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The contrasting chemical reactivity of potent isoelectronic iminopyridine and azopyridine osmium(ii) arene anticancer complexes
Original citation:
Fu, Y. (Ying), Romero, Maria, Habtemariam, Abraha, Snowden, Michael E., Song,
Lijiang, Clarkson, Guy J., Qamar, Bushra, Pizarro, Ana M., Unwin, Patrick R. and Sadler,
Peter J.. (2012) The contrasting chemical reactivity of potent isoelectronic iminopyridine
and azopyridine osmium(ii) arene anticancer complexes. Chemical Science, Vol.3
(No.8). pp. 2485-2494.
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The contrasting chemical reactivity of potent isoelectronic
iminopyridine and azopyridine osmium(II) arene anticancer complexes
Ying Fu, María J. Romero, Abraha Habtemariam, Michael E. Snowden, Lijiang Song, Guy J. Clarkson,
Bushra Qamar, Ana M. Pizarro, Patrick R. Unwin and Peter J. Sadler*
5
Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X
First published on the web Xth XXXXXXXXX 200X
DOI: 10.1039/b000000x
A wide variety of steric and electronic features can be incorporated into transition metal
coordination complexes, offering the prospect of rationally-designed therapeutic agents with novel
10 mechanisms of action. Here we compare the chemical reactivity and anticancer activity of
organometallic OsII complexes [Os(η6-arene)(XY)Z]PF6 where arene = p-cymene or biphenyl, XY =
N,N΄-chelated phenyliminopyridine or phenylazopyridine derivatives, and Z = Cl or I. The X-ray
crystal structure of [Os(η6-p-cym)(Impy-OH)I]PF6⋅0.5CH2Cl2⋅H2O (Impy-OH = 4-[(2pyridinylmethylene)amino]-phenol) is reported. Like the azopyridine complexes we reported
15 recently (Dalton Trans. 2011, 40, 10553-10562), some iminopyridine complexes are also potently
active towards cancer cells (nanomolar IC50 values). However we show that, unlike the azopyridine
complexes, the iminopyridine complexes can undergo aquation, bind to the nucleobase guanine, and
oxidize coenzyme nicotine adenine dinucleotide (NADH). We report the first detection of an Oshydride adduct in aqueous solution by 1H NMR (-4.2 ppm). Active iminopyridine complexes
20 induced a dramatic increase in the levels of reactive oxygen species (ROS) in A549 lung cancer
cells. The anticancer activity may therefore involve interference in the redox signalling pathways in
cancer cells by a novel mechanism.
Introduction
25
Transition metal coordination complexes offer a variety of
electronic and structural features for the design of therapeutic
agents, including the choice of the metal and its oxidation
state, the number and types of coordinated ligands, and their
geometries.1-9 The heavier, third-row (5d)
30 coordination
transition elements in particular tend to exhibit slow ligand
exchange kinetics and so might reach biological target sites
with at least some of the initial ligands still bound.
Organometallic arene and cyclopentadienyl complexes are
10-13
35 attractive
since their potential amphiphilicity can be
beneficial for drug transport and target recognition. Subtle
changes in structure can lead to dramatic changes in chemical
reactivity and biological activity. For example, certain inert
N,N΄-chelated half-sandwich organometallic iodido Os II arene
40 azopyridine complexes exhibit potent in vitro and in vivo
anticancer activity.14, 15 Their mechanism of action is different
from that of N,O-chelated Os II arene picolinate complexes
which can hydrolyze and then bind to DNA bases, 16 with
DNA being a likely target, as it is for cisplatin.17,18 In
II
45 contrast, with the strong π-acceptor azopyridine present, Ru
arene complexes with iodide as the monodentate ligand are
relatively inert towards substitution reactions and appear to be
activated by redox reactions, for example with glutathione.19
Other than glutathione, the couple NADH and oxidized
50 nicotinamide
adenine dinucleotide (NAD+) is also an
important part of the cellular redox balance system.20 Under
This journal is © The Royal Society of Chemistry [year]
physiological conditions, the ratio of [NADH]/[NAD+] in cells
is low.21 The cellular concentration of free NADH is in the
nanomolar range,21 which makes it a potential and sensitive
55 target for anticancer drugs.
Early work by Steckhan22 and Fish23 demonstrated an
efficient process for the regeneration of NADH from NAD +
using formate as an hydride source and rhodium catalysts.
Similarly we have shown that ruthenium arene anticancer
+
60 complexes can reduce NAD
regioselectively to NADH
through transfer hydrogenation.24 Recent studies have shown
that both organometallic Ru II arene complexes and Ir III Cp*
complexes can oxidize NADH to NAD+.25-27 However, there
are no reports of osmium complexes as transfer hydrogenation
65 catalysts in aqueous solution.
There are some intriguing differences between the chemical
properties of organometallic Ru II and OsII arene complexes
even though their three-dimensional structures can be almost
identical.28 For example, complexes of OsII (a low-spin 5d6
16, 29
70 metal ion) are often more inert,
and aqua adducts of OsII
16
arenes are more acidic. Several organometallic osmium
anticancer complexes have been designed based on their
ruthenium analogues.30-32 Studies of both RuII and OsII
azopyridine anticancer complexes have been reported.
75 However, no anticancer studies of ruthenium arene complexes
with iminopyridine ligands have been reported. Osmium(II)
arene phenylazopyridine complexes exhibit anticancer activity
in the nanomolar range in vitro.14 Moreover the complex
([Os(η6-p-cym)(Azpy-NMe2)I]PF6 (FY026) is active against
Journal Name, [year], [vol], 00–00 | 1
coloreactal cancer in vivo and has low toxicity.15 The
exploration of bioisosterism provides a useful approach to the
study of the anticancer activity33 of osmium arene complexes.
Here we compare the chemical reactivity and biological
II
5 activity of Os
arene azopyridine (N=N) complexes with
isoelectronic iminopyridine (HC=N) complexes. Anticancer
activity has been studied in A2780 ovarian cancer cells as
well as in the NCI 60-cell line panel. To investigate possible
redox mechanisms, the production of ROS in lung cancer cells
studied as well as the influence of combined
10 was
administration with the glutathione synthase inhibitor Lbuthionine-sulfoximine (L-BSO).34 Attempts are made to
correlate biological activity with chemical reactivity through
studies of aquation, binding to the nucleobase guanine, and
15 redox reactions with glutathione and NADH. The data suggest
that these osmium arene complexes possess novel mechanisms
of action that can be finely tuned through the choice of the
ligands.
20
Results
PF6
Arene
R2
X
Os
N
25
R1
N
CH
30
p -cym
bip
70
Complex
1
2
3
4
5
Arene
bip
p-cym
bip
p-cym
bip
R1
H
H
H
H
H
R2
H
H
OH
OH
NMe2
X
I
I
I
I
I
Chelating ligand
Impy
Impy
Impy-OH
Impy-OH
Impy-NMe2
6
p-cym
H
NMe2
I
Impy-NMe2
7
bip
OMe
NMe2
I
OMe-Impy-NMe2
8
9
10
11
12
13
p-cym
bip
p-cym
bip
p-cym
bip
OMe
H
H
H
H
H
NMe2
H
H
OH
OH
NMe2
I
Cl
Cl
Cl
Cl
Cl
OMe-Impy-NMe2
Impy
Impy
Impy-OH
Impy-OH
Impy-NMe2
14
p-cym
H
NMe2
Cl
Impy-NMe2
15
16
bip
p-cym
OMe
OMe
NMe2
NMe2
Cl
Cl
OMe-Impy-NMe2
OMe-Impy-NMe2
Chart 1 Osmium (II) arene iminopyridine complexes
synthesized and studied in this work.
35
2 | Journal Name, [year], [vol], 00–00
Synthesis and characterization
Sixteen osmium(II) arene iminopyridine complexes (Chart
1) of general formula [Os(η6-arene)(R1-Impy-R2)X]PF6 [arene =
40 biphenyl (bip) or p-cymene (p-cym); X = Cl or I] containing
different chelating iminopyridine ligands [Impy (R1= R2= H),
Impy-OH (R1= H, R2= OH), Impy-NMe2 (R1= H, R2= NMe2) and
OMe-Impy-NMe2 (R1= OMe, R2= NMe2)] were synthesized. The
general method involved stirring an osmium dimer and the
chelating iminopyridine ligand (synthesized
45 appropriate
following a literature method)35 in a methanol solution, similar to
that reported previously for the synthesis of the azopyridine
analogues.14 In general, the iminopyridine complexes were
obtained in good yields and were well characterized by 1H-NMR
50 spectroscopy, mass spectrometry and CHN elemental analysis
(for details see ESI).
Half-sandwich organometallic arene complexes containing
an unsymmetrical chelating ligand and a monodentate ligand, are
chiral. 1H NMR spectra recorded before and after adding the
55 chiral anionic shift-reagent ∆–trisphat to a solution of complex 14
in CDCl3, 298 K, showed a splitting of the peaks (Figure S1),
indicating the presence of two enantiomers in ca. 1:1 ratio,
consistent with a previous report on osmium/ruthenium arene
picolinamide anticancer complexes.17 No attempt was made to
60 separate the enantiomers, although the chirality at the metal
centre may affect anticancer activity if the biological target is
chiral.
Recrystallization of [Os(η6-p-cym)(Impy-OH)I]PF6 (4) from
DCM/methanol afforded purple single crystals corresponding to
65 4⋅0.5CH2Cl2⋅H2O suitable for X-ray diffraction (Figure 1).
Crystallographic data are listed in Table S1 and selected bond
lengths and angles in Table S2. The asymmetric unit contains two
crystallographically independent cations [Os(η6-p-cym)(ImpyOH)I]+ comprising of either enantiomer.
75
80
85
Fig. 1 X-ray structure of the cation [Os(η6-p-cym)(ImpyOH)I]+ in crystals of 4⋅0.5CH2Cl2⋅H2O showing the atom
numbering scheme. The thermal ellipsoids are drawn
at 50%
probability. The hydrogen atoms, counterion (PF6 ), and solvent
molecules have been omitted for clarity.
90
In addition, the two enantiomers are partially solvated by one
dichloromethane and two water molecules in the asymmetric unit.
The osmium complex shows a pseudo-octahedral “piano-stool”
structure with the p-cymene ligand π-bonded to the metal ion and
This journal is © The Royal Society of Chemistry [year]
the Impy-OH chelating ligand coordinated through the pyridine
and imine nitrogen atoms. The imine bond adopts an E
conformation, minimising the steric hindrance arising from the
bulky phenol group. The coordination sphere of the OsII ion is
5 completed by a terminal monodentate iodide. The Os−N
(2.077(4)-2.087(4) Å), Os−I (2.7091(4) Å, 2.7247(4) Å) and
Os−arene centroid bond lengths (1.6845(2) Å, 1.6962(2) Å) in
this complex are similar to those of the osmium analogues
previously reported.16 The crystal structure of 4⋅0.5CH2Cl2⋅H2O
6
10 shows differences from the reported crystal structure of [Os(η 14
bip)(Azpy-O)I]⋅0.5H2O. In complex 4⋅0.5CH2Cl2⋅H2O there is
no deprotonation of the hydroxyl group on the chelating ligand
which allows both molecules to establish an hydrogen bond
between the phenolic OH group of the iminopyridine ligand and a
15 water molecule [O115-H11H⋅⋅⋅O400 1.84 Å, O215-H21H⋅⋅⋅O300
1.89 Å] as shown in Figure S2.
Aquation, pKa of aqua complexes and binding to 9ethylguanine
20
The possible activation of these complexes by aquation and
their interaction with 9-ethylguanine (9-EtG), a model DNA
nucleobase, were studied by 1H NMR spectroscopy. It was found
that the osmium iminopyridine complexes [Os(η6-p-cym)(Impy6
25 NMe2)I]PF6 (6) and [Os(η -p-cym)(Impy-NMe2)Cl]PF6 (14), can
undergo aquation. After incubation of the samples at 310 K for 24
h in D2O, the extent of aquation reached 50% and 99%,
respectively. The formation of the aqua product (14A) was
confirmed by the presence of new signals in the 1H NMR spectra
30 shifted with respect to the signals observed for 14 (Figure S3).
These signals were confirmed as corresponding to the aqua
adduct after repeating the experiment in the presence of 500 mM
NaCl where only one set of signals for the chlorido complex was
observed.
35
40
45
50
60
for the coordinated water and 2.3 (pKa 2.6) for the dimethylamine
substituent; (B) Reaction of the hydroxido complex 14A (1 mM)
with 9-ethylguanine (2 mM, 2 mol equiv) in 10%D2O/90%H2O
phosphate buffer, pH* 7.4, followed by 1H NMR. The spectra
were recorded 10 min after preparing the mixture and after
incubation for 18 h and 38 h at 310 K.
65
The pKa value of the aqua adduct (14A) was determined by
H NMR spectroscopy (Figure 2A). The chemical shifts of the 1H
NMR signals gradually shifted upfield with increase in pH* from
c.a. pH* 1.5 to 13. A plot of the chemical shift of the pyridine
*
70 proton Ha against pH was fitted to the Henderson-Hasselbalch
equation, giving a pKa value of 5.2.36 The low pKa value obtained
for the aqua adduct suggests that the hydroxido adduct will be the
predominant species under physiological conditions. The pKa
value of 2.6 for the dimethylamine substituent on the
75 iminopyridine chelating ligand Impy-NMe2 is the first reported
pKa of an NMe2 substituent in this type of ligand bound to metal
(Figure 2A).
To study the potential for DNA binding, the reaction of 14A
with 9-EtG in 0.1 M phosphate buffer (10%D2O/90%H2O, pH*
1
80 7.4) was monitored over various time intervals by
H NMR
spectroscopy after incubation of the solution at 310 K. 9-EtG
binding was confirmed by the appearance of a new set of peaks
assignable to a 14-9-EtG adduct shifted downfield in comparison
to the peaks of 14A, while the signals corresponding to
85 coordinated 9-EtG shifted upfield with respect to the free
nucleobase. The extent of binding reached only 14% after 18 h
and increased only slightly to 19% after another 20 h of
incubation at 310 K (Figure 2B).
1
90
Anticancer Activity
The activity of all sixteen osmium arene iminopyridine
complexes towards the A2780 human ovarian cancer cell line
was determined and of selected complexes towards the A549
95 human lung cancer cell line (Table 1). They exhibit a broad range
of anticancer activity with IC50 values (the concentration that
inhibits cell growth by 50%) towards A2780 cells ranging from
0.14 µM for [Os(η6-bip)(Impy-NMe2)I]PF6 (5) to 35.5 µM for
[Os(η6-p-cym)(OMe-Impy-NMe2)I]PF6 (8) (Table 1A). When the
100 ortho -OMe substituent on the pyridine ring in 8 was replaced by
H in [Os(η6-p-cym)(Impy-NMe2)I]PF6 (6), the anticancer activity
increased 44-fold (IC50 decreased from 35.5 µM to 0.80 µM).
This trend was also observed when biphenyl is the arene and the
monodentate ligand is chloride where a 35-fold increase in
105 anticancer activity was observed. Anticancer efficacy was also
observed towards A549 cells. A significant enhancement in
activity was obtained by combination treatment with Lbuthionine-sulfoximine (L-BSO), a specific inhibitor of γglutamylcysteine synthetase (Table 1B). L-BSO depletes
110 intracellular glutathione, which plays an important role in the
maintenance of the redox balance in cancer cells.
55
Fig. 2 (A) Dependence of the 1H NMR chemical shift of the
pyridine ortho proton Ha on pH* in complex 14A. The line
represents a computer best fit giving pKa* values of 5.1 (pKa 5.2)
This journal is © The Royal Society of Chemistry [year]
115
Journal Name, [year], [vol], 00–00 | 3
Table 1 Anticancer activity in vitro towards (A) A2780
human ovarian cancer cells, (B) A549 human lung cancer cells
and combination treatment with 50 µM L-BSO, (C) NCI 60-cell
line screening (data for 58 cell lines) of complexes 6 and 14.
5
(A) A2780 Ovarian Cancer Cells
Complex
IC50(µM)
(1) [Os(η -bip)(Impy)I]PF6
(2) [Os(η6-p-cym)(Impy)I]PF6
(3) [Os(η6-bip)(Impy-OH)I]PF6
(4) [Os(η6-p-cym)(Impy-OH)I]PF6
(5) [Os(η6-bip)(Impy-NMe2)I]PF6
(6) [Os(η6-p-cym)(Impy-NMe2)I]PF6
(7) [Os(η6-bip)(OMe-Impy-NMe2)I]PF6
(8) [Os(η6-p-cym)(Ome-Impy-NMe2)I]PF6
(9) [Os(η6-bip)(Impy)Cl]PF6
(10) [Os(η6-p-cym)(Impy)Cl]PF6
(11) [Os(η6-bip)(Impy-OH)Cl]PF6
(12) [Os(η6-p-cym)(Impy-OH)Cl]PF6
(13) [Os(η6-bip)(Impy-NMe2)Cl]PF6
(14) [Os(η6-p-cym)(Impy-NMe2)Cl]PF6
(15) [Os(η6-bip)(OMe-Impy-NMe2)Cl]PF6
(16) [Os(η6-p-cym)(OMe-Impy-NMe2)Cl]PF6
Cisplatin
18.6 (±0.9)
29.4 (±5.3)
5.4 (±1.1)
31.8 (±3.8)
0.14 (±0.01)
0.80 (±0.05)
9.14 (±0.93)
35.5 (±3.2 )
4.6 (±0.4)
26.2 (±2.8)
2.4 (±1.1)
5.5 (±0.6)
0.44 (±0.01)
1.50 (±0.047)
9.50 (±0.22)
32.9 (±1.2)
1.8 (±0.1)
6
Cancer Institute (DTP of NCI), which includes nine tumour-type
subpanels. The mean values of IC50, TGI (the concentration
which inhibits cell growth by 100 %) and LC50 (the concentration
that kills original cells by 50 %) are listed in Table S3. Both
complexes showed anticancer activity within the same range as
25 cisplatin (Table 1C). Complexes 6 and 14 showed a broad
spectrum of activity, with IC50 values ranging from 0.5 µM to
more than 100 µM, and a particular selectivity for melanoma and
breast cancer cells. The high activity towards MDA-MB-468
breast cancer cells is particularly notable, with some IC50 values
30 in the nanomolar-micromolar range (0.46 µM for 6 and 2.06 µM
for 14).
Both complex 14 and cisplatin (CDDP) showed higher
activity towards A549 human lung cancer cells (8.7 µM for
cisplatin and 6.7 µM for 14, Figure 3) compared to MRC-5
35 human fetal lung fibroblast-like cells (IC50 values of 16.6 µM and
51.3 µM, respectively). Hence the ratio of IC50 values in normal
cells compared to this cancer cell line is ca. 2 for cisplatin but
increases to ca. 8 for complex 14.
20
40
45
(B) A549 Lung Cancer Cells
50
Complex
IC50(µM)
IC50(µM) with L-BSO
3
4.55(±1.09)
1.59(±0.36)
6
3.7(±0.2)
0.7 (±0.1)
9
>100
25.88(±12.29)
10
0.9(±0.09)
NAa
13
2.55(±1.35)
NAa
14
Cisplatin
6.65(±0.06)
8.68(±2.11)
2.34(±1.18)
NAa
55
60
10
(C) NCI 60-Cell Line
Complexb
IC50(µM)
TGI(µM)
LC50(µM)
6
3.72
13.2
49
8.3
33.9
75.8
14
b
Cisplatin
10.3
50.7
90.5
a
NA = not acquired. b Data from NCI/DTP screening. 6 =
NSC755639; 14 = NSC755640. Mean-graph midpoint (MGMID) for IC50, TGI and LC50 values of NCI all cell panels.
15
The potency of complexes [Os(η6-p-cym)(Impy-NMe2)I]PF6
(6) and [Os(η6-p-cym)(Impy-NMe2)Cl]PF6 (14) towards A2780
cancer cells was similar to that of the anticancer drug cisplatin
and so were further screened in the human tumour 60-cell line
panel of the Developmental Therapeutics Program of the National
4 | Journal Name, [year], [vol], 00–00
Fig. 3 In vitro cytotoxicity data for cisplatin and complex 14
towards A549 human lung cancer cells and MRC-5 human fetal
lung fibroblast-like cells.
Induction of Reactive Oxygen Species (ROS) in Lung Cancer
Cells
OsII arene azopyridine complexes can increase ROS levels in
cancer cells.14 Consequently, the effect of iminopyridine
complexes on cellular ROS levels was also investigated. The
65 level of ROS induced in A549 human lung cancer cells by
osmium complexes 6 and 14 was monitored using the probe 2,7dichlorodihydrofluorescein-diacetate
(DCFH-DA).
This
hydrolyzes to 2,7-dichlorodihydrofluorescein (DCFH) in live
cells, and in turn is oxidized to 2,7-dichlorofluorescein (DCF) in
37, 38
70 the presence of ROS, exhibiting a green fluorescence.
Using
this probe, the level of general oxidative stress induced in A549
cells by 6 and 14 was determined. The accumulation of ROS
during combined exposure to 6 or 14 with L-BSO was also
studied.
75
This journal is © The Royal Society of Chemistry [year]
60
still observable after 30 h. However, no hydride peak was
detected after 45 h. This suggested that the oxidation of NADH in
the presence of 14 occurs via hydride transfer to Os but the
resulting Os-H adduct is unstable and decomposes.
5
65
10
70
15
Fig. 4 Time dependence of the ROS level in A549 human
lung cancer cells after treatment with 6 (4 µM), 14 (4 µM), LBSO (50 µM), 6 or 14 combined with L-BSO (50 µM), and H2O2
(10 µM, positive control) during 4 h at 310 K. C= control.
In A549 cells, a relative increase in DCF fluorescence was
detected over a period of 4 h after treating the cells with 6 (4
µM), 14 (4 µM), 6 (4 µM) plus L-BSO (50 µM), 14 (4 µM) plus
L-BSO (50 µM) or 10 µM H2O2 for comparison (Figure 4). After
treatment only with 6 or 14, the ROS level increased dramatically
compared to the control and even the positive control (H2O2, 10
25 µM). This level increased further in the presence of L-BSO. The
further increase of ROS by the combination treatment may
explain why L-BSO can enhance the cytotoxicity of complex 6
and 14 towards A549 cancer cells significantly (Table 1B). LBSO alone at a dose of 50 µM had no significant effect on the
30 increase of ROS (C+L-BSO, Fig.4) and the growth of A549
cancer cells, but greatly enhanced the cytotoxicity of osmium
iminopyridine complexes towards A549 cancer cells.
20
Oxidation of NADH to NAD+
35
After observing the increase in ROS levels in cells induced
by osmium iminopyridine complexes, we investigated reactions
of the complexes with potential cellular reducing agents. It was
found that 6 and 14 do not catalytically oxidize GSH, in contrast
19
40 to their ruthenium azopyridine iodido analogues (Figure S4).
+
Since NADH/NAD is an important redox couple which
maintains the redox balance in cells, reactions with NADH were
also studied. Reactions of NADH with complexes 6, 8, 14, 14A
and 16 were followed by 1H NMR spectroscopy. These
+
45 complexes oxidized NADH to NAD to different extents: 2.0 ±
0.2 mol equiv of NADH per mol of 14A, 1.6 ±0.1 for 6 and 14,
and 1.2 ±0.1 for 8 and 16, after incubation in a
10%MeOD/90%D2O phosphate buffer solution (pH* 7.4) for 24
h at 310 K. Under these conditions the azopyridine complex
6
50 [Os(η -p-cym)(Azpy-NMe2)I]PF6
(FY026) does not oxidize
NADH (Figures S5 and S6). The control NADH was stable
towards NAD+ formation under the same conditions at milimolar
concentrations. Since more than one mol equiv of NADH was
oxidized per mol equiv of osmium complex, this implies a
1
55 catalytic mechanism. The H NMR spectrum for the reaction with
14A at 310 K (10%D2O/90%H2O phosphate buffer) gave a weak
signal at –4.2 ppm (Figure 5) assignable to an osmium hydride
adduct. This signal was detected after 27 h of reaction and was
This journal is © The Royal Society of Chemistry [year]
75
Fig. 5 Decay of the Os-H 1H NMR peak from reaction of the
hydroxido complex 14A (2.5 mM) with 4 mol equiv of NADH in
*
80 10%D2O/90%H2O phosphate buffer, pH
7.4 after different
periods of incubation at 310 K.
LC-MS was also employed to analyse the products from the
reaction between NADH (0.5 mM) and 14A (0.5 mM) after
85 incubation at 310 K for 24 h. The decrease in intensity of the
peak with a retention time of ca. 11.5 min (NADH; calcd for
C21H30N7O14P2 m/z = 666.1, found m/z = 666.1) and the
appearance of a new peak with a retention time of 4.8-5.2 min
(NAD+ calcd for C21H28N7O14P2 m/z = 664.1, found m/z = 664.1)
90 were observed after the reaction with 14A (Figure S7).
The reaction of the chlorido complex 14 with NADH (8 mol
equiv) was monitored by UV-Vis over 18 h at 310 K in a
phosphate buffer pH 7.4 (Figure 6). The kinetic experiment
showed an isosbestic point at 300 nm corresponding to the
+
95 oxidation of NADH to NAD and another isosbestic point at 432
nm related to the aquation of 14, suggesting that the aquation of
the chlorido complex 14 accompanies the oxidation. The
formation of NAD+ was confirmed by a decrease in intensity of
the characteristic NADH band at 338 nm and the simultaneous
100 increase in intensity at 260 nm.
105
110
115
Fig. 6 (A) UV-Vis spectra for the reaction between the
chlorido complex 14 (0.025 mM) and NADH (8 mol equiv) in 1
mM phosphate buffer (pH* 7.4) during 18 h at 310 K. The spectra
Journal Name, [year], [vol], 00–00 | 5
were recorded at intervals of 20 min. Inset: (B) Dependence of
the turnover number (TON) of complex 14 (0.025 mM) on the
mol ratio of NADH. The reaction was followed by the decrease in
intensity of the NADH absorption band at 338 nm.
5
The dependence of the oxidative activity of 14 on the
concentration of NADH in solution was studied by following the
conversion of 2 and 8 mol equivalents of NADH to NAD+
(Figure S8A) under the same conditions described above. The
10 rate of reaction of 14 (25 µM) was similar for reactions of 2 and 8
mol equiv NADH suggesting that the formation of an initial
adduct is rate-limiting. An increase in NADH concentration
resulted in higher turnover numbers for complex 14 (Figure 6B).
The NAD+/NADH ratio after 18 h was determined to be 0.14
15 for the reaction between 14 and 2 equiv of NADH, 0.11 for 4
equiv, 0.12 for 6 equiv and 0.13 for 8 equiv, respectively. The
rate of oxidation of NADH (8 mol equiv) was the same in the
presence of either 25 µM or 10 µM 14 (Figure S8B). The control
solutions showed a slight decomposition of the NADH under
20 these conditions attributable to the effect of phosphate buffer,
consistent with previous reports.39, 40
Since the instability of the osmium hydride adduct formed
by reaction with NADH might be due to protonation and
liberation of H2,41 an attempt was made to detect H2 as a product
25 by gas chromatography. However, none was detected (for
conditions see ESI).
The reaction between osmium complex 14 (0.1 mM) and
NADH (0.4 mM) under aerobic and anaerobic (bubbling argon
for 10 min) conditions was compared as monitored by 1H NMR
30 spectroscopy (18-h incubation, 310 K in 10%MeOD/90%D2O
phosphate buffer, pH* 7.4). Under argon there was almost no
conversion of NADH to NAD+ (3 ± 0.5% compared to 17 ± 1%
in air; Figure 7). It can therefore be concluded that O2 plays a role
in the conversion of NADH to NAD+.
35
After saturating a solution containing 14 (0.1 mM) and
NADH (0.5 mM) with O2 (bubbling for 10 min), the conversion
+
60 of NADH to NAD increased from 10% (aerobic) to 25% (O2saturated), whereas such saturation had little effect on a similar
control solution of NADH alone (no significant NAD+ formation
after O2 saturation and incubation for 18 h at 310 K; Figure 8).
65
70
75
80
Fig. 8 1H NMR spectra for the reaction between the osmium
complex 14 (0.1 mM) and NADH (5 mol equiv.) (A) under
oxygen saturation, (B) in air, (C) NADH alone under oxygen
*
85 saturation. Solvent: 10%MeOD/90%D2O phosphate buffer, pH
7.4). Incubation conditions: 18 h at 310 K.
To investigate whether aquation is a necessary step for 14 to
oxidize NADH to NAD+, the reaction of 14 (0.1 mM) and NADH
90 (0.4 mM) was carried out under three conditions: (1) without
NaCl, (2) with 100 mM NaCl (3) with 500 mM NaCl. The
resulting NMR spectra after 24 h incubation at 310 K are shown
in Figure 9.
95
40
100
45
105
50
Fig. 7 1H NMR spectra for the reaction between osmium
complex 14 (0.1 mM) and NADH (4 mol equiv) in
10%MeOD/90%D2O phosphate buffer, pH* 7.4, after 18 h of
55 incubation at 310 K. (A) Under aerobic conditions, and (B) under
argon.
6 | Journal Name, [year], [vol], 00–00
110
Fig. 9 Reactions of complex 14 [Os(η6-p-cym)(ImpyNMe2)Cl]PF6 (0.1 mM) with NADH (0.4 mM) in the absence and
presence of NaCl (to suppress aquation), followed by 1H NMR
115 spectroscopy. (A) 500 mM NaCl; (B) 100 mM NaCl; (C) no
This journal is © The Royal Society of Chemistry [year]
NaCl. Solvent: 10% MeOD-d4/90% D2O phosphate buffer, pH*
7.4). 1H NMR spectra were recorded 24 h after incubation at 310
K. 14A is the aqua complex. The presence of NaCl suppresses
aquation but the extent of oxidation of NADH to NAD+ (40 ±
5 3%) is not affected.
The extent of aquation decreased from 99% to 11% and 1% at
NaCl concentrations of 0 mM, 100 mM and 500 mM,
respectively. After 24 h incubation with NADH at 310 K, the
+
10 same amount of NAD product (40 ± 3%) was observed for each
of these three solutions (Figure 9), this result indicating that
NADH can react directly with the chlorido complex.
Since complex 14 appeared to oxidize NADH by hydride
transfer, we studied the effect of complex 14 on the
+
15 NAD /NADH ratio in human ovarian A2780 cancer cells. After
treatment of the cancer cells with 1.5 µM of complex 14 for 6 h
(see Supporting Information), the NAD+/NADH ratio in cell
lysates increased from 2.54±0.46 to 5.44±0.26. The increase may
suggest that the anticancer activity of these OsII arene
20 iminopyridine complexes can be related to their reactivity with
the cellular reducing agent NADH and the consequent change in
the redox status of the cancer cells.
Electrochemistry
25
Cyclic voltammetry studies of iodido complexes 3 and 6, and
chlorido complexes 10 and 14 together with 3 and 10 showed that
these compounds underwent two irreversible electrochemical
reductions in dimethylformamide with potentials ranging from –
30 0.58 to –0.76 V for the first and –0.82 to –1 V for the second.
Subtle variations in the CV morphology and forward-reverse
peak separations for the different complexes suggests some small
differences in heterogeneous electron transfer kinetics (Figure
S9).
previously
reported
osmium
picolinate
DNA-targeted
complexes,16 which suggests that other targets may be more
important. The contrast between these iminopyridine complexes
and their isoelectronic azopyridines analogues is quite striking.
The azopyridine complexes do not readily hydrolyse and do not
bind to guanine. In accordance with this, the azopyridines appear
65 to exert their anticancer activity partly by redox mechanisms. We
also investigated whether redox mechanisms are important for
iminopyridine complexes (vide infra).
60
70
Structure-Activity Relationships (SAR) for Human Ovarian
Cancer Cells
When the anticancer activity of osmium arene
phenyliminopyridine complexes with various substituents on the
phenyl ring is compared with their phenylazopyridine analogues,
75 the correlation coefficient is low (Figure 10), which suggests that
the mechanism of action is different for these two families of
osmium anticancer complexes. For a subset of 8 of the 11
complexes however, there is a stronger correlation, Figure 10.
The iminopyridine complexes bearing a biphenyl arene exhibit
80 higher anticancer activity towards A2780 cells than their pcymene analogues (Table 1, Chart 1). It seems likely that the
greater hydrophobicity of the biphenyl complexes enhances cell
uptake, although intercalation into DNA may also contribute if
DNA is a target.43
85
90
35
Discussion
95
Aquation and Binding to 9-Ethylguanine
The chlorido complex 14 [Os(η6-p-cym)(Impy-NMe2)Cl]PF6
hydrolysed almost completely after 24 h in water at 310 K,
whereas the iodido complex 6 [Os(η6-p-cym)(Impy-NMe2)I]PF6
was only ca. 50% hydrolysed. This is consistent with the
expected strengthening of the Os-halide bond for the heavier
45 halides. However, both these complexes are much less stable in
water than the analogous azopyridine complexes.14
The pKa of the aqua adduct 14A was determined to be 5.2,
which implies that the major product of aquation at physiological
pH (7.4) will be the hydroxido adduct. This may account for the
50 low extent and slowness of binding to the nucleobase 9-ethyl
guanine (only 19% after 38 h of incubation at 310K, Figure 2),
since Os-OH bonds are expected to be much less reactive than
Os-OH2 bonds. We chose to study guanine as the nucleobase
since G N7 is the most electron-dense site on DNA and is a
42
55 known target for platinum anticancer drugs.
The findings for guanine binding suggest that DNA (and
RNA) could be a target for osmium iminopyridine complexes, but
the extent of binding of 14 to 9-EtG was much less than for the
40
This journal is © The Royal Society of Chemistry [year]
100
105
110
115
Fig. 10 Correlations of the IC50 values of osmium arene
iminopyridine complexes with their azopyridine analogues. The
Journal Name, [year], [vol], 00–00 | 7
numbers in brackets refer to iminopyridine complexes (see Chart
1). Complexes with biphenyl arene (red) and para-cymene (blue)
arene are labelled with different colours; chlorido complexes are
indicated by Cl in the brackets; different substituents on the
5 chelating ligands are designated using different shapes: R= H
(square), R= OH (circle) and R= NMe2 (triangle). Overall the
correlation is poor (R2= 0.0673) although if outliers 4, 9 and 13
are ignored, then there is a reasonable correlation for the
remaining 8 complexes (R2= 0.9395).
10
In general, the chlorido iminopyridine complexes are as
active or more active than their iodido analogues. This contrasts
with the azopyridine series for which the opposite is true, except
for those with a –NMe2 electron-donating substituent in the
15 phenyl ring of the phenyliminopyridine ligand. For the inert
azopyridine complexes, addition of an ortho substituent such as
CF3 or Cl on the pyridine ring maintained or increased the
anticancer activity. However, in the present case, ortho
functionalization of the pyridine ring with a methoxy group
20 decreased the anticancer activity significantly (Table 1). Unlike
the azopyridine complexes, the Os-Cl/I bond is reactive in
iminopyridine complexes and an ortho substituent may hinder the
reactivity. Substitution reactions at the osmium centre may
therefore be important in the mechanism of action of
25 iminopyridine complexes.
Additionally, it is notable that 6 and 14 are more potent
anticancer complexes than previously-reported ruthenium and
osmium arene complexes which bind more strongly to DNA
bases and appear to have DNA as a major target.16, 44 Redox
30 mediated mechanisms of activity may be more effective since
they can affect multi-specific targets in cells.
Accumulation of ROS in Cancer Cells
ROS are highly reactive O2 metabolites that include
superoxide radicals (O2-·), hydrogen peroxide (H2O2) and
hydroxyl radicals (OH·).45 Because cancer cells have
increased ROS levels compared to normal cells, this
difference can be exploited as a biochemical basis for
46, 47
40 therapeutic selectivity.
Increased ROS levels in cancer
cells can reach a threshold and induce cell death but such
increases can be tolerated by normal cells.30 This may explain
why complex 14 is 8-fold more cytotoxic towards A549
human lung cancer cells compared to normal human fetal lung
45 fibroblast-like MRC-5 cells (Fig. 3). Although ROS are
generally believed to be toxic to cells, they act as a ‘doubleedged sword’ for the proliferation of cells. When the ROS
level in cells is up-regulated on a small scale, ROS may
contribute to the proliferation; if the ROS level is raised
50 further, it can cause DNA damage which will induce cell
cycle arrest and apoptosis.48 Therefore selectivity to cancer
cells rather than normal cells can be achieved by increasing
the ROS levels because the basal ROS levels in cancer cells
are higher than in normal cells. These increases in ROS levels
55 provide a basis for a strategy involving combination treatment
of 6 or 14 with the glutathione synthesis inhibitor buthionine
sulfoximine (L-BSO), Fig. 4.
35
8 | Journal Name, [year], [vol], 00–00
Because the GSH concentration is 100-10,000 fold higher in
cells compared to other reductants (e.g. NADH, NADPH and
60 thioredoxin), the level of GSH usually determines the steady-state
value of intracellular redox potentials.49 However, complexes 6
and 14 do not readily oxidize GSH unlike ruthenium azopyridine
analogues.14 The half-wave reduction potentials of osmium
complexes 3, 6, 10 and 14 in dimethylformamide of –0.58 to –
65 0.76 V and –0.82 to –1 V are more negative than those of the
ruthenium azopyridine complexes,19 indicating that the
iminopyridine complexes are more difficult to reduce, perhaps
explaining why no reaction was observed with GSH (Figures S4
and S9). In contrast, novel reactions with the reduced coenzyme
70 NADH, a stronger reducing agent than GSH, were observed.
Oxidation of NADH to NAD+
Apart from a role in the generation of ROS, the facile
oxidation of NADH may help to rationalize the surprisingly good
anticancer activity of these OsII iminopyridine complexes.
NMR, LC-MS and UV-Vis studies showed that osmium
iminopyridine complexes can oxidize NADH to NAD+. The
reaction appears to involve the transfer of hydride from NADH to
80 the Os centre with displacement of the halide ligand (I for 6 and
8, and Cl for 14 and 16) or aqua ligand. During the conversion of
NADH to NAD+ in the presence of 14A, an osmium-hydride peak
was detected by 1H NMR at –4.2 ppm (Figure 5). This value is
consistent with previous reports of Os-H adducts.50, 51 The
organometallic hydride osmium(II) compounds
85 reported
containing arene52, 53 and/or imine ligands54, 55 showed Os-H
signals ranging from -9 ppm to -13 ppm (terminal hydride) and
from -15 ppm to -20 ppm (bridging hydride). In all the reported
compounds, the hydride peaks were detected for complexes in
90 deuterated organic solvents such as CD2Cl2, acetone-d6 or C6D6.
The current work appears to be the first detection of an
osmium(II) hydride NMR peak for an osmium hydride in an
aqueous solution.56-64 These findings suggest that osmium arene
complexes can modulate the redox balance in cells by a novel
95 mechanism.
1
H NMR studies show that complexes 6 and 14 are more
effective in oxidising NADH than 8 and 16 after 24 h incubation
at 310K (Figure S5). This may be due to the steric hindrance
exerted by the methoxy substituent in the ortho-position of the
100 iminopyridine chelating ligand for 8 and 16. This trend is also
observed for the anticancer activity of these compounds: 6 and 14
are ca. 20 times more potent than 8 and 16. It is possible therefore
that NADH oxidation is involved in the mechanism of anticancer
activity of these osmium iminopyridine anticancer complexes.
A possible mechanism for the oxidation of NADH in the
105
presence of these iminopyridine complexes involves hydride
transfer to Os followed by protonation of bound hydride,65
liberation of H2, and regeneration of the aqua/hydroxido Os
adduct, similar to that of IrIII cyclopentadienyl complexes studied
27
110 in our laboratory.
However, no H2 was detected after the
incubation of NADH with 14 or 14A for 24 h at 310 K (100 mM
phosphate buffer, pH 7.2). The presence of O2 did appear to
influence the course of the oxidation significantly, being inhibited
under argon and enhanced by O2 saturation (Figures 7 and 8).
115 One possibility is that hydride is transferred from Os-H to O2 to
75
This journal is © The Royal Society of Chemistry [year]
form H2O2,37 giving a cycle such as that shown in Scheme 1.
However attempts to detect H2O2 production electrochemically
were unsuccessful (for conditions see ESI).
Suppression of aquation of the chlorido complex 14 still led
5 to the oxidation of NADH (Figure 9) suggesting that aquation is
not an essential step in the mechanism and that hydride transfer
can occur with direct chloride displacement.
O
H 2N
N
10
H
X
X-
Os
H 2O 2
N
N
Ribo
ADP
H
N
NADH
CH
X-
O2
(X- = OH -, Cl- , I-)
2
15
N
N
H
Os
H2N
N
N
Os
O
N
H
CH
N
N
CH
H
dinucleotide (NADH). The Os II arene iminopyridine
60 compounds followed opposite trends in redox reactivity
compared to the azo analogues: the iminopyridine complexes
were inert toward GSH but they could oxidize NADH to
different extents depending on the substituents on the
chelating ligand. A mechanism for the oxidation of NADH to
+
65 NAD
through the formation of an Os-hydride adduct in
aqueous solution detected by 1H NMR (singlet at -4.2 ppm) is
proposed. Furthermore, an increase in the ratio of
NAD+/NADH in human ovarian A2780 cancer cells after
treatment with the chlorido complex [Os(η6-p-cym)(Impy70 NMe 2 )Cl]PF 6 was found.
We conclude from these studies that the anticancer activity of
these osmium arene iminopyridine complexes may involve the
modulation of redox pathways in cancer cells by a novel
mechanism. In addition, we have shown that the rational
II
75 design of the chelating ligands and structure of the Os
arene
complexes plays an important role in controlling the reactivity
and biological behaviour of these anticancer drugs.
b
Ri
o
20
A
DP
O
NH 2
ADP
Ribo
80
N
Table 2 Comparison between the biological and chemical
behaviour of osmium(II) arene iminopyridine complexes and
azopyridine complexes.
NAD+
25
Scheme 1 A possible mechanism for the oxidation of NADH
to NAD+ catalysed by an OsII arene iminopyridine complex, X- =
OH-, Cl- or I-.23, 27
[Os(η6-arene)(XY)Z]+ Complexes
XY =
30
Property
Iminopyridine
Azopyridine
Activity to A2780 cells
Nanomolar
Nanomolar
Increase in ROS
Yes
Yes
Oxidation of GSH
No
No
Oxidation of NADH
Yes
No
Aquation
Yes
No
Binding to 9-EtG
Yes
No
Conclusions
In this work, we have prepared new OsII arene iminopyridine
35 anticancer complexes to explore the effect of bioisosterism on
the biological activity of our previously studied OsII arene
azopyridine complexes. We have contrasted the chemical
reactivity and anticancer activity of isoelectronic
organometallic Os II complexes [Os(η6-arene)(XY)Z]PF6
arene = p-cym or bip, XY = N,N΄-chelated
40 where
phenyliminopyridine or phenylazopyridine derivatives, and Z
= Cl or I. The X-ray crystal structure of [Os(η6-p-cym)(ImpyOH)I]PF6·0.5CH2Cl2·H2O is reported. Some OsII arene
iminopyridine complexes are potently active towards cancer
45 cells (nanomolar IC 50 values), like the azopyridine complexes
we reported recently.66 Nevertheless, unlike the azopyridine
analogues, the iminopyridine complexes hydrolyse in aqueous
solution and bind to the model nucleobase 9-ethylguanine,
suggesting that DNA could be a possible target for these
compounds, although other targets are not
50 anticancer
excluded.
Additionally, we have found that active iminopyridine
complexes induce a dramatic increase in the levels of reactive
oxygen species (ROS) in A549 lung cancer cells. For this
we investigated the possibility that these Os II
55 reason,
complexes can be involved in biologically-relevant redox
chemistry, for example with the cellular reducing agents
glutathione (GSH) and reduced coenzyme nicotine adenine
This journal is © The Royal Society of Chemistry [year]
Acknowledgements
85
We thank the ERC (grant no. 247450 BIOINCMED), EPSRC and
Science City/EU ERDF/AWM (MaXis mass spectrometer and
the X-ray diffractometer) for funding. M. J. R. thanks the
Ministerio de Educación-FECYT (Spain) for her postdoctoral
90 fellowship. We thank colleagues in the EC COST Action D39 for
stimulating discussions, Dr. Michael Khan (Life Sciences) for
provision of facilities for cell culture, Joan Soldevila for
discussions on catalytic reactions, Prof. Timothy D. H. Bugg and
Mr. Darren Braddick for use of the microplate reader, Dr.
95 Andrew Crombie and Prof. Colin Murrell for use of gas
chromatography, Dr. Salzitsa Anastasova-Ivanov and Prof.
Journal Name, [year], [vol], 00–00 | 9
23.
Pankaj Vadgama (QMW University of London) for H2O2
detection by electrochemistry, and the National Cancer Institute
(NCI) for 60-cancer-cell-line screening.
24.
75
5
Notes and references
Department of Chemistry, University of Warwick, Gibbet Hill Road,
Coventry CV4 7AL, UK. Tel: +44-2476523818; Fax: +44-2476523819.
Email: P.J.Sadler@warwick.ac.uk
† Electronic Supplementary Information (ESI) available: Experimental
10 Section
(synthesis and characterisation of complexes), Methods
(electrochemistry, LC-MS, GC, X-ray crystallography, Cell culture and
IC50 determinations, ROS detection, NAD+/NADH ratio in cells),
Instrumentation, Tables S1 and S2 of crystallographic data, Table S3 of
NCI 60-cell line anticancer data, and Figures S1-S9. CCDC reference
15 number 867420. See DOI: 10.1039/b000000x/.
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