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The synthesis, structural characterization, and in vitro anti-cancer activity of chloro(p-cymene) complexes of ruthenium(II) containing a disulfoxide ligand
Inorganica Chimica Acta 352 (2003) 238 /246
www.elsevier.com/locate/ica
The synthesis, structural characterization, and in vitro anti-cancer
activity of chloro(p-cymene) complexes of ruthenium(II) containing a
disulfoxide ligand
Lynsey A. Huxham, Elizabeth L.S. Cheu, Brian O. Patrick, Brian R. James *
Department of Chemistry, University of British Columbia, Vancouver, BC Canada V6T 1Z1
Received 6 November 2002; accepted 9 January 2003
This paper is dedicated to Professor Martin Bennett, the connoisseur of Ru /arene chemistry, on the occasion of his retirement (65th birthday);
one of us (B.R.J.) has known Martin for more than one half-life
Abstract
Two diruthenium(II) complexes [RuCl2(p -cymene)]2(m-BESE) (1), [RuCl2(p -cymene)]2(m-BESP) (2), and the mononuclear salt
[RuCl(p -cymene)(BESE)]PF6 (3), containing the disulfoxides BESE and BESP, were synthesized and characterized by elemental
analysis, and NMR and IR spectroscopies, and were shown to contain S-bound sulfoxide groups; the disulfoxides are
EtS(O)(CH2)n S(O)Et, where n /2 (BESE) or 3 (BESP). Complexes 1 and 3 were also characterized by X-ray crystallography.
Preliminary in vitro tests of 1 and 3 were conducted using the MTT assay, which measures mitochondrial dehydrogenase activity as
an indication of cell viability; these complexes showed in vitro anti-cancer activity against a human mammary cancer cell line
(MDA-MB-435s) with IC50 values of 360 and 55 mM, respectively.
# 2003 Elsevier B.V. All rights reserved.
Keywords: Crystal structures; Ruthenium complexes; p -Cymene complexes; Disulfoxide ligands; Anti-cancer activity
1. Introduction
The Pt-based drugs cisplatin and carboplatin are
widely used anti-cancer agents [1], however, they are
associated with high toxicity and some tumours resist
these drugs. The exploration of Ru complexes for use as
anti-cancer agents was initiated in attempts to find less
toxic and more specific drugs [2]. An example of
specificity is shown by the proposed behaviour of
Ru(III) complexes, which suggests they may have low
toxicity, as they bind transferrin, and therefore may
target malignant cells as these up-regulate the expression
of transferrin receptors on the cell membrane due to an
increased iron requirement [2,3]. Some of the initial
biological studies with Ru complexes involved cis - and
trans -RuCl2(DMSO)4, and suggested these have anti-
* Corresponding author. Tel.: /1-604-822 6645; fax: /1-604-822
2847.
E-mail address: brj@chem.ubc.ca (B.R. James).
0020-1693/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0020-1693(03)00155-5
cancer properties and specifically anti-metastatic properties at high, yet relatively non-toxic, doses [4 /7].
Complexes such as Na[trans -RuCl4(R2SO)(L)] and
mer ,cis -RuCl3(R2SO)2(L) (L/NH3 or imidazole;
R2SO /DMSO, and TMSO) have been synthesized as
potential anti-cancer agents [8], and some initial work in
this laboratory investigated RuCl2(R2SO)2(nitroimidazole)2 complexes and their potential as radiosensitizers
[9,10]. The range of Ru(sulfoxide) complexes was then
extended to include disulfoxide complexes, both to
examine their in vitro activity [11] and to reduce the
number of possible isomers formed during the preparation of RuCl2(R2SO)2(nitroimidazole)2 complexes.
Thus, Yapp et al. characterized crystallographically
disulfoxide complexes of the formula cis -RuCl2L2
[L /1,2-bis(ethylsulfinyl)ethane (BESE), and 1,3bis(methylsulfinyl)propane (BMSP)], and trans RuCl2L2 [L /1,2-bis(methylsulfinyl)ethane (BMSE),
and 1,2-bis(propylsulfinyl)ethane (BPSE)], and preliminary in vitro experiments suggested that the trans
complexes accumulate in cells and bind DNA to a
L.A. Huxham et al. / Inorganica Chimica Acta 352 (2003) 238 /246
greater degree than the cis complexes [11]. Further
studies by Cheu in this group have led to the characterization of cis -RuCl2L2 [L/1,2-bis(butylsulfinyl)ethane
(BBSE), 1,2-bis(pentylsulfinyl)ethane (BPeSE), 1,2bis(cyclohexylsulfinyl)ethane (BCySE), and 1,3-bis(ethylsulfinyl)propane (BESP)], and trans -RuCl2L2
(L /BESE and BPSE) [12]. Diruthenium(II) complexes
of the formula [RuCl(L)H2O]2(m-Cl)2 (L /BESE,
BPSE, and BBSE) and a mixed-valence species
(BPSP /1,3-bis(propylsulfi[RuCl(BPSP)]2(m-Cl)3
nyl)propane), of which representative types have been
structurally characterized, have been shown in vitro to
have low toxicity and to bind DNA to a greater degree
than mononuclear sulfoxide complexes [12]. The disulfoxides are of the general formula RS(O)(CH2)n S(O)R,
where R is an alkyl group and n/2 or 3.
Ruthenium(II) arene complexes have been shown to
exhibit biological activity: complexes such as
[RuCl2(C6H6)(metro)],1 where metro /metronidazole
(1-b-hydroxyethyl-2-methyl-5-nitroimidazole) [13], and
[RuCl2(C6H6)(DMSO)] [14] have been studied for topoisomerase II activity and DNA damage ability,
respectively, while reaction of [RuCl2(p -cymene)]2 (mCl2) with adenine (ade H) in the presence of
Ag(CF3SO3) forms [Ru(adenine)(p-cymene)]4(CF3SO3)4
indicating the ability of Ru(p -cymene) complexes to
interact with DNA bases [15]. Following such studies on
Ru /arene systems, attempts in this laboratory to
synthesize water-soluble disulfoxide complexes from
the precursor [RuCl(p-cymene)]2(m-Cl)2 led to the isolation of [RuCl2(p -cymene)]2(m-BESE) (1), [RuCl2(p-cymene)]2(m-BESP) (2) and [RuCl(p -cymene)(BESE)]PF6
(3), work which is described in this paper; 1 is the first
structurally characterized, bridging disulfoxide Ru species. The preliminary in vitro anti-cancer activity of
complexes 1 and 3 using the MTT assay [16] is also
described (see Section 2.3).
During our studies, a report appeared by Dyson and
coworkers on the structurally characterized [RuCl2(p cymene)(pta)] complex (pta /1,3,5-triaza-7-phosphatricycol[3.3.1.1]decane) containing a Ru /P bond; the
complex is water-soluble and exhibits pH-dependent
DNA-binding [17]. Such half-sandwich Ru(II) arene
complexes containing nitrogen ligands were also reported at about the same time by Sadler’s group [18],
who measured IC50 values (concentration of drug
required to reduce the cell culture growth by 50%) for
a human ovarian cancer cell line with several of the
complexes. Significantly, with respect to our paper, in
vitro anti-cancer activity data for the [RuCl(p-cymene)(en)]PF6, an N ,N ?-analogue of our S ,S ? complex 3,
were noted [18] (see Section 3.2).
1
All the arene ligands mentioned in this paper are h6-bonded, and
the h6 description is generally omitted for convenience.
239
2. Experimental
DMSO (Fisher), the dithioethers (Lancaster Synthesis), the deuterated solvents (Cambridge Isotopes), and
all other solvents were used as supplied without further
purification. RuCl3 ×/3H2O was donated by Johnson
Matthey Ltd. and Colonial Metals Inc. The EtS(O)(CH2)n S(O)Et disulfoxides BESE (n /2) and
BESP (n /3) were synthesized by the acid-catalyzed,
DMSO oxidation of the corresponding dithioethers (3,6dithiaoctane and 3,7-dithianonane, respectively), following a literature procedure [19]; BESE (m.p. 148/149 8C)
and BESP (m.p. 127/130 8C) were isolated in the meso form from a mixture of diastereoisomers by three
recrystallizations from EtOH, as we described earlier
[11,12]. Of note, both R ,R - and S ,S -BESE have been
isolated by methodology developed by Khiar’s group
[20], and are readily distinguished from the meso -form
by 1H NMR [21]. [RuCl(p -cymene)]2(m-Cl)2 was synthesized according to the procedure of Bennett et al. using
RuCl3 ×/3H2O and a-phellandrene (Fluka) [22]. All
syntheses were performed under N2, unless otherwise
stated, but all samples and products were stored at room
temperature (r.t., /20 8C) in air. Solution 1H NMR
spectra (D2O or CDCl3) were obtained using a Bruker
AV-300 (300.13 MHz) FT-NMR spectrometer (s /
singlet, bs /broad singlet, d /doublet, t /triplet,
sp /septet, and m/multiplet; all coupling constants
are given in Hz). 1H chemical shifts are given as d
(ppm), with reference to the residual solvent peak as the
internal standard, relative to TMS. Infrared spectra
(KBr, cm 1) were obtained using an ATI Mattson
Genesis Series FTIR instrument. Elemental analyses
were obtained by Mr. P. Borda of this department, using
a Carlo Erba Instruments EA 1108 CHN-O analyzer.
Mass spectral analyses (LSIMS) were also obtained at
the UBC facility. The X-ray crystallographic analyses
are described in Section 2.2.
2.1. Syntheses
2.1.1. [RuCl2(p -cymene)]2(m-BESE) (1)
[RuCl(p-cymene)]2(m-Cl)2 (200 mg, 0.32 mmol) and
BESE (60 mg, 0.32 mmol) were placed under 1 atm N2.
CH2Cl2 (20 ml) was then added, via cannula, to the
solids and the resultant deep red solution stirred for 30
min. The solvent was reduced in volume to 5 ml and
hexanes (10 ml) were added to precipitate the red
complex that was collected and dried in vacuo at
70 8C; yield 164 mg (64%). Attempts to increase the
yield by refluxing or adding excess BESE were unsuccessful. Crystals suitable for X-ray crystallography were
grown from slow evaporation of CH2Cl2 from a
solution of the complex. Anal . Calc. for
C26H42Cl4O2S2Ru2: C, 39.30; H, 5.33. Found: C,
39.46; H, 5.32%. IR nso: 1082, 1110. Mass spectrum
240
L.A. Huxham et al. / Inorganica Chimica Acta 352 (2003) 238 /246
[LSIMS, m /z , matrix: 3-nitrobenzylalcohol (3-NBA)]:
792 [M]. 1H NMR (CDCl3, 300 MHz): d 1.28 (d, p cymene, CH(CH3)2, J 6.90), 1.36 (t, BESE, CH3, J
7.50), 2.27 (s, p -cymene, CH3), 2.82, 3.21 (bs, BESE,
CH2S(O)CH2), 3.05 (sp, p -cymene, CH(CH3)2, J 6.90),
5.55 (bs, p -cymene, (CH3)CHCH), 5.63 (bs, p -cymene,
(CH3)C(CHCH). There are also peaks corresponding to
[RuCl(p-cymene)]2(m-Cl)2, formed via dissociation of 1
(see Section 3.1) at d 1.26 (d, p -cymene, CH(C(CH3)2, J
6.92), 2.14 (s, p -cymene, CH3), 2.90 (sp, p -cymene,
CH(CH3)2, J 6.92), 5.31 (d, p -cymene, (CH3)C(CHCH ,
J 5.94), 5.44 (d, p -cymene, (CH3)C(CHCH, J 5.94).
2.1.2. [RuCl2(p -cymene)]2(m-BESP) (2)
The procedure used for synthesizing 2 is that given for
1 but using [RuCl(p-cymene)]2(m-Cl)2 (50 mg, 0.081
mmol) and BESP (15 mg, 0.081 mmol). The volume of
the solvent was reduced to 2 ml and Et2O (10 ml) was
used to precipitate the red complex that was washed
twice with Et2O, collected, and dried in vacuo at 70 8C;
yield 20 mg (30%). Anal . Calc. for C27H44Cl4O2S2Ru2:
C, 40.11; H, 5.48. Found: C, 39.92; H, 5.57%. IR nso:
1074, 1082, 1090, 1095, 1108, 1116, 1122. Mass spectrum
[LSIMS, m /z, matrix: thioglycerol]: 809 [M ]. 1H NMR
(CDCl3, 300 MHz): d 1.29 (d, p-cymene, CH(CH3)2, J
6.89), 1.34 (t, BESP, CH3, J 7.50), 2.28 (s, p -cymene,
CH3), 2.35 (m, BESP, CH2CH3), 2.80 (bs, BESP,
CH2CH2CH2), 3.05 (m, BESP, CH2CH2CH2), 3.08
(sp, p-cymene, CH(CH3)2, J 6.89), 5.55 (bs, p -cymene,
(CH3)C(CHCH ), 5.63 (bs, p -cymene, (CH3)C(CH /
CH). Again, because of dissociation of 2, peaks due to
[RuCl(p-cymene)]2(m-Cl)2 are seen.
2.1.3. [RuCl(p -cymene)(BESE)]PF6 (3)
2.1.3.1. Method 1. [RuCl(p-cymene)]2(m-Cl)2 (50 mg,
0.081 mmol) and BESE (30 mg, 0.16 mmol) were
dissolved overnight in stirred H2O (20 ml) under 1 atm
N2 or air to give a yellow solution. NH4PF6 (26 mg, 0.16
mmol) was then added and the solution volume reduced
to 5 ml. The mixture was then filtered through Celite to
remove traces of Ru metal, and the filtrate left overnight, when yellow crystalline material formed. This was
filtered off and dried in vacuo at 70 8C; yield 30 mg
(31%). The yield was improved to 62 mg (62%) by
reducing the solvent volume to 1 ml and adding MeOH
(3 ml). X-ray quality needle crystals were grown from
the complex dissolved in a 1:1 mixture of H2O and
MeOH. Anal . Calc. for C16H28ClO2S2RuPF6: C, 32.14;
H, 4.72. Found: C, 32.24; H, 4.77%. IR nso: 1072, 1090,
1107, 1119, 1132, 1142. LM (H2O): 75 V1 cm2 mol 1.
1
H NMR (D2O, 300 MHz): d 1.06 (d, 6H, p -cymene
CH(CH3)2, J 6.93), 1.40 (t, 6H, BESE CH3), 2.06 (s, 3H,
p -cymene, CH3), 2.70 (sp, H, p -cymene, CH(CH3)2),
3.40 /3.70 (m, 8H, BESE CH2S(O) /CH2CH3), 6.28 (s,
4H, p -cymene, CHCH). 1H NMR (CDCl3, 300 MHz):
d 1.24 (d, 6H, p -cymene CH(CH3)2, J 6.93), 1.55 (t, 6H,
BESE CH3), 2.25 (s, 3H, p -cymene CH3), 2.95 (sp, H, pcymene, CH (CH3)2), 3.45 (m), 3.75 (m), 3.52 (s) (8H,
BESE, CH2S(O)CH2CH3), 6.12 (s, 4H, p-cymene,
CHCH).
2.1.3.2. Method 2. Complex 1 (50 mg, 0.063 mmol) was
completely dissolved in H2O (10 ml) and NH4PF6 (21
mg, 0.13 mmol) was added. The solution was concentrated to 5 ml, filtered and left overnight, when yellow
crystals formed. The 1H NMR data agree with those
reported in method 1.
2.2. X-ray crystallographic analyses of 1 and 3
The structures of both 1 and 3 were solved by direct
methods and expanded using Fourier techniques; the Hatoms were included at calculated positions, but not
refined. Selected crystallographic data appear in Table
1. An orange red, block crystal of 1 (/0.30 /0.20 /
0.10 mm3) and a yellow prism crystal of 3 (/0.10 /
0.10 /0.10 mm3) were each mounted on a glass fibre.
All measurements were made on a Rigaku/ADSC CCD
area detector with graphite mono-chromated Mo Ka
radiation (l /0.71069 Å). The cell constants for 1,
based on 10 069 reflections with 2u /3.6 /58.38, corresponded to a primitive triclinic cell with space group P 1̄
Table 1
Selected crystallographic dataa for [RuCl2(p -cymene)]2(m-BESE) (1)
and [RuCl(p -cymene)(BESE)]PF6 (3)
Empirical formula
Formula weight
Crystal system
Space group
Unit cell dimensions
a (Å)
b (Å)
c (Å)
a (8)
b (8)
g (8)
V (Å3)
Z
rcalc (g cm3)
l (Å)
m (cm1)
Observations I /
0.00s (I )
Variables
Observations I /3s (I )
Final R indices [I /
3s (I )]
R indices (all data)
Goodness-of-fit
a
1
3
C26H42O2S2Cl4Ru2
794.69
triclinic
P 1̄ (no. 2)
C16H28O2F6PS2ClRu
598.00
orthorhombic
Pbca (no. 61)
9.7899(4)
12.4235(9)
14.431(1)
75.226(3)
72.818(2)
68.101(3)
1534.9(2)
2
1.72
0.71069
14.91
6266
16.9476(6)
15.1712(4)
35.756(2)
325
5557
R/0.029, Rw /
0.053
R/0.042, Rw /
0.095
1.56
575
5195
R/0.028, Rw /
0.032
R/0.064, Rw /
0.082
0.68
9193(1)
16
1.73
0.71069
11.08
10211
R/SjjFoj/jFcjj/SjFoj, Rw /(S w (SjFoj/jFcj2/(S w /jFoj2)1/2.
L.A. Huxham et al. / Inorganica Chimica Acta 352 (2003) 238 /246
(no. 2). The data were collected at /1009/1 8C to a
maximum 2u value of 58.38 and were collected in 0.508
oscillations with 35.0 s exposures. A sweep of data was
done using f oscillations from 0.0 to 190.08 at x /
/90.08 and a second sweep was performed using v
oscillations between /18.0 and 23.08 at x //90.08.
The crystal to detector distance was 39.17 mm and the
detector swing angle was /5.508. Of the 13880 reflections that were collected, 6270 were unique (Rint /
0.030); equivalent reflections were merged. Data were
collected and processed using the d*TREK program [23].
The linear absorption coefficient, m, for Mo Ka radiation was 14.9 cm 1, and the data were corrected for
Lorentz and polarization effects.
For complex 3, cell constants based on 24 488
reflections with 2u/5.0 /55.98 corresponded to a primitive orthorhombic cell with space group Pbca (no.
61). The data were collected as above in 0.308 oscillations with 28.0 s exposures. A sweep of data was done
using f oscillations from 0.0 to 189.98 at x//90.08
and a second sweep was performed using v oscillations
between /17.0 and 22.98 at x//90.08. The crystal to
detector distance was 38.12 mm and the detector swing
angle was /5.568. The data reduction was generally as
described above with some differences: of the 63 971
reflections collected, 10 959 were unique (Rint /0.092);
m was 11.1 cm 1.
Complex 1 crystallizes with two half-molecules in the
asymmetric unit (i.e. each half-molecule resides on an
inversion centre). The full-matrix least-squares refinement, based on 5557 observed reflections (I /3s(I))
and 325 variable parameters, converged with unweighted (R ) and weighted (Rw) agreement factors of
0.029 and 0.053, respectively.
Complex 3 crystallizes with two 1:1 salt moieties (each
is a different conformation) in the asymmetric unit.
Refinement, based on 5195 observed reflections (I /
3s(I )) and 575 variable parameters, converged with
agreement factors of 0.028 (R ) and 0.032 (Rw).
2.3. Biological MTT assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) was purchased from Sigma. All media
and solutions were purchased sterile, or were sterilized
through 0.2 mm filters (150 ml flask: 33 mm neck,
Corning) and kept under sterile conditions before use.
A need to determine the sensitivity of specific tumors
and individualize therapy has led to the development of
a number of in vitro assays, one of which is the MTT
assay that measures mitochondrial dehydrogenase activity as a reflection of cell viability; this shows
considerable promise in the screening and evaluation
of potential new anti-cancer agents [16,24,25]. The
yellow tetrazolium form of MTT is reduced, in active
cells, by mitochondrial dehydrogenases to form purple
241
formazan crystals [16]. A colourimetric, optical density
(OD) determination then quantifies the percentage of
viable cells (i.e. metabolically active after incubation
with the test complex). The growth inhibitory effect of
the added complex is expressed as a percentage of the
control processed at the same time [(OD of treated
sample/OD of control)/100]. The percentages are then
graphed for each concentration tested in order to create
a dosage effect curve, and the curves are analyzed for the
IC50 value.
The cells used were obtained from a human mammary
cell line MDA-MB-435s. The cells were routinely
maintained with Dulbecco’s Modified Eagle’s medium
(Stem Cell Technologies) supplemented with 10% fetal
bovine serum, at 37 8C in a NAPCO water-jacketed,
CO2-incubator under an atmosphere of 95% air/5%
CO2. The cells were trypsinized biweekly with 0.25%
trypsin/EDTA (2 ml, Gibco) at 37 8C for 3 /4 min and
1 /105 cells were plated in a T-25 flask containing a//
/ medium ( /5 ml). For experiments, 1 /106 cells were
plated in a T-75 flask with a/// medium (20 ml) and
grown for several doubling times, changing the medium
every few days.
For each complex tested, a 96-well plate was prepared
by adding 1 /104 cells in 100 ml of medium to each
experimental and control well. Medium (200 ml) was
then added to one column of wells to serve as a blank.
The outside wells of the plate were filled with sterile H2O
(200 ml), and the plate was incubated for 24 h at 37 8C
under the air/CO2 mixture. At the 23rd hour, the Ru
complex was dissolved in phosphate buffer saline
solution (PBS) (4 8C), and the solution vortexed and
left for 1 h before filtering through a 0.2 mm needle filter
(Nalgen). The stock solution of the complex was then
serially diluted with a/// medium in six-well plates
(Falcon), according to prepared concentrations, and
then added to the experimental wells, starting with the
lowest concentration and ending with the highest
concentration. Medium (100 ml) was then added to the
control wells and the plate was incubated for 68 h, when
the MTT (50 ml of 2.5 mg ml1) was added and the plate
incubated for a further 4 h. The wells were then
aspirated to remove all liquid, and DMSO (150 ml)
was added to each well to dissolve the formazan crystals.
The plates were vortexed, and the proportion of
formazan was quantified by absorbance readings at
570 nm using a Molecular Devices SPECTRAmax
PLUS plate reader.
3. Results and discussion
3.1. Characterization and chemistry of complexes 1 /3
The ORTEP diagram for 1 (Fig. 1) shows two typical
piano-stool type moieties joined by the bridged S-bound
L.A. Huxham et al. / Inorganica Chimica Acta 352 (2003) 238 /246
242
Fig. 1. A molecular structure representation (ORTEP) of one of the crystallographically independent half-molecules of [RuCl2(p -cymene)]2(m-BESE)
(1), with 50% probability thermal ellipsoids shown; H-atoms are omitted for clarity.
disulfoxide meso -BESE; the two sulfur-atoms within
each molecule of the centrosymmetric structure have
opposite chiralities, consistent with synthesis using
meso -BESE. The h6-p-cymene ligands (one at each
Ru) are syn , with the iPr groups pointing in different
Table 2
Selected bond lengths (Å) and bond angles (8) for [RuCl2(p -cymene)]2(m-BESE) (1) and [RuCl(p -cymene)(BESE)]PF6(3)
Bond lengths
S /O
S /C
Ru /Caverage
Ru /Cl
Ru /S
Bond angles
C /S /O
C /S /C
Cl /Ru /S
Ru /S /O
S /Ru /S
Cl /Ru /Cl
1a
3b
1.476(2)
1.483(2)
1.810(3)
1.829(3)
1.801(3)
1.828(3)
2.203
2.209
2.402(7)
2.407(7)
2.394(8)
2.414(7)
2.335(7)
2.345(7)
1.461(3)
1.467(3)
1.791(4)
1.796(4)
1.798(4)
1.807(4)
2.247
108.2(2)
108.4(1)
103.9(1)
107.1(1)
100.2(1)
102.5(1)
85.70(2)
86.89(3)
86.13(2)
86.23(3)
114.15(9)
115.4(1)
2.385(1)
2.288(1)
2.302(1)
108.2(2)
109.1(2)
107.0(2)
107.0(2)
101.0(2)
103.3(2)
87.37(4)
89.40(4)
115.3(1)
118.1(1)
83.73
directions. Selected bond lengths and bond angles of 1
and 3 are given in Table 2. The geometries of the
coordinated sulfoxide and p -cymene moieties are typical
of those well established in Ru(II) /sulfoxide [11,26] and
Ru(II) /p -cymene complexes [17], and the Ru /Cl distances are normal [17,27]. The S /O bond lengths for 1
are similar, for example, to those for the S-bound
sulfoxides in cis -RuCl2(BESE)2 [11].The Ru /S bond
lengths for 1 are just shorter than the sum of the
covalent radii of Ru and S (2.37 Å) [28], suggesting little
p-back-donation between the Ru- and S-atoms. The
Ru /S bond lengths for complex 1 are significantly
longer than the range of values Ru /S (2.271/2.308(8)
Å) seen for cis -RuCl2(BESE)2 [11], perhaps indicating a
general ‘trans ’ influence of the organometallic ligand
greater than that of Cl- or S-bound sulfoxide. The
sulfoxide moieties in 1 have the usual distorted trigonal
pyramid geometry with C /S/C angles of 100.2 and
102.58, and C /S /O angles of 103.9 /108.48 [26]; these
values are close to the average of 100.3(1)8 (mean of 310
values) and 107.34(7)8 (mean of 648 values), respectively, found in metal S-bonded sulfoxide complexes
[26]. The RuCl2(p -cymene) components are essentially
identical to that of RuCl2(p -cymene)(pta) mentioned in
Section 1 [17]. The IR bands at 1082 and 1110 cm 1 are
assigned to nSO of the S-bound sulfoxide [11]. The 1H
NMR spectrum of 1 in CDCl3 at room temperature
shows peaks that correspond to those of [RuCl(p cymene)]2(m-Cl)2 (see Section 2.1.1), implying the presence of an equilibrium (see Eq. (1) and Fig. 4). There
are also downfield shifted signals, corresponding to the
coordinated p-cymene protons of 1: the singlet at d 2.27
is due to the CH3 group (A) (Fig. 2); the aromatic
protons (B and C), seen as broad singlets at d 5.63 and
88.39(3)
90.47(3)
a
The two values arise because of two half-molecules in the
asymmetric unit (Section 2.2).
b
Data are taken from the structural conformer shown in Fig. 3(a).
Fig. 2. The structure of p -cymene showing labelling of the protons.
L.A. Huxham et al. / Inorganica Chimica Acta 352 (2003) 238 /246
5.55, respectively, are assigned based on those reported
by Bennett et al. [22]; the septet at d 3.05 and the
doublet at d 1.28 correspond to the CH proton (D) and
the CH3 protons (E), respectively, of the iPr group. The
triplet at d 1.36 results from the CH3 groups of BESE,
both coordinated and free. The broad peaks at d 2.82
(bs), and 3.21 (bs) correspond to the CH2 protons of
bridged and free BESE.
At room temperature, 1 at 10 3 M in CDCl3
dissociates to /60%; at 243 K, the degree of dissociation is /35%, as estimated by changes in the intensities
of the d 2.27 and 2.14 signals for 1 and [RuCl(p cymene)]2(m-Cl)2, respectively. K for equilibrium (1) at
/20 8C is thus /9 /104 M. Additions of BESE to
CDCl3 solutions of 1 give increasing amounts of 1,
formed in good agreement with such a K value.
243
(and the chloro ligand anti ) to the p -CH3 group of the
h6-p -cymene; the other conformer (Fig. 3(b)) shows the
meso -BESE, but now the S /O groups are syn (and the
chloro ligand anti ) to the iPr group of the cymene. The
bond lengths and angles of both conformers are very
similar, and only those for the conformer shown in Fig.
3(a) are given in Table 2. The S /O bond lengths are
essentially the same as in 1, while the somewhat shorter
Ru /S bond lengths might result from: (a) the S-atoms
not being trans to the centroid of the p -cymene ring (see
above); or (b) a higher degree of p-back-bonding
between the Ru- and S-atoms than in 1 [26,28]. The
C /S /C and C /S /O angles are very much as in 1. The
K
[RuCl2 (p-cymene)]2 (m-BESE) (1) X [RuCl(p-cymene)]2
(m-Cl)2 BESE
(1)
[RuCl2(p -cymene)]2(m-BESP) (2) was prepared by the
method used to synthesize 1, and the structure is
assumed to be analogous to that of 1. The 1H NMR
signals for 2 are at d 1.29 (d), 1.34 (t), 2.28 (s), 2.80 (bs),
2.35 (m), 3.05 (m), 3.08 (sp), 5.55 (bs), and 5.63 (bs) and,
except for the ‘extra’ peak at d 2.35 (which results from
the ‘extra’ CH2 group on the BESP backbone), can be
assigned as for 1. The 1H NMR also shows peaks due to
the p-cymene precursor and some free BESP, again
giving evidence for a dissociative equilibrium analogous
to that shown in Eq. (1). Seven IR bands appear in the
nSO (S-bonded) region suggesting more than one conformer in the solid state (see below for 3). Several other
homo-bimetallic complexes containing a bridging disulfoxide are known, examples including those of Cu(II)
[29], Sn(IV) [30] and Pt(II) [31], but those with an S ,S ?bound disulfoxide are rare; an example is
[PtCl2(PEt3)]2(m-PhS(O)(CH2)2S(O)Ph) [31] but, to our
knowledge, 1 and 2 are the first Ru bridged-disulfoxide
complexes. There are RuII
2 complexes containing (mS ,O /R2SO), with both S- and O-binding from a
monosulfoxide [32].
The yellow [RuCl(p -cymene)(BESE)]PF6 (3) was
synthesized from [RuCl(p -cymene)]2(m-Cl)2, BESE and
NH4PF6 in water, or by dissolving 1 in water and adding
NH4PF6; the complex was isolated by crystallization
from concentrated aqueous solutions or by precipitation
on addition of MeOH. The relatively low yield via
crystallization results from high solubility of the complex in H2O, and also in method 1 some Ru metal is
seen. The conductivity data correspond to the presence
of a 1:1 electrolyte [33]. The ORTEP structures of the two
conformers of the cation found in the asymmetric unit
are shown in Fig. 3 (the PF6 counterion is omitted).
The structure in Fig. 3(a) shows a non-bridging, h2S ,S ?-bound meso -BESE, whose S /O moieties are syn
Fig. 3. ORTEP molecular structures of the two conformers of [RuCl(p cymene)(BESE)] (3) with 50% probability thermal ellipsoids shown,
H-atoms being omitted for clarity; the enantiomorph shown in (a) has
R -chirality at S(1) and S -chirality at S(2), while the enantiomorph
shown in (b) has S -chirality at S(3) and R -chirality at S(4).
244
L.A. Huxham et al. / Inorganica Chimica Acta 352 (2003) 238 /246
Fig. 4. The proposed solution behaviour of [RuCl2(p -cymene)]2(m-BESE) (1), and the subsequent formation of [RuCl(p -cymene)(BESE)]PF6 (3).
bite angle (83.738) of the BESE at the Ru in 3 is
somewhat smaller than those seen (87.69 and 87.198) in
cis -RuCl2(BESE)2 [11]. The Cl /Ru /S angles in 3 are
about 2/38 greater than in 1, while those in RuCl2
(BESE)2 are generally greater still (in the 89.36 /91.838
range) [11].
The 1H NMR spectrum of 3 in D2O shows just one
triplet at d 1.40 for the CH3 groups of the BESE
revealing that they are equivalent and that the molecule
is fluxional in solution with presumed rotation of the p cymene ligand. The eight CH2 protons of the BESE
ligand appear as a broad multiplet between d 3.40 /3.70.
The singlet at d 2.06 corresponds to the coordinated p cymene protons A, and the doublet at d 1.06 and septet
at d 2.70 correspond to the isopropyl protons E and D,
respectively. Unlike in 1 and 2, the aromatic protons B
and C appear as a singlet (d 6.28). A singlet was also
reported for the corresponding protons of the p -cymene
complexes RuX2(p -cymene)L, where X is a halogen and
L is a tertiary phosphine or arsine [22]; no rationale was
offered, and we can offer nothing more constructive
than possible accidental degeneracy. The nSO IR data
are again consistent with S-bound disulfoxide, with the
multitude of bands presumably resulting from the
presence of two conformers and solid state effects.
The precursor for 1 and 2, [RuCl(p-cymene)]2(m-Cl)2,
dissolves in water and is believed to form the cationic
complex {[Ru(p-cymene)]2(m-Cl)3}Cl, as suggested by
Bennett et al. [22]. The 1H NMR spectrum for this
species in D2O shows a singlet at d 2.06 for the A
protons, doublets at d 5.48 and 5.70 for the aromatic
protons C and B, respectively, and a doublet at d 1.19
and septet at d 2.73 for the E and D protons,
respectively. There are also small signals shifted slightly
downfield from each of those noted above, and these
may be due to a species such as [RuCl(p-cymene)(D2O)2] [22,34].
When 1 is dissolved in D2O, the 1H NMR spectrum
shows signals at d 1.07 (d), 1.40 (t), 2.06 (s), 2.71 (sp),
3.40 /3.65 (m), and 6.29 (s), which correspond to those
of the cation of the characterized [RuCl(p -cymene)(BE-
SE)]PF6 (3), and small signals at d 1.17 (t), 2.84 (m), and
3.13 (m), which are those for free BESE; small peaks are
seen also for {[Ru(p -cymene)]2(m-Cl)3}Cl but these
disappear over /24 h. If [RuCl(p -cymene)]2(m-Cl)2 is
placed in D2O and 2 equiv. of BESE are added, the 1H
NMR spectrum shows peaks for free BESE and these
disappear over /24 h with complete generation of
[RuCl(p-cymene)(BESE)]Cl. The 1H NMR data imply
that in water 1 dissociates rapidly to give mainly
[RuCl(p-cymene)(BESE)]Cl and some {[Ru(p-cymene)]2(m-Cl)3}Cl, while the latter species reacts slowly
with BESE to generate the former species. The solution
behaviour for 1 is summarized in Fig. 4.
3.2. Biological MTT data
In the MTT assay, 1 was found to have an IC50 value
of 3609/20 mM, and 3 had a value of 559/15 mM, these
being based on averages of two trials; these numbers
come from data such as those shown in Fig. 5 that
presents graphs of cell viability versus the concentration
of the complexes. Both complexes thus show some anticancer activity, but are less active than cisplatin where
the MTT assay using the same cell line gave a
corresponding IC50 value of /10 mM [35]. Nevertheless,
these preliminary data are considered promising as the
systems are amenable to several approaches of optimization by fine-tuning.
Complex 1 does dissolve in water to generate
[RuCl(p-cymene)(BESE)]Cl, the chloride salt of 3, but
only a maximum of half of the Ru in 1 can be converted
to the ionic form. The nature of the species present in
the phosphate buffer saline solutions is uncertain, but
the higher activity of 3 strongly suggests the involvement
of a cationic species. Recent studies by Sadler’s group
have shown that the closely analogous complex
[RuCl(p-cymene)(en)]PF6 forms adducts (with displacement of the chloro ligand) by the N7 of guanine in DNA
oligonucleotides, and the chloro-cation species and
related complexes exhibit IC50 values of 6 /200 mM for
a human ovarian cancer cell line compared with values
L.A. Huxham et al. / Inorganica Chimica Acta 352 (2003) 238 /246
245
Fig. 5. The MTT assay with [RuCl2(p -cymene)]2(m-BESE) (1) and [RuCl(p -cymene)(BESE)]PF6 (3).
of 0.5 mM for cisplatin and 6 mM for carboplatin [18].
The structures of similar, monofunctional guanine
adducts of other [RuCl(h6-arene)(en)]PF6 complexes
have also been determined (where arene /biphenyl,
5,8,9,10-tetrahydroanthracene, and 9,10 dihydroanthracene) [36]. These new findings, coupled with the earlier
data on Ru(II) /arene complexes mentioned in Section 1
[13 /15], certainly suggest that the mechanism of anticancer activity for Ru(II) /arene complexes involves
interaction of cationic derivatives with DNA, with
presumably subsequent prevention of DNA replication.
4. Supplementary material
Crystallographic data for the structural analysis have
been deposited with the Cambridge Crystallographic
Data Centre, CCDC Nos. 1-200839 and 200840 for
compounds [RuCl2(p -cymene)]2(m-BESE) (1) and
[RuCl(p-cymene)(BESE)]PF6 (3), respectively. Copies
of this information may be obtained free of charge
from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: /44-1223-336-033; e-mail:
deposit@ccdc.cam.ac.uk or www: http://www.ccdc.cam.
ac.uk).
Acknowledgements
We thank the Natural Sciences and Engineering
Research Council of Canada for financial support, and
Dr. Kirsten Skov of the Advanced Therapeutics Department at the British Columbia Cancer Research Centre
for help with the MTT assays.
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