Tuning the reactivity of osmium(II) and ruthenium(II) arene complexes under physiological conditions

Article abstract of DOI:10.1021/ja055886r

The Os^II arene ethylenediamine (en) complexes [(η⁶-biphenyl)Os(en)CI][Z], Z = BPh₄ (4) and BF ₄ (5), are inactive toward A2780 ovarian cancer cells despite 4 being isostructural with an active Ru^II analogue, 4R. Hydrolysis of 5 occurred 40 times more slowly than 4R. The aqua adduct 5A has a low pK _a (6.3) compared to that of [(η⁶-biphenyl)Ru(en) (OH₂)]²⁺ (7.7) and is therefore largely in the hydroxo form at physiological pH. The rate and extent of reaction of 5 with 9-ethylguanine were also less than those of 4R. We replaced the neutral en ligand by anionic acetylacetonate (acac). The complexes [(η⁶- arene)Os(acac)-Cl], arene = biphenyl (6), benzene (7), and p-cymene (8), adopt piano-stool structures similar to those of the Ru^II analogues and form weak dimers through intermolecular (arene)C-H...O(acac) H-bonds. Remarkably, these Os^II acac complexes undergo rapid hydrolysis to produce not only the aqua adduct, [(η⁶-arene)Os(acac)(OH ₂)]⁺, but also the hydroxo-bridged dimer, [(η⁶-arene)Os(μ²-OH)₃Os(η ⁶-arene)]⁺. The pK_a values for the aqua adducts 6A, 7A, and 8A (7.1, 7.3, and 7.6, respectively) are lower than that for [(η⁶-p-cymene)Ru(acac)(OH₂)]⁺ (9.4). Complex 8A rapidly forms adducts with 9-ethylguanine and adenosine, but not with cytidine or thymidine. Despite their reactivity toward nucleobases, complexes 6-8 were inactive toward A549 lung cancer cells. This is attributable to rapid hydrolysis and formation of unreactive hydroxo-bridged dimers which, surprisingly, were the only species present in aqueous solution at biologically relevant concentrations. Hence, the choice of chelating ligand in Os ^II (and Ru^II) arene complexes can have a dramatic effect on hydrolysis behavior and nucleobase binding and provides a means of tuning the reactivity and the potential for discovery of anticancer complexes.

Full text of DOI:10.1021/ja055886r

Published on Web 01/12/2006
Tuning the Reactivity of Osmium(II) and Ruthenium(II) Arene
Complexes under Physiological Conditions
Anna F. A. Peacock,† Abraha Habtemariam,† Rafael Ferna´ndez,† Victoria Walland,†
Francesca P. A. Fabbiani,† Simon Parsons,† Rhona E. Aird,‡ Duncan I. Jodrell,‡ and
Peter J. Sadler*,†
Contribution from the School of Chemistry, UniVersity of Edinburgh, West Mains Road,
Edinburgh EH9 3JJ, U.K., and Cancer Research UK Edinburgh Oncology Unit, UniVersity of
Edinburgh Cancer Research Centre, Edinburgh EH4 2XR, U.K.
Received September 13, 2005; E-mail: P.J.Sadler@ed.ac.uk
Abstract: The OsII arene ethylenediamine (en) complexes [(η6-biphenyl)Os(en)Cl][Z], Z ) BPh4 (4) and
BF4 (5), are inactive toward A2780 ovarian cancer cells despite 4 being isostructural with an active RuII
analogue, 4R. Hydrolysis of 5 occurred 40 times more slowly than 4R. The aqua adduct 5A has a low pKa
(6.3) compared to that of [(η6-biphenyl)Ru(en)(OH2)]2+ (7.7) and is therefore largely in the hydroxo form at
physiological pH. The rate and extent of reaction of 5 with 9-ethylguanine were also less than those of 4R.
We replaced the neutral en ligand by anionic acetylacetonate (acac). The complexes [(η6-arene)Os(acac)-
Cl], arene ) biphenyl (6), benzene (7), and p-cymene (8), adopt piano-stool structures similar to those of
the RuII analogues and form weak dimers through intermolecular (arene)CsH‚‚‚O(acac) H-bonds.
Remarkably, these OsII acac complexes undergo rapid hydrolysis to produce not only the aqua adduct,
[(η6-arene)Os(acac)(OH2)]+, but also the hydroxo-bridged dimer, [(η6-arene)Os(µ2-OH)3Os(η6-arene)]+. The
pKa values for the aqua adducts 6A, 7A, and 8A (7.1, 7.3, and 7.6, respectively) are lower than that for
[(η6-p-cymene)Ru(acac)(OH2)]+ (9.4). Complex 8A rapidly forms adducts with 9-ethylguanine and adenosine,
but not with cytidine or thymidine. Despite their reactivity toward nucleobases, complexes 6-8 were inactive
toward A549 lung cancer cells. This is attributable to rapid hydrolysis and formation of unreactive hydroxo-
bridged dimers which, surprisingly, were the only species present in aqueous solution at biologically relevant
II
concentrations. Hence, the choice of chelating ligand in Os (and RuII) arene complexes can have a dramatic
effect on hydrolysis behavior and nucleobase binding and provides a means of tuning the reactivity and
the potential for discovery of anticancer complexes.
scope for the design of therapeutic and diagnostic agents.6
Certain RuII arene complexes of the type [(η6-arene)Ru(LL)-
Introduction
The serendipitous discovery of the anticancer activity of
cisplatin over 40 years ago1 has stimulated the quest for other
metal-based anticancer agents, especially complexes with fewer
side effects than cisplatin, which circumvent developed and
intrinsic resistance, and which possess a wider range of
anticancer activity. Ruthenium(III) exhibits a spectrum of kinetic
activity similar to that of PtII,2 and two RuIII complexes have
entered clinical trials, trans-[RuCl4(DMSO)(Im)]ImH (NAMI-
A, where Im ) imidazole)3 and trans-[RuCl4(Ind)2]IndH
(KP1019, where Ind ) indazole).4 It has been proposed that
their mode of action involves in vivo reduction of RuIII to the
more reactive RuII.2,5 Organometallic complexes offer a broad
(X)][Z] (where LL is a chelating ligand such as ethylenediamine
(en), X a leaving group such as Cl-, and Z a counterion) exhibit
both in vitro and in vivo activity, in some cases with activity
comparable to that of cisplatin and carboplatin.7,8 Structure-
activity relationships for these monofunctional RuII complexes
with respect to the arene,8 the chelating ligand9 (LL), and the
leaving group10 (X) have been studied. Some related acetyl-
acetonate (acac) complexes [(η6-arene)Ru(acac)(Cl)] are also
cytotoxic to cancer cells.11 As for cisplatin, hydrolysis (substitu-
(6) (a) Fish, R. H.; Jaouen, G Organometallics 2003, 22, 2166-2177. (b)
Melchart, M.; Sadler, P. J. In Bioorganometallics; Jaouen, G., Ed.; Wiley-
VCH: Paris; 2005; Vol. 1, pp 39-64. (c) Yan, Y. K.; Melchart, M.;
Habtemariam, A.; Sadler, P. J. Chem. Commun. 2005, 4764-4776.
(7) Aird, R. E.; Cummings, J.; Ritchie, A. A.; Muir, M.; Morris, R. E.; Chen,
H.; Sadler, P. J.; Jodrell, D. I. Br. J. Cancer 2002, 86, 1652-1657.
(8) Morris, R. E.; Aird, R. E.; Murdoch, P. d. S.; Chen, H.; Cummings, J.;
Hughes, N. D.; Parsons, S.; Parkin, A.; Boyd, G.; Jodrell, D. I.; Sadler, P.
J. J. Med. Chem. 2001, 44, 3616-3621.
† School of Chemistry, University of Edinburgh.
‡ Cancer Research UK Edinburgh Oncology Unit.
(1) Rosenberg, B.; VanCamp, L.; Trosko, J. E.; Mansour, V. H. Nature 1969,
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9
10.1021/ja055886r CCC: $33.50 © 2006 American Chemical Society
J. AM. CHEM. SOC. 2006, 128, 1739-1748
1739

A R T I C L E S
Peacock et al.
using SHELXTL, and H-atoms were placed in geometrically calculated
positions. X-ray crystallographic data for complexes 4, 4R, 6‚0.25CH3-
OH, 7, and 8 are available as Supporting Information and have been
deposited in the Cambridge Crystallographic Data Centre under the
accession numbers CCDC 291587, 291588, 291589, 291590, and
291591, respectively.
(b) NMR Spectroscopy. 1H NMR spectra were acquired on Bruker
DMX 500 (1H ) 500 MHz), AVA 600 (1H ) 600 MHz), or AVA 800
(1H ) 800 MHz) spectrometers using TBI [1H, 13C, X] probe-heads
equipped with z-field gradients. 1H NMR spectra in D2O were typically
acquired with water suppression by Shaka’s method22 or with presatu-
ration. 1H NMR chemical shifts were internally referenced to 1,4-
dioxane (3.75 ppm) for aqueous solutions, (CHD2)(CD3)SO (2.50 ppm)
for DMSO-d6, and CHCl3 (7.26 ppm) for chloroform-d solutions. All
data processing was carried out using XWIN-NMR version 2.0 (Bruker
U.K. Ltd.).
(c) Mass Spectrometry. Electrospray ionization mass spectra (ESI-
MS) were obtained on a Micromass Platform II mass spectrometer,
and D2O/H2O solutions were infused directly. The capillary voltage
was 3.5 V, and the cone voltage was varied between 10 and 45 V,
depending on sensitivity. The source temperature was 353 K. Mass
spectra were recorded with a scan range m/z 200-1200 for positive
ions.
tion of X by H2O) may be an important step in the mechanism
of action of RuII arene complexes12 and the rate-determining
step in DNA binding. Here we explore the chemical and
biological activity of arene en and acac complexes of the heavier
congener OsII. Previous reports on the aqueous chemistry of
osmium arene complexes are limited.13-15 Studies of the
biological activity of osmium complexes appear to have been
largely restricted to osmium carbohydrate polymers which
exhibit antiarthritic activity.16 Third-row transition metal ions
are commonly more inert than those of the first and second
rows. For example, substitution reactions of PtII complexes are
commonly ca. 5 orders of magnitude slower17a than those of its
lighter congener PdII, and similarly OsII is generally found to
be much more inert than its lighter congener RuII.17 Here we
show that the choice of the chelating ligand (LL ) en or acac)
in [(η6-arene)Os(LL)Cl]n+ complexes can have a dramatic effect
on hydrolysis behavior and reactivity toward nucleobases,18
factors which are likely to be important in the design of OsII
arene anticancer complexes.
Experimental Section
(d) pH* Measurement. The pH* (pH meter reading without
correction for effects of D on the glass electrode) values of NMR
samples in D2O were measured at ca. 298 K directly in the NMR tube,
before and after recording NMR spectra, using a Corning 240 pH meter
equipped with an Aldrich micro combination electrode calibrated with
Aldrich buffer solutions at pH 4, 7, and 10.
Materials. 9-Ethylguanine, adenosine, cytidine, thymidine, and
ethylenediamine were purchased from Sigma-Aldrich; deuterated
solvents, ammonium tetraphenylborate, and ammonium tetrafluorobo-
rate from Aldrich; and OsCl3‚nH2O from Alfa Aesar. Ethylenediamine
was distilled over sodium; sodium acetylacetonate monohydrate was
dried in vacuo; and ethanol and methanol were distilled over magnesium/
iodine prior to use.
(e) Hydrolysis. A solution of 5 in 5% MeOD-d4/95% D2O (v/v)
(ca. 1 mM and 298 K) was prepared by dissolution of 5 in MeOD-d4,
followed by rapid dilution with D2O, and 1H NMR spectra were
recorded at various time intervals. The rate of hydrolysis was determined
Complexes 1-8 were synthesized using procedures similar to those
reported previously,8,9 and the details and characterization of the
complexes are given in the Supporting Information. Crystals suitable
for X-ray diffraction were obtained at 253 K for 4 (from a saturated
solution in acetone), 6‚0.25CH3OH (from THF/hexane solution), and
8 (by slow evaporation from an acetone/hexane solution) or at ambient
temperature for 4R (by diffusion of diethyl ether into a dichloromethane
solution) and 7 (by the slow diffusion of diethyl ether into a chloroform
solution).
1
by fitting the data for concentrations (determined by H NMR peak
integrals) versus time using the appropriate equation for pseudo-first-
order kinetics using the program ORIGIN version 5.0 (Microcal
Software Ltd.). Solutions of 6, 7, and 8 at ca. 2 mM concentrations
were made up at 298 K in D2O. Sonication (ca. 10 min) was employed
1
to assist dissolution, and H NMR spectra were recorded at various
1
time intervals. H NMR spectra were also recorded for solutions of
Methods and Instrumentation. (a) X-ray Crystallography. All
diffraction data were collected using a Bruker (Siemens) Smart Apex
CCD diffractometer equipped with an Oxford Cryosystems low-
temperature device operating at 150 K. Absorption corrections for all
data sets were performed with the multiscan procedure SADABS.19
Structures were solved using either Patterson or direct methods
(SHELXL20 or DIRDIF21). Complexes were refined against F or F 2
5-8 in D2O after addition of 1 mol equiv of AgNO3 and removal of
the AgCl precipitate by filtration through a glass wool plug, and after
addition of excess NaCl (to give 0.15 M) to an equilibrium solution of
8 in D2O. The 1H NMR spectrum of 8 after dissolution in a 1 M solution
of NaCl in D2O was also recorded.
To mimic typical biological test conditions (concentrations and
solvents), a 50 µM stock solution of 8 in 0.125% DMSO-d6/99.875%
D2O (v/v) (measured pH* 7.74) was prepared by dissolution of 8 in
DMSO-d6, followed by rapid dilution with D2O. An aliquot of this
stock solution was then diluted with D2O to give a 2 µM Os solution
(12) Wang, F.; Chen, H.; Parsons, S.; Oswald, I. D. H.; Davidson, J. E.; Sadler,
P. J. Chem. Eur. J. 2003, 9, 5810-5820.
(13) Hung, Y.; Kung, W.; Taube, H. Inorg. Chem. 1981, 20, 457-463.
(14) Stebler-Ro¨thlisberger, M.; Hummel, W.; Pittet, P. A.; Bu¨rgi, H. B.; Ludi,
A.; Merbach, A. E. Inorg. Chem. 1988, 27, 1358-1363.
1
(measured pH* 8.27). The 800 MHz H NMR spectra were recorded
(15) Mui, H. D.; Brumaghim, J. L.; Gross, C. L.; Girolami, G. S. Organometallics
1999, 18, 3264-3272.
after ca. 30 min at 298 K. Samples were then incubated at 310 K for
24 h (a typical cell exposure time and temperature) and 1H NMR spectra
recorded at 310 K.
(16) Hinckley, C. C.; Bemiller, J. N.; Strack, L. E.; Russell, L. D. ACS Symp.
Ser. 1983, 209, 421-37.
(17) (a) Tobe, M. L.; Burgess, J. Inorganic Reaction Mechanisms; Addison-
Wesley Longman Inc.: Essex, 1999. (b) Shriver, D. F.; Atkins, P. W.
Inorganic Chemistry, 3rd ed.; Oxford University Press: Oxford, 1999; p
245. (c) Lay, P. A.; Harman, W. D. AdV. Inorg. Chem. 1991, 37, 219-
379. (d) Richens, D. T. The Chemistry of Aqua Ions; Wiley: Chichester,
1997; pp 421-429. (e) Griffith, W. P. In ComprehensiVe Coordination
Chemistry; Wilkinson, G., Ed.; Pergamon: Oxford, 1987; Vol. 4, Ch. 46,
pp 519-633. (f) Ashby, M. T.; Alguindigue, S. S.; Khan, M. A.
Organometallics 2000, 19, 547-552. (g) George, R.; Andersen, J. M.; Moss,
J. R. J. Organomet. Chem. 1995, 505, 131-133. (h) Halpern, J.; Cai, L.;
Desrosiers, P. J.; Lin, Z. J. Chem Soc., Dalton Trans. 1991, 717-723.
(18) Peacock, A. F. A.; Ferna´ndez, R.; Walland, V.; Habtemariam, A.; Fabbiani,
F. P. A.; Parsons, S.; Aird, R.; Jodrell, D. I.; Sadler, P. J. Second
International Symposium on Bioorganometallic Chemistry (ISBOMC’04),
University of Zu¨rich: Zu¨rich, Switzerland, 2004; abstract p 98.
(19) SADABS: Area-Detector Absorption Correction; Siemens Industrial Au-
tomation, Inc.: Madison, WI, 1996.
The pH* of a solution of 8 (ca. 2 mM) in D2O was adjusted from
2.1 to 10.2, and then reversed to 2.5 and retitrated to 11.8, by the
addition of DCl or NaOH, and H NMR spectra were recorded.
(f) Calculation of pKa* Values. For determinations of pKa* values
(pKa values determined for D2O solutions), the pH* values of solutions
1
(20) Sheldrick, G. M. SHELXL-97, Program for the refinement of crystal
structures; University of Gottingen: Gottingen, Federal Republic of
Germany, 1997.
(21) Beurskens, P. T.; Beurskens, G.; Bosman, W. P.; de Gelder, R.; Garcia-
Granda, S.; Gould, R. O.; Israel, R.; Smits, J. M. M. DIRDIF; Crystal-
lography Laboratory, University of Nijmegen: Nijmegen, The Netherlands,
1996.
(22) Hwang, T. L.; Shaka, A. J. J. Magn. Reson. 1995, A112, 275-279.
9
1740 J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006

Tuning the Reactivity of OsII and RuII Arene Complexes
A R T I C L E S
Chart 1. Osmium Arene Complexes Studied in This Work
of 5A, 6A, 7A, and 8A in D2O (formed in situ by dissolution of the
parent chloro complexes 5-8) were varied from ca. 2.5 to ca. 10 by
1
the addition of dilute NaOH, and H NMR spectra were recorded.
The pH titration curves were fitted to the Henderson-Hasselbalch
equation using the program KALEIDAGRAPH,23 with the assumption
that the observed chemical shifts are weighted averages according to
the populations of the protonated and deprotonated species. The errors
in pKa* values are estimated to be ca. (0.04 unit. These pKa* values
can be converted to pKa values by use of the equation pKa ) 0.929pKa*
+ 0.42, suggested by Krezel and Bal,24 for comparison with related
values in the literature.
(g) Interactions with Nucleobases. The reaction of complex 5 or 8
with nucleobases typically involved the addition of a solution containing
1 mol equiv of nucleobase in D2O to an equilibrium solution of 5 or 8
in D2O (which contains largely aqua 5A and chloro 5, or largely aqua
8A and hydroxo-bridged dimer 8B complexes, respectively), or to a
solution of 5A (prepared by the addition of 1 mol equiv of AgNO3 to
a solution of 5 and removal of AgCl by filtration). The pH* value of
the sample was adjusted if necessary with dilute NaOH so as to remain
1
within a physiologically relevant range (close to 7). H NMR spectra
of these solutions were recorded at 298 K at various time intervals.
A pH* titration of a solution containing equimolar 8 and 9-eth-
ylguanine (9-EtG) was carried out from pH* 2 to 9, and 1H NMR
spectra were recorded at 298 K. A solution containing 8 and equimolar
adenosine (Ado) was also titrated at 298 K from pH* 7 to 1 by the
addition of dilute HClO4. For both titrations, the purine H8 chemical
shifts (together with that for H2 of Ado) were plotted against pH* and
the data fitted to the Henderson-Hasselbalch equation.
(h) Cytotoxicity toward A2780 and A549 Human Cancer Cells.
After plating, human ovarian A2780 cancer cells were treated with OsII
complexes on day 3, and human lung A549 cancer cells on day 2, at
concentrations ranging from 2 to 50 µM. Solutions of the OsII complexes
were made up in 0.125% DMSO to assist dissolution. Cells were
exposed to the complexes for 24 h, washed, supplied with fresh medium,
and allowed to grow for three doubling times (72 h), and then the protein
content was measured (proportional to cell survival) using the sulfor-
hodamine B (SRB) assay.25
Results
The complexes studied in this work are shown in Chart 1.
Osmium(II) arene complexes containing chelated en (4, 5) or
acac (6-8) and Cl- as leaving group were synthesized in good
yields via the Cl-bridged dimers, [(η6-arene)OsCl2]2 (1-3).
X-ray Crystallography. We determined the X-ray crystal
structures of [(η6-bip)Os(en)Cl][BPh4] (4), [(η6-bip)Ru(en)Cl]-
[BPh4] (4R),26 [(η6-bip)Os(acac)Cl]‚0.25CH3OH (6‚0.25CH3-
OH), [(η6-bz)Os(acac)Cl] (7), and [(η6-p-cym)Os(acac)Cl] (8).27
All have the familiar pseudo-octahedral “three-legged piano-
stool” geometry, with OsII π-bonded to the arene ligand (the
“seat”) and σ-bonded to a chloride and a chelating ligand (the
three “legs” of the piano stool). Crystallographic data are shown
in Table S1, and selected bond lengths and angles are listed in
Table S2. No intermolecular arene ring stacking is observed
for any of these complexes.
in the same monoclinic unit cell, space group Cc, with almost
identical unit cell dimensions. The BPh4- counterion in both 4
and 4R sandwiches the chelated ethylenediamine, forming
chains with short-range en(NH)‚‚‚C(BPh4) interactions of 3.424-
(5) Å (N(1)‚‚‚C(16)) for 4 and 3.293(6) Å (N(8)‚‚‚C(30)) for
4R (see Figure S1). The metal-Cl (2.4015(9) and 2.4005(10)
Å for 4 and 4R, respectively), metal-N (2.108(3)-2.148(3)
Å), and metal-C (2.149(4)-2.230(4) Å) bond lengths are
similar for the two complexes (Table S2), as is the propeller
twist of the biphenyl ligand (ca. 15°).
The structures of the OsII complexes 6-8 containing chelated
acac are shown in Figure 2. The Os-Cl and Os-O bond lengths
range from 2.4177(10) to 2.4222(10) Å and from 2.062(11) to
2.086(11) Å, respectively (Table S2). Complex 6 crystallizes
with one-fourth solvent molecule of methanol and shows
disorder in the coordinated biphenyl ring, which occupies three
different positions (Figure S2), resulting in a lower quality of
determination. For all of these conformations, the propeller twist
of the biphenyl ligand is ca. 30°, and the pendent phenyl ring
lies above the chelated acac ligand. Complex 7 shows disorder
in the coordinated benzene ring over two different positions
with equal occupancy (Figure S2). Two independent molecules
of 8 crystallize in the same unit cell, but no disorder was
observed for the coordinated p-cymene ring.
The structures of the OsII complex 4 and RuII complex 4R
containing chelated en are shown in Figure 1. They crystallize
(23) KALEIDAGRAPH, version 3.09; Synergy Software: Reading, PA, 1997.
(24) Krezel, A.; Bal, W. J. Inorg. Biochem. 2004, 98, 161-166.
(25) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica,
D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer
Inst. 1990, 82, 1107-1112.
(26) We reported previously the X-ray crystal structure of the PF6 salt8 but were
able to crystallize the OsII complex only as a BPh4 salt. Hence, we have
now determined the X-ray structure of 4 so that direct comparison is
possible.
(27) We reported previously the X-ray structure of the Ru analogue [(η6-p-
cym)Ru(acac)Cl].9
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A R T I C L E S
Peacock et al.
Figure 1. ORTEP diagrams and atom numbering schemes for (A) [(η6-
bip)Os(en)Cl][BPh4] (4) and (B) [(η6-bip)Ru(en)Cl][BPh4] (4R) (50%
probability ellipsoids). The H atoms have been omitted for clarity. The
sandwich formed by the phenyl rings of the counteranion and the en
backbone is apparent.
An interesting feature of the crystal structures of complexes
6-8 is the directional hydrogen bonding ( DsH‚‚‚A 150° <
θ < 180°) between coordinated (arene)H and (acac)O atoms of
adjacent molecules (Table 1), the shortest CH‚‚‚O hydrogen
bond being 2.29 Å for 6. These hydrogen bonds are responsible
for the formation of dimers (Figure S3).
1
Hydrolysis. The H NMR spectrum of the en complex 5,
[(η6-bip)Os(en)Cl][BF4], in 5% MeOD-d4/95% D2O at 298 K
initially contained one major set of peaks. A second set of peaks
increased in intensity with time, reaching equilibrium after ca.
30 h (Figure S4). The new peaks had chemical shifts similar to
those of the aqua complex 5A, prepared by treatment of 5 with
AgNO3, under the same conditions (arene peaks doublet δ 6.28,
triplet δ 6.05, and triplet δ 5.96; pH* 7, Figure S5). Fitting the
hydrolysis data to pseudo-first-order kinetics gave a half-life
of 6.4 ( 0.2 h (298 K) (Figure 3).
In contrast, no peaks for the parent chloro complexes were
1
observed in the H NMR spectra of ca. 2 mM solutions of the
Figure 2. ORTEP diagrams and atom numbering schemes for (A) [(η6-
bip)Os(acac)Cl] (6), (B) [(η6-bz)Os(acac)Cl] (7), and (C) [(η6-p-cym)Os-
(acac)Cl] (8) (50% probability ellipsoids). The H atoms have been omitted
for clarity. Two independent molecules of 8 crystallize in the same unit
cell; only one is shown.
acac complexes 6-8 in D2O at 298 K. Within 10 min of
dissolution, two major sets of peaks were seen in the arene
region: 6A (doublet δ 6.67, triplet δ 6.58, overlapped triplet δ
6.45) and 6B (overlapped doublet δ 6.46, triplet δ 6.25, triplet
δ 6.01), 7A (singlet δ 6.28) and 7B (singlet δ 6.03), and 8A
(doublets at δ 6.28 and δ 6.07) and 8B (doublets at δ 6.04 and
δ 5.82) (Figure 4). Peaks assignable to free acacH (δ 2.26) were
also present in the spectra. The ESI-MS spectra of these NMR
samples contained peaks at m/z 445 (6A) and 741 (6B), 370
(7A) and 591 (7B), and 426 (8A) and 701 (8B), assignable to
the fragment [(η6-arene)Os(acac)]+ arising from 6A, 7A, and
8A (calcd m/z 445.1, 370.1, and 426.1, respectively) and to [(η6-
arene)Os(µ2-OH)3Os(η6-arene)]+ for 6B, 7B, and 8B (calcd m/z
742.1, 590.0, and 701.1, respectively). Further confirmation of
by recording the spectra of solutions of 6, 7, and 8 in D2O after
addition of 1 mol equiv of AgNO3 (and removal of AgCl by
filtration).
The effect of NaCl on the hydrolysis equilibrium for 8 was
investigated. Addition of excess NaCl (to give 0.15 M) to an
equilibrium solution of 8 (1.2 mM) in D2O caused the p-cymene
1H NMR peaks for species 8A to shift from 6.26 and 6.05 ppm
to 6.19 and 5.95 ppm, respectively, and broaden, whereas the
peaks for species 8B at 6.05 and 5.82 ppm remained unchanged.
The resulting spectrum was similar to that obtained on dissolu-
tion of 8 in a 1 M NaCl solution in D2O (see Figure S6).
1
the assignment of the H NMR peaks for 6A, 7A, and 8A to
the aqua complexes [(η6-arene)Os(acac)(OH2)]+ was obtained
9
1742 J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006

Tuning the Reactivity of OsII and RuII Arene Complexes
A R T I C L E S
Table 1. Selected Hydrogen-Bonding Interactions for Complexes
6-8
D
sH‚‚‚
A
H
‚‚‚A (Å)
D‚‚‚A (Å)
D−H‚‚‚A (°)
(A) [(η6-bip)Os(acac)Cl]‚0.25CH3OH (6‚0.25CH3OH)
C42sH42‚‚‚O14 (inter)
C51sH51‚‚‚O54 (inter)
2.50
2.29
3.42(2)
3.23(2)
163
168
(B) [(η6-bz)Os(acac)Cl] (7)
C106sH1051‚‚‚O4 (inter)
C201sH2043‚‚‚O8 (inter)
2.34
2.31
3.332(13)
3.319(9)
169
175
(C) [(η6-p-cym)Os(acac)Cl] (8)
C53sH53‚‚‚O52 (inter)
C411sH41A‚‚‚O14 (inter)
2.56
2.60
3.412(6)
3.565(7)
149
169
Figure 5. Hydrolysis of [(η6-p-cym)Os(acac)Cl] (8) at micromolar
concentrations. Low-field region of the 1H NMR spectra of a 50 µM solution
of 8 in D2O (A) after 30 min at 298 K and (B) at 310 K after 24 h incubation
at 310 K, and of a 2 µM solution of 8 (C) after 30 min at 298 K and (D)
at 310 K after 24 h incubation at 310 K (biological test concentrations and
conditions). Signals at δ 6.04 and 5.82 are assigned as p-cymene arene
resonances of 8B, [(η6-p-cym)Os(µ2-OD)3Os(η6-p-cym)]+. (E) ESI-MS of
sample from (B). The major peak at m/z 704 is assignable to the fragment
[(η6-p-cym)Os(µ2-OD)3Os(η6-p-cym)]+.
Figure 3. Hydrolysis of [(η6-bip)Os(en)Cl]BF4 (5). Time dependence of
the decay of the chloro complex 5 and formation of the aqua/hydroxo adduct
5A in 5% MeOD-d4 95% D2O at 298 K. The curves represent the computer
best-fit for a pseudo-first-order reaction corresponding to an hydrolysis rate
constant of 0.11 h-1 (half-life of 6.4 ( 0.2 h).
Figure 6. Determination of the pKa* values of the aqua complexes [(η6-
arene)Os(LL)(OD2)]n+. Dependence of the 1H NMR chemical shifts of the
coordinated arene ring on pH* for (A) [(η6-bip)Os(en)(OD2)]2+ (5A; b ortho
Ho, 9 meta Hm, [ para Hp), (B) [(η6-bip)Os(acac)(OD2)]+ (6A; b ortho
Ho, 9 meta Hm, 1 para Hp), (C) [(η6-bz)Os(acac)(OD2)]+ (7A), and (D)
[(η6-p-cym)Os(acac)(OD2)]+ (8A). The curves represent the best fits to the
Henderson-Hasselbalch equation and correspond to pKa* values of 6.37
for 5A, 7.28 for 6A, 7.45 for 7A, and 7.84 for 8A.
Figure 4. Hydrolysis of the acac complexes [(η6-arene)Os(acac)Cl]. 1H
NMR spectra of complexes (A) 6 (arene ) bip), (B) 7 (arene ) bz), and
(C) 8 (arene ) p-cym) in D2O recorded 30 min after dissolution at
concentrations of 3.5, 2.1, and 3.4 mM, respectively, and 298 K. Two sets
of peaks appear, corresponding to the aqua species, [(η6-arene)Os(acac)-
(OD2)]+ (6A, 7A, and 8A), and the hydroxo-bridged dimers, [(η6-arene)-
Os(µ2-OD)3Os(η6-arene)]+ (6B, 7B, and 8B).
8 in 0.15 M NaCl in D2O (mimicking the chloride concentration
in cell culture media) at 298 K. Again, the only peaks present
in the spectrum were those for the hydroxo-bridged dimer 8B.
1
The 1H NMR spectra of solutions of 8, [(η6-p-cym)Os(acac)-
Cl], at low (micromolar) concentrations within the range used
in the cytotoxicity tests were recorded. Spectra of 8 at 2 and 50
µM (in 0.125% DMSO-d6/99.875% D2O (v/v), pH* 7.7) after
ca. 30 min at 298 K and 24 h after incubation at 310 K contained
only one set of peaks in the arene region (δ 6.04 and δ 5.82,
Figure 5), assignable to species 8B. Spectra recorded in the
absence of DMSO-d6 were the same. The ESI-MS of an equili-
brium solution (Figure 5) contained a peak at m/z 704, confirm-
ing that the major species present is the hydroxo-bridged dimer,
8B, [(η6-p-cym)Os(µ2-OD)3Os(η6-p-cym)]+ (calcd m/z 703.9).
We also recorded a 1H NMR spectrum of a 50 µM solution of
pH* Dependence. The changes in the H NMR chemical
shifts of the coordinated phenyl ring of 5A present in an
equilibrium solution of 5 in D2O at 298 K (see Figure S4) were
followed with change in pH* over the range 2-9. These data
were fitted to the Henderson-Hasselbalch equation (Figure 6A),
which yielded a pKa* value of 6.37.
1
The pH* dependence of the H NMR chemical shifts of the
coordinated arenes of complexes 6A/B, 7A/B, and 8A/B was
monitored over the range 2-10 (Figure 6). Those for species
6B, 7B and 8B remained unchanged28 with pH* (over the range
2-10), but their intensities increased with increase in pH*
(Figure S7). In contrast, the peaks for 6A, 7A, and 8A shifted
9
J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006 1743

A R T I C L E S
Peacock et al.
Figure 7. Formation of hydroxo-bridged dimer from the aqua complex
[(η6-p-cym)Os(acac)(OD2)]+. 1H NMR spectra recorded during a pH titration
of a solution of the chloro complex 8 in D2O, containing predominantly
the aqua/hydroxo complex 8A ([(η6-p-cym)Os(acac)(OD2/OD)]+/0) and the
dimer 8B ([(η6-p-cym)Os(µ2-OD)3Os(η6-p-cym)]+), showing how the peaks
for 8B increase in intensity with increasing pH* but do not shift, whereas
those for 8A shift to high field (deprotonation, fast exchange between aqua
and hydroxo forms) and decrease in intensity as conversion into the dimer
occurs.
Figure 8. Reaction of 9-ethylguanine with [(η6-bip)Os(en)(OD2/OD)]2+/+
(5A) (at pH* 7.28, mostly in the hydroxo form). Low-field region of the
1H NMR spectrum of 5A (formed by treating a solution of the chloro
complex 5 with 1 mol equiv of AgNO3) after reaction with 2 mol equiv of
9-EtG in D2O at 298 K for 22 h. At this time, 45% of 5A had reacted to
form 9 ([(η6-bip)Os(en)(9-EtG-N7)]2+). Peak labels correspond to the
structures; peak n is from H8 of unbound 9-EtG.
to high field and decreased in intensity with increase in
pH* (Figure 7), the maximum shift change being ca. 0.3 ppm
in each case. These data yielded pKa* values of 7.28, 7.45, and
7.84 for the biphenyl complex 6A, benzene complex 7A, and
p-cymene complex 8A, respectively (Figure 6B-D).
Figure 9. Reaction of 9-ethylguanine with [(η6-p-cym)Os(acac)Cl] (8).
1
Low-field region of the H NMR spectrum of 8 after reaction with 1 mol
equiv of 9-EtG in D2O, 298 K, at equilibrium (within ca. 10 min of reaction).
Peak labels: G, unreacted 9-EtG (corresponds to 40% of total 9-EtG); 10,
[(η6-p-cym)Os(acac)(9-EtG-N7)]+ (60%); 8A, unreacted aqua complex [(η6-
p-cym)Os(acac)(OD2)]+ (7%); 8B, dimer [(η6-p-cym)Os(µ2-OD)3Os(η6-p-
cym)]+ (33%); x, an unidentified product.
1
Analysis of peak integrals of H NMR spectra showed that
millimolar solutions of 8 in D2O at pH* 2.1 contained mostly
8A, and only 4% of 8B. On raising the pH* to 10.2, very little
of 8A remained and the proportion of 8B increased to 78%.
However, on lowering the pH* to 2.5, the amount of 8A present
remained the same but the proportion of 8B fell to 33%, and
peaks for unidentified species x1 and x2 (which were minor
components in the initial solution at pH* 2.1) increased in
intensity (see Figure S8). At pH* 11.8, only peaks for 8B and
free acac were observed (Figure S8).
Addition of 1 mol equiv of 9-EtG in D2O to an equilibrium
solution of the acac complex 8, [(η6-p-cym)Os(acac)Cl], in D2O
(which contained 70% aqua complex 8A and 30% hydroxo-
bridged dimer 8B, together with some free acacH) at 298 K,
pH* 7.50, resulted in a new set of 1H NMR peaks (for species
10, see Figures 9 and S12) within 10 min (by which time
equilibrium had been reached). Peaks for 8A diminished in
intensity, but those for 8B did not change. Based on H8 peak
integrals for free (δ 7.82) and bound (δ 7.76) 9-EtG, 60% of
the 9-EtG reacted to form 10. The major ESI-MS peak for the
equilibrium NMR sample at a low cone voltage (10 V) was at
m/z 605, consistent with the formation of [(η6-p-cym)Os(acac)-
(9-EtG-N7)]+ (10, calcd m/z 605.2) (see Figure 10). To establish
the binding site for OsII on 9-EtG, a pH* titration of the reaction
mixture was monitored by 1H NMR spectroscopy over the range
pH* 2-8 (Figure 11). The chemical shift of H8 for 10 did not
change with pH*, whereas that of free 9-EtG moved to high
field by ca. 1 ppm, corresponding to a pKa* of 3.26, consistent
with values for N7 protonation reported previously.30
Interactions with Nucleobases. Since DNA is a potential
target site for transition metal anticancer complexes,29 reactions
with model nucleobases were studied.
Addition of 1 mol equiv of 9-EtG to an equilibrium solution
of complex 5, [(η6-bip)Os(en)Cl][BF4], in D2O (pH* 7.28) at
1
298 K resulted in no new H NMR peaks after 10 min. After
22 h, 23% of 5 had reacted and a new H8 peak appeared at
8.09 ppm (for species 9, see Figure S9), shifted by 0.27 ppm to
low field relative to that of free 9-EtG. Addition of 2 mol equiv
of 9-EtG to a solution of 5A (pH* 7.0) (prepared by treating a
solution of 5 with 1 mol equiv of AgNO3) also resulted in no
1
new H NMR peaks after 10 min, but after 22 h new peaks
assignable to 9 had appeared, and 45% of 5A had reacted with
9-EtG to form 9 (Figures 8 and S10).
The addition of a solution of 1 mol equiv of adenosine (Ado),
cytidine (Cyt), or thymidine (Thy) to an equilibrium solution
of 5 in D2O at 298 K resulted in no additional 1H NMR peaks
over a period of 24 h (Figure S11).
1
No new H NMR peaks appeared in the spectrum of 8 on
addition of 1 mol equiv of 9-EtG under more alkaline conditions,
where the hydroxo-bridged dimer 8B predominates (pH* 9.9,
>95% 8B), even after 96 h, indicative of the inertness of 8B.
Addition of a solution of 1 mol equiv of Ado to an
equilibrium solution of 8 in D2O (which contain 70% aqua
(28) The intensity of the 1H NMR peak for the acacH methyl protons decreases
at basic pH* as they exchange readily with D from the solvent, as reported
previously: Johnson, D. A. Inorg. Chem. 1966, 5, 1289-1291. The central
γ proton exchanges readily with D2O even at neutral pH*.
(30) Kampf, G.; Kapinos, L. E.; Griesser, R.; Lippert, B.; Sigel, H., J. Chem.
Soc., Perkin Trans. 2 2002, 1320-1327.
(29) Zhang, C. X.; Lippard, S. J. Curr. Opin. Chem. Biol. 2003, 7, 481-489.
9
1744 J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006

Tuning the Reactivity of OsII and RuII Arene Complexes
A R T I C L E S
Figure 10. Characterization of 9-ethylguanine and adenosine adducts by
mass spectrometry. ESI mass spectra for solutions containing [(η6-p-cym)-
Os(acac)Cl] (8) and 1 mol equiv of (A) 9-ethylguanine and (B) adenosine
in D2O. Peaks at m/z 425/426 correspond to 8A ([(η6-p-cym)Os(acac)]+),
m/z 701 to the hydroxo-bridged dimer 8B, and 605 and 694 to 10 ([(η6-
p-cym)Os(acac)(9-EtG)]+) and 11 ([(η6-p-cym)Os(acac)(Ado)]+), respec-
tively.
Figure 12. Reaction of [(η6-p-cym)Os(acac)Cl] (8) with adenosine. (A)
1H NMR spectrum of a D2O solution of 8 and 1 mol equiv of adenosine,
pH* 7.31, 298 K, at equilibrium (within ca. 10 min of reaction). (B)
Dependence of the 1H NMR chemical shifts of Ado H8 and H2 resonances
on pH*. Assignments: 8B, hydroxo-bridged dimer [(η6-p-cym)Os(µ2-OD)3-
Os(η6-p-cym)]+; 8A aqua complex [(η6-p-cym)Os(acac)(OD2)]+; A, unre-
acted adenosine (Ado); d, dioxane, a, free acacH; 11A, major product, [(η6-
p-cym)Os(acac)(Ado-N7)]2+; 11B, minor product, [(η6-p-cym)Os(acac)(Ado-
N1)]2+. Based on peak integration, 66% Ado has reacted to give 56% 11A
and 10% 11B. Binding through N7 lowers the pKa* of N1 by ca. 1.7 pH
units.
Figure 11. Determination of {(η6-p-cym)Os(acac)}+ binding site on
The addition of 1 mol equiv of Cyt or Thy to an equilibrium
solution of 8 in D2O at 298 K resulted in no additional 1H NMR
peaks over a period of 24 h (Figure S13).
Cytotoxicity toward A2780 and A549 Human Cancer
Cells. Complexes 4, 5, and 8 were all nontoxic toward the
human ovarian A2780 cancer cell line at concentrations up to
50 µM (the highest test concentration). The IC50 values are
therefore likely to be >100 µM, and the complexes are inactive.
Similarly complexes 6-8 were found to be inactive against the
human lung A549 cancer cell line.
1
9-ethylguanine. Dependence of the H NMR chemical shift of 9-EtG H8
on pH* for a solution containing 10 ([(η6-p-cym)Os(acac)(9-EtG-N7)]+)
and free 9-EtG. The lack of pH* dependence for 10 over the pH* range
from 5 to 2, where N7 protonates, confirms that binding of 9-EtG to OsII
is through N7.
complex 8A, 30% hydroxo-bridged dimer 8B, and some free
acacH) at 298 K (pH* 7.31) led to the appearance of new peaks
within 10 min (Figure 12A), assignable to a major product 11A
(accounting for 56% of Ado) and a minor product 11B (10%
of Ado). In the H8/H2 region, two new sets of peaks appeared
at 8.50 and 8.31 ppm for 11A and at 8.39 and 8.17 ppm for
11B. ESI-MS studies of this NMR sample at a low cone voltage
(20 V) gave a peak at m/z 694, consistent with [(η6-p-cym)-
Os(acac)(Ado)]+ (11, calcd m/z 694.2) (Figure 10). The binding
sites for {(η6-p-cym)Os(acac)}+ on Ado in 11A and 11B were
assigned with the aid of a 1H NMR pH* titration of the reaction
mixture over the pH* range from 7 to 1 (Figure 12B). The
singlets for H8 and H2 of free Ado shifted to low field with an
associated pKa* of 3.63, a value similar to that reported
previously for N1 protonation,30 whereas the H8 and H2 peaks
for 11B did not shift over this pH* range, consistent with the
presence of N1-bound Ado in this complex, [(η6-p-cym)Os-
(acac)(Ado-N1)]2+. Those for 11A shifted to low field over this
pH* range with an associated pKa* value of 1.92, assignable to
N1 protonation of N7-bound Ado. This corresponds to a
lowering of the pKa* of N1 by 1.7 units for 11A [(η6-p-cym)-
Os(acac)(Ado-N7)]2+ compared to free Ado. Interestingly, the
arene peaks for 11A are greatly broadened (Figure 12A; no
significant sharpening over the temperature range 298-323 K).
Discussion
X-ray Crystallography. A search of the Cambridge Database
revealed that only ca. 200 structures of osmium arene complexes
have been reported, compared to ca. 3000 for ruthenium arene
complexes. The crystal structures of complexes 4 and 6 are the
first to be reported with η6-biphenyl coordination to osmium,
and 4 is the first containing an {(η6-arene)Os(en)} fragment.
The OsII (4) and RuII (4R) en complexes are isostructural in
the solid state. The M-Cl bond lengths are the same (2.40 Å),
even though the ligand exchange rates are different. Similarly,
the OsII acac complex 8 and its RuII analogue [(η6-p-cym)Ru-
(acac)Cl], which we reported previously,9 have similar M-Cl
and M-O bond lengths.
Hydrogen bonds are observed between (arene)H and (acac)O
of adjacent molecules in the crystal structures of osmium
complexes 6-8, giving rise to dimers (Figure S3). Such H-bonds
involving coordinated arenes are becoming increasingly recog-
nized.31 We observed them previously9 for [(η6-p-cym)Ru(acac)-
9
J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006 1745

A R T I C L E S
Peacock et al.
,
Table 2. pKa Values for [(η6-Arene)M(chelate)(OH2)]n+
Cl], and they are also present (but not often noted) in the crystal
structures of other reported OsII arene acac complexes.32
Hydrogen bonds can be distinguished from van der Waals
interactions by their directional nature ( DsH‚‚‚A 150° < θ
< 180°), as they consist of both van der Waals and electrostatic
components.33 CsH‚‚‚O H-bonds are typically given cutoff
values of <2.6 Å.34 They are weaker than classical hydrogen
bonds, and present only in the absence of stronger interactions,
but can direct crystal packing.35 The weak polarization of arene
ring C-H bonds may lead to interactions between the coordi-
nated arene and H-bond acceptors in biological target sites such
as DNA or RNA.
Hydrolysis. There are few previous studies of the aqueous
chemistry of OsII arene complexes.13-15 The hydrolysis of the
OsII en complex 5 in water (Figure 3, half-life of 6.4 h) is ca.
40 times slower than that of its RuII analogue.12 This behavior
is consistent with the much slower ligand exchange rates often
associated with osmium complexes in comparison with those
of ruthenium. However, Stebler-Ro¨thlisberger et al. found that
the OsII and RuII complexes [(η6-bz)M(OH2)3]2+, without a
chelating ligand, possess similar water exchange rates.14 The
presence of a chelating ligand can introduce significant differ-
ences between OsII and RuII.
Changing the chelating ligand from en to acac in OsII arene
complexes had a marked effect on the extent and rate of
hydrolysis, as is also the case for RuII analogues.9 However,
hydroxo-bridged dimers play a dominant role in the hydrolysis
behavior of [(η6-arene)Os(acac)Cl] complexes in water (com-
plexes 6B, 7B, and 8B are products from the hydrolysis of the
bip, bz, and p-cym complexes 6, 7, and 8, respectively), whereas
they are only minor components, and then only at high pH*, in
analogous RuII systems.9 There appear to be no previous reports
of the formation of hydroxo-bridged OsII arene complexes on
dissolution of OsII arenes in water, although the dimers36 have
been synthesized by heating [η6-arene)OsCl2]2 in strongly
alkaline solutions.36c,e X-ray structures of [(η6-arene)Ru(µ2-OH)3-
Ru(η6-arene)]+ have been reported,36d,f-h although the only
X-ray structure of a hydroxo-bridged OsII arene dimer appears
to be that of [(η6-p-cym)Os(µ2-H)(µ2-HCO2)(µ2-OH)Os(η6-p-
cym)]+.36e For (sterically nonhindered) benzene as the arene
ligand, further deprotonation of hydroxo bridges appears to be
possible to give the tetramer [M4(η6-bz)(µ2-OH)4(µ4-O)]2+ (M
) Ru or Os).36a,b,d We found no (ESI-MS) evidence for such
tetramers under the less alkaline conditions used in this work.
In contrast to RuII, the hydroxo-bridged dimer was still
detectable in highly acidic millimolar solutions of OsII acac
complexes (pH* 2).
[(η6-bip)Os(en)(OH2)]2+ (5A), [(η6-bip)Os(acac)(OH2)]+ (6A),
[(η6-bz)Os(acac)(OH2)]+ (7A), [(η6-p-cym)Os(acac)(OH2)]+ (8A),
and Some Ruthenium Arene Aqua Complexes
complex
metal
arene
chelate
pKa
5A
Os
Ru
Ru
Os
Os
Os
Os
Ru
bip
bip
bz
bz
bip
bz
p-cym
p-cym
en
en
en
en
acac
acac
acac
acac
6.34a
7.71b
7.9c
6.3d
6A
7A
8A
7.12a
7.27a
7.63a
9.4d
a pKa values calculated from pKa* according to ref 18. b Reference 12.
c Reference 13. d Reference 9.
Particularly relevant to the biological testing is our finding
that the hydroxo-bridged dimer (8B) is the only species present
on dissolution of [(η6-p-cym)Os(acac)Cl] (8) in water at
micromolar concentrations. The chelated acac ligand is readily
protonated (e.g., by an adjacent coordinated aqua ligand in 8A)
and a good leaving group at acidic or near neutral pH (pKa of
free acacH is 8.837). In strongly basic solution, hydroxide
appears to displace acac. The high stability of the hydroxo-
bridged dimer is further illustrated by the finding that it is still
formed as a major species, even in the presence of a large molar
excess of Cl- (3000-fold, concentrations similar to those in cell
culture media). In contrast, ring-opening of chelated en requires
cleavage of the Os-N bond before protonation can occur, and
en is not readily displaced by hydroxide.
1
We observed separate H NMR peaks for the OH2/OH and
the parent Cl complexes in the spectrum of the OsII en complex
5 on dissolution in D2O. At millimolar concentrations of the
OsII acac complex 8, the 1H NMR chemical shifts are consistent
with the existence largely of the aqua complex 8A (confirmed
by addition of AgNO3), with little of the chloro complex present.
1
Only one set of H NMR peaks was observed for solutions
containing the acac aqua complex 8A and Cl species 8,
suggesting rapid exchange of Cl- and coordinated OH2/OH on
the NMR time scale at 298 K, as observed for ruthenium
analogues.9 Addition of a large excess of chloride shifted and
broadened these signals (Figure S6), suggesting that chloride
binding to {(η6-p-cym)Os(acac)}+ is indeed weak.
pKa* Values of Aqua Complexes. The pKa* values of the
en (η6-arene)OsII aqua complex 5A (6.37) and acac aqua
complexes 6A, 7A, and 8A (7.28, 7.45, and 7.84, Table 2) are
all significantly lower (ca. 1.5 units) than those of analogous
(η6-arene)RuII aqua complexes.9,38 The trend in the lowering
of pKa values for OsII versus RuII complexes is consistent with
that reported previously for ([(η6-bz)MII(OH2)3]2+ and [MII-
(trpy)(bipy)(OH2)]2+ (trpy ) 2,2′,2′′-terpyridine, bipy ) 2,2′-
bipyridine).13,38 The replacement of the neutral chelated en
ligand by an anionic acac raises the pKa* by 0.9 unit. A similar
decrease in acidity is observed for the Ru analogues (∆pKa 1.1
units),9 attributable to the increased electron density on the metal
center. The higher acidity of OsII arene aqua complexes can be
attributed to increased mixing of the δσ* (Os) f σ (OH-)
orbitals.38 The pKa* values of the acac complexes 6A, 7A, and
8A are close to physiological pH (7.4), whereas that of the en
(31) Braga, D.; Grepioni, F.; Desiraju, G. R. J. Organomet. Chem. 1997, 548,
33-44.
(32) Bennett, M. A.; Mitchell T. R. B.; Stevens M. R.; Willis A. C. Can. J.
Chem. 2001, 79, 655-669.
(33) Steiner, T.; Desiraju, G. R. Chem. Commun. 1998, 891-892.
(34) Steiner, T. Chem. Commun. 1997, 727-734.
(35) Desiraju, G. R. Acc. Chem. Res. 1996, 29, 441-449.
(36) (a) Gould, R. O.; Jones, C. L.; Robertson, D. R.; Stephenson, T. A. J.
Chem. Soc., Chem. Commun. 1977, 222-223. (b) Gould, R. O.; Jones, C.
L.; Robertson, D. R.; Tocher, D. A.; Stephenson, T. A. J. Organomet. Chem.
1982, 226, 199-207. (c) Arthur, T.; Robertson, D. R.; Tocher, D. A.;
Stephenson, T. A. J. Organomet. Chem. 1981, 208, 389-400. (d) Gould,
R. O.; Jones, C. L.; Stephenson, T. A.; Tocher, D. A. J. Organomet. Chem.
1984, 264, 365-378. (e) Cabeza, J. A.; Smith, A. J.; Adams, H.; Maitlis,
P. M. J. Chem. Soc., Dalton Trans. 1986, 1155-1160. (f) Vieille-Petit, L.;
Therrien, B.; Su¨ss-Fink, G. Inorg. Chem. Commun. 2004, 232-234. (g)
Kim, T. D.; McNeese, T. J.; Rheingold, A. L. Inorg. Chem. 1988, 27, 2554-
2555. (h) Artero, V.; Proust, A.; Herson, P.; Gouzerh, P. Chem. Eur. J.
2001, 7, 3901-3910.
(37) Kortly, S.; Sucha, L. Handbook of Chemical Equilibria in Analytical
Chemistry; Ellis Horwood Limited: New York, 1985; p 188.
(38) Takeuchi, K. J.; Thompson, M. S.; Pipes, D. W.; Meyer, T. J. Inorg. Chem.
1984, 23, 1845-1851.
9
1746 J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006

Tuning the Reactivity of OsII and RuII Arene Complexes
A R T I C L E S
complex 5A is significantly lower, and therefore almost all of
the hydrolyzed en complex would be present in the hydroxo
form, [(η6-bip)OsII(en)(OH)]+.
Interactions with Nucleobases. There is little reported work
on interactions of osmium complexes and nucleobases. Osmium-
(II) polypyridyl complexes are known to bind to DNA and
nucleobases,39-41 and recently adducts of [(η6-p-cym)OsCl2-
(PR3)], where PR3 ) pta or pta-Me (pta ) 1,3,5-triaza-7-
phosphatricyclo[3.3.1.1]decane), with the 14 mer ATACATG-
GTACATA have been detected by ESI-MS.42
tions and may account for the inactivity of the OsII acac
complexes toward human cancer cells.
Conclusions
We have shown previously that organometallic RuII arene
complexes such as [(η6-arene)Ru(LL)Cl]+, where arene is, for
example, biphenyl, benzene, or p-cymene and LL is en or acac,
exhibit anticancer activity.8,11 However, we report here that the
OsII analogues are inactive toward A2780 human ovarian and
A549 human lung cancer cells, despite being structurally very
similar in the crystalline state. We have therefore explored
differences in their solution chemistry in an attempt to under-
stand their contrasting biological activities. There are few
previous investigations of the aqueous chemistry of osmium
arenes.13-15 In general, as a third-row transition metal ion, OsII
might be expected to be relatively inert compared to the second-
row ion RuII.17 However, the presence of an arene ligand can
greatly increase the kinetic lability,14 and, importantly, our data
show that the chelating ligand also plays a major role.
With en as the chelating ligand, hydrolysis of the OsII
complex 5 is ca. 40 times slower than that of the RuII analogue,
and the pKa value (6.34) of the resulting aqua adduct is 1.4
units lower. This implies that the less-reactive hydroxo adduct
would predominate at physiological pH (7.4). Indeed, reactions
with 9-EtG were slower and occurred to a lesser extent than
those of the RuII complexes. Since G bases on DNA appear to
be targets for {(η6-arene)Ru(en)}2+, these differences may
account for biological inactivity of the OsII en complexes.
In contrast, the acac complexes [(η6-arene)Os(acac)Cl] hy-
drolyze rapidly (<10 min, 298 K) to give a mixture of a
monoaqua complex, [(η6-arene)Os(acac)(OH2)]+, and a hy-
droxo-bridged dimer, [(η6-arene)Os(µ2-OH)3Os(η6-arene)]+,
with loss of the acac ligand. The pKa values of the coordinated
water molecules in the monoaqua acac biphenyl, benzene, and
p-cymene complexes (6A, 7A, and 8A, 7.1-7.6) are ca. 1.8
units lower than those of the RuII analogues, for which the
hydroxo-bridged dimer is not observed as an hydrolysis product
(except in very basic solutions, and then only as a very minor
product).
The monoaqua acac complex, 8A, reacts with purine bases
(9-EtG and Ado), but not with pyrimidines (Cyt or Thy); this
is the same base selectivity that was observed for the RuII
analogues.9 However, the hydroxo-bridged dimer, [(η6-arene)-
Os(µ2-OH)3Os(η6-arene)]+, was unreactive toward both purine
and pyrimidine bases. This may be the key to the inactivity of
the OsII arene acac complexes, since the hydroxo-bridged dimer
was the only species present in micromolar aqueous solutions
similar to those used in the biological tests.
This work demonstrates how the kinetics of ligand substitu-
tion reactions of OsII arene complexes in aqueous solution can
be controlled by variation of the chelating ligand. Further tuning
of substitution rates may be achievable by variations of the
monodentate leaving group and the arene. Such a systematic
approach should contribute to the rational design of OsII arene
complexes as potential anticancer agents and may be more
widely applicable to organometallic complexes in general.
The OsII en complex 5 reacts only slowly with 9-EtG, as does
the aqua complex 5A, and then only to a limited extent (45%
after 22 h at 298 K). No reaction was observed (after 24 h)
with Ado, Cyt, or Thy. The RuII analogue also binds selectively
to guanine when in competition with the other three bases, but
it does bind weakly to Cyt and Thy, highlighting the lower
reactivity of OsII. The aqua acac OsII adduct 8A reacts with
both 9-EtG and Ado rapidly (<10 min), but not with Cyt or
Thy, a behavior similar to that of the RuII analogue.9 Binding
to 9-EtG occurs through N7 and to Ado43 through N7 and N1
(with N7 binding being favored by a factor of ca. 6:1; cf. 4:1
for RuII). Binding to Ado N7 lowers the pKa* of N1 by 1.7
pH* units, compared to 1.3 for RuII.9 This trend is similar to
the lowering of pKa values of water coordinated to OsII
compared to RuII. The selectivity in nucleobase binding of the
OsII en and acac complexes can be rationalized in terms of
H-bonding and nonbonding repulsive interactions between the
chelating ligand and nucleobase substituents in addition to the
electronic properties of the various nucleobase coordination
sites,44 as we have discussed previously for the RuII ana-
logues.9,45 The inertness of the hydroxo-bridged dimer 8B
toward nucleobases is notable.
Cytotoxicity toward A2780 and A549 Human Cancer Cell
Lines. None of the OsII arene en or acac complexes studied
here (Chart 1) were cytotoxic to human ovarian or lung cancer
cells at doses up to 50 µM, in contrast to their RuII analogues,
cf. IC50 value for 4R (analogue of 4) of 5 µM and for [(η6-p-
cym)Ru(acac)Cl] (analogue of 8) of 19 µM.11 The inactivity of
the OsII en complex 5 may be attributable to its much slower
hydrolysis (ca. 40 times) and to the higher acidity of the aqua
complex (ca. 1.4 pKa units) compared to the RuII analogue.12
This results in lower reactivity, e.g., toward nucleobases.
Changing the chelating ligand to acac not only increases the
hydrolysis rate dramatically but also lowers the acidity of the
aqua adduct, such that a significant amount of the aqua complex
(as opposed to the hydroxo adduct) exists at biological pH (7.4).
These aqua acac complexes bind to purine bases, as do the RuII
analogues.9 However, displacement of the coordinated acac from
OsII occurs readily, and largely irreversibly, giving rise to stable
hydroxo-bridged dimers. These inert dimers appear to be the
only species present in solution under biological testing condi-
(39) Content, S.; Kirsch-De Mesmaeker, A. J. Chem. Soc., Faraday Trans. 1997,
93, 1089-1094.
(40) Holmlin, R. E.; Barton, J. K. Inorg. Chem. 1995, 34, 7-8.
(41) Mishima, Y.; Motonaka, J.; Ikeda, S. Anal. Chim. Acta 1997, 345, 45-50.
(42) Dorcier, A.; Dyson, P. J.; Gossens, C.; Rothlisberger, U.; Scopelliti, R.;
Tavernelli, I. Organometallics 2005, 24, 2114-2123.
(43) Binding of {(η6-p-cym)Ru}2+ to (deprotonated) C6NH2 of 9-ethyladenine
and adenine to N6/7 chelate rings has been observed for a cyclic trimer
and tetramer, respectively (Korn, S.; Sheldrick, W. S. Inorg. Chim. Acta
1997, 254, 85-91), but there is no evidence for such binding here.
(44) Lippert, B. Prog. Inorg. Chem. 2005, 54, 385-447.
(45) Chen, H.; Parkinson, J. A.; Morris, R. E.; Sadler, P. J. J. Am. Chem. Soc.
2003, 125, 173-186.
Acknowledgment. We thank the EPSRC and The University
of Edinburgh (studentship for A.F.A.P.), the EC (Marie Curie
Fellowship for R.F.), and The Wellcome Trust for support,
Michael Melchart (University of Edinburgh) for experimental
9
J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006 1747

A R T I C L E S
Peacock et al.
assistance, and members of EC COST Group D20 for stimulat-
ing discussions.
S1-S3), and NMR studies of hydrolysis and binding to
nucleobases (Figures S4-S13); X-ray crystallographic data in
CIF format. This material is available free of charge via the
Internet at http://pubs.acs.org.
Supporting Information Available: Details of the syntheses
and characterization of complexes, crystallographic data, bond
lengths and angles (Tables S1, S2), intermolecular interactions
and disorder in the X-ray structures of 4, 4R, 6, 7, and 8 (Figures
JA055886R
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1748 J. AM. CHEM. SOC. VOL. 128, NO. 5, 2006