Structure-activity relationships for organometallic osmium arene phenylazopyridine complexes with potent anticancer activity

Article abstract of DOI:10.1039/c1dt10937e

We report the synthesis and characterisation of 32 half sandwich phenylazopyridine Os^II arene complexes [Os(η⁶-arene) (phenylazopyridine)X]⁺ in which X is chloride or iodide, the arene is p-cymene or biphenyl and the pyrid

Full text of DOI:10.1039/c1dt10937e

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PAPER
Structure–activity relationships for organometallic osmium arene
phenylazopyridine complexes with potent anticancer activity†
Ying Fu, Abraha Habtemariam, Aida M. B. H. Basri, Darren Braddick, Guy J. Clarkson and Peter J. Sadler*
Received 18th May 2011, Accepted 29th July 2011
DOI: 10.1039/c1dt10937e
We report the synthesis and characterisation of 32 half sandwich phenylazopyridine OsII arene
6
complexes [Os(h -arene)(phenylazopyridine)X]+ in which X is chloride or iodide, the arene is p-cymene
or biphenyl and the pyridine and phenyl rings contain a variety of substituents (F, Cl, Br, I, CF3, OH or
NO2). Ten X-ray crystal structures have been determined. Cytotoxicity towards A2780 human ovarian
cancer cells ranges from high potency at nanomolar concentrations to inactivity. In general the
introduction of an electron-withdrawing group (e.g. F, Cl, Br or I) at specific positions on the pyridine
ring significantly increases cytotoxic activity and aqueous solubility. Changing the arene from p-cymene
to biphenyl and the monodentate ligand X from chloride to iodide also increases the activity
significantly. Activation by hydrolysis and DNA binding appears not to be the major mechanism of
6
action since both the highly active complex [Os(h -bip)(2-F-azpy)I]PF6 (9) and the moderately active
6
complex [Os(h -bip)(3-Cl-azpy)I]PF6 (23) are very stable and inert towards aquation. Studies of
octanol–water partition coefficients (log P) and subcellular distributions of osmium in A2780 human
ovarian cancer cells suggested that cell uptake and targeting to cellular organelles play important roles
in determining activity. Although complex 9 induced the production of reactive oxygen species (ROS) in
A2780 cells, the ROS level did not appear to play a role in the mechanism of anticancer activity. This
class of organometallic osmium complexes has new and unusual features worthy of further exploration
for the design of novel anticancer drugs.
that neatly fits into the active site of the enzyme and promotes
specific outer-sphere contacts between groups on the ligands and
the enzyme. There can also be intriguing differences between
Introduction
The ‘from bench to clinic’ story of cisplatin1 has stimulated
the chemical properties of organometallic RuII and OsII arene
complexes even though their three-dimensional structures are
almost identical. For example, the hydrolysis rate of chlorido arene
complexes of OsII is often ca. 100¥ slower that for RuII, and the
resulting aqua OsII complexes are ca. 1.5 pKa units more acidic.17
Moreover, RuII and OsII complexes can exhibit differences in their
medicinal properties. For example, the osmium analogue of the
ruthenium antimetastatic drug NAMI-A [trans-tetrachloro(1H-
imidazole)(S-dimethylsulfoxide)ruthenate(III)] exhibits better in
vitro anticancer activity.19
The only recognised clinical use of osmium appears to be
for synovectomy in arthritic patients in Scandinavia.20–22 This
involves the local administration of osmium tetroxide (OsO4),
usually considered to be a highly toxic compound. The lack
of reports of long term side effects suggest that osmium itself
can be biocompatible, although clearly this might be dependent
on the exact nature of the compound administered. Osmium
carbohydrate polymers (osmarins) have been also investigated as
potential antiarthritic agents.23
the search for other transition metal anticancer complexes with
improved features. In particular some group 8 organometallic
complexes of iron, ruthenium and osmium show promising
activity.2,3 For example, Jaouen et al. have designed ferricenium
complexes which target hormone receptors in breast cancer cells,4
certain RuII arene complexes are active in vitro and in vivo,5–8 and a
few reports of anticancer active organometallic osmium complexes
have recently appeared.9–15
Osmium complexes are often considered to be relatively inert
(a common characteristic of low-spin d6 metal ions and especially
3rd row transition metals).16,17 Organometallic OsII and RuII arene
complexes can adopt very similar three-dimensional structures.
For example, for the kinase inhibitors designed by Meggers et al.,18
both osmium and ruthenium analogues are part of an inert scaffold
Department of Chemistry, University of Warwick, Gibbet Hill Road,
Coventry, U.K CV4 7AL. E-mail: P.J.Sadler@warwick.ac.uk; Fax: (+44)
024 7652 3819; Tel: (+44) 024 7652 3818
† Electronic supplementary information (ESI) available: Abbreviations
list, synthesis and characterizations of compounds 1–32, Tables S1–S4
and Fig. S1 and Fig. S2. CCDC reference numbers 821519–821528 for
complexes 18, 16, 14, 13, 19, 10, 7, 24, 20, and 26, respectively. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c1dt10937e
Recently, non-organometallic osmium(VI) complexes were re-
ported to show anticancer activity in vivo.12 We have also re-
ported that certain half-sandwich organometallic iodido osmium
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Dalton Trans., 2011, 40, 10553–10562 | 10553
©

phenylazopyridine arene complexes exhibit potent in vitro and
in vivo anticancer activity, higher activity than the clinical drug
cisplatin to a panel of cancer cell lines in vitro.24,25 Similar to
RuII,26 we have found that the introduction of the strong p-acceptor
azopyridine as a ligand in organometallic OsII complexes has a
major effect on their chemical and biological properties.12
For OsII arene picolinate complexes, log P values correlate with
cellular uptake, which indicates that increased lipophilicity favours
uptake by cancer cells, probably through a passive diffusion
pathway, and results in an increase in anticancer activity.27
However, increasing lipophilicity to improve anticancer activity
can result in difficulties with clinical formulation and lower
bioavailability28 Hence there is a need for rational design aimed at
tuning lipophilicity while maintaining anticancer activity.
The goal of anticancer research programs is to identify novel,
synthetically-feasible molecules that exhibit useful anticancer
activity with minimal side-effects. One efficient approach is the
study of bioisosterism, a term coined to describe the modification
of biological activity by isosterism.29–31 Here we report studies of a
series of novel bioisosteres of OsII arene complexes containing
chelated phenylazopyridines with various substituents on the
pyridine ring. We show that cellular reactive oxygen species (ROS)
accumulate but do not lead to the death of cancer cells, suggesting
a mechanism different from that for related complexes with an
unsubstituted pyridine ring and a substituent on the phenyl ring.
In addition, it is found that down-regulation of the cellular GSH
level results in lowering of anticancer activity, a property which has
been found only for the anticancer drug taxol, which kills cancer
cells in a GSH-dependent manner.32
Results
Previously we reported the synthesis and cancer cell cytotoxicity of
twelve osmium(II) phenylazopyridine complexes containing vari-
ous substituents on the phenyl ring.24 The least active compounds
in this class contained unsubstituted phenyl rings.24 In the present
work, we have investigated whether the latter complexes can be
activated by introducing substituents (R) into the pyridine ring in
6
the class [Os(h -p-cym)(R-azpy)X]PF6. We have also investigated
Chart 1 Osmium phenylazopyridine arene complexes studied in this
work.
the effect of changing the arene ligand (from p-cymene to biphenyl)
and the monodentate ligand (X) from Cl to I. The exploration of
such a family of bioisosteres may allow optimisation of properties
and allow discovery of candidates (‘hits’) suitable for preclinical
development.
6
azpy)Cl]PF6 (19), [Os(h -p-cym)(2-Br-azpy)Cl]PF6 (20) and
[Os(h -p-cym)(3-Cl-azpy)Cl]PF6 (26) were also determined by X-
ray crystallography (Fig. 1, Tables S1 and S2†). All adopt the
6
familiar half-sandwich ‘piano-stool’ geometry.
In total, 32 novel complexes were synthesized (Chart 1) in
good yields, with PF6 as the counter anion, and characterized
-
by elemental analysis, ESI-MS and NMR spectroscopy. For ten
complexes, X-ray crystal structures were determined.
Stability and hydrolysis
We investigated the hydrolysis (aquation) of these azopyridine
complexes since this is a potential mechanism for activation
of halido osmium arene complexes in their interactions with
biological targets such as DNA.33 The aqueous behaviour of the
highly active complex 9 [Os(h -bip)(2-F-azpy)I]PF6 and moder-
ately active complex 23 [Os(h -bip)(3-Cl-azpy)I]PF6 was studied
at 310 K. The UV-Vis spectra, showed no change after 24 h,
(Fig. S1†) indicating that complexes 9 and 23 remained stable
and did not hydrolyze over that period, similar to the highly active
azopyridine complexes containing unsubstituted pyridine rings
that we reported previously.24
X-Ray crystal structures
Eighteen novel iodido osmium complexes were synthesized
6
6
6
and the structures of [Os(h -bip)(2-Cl-azpy)I]PF6 (13), [Os(h -
6
6
p-cym)(2-Cl-azpy)I]PF6 (14), [Os(h -p-cym)(2-Br-azpy)I]PF6 (18)
and [Os(h -p-cym)(3-Cl-azpy)I]PF6 (24) were determined by X-
6
ray crystallography. For comparison, fourteen chlorido ana-
logues were synthesized and the structures of the complexes
6
6
[Os(h -bip)(1-Cl-azpy)Cl]PF6 (7), [Os(h -bip)(2-F-azpy)Cl]PF6
6
6
(10), [Os(h -p-cym)(2-Cl-azpy)Cl]PF6 (16), [Os(h -bip)(2-Br-
10554 | Dalton Trans., 2011, 40, 10553–10562
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©

6
6
6
Fig. 1 X-Ray crystal structures of the cations of [Os(h -bip)(1-Cl-azpy)Cl]PF6 (7), [Os(h -bip)(2-F-azpy)Cl]PF6 (10), [Os(h -bip)(2-Cl-azpy)I]PF6
6
6
6
6
(13), [Os(h -p-cym)(2-Cl-azpy)I]PF6 (14), [Os(h -p-cym)(2-Cl-azpy)Cl]PF6 (16), [Os(h -p-cym)(2-Br-azpy)I]PF6 (18), [Os(h -bip)(2-Br-azpy)Cl]PF6 (19),
[Os(h -p-cym)(2-Br-azpy)Cl]PF6 (20), [Os(h -p-cym)(3-Cl-azpy)I]PF6 (24) and [Os(h -p-cym)(3-Cl-azpy)Cl]PF6 (26) with thermal ellipsoids drawn at 50%
6
6
6
probability. The hydrogen atoms, counterions (PF6) and solvent water molecules have been omitted for clarity.
Structure–activity relationships based on bioisosteres
(2) Biphenyl complexes are, in general, 10 times more active
than p-cymene complexes (Table 1C).
We investigated the effect of F, Cl, Br, I and CF3 substituents in
the R1–R4 positions of the pyridine ring of the phenylazopyridine
chelating ligand (Chart 1) on the cytotoxicity of the complexes
towards human ovarian A2780 cancer cells (Table 1A).
(3) The effect of chloride as an electron-withdrawing group
on the pyridine ring depends on its position. Anticancer activity
decreases with pyridine ring substitution position in the order R2
> R1 > R3, with [Os(h -bip)(2-Cl-azpy)I]PF6 (13) being the most
active, (IC50 = 1 mM) (Fig. 2A).
(4) Within the most active series, which contain biphenyl as
the arene and iodide as the monodentate ligand, changing the
electron-withdrawing group at the R2 position leads to increases
in activity in the order Cl < Br £ F < I (Fig. 2B).
(5) Changing the phenyl ring in the phenylazopyridine chelating
ligand to pyridine, does not improve the anticancer activity.
6
Their potency covers a wide range of concentrations, from very
6
high potency with an IC50 of 220 nM for complex 21, [Os(h -
bip)(2-I-azpy)I]PF6, to >100 mM and inactivity for complexes 10,
25, 26 and 30, Table 1. The following trends are observed.
(1) Complexes containing iodide as the monodentate ligand
have a higher activity compared to the chlorido complexes
(Table 1B).
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©

Table 1 (A) IC50 values for A2780 cells for complexes 1–32. (B) Compar-
ison of IC50 values for A2780 cells for monodentate ligand = Cl or I. (C)
Comparison of IC50 values for A2780 cells for arene = p-cym or bip.
Also it is notable that that the osmium(II) chlorido and iodido
complexes with 5-hydroxy-2-(4-nitrophenylazo)pyridine as the
chelating ligand showed similar activity (Table 1A).
(A)
Partition coefficients (log P)
Complex
IC50 (lM)
6
Octanol–water partition coefficients (log P values) provide a
measure of the lipophilicity of compounds and are often a useful
indication of the likely extent of drug uptake by cells.34 We
determined log P values for four iodido complexes 9 (2-F), 13
(2-Cl), 17 (2-Br), and 23 (3-Cl), and two chlorido complexes 19
(2-Br), and 25 (3-Cl), all biphenyl arene complexes, using the
“shake tube method”. The log P values for these complexes (Table
S3†) decrease along the series: complex 19 (monodentate ligand
Cl/substituent 2-Br) > 17 (I/2-Br) > 13 (I/2-Cl) > 23 (I/3-Cl) > 9
(I/2-F)> 25 (Cl/3-Cl) (Fig. 3), ranging from 0.1330 (partitioning
preferentially into octanol) to -1.446 (partitioning preferentially
into water).
(1) [Os(h -bip)(1-CF3-4-Cl-azpy)I]PF6
5.7( 1.0)
10.9( 0.3)
25.2( 0.1)
38.2( 2.8)
3.7( 0.3)
9.0( 4.5)
42.9( 5.4)
24.0( 0.1)
0.63( 0.1)
>100
6.0( 0.4)
13.3( 0.7)
1.0( 0.1)
33.6( 2.0)
>50
6
(2) [Os(h -p-cym)(1-CF3-4-Cl-azpy)I]PF6
6
(3) [Os(h -bip)(1-CF3-4-Cl-azpy)Cl]PF6
6
(4) [Os(h -p-cym)(1-CF3-4-Cl-azpy)Cl]PF6
6
(5) [Os(h -bip)(1-Cl-azpy)I]PF6
6
(6) [Os(h -p-cym)(1-Cl-azpy)I]PF6
6
(7) [Os(h -bip)(1-Cl-azpy)Cl]PF6
6
(8) [Os(h -p-cym)(1-Cl-azpy)Cl]PF6
6
(9) [Os(h -bip)(2-F-azpy)I]PF6
6
(10) [Os(h -bip)(2-F-azpy)Cl]PF6
6
(11) [Os(h -p-cym)(2-F-azpy)I]PF6
6
(12) [Os(h -p-cym)(2-F-azpy)Cl]PF6
6
(13) [Os(h -bip)(2-Cl-azpy)I]PF6
6
(14) [Os(h -p-cym)(2-Cl-azpy)I]PF6
6
(15) [Os(h -bip)(2-Cl-azpy)Cl]PF6
6
(16) [Os(h -p-cym)(2-Cl-azpy)Cl]PF6
30.2( 12.4)
0.59( 0.02)
36.6( 0.9)
>50
6
(17) [Os(h -bip)(2-Br-azpy)I]PF6
6
Cellular uptake and distribution in A2780 cells
(18) [Os(h -p-cym)(2-Br-azpy)I]PF6
6
(19) [Os(h -bip)(2-Br-azpy)Cl]PF6
6
Time-dependent cellular uptake studies of the biphenyl/iodido
complexes 9 [Os(bip)(2-F-azpy)I]PF6 and 23 [Os(bip)(3-Cl-
azpy)I]PF6 showed that the accumulation of 9 is much higher
than that of 23 after 24 h (Fig. 4A) The distribution of Os in
A2780 cells was investigated after incubation with 4 mM complex
9 or 23. The cytosol, membrane-plus-particulate fraction, nucleus
and cytoskeleton fractions were separated and their Os contents
determined by ICP-MS (Fig. 4B). Complex 9 gave rise an uptake
of osmium into the cytosol, membrane plus particulate fraction
and nucleus ca. 23 times higher than for 23 (Fig. 4B, Table. S4†). It
is interesting that for 23 there is a very high percentage of osmium
in the cytoskeleton, up to 43% of the total cellular Os (Fig. S2†).
(20) [Os(h -p-cym)(2-Br-azpy)Cl]PF6
>50
6
(21) [Os(h -bip)(2-I-azpy)I]PF6
0.22( 0.02)
2.4( 0.5)
22.0( 2.0)
48.4( 6.1)
>100
6
(22) [Os(h -p-cym)(2-I-azpy)I]PF6
6
(23) [Os(h -bip)(3-Cl-azpy)I]PF6
6
(24) [Os(h -p-cym)(3-Cl-azpy)I]PF6
6
(25) [Os(h -bip)(3-Cl-azpy)Cl]PF6
6
(26) [Os(h -p-cym)(3-Cl-azpy)Cl]PF6
>100
6
(27) [Os(h -bip)(Abpy))I]PF6
21.3( 6.2)
10.8( 0.11)
22.4( 11.8)
>100
0.29( 0.04)
0.30( 0.05)
6
(28) [Os(h -p-cym)(Abpy))I]PF6
6
(29) [Os(h -bip)(Abpy)Cl]PF6
6
(30) [Os(h -p-cym)(Abpy))Cl]PF6
6
(31) [Os(h - p-cym)(OH-azpy-NO2)I]PF6
6
(32) [Os(h -p-cym)(OH-azpy-NO2)Cl]PF6
(B)
Cl complex/I complex
IC50 (Cl)/IC50 (I)
Detection of ROS in A2780 cancer cells
3/1
4.4
3.5
11.6
2.7
>159
2.2
>50
0.9
>85
>1.4
>4.5
>2.1
1.0
To detect changes in general oxidative stress,35 we determined the
4/2
∑
-
level of reactive oxygen species (ROS, including O2 , OH , H2O2)
in A2780 cells using the probe 2¢,7¢-dichlorodihydrofluorescein-
diacetate (DCFH-DA). When taken up by live cells, DCFH-DA
hydrolyzes to 2¢,7¢-dichlorodihydrofluorescein (DCFH), which in
turn is oxidized to 2¢,7¢-dichlorofluorescein (DCF) in the presence
of ROS and detected by its intense fluorescence.36,37 We investigated
7/5
8/6
10/9
12/11
15/13
16/14
19/17
20/18
25/23
26/24
29/27
6
the change in ROS level induced by 9, [Os(h -bip)(2-F-azpy)I]PF6,
one of the most active compounds. The relative increase in
DCF fluorescence was detected over time after exposure to 9
alone (1 mM), 9 (1 mM) with L-buthionine sulfoximine (L-BSO,
50 mM, an inhibitor of glutamylcysteine synthetase),38 and, for
comparison, H2O2 (50 mM). For 9, an increase in intracellular
ROS level was observed, but it was lower than that observed for
H2O2 (50 mM) after 4 h incubation.
(C)
p-Cym Complex/Bip Complex
IC50 (p-Cym)/IC50 (Bip)
2/1
1.91
1.52
2.43
0.56
>7.5
33.6
62.0
10.9
2.20
0.51
4/3
Exposure of A2780 cells to 9 (1 mM) in the presence of the
6/5
39
antioxidant thiol N-acetyl-L-cysteine (NAC, 50 mM) gave no
8/7
increase in ROS level compared to the control. However, for 9
(1 mM) with L-BSO (50 mM), the increase in ROS level was even
higher than that for H2O2 (50 mM). (Fig. 5A) The increase of
fluorescence over time after exposure of A2780 cells preloaded
with DCFH-DA to 1 mM of 9, indicates that 9 causes a build-up
of ROS inside A2780 cancer cells.
12/10
14/13
18/17
22/21
24/23
28/27
10556 | Dalton Trans., 2011, 40, 10553–10562
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©

Fig. 2 Comparison of the effect of different pyridine ring substitutions on IC50 values for A2780 cells. Different colours were employed to show different
sub-family of these complexes: green (arene = biphenyl, monodentate ligand = iodide), orange (arene = biphenyl, monodentate ligand = chloride), blue
(arene = para-cymene, monodentate ligand = iodide), red (arene = para-cymene, monodentate ligand = chloride). (A) Chloride substituent at R1, R2 and
R3 positions. (B) Fluoride, chloride, bromide or iodide substituent at R2 position.
Table 2 IC50 values for combination treatment with NAC or L-BSO. (A)
Effect of L-BSO treatment (24 h) on IC50 values for A549 human lung
cancer cells. (B) Effect of L-BSO and NAC treatment (24 h) on IC50 values
for IC50 values for A2780 human ovarian cancer cells. IC50 values are the
mean of three experiments and are reported as mean (standard error of
the mean).
Relationship of cytotoxicity to ROS
In order to investigate further the possible involvement of ROS in
the cytotoxicity of the phenylazopyridine osmium arene complexes
studied here, we also studied the combined effects of either
complex 9 or 17 with the reductant NAC which can deplete ROS
or with L-BSO which depletes glutathione levels and can lead to
an increase in levels of ROS in cells.
(A)
Complex
IC50/lM
+ L-BSO IC50/lM
The cytotoxicity assays showed that complex 9 (1 mM) in
combination with the reductant (antioxidant) NAC (50 mM) did
not increase the survival of A2780 cells compared to those treated
with 9 alone for 24 h (Fig. 5B). This suggests that ROS are
not implicated in cell death induced by complex 9. In contrast,
combination treatment with the oxidant L-BSO, which resulted in
an increase in the ROS level, blocked the anticancer activity.
The effect of combination treatment with the reductant (antiox-
idant) NAC (50 mM) on IC50 values for complex 9 was investigated
for both A2780 (ovarian) and A549 (lung) human cancer cell lines
(Table 2). A2780 cells were treated with various concentrations
of 9 or 17 together with NAC (50 mM). Combination treatment
9
1.8 ( 1.4)
3.6 ( 0.2)
>100
30.5 ( 2.6)
17
(B)
Complex
IC50/lM
+ L-NAC IC50/lM
+ L-BSO IC50/lM
9
17
0.63 ( 0.10)
0.59 ( 0.02)
0.97 ( 0.01)
1.14 ( 0.22)
12.6 ( 1.9)
8.4 ( 0.9)
with NAC decreased the cytotoxicity of both 9 and 17, slightly.
However, combination treatment with L-BSO raised the IC50
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Dalton Trans., 2011, 40, 10553–10562 | 10557
©

Fig. 3 Comparison of octanol–water partition coefficients (log P values)
and cytotoxicity towards A2780 cells.
Fig. 5 (A) Relative changes in DCF fluorescence detected over time after
exposure to 1 mM 9, 1 mM 9 with 50 mM NAC, 1 mM 9 with 50 mM L-BSO,
or 50 mM H2O2. For each compound, the fluorescence was averaged over
6 wells (n = 6). (B) Percentage cell survival after 24 h exposure to 50 mM
NAC (C + NAC), 50 mM L-BSO (C + L-BSO), 1 mM 9 (9), 1 mM 9 with
50 mM NAC (9 + NAC), 1 mM 9 with 50 mM L-BSO (9 + L-BSO) and 96 h
recovery for A2780 ovarian cancer cells; effects of combination treatment
6
6
of complex [Os(h -bip)(2-F-azpy)I]PF6 (9) or [Os(h -bip)(2-Br-azpy)I]PF6
(17) with NAC or L-BSO.
Discussion
In our previous studies we found that replacement of the N,N-
6
chelating ligand ethylenediamine by phenylazopyridine in [Ru(h -
arene)(N,N)X]+ complexes introduced some dramatic changes
in their chemical and biological properties.26 Phenylazopyridine
ligands are not only s-donors (like en), but also strong p-
6
acceptors. The chlorido phenylazopyridine complexes [Ru(h -
arene)(azpy)Cl]+ (arene = p-cymene (p-cym), tetrahydronaphtha-
lene (thn), benzene (bz), or biphenyl (bip)) readily underwent
slow decomposition via hydrolysis and/or arene loss. They were
inactive (non-cytotoxic) towards cancer cells but activated by the
introduction of electron-donating substituents into the phenyl
ring, e.g. OH and NMe2. The p-acceptor property of the pheny-
lazopyridine ligand results in highly acidic aqua complexes. For
Fig. 4 Uptake of osmium into A2780 human ovarian cancer cells. (A)
Time dependence for complexes 9 and 23; (B) Osmium distribution of
Os from complexes 9 and 23 in cell fractions: cytosol, membrane plus
particulate fraction, nucleus and cytoskeleton. Data are the mean of three
experiments and are reported as mean standard error of the mean (SEM).
6
example, the pKa of the coordinated water in [(h -p-cym)Ru(azpy-
values for 9 and 17 more than 10 times for A2780 cells. For the
A549 cell line, the IC50 value of 9 was raised from 1.8 mM to more
than 100 mM, and for 17 the IC50 values increased more than 70-
fold (Table 2). Intriguingly, these data resemble those reported for
the effects of combination treatment on the organic drug Paclitaxel
(Taxol) which is in clinical use for the treatment of ovarian, breast
and non-small cell lung cancer. Taxol is known to target tubulin
in microtubules.32
NMe2)OH2]2+ is 4.60.40 Most dramatic was the effect of changing
the monodentate leaving group from chloride to iodide which
conferred on the complexes remarkable inertness towards ligand
substitution.26 Moreover these iodido complexes with substituents
on the phenyl ring were highly cytotoxic to A2780 human ovarian
and A549 human lung cancer cell lines with IC50 values of 2–
6 mM. The mechanism of cytotoxicity appeared to involve ligand-
based redox reactions.26
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These findings for RuII complexes appear to be mirrored to some
extent for OsII complexes. The chlorido OsII phenylazopyridine
complex [Os(h -p-cym)(azpy)Cl]PF6 is inactive towards A2780
targets, as demonstrated for interactions between serum albumin
and biphenyl and p-cymene RuII complexes.43
6
The inertness of the most active complexes to hydrolysis suggests
direct binding to DNA is not the major mechanism of action and
that a novel target for these metal arene complexes is involved.
This was also the conclusion from our recent work on OsII
phenylazopyridine complexes containing unsubstituted pyridine
and substitutions on the phenyl ring.
human ovarian cancer cells but active with electron-donor NMe2
or OH substituents on the phenyl ring.24 The iodido complexes
6
are more active and complexes with moderate activity e.g. [Os(h -
p-cym)(azpy)I]PF6 (IC50 = 10.3 mM in A2780 cell line) become
potently cytotoxic at nanomolar concentrations towards a panel of
human cancer cell lines when substituents are introduced into the
Reactive oxygen species (ROS) play important roles in reg-
ulating cell proliferation, death, and senescence, they can also
play significant roles in the mechanism of action of anticancer
agents.44 We suggested previously that the cytotoxicity mechanism
for osmium arene phenylazopyridine iodido complexes containing
unsubstituted (R1–R4 = H) pyridine rings and their ruthenium
analogues is related to ROS generation.26 However, the complexes
investigated in the current work containing substituted pyridine
rings do not appear to share a mechanism dependent to the forma-
tion of ROS. Down-regulation of ROS by increasing intracellular
GSH, does not block the anticancer activity of 9 and 17. It is also
known that cisplatin resistance is partly related to an increase of
GSH levels,45 and the data for complexes 9 and 17 suggest that they
have potential for the treatment of cisplatin-resistant tumours.46
The mechanism of action of complexes 9 and 17 is GSH-
dependent. L-Buthionine sulfoximine (L-BSO) depletes intracel-
lular glutathione levels by inhibiting the enzyme glutamylcysteine
synthetase. L-BSO is on clinical trial for combination treatment
with Melphalan,47 and increases the cytotoxicity of a number of
therapeutic agents, including cisplatin, especially towards resistant
cancer cell lines.48,49 The only reported exception in which the L-
BSO can block the cytotoxicity of a drug appears to be taxol.32
Our report is apparently only the second in which a blocking of
anticancer activity by L-BSO has been observed.
6
phenyl ring, e.g. IC50 = 140 nM for [Os(h -bip)(azpy-NMe2)I]PF6
towards A2780 ovarian cancer cells.24
The aim of the current work was to investigate the effects on
anticancer activity of introducing substituents into the pyridine
ring especially when the phenyl ring is left unsubstituted. We
synthesised and characterized 32 novel organometallic osmium
phenylazopyridine complexes (Chart 1). All the osmium com-
plexes adopted the familiar piano-stool geometry in the crystalline
state, with bond lengths and angles within the expected ranges.24
Selected bond lengths and angles for these structures are listed in
the supporting information (Table S2†). For the iodido osmium
complexes, the Os–C (arene) bond lengths are in the range of
˚
˚
2.172–2.284 A, and the Os–I bond lengths from 2.7002–2.7063 A
(Table S2†). For the chlorido osmium complexes, the Os–C (arene)
˚
bond lengths are in the range of 2.173–2.286 A, and Os–Cl bond
˚
lengths are 2.3727–2.3954 A (Table S2†). A relatively longer bond
length for Os(1)–N(1)(pyridine) compared to Os(1)–N(8)(azo) was
observed in all the 10 crystal structures. This can be attributed
to the back-bonding competition for the osmium 5d6 electron
density by the phenylazopyridine and arene-acceptor ligands.
A similar trend was also observed for ruthenium arene pheny-
lazopyridine complexes.40 The longest Os(1)–N(1) bond length
6
˚
is 2.124(9) A in [Os(h -bip)(1-Cl-azpy)Cl]PF6 (7), the structure
determined for a complex with an ortho pyridine substituent (Cl),
For the structure activity relationships, it is evident that when
biphenyl is the arene and iodide is the monodentate ligand,
the osmium complexes in this class show the highest anticancer
activity towards A2780 cells, consistent with our previous work on
phenylazopyridine complexes with unsubstituted pyridine rings.24
In our previous work some chlorido complexes were found to be
unstable in aqueous solution which resulted in low anticancer
activity.24,40 Introducing biphenyl as the arene instead of p-
cymene not only provides a potential DNA intercalator but also
increases the hydrophobicity for interaction with proteins which
may contribute to the increased activity.5,50
Consideration of the substituents on the pyridine ring shows
that changing from chloride at R3 to fluoride causes the cellular
accumulation of osmium to increase dramatically and is accom-
panied by an improvement in anticancer activity. The difference
in the extent of cellular accumulation within this family may
contribute to the observed variations in anticancer activity, as
may interactions with targets (which may be proteins).50
To investigate whether there is a link between lipophilicity and
anticancer activity for these complexes, their log P values were
determined; they cover a broad range from -1.446 to 0.1330
(Table S3†). These values can be compared to those for the less
lipophilic clinical PtII drug cisplatin (log P = -2.36)51 and to the
RuIII drug NAMI-A (log P = -0.25)52 which is on clinical trials as
an antimetastatic agent. The sequence of potency towards A2780
human ovarian cancer cells for the osmium phenylazopyridine
complexes is: 17 > 9 > 13 > 23 > 19 > 25, whereas lipophilicity
Table S2.†
6
Three of the structures show p–p stacking. [Os(h -bip)(2-F-
azpy)Cl]PF6 (10) exhibits intramolecular p–p stacking between
the phenyl ring of the biphenyl arene ligand and the phenyl ring of
the phenylazofluoropyridine ligand. There is also intermolecular
p–p stacking involving the same groups, but between symmetry-
6
related molecules. For [Os(h -p-cym)(3-Cl-azpy)I]PF6 (24), there
is p–p stacking between the p-cymene and a phenyl ring in
a symmetry-related molecule. There is an intermolecular p–p
stacking interaction between 2-Cl-azpy ligands that lie in a head-
6
to-tail dimer fashion related by an inversion centre in [Os(h -p-
cym)(2-Cl-azpy)I]PF6 (14). The distances between the planes of
˚
the stacked rings (ca. 3.3 A) are within the normal range for such
interactions.41
Remarkably, electron-withdrawing substituents on the pyridine
ring can give rise to highly potent complexes with IC50 values
in the nano-molar range (Table 1A and Fig. 2). The structure–
activity relationships show that potency is higher for biphenyl
versus p-cymene as the arene (Table 1C), and for iodide compared
to chloride as the monodentate ligand (Table 1B). Biphenyl is
a stronger p-acceptor than p-cymene (back-donation of electron
density from RuII) whereas p-cym is the stronger electron donor.
When RuII arene complexes bind to DNA, the uncoordinated
phenyl ring of bip can intercalate between DNA bases whereas
DNA distortions caused by p-cymene are steric in origin.42 The
arene can also have a major effect on interactions with protein
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decreases in the order 9 (monodentate ligand Cl/pyridine sub-
stituent 2-Br) > 17 (I/2-Br) > 13 (I/2-Cl) > 23 (I/3-Cl) > 9
(I/2-F)> 25 (Cl/3-Cl). Hence there appears to be little correlation
between lipophilicity (log P), and anticancer activity (Fig. 3 and
Table S3†). The introduction of bioisosterism into the design has
led to the discovery of compounds with different pharmaceuti-
cal properties, potential candidates for further pharmacokinetic
studies.
recorded at ca. 310 K in double distilled water from 800 to 200 nm.
Further details are in the SI.†
pH* measurements. pH* (pH meter reading from D2O solu-
tion without correction for effects of deuterium on glass electrode)
values were measured at ambient temperature before the NMR
spectra were recorded, using a Corning 240 pH meter equipped
with a microcombination electrode calibrated with Aldrich buffer
solutions at pH 4, 7 and 10.
The cell distribution studies showed a dramatic increase in
accumulation of osmium in the membrane and particulate fraction
of A2780 cells for 9 compared to 23, and 9 is more than 50 times
more active than 23. Whether the critical target site is in the
membrane and particulate fraction remains to be investigated
further. However, to identify and determine the contribution of
the individual binding sites is a complex task, for example, only ca.
1% of Pt from intracellular cisplatin binds to its target, DNA,53,54
Further work is required to validate the target for this sub-family
of osmium arene phenylazopyridine complexes.
6
X-Ray crystallography. X-Ray diffraction data for [Os(h -
6
bip)(1-Cl-azpy)Cl]PF6 (7) [Os(h -bip)(2-F-azpy)Cl]PF6 (10,
[Os(h -bip)(2-Cl-azpy)I]PF6 (13), [Os(h -p-cym)(2-Cl-azpy)I]PF6
(14), [Os(h -p-cym)(2-Cl-azpy)Cl]PF6 (16), [Os(h -p-cym)(2-Br-
azpy)I]PF6 (18), [Os(h -bip)(2-Br-azpy)Cl]PF6 (19), [Os(h -p-
cym)(2-Br-azpy)Cl]PF6 (20), [Os(h -p-cym)(3-Cl-azpy)I]PF6 (24)
and [Os(h -p-cym)(3-Cl-azpy)Cl]PF6 (26) were obtained on an
Oxford Diffraction Gemini four-circle system with a Ruby CCD
area detector using Mo-Ka radiation.56 Absorption corrections
were applied using ABSPACK. The crystals were mounted in oil
and held at 100(2) K with the Oxford Cryosystem Cryostream
Cobra, except compound 7 for which data was collected at ambient
temperature. The structures were solved by direct methods using
SHELXS (TREF) with additional light atoms found by Fourier
methods.57 Refinement used SHELXL 97.58 H atoms were placed
at geometrically calculated positions and refined riding on their
parent atoms. X-ray crystallographic CIF files for complexes 18,
16, 14, 13, 19, 10, 7, 24, 20, and 26 have been deposited in the
CCDC with reference numbers 821519–821528, respectively.
6
6
6
6
6
6
6
6
Experimental
Materials
OsCl3·3H2O and osmium Specpure Plasma Standard were pur-
chased from Alfa–Aesar. Ethanol and methanol were dried
over Mg/I2 or anhydrous quality was used (Aldrich). All other
reagents used were obtained from commercial suppliers and used
6
as received. The preparations of the starting materials [Os(h -
6
bip)Cl2]2 and [Os(h -p-cym)Cl2]2 have been previously reported.55
Cell cultures. A549 non-small cell lung and A2780 ovarian
human cell lines (ECACC, Salisbury, UK) were cultured in RPMI
1640 cell culture medium supplemented with 1 mM sodium
pyruvate, 2 mM L-glutamine and 10% fetal bovine serum (all from
Sigma).
The synthesis of the phenylazopyridine ligands has been previously
described.26 The A2780 human ovarian carcinoma cell line was
purchased from European Collection of Animal Cell Cultures
(Salisbury, UK), RPMI-1640 media and trypsin were purchased
from Invitrogen, bovine serum from Biosera, penicillin, strepto-
mycin, trichloroacetic acid (TCA) and sulforhodamine B (SRB)
from Sigma–Aldrich, and tris[hydroxymethyl]aminomethane from
Formedium.
Synthesis. Complexes 1–32 were prepared by the same general
method: reaction of the appropriate phenylazopyridine deriva-
6
6
6
tive with the dimers; [Os(h -bip)Cl2]2, [Os(h -bip)I2]2, [Os(h -p-
6
cym)Cl2]2 or [Os(h -p-cym)I2]2. The purities of all compounds (1–
Instrumentation and methods
32) prepared were determined to be ≥95% by elemental analysis.
Single crystals suitable for X-ray diffraction were obtained by
crystallization from methanol solutions at 253 K. The details
of the syntheses and characterizations are in the Supporting
Information.†
NMR spectroscopy. 1H NMR spectra were acquired in 5 mm
NMR tubes at 298 K on either Bruker DPX-400, Bruker DRX-
500 or Bruker AV II 700 spectrometers. 1H NMR chemical shifts
were referenced to acetone-d6 (2.09 ppm). All data processing
was carried out using MestReC or TOPSPIN version 2.0 (Bruker
U.K. Ltd.).
Methods
Determination of IC50 values. The concentrations of the os-
mium complexes that inhibit 50% of the proliferation of human
ovarian A2780 cancer cells were determined using the sulforho-
damine B assay.59 A2780 cells were seeded in 96-well plate (Falcon)
at 5000 cells/well, after the incubation for 48 h. The complexes
were solubilised in DMSO (Sigma) to provide 10 mM stock
solutions. These were serially diluted by cell culture media to
give concentrations four-fold greater than the final concentrations
for the assay. The complexes diluted in cell culture media were
added to the 96-well plate with cells in triplicate. The final DMSO
concentration in each well was no more than 1% (v/v). The
media containing the complexes were removed after 24 h. The
cells were washed with phosphate buffered saline once and cell
Electrospray ionisation mass spectrometry (ESI-MS). Spectra
were obtained by preparing the samples in 50% CH3CN and 50%
H2O (v/v) and infusing into the mass spectrometer (Varian 4000).
The mass spectra were recorded with a scan range of m/z 500–1000
for positive ions.
Elemental analysis. Elemental analysis (carbon, hydrogen,
and nitrogen) was carried out through Warwick Analytical Service
using an Exeter analytical elemental analyzer (CE440).
UV-Vis spectroscopy. UV-Vis spectra were recorded on a Cary
50-Bio spectrophotometer using 1 cm path-length quartz cuvettes
(0.5 mL) and a PTP1 Peltier temperature controller. Spectra were
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culture medium was added (150 mL/well). The cells were then
allowed to grow for a further 72 h. The surviving cells were fixed
by adding 150 mL/well of 50% (w/v) trichloroacetic acid and
incubated for 1 h in a refrigerator (277 K). The plates were washed
with tap water three times and dried under a flow of warm air,
0.4% sulforhodamine B (Sigma) solution (100 mL/well) was added,
followed by washing with 1% acetic acid five times and drying
under a flow of warm air. The dye was dissolved in 10 mM Tris
buffer (200 mL/well). The absorbance of each well was determined
using a Multiskan Ascent plate reader (Labsystems) at 540 nm.
The absorbance of SRB in each well is directly proportional
to the cell number. Then the absorbance was plotted against
concentration and the IC50 determined by using Origin software.
deionized water to obtain 6% HNO3 sample solutions. A blank
and the standards were loaded into the sample tray and were run
from the lowest to the highest concentration in a ‘no gas’ mode,
followed by the samples.
Separation of cell fractions. The A2780 cells were seeded into
Petri dishes at a concentration of 5 ¥106 cells/dish. After 24 h
incubation, osmium compounds 9 (4 mM) and 23 (4 mM) were
added. The cells were harvested after a further incubation with
osmium compound for 24 h. Then the four cell fractions (cytosol,
membrane plus particulate fraction, nucleus and cytoskeleton)
were separated following the protocol described for the kit
(BioVision, Inc., USA). The concentrations of osmium in different
fractions were measured by ICP-MS after digestion following the
same method as for the cellular uptake study.
NAC (N-acetyl-L-cysteine) or L-BSO combination treatments.
A2780 cells were treated with 50 mM NAC or L-BSO and various
concentrations of osmium complexes for 24 h. Then NAC/L-
BSO and osmium complexes were removed at the same time and
cells washed with PBS once, then incubated for a further 72 h for
recovery. Cell viability was determined using the SRB assay as
described above.
Detection of ROS. The vial of DCFH-DA was opened under
N2 protection, and contents dissolved in DMSO to give a 10 mM
stock solution. A2780 cells were seeded (5000 cells/well) into
black 96-well plates and incubated for 24 h at 310 K, 5% CO2,
high humidity. Cells were loaded with DCFH-DA (10 mM) and
incubated for 30 min. The probe was removed and PBS was used
to wash the cells twice. The cells were then kept in PBS solution
and osmium compound 9 (4 mM), or NAC (50 mM) with 9 (4 mM),
or L-BSO (50 mM) with 9 (4 mM) were added. Hydrogen peroxide
(50 mM) was added as the positive control. The fluorescence was
recorded over a period of 4 h at 310 K by excitation at 480 nm and
emission at 530 nm on a TECAN plate reader.
Partition coefficient (log P) and cellular uptake
Determination of log P. Octanol–saturated water and water–
saturated octanol were prepared by stirring the mixture for 24 h.
Aliquots of stock solutions of osmium complexes in octanol–
saturated water (2 mL) were added to the same volumes of water–
saturated octanol (2 mL) and shaken in an IKA Vibrax VXC
basic shaker for 4 h at a speed of 500 g min-1 after partition. The
aqueous layer was transferred into test tubes for osmium analysis.
Aqueous samples before and after partitioning were diluted with
3.6% HNO3 to the appropriate range for analysis by ICP-MS
calibrated with aqueous standards (osmium, 0.1–400 ppb). These
procedures were carried out at ambient temperature (ca. 298 K).
Log P values of osmium complexes were calculated using the
equation log Poct = log([Os]oct/[Os]aq).
Abbreviations.
NAC
N-acetyl-L-cysteine
bpy
2,2¢-bipyridine
L-BSO
L-buthionine-[S,R]-sulfoximine
5-bromo-2-(phenylazo)pyridine
5-iodido-2-(phenylazo)pyridine
3-chloro-6-trifluoromethyl-2-
(phenylazo)pyridine
2-Br-azpy
2-I-azpy
1-CF3-4-Cl-azpy
ICP-MS instrumentation and calibration. ICP-MS analyses
were carried out on an Agilent Technologies 7500 series ICP-
MS instrument. The water used for ICP-MS analysis was double
deionized using a USF Elga UHQ PS water deionizer. The osmium
Specpure plasma standard was diluted in double deionized water
to 20 ppm. The osmium standards for calibration were freshly
prepared by diluting this stock solution with 3.6% HNO3 in double
deionized water. The concentrations used were 400, 200, 100, 50,
25, 10, 5, 1, 0.5 and 0.1 ppb.
1-Cl-azpy
2-F-azpy
2-Cl-azpy
3-Cl-azpy
Abpy
6-chloro-2-(phenylazo)pyridine
5-fluoro-2-(phenylazo)pyridine
5-chloro-2-(phenylazo)pyridine
4-chloro-2-(phenylazo)pyridine
2,2¢-azobispyridine
OH-azpy-NO2
ROS
4-hydroxy-2-(4-nitrophenylazo)pyridine
reactive oxygen species
Acknowledgements
Cellular uptake. A2780 cells were seeded 106 cells/well in 6-
well plates. After 24 h incubation, cells were exposed to 4 mM
osmium complex. After 1 h, 2 h, 4 h and 24 h of drug exposure, the
drug-containing medium was removed. Then samples were washed
with PBS twice, trypsinized, collected and stored at 253 K until
ICP-MS analysis for osmium content. The numbers of cells were
counted using a cytometer. The whole cell pellets were digested as
described below. Firstly, 0.5 mL of freshly distilled 72% HNO3 was
added to each 1 mL cells pellets, and the samples were transferred
into Wheaton V-Vials. The vials were heated in an oven at 373 K
for 16 h to digest the samples fully, allowed to cool, and then
transferred to Falcon tubes. The vials were washed with double
deionized water three times and diluted 10 times with double
We thank Dr Michael Khan (Life Sciences) and Dr Ana Pizarro
(Chemistry) for provision of facilities for cell culture, Professor
Tim Bugg for use of a microplate reader, Dr Lijiang Song and Mr
Philip R. Aston for assistance with ICP-MS and the ERC (247450
BIOINCMED), EPSRC and Science City/EC ERDF/AWM for
funding.
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