Tuning the cytotoxicity of ruthenium(ii) para-cymene complexes by mono-substitution at a triphenylphosphine/phenoxydiphenylphosphine ligand

Article abstract of DOI:10.1039/c7dt03385k

The new complexes [RuCl₂(η⁶-p-cymene)(κP-Ph₂PR)] [R = 4-C₆H₄OSiMe₂^tBu, 1; R = 4-C₆H₄Br, 2; R = OC(O)CHCl₂, 3; R = OPh, 4; R = O(2-C₆H₄SiMe₂^tBu), 5] and [Ru(C₂O₄)(η⁶-p-cymene){κP-Ph₂PO(2-C₆H₄(SiMe₂^tBu))}], 6, were obtained in 83-98% yield from Ru(ii) arene precursors by three different synthetic strategies. The unprecedented phosphine Ph₂P(O(2-C₆H₄SiMe₂^tBu)) was synthesized in 86% yield from 2-C₆H₄Br(OSiMe₂^tBu) and Ph₂PCl, via intramolecular oxygen to carbon 1,3 migration of the silyl group (retro-Brook rearrangement). All the complexes were fully characterized by analytical and spectroscopic methods, and by single crystal X-ray diffraction in the cases of 3, 4, 5 and 6. Complexes 1-6 and the model compounds [RuCl₂(η⁶-p-cymene)(κP-PPh₃)] (Ru-PPh₃) and [Ru(C₂O₄)(η⁶-p-cymene)(κP-PPh₃)] (Ru-PPh₃-O) underwent slow degradation in chloroform solutions upon air contact; the mixed valence complex [(η⁶-p-cymene)Ru(μ-Cl)₃RuCl₂(κP-PPh₃)], 7, was isolated from a solution of Ru-PPh₃ in CHCl₃, and X-ray identified. The antiproliferative activity of 1-6 and Ru-PPh₃, Ru-PPh₃-O and [RuCl₂(η⁶-p-cymene)(κP-PTA)] (RAPTA-C) was assessed towards the triple-negative breast cancer cell line MDA-MB-231, the ovarian carcinoma cell line A2780 and human skin fibroblasts (HSF). Complexes 1, 2, 5 and 6 displayed IC₅₀ values significantly lower than that of cisplatin, with 2 providing a more potent cytotoxic effect on MDA-MB-231 and A2780 cancer cells compared to the noncancerous cell line (HSF). The stability of all complexes in DMSO/water solution was elucidated by NMR and conductivity measurements, and in particular ³⁵Cl NMR spectroscopy was helpful to check the possible chloride dissociation. The stability studies suggest that the cytotoxic activity in vitro of the compounds is mainly ascribable to Ru(ii) species still bound to the phosphorus ligand.

Full text of DOI:10.1039/c7dt03385k

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Tuning the cytotoxicity of ruthenium(II) para-
cymene complexes by mono-substitution at a
triphenylphosphine/phenoxydiphenylphosphine
ligand†
Cite this: DOI: 10.1039/c7dt03385k
c
Lorenzo Biancalana,a Stefano Zacchini, b Nicola Ferri,
Maria Giovanna Lupo, c Guido Pampalonia and Fabio Marchetti
*
a
The new complexes [RuCl2(η6-p-cymene)(κP-Ph2PR)] [R = 4-C6H4OSiMe2 Bu, 1; R = 4-C6H4Br, 2; R =
t
OC(vO)CHCl2, 3; R = OPh, 4; R = O(2-C6H4SiMe2 Bu), 5] and [Ru(C2O4)(η6-p-cymene){κP-Ph2PO(2-
t
t
C6H4(SiMe2 Bu))}], 6, were obtained in 83–98% yield from Ru(II) arene precursors by three different syn-
t
thetic strategies. The unprecedented phosphine Ph2P(O(2-C6H4SiMe2 Bu)) was synthesized in 86% yield
t
from 2-C6H4Br(OSiMe2 Bu) and Ph2PCl, via intramolecular oxygen to carbon 1,3 migration of the silyl
group (retro-Brook rearrangement). All the complexes were fully characterized by analytical and spectro-
scopic methods, and by single crystal X-ray diffraction in the cases of 3, 4, 5 and 6. Complexes 1–6 and
the model compounds [RuCl2(η6-p-cymene)(κP-PPh3)] (Ru-PPh3) and [Ru(C2O4)(η6-p-cymene)(κP-PPh3)]
(Ru-PPh3-O) underwent slow degradation in chloroform solutions upon air contact; the mixed valence
complex [(η6-p-cymene)Ru(μ-Cl)3RuCl2(κP-PPh3)], 7, was isolated from a solution of Ru-PPh3 in CHCl3,
and X-ray identified. The antiproliferative activity of 1–6 and Ru-PPh3, Ru-PPh3-O and [RuCl2(η6-p-
cymene)(κP-PTA)] (RAPTA-C) was assessed towards the triple-negative breast cancer cell line
MDA-MB-231, the ovarian carcinoma cell line A2780 and human skin fibroblasts (HSF). Complexes 1, 2, 5
and 6 displayed IC50 values significantly lower than that of cisplatin, with 2 providing a more potent cyto-
toxic effect on MDA-MB-231 and A2780 cancer cells compared to the noncancerous cell line (HSF). The
stability of all complexes in DMSO/water solution was elucidated by NMR and conductivity measure-
ments, and in particular 35Cl NMR spectroscopy was helpful to check the possible chloride dissociation.
The stability studies suggest that the cytotoxic activity in vitro of the compounds is mainly ascribable to
Ru(II) species still bound to the phosphorus ligand.
Received 12th September 2017,
Accepted 15th November 2017
DOI: 10.1039/c7dt03385k
rsc.li/dalton
in clinical treatments.2 Ruthenium compounds have been
intensively investigated in this setting,3 and especially Ru(II)
Introduction
Cancer represents the first cause of death in economically arene complexes have aroused great interest.4 In particular,
developed countries, and the second in developing countries.1 [RuCl2(η6-arene)(κP-PTA)] (RAPTA complexes),5 containing the
A huge effort of scientific research is aimed to obtain new amphiphilic phosphorus ligand 1,3,5-triaza-7-phosphatricyclo
effective and selective metal drugs in view of overcoming the [3.3.1.1]decane
(PTA),
and
[RuCl(η6-arene)(κ2N-
severe toxicity and acquired resistance issues associated with NH2CH2CH2NH2)][PF6],6 containing a bidentate ethylene-1,2-
the use of platinum chemotherapics, which are currently used diamine ligand, have emerged as promising anticancer agents
and are pointing to clinical trials (Fig. 1).7 With specific refer-
aDipartimento di Chimica e Chimica Industriale, Università di Pisa, Via G. Moruzzi
13, I-56124 Pisa, Italy. E-mail: fabio.marchetti1974@unipi.it
bDipartimento di Chimica Industriale “Toso Montanari”, Università di Bologna,
Viale Risorgimento 4, I-40136 Bologna, Italy
cDipartimento di Scienze del Farmaco, Università degli Studi di Padova,
Via Marzolo 5, 35131 Padova, Italy
†Electronic supplementary information (ESI) available: CCDC 1571529 (3),
1571530 (4), 1571531 (5) and 1571532 (6) contain the supplementary crystallo-
graphic data for the X-ray studies reported in this paper. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/c7dt03385k
Fig. 1 Ruthenium(II) arene anticancer complexes.
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ence to RAPTA complexes, these display excellent antimeta- have been considered in the present work in view of the
static and antiangiogenic behaviour in vivo and are able to peculiar properties that organosilicon compounds may supply
reduce the growth of certain primary tumours.8 These features to pharmaceuticals.20
have stimulated the search for the anticancer potential of
On account of the fact that platinum drugs are still the only
many other similar compounds, and RAPTA-analogues bearing viable options for the treatment of triple negative breast
various alkyl/aryl-phosphine ligands in the place of PTA have cancers and ovarian carcinomas, the new complexes have been
been screened for their activity.9 Noteworthy results have been assessed for their in vitro antiproliferative activity towards
achieved by Dyson and co-workers with perfluoro-substituted MDA-MB-231 (triple negative breast cancer) and A2780
trialkylphosphines, supplying thermotropic behaviour to the (ovarian carcinoma) cancer cell lines, and human skin fibro-
respective complexes,10 and carbohydrate-modified 3,5,6-bicy- blasts (HSF) as non-transformed primary cell line.
clophosphites, the resulting complexes showing a certain
degree of selectivity against several cancer cells compared to
non tumorigenic cell line.11
Results and discussion
Synthesis and characterization of compounds
On the other hand, the introduction of triphenylphosphine
as a ligand, being considerably more hydrophobic than PTA,
usually leads to higher levels of cytotoxicity in vitro, although The reactions of the dinuclear compound [RuCl2(η6-p-
at the expense of selectivity in some cases.12 For instance, cymene)]2 with Ph2P(4-C6H4OSiMe2 Bu), Ph2P(4-C6H4Br), Ph2P
t
[RuCl(η6-p-cymene)(py*)(PPh3)]+, containing a substituted pyri- (OPh) and Ph2P(O(2-C6H4SiMe2 Bu)), respectively, were con-
t
dine ligand (py*), exhibited activity against human leukemia ducted in chlorinated solvents at ambient temperature or
tumour cell line comparable to that obtained with cisplatin.13 above, and afforded the novel complexes 1–2 and 4–5, in good
Moreover, Hartinger and co-workers recently reported that the to excellent yields (Scheme 1b). The phosphine Ph2P(4-
t
incorporation of PPh3 (or PTA) in Ru(II) arene complexes con- C6H4OSiMe2 Bu) is an intermediate product along the con-
taining a bidentate oxygen co-ligand (3-hydroxy-4-pyridone or venient synthesis of Ph2P(4-C6H4OH), the tert-butyl dimethyl
3-hydroxy-4-pyrone) determined a dramatic increase of the silyl moiety being
a
protecting group.16a,21 Ph2P(O(2-
t
cytotoxic activity on different cell lines,14 and a similar C6H4SiMe2 Bu)) is a unprecedented compound, and was syn-
outcome was observed for [Ru(η6-benzene)(κN-letrozole) thesized in 86% yield from the aryl-silylether 2-C6H4Br
(PPh3)][BF4] vs. [Ru(η6-benzene)(κN-letrozole)2][BF4].15 It has (OSiMe2 Bu) and n-BuLi/Ph2PCl via [1,3] retro-Brook rearrange-
t
been demonstrated that the presence of a triphenylphosphine ment (Scheme 1d).22
ligand is important also to facilitate the binding of the Ru
complex to DNA and then distort its secondary and tertiary tion of [RuCl2(η6-p-cymene){κP-Ph2P(4-C6H4OH)}] with dichlor-
structure.13
oacetic acid (a bioactive molecule, vide infra) through EDCI/
Complex 3 was obtained in 83% yield by direct esterifica-
The investigation on Ru(II) arene compounds bearing sub- DMAP protocol (Scheme 1a). This procedure, negating the
stituted triphenylphosphine ligands has been limitedly devel- necessity of protecting strategies towards the labile Ru–Cl
oped. More precisely, Ph2P(4-C6H4CO2H) and Ph2P(4-C6H4OH) bonds, is convenient on account of the fact it does not require
have been used to incorporate a variety of bioactive carboxylic the manipulation of non coordinated, air sensitive Ph2P(4-
acids within the Ru(II) para-cymene scaffold, through esterifi- C6H4OH).16a Complex 6, differing from 5 in that two chloride
cation reactions.16 The resulting [RuCl2(η6-p-cymene)(κP- ligands are replaced with an oxalate, was straightforwardly pre-
Ph2PArBIO)] complexes display variable activity towards A2780 pared from [Ru(C2O4)(η6-p-cymene)(H2O)] and Ph2P(O(2-
t
and A2780cisR cancer cell lines, the degree of activity being C6H4SiMe2 Bu)) (Scheme 1c).
significantly influenced by the nature of the bioactive
fragment.
All the complexes 1–6 and the ligand Ph2P(O(2-
C6H4SiMe2 Bu)) were characterized by analytical methods, IR
t
Herein, we present the synthesis and the full characteriz- and NMR spectroscopy (see Table 1S provided as ESI†). In the
ation of six new Ru(II)-p-cymene complexes with differently IR spectrum of 3 (solid state), the ester group manifests itself
mono-substituted triphenylphosphine or phenoxydiphenyl- with a broad band around 1770 cm−1, while the carbonyl func-
phosphine ligands, including a dichloroacetic acid functiona- tions of 6 have been detected at 1666–1697 cm−1. In 4–5 and
t
lized triphenylphosphine, a silyl ether substituted triphenyl- Ph2P(O(2-C6H4SiMe2 Bu)), a strong absorption in the range
phosphine and a unprecedented silyl phenoxydiphenylpho- 870–892 cm−1 has been attributed to the stretching vibration
sphine. Dichloroacetic acid is a commercially available inhibi- of the P–O moiety. The dichloroacetate group in 3 is featured
tor of pyruvate dehydrogenase kinase, able to enhance cellular also by the 13C NMR resonances occurring at 162.5 (CvO) and
apoptosis.17 The incorporation of dichloroacetic acid in plati- 64.2 (CHCl2) ppm. The 31P{1H} NMR spectra of 1–3 display a
num based drugs was reported to provide several favourable typical singlet at ca. 24 ppm; the 31P NMR resonance related to
effects,18 and especially the Pt(IV) compound called “mitapla- the aryloxy-diphenylphosphine compounds 4, 5 and Ph2P(O(2-
t
tin” displayed excellent characteristics of cytotoxicity and C6H4SiMe2 Bu)), in CDCl3 solution, falls at significantly lower
selectivity.19 To the best of our knowledge, dichloroacetic acid fields. The phosphorus nucleus of Ph2P(O(2-C6H4SiMe2 Bu))
t
has not been tethered to ruthenium complexes for medicinal resonates at 108.3 ppm in the uncoordinated molecule, and at
applications hitherto. Silicon-containing phosphine ligands 120.3 ppm in 5. The 29Si NMR spectra of 5 and Ph2P(O(2-
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Scheme 1 Synthetic routes to ruthenium triphenylphosphine complexes (a–c) and Ph2P(O(2-C6H4SiMe2tBu)) ligand (d). Yields are given in
parentheses.
Fig. 2 Molecular
structure
of
[RuCl2(η6-p-cymene){κP-Ph2P(4-
C6H4OCOCHCl2)}], 3. Displacement ellipsoids are at the 30% probability
level. H-atoms have been omitted for clarity.
Fig. 3 Molecular structure of [RuCl2(η6-p-cymene){κP-Ph2P(OPh)}], 4.
Displacement ellipsoids are at the 30% probability level. H-atoms have
been omitted for clarity.
t
C6H4SiMe2 Bu)), comprising a carbon-bound silicon atom,
consist of a singlet at approximately 3 ppm; on the other hand,
the resonance related to the oxygen-bound silicon nucleus
falls at 21.7 ppm in 1.
ESI (Tables 12S–15S†). Compounds 3–6 comprise the expected
Crystals suitable to X-ray analysis were collected for 3, 4, 5 three-leg piano-stool geometry typical of other Ru(II)-arene
and 6; views of the ORTEP molecular structures are shown in compounds,23 and the bonding parameters around the Ru(II)
Fig. 2–5, while relevant bonding parameters are provided as centres are similar to those reported for related [RuCl2(p-
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Scheme 2 Degradation route of Ru(II)-arene triphenylphosphine
complex in chloroform solution upon air contact.
the reaction solution, and then identified by X-ray diffraction
(Scheme 2). Complexes belonging to the family [Ru(μ-Cl)3(η6-p-
cymene)RuCl2(κP-PR3)] (R = Ph, Cy, n-Bu) were previously
reported as prepared from [RuCl2(η6-p-cymene)]2 and the
appropriate phosphine, upon ethylene addition.26
Instability of Ru(η6-arene) derivatives in chlorinated sol-
vents is documented in the literature27 and possibly associated
to the decomposition of the solvent into HCl and carbon rad-
icals.28 Interestingly, Ru(II) complexes have been used as cata-
lysts for the photodegradation of chloroform and other chlori-
nated compounds.28b,29
Fig. 4 Molecular structure of [RuCl2(η6-p-cymene){κP-Ph2PO(2-
C6H4(SiMe2tBu))}], 5. Displacement ellipsoids are at the 30% probability
level. H-atoms have been omitted for clarity.
Although Ru(II) arene complexes with phosphine ligands
may undergo, in chloroform in contact with air, degradation
routes including Ru(II) to Ru(III) oxidation, the same com-
pounds are not expected to be engaged in redox processes in
the course of the in vitro cytotoxicity analyses (vide infra). In
fact, former electrochemical studies on a variety of Ru(II) arene
compounds did not evidence redox activity within a biologi-
cally relevant range of potentials.16b,30
In vitro cytotoxicity studies
The ability of the newly prepared compounds 1–6 to inhibit
cell growth was evaluated against the triple-negative breast
cancer cell line MDA-MB-231 (Fig. 6A and Table 1), and the
human ovarian carcinoma cell line A2780 (Fig. 6B and
Table 1). The analyses were extended to [RuCl2(η6-p-cymene)
(κP-PPh3)] (Ru-PPh3), [Ru(C2O4)(η6-p-cymene)(κP-PPh3)] (Ru-
PPh3-O) and [RuCl2(η6-p-cymene)(κP-PTA)] (RAPTA-C), as refer-
ence compounds. Cells were incubated with increasing con-
centration of compounds dissolved in DMSO.
Comparable cytotoxic effects were ascertained for the
respective compounds on the two tumoral cell lines. In par-
ticular, 1, 2, 5 and 6 showed a considerable cytotoxic activity,
although a tendency to stimulate the growth of MDA-MB-231
Fig. 5 Molecular structure of [Ru(C2O4)(η6-p-cymene){κP-Ph2PO(2-
C6H4(SiMe2tBu))}], 6. Displacement ellipsoids are at the 50% probability
level. H-atoms have been omitted for clarity.
cymene)(phosphine)] and [Ru(C2O4)(p-cymene)(phosphine)] cells was observed for 2 and 5 at low concentrations (see
structures.24
Fig. 6). The IC50 values are substantially lower than the values
Complexes 1–6 are air stable and well soluble in chlorinated obtained with cisplatin on the same cell lines under the same
solvents, but not indefinitely stable in chloroform upon air experimental conditions. Otherwise, 3, 4, Ru-PPh3, Ru-PPh3-O
contact. According to IR/NMR experiments (see ESI† for and RAPTA-C did not reduce the cell viability by more than
details), 1 and 6 in chloroform completely degraded after two 50% at the maximal tested concentration (50 µM). It should be
weeks, the red/yellow solutions progressively turning to green noted that RAPTA-C was previously found to be non cytotoxic
and affording a complicated mixture of species including against a panel of cell lines.5
p-cymene. A similar behaviour was found for [RuCl2(η6-p-
These results clearly indicate that the mono-substitution of
cymene)(κP-PPh3)] (Ru-PPh3); in this case, some green crystals one phenyl ring, in PPh3 or PPh2(OPh), can lead to a signifi-
of the dinuclear, mixed valence RuII-RuIII compound [Ru cant increase in the cytotoxicity of the resulting complexes
(μ-Cl)3(η6-p-cymene)RuCl2(κP-PPh3)], 7,25 were isolated from (compare Ru-PPh3 with 1–2, and 4 with 5–6). The relatively
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Fig. 6 Cytotoxic activity of 1–6, Ru-PPh3, Ru-PPh3-O and RAPTA-C towards: (A) MDA-MB-231 cell line; (B) A2780 cell line. Each bar represents the
mean SD of three independent experiments. Inhibitors versus control: *P < 0.05; **P < 0.01; ***P < 0.001.
pyran rings, was previously indicated as an enhancer of cyto-
toxicity against HL60 human leukemia cells and MCF7 breast
cancer cells in vitro, due to its lipophilicity favouring the cellu-
lar uptake of the drug.31
Table 1 IC50 values (μM) determined for 1–6, Ru-PPh3, Ru-PPh3-O,
RAPTA-C and cisplatin, on human breast (MDA-MB-231) cancer cells
and human skin fibroblasts (HSF) cells at 48 h. Values are given as the
mean SD. N/T not tested
It is possible that the drop of activity observed on moving
from 1–2 to 3 is related to some unexpected effect associated
with dichloroacetic acid,19 which may be released from 3
inside the cells by means of intracellular esterases.32
Compounds homologous to 3, [RuCl2(η6-p-cymene)(κP-
Ph2PArBIO)], derivatized with bioactive carboxylic acids
different from CHCl2CO2H, displayed variable cytotoxic activity
towards A2780 and A2780cisR cancer cell lines (see
Introduction) and, in one case (derivatization with
Indomethacin), not appreciable activity.16a
Compound
MDA-MB-231
A2780
HSF
1
2
3
4
5
6
9.2 1.1
13.9 1.2
>50
3.6 0.6
7.0 1.6
>50
7.2 1.0
31.9 1.1
N/T
>50
>50
N/T
29.1 1.4
14.4 1.2
>50
>50
>50
9.4 2.6
7.6 1.2
>50
>50
>50
7.8 1.1
12.5 1.0
N/T
N/T
N/T
Ru-PPh3
Ru-PPh3-O
RAPTA-C
Cisplatin
59.4(ref. 33)
31.5
N/T
We further explored the possible effect of 3 on cell viability
by analysing the cell cycle progression after 24 h incubation of
high activity exhibited by 1, 5 and 6 could be somehow related A2780 cells (Table 2). We detected a lowering in the percentage
to the presence of the tert-butyl dimethyl silyl substituent. As a of cells in S and G2/M phase compared to untreated cells,
matter of fact, tert-butyl dimethyl silyl, tethered to tetrahydro- suggesting a possible interference of 3 with the progression
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tion) display sharp 35Cl resonances at 0.0 ppm (reference) and
−22.7 ppm, respectively (see Experimental). In agreement with
literature reports,34 we clearly detected fast chloride/water
exchange on RAPTA-C in D2O solution by 35Cl NMR
(Scheme 3a). On the other hand, 35Cl NMR indicated no Cl−/
solvent exchange when RAPTA-C was dissolved in CD3OD, and
even in CD3OD/D2O 9 : 1 and DMSO-d6/D2O 9 : 1 mixtures
(Scheme 3b). Under these conditions, a single set of 1H and
31P NMR resonances was observed, in accordance with the
RAPTA-C structure. Conversely, when one equivalent of AgNO3
was added to the CD3OD/D2O 9 : 1 solution (Cl− abstraction,
Scheme 3c), the 1H NMR pattern was in agreement with the
structure [RuCl(η6-p-cymene)(Solv)(PTA)]+ (stereogenic Ru-
centre), and a marked increase in molar conductivity was
measured too (from 35 to 81 S cm2 mol−1).
Table 2 Effect of complex 3 on cell cycle progression of A2780 cell
line. Cells were incubated for 24 h either in the presence or in the
absence of 3 (50 µM). At the end of this incubation period, the cell cycle
analysis was performed
Compound
G0/G1 phase (%)
S phase (%)
G2/M phase (%)
Control
3
T-Test
60.5
78.9
p < 0.001
20.1
4.5
p < 0.001
19.4
16.7
P = 0.04
from G1 to S phase. No sub G0 cells were observed, in agree-
ment with the relatively low cytotoxicity exhibited by the
complex and the absence of induction of apoptosis, under the
employed experimental conditions.
In order to assess the possible selectivity of the more active
compounds (1, 2, 5 and 6) towards cancer cells rather than
non transformed cells, we extended the analysis of the antipro-
liferative activity to human skin fibroblasts (HSF). Complexes
1, 5 and 6 are not endowed with cancer cell selectivity, i.e. they
are cytotoxic also to the HSF. Conversely, a moderate selectivity
has been observed in the case of the Br-functionalized
complex 2 [selectivity indexes are 2.3 (HSF/MDA-MB-231) and
4.6 (HSF/A2780)]: according to this result, the inclusion of
Ph2P(4-C6H4Br) in ruthenium arene compounds might rep-
resent a privileged choice with respect to the use of PPh3 (see
Introduction).
Due to limited solubility in water, stability studies on
[RuCl2(η6-p-cymene)]2, Ru-PPh3, Ru-PPh3-O and 1–6 were
carried out in DMSO/water 9 : 1 solutions. An overview of the
different NMR identified species after 72 h is shown in
Scheme 4, while the fraction of the starting material detected
after 0, 24 and 48 h is given in Table 3. Analogously to what
found for RAPTA-C in DMSO-d6/D2O 9 : 1, 35Cl NMR experi-
ments conducted on freshly prepared solutions suggested to
rule out fast chloride dissociation from 1–3, Ru-PPh3 and
1
[RuCl2(η6-p-cymene)]2.35 Coherently, the respective H and 31P
spectra, displaying a single set of resonances for the p-cymene
ligand (Cs symmetry), did not modify upon addition of 0.15 M
NaCl. Moreover, molar conductivity of the DMSO : water 9 : 1
solutions was significantly lower (≈20–25 S cm2 mol−1) than
Chloride/solvent exchange on model compounds and stability
of 1–6 in aqueous solution
expected for a 1 : 1 electrolyte in this solvent (≈50 S cm2 mol−1
,
see ESI†). The set of signals (1H, 31P) belonging to the starting
Ru-dichlorido complex persisted throughout the stability
experiments (37 °C, 72 h), and the only other set of observed
We performed NMR experiments (1H, 31P, 35Cl) and conduc-
tivity measurements to assess the stability of 1–6 and model
compounds under pseudo-physiological conditions (see also
Experimental section and ESI†). In particular, 35Cl NMR spec-
troscopy revealed to be a helpful tool to detect the solvolysis of
the Ru–Cl bonds. 35Cl is a quadrupolar nucleus, thus co-
valently bound chlorines give raise to broad resonances in the
35Cl NMR spectrum and are difficult to observe in a reasonable
time. Otherwise, the chloride anion can be readily recognized:
for instance, NaCl (D2O solution) and [Et3NH]Cl (CD3OD solu-
1
Ru-arene H signals was due to the formation of [RuCl2(η6-p-
cymene)(DMSO)] (see Scheme 4).
The dimeric compound [RuCl2(η6-p-cymene)]2 was cleanly
cleaved into [RuCl2(η6-p-cymene)(DMSO)], S1, in DMSO/water
9 : 1 as well as in pure DMSO.36 It should be mentioned that a
sharp 35Cl signal was previously reported for [RuCl2(η6-C6Et6)]2
in methanol solution at −50 °C, but not at ambient tempera-
Scheme 3 Behaviour of RAPTA-C in aqueous solutions; presence/absence of Cl− ascertained by 35Cl NMR spectroscopy.
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Scheme 4 Compounds detected in DMSO-d6/D2O solutions of 1–6, [RuCl2(η6-p-cymene)]2, Ru-PPh3, Ru-PPh3-O and RAPTA-C maintained at
37 °C for 72 h.
ture. Such phenomenon was attributed to the existence of the phosphine release was significantly inhibited; the solvato-
equilibrium [RuCl2(η6-C6Et6)]2 ⇄ [Ru2Cl3(η6-C6Et6)]Cl, shifting species [Ru(C2O4)(η6-p-cymene)(DMSO)], S2, was formed from
in favour of the ionic species at low temperature, rather than 6 in low amount only after 72 h.
to a possible fast chloride/solvent exchange on the NMR
timescale.27b
It seems reasonable that, in accordance with the behaviour
of RAPTA-C (Scheme 3) and some water-soluble [RuCl2(η6-p-
Progressive phosphine/DMSO exchange was detected for cymene)(PAr3)] complexes,37 1–6 may undergo Cl−/solvent
1–5 and Ru-PPh3 during 72 h. In 6 and Ru-PPh3-O, lacking of exchange more easily in diluted (micromolar) solutions with a
chloride ligands but containing a bis-carboxylate group, the higher relative content of water, as is the case of cell culture.
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35Cl NMR spectroscopy is helpful to study the possible solvoly-
sis of Ru–Cl bonds, which represents a largely accepted mecha-
nism for drug activation.
Table 3 Fraction of complexes 1–6, Ru-PPh3, Ru-PPh3-O and
RAPTA-C in DMSO-d6/D2O at 37 °C after 24 h and 48 h; % values are
based on 1H NMR spectroscopy (dimethyl sulfone as internal standard)
% (1H NMR vs. internal standard)
Compound
t = 0
t = 23.5 h
t = 48 h
Experimental section
General experimental details
1
2
3
4
5
6
97
97
95
94
96
100
98
99
70
67
56
25
33
75
77
77
90a
24
45
37
14
6
56
56
74
63b
RuCl3·3H2O (99.9%) was purchased from Strem, while all the
other reactants were obtained from Alfa Aesar, Sigma Aldrich
or TCI Europe, and were of the highest purity available. The
following reagents were stored under nitrogen or argon as
received: 2-bromophenol (under protection from the light), tri-
ethylamine, tert-butyldimethylsilyl chloride (TBDMSCl), butyl
lithium (2.5 M solution in hexanes, 4 °C), chlorodiphenyl-
phosphine, phenoxydiphenylphosphine (4 °C), PCl5, oxalyl
chloride (4 °C), ethyl(diisopropylamino)carboxydiimide hydro-
chloride (EDCI·HCl, −20 °C). Dichloroacetic acid was distilled
under reduced pressure, dried under vacuum over P2O5 and
stored under nitrogen. Ph2P(4-C6H4Br),41 [Ru(C2O4)(η6-p-
cymene)(H2O)],24c [RuCl2(η6-p-cymene)(κP-PPh3)],42 and [Ru
(C2O4)(η6-p-cymene)(κP-PPh3)]43 were prepared according to
modified literature procedures (see ESI†). [RuCl2(η6-p-
Ru-PPh3
Ru-PPh3-O
RAPTA-C
100
a t = 16.5 h. b t = 40.5 h.
This process is expected to afford cationic species, basically
[RuCl(η6-p-cymene)(H2O)(PPh2R)]+, whereby the solvolysis of
the phosphine ligand should be inhibited with respect to what
observed on the parent neutral derivatives, [RuCl2(η6-p-
cymene)(PPh2R)], in DMSO : D2O 9 : 1 (Scheme 4). In other
terms, the degree of phosphine dissociation recognized for the
complexes in DMSO/water is probably overestimated respect to
the real situation of the in vitro trials.
The disruptive effect of DMSO is likely to be responsible
also for the observed, partial release of the p-cymene ligand
from 1–6, Ru-PPh3 and Ru-PPh3-O (Scheme 4).38 Indeed, arene
dissociation from Ru(II) complexes in water is generally not
observed39 unless under UV irradiation.40
cymene)]2,44 [RuCl2(η6-p-cymene)(PTA)] (RAPTA-C),45 Ph2P(4-
t
C6H4OSiMe2 Bu)16a
and
[RuCl2(η6-p-cymene)(Ph2P(4-
C6H4OH))]16a were prepared according to the literature. All
reactions, except the preparation of [Ru(C2O4)(η6-p-cymene)
(H2O)], were carried out under a nitrogen atmosphere using
standard Schlenk techniques and solvents distilled from
appropriate drying agents. Once isolated, o-C6H4Br
t
t
t
Accounting all of these considerations, the cytotoxic activity
of 1–6 is plausibly related for the most part to non dissociated
ruthenium-phosphine species.
(OSiMe2 Bu), Ph2PO(2-C6H4(SiMe2 Bu)), o-C6H4(OH)(SiMe2 Bu)
t
(under protection from the light), Ph2P(4-C6H4OSiMe2 Bu) and
Ph2P(4-C6H4Br) were stored under nitrogen, all the other pro-
ducts being air stable. Compound [Ru(C2O4)(η6-p-cymene)
(H2O)] was either used some days after its preparation or
stored under nitrogen for longer periods. Silica gel (Merck,
70–230 mesh) was dried at 150 °C overnight and stored under
Conclusions
Since a variety of ruthenium(II) arene complexes containing a nitrogen. NMR spectra were recorded at 298 K on a Bruker
phosphine ligand have aroused interest for their possible anti- Avance II DRX400 instrument equipped with a BBFO broad-
cancer activity, we have prepared and characterized a series of band probe. Chemical shifts (expressed in parts per million)
new Ru(II) arene complexes containing a triphenylphosphine are referenced to the residual solvent peaks46 (1H, 13C) or to
or phenoxydiphenylphosphine ligand, variably mono-substi- external standard (31P to 85% H3PO4; 29Si to TMS, 35Cl to 1 M
1
tuted at one phenyl ring. In general, the substitution leads to NaCl in D2O). Spectra were assigned with the assistance of H
an increase of the cytotoxic activity of the complexes {31P}, DEPT-135 spectra and 1H–1H (COSY), 1H–13C (gs-HSQC
(MDA-MB-231 and A2780 cancer cell lines), some of them dis- and gs-HMBC) correlation experiments.47 Infrared spectra of
playing IC50 values much lower than those related to cisplatin. solid samples were recorded on a PerkinElmer Spectrum One
A moderate level of selectivity towards cancer cells respect to FT-IR spectrometer, equipped with a UATR sampling acces-
non tumorous cells has been observed with (4-bromophenyl) sory. Infrared spectra of CH2Cl2 solutions were recorded on a
diphenylphosphine, whose use might become convenient PerkinElmer Spectrum 100 FT-IR spectrometer with a CaF2
when the synthetic design of antitumoral ruthenium arene liquid transmission cell. UV-Vis spectra were recorded on a
compounds includes the incorporation of
a triphenyl- Ultraspec 2100 Pro spectrophotometer with 0.1 cm quartz cuv-
phosphine moiety. According to stability studies carried out in ettes. IR and UV-Vis spectra were processed with Spectragryph
aqueous solutions, we presume that the in vitro cytotoxic software.48 Carbon, hydrogen and nitrogen analysis was per-
activity of the compounds is largely ascribable to phosphine- formed on a Carlo Erba mod. 1106 instrument. Melting/
bound Ru(II) species. Although the cross combination with decomposition temperatures were determined on a STMP3
other techniques seems needed for conclusive information, Stuart scientific instrument with
a capillary apparatus.
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Conductivity measurements49 were carried out at 21 °C using
an XS COND 8 instrument (cell constant = 1.0 cm−1).
Synthesis of new compounds
Synthesis
of
[RuCl2(η6-p-cymene){κP-Ph2P(4-
t
C6H4OSiMe2 Bu)}], 1. In a 25 mL Schlenk tube, [RuCl2(η6-p-
t
Chart 2 Structure of 2 (numbering refers to carbon atoms).
cymene)]2 (254 mg, 0.415 mmol) and Ph2P(4-C6H4OSiMe2 Bu)
(389 mg, 0.991 mmol) were dissolved in CH2Cl2 (10 mL). The
resulting deep red solution was stirred at ambient temperature
for 7.5 hours and the formation of a ruthenium-coordinated
phosphine compound was assessed by TLC, 29Si and 31P NMR.
Therefore volatiles were removed under vacuum and the
residue was suspended in pentane (20 mL). The suspension
was cooled to 0 °C and filtered; the resulting orange-brown
solid was washed with a small volume of cold pentane and
dried under vacuum (45 °C) (Chart 1). Yield: 469 mg, 81%. The
title compound is soluble in DMSO, EtOH, CH2Cl2 and Et2O,
less soluble in pentane/petroleum ether and insoluble in H2O.
Anal. calcd for C34H43Cl2OPRuSi: C, 58.44; H, 6.20; Cl, 10.15.
hexane under vigorous stirring caused the precipitation of the
title compound as a red solid. The suspension was filtered; the
solid was washed with hexane and then with a small volume of
hexane/Et2O mixture (1 : 1 v/v ratio), and finally dried under
vacuum (40 °C) (Chart 2). Yield: 125 mg, 91%. 2 was obtained in
admixture with minor products when the reaction was performed
at ambient temperature. The title compound is soluble in DMSO,
acetone and chlorinated solvents, less soluble in MeOH,
poorly soluble in Et2O and insoluble in H2O and hexane. Anal.
calcd for C28H28BrCl2PRu: C, 51.95; H, 4.36; Cl, 10.95. Found:
C, 52.06; H, 4.23; Cl, 10.84. IR (solid state): ˜ν/cm−1 = 3049w,
2960w, 2925w, 2870w, 1572w, 1556w, 1480 m, 1468m-sh,
1434 m, 1384 m, 1325w, 1277w, 1189 m, 1161w, 1117w,
1094 m, 1070 m, 1058m-sh, 1028w, 1009 m, 951w, 926w, 896w,
869w, 811 m, 799m-sh, 752 m, 725s, 698s. 1H NMR (CDCl3):
Found: C, 58.16; H, 6.25; Cl, 10.02. IR (solid state): ˜ν/cm−1
=
3055w, 2957w, 2929w, 2896w, 2885w, 2857w, 1591 m, 1497s,
1482m-sh, 1471 m, 1435 m, 1401w, 1388w, 1361w, 1257s-br,
1176s (νO–Ar), 1093s, 1057w, 1029w, 1005w, 907s, 838s, 824s-sh,
806s, 781s, 744 m, 719w, 693s, 673m-sh. 1H NMR (CDCl3):
3
3
δ/ppm = 7.83–7.76 (m, 4H, C9-H), 7.70 (pseudo-t, JHH = JHP
=
3
3
δ/ppm = 7.83–7.76 (m, 4H, C9-H), 7.70 (pseudo-t, JHH = JHP
=
9 Hz, 2H, C13-H), 7.40–7.32 (m, 6H, C10-H + C11-H), 6.82
(d, 3JHH = 7.3 Hz, 2H, C14-H), 5.19 (d, 3JHH = 5.8 Hz, 2H, C4-H),
4.98 (d, 3JHH = 5.5 Hz, 2H, C3-H), 2.87 (hept, 3JHH = 6.1 Hz, 1H,
8.9 Hz, 2H, C13-H), 7.47–7.34 (m, 8H, C10-H + C11-H +
3
3
C14-H), 5.21 (d, JHH = 4.9 Hz, 2H, C3-H/C4-H), 4.99 (d, JHH
=
3
4.4 Hz, 2H, C3-H/C4-H), 2.84 (hept, JHH = 6.5 Hz, 1H, C6-H),
3
C6-H), 1.86 (s, 3H, C1-H), 1.10 (d, JHH = 6.9 Hz, 6H, C7-H),
1.85 (s, 3H, C1-H), 1.10 (d, JHH = 6.7 Hz, 6H, C7-H). 13C{1H}
3
0.97 (s, 9H, C18-H), 0.20 (s, 6H, C16-H). 13C{1H} NMR (CDCl3):
2
NMR (CDCl3): δ/ppm = 136.3 (d, JCP = 10 Hz, C13), 134.2 (d,
2
δ/ppm = 157.7 (C15), 136.4 (d, JCP = 11 Hz, C13), 134.5 (d,
2JCP = 10 Hz, C9), 133.8 (d, JCP = 45 Hz, C8), 132.3 (d, JCP
=
1
1
2
4
1JCP = 46 Hz, C8), 134.3 (d, JCP = 9 Hz, C9), 130.2 (d, JCP
=
3
4
46 Hz, C12), 131.0 (d, JCP = 10 Hz, C14), 130.6 (d, JCP = 2 Hz,
C11), 128.3 (d, JCP = 10 Hz, C10), 125.3 (d, JCP = 3 Hz, C15),
3
1
2 Hz, C11), 128.0 (d, JCP = 10 Hz, C10), 125.0 (d, JCP = 50 Hz,
C12), 119.7 (d, JCP = 11 Hz, C14), 111.1 (d, JCP = 3 Hz, C5),
3
4
3
2
2
2
111.5 (d, JCP = 3 Hz, C5), 96.3 (C2), 89.1 (d, JCP = 3 Hz,
2
2
96.0 (C2), 89.1 (d, JCP = 3 Hz, C3), 87.3 (d, JCP = 6 Hz, C4),
30.4 (C6), 25.7 (C17), 22.0 (C7), 18.3 (C18), 17.9 (C1), −4.5
(C16). 31P{1H} NMR (CDCl3): δ/ppm = 23.2. 29Si{1H} NMR
(CDCl3): δ/ppm = 21.7.
2
C3/C4), 87.4 (d, JCP = 5 Hz, C3/C4), 30.4 (C6), 22.0 (C7), 17.9
(C1). 31P{1H} NMR (CDCl3): δ/ppm = 24.4.
Synthesis of [RuCl2(η6-p-cymene){κP-Ph2P(4-C6H4OCOCHCl2)}],
3. In a 25 mL Schlenk tube, CH2Cl2 (6 mL), Cl2CHCO2H
(30 µL, 0.36 mmol), [RuCl2(η6-p-cymene){κP-Ph2P(4-C6H4OH)}]
(75 mg, 0.13 mmol), EDCI·HCl (97 mg, 0.51 mmol) and DMAP
(6 mg, 0.05 mmol) were introduced in the order given. The
resulting red solution was stirred at ambient temperature and
Synthesis of [RuCl2(η6-p-cymene){κP-Ph2P(4-C6H4Br)}], 2. In
a
25 mL Schlenk tube, [RuCl2(η6-p-cymene)]2 (65 mg,
0.106 mmol) and Ph2P(4-C6H4Br) (79 mg, 0.232 mmol) were
dissolved in CHCl3 (12 mL). The resulting red solution was
heated at reflux for 15 hours and the progress of reaction was
checked by TLC. The reaction mixture was cooled to ambient
temperature, volatiles were removed under vacuum and the
residue was dissolved in a small volume of CH2Cl2. Addition of
1
aliquots of the solution were taken for H and 31P NMR ana-
lysis. After 2.5 hours, volatiles were removed under vacuum.
Shortly afterwards, the residue was dissolved in a small
volume of CH2Cl2, the solution was diluted with EtOAc
(10 mL) and then extracted with water (3 × 20 mL). Volatiles
were removed under vacuum from the organic phase and the
residue was dissolved in a small volume of DCM/Et2O (1 : 1
v/v). Hexane addition under intense stirring caused the pre-
cipitation of the title compound as a red-brown powder. The
suspension was filtered and the solid was washed with hexane
and dried under vacuum (40 °C) (Chart 3). Yield: 75 mg, 83%.
Note: it is important for the workup to be performed immedi-
ately after the end of the reaction. The title compound is
Chart 1 Structure of 1 (numbering refers to carbon atoms).
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Chart 3 Structure of 3 (numbering refers to carbon atoms).
Chart 4 Structure of 4 (numbering refers to carbon atoms).
red solid was washed with petroleum ether and dried under
vacuum (40 °C) (Chart 4). Yield: 251 mg, 98%. The title com-
pound is soluble in DMSO and chlorinated solvents, less soluble
in acetone and EtOH and insoluble in H2O and hydrocarbons.
Crystals suitable for X-ray analysis were obtained from CH2Cl2
solutions of 4 layered with either Et2O or hexane, and settled
aside at −20 °C. Anal. calcd for C28H29Cl2OPRu: C, 57.54; H,
5.00; Cl, 12.13. Found: C, 57.46; H, 4.92; Cl, 12.21. IR (solid
state): ˜ν/cm−1 = 3055w, 2965w, 2874w, 1589 m, 1491 m,
1481 m, 1435 m, 1389w, 1374w, 1288w, 1238w, 1208s (νO–Ar),
1185 m, 1174 m, 1158w, 1104m-sh, 1093 m, 1075w, 1056w,
1028w, 998w, 956w, 926w, 889s (νP–O), 859 m, 826w, 798w,
soluble in chlorinated solvents and DMSO, less soluble in
Et2O, insoluble in hexane and water. X-ray quality crystals of
3·acetone were obtained from an acetone solution of 3 layered
with heptane and settled aside at −20 °C. Anal. calcd for
C30H29Cl4O2PRu: C, 51.81; H, 4.20; Cl, 20.39. Found: C, 51.67;
H, 4.28; Cl, 20.26. IR (solid state): ˜ν/cm−1 = 3055w, 2962w,
2929w-sh, 2870w, 1785m-sh and 1768
m (νC=O), 1587w,
1493 m, 1483 m, 1471w, 1435 m, 1396w, 1386w, 1289 m,
1234 m, 1205 m, 1168s (νO–Ar), 1139 m, 1093 m, 1057w, 1028w,
1017 m, 999w, 938w, 858 m, 822 m, 798 m, 747 m, 695s. 1H
3
NMR (CDCl3): δ/ppm = 7.90 (pseudo-t, JHH
=
3JHP = 8.2 Hz,
3
2H, C13-H), 7.81 (pseudo-t, JHH
=
3JHP = 8.2 Hz, 4H, C9-H),
7.46–7.36 (m, 6H, C10-H + C11-H), 7.12 (d, JHH = 7.5 Hz, 2H,
1
3
764s, 750s, 727s, 708s, 697s, 689s. H NMR (CDCl3): δ/ppm =
3
8.04–8.00 (m, 4H, C9-H), 7.38–7.31 (m, 6H, C10-H and C11-H),
C14-H), 6.13 (s, 1H, C17-H), 5.20 (d, JHH = 4.2 Hz, 2H, C3-
3
3
7.31–7.22 (m, 4H, C13-H + C14-H), 7.04 (t, JHH = 7.0 Hz, 1H,
H/C4-H), 5.01 (d, JHH = 3.7 Hz, 2H, C3-H/C4-H), 2.81 (hept,
3
3JHH = 6.1 Hz, 1H, C6-H), 1.85 (s, 3H, C1-H), 1.09 (d, JHH = 6.1
3
C15-H), 5.26 (pseudo-q, JHH = 5.8 Hz, 4H, C3-H + C4-H), 2.54
3
Hz, 6H, C7-H). 13C{1H} NMR (CDCl3): δ/ppm = 162.5 (C16),
(hept, JHH = 6.6 Hz, 1H, C6-H), 1.50 (s, 3H, C1-H), 0.85 (d,
3JHH = 6.8 Hz, 6H, C7-H). 13C{1H} NMR (CDCl3): δ/ppm = 152.9
4
2
151.4 (d, JCP = 2 Hz, C15), 136.3 (d, JCP = 10 Hz, C13), 134.2
(d, 2JCP = 10 Hz, C9), 133.8 (d, 1JCP = 45 Hz, C8), 132.0 (d, 1JCP
(d, 2JCP = 5 Hz, C12), 136.2 (d, 1JCP = 48 Hz, C8), 132.1 (d, 2JCP
=
=
4
4
3
11 Hz, C9), 130.9 (d, JCP = 1 Hz, C11), 129.8 (C14), 128.0 (d,
47 Hz, C12), 130.6 (d, JCP = 1 Hz, C11), 128.3 (d, JCP = 10 Hz,
C10), 120.1 (d, 3JCP = 11 Hz, C14), 111.3 (C5), 96.2 (C2), 89.2 (d,
3
3JCP = 10 Hz, C10), 123.7 (C15), 120.8 (d, JCP = 6 Hz, C13),
110.5 (C5), 97.3 (C2), 91.4 (d, 2JCP = 4 Hz, C3/C4), 87.6 (d, 2JCP
=
2JCP = 2 Hz, C3/C4), 87.3 (d, JCP = 5 Hz, C3/C4), 64.2 (C17),
2
5.8 Hz, C3/C4), 30.1 (C6), 21.5 (C7), 17.3 (C1). 31P{1H} NMR
30.3 (C6), 21.9 (C7), 17.8 (C1). 31P{1H} NMR (CDCl3): δ/ppm =
24.0. Freshly-prepared solutions of 3 in CDCl3 and DMSO-d6
show broadening of NMR resonances, probably due to associ-
(CDCl3): δ/ppm = 113.7.
Synthesis and characterization of Ph2P(O(2-C6H4SiMe2 Bu)).
t
Step 1.50 In a 100 mL Schlenk tube, Et3N (1.8 mL, 13 mmol)
and TBDMSCl (1.88 g, 12.5 mmol) were added to a solution of
2-bromophenol (1.0 mL, 9.3 mmol) in CH2Cl2 (15 mL). The
pale yellow solution was stirred at ambient temperature for
4.5 hours under protection from the light and the progress of
reaction was monitored by TLC. The resulting pale yellow sus-
pension was extracted with H2O (3 × 20 mL), then the volatiles
were removed from the organic phase under vacuum (50 °C).
1
ation phenomena. A regular H spectrum can be obtained after
1
some hours at ambient temperature. H NMR (CD3OD): δ/ppm
= 7.91 (pseudo-t, 3JHH = 3JHP = 9.1 Hz, 2H, C13-H), 7.88–7.80 (m,
3
4H, C9-H), 7.52–7.36 (m, 6H, C10-H + C11-H), 7.19 (d, JHH
=
=
3
7.4 Hz, 2H, C14-H), 6.71 (s, 1H, C17-H), 5.32 (d, JHH
5.5 Hz, 2H, C3-H/C4-H), 5.22 (d, 3JHH = 4.9 Hz, 2H, C3-H/C4-H),
3
2.63 (hept, JHH = 7.1 Hz, 1H, C6-H), 1.88 (s, 3H, C1-H), 1.07
(d, JHH = 6.7 Hz, 6H, C7-H). 31P{1H} NMR (CD3OD): δ/ppm =
3
t
The product 2-C6H4(Br)(OSiMe2 Bu) was obtained as a pale
24.0.
yellow liquid. Yield: quantitative. 1H NMR (CDCl3): δ/ppm =
Solutions containing Cl2CHCOCl, freshly prepared from
dichloroacetic acid (see ESI†), reacted with [RuCl2(η6-p-
cymene)(Ph2P(4-C6H4OH))] and Et3N affording mixtures of pro-
ducts including 3.
3
4
3
7.63 (dd, JHH = 7.9, JHH = 1.6 Hz, 1H, C8-H), 7.28 (td, JHH
=
=
4
3
4
7.7, JHH = 1.6 Hz, 1H, C6-H), 6.99 (dd, JHH = 8.1, JHH
3
4
1.5 Hz, 1H, C5-H), 6.92 (td, JHH = 7.4, JHH = 1.4 Hz, 1H,
C7-H), 1.17 (s, 9H, C1-H), 0.37 (s, 6H, C3-H). 13C{1H} NMR
(CDCl3): δ/ppm = 152.6 (C4), 133.5 (C8), 128.3 (C6), 122.4 (C7),
120.4 (C5), 115.5 (C9), 26.0 (C1), 18.6 (C2), −4.0 (C3).
Synthesis of [RuCl2(η6-p-cymene)(κP-Ph2POPh)], 4. In a
25 mL Schlenk tube, [RuCl2(η6-p-cymene)]2 (133 mg,
0.218 mmol) and Ph2P(OPh) (145 mg, 0.521 mmol) were dis-
solved in CHCl3 (7 mL). The resulting red solution was stirred
at ambient temperature for 15 hours and the progress of reac-
tion was checked by TLC. Volatiles were removed under
vacuum and the residue was suspended in petroleum ether
(20 mL). The suspension was filtered and the resulting orange-
Step 2. In a 100 mL Schlenk tube, n-BuLi (3.8 mL of a 2.5 M
solution in hexanes, 9.5 mmol) was slowly added (15′) to a
t
colourless solution of 2-C6H4(Br)(OSiMe2 Bu) (2.70 g, 9.4 mmol)
in Et2O (20 mL), at 0 °C under vigorous stirring. The resulting
pale yellow solution was allowed to reach ambient temperature
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Chart 5 Structures of 2-C6H4(Br)(OSiMe2tBu) (left) and Ph2P(O(2-
C6H4SiMe2tBu)) (right) (numbering refers to carbon atoms).
Chart 6 Structure of 5 (numbering refers to carbon atoms).
and stirred for additional 1.5 hours. Therefore Ph2PCl 1435 m, 1427 m, 1378w, 1359w, 1322w, 1292w, 1273w, 1262w,
(1.75 mL, 9.5 mmol) was added dropwise to the solution along 1255w, 1185s (νO–Ar), 1130 m, 1096s, 1078 m, 1058w, 1037w,
five minutes time at 0 °C, and precipitation of a colourless 1006w, 975w, 936w, 909w, 885s (νP–O), 859w-sh, 835 m, 821s,
solid (LiCl) occurred. The resulting suspension was allowed to 807s, 776 m, 758s, 737s, 722w, 705m-sh, 696s, 687s-sh. 1H
3
3
reach ambient temperature and then stirred for additional NMR (CDCl3): δ/ppm = 7.92 (pseudo-t, JHH = JHP = 8 Hz, 4H,
3
3
20 hours. The reaction mixture was concentrated under C9-H), 7.62 (d, JHH = 8.3 Hz, 1H, C13-H), 7.52 (d, JHH = 7.6
reduced pressure, loaded on top of a silica column (h = 4 cm, Hz, 1H, C16-H), 7.39–7.28 (m, 6H, C10-H + C11-H), 7.19 (dt,
d = 3 cm) and eluted with hexane. The title compound was 3JHH = 7.7 Hz, 4JHH = 1.6 Hz, 1H, C14-H), 7.04 (t, 3JHH = 7.2 Hz,
3
3
obtained as a colourless solid after solvent removal under 1H, C15-H), 5.39 (d, JHH = 6.2 Hz, 2H, C4-H), 5.33 (d, JHH
=
vacuum (50 °C) (Chart 5). Yield: 3.12 g, 86%. Anal. calcd for 6.0 Hz, 2H, C3-H), 2.56 (hept, 3JHH = 6.9 Hz, 1H, C6-H), 1.67 (s,
3
C24H29OPSi: C, 73.43; H, 7.45. Found: C, 73.22; H, 7.56. IR (solid 3H, C1-H), 1.08 (s, 9H, C20-H), 0.91 (d, JHH = 7.0 Hz, 6H,
state): ˜ν/cm−1 = 3073w, 3058w, 3005w, 2950w, 2924 m, 2880w, C7-H), 0.40 (s, 6H, C18-H). 13C{1H} NMR (CDCl3): δ/ppm =
2
1
2852 m, 1584 m, 1564w, 1468s, 1432s, 1421m-sh, 1385w, 1360w, 158.2 (d, JCP = 4 Hz, C12), 137.5 (C16), 135.5 (d, JCP = 31 Hz,
2
4
1306w, 1268 m, 1258 m, 1242w, 1200s (νO–Ar), 1183m-sh, C8), 132.6 (d, JCP = 11 Hz, C9), 131.0 (C11), 130.9 (d, JCP
=
3
3
1161w-sh, 1125 m, 1094 m, 1075 m, 1041 m, 1026 m, 1009w, 2 Hz, C14), 127.7 (d, JCP = 11 Hz, C10), 126.0 (d, JCP = 7 Hz,
3
938w, 914w, 870s (νP–O), 848 m, 833s, 821s, 806s, 772s, 757s, C17), 122.6 (C15), 122.0 (d, JCP = 10 Hz, C13), 110.6 (C5), 97.2
1
744s, 731s, 693s, 683s-sh. H NMR (CDCl3): δ/ppm = 7.64–7.58 (C2), 91.1 (C3), 87.4 (C4), 30.3 (C6), 27.5 (C20), 21.7 (C7), 18.1
(m, 4H, C11-H), 7.46–7.42 (m, 7H, C5-H + C12-H + C13-H), 7.31 (C19), 18.0 (C1), −3.5 (C18). 31P{1H} NMR (CDCl3): δ/ppm =
3
4
3
(dt, JHH = 15.4 Hz, JHH = 1.6 Hz, 1H, C7-H), 7.10 (dd, JHH
=
120.3. 29Si{1H} NMR (CDCl3): δ/ppm = 3.0.
4
3
8.1 Hz, JHP = 2.6 Hz, 1H, C8-H), 7.06 (t, JHH = 7.3 Hz, 1H, C6-
Synthesis
of
[Ru(C2O4)(η6-p-cymene){κP-Ph2PO(2-
H), 0.81 (s, 9H, C1-H), 0.14 (s, 1H, C3-H). 13C{1H} NMR (CDCl3): C6H4(SiMe2 Bu))}], 6. In a 25 mL Schlenk tube, freshly pre-
t
δ/ppm = 162.3 (d, JCP = 9 Hz, C9), 140.0 (d, JCP = 17 Hz, C10), pared [Ru(C2O4)(η6-p-cymene)(H2O)] (114 mg, 0.334 mmol)
2
1
2
4
t
136.8 (C5), 131.7 (d, JCP = 23 Hz, C11), 130.8 (d, JCP = 2 Hz, and Ph2P(O(2-C6H4SiMe2 Bu)) (170 mg, 0.433 mmol) were dis-
3
3
C7), 130.0 (C13), 128.5 (d, JCP = 7 Hz, C12), 127.2 (d, JCP
=
solved in CH2Cl2 (5 mL). The reaction mixture was stirred at
3 Hz, C4), 121.7 (C6), 116.0 (d, 3JCP = 23 Hz, C8), 27.1 (C1), 17.7 ambient temperature, affording a yellow-orange solution in
(C2), −4.4 (C3). 31P{1H} NMR (CDCl3): δ/ppm = 108.3. 29Si{1H} 30′. After 18 hours, the progress of reaction was checked by
NMR (CDCl3): δ/ppm = 3.3.
Synthesis of
TLC and volatiles were removed under vacuum. The residue
[RuCl2(η6-p-cymene){κP-Ph2PO(2- was suspended in Et2O (20 mL) and the suspension was fil-
t
C6H4(SiMe2 Bu))}], 5. In a 25 mL Schlenk tube, [RuCl2(η6-p- tered. The resulting golden yellow solid was washed with Et2O
t
cymene)]2 (66 mg, 0.11 mmol) and Ph2P(O(2-C6H4SiMe2 Bu)) and dried under vacuum (40 °C) (Chart 7). Yield: 206 mg, 86%.
(111 mg, 0.283 mmol) were dissolved in CH2Cl2 (6 mL). The The title compound is soluble in DMSO, EtOH and chlorinated
resulting red solution was stirred at ambient temperature for solvents, insoluble in Et2O, hexane and H2O. X-ray quality crys-
14 hours and the progress of reaction was checked by TLC. tals of 6·hexane were obtained from a CH2Cl2 solution of 2
Volatiles were removed under vacuum and the resulting red layered with hexane and settled aside at −20 °C. Anal. calcd for
oily residue was triturated and suspended in hexane (20 mL).
The suspension was filtered and the red solid was washed with
hexane and dried under vacuum (40 °C) (Chart 6). Yield: 129 mg,
86%. The title compound is soluble in DMSO and chlorinated
solvents, poorly soluble in EtOH and Et2O and insoluble in
hexane and H2O. Crystals suitable for X-ray analysis were
obtained from a CHCl3 solution of 5 layered with either hexane
or heptane, and settled aside at −20 °C. Anal. calcd for
C34H43Cl2OPRuSi: C, 58.44; H, 6.20; Cl, 10.15. Found: C, 58.32;
H, 6.12; Cl, 9.97. IR (solid state): ˜ν/cm−1 = 3063w, 3041w,
2955 m, 2925 m, 2882w, 2853w, 1590w, 1568w, 1542w, 1469 m, Chart 7 Structure of 6 (numbering refers to carbon atoms).
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C36H43O5PRuSi: C, 60.40; H, 6.05. Found: C, 60.31; H, 5.96.
IR (solid state): ˜ν/cm−1 = 3058w, 2956w, 2928w, 2856w, 1697s,
1674s and 1666s-sh (νC=O), 1586w, 1564w, 1471 m, 1434m-sh,
1427 m, 1360s, 1257w, 1180s (νO–Ar), 1128 m, 1100 m, 1074 m,
1036w, 1008w, 937w, 892s (νP–O), 835 m, 822 m, 807 m, 778 m,
764 m, 739s, 710m-sh, 697s. 1H NMR (CDCl3): δ/ppm =
3
7.63–7.53 (m, 5H, C9-H + C13-H), 7.45 (dt, JHH = 7.7 Hz,
Chart 8 Structures of RAPTA-C (left) and solvato-complex formed by
Cl-abstraction with AgNO3 (right) (numbering refers to carbon atoms).
4JHH = 1.4 Hz, 1H, C14-H), 7.41–7.31 (m, 7H, C10-H + C11-H +
C16-H), 7.22 (t, 3JHH = 7.3 Hz, 1H, C15-H), 5.31 (d, 3JHH = 5.8 Hz,
2H, C3-H/C4-H), 5.11 (d, JHH = 5.9 Hz, 2H, C3-H/C4-H), 2.26
(hept, JHH = 6.6, 1H, C6-H), 1.79 (s, 3H, C1-H), 1.16 (d, JHH
3
3
3
=
6.9 Hz, 6H, C7-H), 1.06 (s, 9H, C20-H), 0.40 (s, 6H, C18-H). 9 : 1, c = 1.5 × 10−2 mol L−1, acq. time 30 min): δ/ppm = 48.5
13C{1H} NMR (CDCl3): δ/ppm = 165.4 (C21), 159.2 (C12), 137.8 (Δν1/2 = 5.2 × 102 Hz). [Et3NH]Cl. 35Cl NMR (CD3OD, acq. time
(C16), 133.9 (d, JCP = 47 Hz, C8), 131.7 (C11), 131.1 (C14), 1 min): δ/ppm = −22.7 (Δν1/2 = 2.3 × 102 Hz). 35Cl NMR (CDCl3,
1
131.0 (d, JCP = 12 Hz, C9), 128.9 (d, JCP = 11 Hz, C10), 123.5 acq. time 5 min): δ/ppm = 8.4 (Δν1/2 = 1.3 × 103 Hz).
2
3
(C15), 121.6 (d, JCP = 10 Hz, C13), 111.6 (C5), 99.5 (C2), 87.6
[RuCl2(η6-p-cymene)(PTA)] (RAPTA-C). D2O. Orange solution.
3
(C3/C4), 87.3 (d, JCP = 3.7 Hz, C3/C4), 46.0 (C20), 27.3 (C6), 1H NMR: δ/ppm = 6.05 (d, J = 5.8 Hz), 5.96 (d, J = 5.9 Hz), 5.89
22.4 (C7), 18.3 (C1/C19), 18.0 (C1/C19), 8.8 (C18). 31P{1H} NMR (d, J = 5.5 Hz), 5.84 (m-br), 5.79 (d, J = 5.5 Hz), 4.60 (s), 4.57 (s),
2
(CDCl3): δ/ppm = 124.3. 29Si{1H} NMR (CDCl3): δ/ppm = 3.4.
4.33 (s), 4.31 (s), 4.28 (s), 4.22 (s), 2.59 (m), 2.10 (s), 2.03 (s),
1.99 (m), 1.24–1.17 (m). 31P{1H} NMR: δ/ppm = −23.0, −32.6,
−34.1, −35.1 (major). 35Cl NMR (acq. time 1 min): δ/ppm =
Stability studies in DMSO/water solutions
General features (see ESI† for details). Ruthenium com- 0.49 (Δν1/2 = 35 Hz).
plexes were dissolved in DMSO-d6/D2O 9 : 1 v/v (0.8 mL; [Ru] =
CD3OD. Orange-red solution. 1H NMR: δ/ppm = 5.72
3
3
1.5 × 10−2 mol L−1). An aliquot of the resulting solution (d, JHH = 5.5 Hz, 2H, C3-H/C4-H), 5.68 (d, JHH = 5.7 Hz, 2H,
(0.40 mL) was transferred into a NMR tube, maintained at C3-H/C4-H), 4.57 (s, 6H, C9-H), 4.31 (s, 6H, C8-H), 2.66 (hept,
3
37 °C for 72 hours and analyzed by NMR as a function of time. 3JHH = 6.6 Hz, 1H, C6-H), 2.00 (s, 3H, C1-H), 1.21 (d, JHH
=
The remaining solution was diluted up to 4.0 mL with DMSO/ 6.9 Hz, 6H, C7-H). 31P{1H} NMR (CD3OD): δ/ppm = −36.4.
H2O 9 : 1 v/v (final [Ru] = 1.5 × 10−3 mol L−1), maintained at
CD3OD : D2O 9 : 1 v/v. Orange-red solution. Λm (c = 1.5 ×
37 °C for 72 hours and its conductivity was measured as a 10−3 mol L−1) = 35 S cm2 mol−1
.
1H NMR: a single set of
function of time. Both NMR and conductivity measurements signals was observed, with negligible chemical shift variation
were performed upon brief cooling to ambient temperature, with respect to that in CD3OD. 31P{1H} NMR: δ/ppm = −36.1.
then Ru-containing solutions were heated again at 37 °C. 35Cl NMR (acq. time 10 min): no signal.
Dimethyl sulfone (5.5 × 10−3 mol L−1) was used as a reference
CD3OD : D2O 9 : 1 v/v + AgNO3 (1 eq.). Yellow-orange solu-
for 1H NMR spectra (δ/ppm = 2.97 (s, 6H) in DMSO-d6/D2O tion + AgCl precipitate. Λm (c = 1.5 × 10−3 mol L−1) = 81 S cm2
9 : 1 v/v). Molar conductivity (Λm) was calculated with reference mol−1. NMR data indicate quantitative formation of [(η6-p-
3
to the starting material. Percent values of compounds in solu- cymene)RuCl(Solv)(PTA)]+. 1H NMR: δ/ppm = 6.05 (d, JHH
=
3
3
tion are based on 1H NMR spectroscopy and refer to identified 5.9 Hz, 1H), 5.96 (d, JHH = 6.1 Hz, 1H), 5.80 (d, JHH = 5.5 Hz,
3
compounds only. NMR signals in braces {} indicate super- 1H), 5.76 (d, JHH = 5.5 Hz, 1H, C3-H + C3-H′ + C4-H + C4′-H);
3
impositions with other species.
4.63 (s, 6H, C9-H), 4.41–4.29 (m, 6H, C8-H), 2.65 (hept, JHH
=
3
6.5 Hz, 1H, C6-H), 2.07 (s, 3H, C1-H), 1.27 (d, JHH = 6.9 Hz,
3H, C7-H), 1.24 (d, JHH = 6.4 Hz, 3H, C7′-H). 31P{1H} NMR:
Chloride/solvent exchange experiments
3
General procedures. Solutions of RAPTA-C and [RuCl2(η6-p- δ/ppm = −34.9.
cymene)(κP-PPh3)] (Ru-PPh3) in D2O, CD3OD or CD3OD : D2O
DMSO-d6 : D2O 9 : 1 v/v. Orange solution. Λm (c = 1.5 × 10−3
9 : 1 v/v were analyzed by 1H/35Cl/31P NMR spectroscopy and mol L−1) = 24 S cm2 mol−1. H NMR: δ/ppm = 5.72 (d, JHH
=
1
3
3
conductivity measurements. The subsequent addition of 1.0 5.7 Hz, 2H, C3-H/C4-H), 5.69 (d, JHH = 5.7 Hz, 2H, C3-
eq. of AgNO3 was performed from a 0.2 M solution of AgNO3 H/C4-H), 4.41 (s, 6H, C9-H), 4.14 (s, 6H, C8-H), 1.86 (s, 3H, C1-
3
in CD3OD : D2O 9 : 1 v/v. Freshly-prepared solutions of H), 1.09 (d, JHH = 6.8 Hz, 6H, C7-H). 31P{1H} NMR: δ/ppm =
RAPTA-C, Ru-PPh3 and 1–3 in DMSO-d6 : D2O 9 : 1 v/v (cRu
=
−34.1. 35Cl NMR (acq. time 30 min): no signal.
1
1.5 × 10−2 mol L−1) were analysed by 1H/35Cl/31P NMR spec-
DMSO-d6 : D2O 9 : 1 v/v + NaCl. H NMR and 31P{1H} NMR:
troscopy. 1H and 31P NMR spectra were then repeated after the a single set of signals was observed, identical to that without
addition of NaCl (cNaCl = 0.15 mol L−1) (Chart 8).
NaCl.
35Cl NMR reference data. NaCl. 35Cl NMR (D2O, acq. time
[RuCl2(η6-p-cymene)(κP-PPh3)] (Ru-PPh3). CD3OD : D2O 9 : 1
1 min): δ/ppm = 0 (Δν1/2 = 40 Hz). 35Cl NMR (CD3OD, acq. time v/v. Orange-brown solution. Λm (c = 1.5 × 10−3 mol L−1) = 68 S
1 min): δ/ppm = −28.3 (Δν1/2 = 1.7 × 102 Hz). 35Cl NMR cm2 mol−1. 1H NMR: δ/ppm = 7.83–7.76 (m, 4H), 7.53–7.38 (m,
3
3
(DMSO-d6 : D2O 9 : 1, c = 0.11 mol L−1, acq. time 5 min): 6H), 5.33 (d, JHH = 6.1 Hz, 2H), 5.21 (d, JHH = 5.9 Hz, 2H),
3
δ/ppm = 46.4 (Δν1/2 = 5.8 × 102 Hz). 35Cl NMR (DMSO-d6 : D2O 2.72–2.62 (m, 1H), 1.89 (s, 3H), 1.09 (d, JHH = 6.8 Hz, 6H).
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31P{1H} NMR: δ/ppm = 24.5. 35Cl NMR (acq. time 10 min): no refined by a riding model. All non-hydrogen atoms were
signal
CD3OD : D2O 9 : 1 v/v + AgNO3 (1 eq.). Yellow solution + pre-
refined with anisotropic displacement parameters.
cipitate. Λm (c = 1.5 × 10−3 mol L−1) = 103 S cm2 mol−1. Two
major sets of signals, in ca. 1 : 1 ratio, were identified, along
with other minor products. First set (identical to that of Ru-
PPh3). 1H NMR: δ/ppm = 7.82–7.76 (m, 4H), 7.54–7.38 (m, 6H),
5.36 (d, 3JHH = 6.2 Hz, 2H), 5.21 (d, 3JHH = 5.7 Hz, 2H), 2.69 (m,
In vitro cytotoxicity studies
Reagents. Eagle’s minimum essential medium (MEM) was
purchased from Sigma Aldrich, while trypsin-EDTA, penicillin,
streptomycin, sodium pyruvate, non-essential amino acid solu-
tion, fetal calf serum (FCS), plates and Petri dishes were pur-
chased from EuroClone. The compounds were dissolved in
dimethyl sulfoxide (DMSO) before performing each experi-
ment. The maximal concentration utilized was 50 µM, due to
limited water solubility; cisplatin was tested up to 100 µM.33
The same volume of solvent was added to control conditions
and did not exceed 0.25% v/v.
Cell culture. Human triple negative cancer cell line
MDA-MB-231, human ovarian carcinoma cell line A2780 and
Human Skin Fibroblasts (HSF) were cultured in MEM sup-
plemented with 10% FCS, non-essential amino acids, and
penicillin/streptomycin at 37 °C in a humidified atmosphere
(5% CO2 and 95% air).
Cell viability assay. Sulphorhodamine B (SRB) assay was per-
formed to assess the cell viability after treatments. 5 × 103 cells
per well were seeded in a 96-well tray in triplicate. After 24 h of
incubation, the cells were treated with different concentrations
of compounds. SRB assay were performed after 48 h according
to the method of Skehan et al.53
Cell cycle analysis. Cell cycle analysis was performed as pre-
viously described.54 In brief, cells were seeded (250 000/35 mm
petri dish) and incubated with DMEM supplemented with
10% FCS; 24 h later the medium was replaced with one con-
taining 10% FCS in the presence or absence (control) of 50 µM
of complex 3, and the incubation was continued for a further
24 h. At the end of this incubation period cells were re-sus-
pended with permeabilizing buffer (NaCl 100 mM, TRIS pH
7.4 150 mM; CaCl2 1 mM; MgCl2 0.5 mM; NP-40 0.1% contain-
ing 5 mM propidium iodide and 40 mg ml−1 of RNAse A) and
the DNA nuclear content was analyzed with FACScan™ flow
cytometer and BD CellQuest™ (both from BD Biosciences, San
Jose, CA, USA).
3
3JHH = 7.1 Hz, 1H), 1.89 (s, 3H), 1.11 (d, JHH = 6.9 Hz, 6H).
31P{1H} NMR: δ/ppm = 24.9. Second set. 1H NMR: δ/ppm =
7.70–7.63 (m), {7.54–7.38 (m)}, 5.70 (s-br, 2H), 5.31 (s-br, 1H),
3
5.15 (s-br, 1H), 2.79 (hept, JHH = 6.4 Hz, 1H), 1.82 (s-br, 3H),
1.29 (s-br, 6H). 31P{1H} NMR: δ/ppm = 31.2 (m-br).
DMSO-d6 : D2O 9 : 1 v/v. 1H NMR: δ/ppm = 7.77–7.66 (m,
6H), 7.46–7.35 (m, 9H), 5.27 (d, J = 5.9 Hz, 2H), 5.21 (d, J = 5.6
Hz, 2H), 1.74 (s, 3H), 0.93 (d, J = 6.8 Hz, 6H). 31P{1H} NMR:
δ/ppm = 24.2. 35Cl NMR (acq. time 30 min): no signal.
1
DMSO-d6 : D2O 9 : 1 v/v + NaCl. H NMR and 31P{1H} NMR:
a single set of signals was observed, identical to that without
NaCl.
[RuCl2(η6-p-cymene){κP-Ph2P(4-C6H4O-R)}], R = OSiMe2 Bu
t
t
(1), Br (2), OCOCHCl2 (3). R = OSiMe2 Bu, 1. DMSO-d6 : D2O
9 : 1 v/v. 1H NMR: δ/ppm = 7.73–7.66 (m, 4H), 7.60 (t, J =
9.2 Hz, 2H), 7.44–7.33 (m, 6H), 6.85 (d, J = 7.9 Hz, 2H), 5.27 (d,
J = 6.0 Hz, 2H), 5.20 (d, J = 5.7 Hz, 2H), 1.74 (s, 3H), 1.00–0.85
(m, 15H), 0.18 (s, 6H). 31P{1H} NMR (DMSO-d6 : D2O 9 : 1):
δ/ppm = 23.2. 35Cl NMR (acq. time 30 min): no signal.
1
DMSO-d6 : D2O 9 : 1 v/v + NaCl. H NMR and 31P{1H} NMR:
a single set of signals was observed, identical to that without
NaCl.
R = Br, 2. DMSO-d6 : D2O 9 : 1 v/v. 1H NMR: δ/ppm =
7.74–7.67 (m, 4H), 7.65 (t, J = 9.0 Hz, 2H), 7.54 (d, J = 7.6 Hz,
2H), 7.50–7.40 (m, 6H), 5.30 (d, J = 6.0 Hz, 2H), 5.23 (d, J =
5.8 Hz, 2H), 1.74 (s, 3H), 0.93 (d, J = 6.9 Hz, 6H). 31P{1H} NMR:
δ/ppm = 24.5. 35Cl NMR (acq. time 30 min): no signal.
1
DMSO-d6 : D2O 9 : 1 v/v + NaCl. H NMR and 31P{1H} NMR:
a single set of signals was observed, identical to that without
NaCl.
1
R = OCOCHCl2, 3. DMSO-d6 : D2O 9 : 1 v/v. H NMR: δ/ppm
= 7.72–7.65 (m, 4H), 7.54 (t, J = 8.8 Hz, 2H), 7.46–7.32 (m, 6H),
6.77 (d, J = 7.6 Hz, 2H), 6.37 (s, 1H), 5.26 (d, J = 5.3 Hz, 2H),
5.17 (d, J = 4.7 Hz, 2H), 1.73 (s, 3H), 0.94 (d, J = 6.5 Hz, 6H).
31P{1H} NMR (DMSO-d6 : D2O 9 : 1): δ/ppm = 23.1.
Statistical analysis. Experimental data are expressed as
mean S.D. The effects of the complexes versus control were
analyzed by two-tailed Student’s t test for unpaired data. The
concentration of compounds required to reduce cell viability
by 50% (IC50) was calculated by nonlinear regression curve
(GraphPad Prism, Version 5.01).
1
DMSO-d6 : D2O 9 : 1 v/v + NaCl. H NMR and 31P{1H} NMR:
a single set of signals was observed, identical to that without
NaCl.
X-ray crystallography
Conflicts of interest
Crystal data and collection details for 3·CH3COCH3, 4, 5 and
6·C6H14 are reported in Table 16S.† Data were recorded on a
Bruker APEX II diffractometer equipped with a CCD detector
using Mo-Kα radiation. Data were corrected for Lorentz polariz-
ation and absorption effects (empirical absorption correction
SADABS).51 The structures were solved by direct methods and
There are no conflicts to declare.
Acknowledgements
refined by full-matrix least-squares based on all data using We gratefully thank the University of Pisa for financial support
F2.52 Hydrogen atoms were fixed at calculated positions and (PRA_2017_25, “composti di metalli di transizione come possibili
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agenti antitumorali”). Dr Luigi Quintieri (University of Padova,
Department of Pharmaceutical and Pharmacological Sciences) is
gratefully acknowledged for kindly providing the A2780 cell line.
7 Z. Adhireksan, G. E. Davey, P. Campomanes, M. Groessl,
C. M. Clavel, H. Yu, A. A. Nazarov, C. Hui Fang Yeo,
W. H. Ang, P. Dröge, U. Rothlisberger, P. J. Dyson and
C. A. Davey, Nat. Commun., 2014, 5, 3462.
8 A. Weiss, R. H. Berndsen, M. Dubois, C. Muller, R. Schibli,
A. W. Griffioen, P. J. Dyson and P. Nowak-Sliwinska, Chem.
Sci., 2014, 5, 4742–4748.
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