Ruthenium(ii) arene PTA (RAPTA) complexes: Impact of enantiomerically pure chiral ligands

Article abstract of DOI:10.1039/c2dt32333h

Organometallic ruthenium(ii) arene complexes containing the PTA ligand ([Ru(η⁶-arene)Cl₂(PTA)], PTA = 1,3,5-triaza-7- phosphatricyclo-[3.3.1.1]decane, termed RAPTA) show pharmacologically relevant anti-tumour properties in vitro. Two new enantiomeric pairs of RAPTA compounds, containing the chiral arene (R)- or (S)-2-phenyl-N-(1-phenylethylene)acetamide and either dichlorido or oxalato ligands were synthesised and fully characterised. The stability of the complexes towards hydrolysis was assessed and the dichlorido complexes were found to be more stable towards hydrolysis than the prototype complex RAPTA-C, ([Ru(η⁶-p-cymene)Cl ₂(PTA)]). The cytotoxicity of the compounds towards human ovarian cancer cells is moderate to good with a degree of selectivity towards the cancer cells over healthy cells. More significantly, for the first time we were able to establish the influence of a bulky, chiral group attached to the arene on the cytotoxicity of this class of compound, with the S-enantiomer being more cytotoxic than the R-enantiomer. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2dt32333h

Dalton
Transactions
PAPER
Ruthenium(II) arene PTA (RAPTA) complexes: impact of
enantiomerically pure chiral ligands†
Cite this: Dalton Trans., 2013, 42, 2008
Kelly J. Kilpin, Shona M. Cammack, Catherine M. Clavel and Paul J. Dyson*
Organometallic ruthenium(II) arene complexes containing the PTA ligand ([Ru(η6-arene)Cl2(PTA)], PTA =
1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane, termed RAPTA) show pharmacologically relevant anti-
tumour properties in vitro. Two new enantiomeric pairs of RAPTA compounds, containing the chiral
arene (R)- or (S)-2-phenyl-N-(1-phenylethylene)acetamide and either dichlorido or oxalato ligands were
synthesised and fully characterised. The stability of the complexes towards hydrolysis was assessed and
the dichlorido complexes were found to be more stable towards hydrolysis than the prototype complex
RAPTA-C, ([Ru(η6-p-cymene)Cl2(PTA)]). The cytotoxicity of the compounds towards human ovarian cancer
cells is moderate to good with a degree of selectivity towards the cancer cells over healthy cells. More sig-
nificantly, for the first time we were able to establish the influence of a bulky, chiral group attached to
the arene on the cytotoxicity of this class of compound, with the S-enantiomer being more cytotoxic
than the R-enantiomer.
Received 3rd October 2012,
Accepted 9th November 2012
DOI: 10.1039/c2dt32333h
www.rsc.org/dalton
Introduction
Following the discovery of cisplatin in the late 1960s, a vast
amount of research has been directed towards the develop-
ment of new metal-based chemotherapeutic agents.1,2
Although thousands of new metal-based anti-cancer drugs
have been proposed, with a number entering into clinical
trials, only three (platinum-based) are currently in widespread
clinical use.3 This low success rate has been attributed to
various problems such as high levels of in vivo toxicity, drug
resistance and low aqueous solubility.4
With respect to overcoming these problematic issues,
ruthenium-based complexes have been developed that show
considerable promise,5,6 in particular the Ru(III) coordination
complexes NAMI-A and KP1019 (Fig. 1), which have progressed
into clinical trials.7 We are interested in organometallic Ru(II)
arene complexes, typified by the prototype molecule RAPTA-C
(Fig. 1), which although possesses low in vitro cytotoxicity,
shows selectivity towards tumours in vivo.8 In general, RAPTA
complexes contain a face-capping arene, two labile chloride
Fig. 1 Ruthenium(III) complexes currently in clinical trials, NAMI-A and KP1019,
the Ru(II) arene complex, RAPTA-C and Oxaliplatin.
amenable to structural modifications on the arene ring, or at
the Ru centre (by substitution of either the PTA or chloride
ligands).9 Although a number of the aforementioned modifi-
cations have been carried out, and their effect on cytotoxicity
determined,10–20 a detailed study into the biological effects of
chiral ancillary ligands has yet to be conducted for RAPTA-type
compounds, and Ru(II) arene compounds more generally, even
though examples of Ru(II) chiral–arene complexes have been
reported.21,22
ligands and
a PTA (1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]-
decane) ligand which imparts biologically favourable aqueous
solubility to the compounds owing to its amphilic nature.
Additionally, the robust nature of the RAPTA scaffold is
Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de
Lausanne (EPFL), CH-1015 Lausanne, Switzerland. E-mail: paul.dyson@epfl.ch;
Fax: +41 (0) 21 693 98 85; Tel: +41 (0) 21 693 98 54
†Electronic supplementary information (ESI) available: Selected NMR spectra
and UV-vis traces. See DOI: 10.1039/c2dt32333h
It is well established that different optical isomers, or enan-
tiomers, of a compound can have markedly different biological
2008 | Dalton Trans., 2013, 42, 2008–2014
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Paper
activities, well-documented by the thalidomide tragedy of the
1980s.23 In addition, the different enantiomers may have
differing pharmacokinetics and affinities towards receptor
molecules, as is the case for the opioid substitute metha-
done.24 This is not only true for organic drugs – the clinically
approved Pt(II) drug oxaliplatin (Fig. 1), which contains the
chiral 1R,2R-cyclohexadiamine ligand is not only more active
than the corresponding enantiomer (with 1S,2S-cyclohexadi-
amine), but also shows increased cellular uptake and DNA
binding properties.25 Only recently, chiral methyl substituted
oxaliplatin derivatives have been reported, and again the
stereochemistry greatly affects the biological properties of the
compound.26,27
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In light of these observations we thought that a comparison
between the activity levels of different enantiomers of RAPTA
compounds may be interesting. In this paper two new enantio-
meric pairs of RAPTA-type complexes are described together
with discussions of their stability towards hydrolysis and
initial investigations into their biological activity.
Results and discussion
The synthesis of the complexes employed in this study was
carried out using the methodology previously described to
modify the RAPTA scaffold, depicted in Scheme 1.9 Briefly,
2-(cyclohexa-1,4-dien-1-yl)acetic acid, 1, obtained via a Birch
reduction of phenyl acetic acid,28 was coupled to the appropri-
ate chiral amine, either R- or S-methylbenzylamine, 2-(R) or
2-(S), with the aid of TBTU (O-(benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium tetrafluoroborate), which suppresses race-
misation.29 An excess of the diene, 3-(R) or 3-(S), was then
heated to reflux in EtOH with RuCl3 to give the dimeric Ru(II)
complexes 4-(R) or 4-(S) as orange solids in good yields.30
Reaction of 4-(R) or 4-(S) with 2 equivalents of PTA at room
temperature affords the RAPTA complexes, 5-(R) or 5-(S), also
in reasonable yields as orange solids.31 The oxalato derivatives,
6-(R) or 6-(S) were obtained as yellow solids by first reacting
the dimer 4-(R) or 4-(S) with a slight excess of silver oxalate,
followed by the removal of AgCl, and subsequent addition of
PTA.12
As with other RAPTA-type complexes, both the dichlorido,
5-(R) or 5-(S), and oxalato, 6-(R) or 6-(S), compounds are
soluble in a range of polar (H2O, MeOH, EtOH, DMSO) and
non-polar (CH2Cl2) solvents, with the oxalato complexes
slightly more soluble than the dichlorido complexes.
All the new compounds were characterised fully and shown
to be analytically pure. The 1H NMR spectra of 4, 5 and 6
(d6-DMSO) showed peaks at 5.3–5.8 ppm, characteristic of
protons on the capping arene ring. Unlike RAPTA-C (and the
oxalato analogue), the 1H and 13C{1H} NMR spectra of the
mononuclear complexes comprise five unique proton environ-
ments, and six unique carbon environments (despite the
apparent mirror plane in the molecules). However, when the
spectra are acquired in D2O this symmetry is not observed,
instead the arene protons all appear to be equivalent. Such an
Scheme 1 Synthetic routes to the dichlorido-5-(R), 5-(S) and oxalato-6-(R),
6-(S) Ru(II) complexes used in this study (a) DIEA, TBTU, CH2Cl2, 18 h, rt; (b)
RuCl3·3H2O, EtOH,
6 h, reflux; (c) PTA, CH2Cl2/MeOH (2 : 1), 3 h, rt; (d)
1. Ag2(C2O4), H2O, 12 h, 2. PTA, MeOH, 3 h.
observation suggests the presence of intramolecular (hydro-
gen) bonding interactions in the molecule, possibly between
the amide functionality and either the dichlorido or oxalato
ligands, which hold it in a favourable conformation and
inhibit rotation around the Ru–arene axis. The 31P{1H} spectra
of 5 and 6 each contain a singlet at −31.6 and −32.0 ppm,
respectively, which agree with values reported previously for
RAPTA complexes.31 The signals characteristic of the amide
functionality differ little between the diene, 3, and complexes
4, 5 and 6, which suggest that it is not coordinated to the
ruthenium centre. This inference was further corroborated by
IR spectroscopy, which showed strong CvO stretching bands
at ca. 1655 cm−1 for 3–6. HR-ESI mass spectrometry further
confirmed the formation of 5 and 6. The dominant peaks in
the spectra are those assigned to the [M + H]+ ion, and in the
case of 5, less intense ions assigned to [M − Cl]+ were also
observed. In all cases, the assignments of the spectra were
aided by comparison of the experimental and theoretical
isotope patterns, arising from ruthenium (and chlorine in 5).
Analysis of the optical rotation of the complexes confirmed the
optical purity of the samples.
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Like cisplatin, RAPTA complexes also undergo hydrolysis in
Dalton Transactions
aqueous solutions and at low chloride concentrations to give
aquated species. At relatively high chloride concentrations this
pathway is suppressed.12,32–34 In order to suppress or reduce
the rate of hydrolysis at the Ru centre, oxalato and carboxylato
complexes, analogous to oxaliplatin and carboplatin, were
developed, and interestingly, these modifications did not sig-
nificantly alter the activity in vitro.12 We decided to synthesize
the oxalato analogues of 5 in order to suppress hydrolysis,
which in turn would inhibit the formation of diastereoisomers
arising from a chiral centre on the ligand and at the ruthe-
nium. In this way, the chirality in the molecule is restricted to
the ligand, to facilitate a direct comparison between the two
enantiomers.
Hydrolysis studies of 5-(S) and 6-(S) were carried out in
both aqueous and saline ([Cl−] = 150 mM) solutions, using
both 31P{1H} NMR and UV-vis spectroscopy to monitor the
changes. As expected, the dichlorido species 5-(S) immediately
begins to undergo hydrolysis in D2O and reaches equilibrium
within 2 hours. In the 31P{1H} spectra, this process was charac-
terised by the loss of the peak assigned to the dichlorido
species 5-(S) at −32.0 ppm, coupled with the appearance of
two new peaks of equal intensity at −29.8 and −29.9 ppm,
assigned to the two diastereoisomers represented by Form I
(Scheme 2). Only upon the addition of excess AgBF4 was a
signal (at −28.1 ppm) attributable to Form II (Scheme 2)
observed (Fig. 2). In saline D2O ([Cl−] = 150 mM) hydrolysis
was completely suppressed, with the peak at −32 ppm, corres-
ponding to the dichlorido form, being the only species after
48 hours.
Fig. 2 Selected 31P{1H} NMR spectra of 5-(S) over 24 h in D2O, and with the
addition of excess AgBF4. (5-(S) = [Ru(η6-arene)(PTA)Cl2], Form I = [Ru(η6-arene)-
(PTA)(OH2)Cl]+, Form II = [Ru(η6-arene)(PTA)(OH)(H2O)]+, arene = 2-phenyl-N-
(1-phenylethylene)acetamide).
The oxalato complexes 6-(S) were completely inert to
hydrolysis in D2O solution, even after 48 hours, however in
saline solution ([Cl−] = 150 mM) a minor peak (16%) corres-
ponding to the dichloride 5-(S) appeared after 2 hours
(Fig. S10, ESI†) suggesting some substitution of oxalate for
chloride in relatively high chloride concentrations.
UV-Vis spectra were recorded in phosphate buffered solu-
tions (10 mM, pH 7.4) in the absence and presence of chloride
([Cl−] = 150 mM). The results obtained corroborate what was
observed by 31P{1H} NMR spectroscopy. As an example, in the
absence of chloride ions, the dichlorido complex 5-(S) dis-
played an initial maximum at 340 nm which over time
decreases with two new maxima at 296 and 380 nm appearing,
indicating hydrolysis of 5-(S) (Fig. 3). After 2 hours the spectra
underwent no further change. In contrast, in phosphate
Fig. 3 UV-vis spectra (270–620 nm) of 5-(S) in phosphate buffer (10 mM,
pH 7.4). The arrows show the direction of the changes of the spectra. Spectra
were recorded at 5 min intervals over a 2 h period.
buffered saline solutions containing 150 mM chloride the spectra of 5-(S) remained unchanged over 48 hours. There
were no observable changes in the spectra of the oxalato
complex 6-(S) over 48 hours regardless of the presence or
absence of chloride. Compared to RAPTA-C under the same
conditions (by both 31P{1H} NMR and UV-vis spectroscopy)
hydrolysis of 5-(S) is considerably slower, as RAPTA-C reached
equilibrium within 15 minutes (Fig. S11, ESI†). Such a differ-
ence in exchange kinetics is not unsurprising considering the
electronic differences in the arene rings.
The cytotoxicity of 5 and 6 was determined against A2780
Scheme 2 Aquation/hydrolysis of 5-(R) or 5-(S) in aqueous solutions.
and A2780cisR cells (cisplatin sensitive and cisplatin resistant
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All the complexes are significantly more cytotoxic than
RAPTA-C and oxaloRAPTA-C with the dichlorido-complexes
retaining in vitro selectivity towards cancer cell lines. There are
also notable differences in cytotoxicity between the S- and
R-enantiomers with the S-enantiomer being more cytotoxic
(depending on the cell line under study). Thus, the develop-
ment of chiral ruthenium(II) arene anticancer compounds
should not be overlooked as the modifications described in
this study result in compounds with improved cytotoxicity and
selectivity profiles.
Table 1 Cytotoxicity (IC50, μM) of selected compounds at 72 h
A2780a
A2780cisRb
HEKc
5-(R)
44.0 1.1
30.1 0.5
353
34.2 0.6
8.7 0.1
511
396.1 2.6
228.3 15.5
252
33.2 0.8
20.6 2.4
656
>1000
5-(S)
254.9 13.0
RAPTA-Ce
6-(R)
N/Ad
32.2 0.9
32.4 4.5
N/Ad
6-(S)
oxaloRAPTA-Ce
a Human ovarian carcinoma cells. b Human ovarian carcinoma cells –
acquired resistance to cisplatin. c Human embryonic kidney cells. d N/A
data not available. e Taken from ref. 35.
human ovarian cancers, respectively) and HEK cells (human
embryonic kidney, a model for non-tumourigenic cells) using
the MTT assay. The results, reported as IC50 concentrations,
are presented in Table 1.
Experimental
PTA (1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane),36 2-(cyclo-
hexa-1,4-dien-1-yl)acetic acid, 128 and silver oxalate12 were syn-
thesised according to literature procedures. All other reagents
were purchased from commercial sources and used without
further purification. Column chromatography was carried out
on a Varian 971-FP Autocolumn using SiO2 Luknova flash
Against the A2780 cell line, all the complexes in this study
are considerably more active than the prototype complexes
RAPTA-C and oxaloRAPTA-C. Also, compound 5-(R) has a
remarkable selectivity profile with an IC50 of 44.0 μM in A2780
cells versus >1000 μM in the healthy HEK cell line. Against the
A27080cisR and HEK cell lines, the dichlorido complexes 5-(R)
and 5-(S) show similar levels of activity compared with
RAPTA-C whereas the oxalato compounds 6-(R) and 6-(S) are
far more active than oxaloRAPTA-C. The reasons for these vast
differences in activity profiles are unclear although the arene
ligand must play a role, possibly via modulation of the rate of
hydrolysis at the ruthenium centre, by altering the bioavailabil-
ity of the complex and/or by enhancing specific interactions
with relevant targets.
It is also noteworthy that the different enantiomers also
give rise to significant differences: 5-(S) is much more cytotoxic
toward HEK cells than 5-(R) (ca. 254 versus >1000 μM, respecti-
vely) and with the oxalato complexes 6, the S-enantiomer is
more active than the R-enantiomer against the A2780 cell line
(8.7 versus 34.2 μM, respectively). Across the other cell lines
and complexes, the S-enantiomers were also slightly more
active (with the exception of 6 against HEK). This may be in
part due to the chiral centre being somewhat remote from the
ruthenium centre. Moreover, racemisation and inversion of
the chiral centre inside the cell cannot be discounted.
columns (40–60 μm). H (400.13 MHz), 31P{1H} (161.98 MHz)
1
and 13C{1H} (100.62 MHz) NMR spectra were recorded on a
Bruker Avance II 400 spectrometer at 298 K. Chemical shifts
are reported in parts per million and referenced to residual
solvent peaks (CDCl3: 1H δ 7.26, 13C δ 77.16; d6-DMSO:
1H δ 2.50, 13C δ 39.52 ppm). Coupling constants (J) are
reported in Hertz (Hz). IR spectra were recorded on a Perkin
Elmer Spectrum 1 Spectrometer and UV-vis experiments were
conducted using 1 cm Quartz Cells (Hellma Analytics) on a
Jasco V-550 spectrometer. High Resolution Electrospray Ioni-
zation mass spectra (HR ESI-MS) were obtained on a Thermo-
Finnigan LCQ Deca XP Plus quadropole ion-trap instrument
operated in positive-ion mode. Specific rotation was
measured on a Jasco P-2000 Polarimeter, with [α]D values given
in 10−1 deg cm2 −1. Elemental analyses were carried out by
g
the microanalytical laboratory at the EPFL. Melting points
were determined using a SMP3 Stuart Melting Point Apparatus
and are uncorrected.
Synthesis of 3-(R) and 3-(S)
2-(Cyclohexa-1,4-dien-1-yl)acetic acid, 1, (2.00 g, 14.48 mmol,
1.0 eq.) and diisopropylethylamine (DIEA) (5.6 mL,
31.84 mmol, 2.2 eq.) were added to dry CH2Cl2 (30 mL) at 0 °C.
With stirring, TBTU (4.64 g, 14.48 mmol, 1.0 eq.) was added
and the reaction stirred for 15 min. (R)-1-Phenylethylamine,
Conclusions
RAPTA complexes are a promising class of Ru(II) anti-cancer 2-(R), or (S)-1-phenylethylamine, 2-(S) (1.8 mL, 14.48 mmol,
agents. In this contribution we report the synthesis, aqueous 1.0 eq.) in dry CH2Cl2 (10 mL) was added dropwise and the
behaviour and cytotoxicity of two enantiomeric pairs of reaction stirred for 18 h at room temperature. The resulting
RAPTA-type compounds containing the chiral arene (R)- or (S)- clear orange solution was washed with water (15 mL), brine
2-phenyl-N-(1-phenylethylene)acetamide and either dichlorido (15 mL) and dried (MgSO4). The solvent was removed in vacuo
or oxalato ligands. The dichlorido-complexes were significantly and the product purified by flash column chromatography
more stable towards hydrolysis in aqueous solution than the (9 : 1 CH2Cl2 : MeOH) to give 3-(R) or 3-(S) as white solids.
parent compound RAPTA-C whereas the oxalato complexes (3-(R): 1.94 g, 55%, 3-(S): 2.40 g, 68%).
completely resisted hydrolysis. This difference can be traced to
3-(R): Mp 114 °C; [α]2D0 = +98° (c 0.03, EtOH); Anal. calcd for
the electronic effects induced at the ruthenium(II) centre by C16H19NO: C 79.62, H 7.94, N 5.81. Found: C 79.35, H 7.86,
the arene ligand.
N 6.04%; IR ν(cm−1): 3282, 3029, 2972, 2883, 2823, 1637, 1541,
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Dalton Transactions
1492, 1444, 1360, 1251, 1014, 962, 743, 697, 658, 559, 501, 484, removed. The solid was redissolved in minimal MeOH and
455; NMR (CDCl3) 1H δ 7.34–7.26 (m, 5H, ArH), 5.93 (d, J = precipitated with Et2O at −4 °C. The solid was isolated by
6 Hz, 1H, NH), 5.72–5.66 (m, 3H, cyclohexadiene CH), 5.13 (m, filtration and washed with Et2O (2 × 5 mL) and dried in vacuo.
1H, CH), 2.92 (s, 2H, CH2), 2.74 (m, 2H, cyclohexadiene CH2), (5-(R): 26 mg, 37%; 5-(S): 30 mg, 43%.)
2.62 (m, 1H, cyclohexadiene CH2), 1.47 (d, J = 7 Hz, 3H, CH3);
13C{1H} δ 169.8 (CvO), 143.3 (Ar–C), 130.2 (diene–C), 128.7 saline); Anal. calcd for C22H29N4OPCl2Ru·0.5H2O: C 45.75,
(Ar–C), 127.3 (Ar–C), 126.1 (Ar–C), 124.0 (diene–C), 123.9 H 5.24, N 9.71. Found: C 45.87, H 5.05, N 9.57%; IR ν(cm−1
5-(R): Mp 197 °C (decomp.); [α]2D0 = −20° (c 0.08, 100 mM
)
(diene–C), 123.8 (diene–C), 48.6 (CH), 45.9 (CH2), 29.1 (cyclo- 3277, 2926, 1655, 1500, 1443, 1406, 1335, 1277, 1239, 1099,
hexadiene CH2), 26.9 (cyclohexadiene CH2), 21.8 (CH3); HR 1010, 970, 944, 866, 799, 757, 742, 698, 577, 540, 524, 455, 516;
1
ESI-MS: m/z = 242.155 [M + H]+ (calc. for C16H20NO 242.150).
NMR (d6-DMSO) H δ 8.67 (d, J = 8 Hz, 1H, NH), 7.31 (m, 4H,
3-(S): Mp 114 °C; [α]2D0 = −98° (c 0.03, EtOH); Anal. calcd for ArH), 7.21 (m, 4H, ArH), 5.80 (m, 2H, Ru–ArH), 5.64 (dd, J = 6,
C16H19NO: C 79.62, H 7.94, N 5.81. Found: C 79.65, H 7.95, 16 Hz, 2H, Ru–ArH), 5.33 (t, J = 6 Hz, 1H, Ru–ArH), 4.89 (m,
N 5.73%; IR ν(cm−1): 3283, 3062, 3029, 2972, 2883, 2823, 1637, 1H, CH), 4.43 (s, 6H, N–CH2–N), 4.19 (s, 6H, P–CH2–N), 3.18 (s,
1540, 1492, 1444, 1360, 1251, 1013, 962, 743, 697, 658, 496, 2H, CH2), 1.35 (d, J = 6 Hz, 3H, CH3); 13C{1H} δ 167.6 (CvO),
1
467, 453; NMR (CDCl3) H δ 7.34–7.26 (m, 5H, ArH), 5.92 (d, 144.4 (Ar–C), 128.2 (Ar–C), 126.6 (Ar–C), 125.9 (Ar–C), 101.7 (d,
J = 6 Hz, 1H, NH), 5.70 (m, 2H, cyclohexadiene CH), 5.63 J = 4 Hz, Ru–C), 88.1 (d, J = 5 Hz, Ru–C), 87.7 (d, J = 5 Hz,
(m, 1H, cyclohexadiene CH), 5.14 (m, 1H, CH), 2.92 (s, 2H, Ru–C), 86.0 (Ru–C), 85.6 (Ru–C), 78.7 (Ru–C), 72.2 (d, J = 7 Hz,
CH2), 2.76 (m, 2H, cyclohexadiene CH2), 2.63 (m, 2H, cyclohexa- N–CH2–N), 51.9 (d, J = 18 Hz, P–CH2–N), 48.2 (CH), 22.5 (CH3)
diene CH2) 1.47 (d, J = 7 Hz, 3H, CH3); 13C{1H} δ 169.8 (CvO), (CH2 under solvent, ca. 39.5); 31P{1H} δ −31.6; HR ESI-MS m/z
143.3 (Ar–C), 130.2 (diene–C), 128.7 (Ar–C), 127.4 (Ar–C), 126.1 = 533.083 (15%, [M − Cl]+, calc. for C22H30ClN4OPRu 533.081),
(Ar–C), 124.0 (diene–C), 123.9 (diene–C), 123.8 (diene–C), 48.6 569.057 [M + H]+ (calc. for C22H30Cl2N4OPRu 569.058).
(CH), 45.9 (CH2), 29.1 (cyclohexadiene CH2), 26.9 (cyclohexa-
diene CH2), 21.9 (CH3); HR ESI-MS: m/z = 242.155 [M + H]+ saline); Anal. calcd for C22H29N4OPCl2Ru·1.5H2O: C 44.36,
5-(S): Mp 197 °C (decomp.); [α]2D0 = +20° (c 0.08, 100 mM
(calc. for C16H20NO 242.150).
H 5.42, N 9.41. Found C 44.36, H 5.25, N 9.94%; IR ν(cm−1
)
3277, 3053, 1657, 1504, 1443, 1406, 1336, 1277, 1240, 1198,
1099, 1010, 971, 944, 896, 866, 810, 799, 757, 742, 699, 576,
Synthesis of 4-(R) and 4-(S)
RuCl3·3H2O (100 mg, 0.42 mmol, 1.0 eq.) and either 3-(R) or 532, 516, 493; NMR (d6-DMSO) 1H δ 8.67 (d, J = 8 Hz, 1H, NH),
3-(S) (400 mg, 1.67 mmol, 4.0 eq.) were refluxed in EtOH 7.29 (m, 4H, ArH), 7.21 (m, 1H, ArH) 5.81 (m, 1H, Ru–ArH),
(100 mL) under nitrogen for 6 h. While hot, the solution was 5.67 (dd, J = 17, 6 Hz, 1H, Ar–RuH), 5.63 (dd, J = 17, 6 Hz, 1H,
filtered and the filtrate reduced in volume (to ca. 30 mL). Ar–RuH), 5.34 (t, J = 6 Hz, Ru–ArH), 4.88 (m, 1H, CH), 4.43 (s,
Storage at −4 °C resulted in the precipitation of 4-(R) or 4-(S), 6H, N–CH2–N), 4.18 (s, 6H, P–CH2–N), 3.18 (s, 2H, CH2), 1.35
which was isolated by filtration, washed with Et2O (2 × 5 mL) (d, J = 7 Hz, 3H, CH3); 13C{1H} δ 167.6 (CvO), 144.4 (Ar–C),
and dried under vacuum. (4-(R): 105 mg, 67%, 4-(S): 88 mg, 128.2 (Ar–C), 126.6 (Ar–C), 125.9 (Ar–C), 101.7 (d, J = 4 Hz,
56%).
Ru–C), 88.3 (d, J = 5 Hz, Ru–C), 87.9 (d, J = 5 Hz, Ru–C), 86.0
4-(R): Mp 231 °C (decomp.); Anal. calcd for C32H34N2O2Cl4 (Ru–C), 85.5 (Ru–C), 78.7 (Ru–C), 72.2 (d, J = 7 Hz, N–CH2–N),
Ru2·H2O: C 45.71, H 4.32, N 3.33. Found: C 45.53, H 4.07, 51.9 (d, J = 18 Hz, P–CH2–N), 48.2 (CH), 22.5 (CH3) (CH2 under
N 3.23%; IR ν(cm−1) 3276, 3036, 2973, 1642, 1542, 1493, 1444, solvent, ca. 39.5); 31P{1H} δ −31.5; HR ESI-MS m/z = 533.088
1342, 1254, 1145, 1018, 962, 872, 755, 695, 532, 499, 492, 476; (12%, [M − Cl]+, calc. for C22H30ClN4OPRu 533.081), 569.058
1
NMR (d6-DMSO) H δ 8.71 (d, J = 8 Hz, 1H, NH), 7.30 (m, 4H, (100%, [M + H]+, calc. for C22H30Cl2N4OPRu 569.058).
ArH), 7.22 (m, 1H, ArH), 6.01 (m, 2H, Ru–ArH), 5.80 (m, 3H,
Ru–ArH), 4.91 (m, 1H, CH), 3.36 (s, 2H, CH2), 1.35 (d, J = 7 Hz,
Synthesis of 6-(R) and 6-(S)
3H, CH3).
4-(R) or 4-(S) (50 mg, 0.06 mmol, 1.0 eq.) was dissolved in
4-(S): Mp 231 °C (decomp.); Anal. calcd for C32H34N2O2Cl4- water (100 mL) and silver oxalate (46 mg, 0.15 mmol, 2.5 eq.)
Ru2·3H2O: C 43.84, H 4.60, N 3.20. Found: C 43.74, H 3.96, added. The reaction was stirred for 12 h under a nitrogen
N 2.98%; IR ν(cm−1) 3274, 3036, 2973, 1641, 1542, 1493, 1444, atmosphere in a foil-covered flask. The silver chloride was
1342, 1252, 1145, 1018, 962, 871, 755, 695, 633, 564, 535, 481; removed by filtration through celite and the solvent removed.
1
NMR (d6-DMSO) H δ 8.72 (d, J = 7 Hz, 1H, NH), 7.31 (m, 4H, The solid was redissolved in MeOH (25 mL) and PTA (23 mg,
ArH), 7.22 (m, 1H, ArH), 6.01 (m, 2H, Ru–ArH), 5.80 (m, 3H, 0.14 mmol, 2.4 eq.) was added and the solution stirred for a
Ru–ArH), 4.91 (m, 1H, CH), 3.37 (s, 2H, CH2), 1.34 (d, J = 7 Hz, further 2 h. The solvent was reduced to ∼5% of the original
3H, CH3).
volume and Et2O (25 mL) was added. The solution was stored
at −4 °C and the yellow precipitate isolated by filtration,
washed with Et2O (2 × 5 mL) and dried in vacuo. (6-(R): 35 mg,
Synthesis of 5-(R) and 5-(S)
4-(R) or 4-(S) (50 mg, 0.06 mmol, 1.0 eq.) was dissolved in 49%, 6-(S): 33 mg, 46%.)
CH2Cl2 and MeOH (60 mL, 2 : 1). PTA (19 mg, 0.12 mmol,
6-(R): Mp 171 °C (decomp.); [α]2D0 = −29° (c 0.05, 100 mM
2.0 eq.) was added and the solution stirred at room temp- saline); Anal. calcd for C24H29N4O5PRu·3H2O: C 44.99, H 5.51,
erature for 3 h. The solution was filtered and the solvent N 8.75. Found: C 44.75, H 4.93, 10.54%; IR ν(cm−1) 3272, 3054,
2012 | Dalton Trans., 2013, 42, 2008–2014
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1696, 1645, 1541, 1494, 1445, 1364, 1284, 1240, 1097, 1012, surviving cells was calculated from the absorbance of
968, 945, 899, 782, 741, 699, 576, 538, 526, 517, 471; NMR untreated control cells. Evaluation is based on means from
1
(d6-DMSO) H δ 8.72 (d, J = 8 Hz, 1H, NH), 7.30 (m, 4H, ArH), two independent experiments, each comprising three micro-
7.23 (m, 1H, ArH), 5.96 (d, J = 2 Hz, 2H, Ru–ArH), 5.79 (dd, J = cultures per concentration level.
6, 19 Hz, 2H, Ru–ArH), 5.48 (t, J = 5 Hz, 1H, Ru–ArH), 4.89 (m,
1H, CH), 4.42 (s, 6H, N–CH2–N), 4.03 (s, 6H, P–CH2–N), 3.20 (s,
2H, CH2), 1.36 (d, J = 7 Hz, 3H, CH3); 13C{1H} δ 167.0 (CvO),
Acknowledgements
164.0 (2 × CvO, oxalato), 144.4 (Ar–C), 128.3 (Ar–C), 126.7 (Ar–
C), 125.9 (Ar–C), 102.4 (d, J = 4 Hz, Ru–C), 87.2 (Ru–C), 87.0
(Ru–C), 86.9 (d, J = 5 Hz, Ru–C), 86.6 (d, J = 4 Hz, Ru–C), 76.4
(Ru–C), 71.8 (d, J = 6 Hz, N–CH2–N), 50.1 (d, J = 18 Hz, P–CH2–
N), 48.2 (CH), 38.5 (CH2), 22.6 (CH3); 31P{1H} δ −32.0;
This work was supported by the New Zealand Foundation of
Research, Science and Technology (Postdoctoral Fellowship,
EPFL1001, to KJK), an Erasmus Grant (Commission of the
European Communities within the framework of the LLP
Erasmus Programme – SMC), and the Swiss National Science
Foundation (CMC). Ms. S. Crot is gratefully acknowledged
for technical assistance and Dr B. S. Murray for helpful
discussions.
HR ESI-MS m/z
= 587.100 (100%, [M +
H]+, calc. for
C24H30N4O5PRu 587.100).
6-(S): Mp 171 °C (decomp.), [α]2D0 = +29° (c 0.05, 100 mM
saline); Anal. calcd for C24H29N4O5PRu·2H2O: C 46.29, H 5.35,
N 9.00. Found: C 46.54, H 4.76, N 8.95%; IR ν(cm−1) 3276,
3060, 1696, 1670, 1654, 1541, 1445, 1364, 1285, 1240, 1098,
1012, 967, 946, 899, 781, 742, 698, 576, 518, 511, 532, 492, 478;
1
NMR (d6-DMSO) H δ 8.71 (d, J = 8 Hz, 1H, NH), 7.30 (m, 4H,
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2014 | Dalton Trans., 2013, 42, 2008–2014
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