Transfer Hydrogenation and Antiproliferative Activity of Tethered Half-Sandwich Organoruthenium Catalysts

Article abstract of DOI:10.1021/acs.organomet.8b00132

We report the synthesis and characterization of four neutral organometallic tethered complexes, [Ru(η⁶-Ph(CH₂)₃-ethylenediamine-N-R)Cl], where R = methanesulfonyl (Ms, 1), toluenesulfonyl (Ts, 2), 4-trifluoromethylbenzenesulfonyl (Tf, 3), and 4-nitrobenzenesulfonyl (Nb, 4), including their X-ray crystal structures. These complexes exhibit moderate antiproliferative activity toward human ovarian, lung, hepatocellular, and breast cancer cell lines. Complex 2 in particular exhibits a low cross-resistance with cisplatin. The complexes show potent catalytic activity in the transfer hydrogenation of NAD⁺ to NADH with formate as hydride donor in aqueous solution (310 K, pH 7). Substituents on the chelated ligand decreased the turnover frequency in the order Nb > Tf > Ts > Ms. An enhancement of antiproliferative activity (up to 22%) was observed on coadministration with nontoxic concentrations of sodium formate (0.5-2 mM). Complex 2 binds to nucleobase guanine (9-EtG), but DNA appears not to be the target, as little binding to calf thymus DNA or bacterial plasmid DNA was observed. In addition, complex 2 reacts rapidly with glutathione (GSH), which might hamper transfer hydrogenation reactions in cells. Complex 2 induced a dose-dependent G₁ cell cycle arrest after 24 h exposure in A2780 human ovarian cancer cells while promoting an increase in reactive oxygen species (ROS), which is likely to contribute to its antiproliferative activity.

Full text of DOI:10.1021/acs.organomet.8b00132

Article
Cite This: Organometallics XXXX, XXX, XXX−XXX
Transfer Hydrogenation and Antiproliferative Activity of Tethered
Half-Sandwich Organoruthenium Catalysts
Feng Chen,† Isolda Romero-Canelon,†,‡ Joan J. Soldevila-Barreda,† Ji-Inn Song,† James P. C. Coverdale,†
́
Guy J. Clarkson,† Jana Kasparkova,§ Abraha Habtemariam,† Martin Wills,† Viktor Brabec,§
and Peter J. Sadler*,†
†Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, U.K.
‡School of Pharmacy, Institute of Clinical Sciences, University of Birmingham, Birmingham B15 2TT, U.K.
§Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ-61265 Brno, Czech Republic
S
* Supporting Information
ABSTRACT: We report the synthesis and characterization of four
neutral organometallic tethered complexes, [Ru(η6-Ph(CH2)3-ethyl-
enediamine-N-R)Cl], where R = methanesulfonyl (Ms, 1), toluene-
sulfonyl (Ts, 2), 4-trifluoromethylbenzenesulfonyl (Tf, 3), and 4-
nitrobenzenesulfonyl (Nb, 4), including their X-ray crystal structures.
These complexes exhibit moderate antiproliferative activity toward
human ovarian, lung, hepatocellular, and breast cancer cell lines.
Complex 2 in particular exhibits a low cross-resistance with cisplatin.
The complexes show potent catalytic activity in the transfer hydro-
genation of NAD+ to NADH with formate as hydride donor in aqueous
solution (310 K, pH 7). Substituents on the chelated ligand decreased
the turnover frequency in the order Nb > Tf > Ts > Ms. An
enhancement of antiproliferative activity (up to 22%) was observed on
coadministration with nontoxic concentrations of sodium formate (0.5−2 mM). Complex 2 binds to nucleobase guanine (9-
EtG), but DNA appears not to be the target, as little binding to calf thymus DNA or bacterial plasmid DNA was observed. In
addition, complex 2 reacts rapidly with glutathione (GSH), which might hamper transfer hydrogenation reactions in cells.
Complex 2 induced a dose-dependent G1 cell cycle arrest after 24 h exposure in A2780 human ovarian cancer cells while
promoting an increase in reactive oxygen species (ROS), which is likely to contribute to its antiproliferative activity.
amine; RM175 in Figure 1) is believed to target DNA and can
bind to guanine bases accompanied by arene intercalation. It can
also induce oxidation of bound glutathione (GSH), which can be
displaced by guanine, providing a redox-mediated route to DNA
binding.27,28
INTRODUCTION
■
The clinical anticancer drug cisplatin arose from the serendip-
itous discovery of its biological anticancer activity by Rosenberg
et al. about 50 years ago.1 Since then, anticancer complexes based
on other platinum-group metals (Ru,2−4 Rh,5−8 Os,9−11 Ir,12,13
and Pd14−17) have been studied. Ruthenium complexes have
shown promising potential with relatively low toxicity and might
provide alternatives to platinum drugs. These Ru complexes also
have the potential to overcome the severe side effects and drug
resistance which are problems with some platinum-based
chemotherapeutics.18,19 The two RuIII complexes NAMI-A and
KP-1019 (Figure 1) have entered phase II clinical trials, the
former as an antimetastatic agent.20−22 The mode of action of
NAMI-A and KP-1019 in cancer cells is not yet understood, but
the reduction of RuIII to RuII is a plausible pathway for their
activation.23,24 The RuII complex [Ru(η6-p-cym)Cl2(PTA)] (p-
Organometallic half-sandwich RuII complexes also exhibit
catalytic activity in transfer hydrogenation (TH) reactions using
a variety of reducing agents as a hydride source (e.g., H2,
isopropyl alcohol, and sodium formate).29−32 The Noyori-type
RuII complex [(η6-p-cym)Ru(TsDPEN)Cl] (TsDPEN = (R,R)-
N-(p-toluenesulfonyl)-1,2-diphenylethylenediamine) is an effi-
cient catalyst for the asymmetric transfer hydrogenation of
ketones and amines with high yields and enantiomeric excesses
using isopropyl alcohol or formic acid as hydride source (Figure
1).33,34 The sulfonyl RuII complex [(η6-arene)Ru(TsEn)Cl]
(TsEn = toluenesulfonylethylenediamine) is a more water
soluble catalyst and, under biologically relevant conditions, can
reduce the coenzyme nicotinamide adenine dinucleotide
(NAD+) in vitro and in cells using nontoxic doses of sodium
c y m
= p a r a - c y m e n e , P T A = 1 , 3 , 5 - t r i a z a - 7 -
phosphatricyclo[3.3.1.1]decane; RAPTA in Figure 1) also
exhibits promising antimetastatic effects in vitro and in vivo25
and has antiangiogenic activity toward chicken chorioallantoic
membranes with low dose-dependent antiproliferative activity.26
[(η6-biph)Ru(en)Cl]PF6 (biph = biphenyl, en = ethylenedi-
Received: March 5, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.organomet.8b00132
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ray crystal structures, and investigated their catalytic TH
reduction of NAD+ to NADH using sodium formate as a hydride
source under biologically relevant conditions. The interaction of
complex 2 with the abundant intracellular tripeptide γ-L-Glu-L-
Cys-Gly (GSH) and the effect of GSH on catalytic TH reduction
of NAD+ were also studied. We also investigated the effect of
nontoxic concentrations of formate on the antiproliferative
activity of these complexes in several human cancer cell lines, the
induction of reactive oxygen species (ROS), and changes in
integrity of their cell membranes.
RESULTS
■
Synthesis and Characterization. The four neutral tethered
RuII complexes [Ru(η6-Ph(CH2)3-ethylenediamine-N-R)Cl],
where R = Ms (1), Ts (2), Tf (3), and Nb (4), were synthesized
following a literature method for related complexes (Scheme 1).
They were characterized by elemental analysis (CHN), high-
resolution mass spectrometry, and NMR (1H, 13C, and 19F)
spectroscopy (Figures S1−S9 in the Supporting Information).
Crystals of complexes 1−4 suitable for X-ray diffraction were
obtained from a slow diffusion of diethyl ether into methanol
solutions of the complexes at ambient temperature. The
complexes adopt the expected pseudo-tetrahedral geometry
with η6-phenyl ring occupying three Ru coordination sites,
together with nitrogen atoms of the diamine ligand (bond
lengths 2.11−2.15 Å) and a monodentate chloride; ethylenedi-
amine ligands are deprotonated and bound as monoanionic
bidentate ligands. Generally, the Ru−N(H) bond distance
(range 2.140−2.149 Å) is slightly longer than that of Ru−N(−)
(2.112−2.121 Å). The η6-phenyl ring and ethylenediamine are
linked by a three-carbon tether chain. The structures are shown
in Figure 3. Selected bond lengths (Å) and bond angles (deg) are
given in Table 1 and X-ray crystallographic data in Tables S1−S5.
pKa* Determination and Interaction with Guanine. The
pKa* value of the aqua adduct 2a (from complex 2) in MeOD-
d4/D2O (1/9, v/v) was determined by 1H NMR at 310 K (Figure
S10A in the Supporting Information) by titration over the pH*
(meter reading) range from 2 to 12 and plots of the chemical shift
of a tosyl proton as a function of pH* fitted to the Henderson−
Hasselbalch equation. The pKa* value of aqua complex 2a was
found to be 9.52 0.03; a second pKa* value of <2 was too low to
be determined.
Figure 1. Organometallic half-sandwich RuII and RuIII (NAMI-A and
KP-1019) anticancer agents and catalytic transfer hydrogenation
catalysts.
formate as hydride donor (JS2 in Figure 1).35−37 Mammalian
cells can often tolerate millimolar levels of formate without
observed toxicity.35,37
Tethered RuII half-sandwich compounds in which the η6-arene
ring and a diamine ligand are connected through a three (or four-
)-atom chain have a “locked” arene ring, providing control over
the spatial positions of the substituents on the ethylenediamine
ligands, and have enhanced stability.38,39 Wills et al. have
reported a series of tethered RuII η6-arene complexes and used
them as efficient catalysts in the transfer hydrogenation reactions
of ketones and amines.40,41 However, there have been few
investigations on the antiproliferative activity of tethered RuII
complexes. Recently, chiral tethered RuII complexes (two
enantiomers) were synthesized and found to have potent
antiproliferative activity toward a panel of NCI-60 cancer cell
lines (IC50 against A2780 ovarian cancer cells as low as 1.1 μM;
Figure 2). Interestingly, their potency increased by up to 25%
upon coincubation of the cancer cells with formate.42
The interaction of complex 2 with the DNA nucleobase model
9-ethylguanine (9-EtG; Figure S10B in the Supporting
1
Information) was studied by H NMR spectroscopy. Complex
Here we have synthesized and characterized the water-soluble
tethered RuII complexes [Ru(η6-Ph(CH2)3-ethylenediamine-N-
R)Cl], where R = methanesulfonyl (Ms, 1), toluenesulfonyl (Ts,
2),43 4-trifluoromethylbenzenesulfonyl (Tf, 3), or 4-nitro-
benzenesulfonyl (Nb, 4), including determination of their X-
2 (2 mM in MeOD-d4/D2O, 1/9 (v/v)) reacted rapidly with 9-
EtG (1 mM in D2O, within 10 min) at 310 K. The adduct 2-9-
EtG gave rise to a new set of η6-arene peaks (Figure S10B in the
Supporting Information), with up to 90% yield of 2-9-EtG when
a 1.5 mol equiv 9-EtG solution was added.
A preliminary 1H NMR study indicated that histidine can also
displace the chloride ligand on 2 and form a stable adduct (Figure
S10C in the Supporting Information), as might be expected,
since, like guanine, it also contains an imidazole ring.
Transfer Hydrogenation Reactions. Catalytic transfer
hydrogenation reactions of complexes 1−4 and sodium formate
as hydride donor for conversion of nicotinamide adenine
dinucleotide (NAD+) to NADH were studied in aqueous
media by UV−visible spectroscopy by following the absorbance
1
at 340 nm for NADH and by H NMR spectroscopy (20%
DMSO-d6 in D2O) by monitoring peaks corresponding to 1,4-
NADH. The reactions were performed in the mixed solvent
DMSO-d6/D2O (1/4, v/v) to ensure the solubility of these
Figure 2. Enantiomers of chiral tethered RuII TsDPEN complexes.
B
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a
Scheme 1. Synthesis Route for Tethered RuII Complexes 1−4
a
DME = 1,2-dimethoxyethane.
Figure 3. ORTEP diagrams for RuII complexes 1−4. Thermal ellipsoids are shown at the 50% probability level. All hydrogen atoms have been omitted
for clarity.
Table 1. Selected Bond Lengths (Å) and Angles (deg) for Complexes 1−4
1
2
3
4
a
Ru1−N(−)
2.121(2)
2.146(2)
2.4174(6)
1.654
2.1181(14)
2.1490(14)
2.4234(4)
1.657
2.1202(16)
2.1485(16)
2.4243(4)
1.658
2.1124(18)
2.140(2)
2.4142(6)
1.653
b
Ru1−N(H)
Ru1−Cl1
Ru1−arene (centroid)
N(−)−Ru1−N(H)
N(−)−Ru1−Cl1
78.77(8)
88.17(6)
83.20(6)
78.87(5)
88.29(4)
83.02(4)
78.87(6)
87.33(4)
83.56(5)
78.54(7)
87.24(5)
83.43(6)
N(H)−Ru1−Cl1
a
b
N(−) corresponds to N103(1), N9(2,3), and N8(4). N(H) corresponds to N106(1), N12(2,3), and N11(4).
complexes, although it was found earlier that the rates of such TH
reactions are enhanced in methanol, which like DMSO is a
solvent of lower polarity in comparison to water.36
All of the tethered RuII complexes exhibited potent catalytic
activity, with TOFs by UV−vis and NMR spectroscopy in the
ranges 3.7−8.9 and 5.8−9.9 h−1, respectively (Table 2), following
the order 1 < 2 < 3 < 4, suggesting that more strongly electron
C
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withdrawing groups on the ethylenediamine ligand facilitate
hydride transfer between formate and NAD+.
by LC-MS, which revealed that the 2-SG adduct was formed
rapidly when 2 was mixed with GSH (Figure S13 in the
Supporting Information). The eluents are shown in Figure S14 in
the Supporting Information.
Table 2. TOFs (h−1) for Transfer Hydrogenation Reactions of
NAD+ to NADH using Complexes 1−4 as Catalysts and
Formate as Hydride Donor
Next, the time dependence of the reaction of complex 2 with
GSH was studied under similar conditions: 2 mM 2 in MeOD-
1
d4/D2O (2/8 v/v) with 20 mM GSH, monitored by H NMR
complex
R
TOF (NMR)
TOF (UV−vis)
spectroscopy from 5 min to 24 h (Figure 4). As in the above
experiments, a new set of low-field resonances appeared
immediately (H1′−H6′) in the presence of excess GSH (10
mol equiv), but with time, the low-field resonances H1′−H6′
decreased gradually and disappeared after 24 h (Figure 4);
meanwhile, another two new sets of peaks slowly appeared. The
2D NMR (COSY) spectrum suggested that the 2-SG adduct
degraded with time to release the neutral free ligand η6-
Ph(CH2)3-ethylenediamine-N(H)-Ts (assigned to one of the
two sets of low-field peaks, Figure S15 in the Supporting
Information) with up to 70% of decomposition observed within
5 h incubation at 310 K, as shown in Figure S16 in the Supporting
Information. Such liberation of free ligand was also detected by
LC-MS at 333.21 m/z (calculated [ligand + H]+ m/z 333.16,
Figure S13 in the Supporting Information). Complex 1 (2 mM in
MeOD-d4/D2O (1/4 v/v), pH* 7) showed a behavior in
reactions with GSH (10 mol equiv) similar to that of complex 2.
1-SG adduct formed rapidly but decomposed after 24 h
incubation at 310 K. Complexes 3 and 4 (2 mM in DMSO-d6/
D2O (1/4 v/v)) also reacted with GSH, but the SG adduct
1
2
3
4
Ms
Ts
Tf
3.79 0.05
4.7 0.1
8.9 0.3
8.5 0.2
5.8 0.2
8.3 0.1
8.69 0.07
9.9 0.2
Nb
The TH reactions were also studied by NMR in solutions with
a higher DMSO-d6/D2O ratio. Above 20% DMSO, the rate
increased markedly (Figure S11 in the Supporting Information),
and that with 80% DMSO was too fast to follow by NMR.
Interaction with Glutathione (GSH). The reaction of
complex 2 ([Ru(η6-Ph(CH2)3-ethylenediamine-N-Ts)Cl]) with
GSH was initially monitored by 1H NMR. As shown in Figure S2
in the Supporting Information, the low-field η6-phenyl peaks of
complex 2 decreased gradually, and a new set of triplets (H1′ and
H2′) emerged when 0.2 and 0.5 mol equiv of GSH were added.
The low-field resonances of the tosyl protons of complex 2 (H1
and H2) disappeared and a new set of peaks (H1′−H7′)
appeared when 1 mol equiv or more of GSH was added (Figure
S12 in the Supporting Information). The reaction was confirmed
Figure 4. Dependence on time of the interaction of complex 2 (2 mM in MeOD-d4/H2O (2/8 v/v)) with GSH (20 mM, in D2O, pH* adjusted to 7.2
0.1), monitored by 1H NMR (600 MHz) at 310 K. The two sets of peaks for H1′−H7′ can be assigned to diastereomers; free ligand resonances in the
low-field region are shown in the dashed red box.
D
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Table 3. In Vitro Anticancer Activity of Complexes 1−4 Against Various Cell Lines
a
cell line IC50 (μM)
b
complex
A2780
1
A2780Cis
>50
A549
1
HEPG2
MCF 7
MRC 5
RI
1
23
33
27.8 0.1
28.9 0.9
31
1
1
3
1
>2
2
2
7.3 0.4
>50
15
1
37.6 0.6
26
4
33
24
2
3
38
28
26
3
>50
>50
31
30
2
1
>50
23
n.d.
>3
11
4
16.0 0.3
1.20 0.02
4
9.9 0.5
7.3 0.2
CDDP
13.4 0.3
3.1 0.1
5.7 0.9
12.8 0.3
a
Data are shown as means standard deviations (STD), from duplicates of triplicates; cell viability was assessed after 24 h drug exposure and 72 h
recovery in drug-free medium. Human cell lines: A2780, ovarian cancer; A2780Cis, cisplatin-resistant ovarian cancer; A549, lung cancer; HEPG2,
b
hepatocellular cancer; MCF7, breast cancer; MRC5, normal lung fibroblasts. RI denotes the resistance index between A2780 ovarian cancer cells
and its resistant derivative A2780Cis. n.d. = not determined.
appeared to be stable under these conditions, since the 1H NMR
spectra appeared to be unchanged after 24 h incubation at 310 K
(data not shown). Complexes 3 and 4 both have electron-
withdrawing groups on the phenyl ring of the sulfonamide
substituent.
Reduction of NAD+ by TH in the Presence of GSH. Due
to the thiophilic nature and instability of complex 2 in the
presence of GSH, the influence of GSH on the conversion of
NAD+ to NADH by TH from complex 2 with formate as the
1
hydride source was investigated. H NMR spectra (MeOD-d4/
D2O (2/8 v/v)), at pH* 7.2 and 310 K with complex 2, NAD+,
GSH, and sodium formate in the mole ratio of 1/4/X/25 (where
X = 0.5, 1, 2 and 5, respectively) were recorded every 5 min. The
turnover frequency of NAD+ to NADH decreased slightly in the
presence of 0.5 mol equiv of GSH (TOF 4.27
0.05 h−1);
Figure 5. Cellular Ru accumulation in A2780 and A2780Cis ovarian
cancer cells exposed to equipotent IC50 concentrations of complexes 2
and 4.
however, the TOF decreased dramatically to 1.35 h−1 when 1.0
mol equiv of GSH was present. The hydride transfer reaction
completely stopped when excess GSH was added (2 or 5 mol
equiv).
cells, while the cellular Ru content from complex 2 was much
lower: 8.8 0.9 and 7 1 ng per million cells, respectively.
Cell Cycle Arrest. Since complex 2 [Ru(η6-Ph(CH2)3-
ethylenediamine-N-Ts)Cl] binds to the nucleobase guanine (9-
EtG), it was possible that DNA might be a target for anticancer
activity. In order to investigate this, the effects on the cell cycle of
A2780 cells exposed to complex 2 at IC50 and 2 × IC50
concentrations for 24 h were studied, using propidium iodide
staining, and measured using flow cytometry (Figure 6). In
comparison to negative control populations, a statistically
significant increase of the cell population in the G1 phase was
Antiproliferative Activity. The antiproliferative activity of
tethered RuII complexes 1−4 against human ovarian (A2780),
lung (A549), hepatocellular (HEPG2), breast (MCF7), and
cisplatin-resistant ovarian (A2780Cis) cancer cell lines and
normal human lung fibroblast cells (MRC5) was determined
(Table 3) and compared to that of the clinical drug cisplatin
(CDDP). Complexes 1−4 exhibited good to moderate
anticancer activity against all of these cancer cell lines, with
IC50 values in the range of 7.3−>50 μM. Complex 2 displayed
good anticancer activity against A2780 and cisplatin-resistant
A2780 cancer cells, with IC50 values of 7.3 and 15 μM,
respectively. Remarkably, this complex exhibits a resistance index
(RI) of only 2 in comparison to 11 for cisplatin. The RI is the
ratio of the activity (IC50) toward the resistant cell line in
comparison to the parental line and gives an indication of the
capacity of a complex to overcome platinum resistance.
Furthermore, complex 4 exhibited anticancer activity against
MCF7 cancer cells similar to that of cisplatin, with an IC50 value
of 9.9 μM. The antiproliferative activity in A549 lung and
HEPG2 hepatocellular cancer cells has minimal variation among
complexes 1−4, with all IC50 values averaging 32 and 25 μM,
respectively. Corresponding values for the normal fibroblast cell
line MRC5 are given in Table 3.
Cellular Accumulation of Ru. Cellular Ru accumulation
from administration of the most promising complexes 2 and 4 in
A2780 human ovarian cancer cells and A2780Cis cells was
determined. Complex 4 showed a higher cellular Ru accumu-
lation in comparison to complex 2 in both A2780 and A2780Cis
(Figure 5). The cellular Ru contents for complex 4 were 150 38
ng per million A2780 cells and 241 10 ng per million A2780Cis
Figure 6. Cell cycle arrest analysis of A2780 human ovarian cancer cells
after 24 h of exposure to complex 2 at 310 K at IC50 and 2 × IC50
concentrations. Cell staining for flow cytometry was carried out using
PI/RNase. p Values were calculated after a t-test against the negative
control data: *p <0.05, **p <0.01.
E
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observed, with percentages increasing to 75
1% at IC50
not bind strongly to high-molecular-mass DNA under the
experimental conditions used.
concentration and 85 3% population at 2 × IC50 concentration.
This evidence of G1 arrest with lack of an accumulation of cells in
the S phase might suggest that DNA is not a major target and that
complex 2 could have a cytostatic effect on ovarian cancer cells.
Nonetheless, studies of the interaction of complex 2 with DNA
were carried out to investigate further (vide infra).
ROS Induction in A2780 Human Ovarian Cancer Cells.
The level of reactive oxygen species (ROS) in A2780 human
ovarian cancer cells induced by exposure to complex 2 was
determined at IC50 concentration by flow cytometry fluorescence
analysis (Figure 7). The total level of oxidative stress, including
In further experiments, solutions containing plasmid DNA
pBR322 (28 μg mL−1) and complex JS2 or complex 2 in various
molar ratios (ri = 0.05−1) were incubated in 0.01 M NaClO4 at
310 K for 24 h in the dark. Subsequently the samples were
directly mixed with the loading buffer and loaded onto a 1%
agarose gel running at 298 K in the dark with Tris-acetate-EDTA
(TAE) buffer, and the voltage was set at 25 V. There was no
separation step before the samples were loaded onto the gel to
remove weakly bound complex, if any. The gels were then stained
with EtBr, followed by photography with a transilluminator.
As seen in Figure S17, no significant changes in the mobilities
of supercoiled (SC) or open circle forms (OC) were observed
even at very high concentrations of Ru compounds (ri = 1),
indicating that these complexes do not unwind DNA and do not
form DNA adducts. No changes in intensities of SC and OC
forms also indicated that the Ru complexes do not cleave DNA in
the dark.
Binding to Short Single- or Double-Stranded Oligonu-
cleotides. Binding of complex 2 to short single- or double-
stranded synthetic oligonucleotides was also investigated. A 50-
mer oligonucleotide (single- or double-stranded) was incubated
with complex 2 or complex JS2 (ri = 0.5, concentration of
oligonucleotide related to phosphates) in 0.05 M NaClO4 at 310
K in the dark. After 24 h the reaction was stopped, and samples
were exhaustively dialyzed against water. The ruthenium
contents in these samples were determined by FAAS, and the
concentrations of DNA were determined by absorption
spectroscopy. No Ru associated with single- or double-stranded
oligonucleotides treated with complex JS2 was found, whereas
3−4% of Ru was bound to single-stranded oligonucleotide on
incubation with complex 2, but no detectable amount of Ru was
bound to double-stranded oligonucleotide.
Figure 7. ROS induction in A2780 cancer cells exposed to complex 2.
The FL1 channel detects total oxidative stress, and the FL2 channel
detects superoxide production. Complex 2 is shown in red and negative
control in blue. p Values were calculated after a t-test against the negative
control data: *p <0.05, **p <0.01.
H2O2, peroxy and hydroxyl radical, peroxynitrite, NO, and
superoxide production, was monitored using the green channel
FL1 and orange channel FL2, respectively. Increased ROS levels
were detected in the majority of the population of A2780 cells,
with up to 82% of cells exhibiting high fluorescence in the FL1-
green channel. There is only a minimal increase in the level of
cellular superoxide (Table S6 in the Supporting Information).
Cell Membrane Integrity. We further investigated the effect
of complex 2 on the cellular membrane integrity of A2780
ovarian cancer cells using flow cytometry analysis of cells exposed
for 24 h to the ruthenium complex and stained in the dark with
propidium iodide. This experiment did not include fixation of the
cells prior to staining. The results show that there are no induced
changes in the membrane integrity of cancer cells, as there are no
statistical differences between the drug-exposed and negative
control cells (Table S7 in the Supporting Information).
Effect of Formate on Antiproliferative Activity. The
antiproliferative activity of tethered complexes 1−4 against
A2780 human ovarian cancer cells in the presence of sodium
formate was determined (Figure 8). Experiments included three
sets of controls: the first, negative controls, consisted of
Calf Thymus DNA and Bacterial Plasmid DNA. The
interaction of complex 2 ([Ru(η6-Ph(CH2)3-ethylenediamine-
N-Ts)Cl]) with double-helical calf thymus DNA (ct-DNA) and
bacterial plasmid DNA was studied and compared to that of
nontethered [(η6-p-cym)Ru(TsEn)Cl] (JS2 in Figure 1).37
Double-helical ct-DNA at a concentration of 32 μg mL−1 was
incubated with JS2 or complex 2 at ri values of 0.1 and 0.5 in 10
mM NaClO4 at 310 K (ri = the molar ratio of free ruthenium
complex to nucleotide phosphates at the onset of incubation with
DNA). The reaction was terminated after 24 h of incubation, and
samples were exhaustively dialyzed against water. The ruthenium
content in these samples was determined by flameless atomic
absorption spectrometry (FAAS) and the concentration of DNA
by absorption spectrophotometry. No detectable amount of
ruthenium was found in samples of DNA treated with JS2 or
complex 2, even at very high ri. Therefore, these compounds do
Figure 8. Cell viability of A2780 ovarian cancer cells on exposure for 24
h to complexes 1−4 (at equipotent 1/3 × IC50 concentrations) and
sodium formate at concentrations of 0, 0.5, 1.0, and 2.0 mM. p Values
were calculated after a t-test against the negative control data: *p <0.05,
**p <0.01.
F
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untreated cells (only vehicle exposure), a second set was exposed
to three concentrations of sodium formate (0.5, 1, and 2 mM),
and a third set was exposed to cisplatin as positive controls. The
results indicate that formate alone is not toxic toward A2780
ovarian cancer cells under the conditions used, as the statistical
difference between the first two controls was never significant
(Table S8 in the Supporting Information). A2780 cancer cells
were incubated with equipotent concentrations of complexes 1−
4 (1/3 × IC50) and three concentrations of sodium formate (0.5,
1, and 2 mM) for 24 h. Following 72 h of recovery time in drug-
free medium, cell survival was evaluated using the Sulforhod-
amine B colorimetric assay. A decrease in cell viability was
observed, and importantly this was greater with increasing
concentrations of sodium formate. Complex 4 showed the least
effect on cell viability, decreasing from 95% to 81%, while
complex 1 exhibited the highest changes, varying from 96% to
74% when 2 mM formate was coadministered. Overall, the
percentage of cell viability reduction which accompanies formate
coadministration follows the order 1 > 2 > 3 > 4 (percentages of
cell survival are given Table S8 in the Supporting Information).
introduction of a sulfonyl functional group raised the TOF to
2.88 h−1 (JS2 in Figure 1).35 In this work, complexes 1−4
displayed more potent catalytic activity toward TH of NAD+ to
NADH. The reaction rate for tosylated complex 2 is ca. 25.8×
and 1.6× faster than those for Ru-en and Ru-TsEn complexes
and is comparable to that of the RhIII complex [(η5-Cp*)Rh-
(bipy)Cl]PF6.54,55 This may be facilitated by the longer Ru−
N(H) and shorter Ru−N(−) bond lengths allowing the tethered
complex to approach NAD+ more closely in the TH catalytic
cycle, together with the ease of hydrolysis of the tethered
complexes. In general, the TOF values determined by UV−vis
are similar to those determined by NMR spectroscopy (Table 2),
the small difference probably arising from the mole ratios of
formate used, since the TOF increases with an increase in the
molar ratio of formate.36 It appears that the presence of the
electron-withdrawing sulfonamide on the chelating ligand gives
rise to higher catalytic activity, consistent with the previous
reported TH reduction of aldehydes and quinoxalines.36,56,57
As a major peptide in cells, glutathione (γ-L-Glu-L-Cys-Gly,
GSH) plays a significant role in cell metabolism: e.g., in the
maintenance of cellular redox state and signal transduction.58 It
functions as an important reducing agent (GSH/GSSG couple)
and has a high affinity for transition-metal complexes.59 Acquired
drug resistance in cancer cells is often associated with
overexpression of GSH, which can act as a detoxification
agent.60 Complex 2 (2 mM, MeOD-d4/H2O (1/4 v/v)) reacted
rapidly with GSH (0.5−10 mol equiv, pH* 7.1, 310 K; Figure S2
in the Supporting Information) to form the adduct 2-SG, but this
decomposed within 24 h, as shown in Figure 4, at 310 K in
aqueous solution with eventual loss of the chelated tethered
sulfonyl-ethylenediamine ligand (Figure 4 and Figure S15).
Complex 1 reacted with GSH similarly to complex 2. Complexes
3 and 4 also reacted rapidly under NMR conditions (millimolar
concentrations) but appeared to form more stable 3-SG and 4-
SG complexes. This high thiol affinity may mean that these
tethered Ru complexes bind rapidly to GSH on entering cells,
blocking the approach of Ru complex to DNA, and the
decomposition of the adduct may lead to metabolites that are
toxic to cells.59 This may partially explain why TH reduction of
NAD+ was hampered in the presence of GSH and only a limited
increase in potency is observed in A2780 cancer cells exposed to
complex 2 and sodium formate, in comparison to the Ru complex
[(η6-p-cym)Ru(TsEn)Cl], even though the TH reduction of
NAD+ was believed to be taking place in the cancer cells.37
Cisplatin is frequently used clinically in combination chemo-
therapy, especially for ovarian and testicular cancers.61 However,
poor 5 year survival rates in ovarian cancer patients are partly
attributable to the development of drug resistance.62 Complex 2
showed much lower cross-resistance with cisplatin (resistance
index ca. 2 versus 11 for cisplatin) despite the low cell uptake in
resistant human ovarian A2780Cis cancer cells. Furthermore, the
higher selectivity index between MRC5 normal cells and
A2780Cis cancer cells (2.6 for complex 2 versus 0.95 for
cisplatin), indicates that complex 2 might be able to overcome
cisplatin resistance with fewer side effects.
DISCUSSION
■
The X-ray crystal structures of complexes 1−4 show that they
adopt the well-known “piano-stool” geometry, with nitrogens of
the diamine and a chloride bound to the metal center forming the
three legs and a phenyl ring forming the seat, being linked to the
ethylenediamine by a three-carbon tether.44,45 Complexes 1−4
all have similar tethered structures. The length of the bond
between Ru and the deprotonated N is within the range 2.112−
2.121 Å, shorter than in the chiral tethered complex (R,R)-
[Ru(η6-Ph(CH2)3-TsDPEN-N(H))Cl] (2.144(3) Å) and [(η6-
p-cym)Ru(TsDPEN)Cl] (2.139(6) Å), while the Ru−N(H)
bond length is within the range 2.140−2.149 Å, which is longer
than those in the latter two complexes (2.134(3) and 2.105(6) Å,
respectively).38,40,46 Complexes 1−4 have Ru−N(−) and Ru−
N(H) bond lengths very similar to those in the complex [(η6-
hmb)Ru(TsEn)Cl] (hmb = hexamethylbenzene, 2.129(3) and
2.141(3) Å, respectively).36 The N−Ru−N angles are in the
range 78.54−78.87°, close to those in the chiral tethered Ru
complex (R,R)-[Ru(η6-Ph(CH2)3-TsDPEN-N(H))Cl].38 The
remaining bond lengths and angles show no significant difference
in comparison to either tethered or nontethered Ru sulfonyl
ethylenediamine complexes.
Complex 2 reacted rapidly with guanine (9-EtG), as studied by
NMR at millimolar concentrations.28 However, at lower
concentrations (micromolar), little binding to DNA was
observed when calf thymus and bacterial plasma DNA were
exposed to complex 2, consistent with results reported previously
for the nontethered RuII complex [(η6-p-cym)Ru(TsEn)Cl]
(JS2), implying that DNA is not likely to be a target for Ru
sulfonamide complexes.37 The binding to L-His (as well as L-Cys)
suggests that proteins and enzymes may also be targets for these
complexes.
Hydride transfer between coenzyme NAD+ and NADH plays a
pivotal role in cell metabolism;47 this pair of coenzymes is
believed to be involved in over 400 cellular reactions.48 Studies of
TH reactions for conversion of NAD+ to NADH catalyzed by
transition-metal complexes were initiated by Fish and
Steckhan.49−51 The use of RuII catalysts to mimic the cellular
reaction and achieve TH reduction of NAD+ under biologically
relevant conditions has been well studied.52,53 The en complex
RM175 showed strong DNA affinity but low catalytic efficiency
toward TH reduction of NAD+ (TOF, 0.18 h−1);35 whereas the
Cellular accumulation is an important factor in drug
cytotoxicity.63 Cellular accumulation of Ru from complexes 2
and 4 in A2780 and A2780Cis cancer cells did not correlate with
their cytotoxicity (IC50). The cellular accumulation of Ru from
complex 2 is similar in A2780 vs A2780 Cis cells, yet its IC50
varies between 7.3 and 15 μM. In comparison, complex 4 loses its
activity in the resistant cell line, yet its cellular accumulation is
notably increased. TOFs for NAD+ TH by formate for complexes
G
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1−4 do not correlate with the reduction of cell viability induced
by 2 mM sodium formate, following the order 4 < 3 < 2 < 1. This
may imply that factors other than the catalytic conversion of
NAD+ to NADH are also involved in the processes which
determine the cellular mechanism of antiproliferative activity.
In contrast to the negative control (A2780 cells not treated,
Figure 6), complex 2 can induce concentration-dependent G1
cell cycle arrest, which inhibits cell division. Previously, RuII
complexes have been reported to induce G1 arrest, e.g., the Ru-
Norharman complex [Ru(bipy)2(9H-pyrido-[3,4-b]indole)2]2+
and [Ru(η6-p-cym)(p-Impy-NMe2)Cl].64,65 Clinical anticancer
drugs, for instance clotrimazole and paclitaxel, can also induce G1
cell cycle arrest.66,67 Paclitaxel can inhibit cell proliferation by
activation of p53 tumor suppressor gene.67 Microtubules are also
potential targets. They are important cycloskeletal polymers,
which can form a constantly reorganized solid backbone that
serves as a polarity information source, to separate chromosomes
through cell division.68 The recently reported RuII complexes
[Ru(η6-Ph(CH2)3-TsDPEN-N(H))Cl] (R,R or S,S) distribute
mainly in the cycloskeleton and can effectively target and inhibit
microtubule polymerization.42 In view of its structural similarity,
complex 2 might also potentially target microtubules and
subsequently trigger G1 cell arrest.
DMSO increased the rate (Figure S11 in the Supporting
Information), as was found earlier for MeOH in related
systems.36 It is also possible that these tethered RuII complexes
can target proteins and enzymes in cells, binding e.g., to histidine
as well as the cysteine residues (Figures S10 and S12). However,
the contribution of such interactions to the biological activity
remains to be investigated.
Concentration-dependent G1 cell cycle arrest was observed on
exposure of A2780 cells to complex 2. In addition, complex 2 can
induce a high level of intracellular ROS, which may provide a
basis for killing cancer cells.
EXPERIMENTAL SECTION
■
Materials. Ruthenium trichloride was purchased from Precious
Metals Online (PMO Pty Ltd.). β-Nicotinamide adenine dinucleotide
hydrate (NAD+) was purchased from Sigma-Aldrich. Methylsulfonyl
chloride, toluenesulfonyl chloride, 4-trifluoromethylbenzenesulfonyl
chloride, and 4-nitrobenzenesulfonyl chloride were obtained from
Fluka and Sigma-Aldrich. Glutathione was obtained from Alfa Aesar.
The A2780 ovarian, A2780 Cis ovarian, A549 lung, HEPG2
hepatocellular, and MCF7 breast human adenocarcinoma cell lines as
well as MRC5 human fibroblast cells were purchased from the European
Collection of Animal Cell Culture (ECACC, Salisbury, U.K.).
Propidium iodide (>94%) and RNase A were obtained from Sigma-
Aldrich.
ROS are important factors in cell signaling and can control cell
survival, cell proliferation, and the maintenance of cell redox
homeostasis.69 A moderate level of intracellular ROS would
encourage the growth of cancer cells; however, higher levels will
cause damage and even induce apoptosis of cancer cells.70
Complex 2 significantly increased the ROS level in A2780 cancer
cells at IC50 concentrations, giving over 80% of cancer cells total
oxidative stress, which may contribute to cell death.
Syntheses. To a stirred solution of 3-(1,4-cyclohexadien-1-yl)-1-
propanol (1.21 g, 9.18 mmol) in DCM (25 mL) was added NEt3 (2.7
mL, 19.28 mmol), and the resulting solution was cooled to 273 K. A
solution of methanesulfonyl chloride (1.1 mL, 13.8 mmol) was added
over a period of 20 min, and the internal temperature was kept at 278 K.
After 30 min, the solution was warmed to ambient temperature and
stirred overnight. The reaction solution was quenched with saturated
NaHCO3 solution, washed with water and brine, and dried over
Mg2SO4. The mesylate product was carried forward directly to the next
step. A solution of the mesylate derivative in 10 mL of 1,2-
dimethoxyethane (DME) was added slowly over a period of 5 min to
a stirred solution of monosulfonated ethylenediamine (9.25 mmol) in
1,2-dimethoxyethane (20 mL) and NEt3 (2.7 mL, 19.43 mmol) at 333 K.
The resulting solution was heated to 353 K and stirred overnight. The
reaction was quenched with saturated NaHCO3 solution. The reaction
mixture was worked up with water and brine and dried over Mg2SO4.
The desired ligands were isolated as yellow oils by silica column
chromatography with EtOAc and hexane as eluents.
To a stirred solution of tethered ethylenediamine ligands (0.808
mmol) in anhydrous EtOH (15 mL) was added concentrated HCl (0.12
mL, 35%, 1.21 mmol) at 273 K. The solution was heated at 333 K for 30
min. After this time the solution was heated to 348 K and a solution of
RuCl3 (0.110 g, 0.533 mmol) in EtOH (15 mL) and water (0.5 mL) was
added dropwise over 20 min. The solution was stirred at 348 K overnight
and cooled, hexane (60 mL) added with vigorous stirring, and the
resulting solid collected by filtration. The solid (dimer precursor) was
then washed with hexane and dried under high vacuum to give a dark
brown solid. The filtrate was concentrated to give an orange powder.
To a stirred solution of dimer precursors (0.22 mmol) in DCM (50
mL) at 273 K was added N,N-diisopropylethylamine (3.0 mL, 1.70
mmol), and the solution was stirred at ambient temperature for 2 h. The
solution was then filtered over Celite, and the DCM was removed by
rotary evaporation. EtOH was added to the resulting paste and stored in
the freezer for 3 h before the cold solution was filtered, and the orange
precipitate was collected. The precipitate was washed with further
portions of cold EtOH. The desired ruthenium complex was isolated by
silica column chromatography with MeOH and DCM (1/10 v/v).
[Ru(η6-Ph(CH2)3-ethylenediamine-N-Ms)Cl] (1). The general syn-
thesis of tethered Ru complexes followed a reported protocol.43 Yield:
47 mg (60%). 1H NMR (400 MHz, MeOD-d4): δH 1.98−2.04 (m, 1H),
2.29−2.41 (m, 4H), 2.48−2.51 (m, 1H), 2.76 (s, 3H), 2.79−2.82 (m,
2H), 2.87 (d, 1H, J = 7.7 Hz), 3.24−3.28 (m, 1H), 5.14−5.20 (m, 2H),
5.71−5.72 (m, 1H), 5.83−5.88 (m, 2H). 13C NMR (125.7 MHz,
CONCLUSIONS
■
The four tethered RuII catalysts 1−4 have been synthesized and
their structures determined by X-ray crystallography. All of the
complexes exhibit potent reductive catalytic transfer hydro-
genation activity using formate as the hydride source in aqueous
media, as shown by the conversion of coenzyme NAD+ to
NADH, and follow the general reactivity trend 1 < 2 < 3 < 4.
Coincubation of complexes 1−4 with A2780 cancer cells in the
presence of formate resulted in the dose-dependent reduction of
cell viability (Figure 8). Formate alone under similar conditions
had no effect on cell viability. Such a combination of catalyst and
nontoxic cocatalyst may provide a promising strategy for the
design of new drugs which can be used at low concentrations and
have new mechanisms of action that are effective against resistant
cancers.
These complexes exhibit moderate to good anticancer activity
toward A2780, A2780 Cis, A549, MCF7, and HEPG2 cancer cell
lines. Some of the complexes displayed cytotoxicity comparable
to that of the clinically used drug cisplatin. Complex 2 ([Ru(η6-
Ph(CH2)3-ethylenediamine-N-Ts)Cl]) in particular exhibits a
low cross-resistance with cisplatin toward A2780 human ovarian
cancer cells and unlike cisplatin does not appear to involve a
DNA-targeting mechanism of action.
Complex 2 reacts rapidly with GSH to form a 2-SG adduct,
which can effectively block the TH reduction of NAD+. At
millimolar NMR concentrations, the chelated tethered ligand
was readily displaced by excess GSH, but such reactions may be
much slower in cells where the Ru concentration would be ca.
200× lower. It is possible that these TH reactions are faster in
cells if they take place in compartments where the dielectric
constant is lower than that of water, since higher percentages of
H
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DMSO-d6): δC 33.3, 43.8, 52.7, 56.8, 62.0, 78.5, 82.1, 82.9, 95.3, 98.0,
105.5. HR-MS: calcd for [C13H22ClN2O2SRu]+ m/z 357.0211, found
m/z 357.0211. Anal. Calcd for [C12H19ClN2O2RuS(H2O)0.6]: C, 35.79;
H, 5.06; N, 6.96. Found: C, 35.83; H, 4.91; N, 6.94.
ShelXT72 structure solution program using direct methods and refined
with the ShelXL73 refinement package using least-squares minimization.
The data were processed by the modeling program Mercury 3.8. X-ray
crystallographic data for complexes 1−4 have been deposited in the
Cambridge Crystallographic Data Center (CCDC) under the
deposition numbers CCDC 1823316−1823319 (complexes 4, 3, 2,
and 1, respectively).
ICP-MS/-OES Determination. ICP-MS and ICP-OES analyses were
carried out on Agilent Technologies 7500 series and PerkinElmer
Optima 5300 DV series instruments, respectively. The double-deionized
(DDW) water used for both analyses was from a Millipore Milli-Q water
purification system and a USF Elga UHQ water deionizer. The
[Ru(η6-Ph(CH2)3-ethylenediamine-N-Ts)Cl] (2). The synthesis of
complex 2 (but not its X-ray structure) was reported previously by one
of us.43 The method described above for complex 1 was used.
Recrystallization from methanol resulted in a bright red solid. Yield: 63
mg (63%). 1H NMR (400 MHz, CDCl3): δH 7.73 (d, 2H, J = 8.2 Hz),
7.15 (d, 2H, J = 8.2 Hz), 6.36 (t, 1H, J = 5.6 Hz), 5.96 (t, 1H, J = 5.6 Hz),
5.83 (t, 1H, J = 5.8 Hz), 5.01 (d, 1H, J = 5.6 Hz), 4.91 (d, 1H, J = 5.8 Hz),
3.79 (s, 1H), 3.30−3.23 (m, 1H), 3.03 (dd, 1H, J = 11.5 Hz, 4.2 Hz),
2.79−2.71 (m, 1H), 2.66−2.62 (m, 1H), 2.43−2.36 (m, 3H), 2.34 (s,
3H), 2.27−2.19 (m, 2H), 2.08−1.99 (m, 1H). 13C NMR (125.7 MHz,
DMSO-d6): δC 26.1, 33.2, 33.5, 52.2, 56.7, 62.0, 78.9, 82.1, 83.2, 94.5,
97.6, 104.9, 132.1, 133.6, 144.5, 146.9. HR-MS: calcd for
[C18H23N2O2RuS]+ m/z 433.0524, found m/z 433.0522. Anal. Calcd
for [C18H23ClN2O2RuS(H2O)0.1]: C, 46.02; H, 4.98; N, 5.96. Found: C,
46.03; H, 4.92; N, 5.93.
Ruthenium Specpure plasma standard (ruthenium chloride, 1004
5
μg/mL in 10% v/v hydrochloric acid) was diluted with 3.6% v/v HNO3,
and calibrants were prepared freshly at concentrations of 0.1−500 ppb
for ICP-MS and 50−700 ppb for ICP-OES. In particular, calibration
standards for ICP-OES analysis were adjusted with standard sodium
chloride (TraceSELECT) solution to match the sample salinity. The
instrument was set to detect 101Ru with typical detection using no-gas
mode for ICP-MS analysis.
TOFs Determined by UV−Vis Spectroscopy. Complexes 1−4 were
dissolved in DMSO/H2O (1/9 v/v) (84 μM) in a glass vial. Solutions of
sodium formate (102 mM) and NAD+ in H2O (510 μM) were also
prepared and then mixed at 310 K. In a typical experiment, an aliquot of
330 μL from each solution was placed in a 1 mL cuvette and the pH
adjusted to 7.2 before the sample was introduced into the UV−vis
instrument, giving a total volume of 1 mL (final concentrations were Ru
complex 28 μM, NAD+ 170 μM, NaHCO2 34 mM, molar ratio 1/6/
1200). UV spectra were recorded every 5 min until completion of the
reaction. The spectrum was monitored for an increase in the band at 340
nm, which corresponds to the absorption of NADH.
TOFs Determined by NMR. Complexes 1−4 were dissolved in
DMSO-d6/D2O (3/2 v/v; 1.4 mM) in a glass vial. Aliquots (200 μL) of
solutions of sodium formate (35 mM) and NAD+ (5.6 mM) in D2O
were added, and the pH* was adjusted to 7.2 0.1, for a final volume of
0.64 mL (Ru complex 0.44 mM, NAD+ 1.75 mM, NaHCO2 10.94 mM;
molar ratio 1/4/25). The solution was transferred to a 5 mm NMR tube,
and a 1H NMR spectrum was recorded at 310 K every 162 s until the
completion of the reaction. Molar ratios of NAD+ and NADH were
determined by integrating the peaks corresponding to resonances for
NAD+ (9.33 ppm) and 1,4-NADH (6.96 ppm). The turnover number
(TON) for the reaction was calculated
[Ru(η6-Ph(CH2)3-ethylenediamine-N-Tf)Cl] (3). Complex 3 was
obtained following the method described above for complex 1.
Recrystallization from methanol resulted in a brownish-red solid.
Yield: 38 mg (36%). 1H NMR (400 MHz, DMSO-d6): δH 1.84−1.86 (m,
1H), 2.10 (t, 1H, J = 11.2 Hz), 2.25 (t, 2H, J = 9.7 Hz), 2.33−2.46 (m,
2H), 2.64 (d, 1H, J = 8.9 Hz), 2.75 (q, 1H, J = 9.7 Hz, 21.9 Hz,), 2.85 (d,
1H, J = 8.0 Hz), 3.14 (s, 1H) (broad single peak), 4.35 (s, 1H) (broad
single peak), 5.27 (d, 1H, J = 4.9 Hz), 5.40 (d, 1H, J = 5.6 Hz), 5.83−5.89
(m, 2H), 5.97 (t, 1H, J = 5.1 Hz), 7.75 (d, 2H, J = 8.0 Hz), 7.93 (d, 2H, J
= 8.0 Hz). 13C NMR (125.7 MHz, DMSO-d6): δC 33.2, 33.4, 52.3, 56.7,
61.9, 79.2, 81.9, 83.6, 94.8, 97.8, 105.3, 130.1, 132.9, 134.8, 135.1, 135.0.
19F NMR (376.4 MHz, DMSO-d6): δF −61.02. HR-MS: calcd for
[C18H20N2F3O2SRu]+ m/z 487.0241, found m/z 487.0240. Anal. Calcd
for [C18H20ClF3N2O2RuS(H2O)0.1]: C, 41.28; H, 3.89; N, 5.35. Found:
C, 41.20; H, 3.65; N, 5.26.
[Ru(η6-Ph(CH2)3-ethylenediamine-N-Nb)Cl] (4). Complex 4 was
obtained following the method described above for complex 1.
Recrystallization from methanol and diethyl ether resulted in a bright
red solid. Yield: 44 mg (43%). 1H NMR (300 MHz, CDCl3): δH 2.00−
2.08 (m, 1H), 2.14−2.58 (m, 2H), 2.34−2.50 (m, 3H), 2.69−2.82 (m,
2H), 3.12 (dd, 1H, J = 5.5 Hz, 14.8 Hz), 3.23−3.32 (m, 1H), 3.70 (s,
1H) broad single peak, 4.92 (d, 1H, J = 7.9 Hz), 5.00 (d, 1H, J = 7.6 Hz),
5.86 (t, 1H, J = 7.6 Hz,), 5.96 (t, 1H, J = 8.1 Hz), 6.30 (t, 1H, J = 7.3 Hz),
7.96 (d, 2H, J = 11.6 Hz), 8.18 (d, 2H, J = 11.8 Hz). 13C NMR (125.7
MHz, DMSO-d6): δC 33.1, 33.5, 52.4, 56.7, 61.8, 79.3, 81.9, 83.8, 94.8,
97.8, 105.5, 128.4, 133.4, 153.1, 155.0. HR-MS: calcd for
[C17H20N3O4SRu]+ m/z 464.0218, found m/z 464.0216. Anal. Calcd
for [C17H20ClN3O2RuS(H2O)0.5]: C, 40.20; H, 4.17; N, 8.27. Found: C,
40.26; H, 3.89; N, 8.04.
[NAD+]
I6.96
TON =
I6.96 + I9.93 [catalyst]
where In is the integral of the signal at n ppm and [NAD+] is the
concentration of NAD+ at the start of the reaction.
Instruments and Methods. NMR Spectroscopy. 1H NMR spectra
were obtained on either a Bruker HD-400, or a AVIII 600 spectrometer
at 298 or 310 K. Data were processed by Topspin-NMR version 3.5pl7
(Bruker U.K. Ltd.), with NMR proton chemical shifts internally
referenced to TMS via 1,4-dioxane in D2O (δ 3.75) or residual MeOD-
d4 (δ 3.31 ppm), DMSO-d6 (δ 2.50 ppm) or CDCl3 (δ 7.26 ppm). 1D
spectra were recorded using standard pulse sequences. NMR spectra
were acquired with 16 transients into 32 k data points over a spectral
width of 14 ppm and 32 transients into 32 k data points over a spectral
width of 30 ppm with 2 s relaxation delay for the kinetic experiments.
High-Resolution Mass Spectrometry (HRMS). All samples were
prepared in methanol. High-resolution mass spectrometry data were
obtained on a Bruker Maxis Plus Q-TOF instrument.
Elemental Analysis. Elemental analyses were performed by Warwick
Analytical Service using an Exeter Analytical elemental analyzer
(CE440).
X-ray Crystallography. Single crystals of 1−4 were obtained from a
slow diffusion of diethyl ether into methanol solutions of the complexes.
Suitable crystals were selected and mounted on a glass fiber with
Fromblin oil and placed on an Xcalibur Gemini diffractometer with a
Ruby CCD area detector. The crystals were kept at 150 2 K during
data collection. Using Olex2,71 the structure was solved with the
Reaction with GSH. Reactions of complex 2 (2 mM, MeOD-d4/D2O
(2/8 v/v)) and GSH (in D2O) in the mole ratio of 1/X, where X = 0.2,
0.5, 1, 2, 5, and 10, were studied by 1H NMR (600 MHz) spectroscopy
under conditions of MeOD-d4/D2O (1/9 v/v), pH* 7.2, and 310 K.
Each reaction was complete within 10 min. A second set of experiments
investigated the time dependence of reactions of 2 with GSH (20 mM)
in the mole ratio of 1/20, monitored by 1H NMR (600 MHz)
spectroscopy under conditions similar to those above (pH* 7.2, 310 K),
from 5 min to 24 h.
Cell Culture. A2780 and A2780Cis human ovarian, A549 human lung,
HEPG 2 human hepatocellular, MCF 7 human breast cancer, and MRC
5 human normal fibroblast cell lines were grown in Roswell Park
Memorial Institute medium (RPMI-1640) supplemented with 10% of
fetal calf serum, 1% of 2 mM glutamine, and 1% penicillin/streptomycin.
All cells were grown as adherent monolayers at 310 K with 5% CO2
humidified, and all cells were passaged at ca. 80% confluency.
IC50 and in Vitro Cytotoxicity Determination. The antiproliferative
activity and cytotoxicity of complexes 1−4 were determined in five
different cancer cell lines and one human normal cell line. In general,
about 5000 cells per well were seeded in 96-well plates. The plates were
preincubated with drug-free medium at 310 K for 48 h before adding the
tested compounds (various concentrations). Stock solutions of
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complexes 1−4 were prepared in DMSO/cell culture medium, and exact
complex concentrations were determined by ICP-OES. After 24 h drug
exposure, supernatants were removed by suction and each well was
washed with PBS. A further cell recovery for 72 h was allowed in drug-
free medium at 310 K. The sulforhodamine B (SRB) assay was used to
determine cell viability. IC50 values, as the concentration that causes 50%
cell death, were determined as duplicates of triplicates in two
independent sets of experiments, and their standard deviations were
calculated.
Cellular Ru Accumulation. Cellular Ru accumulations for complexes
2 and 4 were determined on A2780 ovarian and A2780 cisplatin-
resistant ovarian cancer cells. Approximately 2 × 106 cells were seeded in
a six-well plate and preincubated in drug-free medium at 310 K for 24 h,
and stock solutions of both complexes were prepared in a mixture of
DMSO/cell culture medium and their accurate Ru concentrations were
determined by ICP-OES. Working solutions were then obtained by
dilution in cell culture medium. Both cancer cell lines were exposed to
complexes 2 and 4 for 24 h, at equipotent IC50 concentrations. The
experiment did not involve recovery time in drug-free medium. After
this time, cells were treated with trypsin and counted and cell pellets
were collected. Cell pellets were digested overnight in 200 μL
concentrated nitric acid (73%) at 353 K; the resulting solutions were
diluted with double-distilled water to a final concentration of 3.6%
HNO3 (v/v), and the amount of Ru taken up by the cells was
determined by ICP-MS (Agilent technologies 7500 series). Data
acquisition was carried out in ICP-MS top B.03.05 and processed on
offline Data analysis B.03.05. These experiments did not include any cell
recovery time in drug-free media; they were carried out in triplicate, and
the standard deviations were calculated. Statistical significances of
variations were determined using Welch’s t-tests.
Cytometer and the histograms were analyzed using Flowjo software and
their standard deviations were calculated.
Coincubation of Ru Complexes with Formate. Cell viability assays
of complexes 1−4 were carried out with A2780 ovarian cancer cells with
sodium formate. These experiments were performed with the following
modifications: a fixed concentration of each Ru complex equal to 1/3 ×
IC50 was used in coadministration with three different concentrations of
sodium formate (0.5, 1.0, and 2.0 mM). Drug stock solutions (ca. 100
μM) were prepared as described for in vitro growth inhibition assays and
then further diluted with media until working concentrations were
achieved. Separately, a stock solution of sodium formate was prepared in
saline. The complex and formate solutions were added to each well
independently within 5 min of each other. All other experimental
conditions were kept unchanged (drug exposure, cell recovery time, and
end point assay used). The standard deviations were calculated.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.organo-
met.8b00132.
Tables giving crystallographic data, induction of ROS and
superoxide determined by flow cytometry, cell membrane
integrity, and viability of A2780 ovarian cancer cells
exposed to formate and complexes 1−4 and figures giving
the determination of pKa of aqua complex 2a and reaction
with 9-EtG, reaction of 2a with various mol equiv of GSH
1
monitored by NMR and by HPLC, 2D COSY H NMR
Cell Cycle Analysis. A2780 cells at 1.5 × 106 cells per well were seeded
in a six-well plate. Cells were preincubated in drug-free media at 310 K
for 24 h, after which drugs were added at equipotent concentration equal
to the IC50 value. After 24 h of drug exposure, supernatants were
removed by suction and cells were washed with PBS. Finally, cells were
harvested using trypsin-EDTA and fixed for 2 h using cold 70% ethanol.
DNA staining was achieved by resuspending the cell pellets in PBS
containing propidium iodide (PI) and RNase. Cell pellets were washed
and resuspended in PBS before being analyzed in a Becton Dickinson
FACScan flow cytometer using excitation of DNA-bound PI at 536 nm,
with emission at 617 nm. Data were processed with Flowjo software
using a Watson (Pragmatic) fitting model. Welch’s t-tests were carried
out to determine the statistical variations.
spectrum for reaction of complex 2 with GSH, depend-
ence of release of free sulfonylethylenediame ligand on
time, and interaction of complexes with bacterial plasmid
DNA (PDF)
Accession Codes
CCDC 1823316−1823319 contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2
1EZ, UK; fax: +44 1223 336033.
ROS Determination. Flow cytometry analysis of ROS/superoxide
generation in A2780 cells caused by exposure to complex 2 was carried
out using the Total ROS/Superoxide detection kit (Enzo-Life Sciences)
according to the instructions. A total of 1.5 × 106 A2780 cells per well
were seeded in a six-well plate. Cells were preincubated in drug-free
media at 310 K for 24 h in a 5% CO2 humidified atmosphere, and then
drugs were added to triplicates at IC50 concentration. After 24 h of drug
exposure, supernatants were removed by suction and cells were washed
and harvested. Staining was achieved by resuspending the cell pellets in
buffer containing the orange/green fluorescent reagents. Cells were
analyzed in a Becton Dickinson FACScan flow cytometer using FL1
channel Ex/Em 490/525 nm for the oxidative stress and FL2 channel
Ex/Em 550/620 nm for superoxide detection. Positive controls were
obtained by exposure of cells to pyocyanin for 30 min. Data were
processed using Flowjo software. At all times, samples were kept under
dark conditions to avoid light-induced ROS production. Welch’s t-tests
were carried out to establish statistical significance of the variations.
Cell Membrane Integrity Determination. Flow cytometry analysis of
the cellular membrane integrity of A2780 cells caused by exposure to
complex 2 was carried out using flow cytometry and propidium iodide
staining. Briefly, A2780 cells were seeded in six-well plates (1.5 × 106
cells per well) and preincubated for 24 h in drug-free media at 310 K,
after which they were exposed to complex 2 at IC50 concentration. Cells
were harvested using trypsin and stained in the dark using a mixture of
propidium iodide and RNase without previous fixation of the cells. After
staining, cell pellets were analyzed in a Becton Dickinson FACScan Flow
AUTHOR INFORMATION
Corresponding Author
*E-mail for P.J.S.: p.j.sadler@warwick.ac.uk.
ORCID
Jana Kasparkova: 0000-0002-5279-5381
Martin Wills: 0000-0002-1646-2379
Viktor Brabec: 0000-0002-8233-1393
Peter J. Sadler: 0000-0001-9160-1941
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the ERC (grant no. 247450) and EPSRC (grant no.
EP/F034210/1), the China Scholarship Council (CSC) for a
scholarship for F.C., and Bruker Daltonics and Warwick
Collaborative Postgraduate Research Scholarship (WCPRS)
for funding for J.P.C.C. This work was also supported by the
Czech Science Foundation (Grant 17-09436S). We also thank
Dr. Ivan Prokes, Dr. Lijiang Song, and Mr. Philip Aston
(University of Warwick) for assistance with NMR and MS
measurements , and Hannah Bridgewater for help with cell
culture.
J
DOI: 10.1021/acs.organomet.8b00132
Organometallics XXXX, XXX, XXX−XXX

Organometallics
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