Ruthenium arene complexes with triphenylphosphane ligands: Cytotoxicity towards pancreatic cancer cells, interaction with model proteins, and effect of ethacrynic acid substitution

Article abstract of DOI:10.1039/c7nj02300f

The ruthenium-arene complexes [(η⁶-p-cymene)RuCl₂(κP-PPh₂(4-C₆H₄CO₂H))], 1, [(η⁶-p-cymene)RuCl(κ²P,O-PPh₂(2-C₆H₄CO₂))], 2, [(η⁶-p-cymene)RuCl₂(κP-PPh₂(2-C₆H₄OCO-EA))], 3, and [(η⁶-p-cymene)RuCl₂(κP-PPh₂(4-C₆H₄CO₂CH₂CH₂OCO-EA))], 4 (EA-CO₂H = ethacrynic acid), were synthesized in good to high yields and characterized by analytical techniques, IR, UV-Vis and multinuclear NMR spectroscopy, and single crystal X-ray diffraction in the cases of 1 and 2. The unstable compounds [(η⁶-arene)RuCl₂(κP-PPh₂(2-C₆H₄CO₂CH₂CH₂OCO-EA))] (arene = p-cymene, 5a; arene = C₆H₆, 5b) were obtained and identified in solution by NMR. Electrochemical and spectro-electrochemical experiments were performed in order to assess the redox behaviour of 1-4 in CH₂Cl₂. The in vitro cytotoxicity of 1-4 was determined on the human pancreatic cancer cell line BxPC3 and the mouse embryo fibroblast Balb/3T3 Clone A31 cell line, the latter acting as a model for normal cells. Furthermore, the interaction of 1, 3 and 4 with two model proteins was investigated by high resolution ESI-MS.

Full text of DOI:10.1039/c7nj02300f

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The role of atropisomers on the photo-reactivity and fatigue of
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Ruthenium Arene Complexes with Triphenylphosphane Ligands:
Cytotoxic Activity Towards Pancreatic Cancer Cells, Interaction with
Model Proteins, and Critical Effect of Ethacrynic Acid Substitution
Lorenzo Biancalana,a Alessandro Pratesi,b Federica Chiellini,a Stefano Zacchini,c Tiziana Funaioli,a,*
Chiara Gabbiani,a,* Fabio Marchetti,a,*
a Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via G. Moruzzi 13, I-56124 Pisa,
Italy.
b
MetMed, Dipartimento di Chimica “Ugo Schiff”, Università di Firenze, Via della Lastruccia 3, I-
50019 Sesto Fiorentino, Italy.
c Dipartimento di Chimica Industriale “Toso Montanari”, Università di Bologna, Viale Risorgimento 4,
I-40136 Bologna, Italy.
Corresponding Authors
*E-mail addresses: fabio.marchetti1974@unipi.it; tiziana.funaioli@unipi.it; chiara.gabbiani@unipi.it.
1

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Abstract
The ruthenium-arene complexes [(η6-p-cymene)RuCl2(κP-PPh2(4-C6H4CO2H))], 1, [(η6-p-
cymene)RuCl(κ2P,O-PPh2(2-C6H4CO2))], 2, [(η6-p-cymene)RuCl2(κP-PPh2(2-C6H4OCO-EA))], 3, and
[(η6-p-cymene)RuCl2(κP-PPh2(4-C6H4CO2CH2CH2OCO-EA))], 4 (EA-CO2H = ethacrynic acid), were
synthesized in good to high yields and characterized by analytical techniques, IR, UV-Vis and
multinuclear NMR spectroscopy, and single crystal X-ray diffraction in the cases of 1 and 2. The
unstable compounds [(η6-arene)RuCl2(κP-PPh2(2-C6H4CO2CH2CH2OCO-EA))] (arene = p-cymene,
5a; arene = C6H6, 5b) were obtained and NMR identified in solution. Electrochemical and spectro-
electrochemical experiments were performed in order to assess the redox behaviour of 1-4 in CH2Cl2.
The in vitro cytotoxicity of 1-4 was determined on the human pancreatic cancer cell line BxPC3 and
the mouse embryo fibroblasts Balb/3T3 Clone A31 cell line, the latter acting as a model for normal
cells. Furthermore, the interaction of 1, 3 and 4 with two model proteins was investigated by high
resolution ESI-MS.
Keywords: metal-based drugs, ruthenium arene complexes, ethacrynic acid, pancreatic cancer, protein
interaction, mass spectrometry.
Introduction
Ruthenium complexes have emerged as promising candidates to overcome the limitations associated
with the currently used metal based (platinum based) anticancer drugs, and two of them, i.e.
[indazoleH][trans-Ru(N-indazole)2Cl4]
(KP1019)
and
[imidazoleH][trans-Ru(N-imidazole)(S-
DMSO)Cl4] (NAMI-A), proceeded to the clinical stage of drug development and successfully passed
phase I trials (Figure 1).1 The mechanism of action of these ionic species is believed to involve a single
electron reduction process of the Ru(III) centre occurring in the tumour environment.1,2 Following
2

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these findings, a large variety of ruthenium(II) complexes have been investigated as possible anticancer
agents, and especially those complexes based on the [Ru(η6-arene)Cl2] scaffold have aroused a
considerable interest.3 Within this family of compounds, RAPTA-C, containing 1,3,5-triaza-7-
phosphatricyclo[3.3.1.1]decane (PTA) as a ligand (Figure 1), displays a significant antitumor activity in
vivo and points towards clinical investigation.4 In the attempt to modulate the properties of the
ruthenium species, several complexes have been prepared by replacement of PTA with a
triphenylphosphane derived ligand.5 Although the lipophilicity of the triphenylphosphane frame may
result in a decrease of the water solubility, the introduction of this group usually leads to enhanced
cellular uptake and cytotoxicity, possibly favoured by intercalation to DNA.5b,6
Figure 1. Structures of ruthenium complexes with known antitumor activity.
One of the most common strategies to increase the anticancer activity of metal complexes consists in
the inclusion of organic fragments with a known biological function.2,7 Ethacrynic acid (EA-CO2H) is a
biologically active molecule acting as inhibitor of glutathione transferases (GSTs), i.e. a group of
cytosolic detoxification enzymes partially responsible for the drug resistance in primary and metastatic
tumours.8 It has been demonstrated that pancreatic cancer cells display an over-expressed GSTs
detoxification system.9 Ethacrynic acid has been included within ruthenium(II)-arene complexes using
3

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some different synthetic approaches,8,10 and also platinum(IV) complexes have been modified with
ethacrynic acid.11 The ruthenium compounds have shown the targeted property to inactivate GSTs and
determine apoptosis towards ovarian carcinoma, even in the cisplatin resistant cell line.12
Only a limited number of ruthenium compounds have been considered as possible drugs against the
pancreatic cancer,13 including Ru(II) arene compounds, and to the best of our knowledge no
information regarding triphenylphosphane derivatives have appeared in the literature so far.3e,14 It
should be remarked that the research pointing to new, effective drugs against pancreatic cancer, i.e. one
of the deadliest cancers in western countries with a low median survival rate, is of great importance.15
Indeed pancreatic ductal adenocarcinoma (PDAC), accounting for over 90% of pancreatic cancer cases,
develops from the exocrine cells of pancreas, and has usually metastasized by the time it is diagnosed;
at that stage, only few clinical treatments can be carried out, mostly palliative and determining severe
side effects.16
Herein, a series of ruthenium(II)-arene complexes containing triphenylphosphane ligands, two of
them functionalized with ethacrynic acid, have been synthesized, fully characterized and investigated
for their in vitro cytotoxic activity towards the human pancreatic cancer cell line BxPC3 and the mouse
embryo fibroblast Balb/3T3 Clone A31 cell line. Additional experiments have been carried out to shed
some light on the mechanism of action of the compounds, i.e. electrochemical and
spectroelectrochemical analyses, stability studies (NMR, UV-Vis, conductivity) in aqueous medium
and studies of reactivity with two small proteins, namely hen egg white lysozyme (HEWL) and horse
heart cytochrome c (Cyt-c). These two proteins are suitable to ESI-MS analyses 17 due to their size and
overall properties, and, thus, are often used as “model proteins” for metalation studies.18 It should be
mentioned that cytochrome c is importantly involved in apoptotic pathways,19 while lysozyme is
relevant in certain defence mechanisms.20
4

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Results and discussion
1. Synthesis and characterization of compounds.
The previously reported triphenylphosphane-ethacrynic acid conjugate L2 was prepared by a
modification of the published procedure,10a,21 while the new ligands PPh2(2-C6H4OCO-EA), L1 and
PPh2(2-C6H4CO2CH2CH2OCO-EA), L3, were isolated from the condensation of ethacrynic acid (EA-
CO2H)
with
(2-hydroxyphenyl)diphenylphosphane
or
EA-CO2(CH2)2OH
with
2-
(diphenylphosphino)benzoic acid, respectively (Scheme 1). The synthesis and chromatographic
purification of L1 and L3 required the use of a strictly inert atmosphere, then these air sensitive
products were characterized by analytical and spectroscopic techniques.
Scheme 1. Synthesis of triphenylphosphane ligands functionalized with ethacrynic acid (EA-CO2H).
The ruthenium arene complexes 1-4 were synthesized by allowing the dimeric precursor [RuCl2(p-
cymene)]2 to react in CH2Cl2 or CHCl3 at reflux conditions with an excess of the appropriate
phosphane ligand, i.e. PPh2(4-C6H4CO2H), PPh2(2-C6H4CO2H), L1 and L2 respectively (Scheme 2),
and then isolated after work up in 65-90% yields. The formation of 2 is accompanied by HCl evolution,
leading to bidentate coordination of the resulting phosphino-carboxylate moiety. Compound 2 was
alternatively obtained by the reaction of [RuCl2(p-cymene)]2 with K[PPh2(2-C6H4CO2)], affording KCl
5

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as the side product. Complexes 2-3 are unprecedented, whereas 4,10a for which improved synthetic
procedure and characterization are given here, and 1 22 have been already reported.
Scheme 2. Synthesis of ruthenium complexes with triphenylphosphane derived ligands.
The reactions of L3 with [(arene)RuCl2]2 (arene = p-cymene, C6H6) proceeded with the initial
formation of the expected products 5a-b (Scheme 3), which were identified in the reaction solution by
NMR spectroscopy. However 5a-b revealed to be unsuitable to biological analyses: an extensive
decomposition with release of the arene ligand was observed in the reaction medium, and the
compounds were not isolated in the solid state.
6

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Scheme 3. Formation of unstable Ru(II) phosphane complexes (arene = p-cymene, 5a; C6H6, 5b).
31
Selected spectroscopic data for 1-5 and related phosphane ligands are compiled in Table S1. The P
NMR spectra of 1-5 (in CDCl3) consists of one resonance due to the ruthenium bound phosphorous,
which is found significantly low field shifted with reference to the corresponding non-coordinated
phosphane (e.g., δ = 25.0 ppm in 3 and δ = −16.9 ppm in L1). More in detail, the 31P resonance falls at
30.5 ppm in 2, containing a bidentate P,O-ligand, while it occurs around 25 ppm in 1, 3, 4 and 5a, all of
these compounds featured by a monodentate phosphane ligand. Complex 2 is a racemate, due to the
presence of a stereogenic ruthenium centre, and the two phenyl substituents are diastereotopic. The
10a,21
NMR resonances of the ethacrynic acid fragment in 3-5
and the resonances of the p-cymene
ligand 10a,23 in 1-4 and 5a match those previously reported for analogous compounds.
The stable products 1-4 were further characterized by analytical methods and IR and UV-Vis
spectroscopy. The IR carbonyl stretching vibration due to the monodentate benzoate group in 2 has
been detected at 1604 cm−1, in analogy to the case of a strictly related Ru complex.24
X-ray quality crystals of 1 and 2 could be collected from CH2Cl2/hexane mixtures settled at 4°C: views
of the ORTEP molecular structures are shown in Figures S1 and 2, while relevant bonding parameters
are given in Tables S2 and 1, respectively.
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Figure 2. Molecular structure of [(η6-p-cymene)RuCl(κ2P,O-PPh2(2-C6H4CO2))], 2. Displacement ellipsoids are at
the 50% probability level. H-atoms have been omitted for clarity.
Table 1. Selected bond distances (Å) and angles (°) for 2.
Ru(1)−C(1)
Ru(1)−C(3)
Ru(1)−C(5)
Ru(1)−Cl(1)
Ru(1)−P(1)
C(11)−O(1)
2.219(7)
2.196(7)
2.254(8)
2.4197(19)
2.3168(19)
1.289(9)
Ru(1)−C(2)
Ru(1)−C(4)
Ru(1)−C(6)
Ru(1)−O(1)
C(12)−C(11)
C(11)−O(2)
2.183(7)
2.191(7)
2.269(7)
2.096(5)
1.537(10)
1.235(8)
Cl(1)-Ru(1)−O(1)
O(1)-Ru(1)−P(1)
87.31(15)
81.26(15)
Cl(1)−Ru(1)−P(1) 87.80(7)
C(12)−C(11)−O(1) 121.3(6)
O(1)−C(11)−O(2) 121.3(7)
P(1)−C(17)−C(12) 121.4(5)
C(12)-C(11)−O(2) 117.2(7)
Ru(1)-P(1)−C(17) 111.0(2)
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C(17)-C(12)−C(11) 125.1(6)
C(11)−O(1)−Ru(1) 133.5(4)
Compounds 1 and 2 adopt the typical three-leg piano-stool geometry,25 and the bonding parameters
around the Ru(II) centre are similar to those previously reported for related compounds.10,26 The
coordination mode and bonding parameters for the κ2P,O-PPh2(2-C6H4CO2) ligand in 2 are in keeping
with previous Ru-complexes displaying the same ligand.24,27
2. Electrochemistry and Spectroelectrochemistry.
On considering that the biological activity of ruthenium compounds has been often associated with
their involvement in redox processes,28 the electrochemical behaviour of 1-4, investigated for their
antiproliferative activity (see below), and EA-CO2H was ascertained by cyclic voltammetry (see
Experimental for details). The studied compounds are well soluble in dichloromethane, moreover this
solvent offers a wide electrochemical window. The formal electrode potentials for the observed
electron transfers are compiled in Table 2, while the CV profiles are shown in Figure 3.
Table 2. Redox properties of 1-4 and EA-CO2H in 0.2 M [nBu4N][PF6] CH2Cl2 solution.
Oxidation
Reduction
Stability window
b
c
E°' a
∆Ep
type
Epc
type
∆E d
EA-CO2H
none in the solvent window
-2.14
-1.81
-1.62
irrevers.
irrevers.
irrevers.
irrevers.
irrevers.
b
-
1
2
3
4
+1.12
+1.32 c
+1.15
+1.11
92
-
revers.
2.93
2.94
2.83
2.80
irrevers.
109 revers. on CV time-scale -1.68
84
revers. on CV time-scale -1.69
a
Formal electrode potential (V) vs. SCE, calculated as E°' = ½(Epa + Epdc). Peak-to-peak separation (mV)
measured at 0.1 V·s-1. Peak potential (V) for an irreversible process. Potential difference (V) between
c
oxidation and reduction processes.
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75
60
45
30
15
0
30
15
0
60
45
30
15
0
(a)
(b)
(c)
-15
-30
-45
-60
-15
-30
-45
-15
-30
*
+ 2.00
+ 1.00
0.00
-1.00
-2.00
+ 2.00
+ 1.00
0.00
-1.00
-2.00
+ 2.00
+ 1.00
0.00
-1.00
-2.00
E / V vs SCE
E / V vs SCE
E / V vs SCE
60
45
60
45
30
15
0
(d)
(e)
30
15
0
-15
-30
-45
-60
*
-15
-30
-45
*
+ 2.00
+ 1.00
0.00
-1.00
-2.00
+ 2.00
+ 1.00
0.00
-1.00
-2.00
E / V vs SCE
E / V vs SCE
Figure 3. Cyclic voltammograms of 1 (a, 4.1·10-3 M), 2 (b, 6.9ꢀ10-4 M), 3 (c, 4.5·10-3 M), 4 (d, 2.6·10-3 M) and
EA-CO2H (e, 2.7·10-3 M) recorded at a Pt electrode in 0.2 M [nBu4N][PF6] CH2Cl2 solution; scan rate = 0.1 V·s-1.
The peaks marked with (*) are due to the electrolyte solution. Arrows indicate scan direction.
Ethacrynic acid displayed no oxidation process, while a single irreversible reduction was observed at
very low potential (‒2.14 V). On the other hand, 1, 3 and 4 underwent a reversible oxidation process at
ca. +1.1 V.29 Similar values were previously reported for related [(η6-p-cymene)RuCl2(PPh2R)]
complexes, containing a monodentate phosphane ligand, and were attributed to a reversible or quasi-
reversible Ru2+ → Ru3+ oxidation.30 Conversely, the P,O chelate compound 2 showed a weak oxidation
wave at +1.32 V, and this process is irreversible even at higher scan rates (up to 1.00 V·s−1). The
monoelectronic nature of the oxidation process was assessed for 1 as a representative compound, with a
combination of controlled-potential coulometry and linear sweep voltammetry (see Experimental for
details). In the cathodic region, 1-4 displayed a single irreversible reduction around ‒1.7 V,
electrochemically independent on the Ru2+/Ru3+ oxidation discussed above. It was not possible to
determine the number of electrons involved in the reduction as the waves occurred at the upper limit of
10

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the potential window provided by the solvent. Concerning related Ru(II)-arene complexes, this process
has been assigned to a metal-centred reduction.30b,31
The UV-Vis and IR variations related to the electrochemical oxidation of 1, 3 and 4 were investigated
(see Figure S2 given as Supporting Information). Despite the oxidation processes of 3 and 4 are
reversible in the CV timescale, the electro-generated species 3+ and 4+ partially decomposed during the
longer times required for the macro-electrolysis experiments. Indeed, when the scan rate was reversed
some minutes after oxidation was completed, the UV and IR bands became broader and less intense.
On the other hand, 1 was cycled several times between positive and negative potentials and no
significant change in the UV-Vis and IR spectra was observed. IR carbonyl stretching bands moved to
higher wavenumbers on going from the neutral species 1, 3, 4 to the corresponding cations (Figure S2
and Table S3). The main variations for the ethacrynic acid-functionalized complexes 3 - 4 are those
related to the C=O stretching vibration of the ester group closest to the metal centre: the original bands
at 1725 cm−1 (4) and 1782 cm−1 (3) were gradually replaced by new bands at 1729 cm−1 (4+) and 1793
cm−1 (3+), respectively. Similarly, the benzoic acid band of 1, at 1697 cm−1, was replaced on oxidation
by an absorption at 1703 cm−1. The removal of one electron from 1, 3 and 4 produced a markedly
different pattern in the respective UV-Vis spectra (Figure S2): more precisely, the 370-375 nm band
was replaced by four new bands at 315, 355, 420 and 515 nm.
In conclusion, 1-4 exhibit a comparable electrochemical behaviour, with minor differences imputable
to the coordination mode of the phosphane ligand (mono- or bidentate) and the nature of the substituent
on the triphenylphosphane frame (carboxylic acid or ester). However, no redox activity has been
detected within a biologically relevant range of potentials;32 therefore, despite these findings refer to
dichloromethane solutions, the complexes are reasonably expected not to be engaged in redox
processes in physiological environment, and the Ru(II) oxidation state to be preserved.
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3. Stability studies.
NMR and UV-Vis spectroscopy and conductivity measurements were used to assess the stability of
the complexes 1-4 in DMSO/water 9:1 solutions at 37 °C as a function of time. The high relative
content of DMSO in the solvent mixture was required in order to obtain 1·10-2 mol·L-1 solutions of Ru
compounds, this concentration being suitable for NMR analysis. Parallel NMR and UV-Vis analyses
were conducted on 1-4 in 0.11 M sodium chloride DMSO/water solutions. This chloride concentration
resembles that present in the medium used for in vitro tests, and that typical of blood. The details of the
experiments are reported in the SI, and the compounds identified in the distinct cases after 72 h are
shown in Scheme 4. Only a fraction, ranging from 45% (2 and 4) to 24% (1 and 3), of the starting
ruthenium complexes was still persistent in solution after 72 h in the absence of NaCl (Table 3).
Interestingly, the addition of 0.11 M NaCl significantly suppressed the decomposition of 1-3 (e.g. 49%
1
31
of 3 detected after 72 h), see Table 3, without causing any variation to the H and P set of signals.
Degradation pathways of 1-4 include the dissociation of the phosphane and p-cymene ligands. Thus, 4-
(diphenylphosphino)benzoic acid, L1 and L2 were recognized in significant amounts, in admixture
with the respective phosphane-oxides. The NMR data suggest that no hydrolytic cleavage of the ester
bonds occurred, i.e. the ethacrynic acid fragment is preserved within the relevant phosphane. On the
other hand, minor amounts of non coordinated 2-(diphenylphosphino)benzoate (identified as
phosphane oxide) was released from 2. This feature is presumably a consequence of the bidentate
coordination providing additional stability to the Ru-P linkage.
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Scheme 4. Compounds detected in DMSO/H2O and DMSO/H2O/NaCl solutions of 1-4 maintained at 37 °C; %
values are based on 1H NMR spectroscopy and refer to identified compounds only.
Table 3. Fraction of complexes 1-4 in DMSO/H2O and DMSO/H2O/NaCl solutions at 37 °C; % values are based
on 1H NMR spectroscopy (dimethyl sulfone as internal standard, 1:1 molar ratio vs Ru).
% (result of NaCl experiment in parenthesis)
Compound
t = 0
t = 25.5 h
59 (67)
72 (71)
39 (62)
72 (66)
t = 72 h
23 (35)
45 (52)
24 (49)
45 (39)
1
2
3
4
90 (95)
96 (97)
64 (62)
82 (82)
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4. Cytotoxicity studies.
The ability to inhibit cell growth of the complexes 1-4, selected phosphane(oxide) species, ethacrynic
acid and the reference compounds RAPTA-C and cisplatin was evaluated in vitro against the human
pancreatic cancer cell line BxPC3 and the mouse embryo fibroblast Balb/3T3 Clone A31 cell line, the
latter representing a model for non-tumour cells. Since the compounds are almost insoluble in water,
stock solutions were prepared in DMSO and then diluted with cell culture media.
The obtained IC50 values are visualized in Table 4. The ethacrynic acid-functionalized compounds 3
and 4 displayed an enhanced antiproliferative activity with respect to 1 and 2, and ethacrynic acid itself.
Therefore, a synergic effect can be envisaged as obtained by linking ethacrynic acid to the ruthenium
arene triphenylphosphane frame. It is plausible that the ethacrynic acid moiety contributes to the
anticancer effect via the inhibition of GSTs, which sensitizes the cancer cells towards the ruthenium
species; intracellular esterases might be implicated in separating the EA-CO2H fragment from the
ligand/complex, as previously proposed.12,33
Compound 1 did not show appreciable cytotoxicity, in agreement with a recent investigation on other
cell lines.22 The IC50 values of PPh2(4-C6H4CO2H), [PPh2(2-C6H4CO2)]− (as K+ salt) and L1 were
found to be lower than the maximum tested concentration, conversely the phosphane oxide L2=O
exhibited some cytotoxic activity, all of these species being generated from the respective complexes in
aqueous medium (see Stability Studies section); likewise L2=O, also L2 is expected to exert a
cytotoxic behaviour.10 On account of the fact that K[PPh2(2-C6H4CO2)], L1 and L2=O display
significantly higher IC50 values compared to the respective complexes 2-4 (see above), it may be
assumed that the antiproliferative activity of 2-4 is essentially due to the intact ruthenium-phosphane
species.
Compounds 2 and 3 display a moderate selectivity towards the BxPC3 cell line with respect to the non
tumoral cell line, and a reverse situation was found for 4. On comparing the data related to 3 and 4, it
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can be concluded that the site and chemical nature of the binding of the ethacrynic acid moiety to the
phosphane ligand (Scheme 2) may play a significant role in the cytotoxic activity of the resulting
complexes. The IC50 values obtained for 3 appear promising when compared to those obtained for
cisplatin on the same cell lines.
Table 4. IC50 values (ꢀM) of tested compounds.
Compound
IC50 (ꢀM) SD
Balb/3T3
BxPC3
>80
CloneA31
1
>80
2
31
5
52
3
3
4
7.7 1.4
13.4 0.4
>80
14.1 0.3
8.6 0.5
>80
PPh2(4-C6H4CO2H)
K[PPh2(2-C6H4CO2)]
L1
>80
>80
>80
>80
O=L2
34
7
49
>80
13
8
2
EA-CO2H
cisplatin
RAPTA-C
>80
11.0 0.3
> 500
-
BxPC3 = human pancreatic cancer cell line; Balb/3T3 CloneA31 = mouse embryo fibroblast cell line.
5. Interaction of complexes with model proteins.
To gain a deeper insight into the reactivity of the compounds with potential biomolecular targets, 1, 3
and 4 were allowed to react with HEWL and Cyt-c. A three fold molar excess of each complex was
incubated with the selected protein in an ammonium acetate buffer at 37 °C, and the resulting adducts
were analyzed by ESI–MS as a function of time up to 72 h, according to a previously described
protocol.18a Thus, the interaction of 1, 3 and 4 with cytochrome c resulted in a fragmentation of the
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metal complexes, leading to the formation of the adduct cyt-Ru-cym (peak at m/z 12592 Da), consisting
of cytochrome c added of the Ru(η6-p-cymene) moiety. It is noteworthy that the same adduct was
recognized as the prevalent product of the interaction between cytochrome c and other ruthenium arene
compounds, including RAPTA-C.34 However, some differences have been observed within compounds
1, 3 and 4. In the case of 1, the reaction did not reach completion, and the intact protein was detected in
a significant amount after 72 h of incubation (Figure 4). Conversely, in the case of 3, the formation of
cyt-Ru-cym was almost completed after 24 h, and only a small amount of protein remained unreacted
(Figure 5). A further minor peak was assigned to cyt-Ru-cym with the addition of ethacrynic acid (m/z
12895 Da).
12358.32160
12592.32086
Cyt-c
12592.34700
100
Cyt-c + Ru-p-Cymene
80
12358.34774
24 h
60
40
20
72 h
0
12400
12600
12800
m/z
13000
13200
Figure 4. Deconvoluted ESI–MS spectra of cytochrome c in ammonium acetate buffer (pH 6.8), incubated with
complex
1
, after 24 and 72 h respectively, at 37 °C (protein concentration = 10-4 M;
1/protein molar ratio = 3).
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12592.34977
Cyt-c + Ru-p-Cymene
12592.30621
100
80
60
40
20
0
Cyt-c + Ru-p-Cymene + ethacrynic ac.
Cyt-c
12895.35737
24 h
12358.30890
12400
12895.31483
72 h
12600
12800
13000
m/z
13200
13400
Figure 5. Deconvoluted ESI–MS spectra of cytochrome c in ammonium acetate buffer (pH 6.8), incubated with
complex /protein molar ratio = 3).
, after 24 and 72 h respectively, at 37 °C (protein concentration = 10-4 M;
3
3
Also in the case of 4, the formation of the adduct cyt-Ru-cym (see above) occurred with extensive
consumption of the ruthenium species, although this process was slower than in the case of 3 (Figure
6). In fact, a significant amount of unreacted protein was still present after 24 h, and a peak attributed to
the adduct cyt-[Ru(DMSO)(H2O)] was also detected.
Stability studies in DMSO/water solution have indicated that these Ru complexes undergo a slow
dissociation of the phosphane ligand (see Stability Studies section), and this fact may be associated to
the possibility of effective binding of the Ru(p-cymene) fragment to the protein side chain, as observed
in the case of Cyt-c.
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12592.31515
Cyt-c + Ru-p-Cymene
Cyt-c
12592.33442
100
80
60
40
20
0
Cyt-c + Ru-p-Cymene+DMSO+H2O
12358.31779
12629.26006
12396.26475
12690.29214
24 h
12824.32023
12359.33691
12400
12826.33158
12800 12900
12653.36830
12700
72 h
12500
12600
m/z
Figure 6. Deconvoluted ESI–MS spectra of cytochrome c in ammonium acetate buffer (pH 6.8), incubated with
complex /protein molar ratio = 3).
, after 24 and 72 h respectively, at 37 °C (protein concentration = 10-4 M;
4
4
Additional ESI-MS studies were performed to elucidate the reactivity of 1, 3 and 4 with lysozyme. A
markedly lower reactivity of the complexes was ascertained compared to the analogous reactions with
cytochrome c (see Figure S3).
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Conclusions
Some ruthenium arene complexes containing different triphenylphosphane ligands have been prepared,
structurally characterized and evaluated in vitro as possible drugs against pancreatic adenocarcinoma.
The incorporation of ethacrynic acid, i.e. a bioactive molecule implicated in resistance mechanisms, via
the phosphane ligand has provided a synergic effect enhancing the antiproliferative activity of the
complexes. Electrochemical and spectroscopic experiments and ESI-MS studies on the interaction with
model proteins have given insight into the behaviour of the complexes in biological environment. The
complexes functionalized with ethacrynic acid undergo slow and progressive dissociation of the
phosphane ligand, possibly converting into the relevant phosphane oxide, these uncoordinated species
providing minor contribution to the cytotoxic activity. The enhanced phosphane release seems to be
related to the possibility of effective binding of the ruthenium-arene fragment to a model protein
(cytochrome c). It is noteworthy that the stability of the ethacrynic acid functionalized complexes, their
anticancer behaviour and the adduct formation with proteins may be tuned by an appropriate, synthetic
choice of the site and linkage of the bioactive moiety to the phosphane.
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Experimental
[(η6-p-cymene)RuCl2]2 and [(η6-C6H6)RuCl2]2 were prepared according to the literature from
RuCl3·xH2O (99.9%, Alfa Aesar) and α-phellandrene or 1,3-cyclohexadiene, respectively (Sigma
Aldrich).35 The other organic reactants were obtained from Alfa Aesar, Sigma Aldrich or TCI Europe
and were of the highest purity available. Ethylene glycol, 4-(diphenylphosphino)benzoic acid, 2-
(diphenylphosphino)benzoic acid, (2-hydroxyphenyl)diphenylphosphane, ethacrynic acid (EA-CO2H)
and ethyl(diisopropylamino)carboxydiimide hydrochloride (EDCI·HCl) were stored under nitrogen as
received. Synthesis of ligands and complexes was carried out under a nitrogen atmosphere using
standard Schlenk techniques. Solvents were distilled from appropriate drying agents. Once isolated, L1,
L2, L3 and K[PPh2(2-C6H4CO2)] were stored under nitrogen. Silica gel (Merck, 70-230 mesh) was
dried at 150 °C overnight and stored under nitrogen. NMR spectra were recorded at 298 K on a Bruker
Avance II DRX400 instrument equipped with a BBFO broadband probe. Chemical shifts (expressed in
parts per million) are referenced to the residual solvent peaks (1H, 13C{1H}) or to external standard
(31P{1H} to 85% H3PO4). Spectra were assigned with the assistance of DEPT-135, 1H-1H (COSY) and
1H-13C (gs-HSQC and gs-HMBC) correlation experiments.36 Infrared spectra of solid samples were
recorded on a Perkin Elmer Spectrum One FT-IR spectrometer, equipped with a UATR sampling
accessory. Infrared spectra of liquid samples were recorded on a Perkin Elmer Spectrum 100 FT-IR
spectrometer with a CaF2 liquid transmission cell. UV-Vis spectra were recorded on a Ultraspec 2100
Pro spectrophotometer with 0.1 cm quartz cuvettes. Carbon and hydrogen analysis was performed on a
Carlo Erba mod. 1106 instrument. Melting points and decomposition temperatures were determined on
a STMP3 Stuart scientific instrument with a capillary apparatus. Conductivity measurements were
carried out at 21°C using an XS COND 8 instrument (cell constant = 1.0 cm−1).37
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1) Synthesis and characterization of compounds.
Synthesis of EA-CO2(CH2)2OH.
Chart 1. Structure of EA-CO2(CH2)2OH (numbering refers to carbon atoms).
The title compound was prepared according to a modified literature procedure.10a A solution of EA-
CO2H (314 mg, 1.03 mmol) and ethylene glycol (0.60 mL, 11 mmol) in THF (8 mL) was treated with
EDCI·HCl (235 mg, 1.23 mmol) and DMAP (24 mg, 0.19 mmol). The colourless suspension (oil +
solution) was stirred at room temperature and the progress of reaction was monitored with TLC. After
2.5 hours, volatiles were removed under vacuum. The colourless residue was dissolved in CH2Cl2 and
extracted with water (x 3). The organic phase was concentrated under vacuum and loaded on top of a
silica column. Impurities were eluted with CH2Cl2, then EA-CO2(CH2)2OH was collected with
CH2Cl2/Et2O (1:1 v/v). Solvent removal under vacuum (room temperature) yielded EA-CO2(CH2)2OH
as an air stable, colourless crystalline solid. Yield: 229 mg, 64%. Anal. calcd. for C15H16Cl2O5: C,
51.89; H, 4.65. Found: C, 51.63; H, 4.71. IR (solid state): ῦ/cm-1 = 3512w (νO-H), 3089w, 2976w,
2965w, 2940w, 2921w, 2881w, 1737s (νC13=O), 1662s (νC5=O), 1625w, 1587m (νC3=C4), 1559w, 1472m,
1445m, 1414m, 1383m, 1359m, 1292m, 1258m, 1230s, 1205s, 1124m, 1076s, 1001m, 943m, 892m,
1
842m, 810m, 769m, 736m. UV-Vis (CH2Cl2): λmax/nm (ε/M−1·cm−1) = 267 (4.8·103). H NMR
(CDCl3): δ/ppm = 7.14 (d, 3JHH = 8.5 Hz, 1H, C11-H), 6.81 (d, 3JHH = 8.5 Hz, 1H, C10-H), 5.95 (s, 1H,
C4-H), 5.61 (s, 1H, C4-H'), 4.82 (s, 2H, C12-H), 4.39–4.33 (m, 2H, C14-H), 3.89–3.84 (m, 2H, C15-
H), 2.47 (q, 3JHH = 7.4 Hz, 2H, C2-H), 1.83 (s, 1H, OH), 1.15 (t, 3JHH = 7.4 Hz, 3H, C1-H).
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Synthesis of PPh2(2-C6H4OCO-EA), L1.
Chart 2. Structure of PPh2(2-C6H4OCO-EA), L1 (numbering refers to carbon atoms).
A solution of (2-hydroxyphenyl)diphenylphosphane (206 mg, 0.739 mmol) and EA-CO2H (247 mg,
0.813 mmol) in CH2Cl2 (15 mL) was treated with EDCI·HCl (156 mg, 0.816 mmol) and then with
DMAP (20 mg, 0.16 mmol). The resulting colourless solution was stirred at room temperature for 24
hours. Volatiles were removed under vacuum and a colourless foamy solid was obtained. The solid was
washed with hexane then suspended in boiling Et2O (20 mL) for 1 hour. The liquid was filtered and
collected into another flask and the extraction procedure was repeated three times. L1 was obtained as
an air sensitive, colourless solid after solvent removal under vacuum. Yield: 358 mg, 86%. Anal. calcd.
for C31H25Cl2O4P: C, 66.09; H, 4.47. Found: C, 66.22; H, 4.38. Mp: 58°C (colourless liquid). IR (solid
state): ῦ/cm−1 = 3059w, 2966w, 2933w, 2874w, 1779m (νC13=O), 1663s (νC5=O), 1624w-sh, 1584s
(νC3=C4), 1466s, 1435s, 1384m, 1339w, 1292m, 1259s, 1228m, 1219m, 1186m, 1155s, 1118m, 1076s,
1
1028m-sh, 1000m, 946w, 911w, 802m, 743s, 694s. H NMR (CDCl3): δ/ppm = 7.43–7.25 (m, 11H,
Ph), 7.24–7.16 (m, 2H, Ph), 7.04 (d, 3JHH = 8.5 Hz, 1H, C11-H), 6.88–6.83 (m, 1H, Ph), 6.66 (d, 3JHH
=
3
8.7 Hz, 1H, C10-H), 5.93 (s, 1H, C4-H), 5.58 (s, 1H, C4-H'), 4.69 (s, 2H, C12-H), 2.47 (q, JHH = 7.5
Hz, 2H, C2-H), 1.15 (t, 3JHH = 7.4 Hz, 3H, C1-H). 13C{1H} NMR (CDCl3): δ/ppm = 196.0 (C5), 166.1
2
2
(C13), 155.5 (C9), 152.2 (d, JCP = 14.4 Hz, C14), 150.3 (C3), 135.4 (d, JCP = 8.6 Hz, C18), 134.2
2
3
(C8), 134.0 (d, JCP = 20.5 Hz, C21), 131.5 (C7), 130.4 (C16), 129.4 (C23), 128.9 (d, JCP = 6.9 Hz,
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C22), 128.7 (C4), 127.0 (C11/C17), 126.9 (C11/C17), 122.4 (C6 + C15), 111.5 (C10), 66.0 (C12), 23.6
(C2), 12.6 (C1). 31P{1H} NMR (CDCl3): δ/ppm = – 16.9.
Synthesis of PPh2(4-C6H4CO2CH2CH2OCO-EA), L2.
Chart 3. Structure of PPh2(4-C6H4CO2CH2CH2OCO-EA), L2 (numbering refers to carbon atoms).
The title compound was prepared according to a modified literature procedure.10a A solution of 4-
(diphenylphosphino)benzoic acid (131 mg, 0.426 mmol) and EA-CO2(CH2)2OH (124 mg, 0.355 mmol)
in CH2Cl2 (20 mL) was treated with EDCI·HCl (102 mg, 0.533 mmol) and DMAP (9.2 mg, 0.075
mmol). The resulting pale yellow solution was stirred at room temperature and the progress of the
reaction was monitored by TLC. After 6.5 hours, the volume was reduced under vacuum to 2-3 mL,
then Et2O was slowly added. The colourless milky suspension was filtered on a short silica pad and
eluted with Et2O. Volatiles were removed under vacuum and compound L2 was obtained as an air
sensitive, colourless sticky solid. Yield: 178 mg, 79%. Anal. calcd. for C34H29Cl2O6P: C, 64.26; H,
4.60. Found: C, 64.07; H, 4.65. IR (solid state)10a: ῦ/cm−1 = 1763m (νC13=O), 1718s (νC16=O), 1665m
(νC5=O), 1584m (νC3=C4). 1H NMR (CDCl3): δ/ppm = 7.95 (dd, 3JHH = 8.4 Hz, 4JHP =1.4 Hz, 2H, C18-H),
3
3
7.38–7.29 (m, 12H, C19-H + Ph2P), 7.07 (d, JHH = 8.5 Hz, 1H, C11-H), 6.79 (d, JHH = 8.5 Hz, 1H,
C10-H), 5.91 (s, 1H, C4-H), 5.56 (s, 1H, C4-H'), 4.78 (s, 2H, C12-H), 4.55 (s, 4H, C14-H + C15-H),
2.46 (q, 3JHH = 7.4 Hz, 2H, C2-H), 1.14 (t, 3JHH = 7.4 Hz, 3H, C1-H). 31P{1H} NMR (CDCl3): δ/ppm =
– 4.9.
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Synthesis of PPh2(2-C6H4CO2CH2CH2OCO-EA), L3.
Chart 4. Structure of PPh2(2-C6H4CO2CH2CH2OCO-EA), L3 (numbering refers to carbon atoms).
The title compound was prepared as described for L2, using 2-(diphenylphosphino)benzoic acid (240
mg, 0.783 mmol), EA-CO2(CH2)2OH (227 mg, 0.564 mmol), EDCI·HCl (188 mg, 0.980 mmol) and
DMAP (17 mg, 0.14 mmol). The resulting pale yellow solution was stirred at room temperature and the
progress of the reaction was monitored by TLC. After 14 hours, the volume was reduced under vacuum
to 2-3 mL, then Et2O (10 mL) was slowly added. The resulting suspension was loaded on a short silica
column (h 2 cm, d 3 cm) equipped with an external glass jacket filled with a NaCl/ice mixture (T = -
14°C). Compound L3 was obtained as an air-sensitive, pale yellow solid after elution with Et2O (50
mL) and solvent removal under vacuum. Yield: 183 mg, 51%. Anal. calcd. for C34H29Cl2O6P: C, 64.26;
1
H, 4.60. Found: C, 64.10; H, 4.70. H NMR (CDCl3): δ/ppm = 8.12–8.05 (m, 1H, C18-H), 7.47–7.41
(m, 2H, C19-H + C20-H), 7.38–7.30 (m, 6H, C25-H + C26-H), 7.30–7.22 (m, 4H, C24-H), 7.02 (d,
3
3JHH = 8.5 Hz, 1H, C11-H), 7.00–6.93 (m, 1H, C21-H), 6.81 (d, JHH = 8.5 Hz, 1H, C10-H), 5.94 (s,
1H, C4-H), 5.60 (s, 1H, C4-H’), 4.72 (s, 2H, C12-H), 4.47–4.44 (m, 2H, C15-H), 4.43–4.40 (m, 2H,
C14-H), 2.49 (q, 3JHH = 7.4 Hz, 2H, C2-H), 1.16 (t, 3JHH = 7.4 Hz, 3H, C1-H). 13C{1H} NMR (CDCl3):
1
δ/ppm = 195.9 (C5), 167.7 (C13), 166.6 (C16), 155.5 (C9), 150.3 (C3), 140.7 (d, JCP = 27.1 Hz, C22-
H), 137.8 (d, 1JCP = 10.9 Hz, C23-H), 134.7 (C21), 134.0 (d, 2JCP = 20.5 Hz, C24-H), 133.7 (C8), 132.5
3
(C20), 131.5 (C7), 130.9 (C18), 128.8 (C26), 128.7 (C4), 128.6 (d, JCP = 6.7 Hz, C25), 128.5 (C19),
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127.0 (C11), 123.3 (C6), 111.1 (C10), 66.1 (C12), 63.3 (C14), 62.4 (C15), 23.5 (C2), 12.5 (C1).
31P{1H} NMR (CDCl3): δ/ppm = – 4.5.
Synthesis of [(η6-p-cymene)RuCl2(κP-PPh2(4-C6H4CO2H))], 1.
Chart 5. Structure of 1 (numbering refers to carbon atoms).
[(η6-p-cymene)RuCl2]2 (356 mg, 0.58 mmol) and 4-(diphenylphosphino)benzoic acid (428 mg, 1.40
mmol) were dissolved in CH2Cl2 (15 mL). The resulting dark red solution was heated at reflux and the
progress of reaction was monitored by TLC. After 4 hours, the red solution was cooled to room
temperature and volatiles were removed under vacuum. The residue was dissolved in a small volume of
CH2Cl2 and loaded on top of a silica column. Impurities were eluted with Et2O; then a red band was
collected with Me2CO/EtOH (1:1 v/v). Complex 1 was finally obtained an air-stable red-brown powder
after solvent removal under vacuum (40 °C). Yield: 485 mg, 68%. Compound 1 is soluble in DMSO,
acetone and chlorinated solvents, slightly soluble in Et2O and insoluble in H2O. Crystals suitable for X-
ray analysis were obtained by slow diffusion of hexane into a CH2Cl2 solution of 1 at 4°C. Anal. calcd.
for C29H29Cl2O2PRu: C, 56.87; H, 4.77. Found: C, 56.59; H, 4.76. Mp: 160-170°C (red liquid),
decomp. at 215-220°C (black liquid). IR (solid state): ῦ/cm-1 = 3600-3200w-br, 3055m, 2962m,
2872m, 1715s, 1692s (νC16=O), 1599m, 1561w, 1483w, 1470w, 1435s, 1395m, 1373m-sh, 1318w,
1276m-br, 1217m, 1184m, 1116w, 1090s, 1058w, 1030w, 1018w, 999w, 909m, 857m, 799w, 767m,
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747m, 728m, 695s, 675s-sh. IR (CH2Cl2): ῦ/cm-1 = 1733m and 1697s (νC16=O), 1600m, 1560w. UV-Vis
(CH2Cl2): λmax/nm (ε/M−1·cm−1) = 250 (2.7·104), 375 (1.8·103), 475sh (5.2·102). Λm (CH2Cl2) = 0.31
S·cm2·mol–1. 1H NMR (CDCl3): δ/ppm = 8.03–7.99 (m, 2H, C14-H), 7.97–7.91 (m, 2H, C13-H), 7.86–
2
7.80 (m, 4H, C9-H), 7.49–7.37 (m, 6H, C10-H + C11-H), 6.30 (br, 1H, OH), 5.24 (d, JHH = 5.6 Hz,
3
2H, C4-H), 5.00 (d, 2JHH = 5.2 Hz, 2H, C3-H), 2.87 (hept, JHH = 6.7 Hz, 1H, C6-H), 1.87 (s, 3H, C1-
1
H), 1.12 (d, 3JHH = 6.9 Hz, 6H, C7-H). 13C{1H} NMR (CDCl3): δ/ppm = 170.4 (C16), 140.0 (d, JCP
=
43.7 Hz, C12), 134.3 (d, 2JCP = 11.5 Hz, C13), 134.2 (d, 2JCP = 10.0 Hz, C9), 133.1 (d, 1JCP = 45.1 Hz,
3
3
C8), 130.6–130.5 (m, C11 + C15), 129.0 (d, JCP = 9.9 Hz, C14), 128.2 (d, JCP = 9.9 Hz, C10-H),
2
2
111.3 (C5), 96.3 (C2), 88.9 (d, JCP = 2.1 Hz, C3), 87.3 (d, JCP = 5.3 Hz, C4), 30.2 (C6), 21.8 (C7),
17.7 (C1). 31P{1H} NMR (CDCl3): δ/ppm = 25.3.
Synthesis of [(η6-p-cymene)RuCl(κ2P,O-PPh2(2-C6H4CO2))], 2.
Chart 6. Structure of 2 (numbering refers to carbon atoms).
[(η6-p-cymene)RuCl2]2 (190 mg, 0.311 mmol) and 2-(diphenylphosphino)benzoic acid (232 mg, 0.757
mmol) were dissolved in CH2Cl2 (20 mL). The resulting brick red solution was heated at reflux and the
progress of reaction was monitored by TLC and 31P NMR spectroscopy. Evolution of HCl was detected
with a pH paper placed on top of the reflux condenser. After 4 hours, the orange-red solution was
cooled to room temperature and volatiles were removed under vacuum. The orange residue was
suspended in Et2O and filtered. The solid was dissolved in CH2Cl2 then iPrOH (1-2 mL) and Et2O were
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added with intense stirring. Compound 2 readily precipitated as an air-stable orange-brown powder.
The solid was filtered, washed with Et2O (3x5 mL) and dried under vacuum (40°C). Yield: 269 mg,
75%. The precipitation procedure was repeated on the filtrate solution (90% yield).
Alternative procedure: K[PPh2(2-C6H4CO2)] (74 mg, 0.213 mmol) was added to a suspension of [(η6-p-
i
cymene)RuCl2]2 (56 mg, 0.091 mmol) in PrOH (5 mL). The reaction mixture was stirred at room
temperature for 30 hours and the progress of reaction was monitored by TLC. Therefore, the orange
suspension was filtered, the colourless precipitate washed with CH2Cl2 and volatiles were removed
under vacuum from the filtrate solution. The residue was dissolved into a small volume of CH2Cl2 and
loaded on top of a silica column. An orange band was eluted with CH2Cl2:THF (progressively
decreasing v/v ratio). Complex 2 was finally obtained as an orange-brown powder after solvent removal
under vacuo (40°C). Yield: 59 mg, 57%.
Compound 2 is soluble in MeOH and DMSO, moderately soluble in chlorinated solvents and insoluble
in H2O. Crystals suitable for X-ray analysis were obtained from CHCl3 solutions of 2 layered with
hexane or Et2O and settled aside at -20°C. Anal. calcd. for C29H28ClO2PRu: C, 60.47; H, 4.90. Found:
C, 60.12; H, 5.07. Mp: decomp. at 130°C (dark brown solid). IR (solid state): ῦ/cm-1 = 3053w, 2962w,
2926w, 2869w, 1619s-sh, 1604s (νCO2,as), 1583m, 1557m, 1482m, 1469m, 1434m, 1386m, 1328s
(νCO2,s), 1284m, 1253m, 1186w, 1160w, 1143w, 1118m, 1096m, 1060m, 1030w, 998w, 888w, 846m,
800w, 747m, 728m, 694s. UV-Vis (CH2Cl2): λmax/nm (ε/M−1·cm−1) = 350 (1.5·103). Λm (CH2Cl2) =
1
0.92 S·cm2·mol–1. H NMR (CDCl3): δ/ppm = 8.23 (dd, J = 7.1, 4.5 Hz, 1H, C16-H), 7.97 (dd, J =
11.0, 7.8 Hz, 2H, C9-H), 7.68–7.63 (m, 1H, C11-H), 7.59–7.51 (m, 5H, C10-H + C10'-H + C11'-H),
7.51–7.42 (m, 3H, C9'-H + C15-H), 7.33 (t, J = 7.5 Hz, 1H, C14-H), 6.83 (dd, J = 11.1, 7.8 Hz, 1H,
C13-H), 6.08 (s-br, 1H, C4-H), 5.75 (s-br, 1H, C3-H), 5.53 (s-br, 1H, C3'-H), 5.19 (s-br, 1H, C4'-H),
3
2.78–2.65 (m, 1H, C6-H), 1.97 (s, 3H, C1-H), 1.06, 0.60 (d, JHH = 6.8 Hz, 3H, C7-H + C7'-H).
2
13C{1H} NMR (CDCl3): δ/ppm = 172.3 (C18), 138.6–138.1 (m, C17), 135.3 (d, JCP = 10.6 Hz, C9),
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1
3
2
133.2 (d, JCP = 49.3 Hz, C8'), 132.3 (d, JCP = 2.5 Hz, C16) 132.2 (d, JCP = 10.5 Hz, C9'), 132.1
(C11), 131.7 (C13), 131.1 (d, JCP = 1.5 Hz, C14 or C15), 131.0 (d, JCP = 1.7 Hz, C14 or C15), 129.6 (d,
3
3
1JCP = 44.7 Hz, C8), 129.3 (d, JCP = 9.7 Hz, C10 or C10'), 129.1 (d, JCP = 10.3 Hz, C10 or C10'),
1
128.1 (d, JCP = 49.6 Hz, C12), 108.9 (C5), 97.1 (br, C3), 92.0 (C2), 90.9 (C4), 85.0 (C3'), 84.3 (C4'),
29.7 (C6), 23.0, 19.1 (C7 + C7'), 17.7 (C1). 31P{1H} NMR (CDCl3): δ/ppm = 30.5.
Synthesis of [(η6-p-cymene)RuCl2(κP-PPh2(2-C6H4OCO-EA))], 3.
Chart 7. Structure of 3 (numbering refers to carbon atoms).
[(η6-p-cymene)RuCl2]2 (156 mg, 0.254 mmol) and L1 (358 mg, 0.636 mmol) were dissolved in CHCl3
(25 mL) and the resulting brick red solution was heated at reflux for 20 hours. The final dark brown
solution was cooled to room temperature and then volatiles were removed under vacuum. The dark
residue was suspended in Et2O and filtered. The solid was then dissolved in CH2Cl2 (4 mL), and
Et2O/hexane 2:1 v/v (30 mL) was added with intense stirring. Compound 3 precipitated as an air-stable
brown-red powder from a dark green solution. The solid was filtered, washed with hexane (3x5 mL)
and dried under vacuum (room temperature). Yield: 285 mg, 65%. Compound 3 is soluble in DMSO
and chlorinated solvents, less soluble in acetone and MeOH and insoluble in H2O. Anal. calcd. for
C41H39Cl4O4PRu: C, 56.63; H, 4.52. Found: C, 56.50; H, 4.69. Mp: decomp. at 184-188°C (black
liquid). IR (solid state): ῦ/cm-1 = 3053w, 2970w, 2934w, 2875w, 1782s (νC18=O), 1663m (νC26=O),
28

Page 29 of 45
New Journal of Chemistry
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DOI: 10.1039/C7NJ02300F
1625w, 1582m (νC27=C28), 1466m, 1436s, 1379m, 1340w, 1303m, 1264m, 1201s, 1151s, 1115m, 1074s,
1031m, 1000m, 937w, 896w, 853w, 817m, 801m, 780m, 749m, 696s. IR (CH2Cl2): ῦ/cm-1 = 1782s
(νC18=O), 1667s (νC26=O), 1587s (νC27=C28). UV-Vis (CH2Cl2): λmax/nm (ε/M−1·cm−1) = 250 (3.2·104),
1
370 (1.9·103), 470sh (6.2·102). Λm (CH2Cl2) = 0.27 S·cm2·mol–1. H NMR (CDCl3): δ/ppm = 7.75–
7.67 (m, 4H, C9-H), 7.65–7.57 (m, 1H, C14-H), 7.53–7.42 (m, 1H, C15-H), 7.39–7.23 (m, 8H, C10-H,
C11-H, C13-H + C15-H), 6.94 (d, 3JHH = 8.3 Hz, 1H, C24-H), 6.44 (d, 3JHH = 8.3 Hz, 1H, C25-H), 5.92
(s, 1H, C28-H), 5.55 (s, 1H, C28-H'), 5.31 (s, 2H, C4-H), 4.95 (s, 2H, C3-H), 4.54 (s, 2H, C19-H), 2.94
3
(hept, 3JHH = 6.3 Hz, 1H, C6-H), 2.45 (q, 3JHH = 7.2 Hz, 2H, C29-H), 1.88 (s, 3H, C1-H), 1.24 (d, JHH
3
= 6.7 Hz, 6H, C7-H), 1.13 (t, JHH = 7.4 Hz, 3H, C30-H). 13C{1H} NMR (CDCl3): δ/ppm = 195.9
2
(C26), 165.9 (C18), 155.2 (C20), 151.5 (C17), 150.3 (C27), 135.5 (d, JCP = 6.9 Hz, C13), 134.6 (d,
2JCP = 9.7 Hz, C9), 133.5 (C21), 132.5 (C15), 131.1 (C22), 130.7 (d, 1JCP = 43.4 Hz, C8), 130.5 (C11),
128.6 (C28), 128.0 (d, 3JCP = 10.3 Hz, C10), 127.2 (d, 1JCP = 39.8 Hz, C12), 127.1 (C24), 125.9 (d, 3JCP
3
2
= 8.3 Hz, C14), 123.5 (d, JCP = 3.4 Hz, C16), 122.9 (C23), 112.4 (d, JCP = 3.6 Hz, C5), 111.5 (C25),
2
97.8 (C2), 87.9 (d, JCP = 3.4 Hz, C4), 87.7 (C3), 66.6 (C19), 30.5 (C6), 23.5 (C29), 22.1 (C7), 18.2
(C1), 12.5 (C30). 31P{1H} NMR (CDCl3): δ/ppm = 25.0.
Synthesis of [(η6-p-cymene)RuCl2(κP-PPh2(4-C6H4CO2CH2CH2OCO-EA))], 4.
Chart 8. Structure of 4 (numbering refers to carbon atoms).
29