Synthesis and antiproliferative activity of the heterobimetallic complexes [RuClCp(PPh3)-μ-dmoPTA-1κP:2κ2N,N′- MCl2] (M = Co, Ni, Zn; DmoPTA = 3,7-dimethyl-1,3,7-triaza-5- phosphabicyclo[3.3.1]nonane)

Article abstract of DOI:10.1039/c3dt50829c

The synthesis and characterization of three novel heterobimetallic derivatives of general formula [RuClCp(PPh₃)-μ-dmoPTA-1κP: 2κ²N,N′-MCl₂] (M = Co (1), Ni (2) and Zn (3), dmoPTA = 3,7-dimethyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) are described. The sizes of their diffusing units are in agreement with the solid-state structures of 2 and 3, supporting a similar solid-state structure for complex 1. The diamagnetic ruthenium-zinc derivative 3 was fully characterized in solution at 193 K by NMR. Electrochemical properties of the complexes were investigated by cyclic voltammetry, supporting the influence of the {MCl₂} moiety on the electronic properties of the Ru-unit. For the three complexes the GI ₅₀ values were in the range 0.8-6.5 μM and comparable to those of the standard anticancer drug cis-platin in the same cell lines.

Full text of DOI:10.1039/c3dt50829c

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PAPER
Synthesis and antiproliferative activity of the
heterobimetallic complexes [RuClCp(PPh3)-
μ-dmoPTA-1κP:2κ2N,N’-MCl2] (M = Co, Ni, Zn; dmoPTA
= 3,7-dimethyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]-
nonane)†
Cite this: Dalton Trans., 2013, 42, 11212
Manuel Serrano-Ruiz,a Luis Manuel Aguilera-Sáez,a Pablo Lorenzo-Luis,b
José M. Padrónc and Antonio Romerosa*a
The synthesis and characterization of three novel heterobimetallic derivatives of general formula
[RuClCp(PPh3)-μ-dmoPTA-1κP:2κ2N,N’-MCl2] (M = Co (1), Ni (2) and Zn (3), dmoPTA = 3,7-dimethyl-1,3,7-
triaza-5-phosphabicyclo[3.3.1]nonane) are described. The sizes of their diffusing units are in agreement
with the solid-state structures of 2 and 3, supporting a similar solid-state structure for complex 1. The
diamagnetic ruthenium–zinc derivative 3 was fully characterized in solution at 193 K by NMR. Electro-
chemical properties of the complexes were investigated by cyclic voltammetry, supporting the influence
of the {MCl2} moiety on the electronic properties of the Ru-unit. For the three complexes the GI50 values
were in the range 0.8–6.5 μM and comparable to those of the standard anticancer drug cis-platin in the
same cell lines.
Received 27th March 2013,
Accepted 23rd May 2013
DOI: 10.1039/c3dt50829c
www.rsc.org/dalton
1. Introduction
The wide coordination possibilities and interesting properties
of the water soluble phosphines1 have induced us to focus
our research activity during the last few years in synthesizing
new water soluble complexes containing this kind of ligand.
Among the known aqua-soluble phosphines we have been
interested in studying the coordinative properties of the air
stable and economically obtained ligand 1,3,5-triaza-7-phos-
phaadamantane (PTA) and its derivatives,2 some of them
(Scheme 1) featuring high water solubility and interesting
chemical and physical properties.3
The interesting ligand dmPTA was synthesized by us only a
few years ago along with the new water-soluble phosphine
ligands dmoPTA and HdmoPTA (dmPTA = N,N′-dimethyl-1,3,5-
triaza-7-phosphaadamantane; dmoPTA = 3,7-dimethyl-1,3,7-
triaza-5-phosphabicyclo[3.3.1]nonane; HdmoPTA = 3,7-H-3,7-
Scheme 1 PTA molecule and some of its derivatives.
aÁrea de Química Inorgánica, Facultad de Ciencias Experimentales, Universidad
de Almería, 04071 Almería, Spain. E-mail: romerosa@ual.es
bDepartamento de Química Inorgánica, Facultad de Química, Universidad de La
Scheme 2 Ligands dmPTA, HdmoPTA and dmoPTA.
Laguna, La Laguna, Tenerife, Spain. E-mail: plorenzo@ull.es
cBioLab, Instituto Universitario de Bio-Orgánica “Antonio González” (IUBO-AG)
Universidad de La Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La Laguna,
Spain. E-mail: jmpadron@ull.es
†CCDC 929129 (2) and 929128 (3). For crystallographic data in CIF or other elec-
tronic format see DOI: 10.1039/c3dt50829c
dimethyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) (Scheme 2).
These ligands showed interesting coordinative properties pro-
viding a way to obtain heterometallic complexes in which it is
11212 | Dalton Trans., 2013, 42, 11212–11219
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possible to combine the properties of different metal centres 270 and 150 ppm, respectively, by reference to the same
such as in [RuClCp(PPh3)-μ-dmoPTA-1κP:2κ2N,N′-M(acac-κ2O,- signals in the starting complex [RuClCp(HdmoPTA)(PPh3)]-
O′)2] (M = Co, Ni, Zn).4
(OSO2CF3) (δP = −2.93 ppm),4c which are much more larger
Remarkably, the complexes containing dmoPTA have chemical shifts than those observed for the bimetallic com-
shown a better anticancer activity than cis-platin as well as in pounds containing acac-ligand (ca. 50 ppm).4a,b For the same
cell lines sensitive to this complex.5 To have more information reason the 1H NMR of dmoPTA were observed as broad un-
on the influence of the second metal bonded to the dmoPTA resolved peaks in an extremely wide spectral region (1: 140 to
in the chemical properties of the {RuCpCl(dmoPTA)(PPh3)}- −15 ppm; 2: 70 to −5 ppm). It was not possible to obtain
moiety, the new bimetallic complexes [RuClCp(PPh3)- enough quality 13C{1H} NMR spectra for complexes 1 and 2 as
μ-dmoPTA-1κP:2κ2N,N′-MCl2] (M = Co (1), Ni (2) and Zn (3)) they are not soluble enough in the solvents used and also
were synthesized, containing the simple and more reactive probably by the effect on the signals of the carbons closer to
{MCl2}-unit. These complexes were fully characterized and the paramagnetic metal. In contrast, well-shaped and resolved
their anticancer activity assessed on a representative panel of signals were observed in the 1H NMR spectrum of the diamag-
human solid tumor cell lines.
netic complex 3.
We monitored the spin echo intensity of selected PPh3
signals that are briefly influenced by the paramagnetism of
the metal bonded to dmoPTA-NMe atoms. The experiments
were carried out in CDCl3 as this is the solvent in which the
three compounds are soluble and stable. The structurally
related [RuClCp(PTA)(PPh3)]6 derivative was used as a bench-
mark as well as the parent bimetallic complexes [RuClCp-
(PPh3)-μ-dmoPTA-1κP:2κ2N,N′-M(acac-κ2O,O′)2] (M = Co (a), Ni
(b), Zn (c)).4a Calculated diffusion coefficients (D) are given in
Table 1.7 The obtained diffusion coefficients for a–c are
similar while a bigger diffusion coefficient for the benchmark
complex [RuClCp(PTA)(PPh3)] was observed. As far as the
hydrodynamic radii (rH) are concerned, they were calculated by
using the Stokes–Einstein equation (arbitrarily assuming a
spherical shape for the diffusing species) and are also listed in
Table 1.7 Several studies on the hydrodynamic dimensions of
the molecules have been carried out, including metal com-
plexes with the PTA-molecule,8 indicating the convenience of
this kind of study to determine the behavior and dimensions
of the complexes in solution. Interestingly, binuclear com-
plexes 1–3 exhibit very similar rH in agreement with the strong
similarity solid-state structures of 2 and 3, and regardless of
the nature of the metal bonded to the dmoPTA-NMe atoms.
The coefficient diffusion of the starting complex [RuClCp-
(HdmoPTA)(PPh3)](OSO2CF3) is bigger than that for complex 1
2. Results and discussion
2.1. Synthesis and characterization of 1–3
The bimetallic complexes 1–3 were synthesized by a similar
procedure (Scheme 3).
The proton between the two HdmoPTA-NMe atoms in
[RuClCp(HdmoPTA)(PPh3)](OSO2CF3) was removed with pot-
assium tert-butoxide and the resulting deprotonated complex
[RuClCp(dmoPTA-κP)(PPh3)] reacted with MCl2 (M = Co, Ni
and Zn) to give rise 1, 2 and 3, respectively. The single crystal
X-ray structures of 2 and 3 (vide infra) show that the {RuClCp-
(dmoPTA-κP)}-moiety is bonded through its dmoPTA-NMe
atoms to the metal of {NiCl2} and {ZnCl2}-units, respectively.
The properties of the complexes 1–3 are in agreement with
those of the previously studied bimetallic complexes [RuClCp-
(PPh3)-μ-dmoPTA-1κP:2κ2N,N′-M(acac-κ2O,O′)2] (M = Co (a), Ni
(b), Zn (c)).4a,b In particular, for complexes 1–3 it is also clear
that the ruthenium atom of the {RuCpCl(dmoPTA)(PPh3)}-
moiety is sensitive to the metal bonded to the dmoPTA-NMe
.
Additionally, the comparison of the properties of 1–3 with
those of bimetallic complexes containing acac-ligand also
suggests that the ancillary ligands on the metal unit have
some influence on the ruthenium atom.
NMR spectroscopic studies of 1–3 show that solid-state
structures are preserved in solution. Indeed the 31P{1H} NMR
spectra of the complex 3 shows two doublets with 2JPP in agree-
ment with the pseudo-cis arrangement of the dmoPTA and
PPh3 around the Ru-center. The 31P{1H} NMR for PPh3 reson-
ance arises at about 50 ppm while that for dmoPTA is a broad
and unresolved signal in both 1 and 2, the complexes contain-
ing the paramagnetic metals. The chemical shift for
dmoPTA-P atom of 1 and 2 were low field shifted of more than
but smaller than those of complexes
2 and 3. As a
Table 1 Diffusion coefficient (D) and hydrodynamic radii (rH) for 1–3 com-
plexes, [RuClCp(PPh3)-μ-dmoPTA-1κP:2κ2N,N’-M(acac-κ2O,O’)2] [M = Co (a), Ni
(b), Zn (c)], [RuClCp(HdmoPTA)(PPh3)](OSO2CF3) and [RuCpCl(PPh3)(PTA)]a
Complex
D (× 106 cm2 s−1
)
rH (Å)
1
2
3
a
b
c
6.15
6.68
7.15
6.81
6.92
6.82
6.28
7.26
6.5
6.0
5.6
5.9
5.8
5.9
6.4
5.5
[RuClCp(HdmoPTA)(PPh3)]+
[RuCpCl(PTA)(PPh3)]
a Measurements were obtained in CDCl3 0.02 M at 298 K (viscosity
η = 0.543 Kg m−1 s−1 at 298 K, from http://www.bruker-biospin.com).
The experimental error was estimated to be 2%.7
Scheme 3 Synthetic procedure for 1–3.
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consequence, the hydrodynamic radii of the starting complex
is somewhat smaller than that for 1 but significantly larger
than those for complexes 2 and 3.
Complex 3 was fully characterized by NMR in order to
compare its structural behavior in solution with the parent
complex [RuClCp(PPh3)-μ-dmoPTA-1κP:2κ2N,N′-Zn(acac-κ2O,-
1
O′)2].4a The H and 31P{1H} NMR spectra at room temperature
and at −60 °C showed that there was no exchange between the
ligands around the metal center {ZnCl2}, in contrast to that
found for the parent complex containing the {Zn(acac)2}-
moiety. The 1H,1H COSY and 1H,13C HSQC spectra of 3 were
necessary for reaching the correct assignment of the signals
1
in H NMR and 13C{1H} NMR spectra. In solution the NMR of
Fig. 2 Perspective view of the isostructural complexes 2 and 3, showing the
three six-membered rings around the metal with a pseudo-chair conformation.
For the sake of clarity, only methyl hydrogen atoms are included. Dashed lines
represent the significant intermolecular interactions.
complex 3 is more complicated than expected as one of the
dmoPTA ligands has lost the C2 symmetry.
2.2. Electrochemical properties
Table 2. The Cp-rings are essentially planar, the largest devi-
ation from the overall-plan-Cp being 0.0071 Å (C52) for 2 and
0.0150 Å (C53) for 3. The angle between the Cpcent plane and
the P1–Ru1–P2 plane was found to be 53.8(8)° for 2, which is
virtually identical to that observed in 3 (53.7(2)°), but consider-
ably shorter than in the bimetallic complexes [RuClCp(PPh3)-
μ-dmoPTA-1κP:2κ2N,N′-M(acac-κ2O,O′)2] (M = Co, Ni or Zn)4a,b
(range 56.62(4)–56.85(0) Å, mean value 56.73°). The Ru1–Cpcent
distance is 1.853(2) Å for 2 and 1.845(5) Å for 3, near that
expected for similar complexes containing {M(acac)2} moieties
(range 1.836(1)–1.855(1) Å, mean value 1.843 Å).4a,b The rest of
the bond distances found are in agreement with other piano
stool complexes described by our research group.4,9 Besides
the pseudo-octahedral geometry around the ruthenium atom,
two six-membered rings [(M–N–C–P–C–N) + (M–N–C–N–C–N)]
are formed upon chelation of the {RuClCp(PPh3)(dmoPTA-
1κP)}-entity to the Ni or Zn centers (Fig. 2). The six-membered
rings around the metal, with a pseudo-chair conformation, are
completed with two coordinated chloride atoms as additional
ligands. In regard to the coordination of the metal, a little dis-
tortion from the tetrahedral geometry is represented by the
spread of the Cl–M–NMe-dmoPTA angles between 105.3(6)° for
2 and 112.3(1)° for 3 (Table 2). Besides the bond lengths
between dmoPTA-NMe and Ni or Zn atoms are significantly
shorter (ca. 0.2 Å) than that observed in the bimetallic com-
plexes (2.182(6)–2.278(7) Å, mean value 2.226 Å)4a,b probably
As observed for parent complexes [RuClCp(PPh3)-μ-dmoPTA-
1κP:2κ2N,N′-M(acac-κ2O,O′)2] (M = Co (a), Ni (b), Zn (c)),4a the
dichloride-metal moiety coordinated to the dmoPTA-NMe
atoms has a clear influence on the electrochemical properties
of {RuClCp(dmoPTA)} (Fig. 1). Complex 1 shows one reversible
ruthenium-centred monoelectronic wave at 840(5) mV (ΔEp
16 mV, icat/ian = 1; ian2/ν vs. ν constant). Similarly, complexes 2
and 3 display redox waves at 770(5) mV (ΔEp = 25 mV, icat/ian
=
=
1; ian2/ν vs. ν constant) and 798(5) mV (ΔEp = 22 mV, icat/ian = 1;
ian2/ν vs. ν constant) indicating that these processes are also
reversible and can reasonably be attributed to the Ru(II)/Ru(III)
couple. Under similar experimental conditions,4a reversible
one-electron processes were observed for [RuClCp(HdmoPTA)-
(PPh3)](OSO2CF3) at 710 mV and [RuClCp(PPh3)-μ-dmoPTA-
1κP:2κ2N,N′-M(acac-κ2O,O′)2] at 915 mV (M = Co), 812 mV (M =
Ni) and 900 mV (M = Zn).
Therefore the different metals bonded to the dmoPTA-NMe
atoms but also the ligands bonded to this metal influence the
electrochemical properties of the Ru atom.
2.3. Crystal structures of 2 and 3
A perspective view of isostructural complexes 2 and 3 is shown
in Fig. 2. Selected bond distances and angles are reported in
Table 2 Selected bond lengths [Å] and angles [°] for 2 and 3
2
3
Ru1–P1
Ru1–P2
Ru1–Cl1
Ru1–Cpcent
Cp–ML2
Ni–N41
Ni–N42
Cl2–Ni–N41
Cl2–Ni–N42
Cl3–Ni–N42
2.297(7)
2.279(7)
2.445(7)
1.853(2)
53.8(8)
2.008(2)
2.028(2)
108.9(6)
105.3(6)
109.6(6)
Ru1–P1
Ru1–P2
Ru1–Cl1
Ru1–Cpcent
Cp–ML2
Zn–N1
2.295(1)
2.276(2)
2.447(2)
1.845(5)
53.7(2)
2.081(4)
2.067(5)
110.4(1)
108.9(1)
112.3(1)
Zn–N2
Cl2–Zn–N1
Cl3–Zn–N1
Cl3–Zn–N2
Fig. 1 Cyclic voltammograms in CH2Cl2 of 1 (black line), 2 (red line) and 3
(green line).
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due to the trans influence of the acac-ligand. The dmoPTA modulating the antiproliferative activity of the complexes.
torsion angles for both complexes lie in the range 53.8(6)– Complex 3 displayed the smallest cell activity but also the
55.9(3)°. The mean value (54.9°) is somewhat greater than smallest diffusion radius as well as being the only one stable
those observed for complexes containing acac-ligand (52.2(1)– in DMSO–H2O. At present, the mechanism of action of these
54.9(2)°, mean value 53.7°),4a,b and significantly less than compounds remains unclear. Ongoing studies will clarify this,
dmPTA (58.0°) and HdmoPTA (av. 56.8°).4c In 2 and 3 the inter- and will be reported elsewhere, as for example studies focused
molecular interactions play a precise role in supporting the on providing information on the interaction of the complexes
network as responsible for the crystal packing, due to close with purines and the synthesis of parent bimetallic complexes
contacts between the hydrogen on the PPh3 and dmoPTA- containing antiproliferative active metals such as Pt.
groups and the chloride atoms on the coordination of the
metals (H⋯Cl, 2.800–2.896 Å, Fig. 2). These close contacts are
3. Conclusions
Three novel bimetallic [RuClCp(PPh3)-μ-dmoPTA-1κP:2κ2N,N′-
MCl2] (M = Co (1), Ni (2), Zn (3); dmoPTA = 3,7-dimethyl-1,3,7-
less than the sum of the van der Waals radii H (1.20 Å) and
Cl (1.75 Å).4,9
2.4. Antiproliferative activity
triaza-5-phosphabicyclo[3.3.1]nonane) complexes showed
a
similar size by diffusing experiments in CDCl3 in agreement
with their solid-state structures. The ruthenium–zinc derivative
3 is not involved in an equilibrium with other species in solu-
tion, preserving its solid state structure. The electrochemical
properties of the {RuClCp(dmoPTA)(PPh3)}-moiety is clearly
dependent on the {MCl2}-unit bonded to the dmoPTA-NMe
atoms. Comparison of the obtained results with the electrochem-
ical properties found for previously published parent com-
plexes [RuClCp(PPh3)-μ-dmoPTA-1κP:2κ2N,N′-M(acac-κ2O,O′)2]
(M = Co (a), Ni (b), Zn (c)) confirms the suspicion that the
coordination of a metal to the dmoPTA-NMe atoms leads to a
new PTA-resembling ligand whose properties can be tuned by
the adequate selection of the metal, its oxidation state and the
ancillary ligands. The GI50 values obtained for the three com-
plexes were in the range 0.8–6.5 μM. Although there are small
differences in activity those for 1 and 2 are larger than that for
3. The obtained results support that the Ru(II)-moiety is the
most antiproliferative active part of the molecule. At present,
the mechanism of action of these compounds remains unclear
but efforts are targeted to clarify it and to synthesize new
bimetallic complexes with possibly improved antiproliferative
properties.
The antiproliferative activity of compounds 1–3 was studied in
the panel of human solid tumour cells HBL-100, HeLa,
SW1573, and WiDr. The results expressed as GI50 (Table 3)
were obtained using the SRB assay10 after 48 hours of exposure
to the drugs. Overall, the data show that all complexes induce
growth inhibition in all cell lines with GI50 values in the range
0.8–6.5 μM. When compared to the reference drug cis-platin,
compounds 1–3 showed a superior activity profile. In fact, the
Ru(II) complexes were more active against the breast T-47D and
the colon WiDr cells, which were less affected by the exposure
to cis-platin. This observation is consistent with previous
studies in which these cells proved to be more drug resistant.5
The results point out that the three compounds are active in
both drug-sensitive (HBL-100) and drug-resistant (T-47D and
WiDr) cell lines. Although the differences in activity are small
we can observe that compounds 1 and 2 show more potent
activity than the ruthenium–zinc complex 3. Complexes 1 and
2 are transformed quickly in DMSO–water to provide the
complex5 [RuClCp(HdmoPTA)(PPh3)]+ while 3 is stable enough
over the antiproliferative experiment time. Therefore it can be
assumed that the observed anticancer activity of 1 and 2 is
really that for the [RuClCp(HdmoPTA)(PPh3)]+ complex. The
parent bimetallic complexes containing the acac-ligand, which
are stable in a mixture DMSO–water, and the complex [RuClCp-
4. Experimental section
4.1. Materials and methods
(HdmoPTA)(PPh3)](OSO2CF3) showed
activity.5
a somewhat smaller
This fact supports that the main antiproliferative active site All chemicals were reagent grade and, unless otherwise stated,
in the bimetallic complexes is the ruthenium atom but the were used as received by commercial suppliers. All reactions
influence of the other metal unit must not be excluded, were carried out in a pure argon atmosphere by using standard
Table 3 GI50 values of complexes 1–3, [RuClCp(HdmoPTA)(PPh3)]+ and cis-platin against human solid tumor cells
Cell line (type)a
Compound
HBL-100 (breast)
T-47D (breast)
SW1573 (lung)
HeLa (cervix)
WiDr (colon)
1
2
1.0 ( 0.2)
0.8 ( 0.2)
3.5 ( 0.7)
—
1.4 ( 0.2)
1.2 ( 0.2)
6.5 ( 3.0)
1.9 ( 0.5)
1.5 ( 2.3)
1.1 ( 0.1)
1.0 ( 0.1)
4.6 ( 0.1)
1.5 ( 0.1)
3.0 ( 0.4)
1.3 ( 0.1)
1.4 ( 1.1)
4.0 ( 0.1)
2.6 ( 0.2)
2.0 ( 0.3)
1.5 ( 0.3)
1.1 ( 0.2)
5.7 ( 1.4)
1.7 ( 0.4)
2.6 ( 5.3)
3
[RuClCp(HdmoPTA)(PPh3)]+
cis-platin
1.9 ( 0.2)
a Values are given in μM and are means of three experiments ( standard deviation).
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Schlenk-tube techniques with freshly distilled and oxygen-free
solvents.
31P{1H} NMR (121.49 MHz, 22 °C, DMSO-d6): δ(ppm) 54.12
(bs, PPh3), 220 (vbs, dmoPTA). UV-vis (solid state, 22 °C)
The hydrosoluble phosphine-PTA and the complexes λmax/nm: 370 (s), 553 (m), 574 (m), 627 (m), 901 (w), 1275 (m).
[RuClCp(PTA)(PPh3)] and [RuClCp(HdmoPTA)(PPh3)] (OSO2CF3) UV-vis (CHCl3, 22 °C) λmax/nm (ε/dm3 mol−1 cm−1): 241
were prepared as described in the literature.4,6,9 1H and (16 420), 329 (2512), 358 (2000), 418 (864), 549 (147), 568 (229),
13C{1H} NMR spectra were recorded on Bruker AV300 and 632 (411). Cyclic voltammetry (CH2Cl2, 22 °C): Eox = 0.92 mV;
AV500 spectrometers operating at 300.13 and 500.13 MHz (1H), Ered = 0.76 mV; E1/2 = 0.84 mV.
1
1
respectively. The H,1H 2D COSY and H,13C{1H} HSQC NMR
experiments were routinely conducted on the Bruker DRX300 Yield: 0.250 g, 64%. S25 °C, CHCl = 16.7 mg mL−1, S25 °C, H O
instruments in the absolute magnitude mode using a 45 or 1.0 mg mL−1
Elemental analysis for powder sample
4.1.3. [RuClCp(PPh3)-μ-dmoPTA-1κP:2κ2N,N′-NiCl2]
(2).
=
3
2
.
90° pulse after the increment delay. Peak positions are relative C30H36N3P2Cl3RuNi (766.7 g mol−1): Found C 46.98; H 4.70; N
to tetramethylsilane and were calibrated against the residual 5.62%; calcd C 46.99; H 4.73; N 5.48%. IR (KBr, cm−1): ν(CarH)
solvent resonance (1H) or the deuterated solvent multiplet 3071, 3057; ν(CH) 2961, 2915, 2861; δas(CH) 1434 (m); ν(C–N)
(13C{1H}). Chemical shifts for 31P{1H} NMR spectra were 1029 (m), 1071 (m); δoop(CarH) 757 (m), 745 (m); δoop(CvCar)
measured relative to external 85% H3PO4. Infrared spectra 696 (s). 1H NMR (300.13 MHz, 22 °C, CDCl3): δ (ppm)
were recorded on KBr disks using a Bruker Vertex 70 FT-IR 7.35–7.47 (bs, PPh3, 15H), 7.57 (bs, Cp, 5H), 60–74 (bm,
spectrometer, and the intensity of the bands is indicated as: dmoPTA, 16H). 13C{1H} NMR (75.47 MHz, 22 °C, CDCl3):
s (strong), m (medium) and weak (w). UV-vis spectra were δ(ppm) 80.3 (s, Cp), 128–139 (m, PPh3). 31P{1H} NMR
2
obtained with a Jasco V-650 spectrometer from solutions of (121.49 MHz, 22 °C, CDCl3): δ(ppm) 47.3 (d, JPP = 40.5 Hz,
10−3–10−5 M in quartz cuvettes (1 cm optical path). Specific PPh3), 152 (bm, dmoPTA). 31P{1H} NMR (121.49 MHz, 22 °C,
absorption coefficients were obtained by linear regression over DMSO-d6): δ(ppm) 49.38 (bs, PPh3), 81 (vbs, dmoPTA). UV-vis
5–7 points (R2 ≥ 0.999). Elemental analyses (C, H, N, S) were (solid state, 22 °C) λmax/nm: 373 (s), 840 (m), 851 (m), 882 (m),
performed on
analyzer.
a
Fisons Instrument EA 1108 elemental 948 (m). UV-vis (CHCl3, 22 °C, nm) λmax/nm (ε/dm3 mol−1
cm−1): 242 (21 289), 336 (2345), 359 (2418), 417 (987). Cyclic
4.1.1. Synthesis of [RuClCp(PPh3)-μ-dmoPTA-1κP:2κ2N,N′- voltammetry (CH2Cl2, 22 °C): Eox = 0.908 mV; Ered = 0.688 mV;
MCl2] (M = Co (1), Ni (2), Zn (3)). Complexes 1–3 were E1/2 = 0.798 mV.
obtained by using a similar procedure. Potassium tert-butoxide
4.1.4. [RuClCp(PPh3)-μ-dmoPTA-1κP:2κ2N,N′-ZnCl2]
(3).
=
(0.051 g, 0.456 mmol for 1, 3; 0.059 g, 0.525 mmol for 2) was Yield: 0.235 g, 69%. S25 °C, CHCl = 16.7 mg mL−1, S25 °C, H O
3
2
added into a solution of [RuClCp(HdmoPTA)(PPh3)](OSO2CF3) 1.0 mg mL−1. Elemental analysis C30H36N3P2Cl3RuZn (773.39 g
(0.359 g, 0.456 mmol for 1; 0.411 g, 0.522 mmol for 2; 0.356 g, mol−1): Found C 46.33; H 4.86; N 5.31%; calcd C 46.59; H 4.70;
0.453 mmol for 3) in 150 mL of EtOH. The resulting mixture N 5.43%. IR (KBr, cm−1): ν(CarH) 3071, 3057; ν(CH) 2961 (m),
was stirred at room temperature. When the reagents were com- 2915 (m), 2861 (m); δas(CH) 1434 (m); ν(C–N) 1029 (m), 1071
1
pletely dissolved, the respective metal chloride (CoCl2: 0.058 g, (m); δfdp(CarH) 757 (m), 745 (m); δfdp(CvCar) 696 (s). H NMR
0.444 mmol for 1; NiCl2·6H2O: 0.123 g, 0.517 mmol for 2; (500.13 MHz, 22 °C, CDCl3): δ (ppm) 7.38–7.56 (m, aromatic
2
ZnCl2: 0.060 g, 0.440 mmol for 3) was added. The resulting protons, 15 H), 4.53 (s, Cp, 5H), 4.38 (d, JHiHc = 12.2 Hz, Hi),
2
2
mixture was refluxed for 3 h for 1 and 3 while 6 h were needed 4.33 (d, JHgHb = 12.1 Hz, JHjHe = 12.1 Hz, Hg y Hj), 4.1
2
4
4
for synthesizing 2 in good yield. Then, the solvent was reduced (ABEFX system, JHhHd = 13.7 Hz, JHhHa = 1.85 Hz, JHhHj
=
2
2
to 5 mL at room temperature under vacuum. The obtained 1.1 Hz, JPH = 3.4 Hz, Hh), 3.58 (d, JHcHi = 12.1 Hz, Hc), 3.56
green (1), brown (2) and yellow (3) powders were filtered, (ABX system, JHbHg = 13.9 Hz, JPH = 2.2 Hz, Hb), 3.33 (ABEX
washed with cold diethyl ether (2 × 5 mL) and vacuum dried. system, JHaHf = 15.3 Hz, JHaHh = 1.8 Hz, JPH = 2.55 Hz, Ha),
The obtained solids were dissolved into 10 mL of CHCl3 and 3.08 (d, JHeHj = 11.9 Hz, He), 2.75 (ABEX system, JHdHh
2
2
2
4
2
2
2
=
4
2
filtered through Celite. The solutions were concentrated to 13.8 Hz, JHdHb = 1.3 Hz, JPH = 2.3 Hz, Hd), 2.351–2.461 (s + s,
2
4
2 mL and then 10 mL of EtO2 were added. The precipitates CH3N, 6H), 1.79 (ABEX system, JHfHa = 15.3 Hz, JHfHg
=
2
were filtered and dried under vacuum.
2.1 Hz, JPH = 3.4 Hz, Hf). 13C{1H} NMR (75.47 MHz, 22 °C,
4.1.2. [RuClCp(PPh3)-μ-dmoPTA-1κP:2κ2N,N′-CoCl2]
Yield: 0.255 g, 75%. S25 °C,CHCl = 12.5 mg mL−1, S25 °C,H O
(1). CDCl3): δ (ppm) 44.9 (d, JCP = 4.8 Hz, NMe′), 44.6 (d,
3
1
=
3JCP = 4.9 Hz, NMe′′), 45.1 (d, JCP = 21.0 Hz, C1), 58.2
3
2
1.0 mg mL−1. Elemental analysis C30H36N3P2Cl3RuCo (766.94 g (dd, JCPdmoPTA = 14.5 Hz, JCPPPh3 = 4.8 Hz, C2), 58.5 (d,
1
3
mol−1): Found C 46.72; H 4.71; N 5.39%; calcd C 46.98; H 4.73; 1JCPdmoPTA = 13.4 Hz, JCPPPh3 = 1.2 Hz, C3), 74.9 (d, JCP
=
3
3
N 5.48%. IR (KBr, cm−1): ν(ArH) 3071, 3057; ν(CH) 2961, 2915, 2.9 Hz, C5), 75.3 (d, JCP = 2.8 Hz, C4), 79.8 (t, JCP = 2.2 Hz,
3
2
2861; δas(CH3), δ (CH2) 1434 (m); ν(C–N) 1029 (m), 1071 (m); Cp), 128–138.1 (m, PPh3). 31P{1H} NMR (121.49 MHz, 22 °C,
δfdp(CarH) 757 (m), 745 (m); δfdp(CvCar) 696 (s). 1H NMR CDCl3): δ (ppm) 2.56 (d, JPP = 41.7 Hz, dmoPTA), 46.4 (d,
2
(300.13 MHz, 25 °C, CDCl3): δ (ppm) 7.0–7.6 (bs, PPh3, 15 H), 2JPP = 41.7 Hz, PPh3). 31P{1H} NMR (121.49 MHz, 22 °C, DMSO-
10 (bs, Cp, 5 H), 94–138 (bm, dmoPTA, 16 H). 13C{1H} NMR d6): δ(ppm) 47.01 (d, JPP = 44 Hz, PPh3), −1.49 (d, JPP
=
2
2
(75.47 MHz, 22 °C, CDCl3): δ (ppm) 92.8 (s, Cp), 128–139 (m, 44 Hz, dmoPTA). The labels for NMR assignation are showed
aromatic). 31P{1H} NMR (121.49 MHz, 22 °C, CDCl3): δ (ppm) in Scheme 4. UV-vis (solid state, 22 °C) λmax/nm: 374. UV-vis
50.28 (d, 2JPP = 33.3 Hz, PPh3), 277 (bs, dmoPTA).
(CHCl3, 22 °C) λmax/nm (ε/dm3 mol−1 cm−1): 241 (15 061), 336
11216 | Dalton Trans., 2013, 42, 11212–11219
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coefficients of >0.999 and 6–8 points were used for regression
analysis. The value for δ parameter was set to 1.75–2 ms.
Additionally three different measurements with different
diffusion parameters (Δ = 25–100 ms) were always carried out
to check the reproducibility of the data obtained. The gradient
strength was increased in 10–15% steps from 10%. A measure-
ment of 1H NMR spectrum and T1 was carried out before
each diffusion experiment and the recovery delay (ca. 5–8 s) set
to five times T1.
Scheme 4
The number of scans varied between 32 and 64 per
increment. Typical experiment duration is 1.5–3 h.
(1866), 360 (1927), 421 (741). Cyclic voltammetry (CH2Cl2,
22 °C): Eox = 0.899 mV; Ered = 0.65 mV; E1/2 = 0.77 mV.
4.5. Cyclic voltammetry experiments
4.2. Stability of 1–3 in CDCl3
Electrochemical experiments were recorded with a VersaSTAT3
apparatus. A standard disposition for the measurement cell
was used including three-electrode glass cell consisting of
a platinum-disk working electrode, a platinum-wire auxiliary
electrode and an Ag reference electrode. The supporting
electrolyte solution (NBu4PF6, 0.1 M) was scanned over the
solvent window to verify the absence of electro-active
impurities. A similar concentration of the analyte (2.5 10–4 M)
was employed in all the measurements and HPLC grade
CH2Cl2 as solvent.
Into a 5 mm NMR tube were dissolved 10 mg of 1–3 under N2
atmosphere in 0.5 mL of CDCl3. The solution was recorded by
1H and 31P{1H} NMR at room temperature and 40 °C. After
2 days no significant decomposition of the complexes was
observed at both temperatures and then air was bubbled into
the solutions. The compounds were fully decomposed after
3 days at room temperature and 1 day at 40 °C.
4.3. Stability of 1–3 with DMSO and water
The complexes (10 mg) were dissolved in a mixture of 0.5 mL
of DMSO-d6 and the resulting solution studied by NMR
at room temperature and 40 °C. The three complexes were
stable at room temperature for more than 1 day while
some decomposition (<5%) is observed at 40 °C after 12 h.
Nevertheless, when 0.1 mL of H2O was introduced in the solu-
tion, 1 was decomposed quickly at room temperature (5 min)
while 2 needed more than 0.2 mL of H2O to be decomposed
within 10 min and 3 decomposed partially (<20%) with 0.5 mL
after 15 h. The complete decomposition of 3 was achieved
after 3 days at room temperature and within 2 days at 40 °C.
The resulting final compound obtained from the three com-
plexes was characterized by NMR as the ruthenium complex
[RuClCp(HdmoPTA)(PPh3)], which is stable under the reaction
conditions for more than 3 days. The UV-vis absorption
analysis of the resulting solutions showed the absorption
bands of CoCl2 (1) and NiCl2 (2) in water.11
4.6. X-ray analysis
Single crystals with suitable dimensions were mounted on
a glass fiber with cyanoacrylate at room temperature. Data
collection was performed on a Bruker APEX diffractometer
(XDIFRACT service of the University of Almeria) in the range
4.668 ≤ 2θ ≤ 43.593 and 4.682 ≤ 2θ ≤ 50.03 for 2 and 3. Data
were collected at 100(2) K using graphite-monochromatized
Mo-Kα (λ = 0.71073) in the range −17 ≤ h ≤ 17, −11 ≤ k ≤ 11,
−26 ≤ l ≤ 20 for 2 and −14 ≤ h ≤ 17, −11 ≤ k ≤ 11, −26 ≤ l ≤
26 for 3. Lorentz and polarization corrections were applied
to 17 306 for 2 and 5506 for 3 reflections collected of which
4762 for 2 and 4085 for 3 were unique with I0 > 2σ(I0). The
structures were determined by direct methods (SIR97 or
SHELXS-XTL)12,13 and refined by least-squares procedures
on F2 (SHELX-XTL).13 The function minimized during the
refinement was w = 1/[σ2(F0 ) + (0.0368P)2 + 0.0000P] for 2 and
2
w = 1/[σ2(F0 ) + (0.0436P)2 + 0.0000P] for 3. The final geometri-
2
4.4. Diffusion measurements
cal calculations, the graphical manipulations and the analysis
The diffusion experiments were performed in CDCl3 by the of H-bond network and other crystallographic calculations
stimulated echo pulse sequence without spinning. The shape were carried out with SHELXS-XTL package.13 In the final
of the gradient pulse was rectangular and its strength varied least squares cycles all non-H atoms were allowed to vibrate
automatically in the course of the experiments. The diffusion anisotropically. The hydrogen atoms were located at the calcu-
coefficients (D, Table 1) were determined from the slope of lated positions and assigned a fixed displacement and con-
the regression line ln(I/I0) versus G2 according to ln(I/I0) = strained to ideal geometry. Crystal data and data collection
−(γδ)2(Δ − δ/3)DG2, in which: I and I0 are the observed spin details are given in Table 4.
echo intensity with and without gradients, respectively; G is
the gradient strength; Δ is the delay between the mid-points of
4.7. Growth inhibition assays
the gradients; D is the diffusion coefficient; δ is the gradient The human solid tumor cell lines HBL-100, HeLa, SW1573,
length. The calibration of the gradients was carried out by a T-47D and WiDr were used in this study. These cell lines
diffusion measurement of HDO in D2O. The experimental were a kind gift from Prof. G. J. Peters (VU Medical Center,
error in the D values was estimated to be 2%.7 All the data Amsterdam, Netherlands). Cells were maintained in 25 cm2
leading to the reported D values afforded lines with correlation culture flasks in RPMI 1640 supplemented with 5% heat
This journal is © The Royal Society of Chemistry 2013
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Dalton Transactions
L. M. Aguilera are grateful to Excellence project P09-FQM-5402
Table 4 Selected crystallographic data for 2 and 3
for
a postdoctoral and a technic-predoctoral contract,
2
3
respectively.
Empirical formula
Formula weight
Cryst dimensions (mm)
C
30H36Cl3N3P2RuNi
C30H36Cl3N3P2RuZn
773.35
0.099 × 0.083 ×
0.065
766.69
0.100 × 0.085 ×
0.070
Cryst system
Space group
a [Å]
b [Å]
c [Å]
Monoclinic
P21/n
14.8944(9)
9.4708(6)
22.5306(14)
102.3250(10)
4
3105.0(3)
1.640
1.480
Monoclinic
P21/n
14.847(4)
9.499(2)
22.587(6)
102.151(6)
4
3114.1(13)
1.650
1.642
Notes and references
1 (a) Aqueous-Phase Organometallic Catalysis. Concepts and
Applications, ed. B. Cornils and W. A. Herrmann, Wiley-
VCH, Weinheim, Germany, 2nd edn, 2004, pp. 52–59;
(b) F. Joó, Aqueous Organometallic Catalysis, Kluwer,
Dordrecht, The Netherlands, 2001, pp. 32–39; (c) Aqueous
Organometallic Chemistry and Catalysis, ed. I. T. Horváth
and F. Joó, NATO ASI 3/5, Kluwer, Dordrecht, 1995, vol. 5,
pp. 61–80; (d) S. Bolaño, M. M. Rodríguez-Rocha, J. Bravo,
J. Castro, E. Oñate and M. Peruzzini, Organometallics, 2009,
28, 6020; (e) F. Mohr, E. Cerrada and M. Laguna, Organo-
metallics, 2006, 25, 644; (f) F. Mohr, S. Sanz,
E. R. T. Tiekink and M. Laguna, Organometallics, 2006, 25,
3084; (g) B. J. Frost and C. A. Mebi, Organometallics, 2004,
23, 5317; (h) D. J. Darensbourg, C. G. Ortiz and
J. W. Kamplain, Organometallics, 2004, 23, 1747;
(i) D. J. Darensbourg, F. Joó, M. Kannisto, A. Kathó,
J. H. Reibenspies and D. J. Daigle, Inorg. Chem., 1994, 33,
200.
β [°]
Z
Volume [Å3]
ρcalcd. [mg m−3
]
μ [mm−1
F(000)
]
1560
5470/0/363
1568
5506/0/363
Data/restrains/
parameters
R1 [I > 2σ(I)] (all)
wR2 [I > 2σ(I)] (all)
GOF
0.0284 (0.0335)
0.0685 (0.0704)
0.978
0.0558, 0.1055
0.0833, 0.1150
1.02
Δρ [e Å−3
]
0.960; −0.430
1.053; −0.698
inactivated fetal calf serum and 2 mM L-glutamine in a 37 °C,
5% CO2, 95% humidified air incubator. Exponentially growing
cells were trypsinized and re-suspended in antibiotic contain-
ing medium (100 units of penicillin G and 0.1 mg of strepto-
mycin per mL). Single cell suspensions displaying >97%
viability by trypan blue dye exclusion were subsequently
counted. After counting, dilutions were made to give the
appropriate cell densities for inoculation onto 96-well microti-
ter plates. Cells were inoculated in a volume of 100 μL per well
at densities 10 000 (HBL-100, HeLa and SW1573) of 15 000
(T-47D), and 20 000 (WiDr) cells per well, based on their dou-
bling times. Compounds were initially dissolved in DMSO at
400 times the desired final maximum test concentration.
Control cells were exposed to an equivalent concentration of
DMSO (0.25% v/v, negative control). Each agent was tested
in triplicate at different dilutions in the range of 1–100 μM.
The drug treatment was started on day 1 after plating. Drug
incubation times were 48 h, after which time cells were precipi-
tated with 25 μL ice-cold TCA (50% w/v) and fixed for 60 min
at 4 °C. Then the SRB assay was performed.10 The optical
density (OD) of each well was measured at 492 nm, using
BioTek’s PowerWave XS Absorbance Microplate Reader. Values
were corrected for background OD from wells only containing
medium.
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Acknowledgements
Financial support co-financed by the EU FEDER: the Spanish
MINECO (CTQ2010-20952), the Spanish Instituto de Salud
Carlos III (PI11/00840) and Junta de Andalucía through PAI
(research teams FQM-317) and Excellence Projects
P07-FQM-03092 and P09-FQM-5402. Thanks are also given to
COST Action CM0802 (WG2, WG3, WG4). M. Serrano-Ruiz and
5 C. Ríos-Luci, L. G. León, A. Mena-Cruz, E. Pérez-Roth,
P. Lorenzo-Luis, A. Romerosa and J. M. Padrón, Bioorg.
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F. García-Maroto, Inorg. Chem., 2006, 45, 1289.
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12 A. Altamore, G. Cascarano, C. Giacovazzo, A. Guagliardi,
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This journal is © The Royal Society of Chemistry 2013
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