Pyridine analogues of the antimetastatic Ru(III) complex NAMI-A targeting non-covalent interactions with albumin

Article abstract of DOI:10.1021/ic202029e

A series of pyridine-based derivatives of the antimetastatic Ru(III) complex imidazolium [trans-RuCl₄(1H-imidazole)(DMSO-S)] (NAMI-A) have been synthesized along with their sodium-ion compensated analogues. These compounds have been characteriz

Full text of DOI:10.1021/ic202029e

Article
pubs.acs.org/IC
Pyridine Analogues of the Antimetastatic Ru(III) Complex NAMI-A
Targeting Non-Covalent Interactions with Albumin
Michael I. Webb, Ryan A. Chard, Yaser M. Al-Jobory, Michael R. Jones, Edwin W. Y. Wong,
and Charles J. Walsby*
Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada
S
* Supporting Information
ABSTRACT: A series of pyridine-based derivatives of the antimeta-
static Ru(III) complex imidazolium [trans-RuCl4(1H-imidazole)-
(DMSO-S)] (NAMI-A) have been synthesized along with their
sodium-ion compensated analogues. These compounds have been
characterized by X-ray crystallography, electron paramagnetic resonance
(EPR), NMR, and electrochemistry, with the goal of probing their
noncovalent interactions with human serum albumin (hsA). EPR
studies show that the choice of imidazolium ligands and compensating
ions does not strongly influence the rates of ligand exchange processes
in aqueous buffer solutions. By contrast, the rate of formation and
persistence of interactions of the complexes with hsA is found to be
strongly dependent on the properties of the axial ligands. The stability
of noncovalent binding is shown to correlate with the anticipated ability
of the various pyridine ligands to interact with the hydrophobic binding
domains of hsA. These interactions prevent the oligomerization of the complexes in solution and limit the rate of covalent
binding to albumin amino acid side chains. Electrochemical studies demonstrate relatively high reduction potentials for these
complexes, leading to the formation of Ru(II) species in aqueous solutions containing biological reducing agents, such as
ascorbate. However, EPR measurements indicate that while noncovalent interactions with hsA do not prevent reduction, covalent
binding produces persistent mononuclear Ru(III) species under these conditions.
INTRODUCTION
■
Ru(III) complexes have recently drawn increasing attention as
the next generation of metal-based alternatives to platinum-
based anticancer drugs.1−3 Not only do many of these
compounds exhibit potent cytotoxic or antimetastatic activity,
but they are frequently found to have low levels of side effects.
Two of these complexes, imidazolium [trans-RuCl4(1H-
imidazole)(DMSO-S)] (NAMI-A) and indazolium [trans-
RuCl4(1H-indazole)2] (KP1019) (Figure 1), have completed
phase-I clinical trials successfully and are now undergoing
further clinical investigations.4−6
The Ru(III) anticancer compounds with proven clinical
activity are generally negatively charged octahedral complexes
containing axial heterocyclic nitrogen donor ligands and
equatorial chlorides, with charge compensation provided by
protonated nitrogen heterocycles or sodium ions. NAMI-A and
related complexes, with a single axial heterocyclic nitrogen
ligand and dimethylsulfoxide (DMSO) at the other axial
position, were first described by Sava and co-workers,7,8 and
have been intensively studied. Despite modest cytotoxic
activity, these complexes have attracted significant interest
because of their ability to prevent the formation of metastases
and inhibit their growth.9,10 The other major family of active
Ru(III) compounds are bis-azole complexes such as KP1019
and imidazolium [trans-RuCl4(1H-imidazole)2] (KP418) (Fig-
Figure 1. Anticancer Ru(III) complexes.
ure 1), which were initially characterized by Keppler and co-
workers in the early 1990s.11−13 These “Keppler-type”
complexes generally show more conventional antineoplastic
properties.1,3,5 It is now widely accepted that both types of
compounds are prodrugs, which are activated in vivo by
processes that are modulated by their axial ligands.1,3,14
A
variety of studies have demonstrated that complicated ligand
exchange processes occur when these compounds are dissolved
Received: September 16, 2011
Published: December 23, 2011
© 2011 American Chemical Society
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in aqueous media, because of the loss of chloride ligands, and
DMSO in the case of NAMI-A; the azole ligands are kinetically
inert and remain coordinated under physiological conditions.15
Aquation of NAMI-A occurs readily in buffer solutions to give
initially soluble mononuclear Ru(III) products, which are then
converted to insoluble oligomers.15−17 By contrast, aquation of
Keppler-type compounds produces solutions of solely mono-
nuclear complexes. In the case of KP1019, this leads to an
insoluble monoaqua complex which rapidly precipitates at pH
7.4,12,18,19 whereas KP418 remains in solution following
aquation.20,21
Both NAMI-A and Keppler-type Ru(III) anticancer com-
plexes interact readily with serum proteins, particularly human
serum albumin (hsA).17,21−25 Several studies have indicated
that these complexes can bind to hsA following ligand
exchange, via histidine and cysteine side chains to give relatively
stable protein-bound species.3,5,21 Recently, we have used
electron paramagnetic resonance (EPR) spectroscopy to
characterize the formation of noncovalent interactions of
NAMI-A, KP1019, and KP418 with hsA.17,21 Our studies
have demonstrated that the axial heterocyclic ligands play a
dominant role, fundamentally influencing the solution behavior
of the complexes. A particularly important finding of these
studies was that KP1019 forms noncovalent interactions with
hsA more readily than KP418.21 This has been correlated with
the greater ability of the indazole ligands of KP1019 to form
interactions with the hydrophobic domains of hsA, as compared
to the imidazole ligands of KP418. These observations form the
basis of our hypothesis that noncovalent interactions with hsA
can be tuned to control the speciation and transportation of the
complexes in vivo. Specifically, by modifying the heterocyclic
axial ligands of NAMI-A derivatives, we have sought to promote
binding to the hydrophobic binding sites of hsA. This is a novel
drug design strategy which has the potential to accelerate the
formation of initial protein interactions, while limiting covalent
binding. The latter consideration is particularly important since
it impacts on the bioavailability of the compounds.24,26
yl-4-pyridyl-methane (diphenPyr), have the potential to
provide steric, hydrophobic, and π−π stacking interactions
with amino acid side chains. In addition to the NAMI-A
analogues (series a), which have the protonated heterocyclic
ligands for counterions, we have also synthesized their sodium-
ion compensated counterparts (series b), which are analogues
of sodium [trans-RuCl4(1H-imidazole)(DMSO-S), also known
as NAMI.27 This has enabled analysis of the effect of the
counterions on protein interactions, and has improved the
aqueous solubility of the compounds, particularly for the
complexes with highly hydrophobic ligands (3b and 4b). The
synthesis of complexes with different counterions was also
motivated by reports that this has evoked different specificities
for the biomolecule interactions and anticancer activity of
several Ru(III) complexes.28
We have used EPR to characterize the ligand-exchange
processes of these compounds and their interactions with hsA.
These studies demonstrate that the aqueous solution behavior
of the complexes is similar to NAMI-A, with exchange of Cl−
and DMSO ligands. However, the pyridine ligands have
significant and distinct effects on the interactions with hsA.
We observe a trend in the stability of noncovalent binding
which correlates with the increasing ability of ligands 1−4 to
promote interactions with the hydrophobic binding domains of
hsA. These interactions forestall the formation of covalent
interactions and influence the final concentration of hsA-
coordinated Ru(III) species. The reduction potentials of 1a,b−
4a,b are found to be higher than NAMI-A, and noncovalent
interactions do not prevent the reduction of the complexes in
the presence of ascorbic acid, indicating that noncovalently
protein bound Ru(II) species may be generated. By contrast,
coordinated Ru(III) complexes are found to be resistant to
reduction, demonstrating the potential of hsA interactions to
tune the behavior of these compounds in vivo.
EXPERIMENTAL SECTION
■
Materials. The starting compounds RuCl3 (Aldrich), pyridine
(Anachemia), 4-methylpyridine (TCI America), 4-(4-nitrobenzyl)-
pyridine (Alfa Aesar), diphenyl-4-pyridyl-methane (Aldrich), and
DMSO (Caledon Laboratory Chemicals), as well as hsA (Aldrich),
were used as purchased without further purifications. Hydrogen trans-
bis(dimethylsulfoxide)tetrachloro-ruthenate(III), [(DMSO)2H][trans-
Ru(DMSO-S)2Cl4], and sodium trans-bis(dimethylsulfoxide)-
tetrachloro-ruthenate(III), Na[trans-Ru(DMSO-S)2Cl4], were synthe-
sized according to literature procedures.29,30
In this work, we have synthesized and structurally
characterized a series of NAMI-A analogues with pyridine-
based ligands, which were selected for their differing potential
to promote noncovalent protein interactions (Figure 2). The
PyrH[trans-RuCl4(1H-Pyr)(DMSO-S)] (1a). [(DMSO)2H][trans-Ru-
(DMSO-S)2Cl4] (212 mg, 0.38 mmol) was dissolved in methanol (15
mL) at room temperature. Pyridine (123 μL, 1.52 mmol) was added
directly to the reaction mixture. The resulting solution was stirred at
room temperature for 24 h, after which a light orange-yellow
precipitate was observed. The solution was filtered and washed with
methanol (3 × 2 mL) and diethyl ether (3 × 2 mL). Yield: 74%
C12H17N2SOCl4Ru Calc. C 30.01, H 3.57, N 5.83. Found C 30.05, H
3.48, N 5.74. Melting point 179−181 °C (decomp). 1H NMR (D2O):
δ = 8.73, 8.54, 8.00, 4.29, 2.65, −2.92, −14.51. Crystals suitable for X-
ray diffraction were isolated from the reaction filtrate after cooling to
−20 °C for several days.
Na[trans-RuCl4(1H-Pyr)(DMSO-S)] (1b). Na[trans-Ru(DMSO-
S)2Cl4] (105 mg, 0.25 mmol) was dissolved in acetone (8 mL) at
room temperature. Pyridine (60 μL, 1 mmol) was added directly to
the reaction mixture. The resulting solution was mixed for 20 min at
room temperature, after which a clear orange-yellow solution was
observed. The clear solution was stirred at room temperature for an
additional 2.5 h, after which diethyl ether was added until precipitation
was observed. The resulting red precipitate was isolated by suction
filtration and washed with dichloromethane (3 × 2 mL) and diethyl
Figure 2. Pyridine analogues of NAMI-A synthesized in this work with
four different pyridine-based ligands and two types of counterions.
ligands we have chosen, 1, pyridine (Pyr); 2, 4-methylpyridine
(mePyr); 3, 4-(4-nitrobenzyl)pyridine (NbenzPyr); 4, diphen-
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ether (3 × 2 mL). Yield: 77% C7H11NSOCl4RuNa Calc. C 19.87, H
2.62, N 3.31. Found C 19.82, H 2.87, N 3.37. Melting point 206−208
54.92, H 4.79, N 3.59. Melting point 143−145 °C (decomp). 1H
NMR (CDCl3): δ = 8.84, 7.64, 7.52, 6.64, 6.46, 6.24, 5.09, 1.52, 1.01,
−2.40, −13.37.
1
°C (decomp). H NMR (D2O): δ = 4.30, −2.89, −14.51. Crystals
suitable for X-ray diffraction were isolated from the reaction filtrate.
mePyrH[trans-RuCl4(1H-mePyr)(DMSO-S)] (2a). [(DMSO)2H]-
[trans-Ru(DMSO-S)2Cl4] (177 mg, 0.32 mmol) was mixed with
acetone (12 mL) at room temperature. 4-Methylpyridine (123 μL,
1.26 mmol) was added directly to the reaction mixture, which
immediately produced an orange precipitate. The resulting solution
was stirred at room temperature for 3 h, then subsequently filtered and
washed with acetone (2 × 2 mL). Yield: 86% C14H21N2SOCl4Ru Calc.
C 33.08, H 4.16, N 5.51. Found C 33.11, H 4.23, N 5.40. Melting
Na[trans-RuCl4(1H-dibenzPyr)(DMSO-S)] (4b). Na[trans-Ru-
(DMSO-S)2Cl4] (63.8 mg, 0.15 mmol) was dissolved in acetone (5
mL) at room temperature. Diphenyl-4-pyridyl-methane (111 mg, 0.45
mmol) was dissolved in acetone (2 mL) then added dropwise to the
reaction mixture. The solution was stirred at room temperature for 3 h,
resulting in a clear golden-yellow solution. Solvent was removed under
decreased pressure to a minimal volume, then dichloromethane (10
mL) was added until precipitation was observed. The resulting mixture
was filtered, yielding an orange powder which was dried at 75 °C for 3
h. Yield: 58% C20H21NOSCl4RuNa Calc. C 40.76, H 3.59, N 2.38.
Found C 40.40, H 3.41, N 2.07. Melting point 225−227 °C (decomp).
1H NMR (D2O): δ = 6.59, 6.36, 5.38, 4.87, 0.98, −3.13, −14.81.
Crystals suitable for X-ray diffraction were isolated from an ethyl
acetate solution upon cooling to −20 °C for several days.
Crystallographic Structure Determination. Single crystal X-ray
diffraction analysis was performed on a Bruker SMART diffractometer
equipped with an APEX II CCD area detector fixed at a distance of 6.0
cm from the crystal and a Mo Kα fine focus sealed tube (λ = 0.71073
nm) operating at 1.5 kW (50 kV, 30 mA) and filtered with a graphite
monochromator. The temperature was regulated using an Oxford
Cryosystems Cryostream. Structures were solved using direct methods
(SIR92) and refined by least-squares procedures in CRYSTALS.31
Diagrams of 1a,b, 2a,b, 3a,b and 4b were generated by ORTEP-3 for
Windows (v. 2.0032 and rendered using POV-Ray (v. 3.6.1).33 Crystal
data, data collection parameters, and details of structure refinement for
compounds 1a,b, 2a,b, 3a,b and 4b are listed in Table 1.
1
point 182−184 °C (decomp). H NMR (D2O): δ = 8.50, 7.78, 2.57,
−2.63, −3.03, −14.85. Crystals suitable for X-ray diffraction were
isolated from the reaction filtrate after cooling to −20 °C for several
days.
Na[trans-RuCl4(1H-mePyr)(DMSO-S)] (2b). Na[trans-Ru(DMSO-
S)2Cl4] (68.5 mg, 0.16 mmol) was dissolved in acetone (5 mL) at
room temperature. 4-Methylpyridine (47.5 μL, 0.49 mmol) was added
directly to the reaction mixture. The resulting solution was stirred at
room temperature for 3 h, yielding a clear golden-yellow solution.
Solvent was removed under decreased pressure to a minimal volume,
then dichloromethane (10 mL) was added until precipitation was
observed. The resulting mixture was filtered and washed with diethyl
ether (3 × 2 mL). Yield: 81% C8H13NSOCl4RuNa Calc. C 21.98, H
3.00, N 3.20. Found C 21.67, H 3.05, N 3.33. Melting point 199−200
1
°C (decomp). H NMR (D2O): δ = −2.62, −3.01, −14.64. Crystals
suitable for X-ray diffraction were isolated from the reaction filtrate
after cooling to −20 °C for several days.
NbenzpyrH[trans-RuCl4(1H-Nbenzpyr)(DMSO-S)] (3a).
[(DMSO)2H][trans-Ru(DMSO-S)2Cl4] (91 mg, 0.16 mmol) was
dissolved in methanol (6 mL) at room temperature. 4(4-Nitrobenzyl)-
pyridine (138 mg, 0.65 mmol) was dissolved in methanol (3 mL) and
added dropwise to the reaction mixture. The resulting solution was
stirred at room temperature for 3 h, after which a yellow-orange
precipitate was observed. The solution was filtered and washed with
methanol (2 × 2 mL). Yield: 55% C26H27N4O5SCl4Ru Calc. C 41.61,
H 3.63, N 7.47. Found C 41.45, H 3.61, N 7.39. Melting point 165−
167 °C (decomp). 1H NMR (CDCl3): δ = 8.92, 8.53, 7.85, 7.37, 5.48,
4.94, 1.55, −0.41, −1.98, −12.68. Crystals suitable for X-ray diffraction
were obtained by dissolution in acetone, followed by the addition of an
equal amount of dichloromethane with the resulting mixture being left
at room temperature for several hours.
Na[trans-RuCl4(1H-Nbenzpyr)(DMSO-S)] (3b). Na[trans-Ru-
(DMSO-S)2Cl4] (104 mg, 0.25 mmol) was dissolved in acetone (6
mL) at room temperature. 4(4-Nitrobenzyl)pyridine (157 mg, 0.74
mmol) was dissolved in acetone (4 mL) then added dropwise to the
reaction mixture. The resulting solution was stirred at room
temperature for 30 min, after which a clear golden-yellow solution
was observed. Solvent was removed under decreased pressure to a
minimal volume, then dichloromethane (5 mL) and diethyl ether (5
mL) were added until precipitation was observed. The solution was
left standing for 10 min to complete the precipitation and was
subsequently filtered and washed with diethyl ether (3 × 3 mL). Yield:
81% C14H16N2O3SCl4RuNa Calc. C 30.12, H 2.89, N 5.02. Found C
30.07 H 2.80, N 4.98. Melting point 156−158 °C (decomp). 1H NMR
(D2O): δ = 6.75, 4.85, −0.30, −3.05, −14.63. Crystals suitable for X-
ray diffraction were isolated from product dissolved in ethyl acetate at
elevated temperatures followed by gradual cooling to room temper-
ature.
Electrochemical Measurements. Cyclic voltammograms were
recorded on a Princeton Applied Research potentiostat/galvanostat
Model 263A, equipped with a Ag/AgCl (3 M KCl) reference
electrode, a platinum disk working electrode and a platinum disk
counter electrode. All spectra were collected in an aqueous buffer
consisting of 50 mM NaH2PO4, 150 mM NaCl, and 20 mM NaHCO3,
henceforth “physiological buffer”. K4[Fe(CN)6] was used as a
calibration. Measurements were performed using 5 mM concen-
trations of each complex in 5 mL of solvent using a 100 mV/s scan
rate. All scans were conducted under these conditions unless otherwise
noted.
Preparation of EPR Samples. Complexes in Buffer. Com-
pounds were dissolved in physiological buffer to give a concentration
of 3 mM, and incubated at 37 °C. Aliquots of 210 μL were withdrawn
at the following time points: 0, 10, 20, 30, 60, and 120 min. Each
sample was promptly mixed with 30% by volume of glycerol, which
acted as a glassing agent, and frozen in liquid nitrogen.
Complexes with hsA. To 600 μL of a 0.75 mM solution of hsA in
physiological buffer was added 600 μL of a 1.5 mM solution of each
complex also in physiological buffer. The combined solution was then
diluted to 4 mL with physiological buffer and incubated at 37 °C for
one of the following time periods: 0, 10, 20, 30, 60, and 120 min. Each
4 mL solution was concentrated down to a volume of less than 200 μL
using an Amicon centrifugal filter unit (molecular-weight cutoff 30
kDa) by centrifuging at 8 °C and 4500 rpm for 30 min, or until a
volume of less than 200 μL was attained. The resulting filtered product
was then mixed with 90 μL of glycerol and diluted to a final volume of
300 μL with physiological buffer, and finally transferred to an EPR
tube and immediately frozen in liquid nitrogen.
Complexes with Ascorbic Acid in Buffer. A 1 mM solution of each
complex was prepared in physiological buffer at 25 °C . Ascorbic acid
was subsequently added to give a concentration of 1 mM. The
resulting solutions were then incubated at 37 °C for either 0 or 120
min. Aliquots of 210 μL were mixed with 30% glycerol by volume, and
transferred to an EPR tube and immediately frozen in liquid nitrogen.
Complexes with hsA and Ascorbic Acid. A 1 mM solution of each
complex was mixed with a 0.5 mM solution of hsA and incubated at 37
°C for either 0 or 120 min. After incubation and isolation of the
protein bound fractions, ascorbic acid (15 mM) was added to the
mixture, giving an acid concentration of 1 mM. Protein-bound
dibenzPyrH[trans-RuCl4(1H-dibenzPyr)(DMSO-S)] (4a).
[(DMSO)2H][trans-Ru(DMSO-S)2Cl4] (204 mg, 0.37 mmol) was
dissolved in methanol (10 mL) at room temperature. Diphenyl-4-
pyridyl-methane (537 mg, 2.19 mmol) was dissolved in methanol (10
mL) then added dropwise to the reaction mixture. The resulting clear
orange solution was stirred at room temperature for 30 min, affording
a clear golden-yellow solution. Solvent was removed under reduced
pressure until a yellow precipitate was observed. The solution was
subsequently filtered and washed with methanol (3 × 2 mL). Yield:
40% C36H37N2OSCl4Ru Calc. C 54.83, H 4.73, N 3.55. Found C
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fractions were separated by ultrafiltration as described above for the
complexes incubated with hsA in buffer.
EPR Measurements and Simulations. EPR spectra were
collected at X-band (9.3−9.4 GHz) using a Bruker EMXplus
spectrometer with a PremiumX microwave bridge and HS resonator.
Low temperature (20 K) measurements were performed using a
Bruker ER 4112HV helium temperature-control system and
continuous-flow cryostat. So that the intensities of EPR signals from
Ru(III) species in each solution after different incubation times could
be compared, sample preparation and spectroscopic parameters were
unchanged for each experiment. EPR samples (300 μL) for each series
of incubation times were drawn from a common stock solution. The
design of the Bruker cryostat system, which contains a quartz-insert
tube holder, ensures reproducible sample placement within the EPR
resonator. Consequently, variation in instrument sensitivity between
measurements was minimal, and automatic tuning of the spectrometer
gave a Q-factor of 6500
10%. To monitor the accuracy of this
approach, a reference compound 5,10,15,20-tetrakis(4-sulfonatophen-
yl) porphyrinato iron(III) chloride (FeTPPS) was added to several
samples. In each case, the original samples were briefly thawed and 20
μL of a 3 mM solution of FeTPPS in buffer was added. The samples
were then refrozen in liquid nitrogen and their EPR spectra
remeasured. FeTPPS gives an axial EPR spectrum with a characteristic
signal at g⊥ = 5.8, and this feature was used as a reference to compare
the overall intensity of Ru(III) signals at different time points. In
experiments with hsA, the distinctive EPR signal from a minor Fe(III)
human serum transferrin impurity at g = 4.3 also provided a reference
for normalizing the Ru(III) EPR signal sensitivity.
All spectra were simulated using the program Bruker WinEPR
Simfonia which efficiently produces accurate results for most S = 1/2
systems. A manual, iterative fitting procedure was employed to analyze
overlapping spectra observed when multiple Ru(III) species were
present in a particular sample. The Matlab-based simulation package
EasySpin34 was also used to reproduce the simulations of some spectra
and the results were identical to those using WinEPR Simfonia.
RESULTS AND DISCUSSION
■
Synthesis. Compounds 1a−4a and 1b−4b were synthe-
sized using procedures based on those reported for NAMI-A
and NAMI, respectively.30,35 Initial synthesis of the bis-DMSO
complexes, [(DMSO)2H][trans-Ru(DMSO-S)2Cl4], and Na-
[trans-Ru(DMSO-S)2Cl4] was followed by addition of the
ligands 1−4 to give 1a−4a, and 1b−4b respectively in good
yield. The identity and purity of these compounds was
confirmed by elemental analysis, NMR, EPR, and X-ray
crystallography.
Although the original patents describing NAMI-A and related
complexes encompass compounds with a variety of heterocyclic
nitrogen ligands,36 synthesis and, particularly, characterization
of these species has been somewhat limited. In the case of
NAMI-A analogues with different heterocyclic nitrogen donor
ligands, a number of imidazole-based derivatives have been
reported,37,38 along with a modest number of compounds with
other types of nitrogen heterocycles, including thiazoles,39,40
triazoles,41−43 pyridines,7,44−50 and various other azole
ligands.41,44,51 Of the compounds described here, synthesis of
1a,b and 2a,b have been previously reported.7,46,48,50 However,
aside from NMR and UV−vis studies of 2a,50 only 1a has been
fully structurally characterized.46 No NAMI-A derivatives
similar to 3a,b and 4a,b have been reported to date. As
described here, we have characterized all eight compounds
1a,b−4a,b prior to studying their aqueous solution behavior,
protein interactions, and electrochemistry.
Crystal Structures. The structures of compounds 1a,b,
2a,b, 3a,b, and 4b determined by X-ray crystallography are
shown in Figure 3. The significant differences in the solvent
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Figure 3. Crystal structures of compounds 1a,b, 2a,b, 3a,b, and 4b.
preferences of the azole ligands and counterions required
different approaches to obtain crystals suitable for X-ray
diffraction studies. Crystals of complexes 1a, 1b, 2a, and 2b
were all isolated directly from the reaction filtrate after standing
for several days at low temperature. For 3a, dissolution in
acetone followed by the addition of dichloromethane (1:1)
resulted in X-ray quality crystals within minutes. Single crystals
of 3b were obtained by dissolution in ethyl acetate at elevated
temperatures followed by a very gradual cooling period. Despite
numerous attempts using a wide array of conditions and solvent
systems, no crystals suitable for X-ray diffraction studies of
compound 4a were attained. However, crystals of 4b were
obtained from a solution of ethyl acetate after several days at
low temperature. The crystal structure of 1a has been reported
previously,45 and we find metrical parameters that agree with
this earlier study within experimental error. To our knowledge,
none of the other compounds have been characterized
crystallographically.
For all of the structures solved, similar distorted octahedral
geometry around the ruthenium(III) metal center is observed.
In all cases the DMSO ligand is coordinated via the sulfur atom
while the pyridine ligand is coordinated through the
heterocyclic nitrogen. An equatorial plane of four chlorides
completes the coordination sphere. This type of coordination
environment around the ruthenium metal center has been well
documented, and is typical of analogous com-
plexes.7,13,30,37,40,41,47,52
crystallographically characterized complexes (1a,b, 2a,b, 3a,b,
4b), the average Ru−N bond distance (2.11 Å) is similar to the
reported value for NAMI (2.081 Å) and NAMI-A (2.099 Å).
The corresponding average Ru−S bond distances (2.30 Å) are
also very similar to the values for NAMI (2.296 Å) and NAMI-
A (2.293 Å). Ru−Cl bond distances were evaluated individually
across all the complexes, with the resulting average Ru−Cl
bond distance of 2.355 Å close to those of NAMI-A (2.353 Å)
and NAMI (2.342 Å).
In the case of the sodium salts, all of the structures (1b−4b)
were solved as dimers, since the sodium cations exhibit a
distorted octahedral geometry between chloride ligands on
neighboring ruthenium centers and cocrystallize with solvent
molecules. Low temperature collection of diffraction data was
required to prevent desolvation of crystals of 1b, 2b, and 3b,
which otherwise reduced the crystals to powder. Similar
behavior has been reported for crystals of NAMI.30 The
bridging oxygen atoms in 1b and 2b and the free oxygen atom
in 2b were modeled as water molecules. Determination of the
identity of these water molecules was complicated by the
vibrational motion present in both 1b and 2b, which is evident
in the pyridine rings and the anisotropic temperature factors of
the equatorial chlorides. Lastly, for 3b and 4b, cocrystallizing
ethyl acetate molecules were modeled; with relative ease in the
case of 3b and with significant disorder for 4b, which required
two solvent molecules being modeled with partial occupancy.
EPR of Aqueous Solution Behavior. Before studying the
protein interactions of compounds 1a,b−4a,b, it was first
necessary to characterize their ligand-exchange processes in
physiological buffer. Principally, we wanted to determine
whether their fundamental solution behavior was similar, and
The bond lengths for all of the complexes are listed in
Supporting Information, Table S3. The metal−ligand bond
lengths of all the synthesized complexes are similar to NAMI-
A40 and its sodium-compensated analogue NAMI.30 For the
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whether it would play a significant role in the rate of formation
of interactions with hsA.
The paramagnetic Ru(III) (low-spin d5, S = 1/2) centers of
these compounds facilitate EPR studies to determine the
symmetry and ligand fields of the complexes as they undergo
ligand-exchange processes. Analysis of g values and signal
intensities from frozen solutions of the compounds enables
identification of species produced by ligand exchange and
determination of the rates of these processes. Furthermore,
processes that produce diamagnetic species, such as reduction
to give Ru(II) (low-spin d6, S = 0) or dimerization to give
antiferromagnetically coupled bimetallic species, can be
monitored via loss of overall signal intensity because of the
production of “EPR silent” species.
Under the physiologically relevant experimental conditions
used in these studies (pH 7.4, 37 °C), Ru(III) complexes
generally exhibit relatively slow ligand exchange.53 Thus, we
tracked the concentration changes of various Ru(III) complexes
after incubation times up to 2 h, by freezing the solutions in
liquid nitrogen at selected time points. EPR measurements of
these frozen solutions then provide a “snap shot” of the species
present and their relative concentrations. Frequently, ligand
exchange processes gave complex mixtures of Ru(III) species,
which exhibited overlapped EPR spectra, requiring deconvolu-
tion for analysis. This was achieved by simulating the spectra of
each individual species to determine a set of g values and line
widths. Each spectrum was then multiplied by a weighting
factor, correlating with the concentration of the Ru(III) species
in the solution, before adding together the components from all
of the identified species to reproduce the experimental
spectrum. To ensure a unique solution was determined for
the spectral parameters of each overlapped species, simulations
were performed for all of the incubation time points
simultaneously, with only the weighting factor for each spectral
component varied.
To compare the solution behavior of 1a,b−4a,b, the
compounds were incubated in physiological buffer (pH 7.4)
at 37 °C for time periods of 0, 10, 20, 30, 60, and 120 min
before freezing in liquid nitrogen and measurement by EPR.
The resulting EPR spectra are shown in Figure 4 for 1a and 4b,
and in the Supporting Information, Figures S1 and S2 for the
remaining complexes. For each complex a well-resolved uniaxial
spectrum was observed prior to incubation at 37 °C, as
expected for mononuclear low-spin Ru(III) complexes with
tetragonal symmetry, indicating detection of the parent
compound in each case. The g values of the parent compounds,
determined by simulation (Figure 4, right-hand panels and
Supporting Information, Figures S5−S12), are similar with g⊥ =
2.43−2.44 and g|| = 1.75−1.80, as expected since the
substituents on the pyridine ligands do not affect the ligand
field of the Ru(III) ion significantly. In the case of compounds
3a and 4a, the spectra attained before incubation in buffer at 37
°C are not strictly uniaxial. Relatively poor water solubility
results in some aggregated complexes in solution. This
produces a weak, broad EPR signal which distorts the axial
peak from the dissolved complexes. By contrast, their sodium
analogues, 3b and 4b, are readily soluble in aqueous media and
thus do not display this signal.
Figure 4. EPR measurements of 1a and 4b incubated in physiological
buffer at 37 °C, and spectral deconvolution of spectra collected after
10 min of incubation. As shown by the spectral simulations, two
aquated species are generated, with the axially symmetric species 1a-
C3 and 4b-C3, generated by loss of DMSO, dominating in both cases.
Smaller contributions from the lower symmetry species, 1a-C2 and 4b-
C2 are generated by loss of Cl−. Experimental parameters: frequency
= 9.38 GHz, microwave power = 2.0 mW, time constant = 40.96 ms,
modulation amplitude = 6 G, average of five 2-min scans. For EPR
measurements of the solution behavior of other complexes, see
Supporting Information, Figures S1 and S2. For spectral deconvolu-
tion of spectra collected at each incubation time point for each
complex, see Supporting Information, Figures S5−12. For spectral
parameters used in each simulation see Supporting Information, Table
S1.
replaced the signals from the parent compounds, indicating
the loss of one of the axial ligands. Nitrogen donor ligands are
generally considered to be kinetically inert under physiological
conditions (pH 7.4),15 implying that the axial DMSO ligand of
the complexes exchanges with water in each case. However, to
confirm this, we performed NMR measurements of the sodium-
compensated complexes 1b−4b, following incubation in buffer
With incubation at 37 °C, a new uniaxial EPR signal was
observed in measurements of each complex with g⊥ = 2.30−
2.31 and g|| = 1.87−1.88 (EPR spectra labeled as 1a-C3 and 4b-
C3 in Figure 4, and correspondingly for other complexes in
Supporting Information, Figures S5−S12), which steadily
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for 0, 2, and 24 h (Supporting Information, Figures S30−S33).
Only trace amounts of free pyridine ligands were detected even
after 24 h, confirming [trans-RuCl4(1H-L)(H2O)]− as the
dominant product in solution after 10 min of incubation.
Simulations of the spectra obtained after 10 and 20 min of
incubation for each complex at 37 °C reveal a smaller
contribution from a third species, which exhibits a rhombic
EPR spectrum, g = [2.41−2.46, 2.25−2.31, 2.01−2.07] (Figure
4, right-hand panels, Supporting Information, Figures S5−S12).
The lower symmetry species observed are consistent with
aqueous exchange of an equatorial chloride ligand, which
produces a second monoaqua species RuCl3H2O(1H-L)-
(DMSO-S) (1a-C2 and 4b-C2 in Figure 4). The lower signal
intensity from this species in each case indicates that Cl−
exchange occurs concurrently with loss of DMSO, but at a
significantly slower rate. The g values for each compound
(1a,b−4a,b), and their monoaqua derivatives ([1a,b−4a,b]-C2
and [1a,b−4a,b]-C3) are listed in the Supporting Information,
Table S1.
contrast, the sodium analogue, 4b, dissolves readily in buffer
and shows the typical speciation behavior.
The aqueous solution behavior of NAMI-A has been studied
extensively by a variety of techniques, demonstrating that
aquation products and oligomers predominate after incubation
of the complex in aqueous buffer solutions at pH
7.4.15−17,19−21,41,54 We have previously reported EPR character-
ization of NAMI-A in physiological buffer, and find speciation
and ligand-exchange rates that are very similar to those of the
pyridine complexes described in this work.17 This further
demonstrates that although the axial azole ligands influence the
electrochemistry and biomolecule interactions of this family of
compounds, they do not impact significantly on their
fundamental aqueous solution behavior. This is an important
observation since solutions of these types of compounds are
frequently prepared in physiological saline, prior to intravenous
administration. Thus, the time taken between the preparation
of these solutions and infusion determines the actual
compounds delivered to patients, significantly affecting in
vivo behavior and activity.
Further incubation of the compounds in solution leads to a
considerable decrease in the overall EPR signal intensity. Since
these buffer solutions do not contain significant reducing
agents, redox processes leading to EPR silent Ru(II) (d6, S = 0)
complexes are not responsible for this effect. Instead,
oligomerization of the aquation products to give oxo- or
hydroxo-bridged species likely explains the loss of paramagnetic
species, since antiferromagnetic coupling between adjacent
Ru(III) centers can produce species with a net spin of S = 0.
This behavior has been previously reported for NAMI-A, and is
accompanied by a distinct change in the color of the solution
from yellow to dark brown.15,17 A similar phenomenon was also
observed for each of the pyridine complexes described in this
work. Although the EPR signals are significantly attenuated
after longer incubation periods, it is still possible to observe
ongoing changes in the spectra. This indicates a small
proportion of the complexes remain as mononuclear Ru(III)
species and continue exchanging Cl− ligands to give polyaqua
species. However, the very low concentration of these species
indicates that they are unlikely to play a significant role in the
activity of the compounds.
Overall, the rates of ligand exchange processes do not vary
dramatically depending either on the axial ligands or the
counterions. In every case the axial monoaqua complex
dominates after 10 min of incubation at 37 °C, and the overall
signal intensity is significantly reduced after 30 min. The
spectroscopic parameters of each complex are also relatively
similar, as expected given the similar ligand-fields provided by
the axial ligands in each case. Furthermore, changing the
counterions did not affect the EPR spectra noticeably. Thus,
the spectra shown for 1a and 4a in Figure 4 are similar to those
observed for the other compounds (Supporting Information,
Figures S1−S2). The only exception was 4a, which was
relatively insoluble in aqueous media because of its hydro-
phobic dibenzPyr axial ligand and dibenzPyrH counterion,
requiring 20% DMSO for sufficient dissolution. The resulting
EPR spectra, although showing contributions from the parent
compound and monoaqua complexes in solution, are
dominated by a broad signal, which is relatively unchanged
even following longer incubation periods, and likely arises from
aggregated complexes. This was confirmed by measurement of
a powder sample of 4a, which gave a broad signal matching that
seen in the frozen solution experiment (data not shown). By
EPR of Interactions of Complexes with hsA. As the
dominant protein in the circulatory system, hsA has been
identified as the agent responsible for the binding of many
drugs and their delivery to sites of disease.55,56 A number of
studies have characterized covalent interactions of NAMI-A and
Keppler-type complexes with hsA, because of ligand exchange
with amino acid side chains, particularly histidine imida-
zoles.22,25,26,57 There is some debate regarding the effects of
these interactions on the bioavailability and antiproliferative
activity of NAMI-A and other Ru(III) anticancer com-
pounds.24,26 However, recent studies of NAMI-A bound to
bovine serum albumin demonstrate that the protein-bound
complex exhibits high antimetastatic activity.24
The hydrophobic regions in subdomains IIA and IIIA of hsA
are prominent molecular binding sites and are the most
probable locations of noncovalently bound ruthenium
species.56,58 We have previously used EPR methods to show
that NAMI-A, and the monoaqua complex formed by DMSO
exchange, both rapidly form noncovalent interactions with
hsA.17 Although these species are predominant shortly after
dissolution in the presence of hsA, or in whole human serum,
they are steadily converted to covalently bound species with
further incubation via ligand exchange with amino acid side
chains.17
The complexes 1a,b−4a,b were incubated for 0, 10, 20, 30,
60, and 120 min at 37 °C in buffer solutions of hsA. Protein-
bound fractions were then isolated by ultrafiltration, with a
molecular-weight cutoff of 30 kDa, frozen in liquid nitrogen,
and studied by EPR. The resulting spectra for each incubation
time are shown in Figure 5 for 1a and 4a, and in Supporting
Information, Figures S13−S20 for the other complexes. This
approach allows us to observe exclusively hsA-bound Ru(III)
species while excluding contributions from free complexes in
solution, and has been used in earlier studies to identify both
covalent and noncovalent binding interactions of NAMI-A and
the Keppler-type complexes KP1019 and KP418 with hsA.17,21
For each of the pyridine complexes 1a,b−4a,b incubated
with hsA, EPR measurements after short incubation periods
reveal signals that correspond to the parent compounds and the
monoaqua complexes ([1a,b−4a,b]-C2 and [1a,b−4a,b]-C3)
that were observed in the buffer solution experiment described
previously. However, since only protein bound fractions are
present, these signals are due to complexes bound to hsA
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After longer incubation periods the EPR spectra from all of
the pyridine complexes are increasingly dominated by two
overlapping broader signals, which are not observed in the
buffer solution experiments, with g values of g⊥ = 2.33−2.40
and g|| = 1.76, and g = [2.56−2.59, 2.24−2.30, 1.76−1.78]. The
distinct g values of these species indicate new Ru(III) ligand
environments due to protein interactions. This reflects
coordination to amino acid side chains, most probably histidine
imidazoles, as suggested in previous studies.17,21,25,57,59,60 The
hsA protein has upward of 5 surface histidine residues that are
capable of coordinating to exogenous species,61 and previous
studies have demonstrated that up to 5 equivalents of NAMI-A
can bind to the protein.59,60 In our experiments the ratio of
complex to hsA molecules was 2:1. This increases the
possibility of different coordination modes, as compared to a
1:1 ratio, but was not an excessive concentration for
noncovalent interactions. The strong EPR signals observed
from these species indicate protein coordination is highly
favored, likely giving more than one Ru(III) complex
coordinated per protein molecule. Furthermore, EPR measure-
ments of the filtrate after separation of protein bound fractions
did not show detectable concentrations of Ru(III).
As shown by the spectral deconvolution of the EPR spectrum
of 1a after 30 min of incubation with hsA, (Figure 5, right-hand
panel, and for the other pyridine complexes in Supporting
Information, Figures S13−S20), one of these species has a
uniaxial EPR spectrum (1a-hsA-2), indicating coordination at
the labile axial site, while the other has rhombic symmetry (1a-
hsA-1) demonstrating coordination to an equatorial position
previously occupied by chloride in the parent complex. These
observations are similar to those reported for NAMI-A,
indicating that the pyridine complexes have the same types of
covalent interactions with hsA.17 However, as shown by the
corresponding spectra for 4a (Figure 5), the chemical structure
of the pyridine ligands strongly affects the rates that the various
protein-bound species form, and their relative concentrations.
A third protein-coordinated species is also evident for each
complex (1a-hsA-3 and 4a-hsA-3 in Figure 5, and correspond-
ing species in Supporting Information, Figures S13−S20)
which is characterized by a sharp, rhombic EPR spectrum with
g = [2.42, 2.06, 1.96]. Although the EPR signal intensity of this
species is much lower than the main protein-bound species
described above, it forms more rapidly, essentially reaching its
maximum concentration before incubation at 37 °C. The
smaller g-value dispersion and narrow line widths of this species
demonstrate a different ligand environment from the other
protein-coordinated species. Human serum albumin has one
free cysteine (Cys-34) which has a reactive thiol available for
coordination under physiological conditions,62,63 and we
suggest that coordination of the pyridine complexes described
here to this residue is a possible source of this signal. This
species has also been observed for NAMI-A incubated with
hsA, exhibiting a very similar EPR signal.17 Furthermore,
interactions of platinum-based drugs with Cys-34 of hsA have
been reported,62 indicating that this could be a favorable mode
of coordination for ruthenium complexes.
Figure 5. EPR spectra of 1a and 4a incubated with hsA in
physiological buffer at 37 °C, and spectral deconvolution of spectra
collected after 30 min of incubation. Experimental parameters:
frequency = 9.38 GHz, microwave power = 2.0 mW, time constant =
40.96 ms, modulation amplitude = 6 G, average of five 2-min scans.
For EPR measurements of hsA binding behavior of other complexes,
see Supporting Information, Figures S3−S4. For spectral deconvolu-
tion of spectra collected at each incubation time point for each Ru(III)
species, see Supporting Information, Figures S13−S20. For spectral
parameters used in each simulation see Supporting Information, Table
S2.
The incubation time-dependence of the EPR signals from 1a
with hsA closely resembles that of NAMI-A,17 as might be
expected given the similar propensity of the imidazole and
pyridine ligands to form hydrophobic interactions with hsA. In
both cases, EPR measurements reveal the formation of
noncovalently bound species prior to incubation. After 10
min of incubation at 37 °C, these species have mostly
without ligand exchange with the protein, that is, noncovalently
bound species. The strong signals observed in each case, even
prior to incubation at 37 °C, demonstrate that these
interactions form readily with hsA and initially predominate
in solution.
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Figure 6. Relative fractions of covalently and noncovalently hsA bound Ru(III) complexes following 0, 10, and 30 min of incubation at 37 °C, as
determined from integrated EPR signal intensity.
disappeared, and by 20 min they are completely replaced by
covalently bound complexes whose concentration continues to
increase, reaching a maximum after about 1 h (Figure 5).
Almost identical behavior is observed for the sodium
compensated complex, 1b (Supporting Information, Figure
S4b), demonstrating that the counterions do not significantly
influence the protein interactions in this case. These results
indicate that by using fresh solutions of each complex for
intravenous treatment, covalently bound species will rapidly
predominate in vivo. By contrast, the mePyr analogues 2a and
2b, show significant concentrations of the monoaqua complex,
trans-RuCl4(1H-mePyr)(H2O), noncovalently bound to the
protein after 10 min of incubation (Supporting Information,
Figures S15b and S16b), demonstrating that these species are
significantly stabilized toward noncovalent interactions with
hsA by the presence of the methyl group at the 4 position as
compared to 1a and 1b. This effect is even more pronounced in
the other pyridine derivatives, with the NbenzPyr complexes 3a
and 3b showing dominant noncovalent interactions after 20
min of incubation (Supporting Information, Figures S17c−
S18c), and the diphenPyr complexes 4a and 4b found primarily
in noncovalent interactions after at least 60 and 30 min of
incubation with hsA at 37 °C respectively (Figure 5, right-hand
panel, and Supporting Information, Figures S19e−S20d).
The relative concentrations of covalently and noncovalently
hsA-bound Ru(III) complexes was determined for 1a,b−4a,b
over the first 30 min of incubation by first simulating the
overlapping spectral signals using weighting factors, as
described in the previous section (Figure 5 and Supporting
Information, Figures S13−S21). Using the double integration
values of these simulated spectra gave the relative concen-
trations of each of the species present following incubation of
1a,b−4a,b for 0, 10, and 30 min at 37 °C. As summarized in
Figure 6, these calculations reveal a trend in the stability of the
noncovalent interactions of 1a,b−4a,b with hsA that correlates
with the ability of their axial ligands to facilitate interactions
with the hydrophobic binding domains of hsA. As shown by the
relative proportions of covalently and noncovalently bound
Ru(III) species, the axial ligands increase the persistence of
noncovalently bound species according to diphenPyr >
NbenzPyr > mePyr > Pyr. Figure 5 highlights the dramatic
difference between the protein interactions of 1a and 4a. The
Pyr complex (1a) is observed solely as covalently bound species
after 30 min of incubation with hsA, whereas the diphenPyr
complex (4a) still shows a contribution from noncovalently
bound species after 120 min. Spectral deconvolutions for all
protein-bound species at all incubation time points, in addition
to those in Figure 5, are shown in the Supporting Information,
Figures S13−S20.
The sodium compensated analogues of the complexes
described here are desirable in consideration of their
pharmaceutical potential because of the potential toxicity of
the pyridine counterions in 1a−4a. Furthermore, as described
above, the sodium salts also have significantly improved
solubility in aqueous solution, which is particularly important
when highly hydrophobic ligands such as diphenPyr are used.
However, the counterion choice does not seem to significantly
impact the interactions with hsA. The notable exception to this
is in the behavior of the diphenPyr compounds, where the
sodium compensated complex (4b) very rapidly forms
noncovalent interactions with the protein, while the diphen-
pyrH salt (4a) initially exhibits relatively low levels of hsA-
bound species. This is consistent with the relative insolubility of
4a in aqueous solution, which leads to the formation of
aggregated species, as described above. With further incubation,
the signals from noncovalently bound 4a increase significantly,
demonstrating that the protein interactions effectively solubilize
the complex.
While it is clear that increased derivatization of the pyridine
ligands forestalls the formation of covalent interactions with
hsA, by stabilizing noncovalent binding, it also influences the
final concentrations of coordinated Ru(III) species (Figures 5
and 6, Supporting Information, Figures S13−S20). The Pyr
compounds 1a and 1b show strong, and similar intensity, EPR
signals from hsA-coordinated complexes after 2 h of incubation
at 37 °C. Comparison with the mePyr complexes 2a and 2b
shows signal intensities which are 40 and 60% lower,
respectively, after the same incubation time. This effect is
even more pronounced for the NbenzPyr and diphenPyr
analogues with the concentrations of the analogous species
reduced by 70−80%. Formation of covalent interactions is slow,
as expected for ligand exchange reactions under these
conditions,53 but is thermodynamically favored and thus
dominates at longer time periods. However, these data indicate
that stabilizing noncovalent interactions leads to less total hsA-
coordinated Ru(III) species after long incubation times.
In previous studies of the bis-indazole (KP1019) and bis-
imidazole (KP418) Keppler-type complexes (Figure 1), we
have observed significant differences in noncovalent inter-
actions with hsA.21 KP1019 forms noncovalent interactions
with hsA very rapidly, which prevents precipitation, and has
been linked to the low level of side effects observed in clinical
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studies.5,6,21 As with NAMI-A, noncovalently hsA-bound
KP1019 complexes are slowly converted to protein coordinated
species. By contrast, KP418 does not form noncovalent
interactions with hsA, demonstrating that the imidazole ligands
are less favorable toward these types of interactions.21
Interestingly, the aquated derivatives of KP418 do form
noncovalent interactions with hsA, and these interactions are
sufficiently favored that they prevent covalent binding to the
protein. This demonstrates that noncovalent interactions with
hsA are influenced not only by the potential of the axial ligands
to interact directly with the protein, but also by the charge and
perhaps polarity of the complexes.
Effect of Protein Binding on Reductive Stability. The
“activation by reduction” hypothesis for Ru(III) anticancer
compounds postulates that conversion to Ru(II) species in vivo
by biological reducing agents activates them toward ligand
substitution and increases their propensity for binding to
biomolecules.64 In the case of DNA interactions, this process
can then lead to apoptosis.1 This has been proposed as a
targeting strategy for these compounds, since they could be
preferentially reduced in the hypoxic environments frequently
found in tumors.64 Intracellular processes may not be as
relevant to the activity of NAMI-A-type complexes as they are
to Keppler-type complexes. However, modulation of the
oxidation state in blood after intravenous infusion is anticipated
to be important to mediate interactions with many extracellular
biomolecule targets, and is likely an important component of
antimetastatic activity.3,10,24,65
The cyclic voltammograms of compounds 1a,b, 2a,b, and
3a,b were measured in physiological buffer with a Ag/AgCl
working electrode and scan rates of 100 mV/s (Supporting
Information, Figures S34−S39). Because of the lower solubility
of compounds 4a and 4b, they were dissolved in a 1:1 mixture
of DMSO and buffer, before measurement of their cyclic
voltammograms (Supporting Information, Figures S40, S41).
To determine the effect of DMSO on the reduction potentials,
the cyclic voltammograms of NAMI-A were measured in both
buffer (Supporting Information, Figure S42) and 1:1 DMSO/
physiological buffer (Supporting Information, Figure S43).
These measurements indicate a shift in electrochemical
potential to lower values (ca. 160 mV).
each case,66 likely reflecting minor differences in the
experimental methods used.
As pointed out in previous reports,42,64 the reduction
potentials of these types of ruthenium complexes can be
estimated using the additive description of ligand contributions
to E1/2 given by Lever’s equation (eq 1).67
E = S
E + I
L M
∑
M
(1)
The electrochemical ligand parameter, EL, correlates with the
net electron donating character of the constituent ligands of
redox-active metal complexes. SM and IM are paramaters derived
from the empirical correspondence between ∑EL, and
experimental values of E1/2 for a particular redox couple. The
reduction potential has been shown to be dependent on solvent
environment and the overall charge of the complexes
measured.67 Reisner et al. have reported values of SM = 0.88
and IM = 0.46 for Ru(III)(−1) → Ru(II)(−2) in neutral
phosphate buffer solutions.64 Ligand parameters of EL(Cl−) =
−0.24,67 EL(DMSO, S-coordinated) = 0.57,68 EL(Pyr) = 0.25,67
and EL(mePyr) = 0.2367 give, from eq 1, E1/2(1a,b) = 330 mV
and E1/2(2a,b) = 320 mV, in good agreement with our
experimental values (Table 2). Values of EL for NbenzPyr and
dibenzPyr have not been reported previously, but the
experimental values of E1/2 found here indicate they are very
similar to Pyr and mePyr, with EL ≈ 0.25.
The values of E1/2 for the pyridine analogues described here
are significantly more positive than NAMI-A, which has
previously been reported as E1/2 = 260 mV versus NHE in a
0.10 M phosphate buffer system at pH 7.4,69,70 and is found to
be E1/2 = 270 mV under the aqueous buffer conditions used in
this work. The higher reduction potentials of 1a,b−4a,b reflect
the greater net electron donating character of pyridine as
compared to imidazole (EL(imidazole) = 0.0971). These
reduction potentials are readily accessible to biological reducing
agents such as ascorbic acid (E° = +60 mV vs NHE) and
glutathione (E° = −250 mV vs NHE).64 Several earlier studies
have shown that the Ru(III) center of NAMI-A is reduced
readily to Ru(II) in the presence of ascorbic acid.16,17,60 EPR
measurements can be used to follow this process by tracking
the overall loss of signal intensity because of the production of
diamagnetic Ru(II) centers from paramagnetic Ru(III) species.
To study the reduction behavior of the pyridine complexes
described here, we initially incubated each compound with an
equimolar concentration of ascorbic acid in buffer at 37 °C. For
each complex, 1a,b−4a,b, this leads to essentially complete loss
of the EPR signal after less than 10 min of incubation (data not
shown), demonstrating that reduction of almost all Ru(III)
centers occurs very rapidly, as expected given the relatively high
reduction potential of these compounds.
For each complex a one-electron Ru(III) → Ru(II) redox
couple was observed. As shown in Table 2, the values of E1/2 for
Table 2. Reduction Potentials of Compounds 1a,b−4a,b vs
a
NHE
LH+ counterion
1a
2a
3a
4a
b
E1/2 Ru(III/II)
330 (80)
320 (70)
330 (120)
160 (100)
(mV)
Na+ counterion
1b
2b
3b
4b
To determine the influence of protein interactions on the
reduction of the Ru(III) centers of 1a,b−4a,b the complexes
were first incubated with hsA and the resulting solutions were
then mixed with buffered ascorbic acid solutions, to give
solutions with 1:1 molar ratios of ruthenium complexes/
ascorbic acid. In the first of these experiments, protein-bound
fractions of each complex were prepared by briefly mixing each
complex with hsA in buffer at 25 °C, followed by ultrafiltration.
These solutions were then combined with buffered ascorbic
acid and briefly mixed at 25 °C. As described in the previous
section, this initial incubation procedure with hsA produces
significant concentrations of noncovalently bound complexes
for 1a,b−4a,b, as shown by distinctive EPR signals (Figure 5
b
E1/2 Ru(III/II) (mV)
a
330 (70)
320 (70)
330 (80)
170 (90)
Peak-to-peak differences in parentheses (|Epc − Epa|) (mV).
Compounds 4a and 4b measured in 1:1 DMSO/buffer. See main
b
text for solvent-correction to E1/2
.
1a,b−3a,b are very similar, as expected given the similar
donating properties of the pyridine ligands, and fall in the range
of 320−340 mV. Addition of the solvent correction for 4a and
4b gives 320 and 330 mV, respectively. These values are all
slightly higher than previous electrochemical measurements
which report E1/2 for 1b in aqueous solution of 300 mV30 and
for 2a in buffered saline (pH 7.40),50 of 310 mV vs NHE in
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and Supporting Information, Figures S13−S20). However, as
shown by the EPR spectra labeled “0 min” in Figure 7 and
water (EL(H2O) = 0.04), to give the neutral species RuCl3(His-
Im)(1H-L)(H2O), then eq 1 gives E1/2 = −390 mV, and so this
species is expected to be resistant to reduction by ascorbic acid.
This scenario seems reasonable, given the demonstrated
proclivity of these complexes to undergo DMSO exchange.
Although there is some uncertainty in these calculations for a
variety of reasons, they do demonstrate the overall concept that
a combination of ligand exchange with H2O and protein side
chains can produce species that are resistant to reduction under
physiological conditions.
Similar enhancement in reductive stability with protein
binding has also been reported for NAMI-A,17 and this has
been suggested as a method for targeted activation of these
types of compounds.64 Lowering of the reduction potential by
protein binding allows for transport in vivo in the Ru(III)
oxidation state without reduction by physiological reducing
agents. However, the reduction potential found in proliferating
cells (E1/2 ≈ −240 mV72), can be reduced by as much as 100
mV in hypoxic tumor environments.73 This provides the
potential for reduction of Ru(III) species that would otherwise
be redox stabilized under physiological conditions.69 The
production of more labile Ru(II) species then facilitates
coordination to biomolecules and apoptosis.1,64
Figure 7. EPR measurement of 1a and 4a after initial incubation with
hsA in physiological buffer at 37 °C for the times shown; followed by
addition of ascorbic acid. Experimental parameters: frequency = 9.38
GHz, microwave power = 2.0 mW, time constant = 40.96 ms,
modulation amplitude = 6 G, average of five 2-min scans. For EPR
spectra of analogous experiments with other complexes see Supporting
Information, Figure S21.
CONCLUSIONS
■
It is now widely accepted that protein interactions are a key
component of the pro-drug behavior of anticancer Ru(III)
complexes. Binding to hsA is expected to be predominant in
vivo and is thus central to the transport and speciation of these
compounds. As we have shown here using pyridine complexes,
the properties of the axial azole ligand of NAMI-A-like
complexes play a critical role in modulating interactions with
hsA. From EPR studies we have demonstrated that the rates of
ligand exchange in buffer solution are relatively unaffected by
the characteristics of the pyridine ligands. However, these
ligands do control the persistence of noncovalent protein
interactions, through distinct abilities to interact with the
hydrophobic binding domains of hsA. This not only controls
speciation, as compared to buffer solution, but also affects the
concentration of covalently bound mononuclear Ru(III)
species after long incubation periods. As we have shown,
noncovalently bound complexes are readily reduced by ascorbic
acid, while covalently bound complexes are not. Consequently,
it seems likely that increased stability of noncovalent
interactions reduces the concentration of Ru(III) species
found in vivo. Overall, these results indicate that tuning
noncovalent interactions of Ru(III) complexes with hsA
through the selection of suitable axial azole ligands is an
important component of their activity and selectivity, and
should be considered as part of future drug design strategies.
Supporting Information, Figure S21a, the addition of ascorbic
acid reduces these species to give EPR-silent Ru(II) centers,
demonstrating that noncovalent binding to the hydrophobic
binding domains of hsA does not limit interactions with
exogenous reducing agents.
Incubation of 1a,b−4a,b with hsA for 120 min at 37 °C,
gives, almost exclusively, complexes that are covalently protein
bound, as described above, with corresponding EPR spectra
(Figure 5 and Supporting Information, Figures S13−S20). In
contrast to the noncovalently bound species, addition of
ascorbic acid causes no change to the appearance or intensity of
the EPR spectra from these species (Figure 7 and Supporting
Information, Figure S21b). This indicates that covalent binding
to hsA prevents reduction and thus can give mononuclear
Ru(III) species in vivo. This increase in the reductive stability
can be explained by the effect of ligand exchange on E1/2. Since
the complexes are expected to be bound primarily to surface
imidazoles of hsA histidine residues, the effect of covalent
binding on the reduction potential can be estimated from eq 1
using the value of the ligand parameter for imidazole. Histidine
imidazole (His-Im) coordination at the axial position, initially
occupied by DMSO (species 1a-hsA-2 in Figure 5 and
corresponding species in Supporting Information, Figures
S13−S20 for other complexes) gives [trans-RuCl4(1H-L)(His-
Im)]−. Assuming that this Ru(III) species can be described with
a −1 charge, and correspondingly SM = 0.88 and IM = 0.46, then
eq 1 gives E1/2 = −0.90 mV, which is sufficiently negative to
prevent reduction by ascorbic acid. Substitution of a Cl− ligand
for a histidine imidazole gives RuCl3(His-Im)(1H-L)(DMSO-
ASSOCIATED CONTENT
■
S
* Supporting Information
For compounds 1a,b−4a,b: (i) EPR spectra, and spectral
deconvolutions by simulation, for all incubation times, up to 2 h
with buffer, hsA, hsA and ascorbic acid; (ii) tables of g values
and line widths for EPR spectral simulations; (iii) NMR
spectra; (iv) crystallographic data in CIF format and tables of
Ru-ligand bond lengths for each compounds except 4a; (v)
cyclic voltammogram traces. This material is available free of
charge via the Internet at http://pubs.acs.org.
S). This neutral complex is then described by eq 1 with SM
=
0.97 and IM = 0.04,64 which gives E1/2 = 220 mV, and thus this
species is predicted to be readily reduced by ascorbic acid.
However, if the lower-symmetry hsA-coordinated species (such
as 1a-hsA-1 in Figure 5) has histidine imidazole coordination at
an equatorial position, and undergoes DMSO exchange with
964
dx.doi.org/10.1021/ic202029e | Inorg. Chem. 2012, 51, 954−966

Inorganic Chemistry
Article
(14) Alessio, E.; Mestroni, G.; Bergamo, A.; Sava, G. Curr. Top. Med.
Chem. 2004, 4, 1525−1535.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: cwalsby@sfu.ca.
(15) Bacac, M.; Hotze, A. C. G.; van der Schilden, K.; Haasnoot, J.
G.; Pacor, S.; Alessio, E.; Sava, G.; Reedijk, J. J. Inorg. Biochem. 2004,
98, 402−412.
(16) Sava, G.; Bergamo, A.; Zorzet, S.; Gava, B.; Casarsa, C.;
Cocchietto, M.; Furlani, A.; Scarcia, V.; Serli, B.; Iengo, E.; Alessio, E.;
Mestroni, G. Eur. J. Cancer 2002, 38, 427−435.
ACKNOWLEDGMENTS
■
The authors are grateful to Prof. Tim Storr for assistance with
the electrochemical measurements, and Dr. Michael Katz for
assistance with X-ray structures of 1b and 2b. Financial support
was provided by The Natural Sciences and Engineering
Research Council of Canada (NSERC), The Canada
Foundation for Innovation (CFI), and Simon Fraser University.
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