPy ligand of
the complexes. Hydrolysis of all complexes is rapid, but readily reversed by
addition of NaCl. The complexes show preferential binding to 9-ethylguanine
over 9-methyladenine and are active catalysts for the oxidation of NADH to
+
NAD . Antiproliferative activity experiments in A2780 ovarian, MCF-7 breast,
A549 lung, and HCT116 colon cancer cell lines showed IC50 values ranging from 1 to 89 μM, with the most potent complex,
5
5
[
(η -Cp*)Ir(2-(2′-methylphenyl)pyridine)Cl] (13) (A2780 IC = 1.18 μM), being 10× more active than the parent, [(η -
5
0
5 xPh
Cp*)Ir(2-phenylpyridine)Cl], and 2× more active than [(η -Cp )Ir(2-phenylpyridine)Cl]. Intriguingly, contrasting biological
activities are observed between structural isomers despite exhibiting similar chemical reactivity. For pairs of structural isomers
both the nature and position of the functional group can affect the hydrophobicity of the complex. An increase in hydrophobicity
resulted in enhanced cellular-iridium accumulation in A2780 ovarian cells, which generally gave rise to an increase in potency.
The structural isomers [(η -Cp*)Ir(2-(4′-fluorophenyl)pyridine)Cl] (2) and [(η -Cp*)Ir(2-phenyl-5-fluoropyridine)Cl] (4)
preferentially localized in the cytosol > membrane and particulate > nucleus > cytoskeleton. This work highlights the strong
dependence of biological behavior on the nature and position of the substituent on the chelating ligand and shows how this class
of organometallic anticancer complexes can be fine-tuned to increase their potency without using extended cyclopentadienyl
systems.
5
5
INTRODUCTION
Chelated half-sandwich iridium(III) complexes have been
shown to bind to DNA nucleobases via the monodentate site
and can inhibit the synthesis of DNA by DNA polymerases.
■
There is an increasing interest in the design of organometallic
4a
1
anticancer complexes, especially complexes that are active
Some of these complexes also inhibit thioredoxin reductase 1
2
against cisplatin-resistant cancers, and organo-iridium(III)
complexes are showing promise in this regard. We have
7
(
Trx-R). Intriguingly they can also catalyze the oxidation of
3
+
+
NADH to NAD , and modulation of the NAD /NADH ratio
has been demonstrated in A2780 human ovarian cancer cells.
8
reported that the biological activity of half-sandwich penta-
methylcyclopentadienyl (Cp*) iridium(III) complexes can be
increased by the incorporation of phenyl substituents, as in
Disruption of this redox process may contribute to a
9
multitargeting mechanism of action. Recently we reported
xPh
xBiPh
4
5
Cp and Cp
ligands. This increase in activity may be due
that the highly potent and selective C,N-chelated complex [(η -
in part to an increase in hydrophobicity, resulting in enhanced
cellular accumulation. Extended cyclopentadienyl systems have
also been shown to strengthen interactions with DNA via
C
6
H
5
C
6
H
4
Cp*)Ir(2-PhPy)pyridine]PF
6
, which bears pyridine
in place of chloride as a monodentate ligand, can produce H O
upon the catalytic reaction with NADH via hydride transfer to
molecular O2.
2
2
10
4a
intercalation between DNA base pairs. The introduction of
the negatively charged C,N-chelating ligands in place of neutral
N,N ligands has also been shown to improve antiproliferative
The effect of incorporating functionality on the chelating
ligand in half-sandwich iridium(III) anticancer complexes has
5
activity. Again, the increase in activity may be attributed to an
Special Issue: Mike Lappert Memorial Issue
Received: February 3, 2015
increase in hydrophobicity of the resulting neutral complex.
Anionic ligands in place of neutral ligands in other iridium
3e,6
systems have also been explored.
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.organomet.5b00097
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Article
7
,11
not been widely explored.
The aim of the present research
Scheme 1. Synthetic Route for 2-(R′-phenyl)-R-pyridine
5
was to synthesize a novel set of complexes bearing functionally
diverse 2-PhPy ligands. 2-PhPy has shown promise in half-
sandwich iridium anticancer complexes.
Ligands L1−15 (A) and [(η -Cp*)Ir(2-(R′-phenyl)-R-
pyridine)Cl] Complexes 1−15 (B)
5
,12
Synthesis of 15 2-
PhPy ligands that bear either −F, −CHO, −NO , −OH,
2
−
CH OH, or −CH functional groups on different positions
2
3
on the chelating ligand (Chart 1) was accomplished by
5
Chart 1. [(η -Cp*)Ir(2-(R′-phenyl)-R-pyridine)Cl]
Complexes Studied in This Work
complexes of the type [(η -Cp*)Ir(2-PhPy)X]0/+. The
5
1
13
complexes were characterized by H and C NMR spectros-
copy, ESI-MS, CHN analysis, and RP-HPLC. Crystals of
complexes 1 and 2 suitable for structure determination by X-ray
diffraction were obtained by slow evaporation from chloro-
form/hexane at 273 K.
Molecular Structures. The X-ray crystal structures of
5
complexes [(η -Cp*)Ir(2-(2′-fluorophenyl)pyridine)Cl] (1)
5
and [(η -Cp*)Ir(2-(4′-fluorophenyl)pyridine)Cl] (2) were
determined by X-ray diffraction and are shown in Figure 1.
employing Suzuki−Miyaura palladium cross-coupling. The
5
resulting 15 novel iridium(III) Cp* complexes [(η -Cp*)Ir(2-
(
R′-phenyl)-R-pyridine)Cl] (Chart 1) have been investigated
for their aqueous chemistry, electrostatic potential surfaces
(
EPS), interaction with model DNA nucleobases, and ability to
catalyze the oxidation of reduced coenzyme nicotine adenine
+
dinucleotide NADH to NAD . Correlations between relative
hydrophobicity, cellular accumulation of iridium, and anti-
proliferative activity have been studied, along with comparisons
of the cellular distributions of a pair of structural isomers.
Importantly it is apparent that within this series there are
potent complexes even without the presence of extended
cyclopentadienyl systems.
Figure 1. X-ray crystal structures of compounds 1 (A) and 2 (B).
Hydrogen atoms have been omitted for clarity, and thermal ellipsoids
are shown at 50% probability. The disorder observed for complex 1
has been removed for clarity, but is described in the Supporting
Information.
RESULTS
■
5
Synthesis and Characterization of [(η -Cp*)Ir(2-(R′-
X-ray crystallographic data are listed in Table S1, and selected
bond lengths and angles in Table 1. The complexes exhibit the
expected pseudo-octahedral half-sandwich structure, with the
Cp* ring occupying three coordination sites, the 2-PhPy
chelating ligand occupying two sites, and the monodentate
chlorido ligand occupying the sixth coordination position. Ir−
centroid distances are 1.828 and 1.833 Å for 1 and 2,
respectively, while the Ir−C distances of 2.0482(18) and
2.0346(19) Å and Ir−N distances of 2.0797(17) and
2.0931(16) Å show significant variation and Ir−Cl distances
of 2.4056(5) and 2.4026(5) Å show small (>3σ) differences for
complexes 1 and 2, respectively. The bond angles are similar,
with the angle between the chelating ligand and iridium center
(C−Ir−N) being the smallest, 77.84(7)° and 78.13(7)° for 1
and 2, respectively. Weak offset intermolecular π−π stacking
phenyl)-R-pyridine)Cl] Complexes. Suzuki−Miyaura cross-
coupling chemistry was employed following an adapted
1
3
protocol, to synthesize chelating 2-PhPy ligands with
electron-donating or electron-withdrawing substituents,
Scheme 1. They were purified using flash silica gel
chromatography with mixtures of chloroform/ethyl acetate,
yielding 2-(R′-phenyl)-R-pyridine ligands L1−15. The syn-
5
thesis of the corresponding complexes [(η -Cp*)Ir(2-(R′-
phenyl)-R-pyridine)Cl] (1−15) was carried out using an
4b
adapted literature procedure. The synthetic routes for the
complexes are shown in Scheme 1. The introduction of
electron-withdrawing nitro (−NO ) and aldehydic (−CHO)
2
groups and an electron-donating hydroxymethyl group
(
−CH OH) in complexes 5−10 is the first to be reported in
2
B
DOI: 10.1021/acs.organomet.5b00097
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Article
Table 1. Selected Bond Lengths (Å) and Bond Angles (deg)
for Complexes 1 and 2
bond/angle
1
2
Ir−C (Cp* ring)
2.163(2)
2.160(2)
2.1609(19)
2.172(2)
2.2522(19)
2.272(2)
1.833
2
2
2
2
.1675(19)
.177(2)
.239(2)
.247(2)
Ir−C (centroid)
Ir−C
Ir−N
1.828
2.0482(18)
2.0797(17)
2.4056(5)
2.0346(19)
2.0931(16)
2.4026(5)
Ir−Cl
C−Ir−N
C−Ir−Cl
N−Ir−Cl
77.84(7)
85.96(5)
87.60(5)
78.13(7)
85.68(5)
87.30(5)
between the 2-PhPy ligands is observed for both complexes
with a centroid−centroid distance of 4.140 Å in complex 1 and
4
.059 Å in complex 2 (Figure S1).
Electrostatic Potential Surface. The EPSs of complexes
2
, 4, 7, 8, and 11−13 were calculated using DFT methods at
14
the PBE0/Lanl2DZ/6-31+G** level, based on the crystal
structure of complex 2, with functional group modifications
15
being made with GaussView 3.0. The resulting EPS of each
complex is shown in Figure 2. The phenyl ring in the chelating
ligand exhibits a more negative electrostatic potential than the
pyridyl ring due to the deprotonated carbon bound to the Ir
center. There are no major differences in the charge
distribution at the iridium center, Cp* ring, or chlorido ligands
among the complexes, indicating that the substituent on the
chelating ligand causes only a localized effect. Complexes 7 and
8
contain the electron-withdrawing nitro group on the phenyl
and pyridyl ring, respectively, causing a more positive surface.
Complex 13 bears a methyl group on the phenyl ring, which
pushes electron density into the ring, causing a more negative
EPS. The substituents themselves cause the outer EPS of each
complex to alter significantly. The calculated bond lengths at
the metal center are shown in Table S2, which shows minimal
differences among the complexes, with the exception of 13,
which exhibits a shorter Ir−N bond. The HOMO and LUMO
orbitals of complexes 2, 4, 7, 8, and 11−13 and corresponding
orbital energies are shown in Table S3.
Figure 2. Electrostatic potential surfaces for complexes 2, 4, 7, 8, and
1
1−13 (calculated at the PBE0/Lanl2DZ/6-31+G** level), where the
EPSs are shown both in space (with positive and negative regions in
blue and red, respectively) and mapped on electron density (isovalue
0
.04) of the molecules. The electrostatic potential is represented with a
color scale ranging from red (−0.040 au) to blue (+0.250 au). Note
the more negative charge distribution on the phenyl ring of the 2-PhPy
chelating ligand compared with the pyridyl ring. The substituents
affect markedly only the electronic charge density on the chelating
ligand, leaving the iridium center, Cp*, and chlorido ligands relatively
unaffected.
Hydrolysis Studies. The hydrolysis of complexes 1−15
1
(
ca. 500 μM) was studied by H NMR spectroscopy in 26.7%
MeOD-d /73.3% D O at 310 K. Methanol was required to
4
2
1
ensure the solubility of the complexes in aqueous solution.
Hydrolysis equilibrium was reached before the time taken to
acquire the first spectra (15 min), consistent with previous
mol ratios of complex to nucleobase by H NMR spectroscopy
in 26.7% MeOD-d /73.3% D O at 310 K, pH* 7.4, after
4
2
incubation for 15 min and 24 h. All complexes showed 100%
binding to 9-EtG and ca. 50−80% binding to 9-MeA after 15
min, which remained unchanged over a period of 24 h.
Competitive binding studies between 9-EtG and 9-MeA were
also performed under the same conditions. This resulted in
mostly 9-EtG binding for all the complexes (Figure 3).
Complexes 2, 4, and 14 show 5−10% binding to 9-MeA in
the presence of 9-EtG. Binding of 9-EtG to 2 and 4 in the
presence of 4 mM NaCl, where the complexes exist primarily as
the Ir−Cl species, reached 100% after 24 h (Figure S3).
Catalysis of NADH Oxidation. The reaction between
NADH and complexes 2, 4, 11, and 12 was monitored by UV−
vis spectroscopy at varying concentrations of NADH (69, 103,
and 146 μM) with a fixed concentration of complex (2.5 μM)
III
4b
reports of Ir C,N-chelated half-sandwich complexes.
Between 67% and 80% was found to be in the hydrolyzed
form, based on peak integrations. Hydrolysis was confirmed by
the sequential addition of NaCl, showing an increase in the
peaks associated with the Ir-Cl species and a decrease in the
peaks associated with the Ir-OD /OD species. With 4 mM
2
NaCl present, almost all of the Ir-OD /OD species had been
2
converted to the Ir-Cl complexes (Figure S2). Above this
concentration of NaCl, precipitation of the complexes was
observed.
Binding to Nucleobases. Complexes 2, 4, 7, 8, and 11−14
ca. 500 μM) were evaluated for their ability to bind to 9-
(
ethylguanine (9-EtG) and 9-methyladenine (9-MeA) in ca. 1:1
C
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Figure 4. (a) UV−visible spectra for the catalytic oxidation of NADH
+
(
146 μM) to NAD in the presence of complex 2 (2.5 μM) over a 24 h
period in 0.5% MeOH/99.5% H O at 310 K in 5 mM Na HPO /
2
2
4
NaH PO buffer, pH 7.5. (b) First-order kinetic plot. [NADH] = 69
2
4
μM (green), 103 μM (purple), and 146 μM (orange).
In order to probe whether the oxidation proceeds via the
formation of an iridium-hydride species, the reaction of
complex 2 (250 μM) with NADH (750 μM) in 50% MeOD-
Figure 3. (Top) Interaction of complex 2 (ca. 500 μM) with
1
d /50% D O at 310 K was monitored by H NMR
4
2
equimolar amounts of 9-EtG and 9-MeA and competition binding by
1
spectroscopy over a 15 h period. After 15 min, a peak
H NMR spectroscopy (600 MHz, 26.7% MeOD-d /73.3% D O at
4
2
corresponding to an Ir−H species was observed at −14.84 ppm
310 K, pH* 7.4, 24 h). Complex 2 shows 100% binding to 9-EtG (blue
(
Figure S5), which was no longer visible after 15 h. The
triangles) and around 50% binding to 9-MeA (red stars). Complex 2
shows a preference for binding to 9-EtG upon co-incubation with 9-
EtG and 9-MeA. (Bottom) Co-incubation of complexes 2, 4, 7, 8, and
+
conversion of NADH to NAD was also confirmed by the
appearance of a new set of peaks corresponding to the
+
1
1−14 with equimolar amounts of 9-EtG and 9-MeA showing a
formation of NAD .
preference for binding to 9-EtG.
Antiproliferative Activity. The antiproliferative activity of
complexes 1−15 in A2780 (ovarian) cancer cells and
complexes 1, 2, 5, 6, 8, 10, 11, and 14 in HCT-116 (colon),
MCF-7 (breast), and A549 (lung) cancer cells is shown in
over a period of 24 h at 310 K, pH 7.5 (Na HPO /NaH PO
4
2
4
2
4
b
buffer, 5 mM). The decrease in the band at 339 nm was
Table 3. The activity of previously reported iridium
monitored, as it corresponds to conversion of NADH to NAD+
complexes [(η -Cp*)Ir(2-phenylpyridine)Cl] (16) and [(η -
5
5
xPh
(Figure 4a). The data were also the basis for the calculation of
Cp )Ir(2-phenylpyridine)Cl] (17) with no substituents on
the turnover number (TON) and turnover frequency (TOF,
the chelating ligand is also shown for comparison. It is evident
that the presence of substituents on the chelating ligand greatly
affects the antiproliferative activity of the complexes, with IC50
values ranging from 1 to 60 μM in the A2780 cell line alone.
−1
h ) for each complex at different concentrations (Table 2).
After 24 h, the maximum TON was found for each complex at
the highest concentration of NADH (146 μM) and ranged
from 22 to 28. Maximum TOF was also reached at 146 μM
5
[(η -Cp*)Ir(2-(2′-methylphenyl)pyridine)Cl] (13) is the most
−1
NADH, ranging from 1.1 to 3.2 h . The reaction proceeded
potent complex, with an IC50 value of 1.18 μM in A2780
via first-order kinetics with respect to [NADH] (Figure 4b),
ovarian cancer cells, making it more potent than the extended
−4
−1
5
xPh
with an average rate constant k of 3.79 × 10 min for 2 and
cyclopentadienyl complex [(η -Cp )Ir(2-phenylpyridine)Cl].
Complexes 2, 8, 13, and 15 also show similar potency to the
clinical drug cisplatin (CDDP). Interestingly, structural isomers
4
−1
4
.03 × 10− min for 4. However, complexes 11 and 12, which
bear the hydroxyl group, gave a non-first-order reaction with
respect to [NADH] during the first 3 h, followed by the
expected first-order kinetics (exemplified for complex 11 in
Figure S4). These results indicate that each of these complexes
shows catalytic behavior toward the oxidation of NADH.
5
exhibit contrasting activities, for example, complexes [(η -
Cp*)Ir(2-(4′-fluorophenyl)pyridine)Cl] (2) (2.7 μM) and
5
[(η -Cp*)Ir(2-phenyl-5-fluoropyridine)Cl] (4) (>60 μM) and
5
complexes [(η -Cp*)Ir(2-(4′-hydroxyphenyl)pyridine)Cl]
D
DOI: 10.1021/acs.organomet.5b00097
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+
Table 2. Calculated TONs and TOFs for Complexes 2, 4, 11, and 12 for the Catalytic Oxidation of NADH to NAD Monitored
via UV−Visible Spectroscopy at Various Concentrations of NADH (69, 103, and 146 μM) with a Fixed Complex Concentration
a
of 2.5 μM over a 24 h Period in 0.5% MeOH/99.5% H O at 310 K in 5 mM Na HPO /NaH PO Buffer, pH 7.5
2
2
4
2
4
complex
NADH conc (μM)
TON (24 h)
TOF (h−1)b
5
[
[
[
[
(η -Cp*)Ir(2-(4′-fluorophenyl)pyridine)Cl], 2
69
03
46
69
03
46
69
03
46
69
03
46
11
19
22
13
20
22
14
21
28
13
15
22
0.6
1.1
1.2
0.7
1.2
1.1
1.9
2.6
3.2
1.4
1.8
2.6
1
1
5
(η -Cp*)Ir(2-phenyl-5-fluoropyridine)Cl], 4
1
1
5
(η -Cp*)Ir(2-(4′-hydroxyphenyl)pyridine)Cl], 11
1
1
5
(η -Cp*)Ir(2-phenyl-5-hydroxypyridine)Cl], 12
1
1
a
+
b
The conversion of NADH to NAD was followed from the absorption at 339 nm. TOFs were calculated as the gradient of time vs TON during
the first 2 h of the reaction time.
Table 3. IC50 Vaules for Complexes 1−15 in A2780 Cells
Table 4. Retention Times (t ) by RP-HPLC and Cellular-
R
and Complexes 1, 2, 5, 6, 8, 10, 11, and 14 in HCT-116,
Iridium Accumulation (at Equipotent (IC )
5
0
a
MCF-7, and A549 Cells
Concentrations) in A2780 Cells for Complexes 2, 4, 7, 8,
and 11−14
IC50 values (μM)
cellular-Ir (ng Ir ×
complex
A2780
HCT-116
MCF-7
A549
6
complex
2-PhPy ligand
tR (min)
10 A2780 cells)
1
2
3
4
5
6
7
8
9
4.5 ± 0.2
2.7 ± 0.3
>50
3.7 ± 0.3
6.8 ± 0.1
n.d.
9.6 ± 0.4
4.8 ± 0.3
n.d.
10.36 ± 0.07
2.1 ± 0.3
n.d.
2
2-(4′-
fluorophenyl)pyridine
18.74 ± 0.05
13.5 ± 0.3
4
7
2-phenyl-5-fluoropyridine 18.60 ± 0.02
2-(4′-
nitrophenyl)pyridine
11.1 ± 0.1
8 ± 1
>60
n.d.
n.d.
n.d.
18.8 ± 0.2
6.9 ± 0.3
4.4 ± 0.4
24.73 ± 2.30
2.7 ± 0.1
>50
21.3 ± 0.7
18.8 ± 0.5
n.d.
11.6 ± 0.5
6.5 ± 0.3
n.d.
15.8 ± 0.4
5.9 ± 0.1
n.d.
8
2-phenyl-4-nitropyridine
18.0 ± 0.1
15.11 ± 0.03
9.0 ± 0.2
1.6 ± 0.1
11
2-(4′-
hydroxyphenyl)pyridine
27.5 ± 0.9
n.d.
11.4 ± 0.4
n.d.
20.1 ± 0.3
n.d.
1
2
3
2-phenyl-5-
hydroxypyridine
17.8 ± 0.2
20.4 ± 0.2
20.9 ± 0.9
10.2 ± 0.2
18 ± 1
10
11
12
13
14
15
16
17
47 ± 1
57.3 ± 0.9
29.3 ± 0.8
n.d.
47 ± 2
28.6 ± 0.9
n.d.
89 ± 1
56.67 ± 0.04
n.d.
1
2-(2′-
47.3 ± 0.1
13.29 ± 0.88
1.18 ± 0.08
3.9 ± 0.2
1.26 ± 0.01
10.78 ± 1.72
2.14 ± 0.50
1.2 ± 0.2
methylphenyl)pyridine
14
2-phenyl-5-
methylpyridine
16.0 ± 0.3
n.d.
n.d.
n.d.
9.6 ± 0.6
n.d.
3.7 ± 0.1
n.d.
8.7 ± 0.3
n.d.
b
b
n.d.
n.d.
n.d.
Cellular-Iridium Accumulation. Cellular-iridium accumu-
lation in A2780 cells after 24 h exposure to complexes 2, 4, 7, 8,
and 11−14 at equipotent (IC ) concentrations was
determined by ICP-MS and is shown in Table 4. Complex
1 shows the lowest accumulation of 1.58 ng Ir × 10 cells,
n.d.
n.d.
n.d.
cisplatin
5.2 ± 0.3
7.0 ± 0.1
3.5 ± 0.2
a
Drug exposure time was 24 h followed by a 72 h recovery time. All
values were determined as independent duplicates of triplicate
experiments, and the standard deviations were calculated. From ref
b.
5
0
b
6
1
4
while complex 13 exhibits the highest extent of accumulation of
8.46 ng Ir × 10 cells.
6
1
Cellular-Iridium Distribution. Structural isomers 2 and 4
5
(
11) (47.3 μM) and [(η -Cp*)Ir(2-phenyl-5-hydroxypyridine)-
were examined for their cellular-iridium distribution in A2780
human ovarian cancer cells at IC50 concentrations, Figure 5.
The cytosol contains the highest amount of iridium (51.4% for
and 69.2% for 4), followed by the cell membrane and
particulate fractions (28.4% for 2 and 17.9% for 4). Complex 4
localizes in the cytosol to a greater extent than complex 2 (p <
Cl] (12) (13.29 μM).
Relative Hydrophobicity. The relative hydrophobicities of
complexes 2/4, 7/8, 11/12, and 13/14 (four pairs of structural
isomers) were determined by RP-HPLC. The more hydro-
phobic complexes have longer retention times (t ). To ensure
solubility of the complexes, methanol was used (MeOH/H O,
2
R
2
0
.01), which in turn localizes in the cell membrane to a greater
1
:9 v/v) with NaCl (50 mM) to suppress the hydrolysis of the
extent than 4 (p < 0.01). The nuclear fraction contains different
amounts of iridium, with 10.4% and 6.7% (p < 0.01) for
complexes 2 and 4, respectively, while the cytoskeletal fraction
contains similar levels of iridium, with 9.8% and 6.1% (p >
0.05) for 2 and 4, respectively.
complexes in both sample preparation and in the HPLC
solvents. The resulting retention times are shown in Table 4.
Complex 11 shows the shortest retention time (least
hydrophobic) at 15.11 min, while complex 14 shows the
longest retention time (most hydrophobic) at 20.9 min.
E
DOI: 10.1021/acs.organomet.5b00097
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Organometallics
Article
ligands, which implies that the reactivity at the metal center
may remain relatively the same for all complexes. The EPS also
shows that the phenyl ring in the chelating ligand has a more
negative electrostatic potential than the pyridyl ring, due to the
negative charge associated with the deprotonation of the
binding carbon. The electron-withdrawing/electron-donating
nature of the substituents affects the EPS of the chelating ligand
by either diminishing or enhancing the electronic charge
density on the ring system to which the substituent is bound.
The electron-withdrawing −NO and −F groups provide a
2
more positive EPS within the ring, while the electron-donating
−
OH and −CH groups generate a more negative EPS within
3
the ring. The presence of the substituents significantly changes
the EPS of the chelating ligand, with areas of higher electronic
charge density for complexes 2, 4, 7, 8, 11, and 12 due to the
presence of heteroatoms with differing electronegativities. As
weak electrostatic forces are often important for binding and
recognition interactions with biomolecules such as peptides,
Figure 5. Cellular-iridium distribution of structural isomers 2 and 4 in
A2780 human ovarian cancer cells. Complex 4 localizes in the cytosol
to a greater extent than 2, while complex 2 localizes in the membrane
and particulate fractions to a greater extent than 4. Student’s t test at p
<
0.01 level indicated by **.
18
proteins, and enzymes, the nature and position of the
substituent may play a significant role in the interaction of the
complexes studied here with target sites.
DISCUSSION
■
Synthesis and Characterization. The synthesis of
complexes 1−15 was carried out using an adapted literature
Aqueous Chemistry. Hydrolysis studies by 1H NMR
showed that all complexes are converted to an equilibrium
4
b
procedure. It was found that the reported reaction time of 2 h
was not sufficient to provide 1−15 in good yields; therefore it
was extended to 18 h to obtain yields ranging from 16% to 80%.
Low yields were obtained for complexes 9 and 12 (22% and
mixture of Ir-Cl and Ir-OD /OD at 310 K within the time
2
taken to complete the first spectrum (15 min) with 67−80%
existing as the aqua/hydroxido adduct. This is consistent with
previous findings that iridium(III) half-sandwich complexes can
1
6%, respectively) due to their poor solubility in chlorinated
4
a,19
exhibit fast rates of ligand exchange
in comparison to their
solvents, preventing efficient separation of the complexes from
20
non-“piano stool” octahedral counterparts. Previous reports
on the aqua adducts of C,N-chelated half-sandwich iridium(III)
sodium acetate used to deprotonate the carbon on the phenyl
1
ring. The characterization of complexes 1−15 was done by H
1
3
complexes showed that the pK values are in the range 8.3−
NMR, ESI-MS, CHN analysis, HPLC, and in most cases
C
a
4b
1
NMR. The H NMR spectra are characterized by one less
proton in the low-field region compared to the starting 2-PhPy
ligand, due to the deprotonation of the bound carbon on the
8.9; therefore the complexes studied here are likely to exist as
the aqua rather than the hydroxido adduct at physiological pH.
The presence of substituents on the 2-PhPy chelating ligand
does not appear to affect the rate or extent of hydrolysis. The
hydrolysis can be readily reversed by addition of NaCl, and a
concentration of 4 mM NaCl almost completely suppresses
hydrolysis. This would imply that at biologically relevant
chloride concentrations (i.e., in the nucleus) the complexes
would exist as the Ir-Cl rather than aqua species.
1
phenyl ring. No appreciable difference in the H NMR chemical
shift of the Cp* ligand was observed among the functionally
1
3
diverse complexes. The C NMR spectra of fluorine-containing
1
9
1
complexes 1, 3, and 4 show coupling to F with J = 249−
CF
1
6
2
58 Hz, values similar to those previously reported. The ESI-
MS of the complexes always showed an m/z peak
5
+
corresponding to {(η -Cp*)Ir(2-(R′-phenyl)-R-pyridine)} ,
where the chlorido ligand had been removed in the ionization
process to form a detectable positive-ion peak. RP-HPLC was
also utilized to determine the purity of the complexes, and
examples of chromatograms are shown in the Supporting
Information (Figure S7).
Interactions with Nucleobases. DNA is a major target for
21
many transition metal anticancer compounds. The interaction
of complexes 2, 4, 7, 8, and 11−14 with DNA nucleobases 9-
EtG and 9-MeA indicated a strong preference for 9-EtG, with
100% binding after 15 min. Complexes 2 and 4 also exhibited
100% binding to 9-EtG after 15 min even in the presence of 4
mM NaCl, where the complex exists as the Ir-Cl species. All
complexes were able to bind to 9-MeA, but only complexes 2,
4, and 14 formed any significant amount of 9-MeA adducts in
competition binding experiments with 9-EtG, highlighting that
binding to 9-MeA was less strong than to 9-EtG. The weaker
binding of 9-MeA may be attributed to the steric hindrance
5
Complexes [(η -Cp*)Ir(2-(2′-fluorophenyl)pyridine)Cl] (1)
5
and [(η -Cp*)Ir(2-(4′-fluorophenyl)pyridine)Cl] (2) exhibit
the “piano-stool” structure expected for half-sandwich metal
complexes with bond lengths/angles comparable to similar
4
b,17
previously reported structures.
However, the position of
the fluorine on the phenyl ring influences the Ir-chelating ligand
bond lengths. Complex 2 exhibits a shorter Ir−C bond and a
longer Ir−N bond length than 1, suggesting that the binding
affinity of N and C for the Ir center is different. Therefore,
changing the position at which electron density is withdrawn
affects the solid-state structure of the complex. Both complexes
exhibit weak offset π−π stacking between the 2-PhPy ligand,
with a centroid−centroid distance of 4.140 Å for 1 and 4.059 Å
for 2.
caused by the −NH group at the 4 position. Guanine is also
2
known to be a better electron donor than adenine, which may
22
also contribute to the selectivity in 9-EtG binding. Guanine is
therefore a potential binding site on DNA, as it is for
23
21a
cisplatin, and other transition metal anticancer complexes.
Catalysis of NADH Oxidation. The potential for
complexes 2, 4, 11, and 12 to catalytically modulate the
+
NAD /NADH ratio in cells was examined. All exhibited
+
DFT calculations on complexes 2, 4, 7, 8, and 11−13
indicate that the presence of substituents does not affect the
electronic density on the iridium center, Cp* ring, or chlorido
catalytic activity toward the conversion of NADH to NAD by
first-order reaction kinetics with respect to [NADH]. Complex
11 exhibits the best catalytic activity, with a TON of 28 and
F
DOI: 10.1021/acs.organomet.5b00097
Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
TOF of 3.2 h− ([NADH] = 146 μM), while 2 and 4 have the
1
the chelating ligand. Previously, it has been demonstrated that
inclusion of fluorine substituents on the chelating ligand of half-
sandwich ruthenium and osmium complexes generally gave the
−1
lowest TON of 22 and TOF of 1.2 and 1.1 h , respectively.
The presence of the hydroxyl group in 11 and 12 seems to
enhance the catalytic activity. Complexes 11 and 12 also
exhibited an unusual kinetic profile for the first 3 h of reaction
time, followed by the expected first-order kinetics. The
hydroxyl group on complex [(η -Cp*)Ir(2-phenyl-6-hydroxy-
pyridine)Cl] has been previously reported to be integral to the
catalytic activity toward the dehydrogenative oxidation of
28
most potent systems, in contrast to the results obtained here.
It has also been demonstrated that in half-sandwich iridium(III)
complexes bearing picolinamide chelating ligands, the inclusion
of a chlorine atom in the arene ring of the chelating ligand
5
7
increases activity over the unfunctionalized complex. A further
increase in activity was observed upon the introduction of a
second chlorine atom depending on its position. However, the
contrasting activities between structural isomers observed in
this work are much greater than for the picolinamide
complexes.
24
organic compounds, which implies that the hydroxyl group
may be involved in the catalytic cycle. Although the position of
the hydroxyl group in 11 and 12 is situated further from the
5
metal center than in [(η -Cp*)Ir(2-phenyl-6-hydroxypyridine)-
Cl], the unusual kinetics observed may indicate that it is initially
playing a role in the catalytic activity. It has been suggested that
the rate-limiting step in this catalytic reaction is the formation
of the Ir-NADH adduct, leading to first-order reaction kinetics
Relationship between Hydrophobicity, Iridium-Cellu-
lar Accumulation, and Potency. The complexes studied
here are all neutral species in the presence of biologically
relevant concentrations of sodium chloride. Hydrophobicity
8b
29
with respect to [NADH]; therefore the unusual kinetic profile
of 11 and 12 may involve participation of the hydroxyl group
on the chelating ligand. The involvement of the ligand in the
oxidation of NADH in half-sandwich iridium(III) complexes
has not been previously observed. The oxidation of NADH
can promote interaction with the lipophilic cell membrane3
0
and promote hydrophobic interactions with protein targets.
In general, the accumulation and potency of organometallic
complexes in cells increases with an increase in their
23c,31
hydrophobicity.
The relative hydrophobicity of complexes
1
proceeds via formation of an Ir-H species as observed in the H
2, 4, 7, 8, and 11−14 (four pairs of structural isomers) was
determined by RP-HPLC. As RP-HPLC relies on the relative
interaction between the hydrophilic mobile phase and hydro-
phobic stationary phase, determination of the relative hydro-
phobicity of a series of complexes can be based on retention
NMR spectrum, in agreement with previous work on related
8
a,25
complexes.
As these complexes can catalytically convert
+
NADH to NAD , it may be possible to disrupt this important
redox couple in cells, which may have implications for cell
metabolism, including lactate dehydrogenase-catalyzed lactate-
times (t ). It was found that the nature of the substituent on
R
26
10,27
pyruvate conversions, enhancement of ROS levels,
and
the 2-PhPy ligand can dictate the hydrophobicity of the
complex, with hydroxyl complexes 11 and 12 being the least
lipophilic, while complexes 13 and 14, bearing methyl groups,
are the most. Intriguingly, complex 12 appeared to be much
more hydrophobic than the corresponding structural isomer 11,
with retention times of 17.8 and 15.1 min, respectively (Figure
S6). It is apparent that not only the type of substituent but also
its position govern the hydrophobicity of the complex.
reducing the concentration of one of the major electron donors
in the electron transport chain.
Antiproliferative Activity. The antiproliferative activity of
the 15 complexes covered a wide range, from 1 to 60 μM in
A2780, 3.7 to 54.3 μM in HCT-116, 3.7 to 47 μM in MCF-7,
and 2.1 to 89 μM in A549 cells, showing that the substituent on
the chelating ligand has a significant effect on the
antiproliferative activity of the complex. Complexes 2, 8, 13,
and 15 are all just as or more potent than the previously
To examine if structural isomers accumulate in cells to a
different extent, the cellular-iridium accumulation in A2780
cells of 2, 4, 7, 8, and 11−14 was determined at equipotent
(IC ) concentrations. For isomers 2/4 and 13/14 the levels of
xPh
reported phenyl-extended Cp complex 17 (IC = 2.14 μM)
5
0
toward A2780 cells, showing that the addition of functional
50
groups to the 2-PhPy ligand can provide a strategy for
increasing potency rather than functionalizing the Cp ligand.
cellular-iridium accumulation are similar, with only small
differences between them. Isomers 7/8 show no difference
between their respective iridium accumulations. However,
isomers 11/12 show contrasting levels of cellular-iridium
accumulation, with 12 being accumulated 6 times more than
x
In contrast, complexes 3, 4, 7, 9, 10, and 11 are less active
toward A2780 cells than parent complex 16. It is apparent that
the presence of hydroxymethyl groups in complexes 9 and 10
decreases the antiproliferative activity. However, the presence
of methyl groups enhances the activity, making them
potentially interesting for further study. Methyl groups are
inherently more likely to contribute to increased lipophilicity
over hydroxymethyl groups, perhaps indicating that hydro-
phobicity is important for high potency.
6
11 (10.2 vs 1.6 ng Ir × 10 A2780 cells).
The hydrophobicity measurements were correlated with
cellular-iridium accumulation in A2780 cells in order to
investigate if hydrophobicity is important in their ability to
enter cells (Figure 6a,b). There is a clear relationship between a
higher hydrophobicity and an increase in cellular accumulation
2
The fluoro complexes 1 and 2, in which the fluorine atom is
situated on the phenyl ring, have promising activity of 2.7 and
(R = 0.969). Isomers 13 and 14 are the most hydrophobic
complexes and accumulate in A2780 cells to the highest extent,
while complex 11, with the lowest hydrophobicity, showed the
lowest accumulation. It was recently demonstrated that the
4
.4 μM, respectively, whereas complexes 3 and 4, in which the
fluorine substituent is on the pyridyl ring, have IC50 values of
50 μM, demonstrating the contrasting biological activity of the
structural isomers. This difference in activity for isomers is also
observed for complexes 11 (IC50 = 47.3 μM) and 12 (IC50
3.29 μM), which contain hydroxyl groups on the phenyl and
5
>
structurally similar iridium(III) complex [(η -Cp*)Ir(7,8-
benzoquinoline)Cl] is transported into cells by both energy-
=
independent passive diffusion and energy-dependent path-
12
1
ways. Since an increase in hydrophobicity results in an
increase in cellular accumulation, it is likely that passive
diffusion plays at least a part in the mechanism of cellular
accumulation for these complexes, making hydrophobicity an
important design feature.
pyridyl ring, respectively, and for complexes 7 (IC50 = 24.73
μM) and 8 (IC50 = 2.7 μM), which bear a nitro group on the
phenyl and pyridyl ring, respectively. Therefore, not only is the
type of substituent important, but also where it is situated on
G
DOI: 10.1021/acs.organomet.5b00097
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Organometallics
Article
in enhanced cellular accumulation for the more hydrophobic
complex 12, which leads to a more potent complex.
Interestingly, although 2 and 8 exhibit low IC50 values (IC50
=
2.7 μM), they show similar levels of cellular accumulation to
their less potent structural isomers 4 (IC50 > 60 μM) and 7
IC50 = 24.73 μM). As the iridium accumulation is performed
(
at IC50 concentrations, this may indicate that the mechanism
involved in the antiproliferative activity of complexes 2 and 8 is
more efficient over their counterparts 4 and 7.
Cellular-Iridium Distribution of Isomers 2 and 4. The
distribution of iridium in A2780 human ovarian cancer cells
after exposure to structural isomers 2 and 4 follows the order of
localization: cytosol > cell membrane and particulate > nucleus
>
cytoskeleton. Although both isomers followed the same
trend, they do so to different extents. Complex 4 localizes in the
cytosol to a greater extent than 2 (69.2% vs 51.4%), whereas 2
localizes in the cell membrane and particulate to a greater
extent than 4 (28.4% vs 17.9%). The variations in localization
preference arising from this small structural change may reflect
32
the differences observed in antiproliferative activity. For
example, if the biological target of these complexes is in the cell
membrane and particulate fraction, then that may explain why
isomer 2 is more active than 4. As the isomers exhibit
dramatically different IC values yet both bind to guanine and
50
accumulate in the nucleus to similar extents, DNA may not be
the main biological target. It has also been shown that halogen-
bonding interactions could be important in rational drug
33
design; therefore the position of fluorine within the ligand
may influence the way the complexes interact with
biomolecules, leading ultimately to variations in activity.
CONCLUSIONS
Interest in iridium(III) half-sandwich complexes as anticancer
agents is increasing, with new complexes being reported and
■
3
a,7,10,11,34
novel mechanisms of action being discovered.
In this
work we have investigated the effects of electron-donating and
electron-withdrawing substituents on C,N-chelating 2-PhPy
ligands on the physiochemical and biological activity of their
complexes.
DFT calculations show that the nature and position of the
substituent have minimal effects on the electronic charge at the
iridium center, Cp*, or chlorido ligands, but exert localized
changes in the electrostatic potential surface and overall
electron density of the chelating ligand. This finding provides
potential for modulating the way the complexes interact with
biological targets such as proteins and oligonucleotides.
All complexes hydrolyze rapidly at 310 K, but total
suppression of aquation can be achieved with 4 mM NaCl,
implying that under physiological conditions the complexes
would exist as the neutral chlorido species. All complexes bind
strongly to the nucleobase 9-EtG, even in the presence of 4
mM NaCl, indicating that DNA could be a target for these
complexes. The observed catalytic oxidation of NADH might
occur in cells and disturb the redox balance and is an interesting
property of some iridium(III) half-sandwich anticancer
Figure 6. (a) Structures of complexes studied. (b) Plot of relative
hydrophobicity, as indicated by retention times on RP-HPLC, against
cellular accumulation of iridium at IC50 concentrations in A2780 cells,
after exposure to complexes 2, 4, 7, 8, and 11−14, showing a
correlation of hydrophobicity with cellular accumulation. (c)
Comparison of cellular accumulation of iridium and antiproliferative
activity of complexes 2, 4, 7, 8, and 11−14 in A2780 cells, showing
that in general higher cellular accumulation results in more potent
complexes.
The cellular-iridium accumulation was correlated with
antiproliferative activity in A2780 cells, which shows that a
pattern exists between enhanced accumulation and increase in
potency (Figure 6c). Complex 11 exhibits the lowest cellular6
accumulation and second lowest potency (1.58 ng Ir × 10
cells, 47.3 μM), and complex 13 exhibits the highest cellular
8
,35
complexes.
The catalytic activity showed dependence on
the substituent present on the 2-PhPy ligand, whereby hydroxyl
substituents slightly enhanced the catalytic ability of the
complex compared to fluorine. The oxidation of NADH
appears to be mediated through the formation of Ir-H species,
6
accumulation and potency (18.46 ng Ir × 10 cells, 1.18 μM).
1
In the case of structural isomers 11 and 12, the difference in
hydrophobicity of the complexes as a consequence of changing
the position of the hydroxyl group on the 2-PhPy ligand results
as detected by H NMR spectroscopy.
The antiproliferative activity of the complexes is strongly
dependent on the type of substituent and its position on the
H
DOI: 10.1021/acs.organomet.5b00097
Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
5
chelating ligand, giving rise to contrasting activities between
structural isomers despite exhibiting similar chemical behavior.
The presence of substituents on the 2-PhPy ligand could in
some cases generate complexes that are more potent than those
bearing the extended CpxPh ligand, thus providing an alternative
strategy for increasing potency in these systems.
Further physicochemical analysis of these complexes showed
that both the nature and position of substituent can determine
their hydrophobicity and, consequently, affect the accumulation
of Ir in A2780 cells. Correlation analysis showed that an
increase in hydrophobicity results in an increase in cellular-Ir
accumulation, which generally results in an increase in potency.
Cellular-Ir distribution in A2780 cells of a pair of structural
isomers that exhibit contrasting biological activity yet similar
levels of Ir accumulation showed that the position of the
substituent affects the extent of localization in cellular
compartments, perhaps indicating that the less active isomer
does not reach the biological target as efficiently as its active
counterpart.
This study shows that small changes in the functionalization
of the chelating ligands in half-sandwich iridium(III) complexes
can have a profound effect on the biological activity of the
complexes. Importantly, it also highlights that the inclusion of
substituents in 2-PhPy ligands can provide complexes with
enhanced potency without the use of extended cyclo-
pentadienyl ligands, a strategy that could be beneficial for the
development of this class of complexes.
Synthesis of [(η -Cp*)Ir(2-(R′-phenyl)-R-pyridine)Cl] Complexes.
The synthesis of complexes 1−15 was performed using the following
4
b
adapted literature procedure. 2-(R′-phenyl)-R-pyridine (2 molar
equiv) was dissolved in anhydrous dichloromethane, sodium acetate (4
molar equiv) was added in one portion, and the mixture was stirred at
5
ambient temperature under nitrogen for 30 min. [(η -Cp*)IrCl ] (1
2
2
molar equiv) was added in one portion, and the reaction mixture
stirred at ambient temperature for 18 h. The mixture was filtered
through Celite under suction, and the filtrate was concentrated to
dryness on a rotary evaporator. The crude product was recrystallized
from chloroform/hexane at 273 K to yield purified complexes.
Characterization data are shown in the Supporting Information.
Methods and Instrumentation. X-ray Crystallography. Dif-
fraction data for complexes 1 and 2 were obtained on an Oxford
Diffraction Gemini four-circle system with a Ruby CCD area detector
using Mo Kα radiation. Absorption corrections were applied using
37
ABSPACK. The crystals were mounted in oil and held at 100 K with
the Oxford Cryosystem Cobra. The structures were solved by direct
methods using SHELXS (TREF) with additional light atoms found
by Fourier methods. Complexes were refined against F using
3
8
2
3
9
SHELXL.
X-ray crystallographic data for complexes 1 and 2 have been
deposited in the Cambridge Crystallographic Data Centre under the
accession numbers CCDC 1043863 and 1043864, respectively.
NMR Spectroscopy. H NMR spectra were obtained in 5 mm NMR
tubes at 298 K (unless stated otherwise) on either Bruker DPX-300,
1
1
Bruker DPX-400, or AV III 600 NMR spectrometers. H NMR
chemical shifts were internally referenced to (CHD )(CD )SO (2.50
2
3
ppm) for DMSO-d , CHCl (7.26 ppm) for chloroform-d , (CHD )-
6
3
1
2
(CD )CO (2.05 ppm) for acetone-d , and 1,4-dioxane (3.75 ppm) for
3
6
13
aqueous solutions. C NMR data were obtained in 5 mm NMR tubes
1
3
at 298 K on a Bruker AV III-500 HD NMR spectrometer. C NMR
chemical shifts were internally referenced to CDCl (77.16 ppm) for
EXPERIMENTAL SECTION
■
3
Materials. IrCl ·nH O (99%) was purchased from Precious Metals
chloroform-d , (CD ) SO (39.52 ppm) for DMSO-d , and (CD ) CO
3
2
1
3
2
6
3 2
Online (Australia). 1,2,3,4,5-Pentamethylcyclopentadiene, NaCl
(29.84 ppm) for acetone-d .The data were processed using TOPSPIN
(version 2.1 Bruker UK Ltd.).
6
(
>99.999%), tetrakis(triphenylphosphine)palladium (99%), phenyl-
boronic acid, 6-bromo-3-pyridinecarboxaldehyde, 2-bromo-4-nitro-
pyridine, 4-formylphenylboronic acid, 4-(hydroxymethyl)-
phenylboronic acid, 4-hydroxyphenylboronic acid, 2-bromo-4-
hydroxymethylpyridine, 2-bromo-4-methylpyridine, 2-bromo-5-fluoro-
pyridine (97%), p-tolylboronic acid (97%), 4-nitrophenylboronic acid
Mass Spectrometry. ESI-MS were obtained on a Bruker Esquire
2000 ion trap spectrometer. Samples were prepared in either MeOH
or CH CN. The mass spectra were recorded with a scan range of
3
either m/z 50−500 or m/z 400−100 for positive ions. Data were
processed using Data Analysis 3.3 (Bruker Daltonics).
(95%), 9-ethylguanine (≥98%), 9-methyladenine (97%), and β-
Elemental Analysis. CHN elemental analyses were carried out on a
CE-440 elemental analayser by Exeter Analytical (UK) Ltd.
pH Measurements. pH and pH* (pH meter reading without
correction for the effect of deuterium on the glass electrode) were
measured at ambient temperature using a miniLab IQ125 pH meter,
pH sensor, and referenced to KCl gel.
nicotinamide adenine dinucleotide-reduced dipotassium salt (≥95%)
were purchased from Sigma-Aldrich (UK). 2-Bromopyridine, 2-
fluorophenylboronic acid, and 4-fluorophenylboronic acid were
purchased from Fisher Scientific (UK), 2-bromo-5-hydroxypyridine
and 2-bromo-5-methylpyridine were from VWR International Ltd.
(
UK), and 2-bromo-3-fluoropyridine was from Tokyo Chemical
HPLC. Purity measurements by HPLC were performed using the
Agilent 1200 system with a VDW and 100 μL loop. The column used
was an Agilent Zorbax Eclipse Plus C18, 250 × 4.6 mm with a 5 μm
Industry Ltd. (UK). Solvents used for synthesis were of laboratory
grade and used without further purification. Solvents for RP-HPLC
(
water and acetonitrile) were of HPLC grade with added trifluoro-
pore size. Mobile phase used was H O 0.1% TFA/MeCN 0.1% TFA at
2
acetic acid (purchased from Sigma-Aldrich (UK)) for purity
gradients of t = 0 min 10% B, t = 30 min 80% B, t = 40 min 80% B, t =
41 min 10% B, and t = 55 min 10% B over a 55 min period. Flow rate
was 1 mL min , and the detection wavelength was set at 254 nm with
5
measurements. [(η -Cp*)IrCl ] was synthesized according to a
2
2
36
−1
literature procedure.
Syntheses. Synthesis of 2-(R′-phenyl)-R-pyridine Ligands. The
the reference wavelength at either 360 or 510 nm. Sample injections
were half the loop volume (50 μL) with needle washes of MeOH and
synthesis of ligands L1−15 was performed using the following adapted
13
literature procedure. Phenylboronic acid/derivatives (1.4 mol equiv)
and 2-bromopyridine/derivatives (1 mol equiv) were dissolved in
H O between injections. It was assumed that all species in a sample
2
have the same extinction coefficient at 254 nm. All peaks were
1:1:1 (v/v) tetrahydrofuran/water/1 M aqueous sodium carbonate
manually integrated to gain the percentage area. Samples were
solution and stirred at ambient temperature under nitrogen for 1 h.
Tetrakis(triphenylphosphine)palladium (2% cat) was added in one
portion, and the reaction mixture was heated under reflux at 373 K for
dissolved in 2.7% DMSO/97.3% H O at ca. 100 μM.
2
Computation. Calculations were performed using the Guassian 03
1
5
package. Geometry optimization calculations were performed in the
1
4a
24 h. The reaction mixture was cooled to ambient temperature and
gas phase with the hybrid functional PBE1PBE. The LanL2DZ basis
14b
extracted with dichloromethane. The organic layer was washed with
saturated sodium hydrogen carbonate solution and brine, then dried
with magnesium sulfate, and concentrated to dryness, yielding the
crude material. Purification by silica flash chromatography was
performed using chloroform/ethyl acetate, yielding purified products.
Characterization data are shown in the Supporting Information.
set and effective core potential were used for the Ir atom, and the 6-
14c
31+G** basis set was used for all other atoms. The nature of all
stationary points was confirmed by performing a normal-mode
analysis. Electrostatic potential surfaces for complexes 2, 4, 7, 8, and
11−13 are shown both in space (with positive and negative regions in
blue and red, respectively) and mapped on electron density (isovalue
I
DOI: 10.1021/acs.organomet.5b00097
Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
0
.04) of the molecules. The electrostatic potential is represented with a
around 80−90% confluence was reached, using 0.25% v/v trypsin for
A2780 and 0.25% v/v trypsin/EDTA for all other cell lines.
color scale ranging from red (−0.040 au) to blue (+0.250 au).
Inductively Coupled Plasma−Optical Emission Spectrometry
In Vitro Growth Inhibition Assay. Briefly, 96-well plates were used
to seed 5000 cells per well; they were left to preincubate in drug-free
media at 310 K for 48 h before adding various concentrations of the
compounds to be tested. Stock solutions of the Ir complexes were first
prepared in 5% v/v DMSO and a mixture of 0.9% w/v saline and
medium (1:1) following serial dilutions in the corresponding cell
culture medium. A drug exposure period of 24 h was allowed. After
this, the supernatant 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 was used to determine cell
viability. This assay, first developed by Skeham et al. in 1990, is based
on the ability of the sulforhodamine B to bind electrostatically to basic
amino acid residues of proteins from fixed cells. Absorbance
measurements of solubilized dye allow the determination of the
amount of viable treated cells against an untreated control. These
measurements were carried out using a BioRad iMark microplate
reader with a 470 nm filter. IC50 values, as the concentration that
causes 50% of inhibition of cell growth, were determined as duplicates
of triplicates in two independent sets of experiments, and their
standard deviations calculated.
(ICP-OES). ICP-OES analyses were carried out on a PerkinElmer
Optima 5300 DV series ICP-OES instrument. The water used for ICP-
OES analysis was doubly deionized (DDW) using a Millipore Milli-Q
water purification system and a USF Elga UHQ water deionizer. The
iridium Specupure plasma standard (Alfa Asar, 1000 pm in 10% HCl)
was diluted with 2.5% HNO DDW to freshly prepare calibrants at
3
concentrations of 1000, 800, 600, 400, 200, and 100 ppb, which were
spiked with NaCl to match the salt content of the samples being
analyzed.
Inductively Coupled Plasma−Mass Spectrometry (ICP-MS). 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 using a Millipore Milli-Q water purification system and a
USF Elga UHQ water deionizer. The iridium Specupure plasma
standard (Alfa Asar, 1000 pm in 10% HCl) was diluted with 2.5%
HNO DDW to prepare calibrants freshly at concentrations of 100, 80,
3
60, 40, 20, 5, 1, 0.8, and 0.5 ppb. The ICP-MS instrument was set to
193
detect Ir using no gas mode.
Hydrolysis Studies. The aquation of complexes (ca. 500 μM) was
1
monitored by H NMR spectroscopy over 24 h in 26.7% MeOD/
7
All spectra were internally referenced to 1,4-dioxane (3.75 ppm), and
water suppression was performed using Shaka techniques. NaCl was
added to aid assignment of species to either the Ir-OD or Ir-Cl
adducts. Percentages of each adduct were calculated based on H
NMR peak integrations.
Metal Accumulation in Cancer Cells. Metal accumulation studies
for complexes 2, 4, 7, 8, and 11−14 were conducted on the A2780
3.3% D O at 310 K using a Bruker AV III 600 NMR spectrometer.
2
6
4
0
ovarian carcinoma cell line. Briefly, 1.5 × 10 cells were seeded on a
six-well plate, after 24 h of preincubation time in drug-free medium, at
2
1
310 K in a 5% CO humidified atmosphere; the test complexes were
2
added to give final concentrations equal to IC50 and then allowed a
further 24 h of drug exposure under similar conditions. After this time,
cells were treated with trypsin and counted, and cell pellets were
collected. Each pellet was digested overnight in concentrated nitric
acid (73%) at 353 K; the resulting solutions were diluted to 5% v/v
Reactions with DNA Nucleobases. Interactions of complexes (ca.
500 μM) with 9-ethylguanine and 9-methyladenine (ca. 500 μM) were
1
studied by H NMR spectroscopy for periods of 15 min and 24 h after
addition of the nucleobase(s) in 26.7% MeOD/73.3% D O at 310 K
using a Bruker AV III 600 NMR spectrometer.
Hydrophobicity Determinations. Relative hydrophobicity measure-
ments by HPLC were performed using the Agilent 1200 system with a
VDW and 100 μL loop. The column used was an Agilent Zorbax
Eclipse Plus C18, 150 × 4.6 mm with a 5 μm pore size. Mobile phase
used was H O 50 mM NaCl/H O/MeCN 1:1 50 mM NaCl at
2
HNO using doubly deionized water, and the amount of iridium taken
3
up by the cells was determined by ICP-MS, using an Agilent
Technologies 7500 series instrument. The solvent used for all ICP-MS
experiments was DDW with 5% v/v HNO . Metal standards (Ir) were
3
freshly prepared before each experiment. The concentrations used for
the calibration curve were 0, 0.5, 0.8, 1, 5, 20, 40, 60, 80, and 100 ppb.
The isotope detected was 1 Ir; readings were made in duplicate (no-
gas mode). These experiments did not include any cell recovery time
in drug-free media. They were all carried out in triplicate in two sets of
independent experiments, and the standard deviations were calculated.
Metal Distribution in A2780 Cells for Complexes 2 and 4. Cell
pellets were obtained as described above and were fractionated using
the FractionPREP kit from BioVision according to the supplier’s
instructions. Each sample was digested overnight in concentrated nitric
acid (73%), and the amount of Ir taken up by the cells was determined
by ICP-MS. These experiments were all carried out in triplicate, and
the standard deviations were calculated. Statistical significance between
results was established from the Student’s t test, where ** = p < 0.01.
2
2
93
gradients of t = 0−20% B, t = 15 min 100% B, t = 40 min 100% B, t =
4
2 min 20% B, and t = 57 min 20% B over a 57 min period. Flow rate
−1
was 1 mL min , and the detection wavelength was set at 254 nm with
the reference wavelength at 360 nm. Sample injections were half the
loop volume (50 μL) with needle washes of H O between injections.
Samples were dissolved in 10% MeOH/90% H O in 50 mM NaCl at
ca. 100 μM. Reported retention times (t ) and standard deviations
2
2
R
(
SD) are from duplicates of triplicate measurements.
+
Catalytic Oxidation of NADH to NAD . Complexes were evaluated
+
for the catalytic oxidation of NADH to NAD by UV−visible
spectroscopy over a 24 h period in 0.5% MeOH/99.5% H O at 310 K
in 5 mM Na HPO /NaH PO buffer, pH 7.5. The complex
concentration remained fixed at 2.5 μM with NADH concentrations
of 69, 103, 127, and 146 μM. The conversion of NADH to NAD was
followed by absorption at 339 nm (εNADH = 6220 cm M ) to allow
evaluation of kinetic data.
2
2
4
2
4
+
−1
−1
ASSOCIATED CONTENT
■
*
S
Supporting Information
The reaction between complex 2 (250 μM) and NADH (750 μM)
Selected X-ray crystallographic data for complexes 1 and 2
1
in 50% MeOD/50% H O at 310 K, pH* 7.4, was monitored by H
NMR spectroscopy.
2
(
Table S1), calculated bond lengths and HOMO and LUMO
orbital diagrams and energies (Tables S2 and S3), intermo-
lecular stacking features (Figure S1), aqueous chemistry and
nucleobase binding (Figures S2 and S3), NADH reaction
kinetics and hydride formation (Figures S4 and S5), relative
hydrophobicity HPLC traces (Figure S6), and Cartesian
coordinates determined by DFT calculations are available in
the SI. Cartesian coordinates for DFT calculations are also
supplied in .xyz format. These materials are available free of
charge via the Internet at http://pubs.acs.org. X-ray crystallo-
graphic data in CIF format are available from the Cambridge
Crystallographic Data Centre (http://www.ccdc.cam.ac.uk).
Cell Experiments. The cell lines used in this work, A2780 human
ovarian carcinoma, A549 human Caucasian lung carcinoma, HCT116
human colon carcinoma, and MCF7 human Caucasian breast
carcinoma, were obtained from the European Collection of Cell
Cultures (ECACC). A2780 ovarian cells were grown in Roswell Park
Memorial Institute medium (RPMI-1640), A549 and MCF7 in
Dulbecco’s modified Eagle medium (DMEM), and HCT116 in
McCoy’s modified 5A medium. All media were supplemented with
1
0% v/v fetal calf serum, 1% v/v 2 mM glutamine, and 1% v/v
penicillin/streptomycin.
All cells were grown in 75 cm2 culture flasks as adherent
monolayers, and they were split two to three times a week when
J
DOI: 10.1021/acs.organomet.5b00097
Organometallics XXXX, XXX, XXX−XXX

Organometallics
Article
(
7) Almodares, Z.; Lucas, S. J.; Crossley, B. D.; Basri, A. M.; Pask, C.
AUTHOR INFORMATION
■
*
M.; Hebden, A. J.; Phillips, R. M.; McGowan, P. C. Inorg. Chem. 2014,
Corresponding Author
E-mail: P.J.Sadler@warwick.ac.uk.
5
3, 727−736.
(8) (a) Liu, Z.; Deeth, R. J.; Butler, J. S.; Habtemariam, A.; Newton,
M. E.; Sadler, P. J. Angew. Chem., Int. Ed. 2013, 52, 4194−4197.
b) Betanzos-Lara, S.; Liu, Z.; Habtemariam, A.; Pizarro, A. M.;
Qamar, B.; Sadler, P. J. Angew. Chem., Int. Ed. 2012, 51, 3897−3900.
9) Romero-Canelon, I.; Sadler, P. J. Inorg. Chem. 2013, 52, 12276−
2291.
10) Liu, Z.; Romero-Canelon, I.; Qamar, B.; Hearn, J. M.;
Habtemariam, A.; Barry, N. P. E.; Pizarro, A. M.; Clarkson, G. J.;
Sadler, P. J. Angew. Chem., Int. Ed. 2014, 53, 3941−3946.
(11) (a) Ruiz, J.; Rodriguez, V.; Cutillas, N.; Samper, K. G.;
Capdevila, M.; Palacios, O.; Espinosa, A. Dalton Trans. 2012, 41,
12847−12856. (b) Geldmacher, Y.; Splith, K.; Kitanovic, I.; Alborzinia,
H.; Can, S.; Rubbiani, R.; Nazif, M.; Wefelmeier, P.; Prokop, A.; Ott,
Notes
(
The authors declare no competing financial interest.
(
́
1
ACKNOWLEDGMENTS
■
(
́
We thank the ERC (grant no. 247450), EPSRC (grant no. EP/
F034210/1), and Science City (AWM/ERDF) for support and
members of EU COST Action CM1105 for stimulating
discussions. We thank Prof. Rob Deeth for advice on DFT
1
3
calculations, Dr. Ivan Prokes for acquiring C NMR data, and
the Dalton division of the RSC for a travel grant for A.J.M. to
present this research at AsBIC7.
I.; Wo
31−646.
12) Novohradsky, V.; Liu, Z.; Vojtiskova, M.; Sadler, P. J.; Brabec,
V.; Kasparkova, J. Metallomics 2014, 6, 682−690.
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(
DEDICATION
■
(
We dedicate this article to Mike Lappert, who inspired
organometallic chemists over many years.
2
(
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DOI: 10.1021/acs.organomet.5b00097
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