Preparation of new half sandwich ruthenium arene complexes with aminophosphines as potential chemotherapeutics

Journal of Inorganic Biochemistry 117 (2012) 171 –188

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Preparation of new half sandwich ruthenium arene complexes with

aminophosphines as potential chemotherapeutics

Cristina Aliende ^a, Mercedes Pérez-Manrique ^a, Félix A. Jalón ^b, Blanca R. Manzano ^b, Ana M. Rodríguez ^b,

c

d

José Vicente Cuevas ^a, Gustavo Espino ^a,, Mª Ángeles Martínez , Anna Massaguer ,

⁎

Marta González-Bártulos ^d, Rafael de Llorens ^d, Virtudes Moreno ^e

Departamento de Química, Facultad de Ciencias, Universidad de Burgos, Plaza Misael Bañuelos s/n, 09001, Burgos, Spain

a

b

Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Químicas, Universidad de Castilla-La Mancha, Avda. Camilo J. Cela 10, 13071, Ciudad Real, Spain

c

Departament de Química, Universitat de Girona, Campus de Montilivi, s/n, 17071, Girona, Spain

d

Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, s/n, 17071, Girona, Spain

Departament de Química Inorgànica, Universitat de Barcelona, Martí i Franquès 1–11, 08028, Barcelona, Spain

e

a r t i c l e i n f o

a b s t r a c t

Article history:

Aminophosphines 2-(diphenylphosphino)-1-methylimidazole (dpim) and diphenyl-2-pyridylphosphine

(PPh₂py) have been used to prepare two series of Ru(II) arene complexes of formulae [(η⁶-p-cymene)

Ru(κ²-O,O′-X)(κ¹-P-dpim)]Y (series a: 1a·Y–3a·Y) and [(η⁶-p-cymene)Ru(κ²-O,O′-X)(κ¹-P-PPh₂py)]Y

(series b: 1b·Y–3b·Y) (where X=acac, acetylacetonate; bzac, benzoyl acetonate; dbzm, dibenzoyl

methanoate; Y=BF₄, BPh₄). The structures of 1a·BF₄, 1a·BPh₄, 3a·BF₄, 1b·BPh₄and 3b·BPh₄were deter-

mined by X-ray diffraction. The tetraﬂuoroborate derivatives are more soluble in organic solvents than

their tetraphenylborate counterparts. Five BF₄⁻derivatives (all except the unstable 1b·BF₄) were selected

to evaluate the cytotoxic behavior in vitro against the human cancer cell lines MCF-7 (breast cancer) and

CAPAN-1 (pancreatic cancer). 2b·BF₄and 3b·BF₄exhibited IC₅₀values similar to those of cisplatin. Electro-

phoresis and AFM studies showed good correspondence between the biological activity levels of 2b·BF₄and

3b·BF₄and their ability to modify the DNA structure. Hydrolytic studies indicate that aquation could be in-

volved in the activation mechanism of these complexes and conﬁrm that the hydrolysis rate of 3b·BF₄is

higher than that of 3a·BF₄. Thus, the cytotoxic activity trends are explained in terms of the higher reactivity

of derivatives from series b, which in turn is rationalized as being the result of the electronic features of dpim

and PPh₂py established by cyclic voltammetry measurements.

Received 22 March 2012

Received in revised form 31 July 2012

Accepted 31 July 2012

Available online 30 August 2012

Keywords:

Ruthenium(II) arene complexes

Aminophosphines

Anticancer drugs

Cytotoxicity

Metal-based drugs

DNA interaction

1. Introduction

1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane) and related complexes

developed by the group of Dyson, which show selectivity towards

Chemotherapy has become one of the most successful tools in the

battle to treat cancer. Indeed, modern societies enjoy a huge battery of

different medicines, such as cisplatin and its congeners, to cure certain

types of cancer. However, new anticancer drugs are required not only

to widen the number of malignant tumors that can be successfully

treated, but also to improve the selectivity over tumor tissue types [1].

In particular, the family of organometallic Ru(II) arene complexes is

developing very rapidly due to the high activity of some leading com-

plexes against both primary tumors [2–4] and metastasis processes

[5,6]. For instance, the [(η⁶-arene)RuCl(en)]PF₆family, developed by

Sadler and co-workers, shows high cytotoxicity to cancer cells, including

cisplatin-resistant cells, with DNA as the alleged biological target [4,7,8].

Another signiﬁcant case is that of [(η⁶-toluene)RuCl₂(pta)] (pta:

metastatic tumors [9]. It is worth noting that updated perspectives on

this issue have been reported in recent reviews [1,9–12].

The attribution of several advantages to Ru as the metal ion has

led to particular interest in this family of compounds. These advan-

tages include the following: (i) these compounds show lower general

toxicity than Pt drugs, allegedly because they make use of plasma

iron-binding proteins such as transferrin to reach the cells [11],

(ii) most of the Ru complexes can interact with DNA in in vitro models

but display binding modes and action mechanisms that differ from

those of Pt derivatives, and (iii) Ru(II) arene complexes have a versa-

tile stereochemistry that offers multiple possibilities for innovative

design and modiﬁcation, either in the arene or in the three remaining

σ-bonded ligands. Similarly, certain opportunities for enhancement

have also been emphasized with regard to the performance of the

candidates under scrutiny, namely: (i) both selectivity and broaden-

ing of the therapeutic action should be improved [12]; (ii) DNA inter-

action cannot guarantee in vivo activity [13]; and (iii) proteins should

⁎

Corresponding author.

E-mail address: gespino@ubu.es (G. Espino).

http://dx.doi.org/10.1016/j.jinorgbio.2012.07.022

172

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

also be taken into account as pharmacological targets [13]. Thus, in

recent years several innovative strategies have been proposed to

make progress in this ﬁeld and these include the following:

(a) multifunctional chemotherapy, which aims to identify synergisms

between the metallic center and functional ligands [14,15], (b) the

use of multinuclear arene ruthenium compounds that focus on the

modiﬁcation of the pharmacological action mechanism [16], (c) photo-

dynamic therapy (PDT), in which nontoxic photosensitive compounds

(photosensitizers) are used that are ‘in situ’ activated by speciﬁc wave-

lengths of light [17,18], and (d) catalytic oxidation of glutathione to glu-

tathione disulﬁde with complexes of formula [(η⁶-arene)Ru(azpy)I]⁺

(azpy=N,N-dimethylphenyl or hydroxyphenyl-azopyridine), whose

cytotoxic action is based on the accumulation of reactive oxygen species

(ROS) in the cancer cells [19].

In particular, our group is attracted by the possibilities of

multifunctional chemotherapy. Indeed, we believe that the use of

Ru(II) complexes with aminophosphines as functional ligands could pro-

vide novel drugs with enhanced cytotoxic properties. Aminophosphines

are a class of ligands that have been widely used in catalysis, mainly due

to their hemilabile behavior [20]. In this context, we have previously

reported the catalytic activity of complexes of general formula [(η⁶-p-

cym)RuCl₂(κ¹-P-L)] (L=2-(diphenylphosphino)-1-methylimidazole,

dpim, A; or 2-(diphenylphosphino)pyridine, PPh₂py, B) (Scheme 1)

[21]. By contrast, aminophosphines have been underutilized in the

ﬁeld of bioorganometallics, with certain well known exceptions. The

pioneering work of Dyson et al. has focused on the therapeutic potential

of complexes with the hydrophilic phosphine PTA (1,3,5-triaza-7-

phosphaadamantane) [1,9,22]. In particular, they have reported that

complexes of the type [(η⁶-p-cym)Ru(κ²-O,O′-X)(PTA)]Y (p-cym=

p-cymene; X=β-diketonate, Y=BPh₄, BF₄) (see D in Scheme 1) are

water-soluble and display remarkable cytotoxicity in A2780 human

ovarian cancer cells [23]. Previously, Sadler et al. had found that neutral

complexes of the class [(η⁶-p-cym)Ru(κ²-O,O′-X)Cl] (X=β-diketonate)

are highly cytotoxic (see C in Scheme 1) [24]. Thus, inspired by the pre-

vious work of Dyson and Sadler, we present here the synthesis and char-

acterization of a new family of Ru(II)-arene complexes with the general

formula [(η⁶-p-cym)Ru(κ²-O,O′-X)(κ¹-P-L)]Y (where X=β-diketonate,

L=dpim; PPh₂py,; Y=BPh₄, BF₄) and also describe the in vitro antican-

cer activity of these compounds. In addition, we have examined the in-

teraction of the new complexes with DNA. The main objectives of this

work were to compare the role of the aforementioned aminophosphines

in the biological properties of Ru(II) complexes and to establish a rela-

tionship between cytotoxic activity and DNA interaction. Indeed, it was

found that the most active specimens are those that have the highest de-

gree of interaction with the nucleic acid, namely [(η⁶-p-cymene)

Ru(bzac)(PPh₂py)]BF₄(bzac=benzoyl acetonate) and [(η⁶-p-cymene)

Ru(dbzm)(PPh₂py)]BF₄(dbzm=dibenzoyl methanoate).

2. Experimental

2.1. Materials and reagents

Starting materials: [(η⁶-p-cymene)Ru(μ-Cl)Cl]₂, [25,26] dpim, [27]

[(η⁶-p-cymene)Ru(acac)Cl] (1), [23] [(η⁶-p-cymene)Ru(bzac)Cl] (2),

[28,29] [(η⁶-p-cymene)Ru(dbzm)Cl] (3), [24] and [(η⁶-p-cymene)

Ru(hfacac)Cl] (4) [24] were prepared according to literature procedures.

PPh₂py, acetylacetone (acacH), phenyl-1,3-butanedione (bzacH), 1,3-

diphenyl-1,3-propanedione (dbzmH), hexaﬂuoroacetylacetone (hfacH),

NaBF₄, and NaBPh₄, were purchased from Aldrich and used without

further puriﬁcation. Deuterated solvents were obtained from SDS

and Euriso-top. pBR322 plasmid DNA used in the EM and AFM studies

was purchased from Boehringer Mannheim (Germany). Ultrapure aga-

rose was obtained from ECOGEN (Barcelona, Spain). HEPES (N-2-

hydroxyethyl piperazine-N′-2-ethanesulfonic acid) was obtained from

ICN (Madrid). EDTA and Tris–HCl used in EM study were obtained

from Sigma-Aldrich (Madrid, Spain).

2.2. General methods

All synthetic manipulations were carried out under an atmosphere

of dry oxygen-free nitrogen using standard Schlenk techniques. Sol-

vents were distilled from the appropriate drying agents and degassed

before use. Elemental analyses were performed with a Perkin-Elmer

2400 CHN microanalyzer. The analytical data for the new complexes

were obtained from crystalline samples when possible. In some

cases the data were acceptably accurate, but in others the agreement

of calculated and found values for carbon was >0.4%, so that solvent

molecules were introduced in the molecular formulae to improve

agreement. In any case, all the complexes were obtained in enough

analytic purity to be used as starting materials. IR spectra were

recorded on a Nicolet Impact 410 spectrophotometer as KBr pellets

and on a Jasco (650–160 cm⁻¹range) as Nujol mulls deposited on a

polyethylene ﬁlm. Only relevant bands are collected. The intensity

of the peaks is described as: w=weak, m=medium, s=strong,

vs=very strong. Fast atom bombardment mass (FAB MS) spectra

(position of the peaks in DA) were recorded with an Autospec spec-

trometer. NMR spectra were recorded at 298 K (unless otherwise

stated), on a Varian Unity Inova-400 (400 MHz for ¹H; 161.9 MHz

for ³¹P; 100.6 MHz for ¹³C; 376 MHz for ¹⁹F MHz). ¹H and ¹³C{¹H}

chemical shifts were internally referenced to TMS via the residual

¹H and ¹³C signals of CDCl₃(δ=7.26 ppm and δ=77.36 ppm),

according to the values reported by Fulmer et al. [30]. Chemical

shift values are reported in ppm and coupling constants (J) in Hz.

The splitting of proton resonances is deﬁned as s=singlet, d=

doublet, t=triplet, q=quartet, sept=septet, m=multiplet, bs=

broad singlet. All ³¹P resonances were referenced to 85% H₃PO₄at

0 ppm. 2D NMR spectra were recorded using standard pulse–pulse

sequences: COSY (COrrelation SpectroscopY), NOESY (Nuclear

We also established a relationship between the hydrolysis rate of

the new derivatives and the electronic features of the phosphine li-

gand and this could explain the observed cytotoxicity trends.

Scheme 1. Structures of [(η⁶-p-cym)RuCl₂(κ¹-P-L)] (L=dpim, A; or PPh₂py, B), [(η⁶-p-cym)Ru(κ²-O,O′-X)Cl] (C) and [(η⁶-p-cym)Ru(κ²-O,O′-X)(PTA)]Y (D).

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

173

Overhauser Enhancement SpectroscopY), HMQC (Heteronuclear Mul-

tiple Quantum Coherence), HMBC (Heteronuclear Multiple Bond Cor-

Grand Island, NY) at 37 °C in a humidiﬁed atmosphere containing

5% CO₂. The cells were passaged two times per week.

relation). The probe temperature

(

1 K) was controlled by a

standard unit calibrated with a methanol reference. All NMR data

processing was carried out using MestReNova version 6.1.1.

2.6. Cytotoxicity assays

Cyclic voltammetric (CV) experiments were performed in a

IJ-Cambria ICH-660 potentiostat using a three electrode cell. Glassy

carbon disk electrodes (3 mm diameter) from BAS were used as

working electrodes, platinum wire as auxiliary and SSCE (Silver/Silver

Chloride Electrode) as the reference electrode. Cyclic voltammograms

were recorded at 100 mV/s scan rate. The complexes were dissolved

in the corresponding solvents (dichloromethane or acetonitrile)

containing the necessary amount of supporting electrolyte

((n-Bu₄N)(PF₆), TBAH) to yield a 0.1 M ionic strength solution.

The cytotoxicity of the ruthenium complexes in MCF-7 cells and

CAPAN-1 cells was evaluated by the MTT assay. Aliquots of 2000

MCF-7 cells and 7000 CAPAN-1 cells were seeded in 96-well plates

and cultured for 48 h with DMEM medium+10% FBS. Then, the

cells were treated for 48 h at 37 °C with the different ruthenium com-

pounds, freshly dissolved in DMSO and milli-Q water, and then serial-

ly diluted in complete culture medium at concentrations ranging

from 0 μM to 100 μM (DMSO ﬁnal concentration in the culture

mediumb1%). Next, the treatments were removed and the cells

were washed with PBS and incubated for 3 additional hours in the

darkness with 100 μL of fresh culture medium together with 10 μL

of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide) (Sigma-Aldrich, St. Louis, MO, USA). The medium was then

discarded and dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was

added to each well to dissolve the purple formazan crystals. Plates

were agitated at room temperature for 10 min and the absorbance

of each well was determined with an absorbance microplate reader

(ELx800, BioTek, Winooski, USA) at a wavelength of 570 nm. Three

replicates were determined for each treatment, and all complexes

were tested at least in three independent experiments. For each treat-

ment, the cell viability was determined as a percentage of the

untreated control cells, by dividing the mean absorbance of the treat-

ment by the mean absorbance of the untreated cells. The concentra-

tion that reduces cell viability by 50% (IC₅₀) was then established

for each compound.

2.3. X-ray crystallography

The single crystals for 1a·BF₄, 1a·BPh₄, 1b·BPh₄, 2, 3a·BF₄and

3b·BPh₄were mounted on a glass ﬁbber and transferred to a Bruker

X8 APEX II CCD-based diffractometer equipped with a graphite

monochromated MoKα radiation source (λ=0.71073 Å). The highly

redundant datasets were integrated using SAINT [31] and corrected

for Lorentz and polarization effects. The absorption correction was

based on ﬁtting a function to the empirical transmission surface as

sampled by multiple equivalent measurements with the program

SADABS [32].

The software package SHELXTL version 6.10 [33] was used for

space group determination, structure solution and reﬁnement by

full-matrix least-squares methods based on F². A successful solution

by the direct methods provided most non-hydrogen atoms from the

E-map. The remaining non-hydrogen atoms were located in an alter-

nating series of least-squares cycles and difference Fourier maps. All

non-hydrogen atoms were reﬁned with anisotropic displacement co-

efﬁcients unless speciﬁed otherwise. Hydrogen atoms were placed

using a “riding model” and included in the reﬁnement at calculated

positions.

For compound 1b·BPh₄, only crystals of low quality (Rint=0.226)

could be grown. Because of the weak diffraction, reﬂections have

been included only up to θ=21.0° for the reﬁnement and the data

were reﬁned isotropically only to reveal atomic connectivity. For

complex 2, the p-cymene ligand shows rotational disorder. In order

to obtain chemically most reasonable results, the structure reﬁne-

ment was carried out with idealized rigid rings for all phenyl rings.

Each of these rings was idealized and reﬁned in spatial position and

orientation and in its size using AFIX 3 instructions of program

SHELXL97.

CCDC 871855, CCDC 871856, CCDC 871857, CCDC 871858, CCDC

871859, CCDC 871860 contain the supplementary crystallographic

data for this paper. These data can be obtained free of charge from

the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.

uk/data_request/cif.

2.7. Electrophoretic mobility study in agarose gel

The pBR322 plasmid DNA was used for the experiments at a con-

centration of 0.25 μg/μL. Stock solutions of the ruthenium complexes

were freshly prepared at a concentration of 0.1 μg/μL in milli-Q water

with 5% DMSO to facilitate the dissolution of compounds. The sam-

ples, with a ﬁnal volume of 20 μL, were prepared by addition of ali-

quots of the complex solutions to the pBR322 DNA in TE buffer

(50 mM NaCl, 10 mM tris-(hydroxymethyl)aminomethane hydro-

chloride (Tris–HCl), 0.1 mM H₄edta, pH 7.4). The ﬁnal concentration

of DNA was 0.035 μg/μL and the input molar ratio (r_i) of the complex

to nucleotide=0.50. A sample of pBR322 DNA with TE buffer alone

was used as negative control. The samples were incubated for 24 or

48 h at 37 °C, and then 4 μL of loading buffer (6×) (10 mM Tris–

HCl (pH 7.6), 0.03% bromophenol blue, 0.03% xylene cyanol, 60% glyc-

erol and 60 mM EDTA) were added to aliquots of 20 μL of each sam-

ple. The mixtures were electrophoresed in a 0.5% agarose gel in TBE

buffer (Tris–borate–EDTA, 89 mM Tris–borate, 2 mM EDTA, pH 8.3)

for 3.5 h at 25 V. Afterwards, the DNA was dyed with a ethydium bro-

mide solution (0.5 μg/mL in TBE) for 45 min and the DNA bands were

visualized. For comparison purposes, cisplatin was evaluated under

the same experimental conditions.

2.4. Solubility and stability

The ruthenium compounds were soluble in DMSO and aqueous

DMSO. The ruthenium complexes were found sufﬁciently stable in

the solution phases used after 48 h, as evidenced by the ¹H and ³¹P

NMR.

2.8. AFM

The pBR322 plasmid DNA at 0.25 μg/μL concentration was used for

the experiments. Stock solution (0.1 μg/μL) of the complexes in

milli-Q water with 6% DMSO was freshly prepared. Samples (Vf=

40 μL) were prepared by diluting 1 μL of DNA pBR322 and an appro-

priate aliquot of the stock solution (ri=0.50) in HEPES buffer

(N-2-hydroxyethyl piperazine-N′-2-ethanesulfonic acid), 10 mM

MgCl₂, pH=7.4) (DMSO ﬁnal concentrationb1%). The different

solutions as well as milli-Q water were passed through 0.2 nm

FP030/3 ﬁlters (Scheicher&Schueell GmbH, Germany) to provide a

clear background. The resulting solutions were incubated for 24 h or

2.5. Cell lines

The human breast cancer cell line MCF-7 and the human pancreat-

ic cancer cells CAPAN-1were obtained from the American Tissue Cul-

ture Collection (ATTC, Rockville, MD, USA). The cells were maintained

in Dulbecco's modiﬁed eagle's medium (DMEM) supplemented with

10% fetal bovine serum and 1% penicillin–streptomycin (GIBCO BRL,

174

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

48 h at 37 °C. The samples were imaged in a NANOSCOPE III

MULTIMODE AFM (Digital Instrumentals Inc., Santa Barbara) operat-

ing in tapping mode.

1 H, H⁷-cym), 1.75 (s, 6 H, H^c-acac), 1.57 (s, 3 H, H¹⁰-cym), 1.17

3

(d, J_HH=7.0 Hz, 6 H, H^8,9-cym) ppm. ¹³C{¹H} NMR (101 MHz,

1

CDCl₃) δ=188.80 (s, 2C, C^b-acac), 136.69 (d, J_PC=84.5 Hz, 1C,

2

4

C^2′-Im), 132.08 (d, J_PC=9.8 Hz, 4C, C^o-Ph), 131.68 (d, J_PC

=

3

2.9. Cell death assay

2.5 Hz, 2C, C^p-Ph), 131.51 (s, 1C, C^4′-Im), 129.33 (d, J_PC

10.2 Hz, 4C, C^m-Ph), 128.71 (s, 1C, C^5′-Im), 128.26 (d, J_PC

1

The cell death pathway (apoptosis or necrosis) induced by the ru-

thenium compounds was evaluated using the 3b⋅BF₄complex which

demonstrated the highest cytotoxic activity. To this end, exponential-

ly growing CAPAN-1 cells were seeded in a twelve-well plate (5×10⁵

cells/well) and cultured for 24 h with DMEM medium+10% FBS.

Then, the medium was removed and the cells were treated for 24 h

with a 3b⋅BF₄solutions around two and three times the IC₅₀(10

and 20 μM) or with medium alone, as a negative control. Then, the

cell death was evaluated using the Vibrant Apoptosis Assay Kit #2

(Invitrogen, Paisley, UK), according to the manufacturer's instruc-

tions. Cells were harvested, washed in cold PBS and in diluted in 1X

annexin-binding buffer to 1×10⁶cells/mL. Subsequently, cells were

stained with Annexin V and propidium iodide (PI) and analyzed by

ﬂow cytometry using a FACSCalibur ﬂow cytometer (Becton Dickin-

son Immunocytometry Systems, San Jose, CA) equipped with the

CellQuestTM software (Becton Dickinson). 10,000 cells were analyzed

in each experiment.

45.2 Hz, 2C, Cⁱ-Ph), 109.92 (d, J_PC=1.9 Hz, 1C, C¹-cym), 101.13

(s, 1C, C^a-acac), 98.61 (s, 1C, C⁴-cym), 89.33 (d, J_PC=3.5 Hz, 2C,

C^3,5-cym), 87.08 (d, J_PC=4.6 Hz, 2C, C^2,6-cym), 36.05 (s, 1C, N-Me),

30.58 (s, 1C, C⁷-cym), 27.50 (s, 2C, C^c-acac), 22.12 (s, 2C, C^8,9-cym),

16.64 (s, 1C, C¹⁰-cym) ppm. ³¹P{¹H} NMR (162 MHz, CDCl₃) δ=11.43

(s, 1 P, κ¹-dpim) ppm. ¹⁹F NMR (376 MHz, CDCl₃) δ=−154.44

(s, ¹⁰B-F, BF₄⁻), −154.49 (s, ¹¹B-F, BF₄⁻) ppm. FT-IR (KBr, cm⁻¹):

3142, 3122, 3081 (ν_C²_{H, sp}), 2964 (ν_C³_{H, sp}), 1573 (s, ν_{C_O+C_C}), 1515

(s, ν_{C_C+C_O}), 1437 (ν_P-C), 1374 (δ_{s, Me}), 1281, 1054 (s, ν_{d B-F}), 750

(δ_CH,oop), 696 (m, δ_{C_C, oop}), 548 (m, dpim), 518 (dpim), 476 (dpim),

441, 421. MS (FAB+, CHCl₃): m/z (%)=601 (34) [M-BF₄]⁺, 467 (18)

[M-BF₄-cym]⁺, 335 (100) [M-BF₄-dpim]⁺. Molar Conductivity (CH₃CN):

121.3 S·cm²·mol⁻¹. Solubility: soluble in acetone, dichloromethane,

chloroform, methanol, barely soluble in ethanol and insoluble in water.

2.11.2. [(η⁶-p-cymene)Ru(acac)(dpim)]BPh₄(1a·BPh₄)

[(η⁶-p-cym)RuCl(acac)] (37.8 mg, 0.102 mmol); NaBPh₄(37.6 mg,

0.110 mmol), dpim (28.4 mg, 0.107 mmol). Yield: 65 mg

(0.071 mmol, 69.5%). FW (C₅₅H₅₆N₂PO₂RuB)=919.91 g/mol. Anal.

calcd for C₅₅H₅₆N₂PO₂RuB·2H₂O: C, 69.10; H, 6.33; N, 2.93. Found: C,

68.68; H, 6.36; N, 2.94. ¹H NMR (400 MHz, CDCl₃) δ=7.45 (m, 2 H,

H^p-Ph, dpim), 7.39 (m, 9 H, 8 H^o-BPh, H^4′-Im), 7.35 (m, 4 H, H^m-Ph,

2.10. Synthesis of [(η⁶-p-cymene)Ru(bzac)Cl] (2) [28,29]

A suspension of Na₂CO₃(102 mg, 0.651 mmol, 2 equiv.) in ace-

tone (20 mL) was prepared. 1-Phenyl-1,3-butanedione (bzacH)

(106 mg, 0.651 mmol, 2 equiv.) and [(η⁶-p-cymene)RuCl₂]₂(200 mg,

0.326 mmol, 1 equiv.) were added, and the mixture was stirred at

room temperature for 3 h and then ﬁltered. The solvent was removed

under vacuum. The residue was extracted with CH₂Cl₂(4×8 mL), and

the ﬁltrated solution was concentrated under vacuum to 5 mL. Then

n-hexane was added (30 mL) and the mixture was cooled in the freezer

to produce an orange solid. Yield: 191 mg (0.651 mmol, 68.0%).

3

dpim), 7.20–7.13 (m, 4 H, H^o-Ph, dpim), 7.08 (d, J_HH=0.9 Hz, 1 H,

3

H^5′-Im), 7.00 (t, J_HH=7.4 Hz, 8 H, H^m-BPh, BPh₄⁻), 6.85 (m, 4 H,

3

H^p-BPh, BPh₄⁻), 5.65 (d, J_HH=6.2 Hz, 2 H, H^2,6-cym), 5.39 (d, J_HH

=

5.3 Hz, 2 H, H^3,5-cym), 4.66 (s, 1 H, H^a-acac), 2.74 (s, 3 H, Me-Im), 2.30

(sept, J_HH=7.0 Hz, 1 H, H⁷-cym), 1.71 (s, 6 H, H^c-acac), 1.38 (s, 3 H,

3

H¹⁰-cym), 1.13 (d, J_HH=7.0 Hz, 6 H, H^8,9-cym). ¹³C NMR (101 MHz,

3

CDCl₃) δ=188.60 (s, 2C, C^b-acac), 164.45 (q, J_BC=49.3 Hz, 4C,

1

Cⁱ-BPh), 136.51 (q, J_BC=1.3 Hz, 8C, C^o-BPh), 136.51 (d, J_PC=84.6 Hz,

2

1

2.11. Synthesis of novel cationic complexes of general formula [(η⁶-p-

cymene)Ru(κ²-O,O′-X)(L)]Y

1C, C^2′-Im), 131.83 (d, J_PC=9.8 Hz, 4C, C^o-Ph), 131.75 (d, J_PC

=

2

4

2.9 Hz, 2C, C^p-Ph), 131.59 (s, 1C, C^4′-Im), 129.34 (d, J_PC=10.1 Hz, 4C,

3

C^m-Ph), 128.48 (s, 1C, C^5′-Im), 128.06 (d, J_PC=44.9 Hz, 2C, Cⁱ-Ph,

1

3

A procedure based on that described by Dyson et al. was followed

[23]. A solution of the respective starting material (1–3) (0.1616,

mmol) in a (1:1) mixture of degassed solvents (acetone, 10 mL and

dichloromethane, 10 mL) was prepared. Then, the corresponding so-

dium salt (NaBF₄, 0.3232 mmol) or (NaBPh₄, 0.1616 mmol) and

aminophophine (dpim or PPh₂py) (0.1616 mmol) were added. The

mixture was stirred and heated to reﬂux temperature with a hot

plate and then was left to cool slowly with stirring for 15 min. The

heating/cooling cycle was repeated three more times to make a

total reaction time of 1 h. The solution was evaporated to dryness

under vacuum, and the solid residue was extracted with CH₂Cl₂

(3×10 mL). The extracts were ﬁltered to remove the NaCl and the

solvent removed under vacuum again. The solid residue was

dissolved in CH₂Cl₂(5 mL) and ethyl acetate (40 mL) was added.

The mixture was concentrated to ca. 10 mL. Finally hexane (60 mL)

was added and the schlenk was introduced in the freezer. Yellow

solids were obtained after ﬁltration, which were dried under vacuum.

dpim), 125.66 (q, J_BC=2.7 Hz, 8C, C^m-BPh), 121.75 (s, 4C, C^p-BPh),

109.88 (s, 1C, C¹-cym), 101.02 (s, 1C, C^a-acac), 98.06 (s, 1C, C⁴-cym),

89.39 (d,

J

_PC=3.4 Hz, 2C, C^3,5-cym), 86.72 (d, J_PC=4.7 Hz, 2C,

C^2,6-cym), 35.94 (s, 1C, N-Me), 30.53 (s, 1C, C⁷-cym), 27.55 (s, 2C,

C^c-acac), 22.08 (s, 2C, C^8,9-cym), 16.54 (s, 1C, C¹⁰-cym). ³¹P{¹H} NMR

(162 MHz, CDCl₃) δ=10.89 (s, 1 P, κ¹-dpim) ppm. FT-IR (KBr pellet

and nujol mull, cm⁻¹): 3120, 3101, 3056 (m, ν_C²_{H, sp}), 2997, 2984

(ν_C³_{H, sp}), 1576 (s, ν_{C_O+C_C}), 1520 (s, ν_{C_C+C_O}), 1482 (BPh₄⁻),

1437 (ν_P−C), 1374 (δ_{s, Me}), 1276, 1098 (m, ν_P−C), 1032 (m), 781

(m), 748 (m, δ_CH,oop), 733 (s), 704 (s, δ_{C_C, oop}, BPh₄⁻), 612 (m),

546 (m, dpim), 519 (dpim), 477 (dpim), 439 (w), 422 (w), 203

(w, Ru-P). MS (FAB+, CHCl₃): m/z (%)=601 (23) [M-BPh₄]⁺, 467

(9) [M-BPh₄-cym]⁺, 335 (100) [M-BPh₄-dpim]⁺. Molar Conduc-

tivity (CH₃CN): 119.7 S·cm²·mol⁻¹. Solubility: soluble in acetone,

dichloromethane, chloroform, barely soluble in methanol and etha-

nol and insoluble in water.

2.11.3. [(η⁶-p-cymene)Ru(bzac)(dpim)]BF₄(2a·BF₄)

2.11.1. [(η⁶-p-cymene)Ru(acac)(dpim)]BF₄(1a·BF₄)

[(η⁶-p-cym)RuCl(bzac)] (80.8 mg, 0.185 mmol); NaBF₄(40.9 mg,

0.373 mmol), dpim (52.5 mg, 0.197 mmol). Yield: 102.7 mg

(0.137 mmol, 74 %). FW (C₃₆H₃₈N₂PO₂RuBF₄)=749.56 g/mol. Anal.

calcd for C₃₆H₃₈N₂PO₂RuBF₄: C 57.69; H 5.11; N 3.74; Found: C 57.57;

H 4.95; N 3.79. ¹H NMR (400 MHz, CDCl₃) δ=7.60–7.17(m, 17 H, Ph,

[(η⁶-p-cym)RuCl(acac)] (80.0 mg, 0.216 mmol); NaBF₄(48.3 mg,

0.440 mmol), dpim (69.1 mg, 0.260 mmol).Yield: 92 mg (0.13 mmol,

62.0%). FW (C₃₁H₃₆N₂PO₂RuBF₄)=687.49 g/mol. Anal. calcd for

C

₃₁H₃₆N₂PO₂RuBF₄: C, 54.16; H, 5.28; N, 4.07. Found: C, 53.85; H,

5.156; N, 4.224. ¹H NMR (400 MHz, CDCl₃) δ=7.54–7.38 (m, 8 H,

H^4′,5′-Im), 6.04 (d, J_HH=6.0 Hz, 1 H, H⁶-cym), 5.87 (d, J_HH=6.0 Hz,

3

H^m,p-Ph, H^4′,5′-Im), 7.30–7.23 (m, 4 H, H^o-Ph), 5.88 (d, J_HH

=

1 H, H²-cym), 5.81 (d, J_HH=6.0 Hz, 1 H, H³-cym), 5.71 (d, J_HH

=

3

6.3 Hz, 2 H, H^2,6-cym), 5.69 (d, J_HH=6.3 Hz, 2 H, H^3,5-cym), 4.70

6.0 Hz, 1 H, H⁵-cym), 5.38 (s, 1 H, H^a-bzac), 2.95 (s, 3 H, Me-Im), 2.42

3

(s, 1 H, H^a-acac), 2.98 (s, 3 H, Me-Im), 2.37 (sept, J_HH=7.0 Hz,

(sept, J_HH=7.0 Hz, 1 H, H⁷-cym), 1.89 (s, 3 H, Me-bzac), 1.64 (s, 3 H,

3

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

175

3

H¹⁰-cym), 1.23 (d, J_HH=7.0 Hz, 3 H, H⁸-cym), 1.16 (d, J_HH=7.0 Hz,

Solubility: soluble in acetone, chloroform and dichloromethane,

barely soluble in methanol and ethanol, and insoluble in water.

3 H, H⁹-cym) ppm. ¹³C{¹H} NMR (101 MHz, CDCl₃) δ=190.54 (s, 2C,

C^b-bzac), 181.21 (s, 2C, C^b′-bzac), 137.30 (s, 2C, Cⁱ-Ph-bzac), 136.61

(d, ¹J_PC=85.3 Hz, 1C, C^2′-Im), 132.17 (d, ²J_PC=9.8 Hz, 2C, C^o-Ph-dpim),

2.11.5. [(η⁶-p-cymene)Ru(dbzm)(dpim)]BF₄(3a·BF₄)

132.13 (d, J_PC=9.8 Hz, 2C, C^o-Ph-dpim), 131.88 (s, 1C, C^p-Ph-

[(η⁶-p-cym)RuCl(dbzm)] (80.3 mg, 0.162 mmol); NaBF₄(35.5 mg,

2

4

bzac), 131.73 (d, J_PC=2.1 Hz, 1C, C^p-Ph-dpim), 131.68 (d, J_PC

=

0.323 mmol),

dpim

(45.5 mg,

0.162 mmol).Yield:

86.6 mg

2.0 Hz, 1C, C^p-Ph-dpim), 131.50 (s, 1C, C^4′-Im), 129.47 (d, J_PC

(0.107 mmol, 66.0%). FW (C₄₁H₄₀N₂PO₂RuBF₄)=811.63 g/mol. Anal.

calcd for C₄₁H₄₀N₂PO₂RuBF₄: C, 60.67; H, 4.97; N, 3.45. Found: C,

60.77; H, 4.95; N, 3.56. ¹H NMR (400 MHz, CDCl₃) δ=7.63 (d, 4 H,

H^o-Ph, dbzm), 7.52 (m, 2 H, H-Ph), 7.48–7.33 (m, 8 H, H-Ph), 7.27–

3

10.4 Hz, 2C, C^m-Ph-dpim), 129.34 (d, ³J_PC=10.5 Hz, 2C, C^m-Ph-dpim),

128.77 (s, 1C, C^5′-Im), 128.71 (s, 2C, Ph-bzac), 128.30 (d, ¹J_PC=44.9 Hz,

1C, Cⁱ-Ph), 127.77 (d, J_PC=45.7 Hz, 1C, Cⁱ-Ph), 127.08 (s, 2C, Ph-

1

bzac), 110.11 (s, 1C, C¹-cym), 98.53 (s, 1C, C⁴-cym), 97.98 (s,

1C, C^a-bzac), 89.78 (d, J_PC=3.6 Hz, 1C, C⁵-cym), 89.45 (d, J_PC=3.3 Hz,

1C, C³-cym), 87.22 (s, 2C, C^6,2-cym), 36.04 (s, 1C, N-Me), 30.57 (s, 1C,

C⁷-cym), 28.27 (s, 1C, Me-bzac), 22.50 (s, 2C, C⁹-cym), 21.87

(s, 2C, C⁸-cym), 16.86 (s, 1C, C¹⁰-cym) ppm. ³¹P{¹H} NMR (162 MHz,

CDCl₃) δ= 11.51 (s, 1P, κ¹-dpim) ppm. ¹⁹F NMR (376 MHz,

CDCl₃) δ=−154.44 (s, ¹⁰B-F, BF₄⁻), −154.49 (s, ¹¹B-F, BF₄⁻) ppm.

FT-IR (KBr, cm⁻¹): 3060 (ν_C²_{H, sp}), 2963, 2926, 2870 (ν_C³_{H, sp}), 1588

(s, ν_{C_O +C_C}), 1556 (vs, ν_{C_O+C_C}), 1516 (vs, ν_{C_C+C_O}), 1488 (s,

7.16 (m, 6 H, H-Ph), 6.07 (s, 1 H, H^a-dbzm), 6.05 (d, J_HH=6.2 Hz,

3

2 H, H^2,6-cym), 5.82 (d, J_HH=5.9 Hz, 2 H, H^3,5-cym), 2.94 (s, 3 H,

3

Me-Im), 2.50 (sept, J_HH=7.0 Hz, 1 H, H⁷-cym), 1.71 (s, 3 H,

3

H¹⁰-cym), 1.23 (d, J_HH=7.0 Hz, 6 H, H^8,9-cym) ppm. ¹³C{¹H} NMR

(101 MHz, CDCl₃) δ=182.89 (s, 2C, C^b-dbzm), 137.80 (s, 2C,

1

2

Cⁱ-Ph-dbzm), 136.56 (d, J_PC=42.0 Hz, 1C, C^2’-Im), 132.18 (d, J_PC

=

9.9 Hz, 4C, C^o-Ph-dpim), 132.07 (s, 2C, C^p-Ph-dbzm), 131.69 (s, 1C,

C^4′-Im), 131.52 (s, J_PC=2.3 Hz, 2C, C^p-Ph-dpim), 129.45 (d, J_PC

=

4

3

10.2 Hz, 4C, C^m-Ph-dpim), 128.82 (s, 4C, C^m-Ph-dbzm), 128.33 (s, 1C,

1

ν

_{C_C+C_O}), 1450, 1438 (ν_P-C), 1374 (s, δ_{s, Me}) 1057 (s, ν_{d B-F}), 754

C^5′-Im), 127.80 (d, J_PC=46.1 Hz, 2C, Cⁱ-Ph, dpim), 127.09 (s, 4C,

(δ_CH,oop), 698 (m, δ_{C_C, oop}), 547 (m, dpim), 518 (dpim), 477

(dpim), 439. MS (FAB+, CHCl₃): m/z (%)=663 (22) [M-BF₄]⁺, 529

(7) [M-BF₄-cym]⁺, 397 (100) [M-BF₄-dpim]⁺. Molar conductivity

C^o-Ph-dbzm), 110.46 (s, 1C, C¹-cym), 98.36 (s, 1C, C⁴-cym), 95.17

(s, 1C, C^a-dbzm), 89.91 (d, J_PC=3.4 Hz, 2C, C^3,5-cym), 87.32 (d, J_PC

=

4.9 Hz, 2C, C^2,6-cym), 36.02 (s, 1C, N-Me), 30.55 (s, 1C, C⁷-cym), 22.24

(s, 2C, C^8,9-cym), 17.07 (s, 1C, C¹⁰-cym) ppm. ³¹P{¹H} NMR (162 MHz,

CDCl₃) δ=11.44 (s, 1P, κ¹-dpim) ppm. ¹⁹F NMR (376 MHz, CDCl₃)

δ=−154.44 (s, ¹⁰B-F, BF₄⁻), −154.49 (s, ¹¹B-F, BF₄⁻) ppm. FT-IR (KBr,

cm⁻¹): 3060 (ν_C²_{H, sp}), 2963, 2925 (ν_C³_{H, sp}), 1734, 1589 (m, ν_{C_}

_{O+C_C}), 1538 (vs, ν_{C_O+C_C}), 1520 (vs, ν_{C_C+C_O}), 1483 (s, ν_{C_}

_{C+C_O}), 1452, 1439 (ν_P-C), 1373 (s, δ_{s, Me}), 1084, 1059 (s, ν_{d B-F}),

1000, 752 (δ_{CH, oop}), 699 (m, δ_{C_C, oop}), 548 (m, dpim), 517 (dpim),

(CH₃CN): 145.4 S·cm²·mol⁻¹

. Solubility: soluble in acetone,

methanol, ethanol, dichloromethane, chloroform and insoluble in

water.

2.11.4. [(η⁶-p-cymene)Ru(bzac)(dpim)]BPh₄(2a·BPh₄)

(η⁶-p-cym)RuCl(bzac)] (100.1 mg, 0.231 mmol); NaBPh₄(82.9 mg,

0.242 mmol), dpim (65.2 mg, 0.245 mmol). Yield: 200.1 mg

(0.204 mmol, 88.2 %). FW (C₆₀H₅₈N₂PO₂RuB)=981.98 g/mol. Anal.

calcd for C₆₀H₅₈N₂PO₂RuB·2H₂O: C, 70.45; H, 5.68; N, 2.87. Found: C,

70.79; H, 6.14; N, 2.75. ³¹H NMR (400 MHz, CDCl₃) δ=7.55–7.27

(m, 17 H, Ph), 7.20–7.04 (m, 8 H, Ph, H^4′,5′-Im), 7.39 (m, 8 H,

H^o-BPh), 6.98 (t, ³J=7.4 Hz, 8 H, H^m-BPh), 6.83 (t, ³J=7.2 Hz, 4 H,

478 (dpim), 438. MS (FAB+, CHCl₃): m/z (%)=725 (47) [M-BF₄]⁺

,

591 (11) [M-BF₄-cym]⁺, 459 (100) [M-BF₄-dpim]⁺. Molar conductivity

(CH₃CN): 145 S·cm²·mol⁻¹. Solubility: soluble in acetone, methanol,

dichloromethane, chloroform, and barely soluble in ethanol, and insol-

uble in water.

3

H^p-BPh), 5.80 (d, J_HH=6.1 Hz, 1 H, H⁶-cym), 5.65 (d, J_HH=6.0 Hz,

2.11.6. [(η⁶-p-cymene)Ru(dbzm)(dpim)]BPh₄(3a·BPh₄)

1 H, H²-cym), 5.49 (d, J_HH=6.2 Hz, 1 H, H³-cym), 5.41 (d, J_HH

=

[(η⁶-p-cym)RuCl(dbzm)] (77.6 mg, 0.157 mmol); NaBPh₄(56.9 mg,

0.166 mmol), dpim (44.4 mg, 0.167 mmol). Yield: 135.6 mg

(0.130 mmol, 82.8 %). FW (C₆₅H₆₀N₂PO₂RuB)=1044.06 g/mol. Anal.

calcd for C₆₅H₆₀N₂PO₂RuB·H₂O: C, 73.51; H, 5.88; N, 2.64. Found: C,

73.51; H, 5.49; N, 2.68. ¹H NMR (400 MHz, CDCl₃) δ=7.59 (m, 4 H,

H^o-Ph, dbzm), 7.53 (m, 2 H, H^p-Ph, dbzm), 7.45—7.31 (m, 14 H,

H^m-Ph-dbzm, H^o-Ph-BPh, H^p-Ph-dpim), 7.17–7.05 (m, 10 H, H^o,

3

6.1 Hz, 1 H, H⁵-cym), 5.35 (s, 1 H, H^a-bzac), 2.70 (s, 3 H, Me-Im), 2.34

(sept, J_HH=7.0 Hz, 1 H, H⁷-cym), 1.84 (s, 3 H, Me-bzac), 1.45 (s, 3 H,

3

H¹⁰-cym), 1.17 (d, J_HH=6.9 Hz, 3 H, H⁸-cym), 1.12 (d, J_HH

=

3

6.9 Hz, 3 H, H⁹-cym) ppm. ¹³C{¹H} NMR (101 MHz, CDCl₃) δ=

190.17 (s, 1C, C^b-bzac), 181.19 (s, 1C, C^b′-bzac), 164.43 (q, J_CB

=

1

49.6 Hz, 4C, Cⁱ, BPh₄⁻), 137.25 (s, 1C, Cⁱ-Ph-bzac), 136.49 (s, 8C,

C^o-Ph, BPh₄⁻), 136.49 (d, J_PC=80.4 Hz, 1C, C^2′-Im), 131.93 (d, J_PC

=

^m-Ph-dpim, H^4′,5′-Im), 6.96 (t, J_HH=7.4 Hz, 8 H, H^m-BPh, BPh₄⁻), 6.80

1

2

3

9.8 Hz, 2C, C^o-Ph-dpim), 131.87 (d, J_PC=9.8 Hz, 2C, C^o-Ph-dpim),

(t, J_HH=7.4 Hz, 4 H, H^p-BPh, BPh₄^-), 6.05 (s, 1 H, H^a-dbzm), 5.82 (d,

2

3

131.77 (s, 1C, C^p-Ph-bzac), 131.55 (s, 1C, C^p-Ph-dpim), 129.44

³J_HH=6.3 Hz, 2 H, H^2,6-cym), 5.50 (d, J_HH=6.3 Hz, 2 H, H^3,5-cym),

3

(d, J_PC=10.4 Hz, 2C, C^m-Ph-dpim), 129.34 (d, J_PC=10.3 Hz, 2C,

2.69 (s, 3 H, Me-Im), 2.41 (sept, J_HH=6.9 Hz, 1 H, H⁷-cym), 1.49 (s,

3

C^m-Ph-dpim), 128.95 (s, 1C, C^5′-Im), 128.76 (s, 2C, C^m-Ph-bzac),

3 H, H¹⁰-cym), 1.17 (d, J_HH=6.9 Hz, 6 H, H^8,9-cym) ppm. ¹³C{¹H}

3

128.15 (d, J_PC=44.9 Hz, 1C, Cⁱ-Ph), 127.77 (d, J_PC=37.3 Hz, 1C,

Cⁱ-Ph), 127.01 (s, 2C, Ph-bzac), 125.67 (m, 8C, C^m, BPh₄⁻), 109.68

(s, 1C, C¹-cym), 97.91 (s, 1C, C⁴-cym), 97.87 (s, 1C, C^a-bzac), 90.12

(d, J_PC=3.5 Hz, 1C, C⁵-cym), 89.49 (d, J_PC=3.3 Hz, 1C, C³-cym),

86.92 (s, 1C, C⁶-cym), 86.88 (s, 1C, C²-cym), 35.90 (s, 1C, N-Me),

30.50 (s, 1C, C⁷-cym), 28.31 (s, 1C, Me-bzac), 22.45 (s, 2C, C⁹-cym),

21.87 (s, 2C, C⁸-cym), 16.73 (s, 1C, C¹⁰-cym) ppm. C^4′-Im and one

C^p-Ph-dpim could not be assigned. ³¹P{¹H} NMR (162 MHz, CDCl₃)

δ=10.85 (s, 1P, κ¹-dpim) ppm. FT-IR (KBr pellet and nujol

mull, cm⁻¹): 3102 (w), 3055 (m, ν_C²_{H, sp}), 2984 (ν_C³_{H, sp}), 2966,

1588 (s, ν_{C_O+C_C}), 1556 (vs, ν_{C_O+C_C}), 1519 (vs, ν_{C_C+C_O}),

1486 (s, ν_{C_C+C_O}), 1437 (ν_P-C), 1372 (δ_{s, Me}), 1277, 1098 (m,

NMR (101 MHz, CDCl₃) δ=182.77 (s, 2C, C^b-dbzm), 164.44 (q, J_CB

=

1

49.2 Hz, 4C, Cⁱ-Ph, BPh₄⁻), 137.79 (s, 2C, Cⁱ-Ph-dbzm), 136.48 (s, 8C,

C^o-Ph, BPh₄^-), 135.91 (d, J_PC=41.7 Hz, 1C, C^2′-Im), 132.15 (s, 2C,

1

C^p-Ph, dbzm), 131.9 (d, J_PC=9.7 Hz, 4C, C^o-Ph-dpim), 131.55 (s, 1C,

2

C^4′-Im), 131.53 (s, 2C, C^p-Ph-dpim), 129.44 (d, J_PC=10.2 Hz, 4C,

3

C^m-Ph-dpim), 128.83 (s, 4C, C^m-Ph-dbzm), 128.55 (s, 1C, C^5′-Im),

127.69 (d, ¹J_PC=44.9 Hz, 2C, Cⁱ-Ph, dpim), 127.01 (s, 4C, C^o-Ph-dbzm),

125.65 (q, J_CB=2.8 Hz, 8C, C^m-Ph, BPh₄⁻), 121.75 (s, 4C, C^p-Ph, BPh₄^-),

3

109.82 (s, 1C, C¹-cym), 97.54 (s, 1C, C⁴-cym), 95.11 (s, 1C, C^a-dbzm),

90.28 (d,

J

_PC=3.4 Hz, 2C, C^3,5-cym), 86.98 (d, J_PC=4.8 Hz, 2C,

C^2,6-cym), 35.85 (s, 1C, N-Me), 30.44 (s, 1C, C⁷-cym), 22.21 (s, 2C,

C^8,9-cym), 16.91 (s, 1C, C¹⁰-cym) ppm. ³¹P{¹H} NMR (162 MHz,

CDCl₃) δ=10.82 (s, 1P, κ¹-dpim) ppm. FT-IR (KBr pellet and nujol

mull, cm⁻¹): 3112 (w), 3054 (m, ν_C²_{H, sp}), 2999, 2982 (ν_C³_{H, sp}), 2923,

1589 (m, ν_{C_O+C_C}), 1537 (vs, ν_{C_O+C_C}), 1520 (vs, ν_{C_C+C_O}),

1484 (s, ν_{C_C+C_O}), 1451, 1437 (ν_P-C), 1373 (δ_{s, Me}), 1314 (m), 1281,

1231, 1184, 1099 (m, ν_P-C), 1029 (m), 766 (m), 749 (m, δ_CH,oop), 732

ν

_P-C), 1031 (m), 1000, 774 (m), 746 (m, δ_CH,oop), 734 (s), 703 (s,

δ_{C_}_{C, oop}), 613 (m), 547 (m, dpim), 518 (dpim), 477 (dpim), 439

(w), 258 (w), 227 (w), 189 (w, Ru-P). MS (FAB+, CHCl₃): m/z

(%)=663 (9) [M-BPh₄]⁺

,

529 (5) [M-BPh₄-cym]⁺

,

397 (100)

[M-BPh₄-dpim]⁺. Molar conductivity (CH₃CN): 97.1 S·cm²·mol⁻¹

.

176

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

(s), 702 (s, δ_{C_C, oop}, BPh₄⁻), 614 (m), 548 (m, dpim), 519 (dpim), 477

(7) [M-BPh₄-cym]⁺, 335 (100) [M-BF₄-PPh₂py]⁺. Molar conductivity

(CH₃CN): 105.3 S·cm²·mol⁻¹. Solubility: soluble in acetone, chloro-

form and dichloromethane, barely soluble in methanol and ethanol,

and insoluble in water.

(dpim), 439 (w), 437 (w), 209 (w, Ru-P). MS (FAB+, CHCl₃): m/z

(%)=725 (35) [M-BPh₄]⁺

,

591 (10) [M-BPh₄-cym]⁺

,

459 (100)

[M-BPh₄-dpim]⁺. Molar Conductivity (CH₃CN): 115.2 S·cm²·mol⁻¹

.

Solubility: soluble in acetone, chloroform and dichloromethane, barely

soluble in methanol and ethanol, and insoluble in water.

2.11.9. [(η⁶-p-cymene)Ru(bzac)(PPh₂py)]BF₄(2b·BF₄)

[(η⁶-p-cym)RuCl(bzac)] (79.3 mg, 0.183 mmol); NaBF₄(41.4 mg,

0.377 mmol), PPh₂py (51.8 mg, 0.197 mmol). Yield: 94.7 mg

(0.127 mmol, 68.5%). FW (C₃₇H₃₇NPO₂RuBF₄)=746.55 g/mol. Anal.

calcd for C₃₇H₃₇NPO₂RuBF₄·0.5H₂O: C, 58.82; H, 5.07; N, 1.85.

Found: C, 58.81; H, 4.60; N, 2.12. ¹H NMR (400 MHz, CDCl₃) δ=

8.90 (m, 1 H, H^6′-py), 7.72 (m, 1 H, H^4′-py), 7.59–7.22 (m, 17 H,

2.11.7. [(η⁶-p-cymene)Ru(acac)(PPh₂py)]BF₄(1b·BF₄)

[(η⁶-p-cym)RuCl(acac)] (100.3 mg, 0.270 mmol); NaBF₄(60.9 mg,

0.545 mmol), PPh₂py (75.0 mg, 0.286 mmol). Yield: 107.2 mg

(0.157 mmol, 58.0%) FW (C₃₂H₃₅NPO₂RuBF₄)=684.48 g/mol. Anal.

calcd for C₃₂H₃₅NPO₂RuBF₄: C, 56.15; H, 5.15; N, 2.05. Decomposition

prevented from getting a good analysis and a mass spectrum. ¹H NMR

(400 MHz, CDCl₃) δ=8.94 (m, 1 H, H^6′-py), 7.76 (m, 1 H, H^4′-py),

7.53–7.46 (m, 3 H, H^p-Ph, H^5′-py), 7.45–7.36 (m, 8 H, H^o,m-Ph), 7.28

3

H^3′,5′-py, Ph), 5.83 (d, J_HH=6.0 Hz, 1 H, H⁶-cym), 5.68 (m, 2 H,

H^3,2-cym), 5.65 (d, ³J_HH=6.0 Hz, 1 H, H⁵-cym), 5.41 (s, 1 H, H^a-bzac),

3

2.47 (sept, J_HH=6.9 Hz, 1 H, H⁷-cym), 1.96 (s, 3 H, H¹⁰-cym), 1.82

3

(m, 1 H, H^3′-py), 5.71 (d, J_HH=6.2 Hz, 2 H, H^2,6-cym), 5.59 (d, J_HH

=

(s, 3 H, Me-bzac), 1.18 (d, J_HH=6.9 Hz, 3 H, H⁸-cym), 1.15 (d, J_HH

=

6.2 Hz, 2 H, H^3,5-cym), 4.76 (s, 1 H, H^a-acac), 2.43 (sept, J_HH=6.9 Hz,

6.9 Hz, 3 H, H⁹-cym) ppm. ¹³C{¹H} NMR (101 MHz, CDCl₃) δ=190.36

3

1 H, H⁷-cym), 1.88 (s, 3 H, H¹⁰-cym), 1.68 (s, 6 H, H^c-acac), 1.16

(s, 2C, C^b-bzac), 181.08 (s, 2C, C^b′-bzac), 156.53 (d, J_PC=71.7 Hz, 1C,

1

(d, J_HH=6.9 Hz, 6 H, H^8,9-cym) ppm. ¹³C{¹H} NMR (101 MHz, CDCl₃)

C^2′-py), 150.78 (d, J_PC=17.3 Hz, 1C, C^6′-py), 137.20 (s, 1C, Cⁱ-bzac),

3

δ=188.63 (s, 2C, C^b-acac), 134.44 (d, J_PC=9.5 Hz, 4C, C^o-Ph), 131.62

136.88 (d, J_PC=6.7 Hz, 1C, C^4′-py), 134.56 (d, J_PC=8.4 Hz, 2C,

2

3

2

(d, J_PC=2.3 Hz, 2C, C^p-Ph), 128.79 (d, J_PC=10.0 Hz, 4C, C^m-Ph),

C^o-Ph₂P), 134.47 (d, ²J_PC=8.4 Hz, 2C, C^o-Ph₂P), 131.76 (s, 1C, C^p-bzac),

4

3

107.92 (s, 1C, C¹-cym), 100.70 (s, 1C, C^a-acac), 98.76 (s, 1C, C⁴-cym),

131.59 (d, J_PC=2.7 Hz, 1C, C^p-Ph₂P), 131.54 (d, J_PC=2.7 Hz, 1C,

4

2

3

88.94 (d,

J

_PC=3.7 Hz, 2C, C^3,5-cym), 88.32 (d, J_PC=4.2 Hz, 2C,

C^p-Ph₂P), 129.57 (d, J_PC=18.1 Hz, 1C, C^3′-py), 128.85 (d, J_PC

=

C^2,6-cym), 30.69 (s, 1C, C⁷-cym), 27.31 (s, 2C, C^c-acac), 22.05 (s, 2C,

C^8,9-cym), 17.23 (s, 1C, C¹⁰-cym) ppm. Some peaks could not be assigned

due to low solubility and incipient decomposition as shown by appear-

ance of free p-cym signals. ³¹P{¹H} NMR (162 MHz, CDCl₃) δ=28.94

(s, 1P, κ¹-PPh₂py) ppm. ¹⁹F NMR (376 MHz, CDCl₃) δ=−154.45

(s, ¹⁰B-F, BF₄⁻), −154.51 (s, ¹¹B-F, BF₄⁻) ppm. FT-IR (KBr, cm⁻¹):

3056 (ν_C²_{H, sp}), 2960, 2921, 2869 (ν_C³_{H, sp}), 1574 (s, ν_{C_O+C_C}),

1517 (s, ν_{C_C+C_O}), 1436 (ν_P-C), 1378 (δ_{s, Me}), 1276, 1058 (s, ν_d

_B-F), 744 (δ_CH,oop), 696 (δ_{C_C, oop}), 529 (PPh₂py), 511 (PPh₂py),

448. Solubility: soluble in acetone, dichloromethane and chloroform,

and insoluble in water.

10.0 Hz, 2C, C^m-Ph₂P), 128.83 (d, J_PC=10.0 Hz, 2C, C^m-Ph₂P), 128.59

3

(s, 2C, C^m-bzac), 128.44 (d, J_PC=61.5 Hz, 1C, Cⁱ-Ph₂P), 127.05 (s, 2C,

1

C^o-bzac), 125.29 (d, J_PC=1.7 Hz, 1C, C^5′-py), 108.24 (d, J_PC

=

4

1.7 Hz, 1C, C¹-cym), 99.06 (s, 1C, C⁴-cym), 98.18 (s, 1C, C^a-bzac),

89.05 (d, J_PC=3.6 Hz, 1C, CH-cym), 88.91 (d, J_PC=3.7 Hz, 1C,

CH-cym), 88.65 (d, J_PC=4.1 Hz, 1C, CH-cym), 88.51 (d, J_PC=4.3 Hz,

1C, CH-cym), 30.70 (s, 1C, C⁷-cym), 28.05 (s, 1C, Me-bzac), 22.48

(s, 1C, C⁹-cym), 21.74 (s, 1C, C⁸-cym), 17.44 (s, 1C, C¹⁰-cym). The

signal for one Cⁱ-Ph of PPh₂py is missed. ³¹P{¹H} NMR (162 MHz,

CDCl₃) δ=29.02 (s, 1P, κ¹-P-PPh₂py) ppm. ¹⁹F NMR (376 MHz,

CDCl₃) δ=−153.98 (s, ¹⁰B-F, BF₄), −154.03 (s, ¹¹B-F, BF₄⁻

)

ppm. FT-IR (KBr, cm⁻¹): 3060 (ν_C²_{H, sp}), 2964, 2927, 2872 (ν_C³_{H, sp}),

1588 (s, ν_{C_O+C_C}), 1556 (vs, ν_{C_O+C_C}), 1515 (vs, ν_{C_C+C_O}), 1487

(s, ν_{C_C+C_O}), 1450, 1436 (ν_P-C), 1376 (δ_s,CH₃), 1058 (s, ν_{d B-F}), 752

(δδ_CH,oop), 697 (m, δ_{C_C, oop}), 530 (m, PPh₂py), 514 (PPh₂py).

2.11.8. [(η⁶-p-cymene)Ru(acac)(PPh₂py)]BPh₄(1b·BPh₄)

[(η⁶-p-cym)RuCl(acac)] (99.8 mg, 0.270 mmol); NaBPh₄(97.3 mg,

0.284 mmol), PPh₂py (75.8 mg, 0.288 mmol). Yield: 202.3 mg

(0.220 mmol, 81.5 %). FW (C₅₆H₅₅NPO₂RuB)=916.91 g/mol. Anal.

calcd for C₅₆H₅₅NPO₂RuB·2H₂O: C, 70.58; H, 6.24; N, 1.47. Found: C,

70.47; H, 5.83; N, 1.64. ¹H NMR (400 MHz, CDCl₃) δ=8.81 (m, 1 H,

H^6′-py), 7.54 (m, 1 H, H^4′-py), 7.48 (m, 2 H, H^p-PPh), 7.40 (m, 8 H,

H^o-Ph, BPh₄⁻), 7.37–7.28 (m, 9 H, H^o,m-PPh, H^5′-py), 7.13 (m, 1 H,

MS (FAB+, CHCl₃): m/z (%)=660 (31) [M-BF₄]⁺

, 526 (17)

[M-BF₄-cym]⁺, 397 (100) [M-BF₄-PPh₂py]⁺. Molar conductivity

(CH₃CN): 142.6 S·cm²·mol⁻¹. Solubility: soluble in acetone, metha-

nol, dichloromethane, chloroform and barely soluble in ethanol and

insoluble in water.

3

H^3′-py), 6.98 (t, J_HH=7.2 Hz, 8 H, H^m-Ph, BPh₄⁻), 6.84 (t, J_HH

=

7.2 Hz, 4 H, H^p-Ph, BPh₄^-), 5.36 (d, J_HH=6.2 Hz, 2 H, H^2,6-cym), 5.13

2.11.10. [(η⁶-p-cymene)Ru(bzac)(PPh₂py)]BPh₄(2b·BPh₄)

3

(d, J_HH=6.2 Hz, 2 H, H^3,5-cym), 4.73 (s, 1 H, H^a-acac), 2.33 (sept,

[(η⁶-p-cym)RuCl(bzac)] (99.7 mg, 0.230 mmol); NaBPh₄(83.2 mg,

0.243 mmol), PPh₂py (64.9 mg, 0.247 mmol). Yield: 153.1 mg

(0.156 mmol, 67.8%). FW (C₆₁H₅₇NPO₂RuB)=978.98 g/mol. Anal.

calcd for C₆₁H₅₇NPO₂RuB·6H₂O: C, 67.4; H, 6.40; N, 1.29. Found: C,

67.13; H, 5.82; N, 1.48. (bad correspondence, very hygroscopic com-

plex). ¹H NMR (400 MHz, CDCl₃) δ=8.78 (m, 1 H, H^6′-py), 7.52 (m,

1 H, H^4′-py), 7.48 (m, 4 H, H^o-PPh), 7.43–7.28 (m, 18 H, H^o-BPh,

H^m-PPh, H^5′-py, H^o,m,p-bzac), 7.23 (m, Hp-PPh), 7.10 (m, 1 H, H^3′-py),

3

³J_HH=7.0 Hz, 1 H, H⁷-cym), 1.65 (s, 6 H, H^c-acac), 1.63 (s, 3 H,

H¹⁰-cym), 1.09 (d, J_HH=7.0 Hz, 6 H, H^8,9-cym) ppm. ¹³C{¹H} NMR

3

(101 MHz, CDCl₃) δ=188.47 (s, 2C, C^b-acac), 164.46 (q, J_BC

=

1

49.2 Hz, 4C, Cⁱ-BPh, BPh₄^-), 156.56 (d, J_PC=72.03 Hz, 1C, C^2′-py),

1

150.75 (d, J_PC=17.6 Hz, 1C, C^6′-py), 136.71 (d, J_PC=6.3 Hz, 1C,

3

C^4′-py), 136.51 (s, 8C, C^o-BPh, BPh₄^-), 134.31 (d, J_PC=9.6 Hz, 4C,

2

C^o-Ph₂P), 131.68 (d, J_PC=2.5 Hz, 2C, C^p-Ph₂P), 129.20 (d, J_PC

=

4

2

17.1 Hz, 1C, C^3′-py), 128.79 (d, J_PC=10.0 Hz, 4C, C^m-Ph₂P), 128.36

6.96 (t, J_HH=7.1 Hz, 8 H, H^m-BPh), 6.82 (t, J_HH=6.8 Hz, 4 H,

3

(d, J_PC=43.6 Hz, 2C, Cⁱ-Ph₂P), 125.69 (q, J_BC=2.7 Hz, 8C, C^m-BPh,

H^p-BPh), 5.47 (d, J_HH=6.1 Hz, 1 H, H⁶-cym), 5.41 (s, 1 H, H^a-bzac),

1

3

4

3

BPh₄⁻),125.34 (d, J_PC=2.3 Hz, 1C, C^5′-py), 107.56 (s, 1C, C¹-cym),

5.31 (d, J_HH=6.1 Hz, 1 H, H²-cym), 5.20 (d, J_HH=6.1 Hz, 1 H,

101.27 (s, 1C, C^a-acac), 98.01 (s, 1C, C⁴-cym), 88.97 (d, J_PC=3.8 Hz,

2C, C^3,5-cym), 87.89 (d, J_PC=4.3 Hz, 2C, C^2,6-cym), 30.60 (s, 1C,

C⁷-cym), 27.32 (s, 2C, C^c-acac), 21.98 (s, 2C, C^8,9-cym), 17.09 (s, 1C,

C¹⁰-cym) ppm. ³¹P{¹H} NMR (162 MHz, CDCl₃) δ=28.61 (s, 1P,

κ¹-PPh₂py) ppm. FT-IR (KBr pellet and nujol mull, cm⁻¹): 3055 (m),

2999, 2983 (w), 2969, 1573 (s, ν_{C_O+C_C}), 1518 (s, ν_{C_C+C_O}), 1480

(BPh₄⁻), 1434 (ν_P-C), 1372 (δ_{s, Me}), 1275, 1097 (m, ν_P-C), 1032 (m),

743 (m, δ_CH,oop), 733 (s), 707 (s, δ_{C_C, oop}, BPh₄⁻), 614 (m), 533 (m,

PPh₂py), 514 (PPh₂py), 482 (m), 470 (w), 443 (w), 432, 420, 210 (w,

Ru-P), 188. MS (FAB+, CHCl₃): m/z (%)=598 (11) [M-BPh₄]⁺, 464

H³-cym), 5.16 (d, J_HH=6.1 Hz, 1 H, H⁵-cym), 2.36 (sept, J_HH

=

3

6.9 Hz, 1 H, H⁷-cym), 1.78 (s, 3 H, Me-bzac), 1.69 (s, 3 H,

H¹⁰-cym), 1.11 (d, J_HH=6.9 Hz, 3 H, H⁸-cym), 1.09 (d, J_HH

=

3

6.9 Hz, 3 H, H⁹-cym) ppm. Acquisition of the ¹³C{¹H} NMR failed

due to low solubility and incipient decomposition. ³¹P{¹H} NMR

(162 MHz, CDCl₃) δ=28.58 (s, 1P, κ¹-P-PPh₂py) ppm. FT-IR (KBr

pellet and nujol mull, cm⁻¹): 3053 (m), 2997, 2981 (w), 2965,

1587 (s, ν_{C_O+C_C}), 1553 (vs, ν_{C_O+C_C}), 1519 (vs, ν_{C_C+C_O}),

1486 (s, ν_{C_C+C_O}), 1436 (ν_P-C), 1371 (δ_{s, Me}), 1289, 1097 (m,

ν

_P-C), 1030 (m), 1000 (w), 853 (W), 770 (w), 744 (m, δ_CH,oop), 734

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

177

(s), 705 (vs, δ_{C_C, oop}, BPh₄⁻), 613 (m), 526 (m, PPh₂py), 511

C^3,5-cym), 88.29 (d, J_PC=4.3 Hz, 2C, C^2,6-cym), 30.53 (s, 1C,

C⁷-cym), 22.08 (s, 2C, C^8,9-cym), 17.48 (s, 1C, C¹⁰-cym) ppm. ³¹P

{¹H} NMR (162 MHz, CDCl₃) δ=28.41 (s, 1P, κ¹-PPh₂py) ppm.

FT-IR (KBr pellet and nujol mull, cm⁻¹): 3055 (m), 2998, 2983 (w),

2966, 1588 (s, ν_{C_O+C_C}), 1571 (w) 1538 (vs, ν_{C_O+C_C}), 1520

(vs, ν_{C_C+C_O}), 1483 (s, ν_{C_C+C_O}), 1451, 1436 (ν_P-C), 1368 (δ_s,

_Me), 1314, 1230, 1183, 1097 (m, ν_P-C), 1030 (m), 1001 (w), 843

(W), 770 (w), 746 (m, δ_CH,oop), 733 (s), 703 (vs, δ_{C_C, oop}, BPh₄^-), 614

(m), 527 (m, PPh₂py), 512 (PPh₂py), 497 (m), 469 (w), 445 (w), 420,

280, 208 (w, Ru-P), 171. MS (FAB+, CHCl₃): m/z (%)=722 (28)

(PPh₂py), 502 (m), 460 (w), 445 (w), 420, 248, 212 (w, Ru-P), 188.

MS (FAB+, CHCl₃): m/z (%)=660 (22) [M-BPh₄]⁺

, 526 (11)

[M-BPh₄-cym]⁺, 397 (100) [M-BPh₄-PPh₂py]⁺. Molar conductivity

(CH₃CN): 100.4 S·cm²·mol⁻¹. Solubility: soluble in acetone, chloro-

form and dichloromethane, barely soluble in methanol and ethanol,

and insoluble in water.

2.11.11. [(η⁶-p-cymene)Ru(dbzm)(PPh₂py)]BF₄(3b·BF₄)

[(η⁶-p-cym)RuCl(dbzm)] (79.9 mg, 0.162 mmol); NaBF₄(36.1 mg,

0.329 mmol), PPh₂py (45.0 mg, 0.171 mmol). Yield: 61.1 mg

(0.076 mmol, 46.6%). FW (C₄₂H₃₉NPO₂RuBF₄)=808.62 g/mol. Anal.

calcd for C₄₂H₃₉NPO₂RuBF₄: C, 62.39; H, 4.86; N, 1.73. Found: C,

62.17; H, 4.69; N, 1.95. ¹H NMR (400 MHz, CDCl₃) δ=8.84 (m, 1 H,

H^6′-py), 7.67 (m, 1 H, H^4′-py), 7.63–7.58 (m, 4 H, H^o-dbzm), 7.54–7.43

(m, 6 H, H^p-dbzm, H^o-PPh₂py), 7.43–7.37 (m, 7 H, H^5′-py, H^m-dbzm,

H^p-PPh₂py), 7.30–7.20 (m, 5 H, H^3′-py, H^m-PPh₂py), 6.08 (s, 1 H,

[M-BPh₄]⁺, 588 (13) [M-BPh₄-cym]⁺, 459 (100) [M-BPh₄-PPh₂py]⁺

.

Molar conductivity (CH₃CN): 92.3 S·cm²·mol⁻¹. Solubility: soluble in

acetone, chloroform and dichloromethane, barely soluble in methanol

and ethanol, and insoluble in water.

2.12. Computational studies

3

H^a-dbzm), 5.81 (d, J_HH=6.2 Hz, 2 H, H^2,6-cym), 5.74 (d, J_HH

=

Computational modeling of the anion BPh₄⁻and BF₄⁻and single

point calculation for the cation 1a⁺(using the structural data obtained

from the X-ray crystallographic determination) were performed using

the Gaussian 03 program [34], employing density functional theory

with the hybrid method known as B3LYP, in which the Becke

three-parameter exchange functional [35] and the Lee–Yang–Parr cor-

relation functional were used [36]. For all nonmetal atoms, the

contracted 6–31 G (d,p) [37,38] basis set was selected, which included

diffuse functions. For the ruthenium center, the double-ζ basis set

Lanl2dz was used in conjunction with a pseudopotential representing

the core set of electrons [39]. Structures of the anions were geometrical-

ly optimized to an energy minimum. The position on the local potential

energy surface was conﬁrmed through vibration analysis using second

derivatives, where no imaginary frequencies were observed. The elec-

trostatic potential maps, as shown in Fig. 5, were generated from the

calculated electron density and potential using Gaussview [40].

The free energies of solvation were obtained using Barone and Cossi's

implementation of the conductor like polarizable continuum model

(C-PCM).[41,42] The C-PCM calculations were performed with default

parameters as single points on the gas-phase geometries since this has

been shown to give better results than reoptimization [43]. The united

atom topological model applied on atomic radii of the UFF force ﬁeld

(UA0) was used.

6.1 Hz, 2 H, H^3,5-cym), 2.50 (sept, J_HH=6.9 Hz, 1 H, H⁷-cym), 2.02

3

(s, 3 H, H¹⁰-cym), 1.18 (d, J_HH=6.9 Hz, 6 H, H^8,9-cym) ppm. ¹³C

3

NMR (101 MHz, CDCl₃) δ=182.75 (s, 2C, C^b-dbzm), 156.36 (d, J_PC

=

1

71.6 Hz, 1C, C^2′-py), 150.80 (d, ³J_PC=16.9 Hz, 1C, C^6′-py), 137.72 (s, 2C,

3

2

Cⁱ-dbzm), 136.91 (d, J_PC=6.9 Hz, 1C, C^4′-py), 134.57 (d, J_PC=9.7 Hz,

4C, C^o-Ph₂P), 131.94 (s, 2C, C^p-dbzm), 131.55 (d, J_PC=2.5 Hz, 2C,

4

C^p-Ph₂P), 129.65 (d, J_PC=18.3 Hz, 1C, C^3′-py), 128.90 (d, J_PC

=

2

3

10.1 Hz, 4C, C^m-Ph₂P), 128.69 (s, 4C, C^m-dbzm), 128.51 (d, J_PC

1

45.1 Hz, 2C, Cⁱ-Ph₂P), 127.07 (s, 4C, C^o-dbzm), 125.26 (d, J_PC=2.6 Hz,

1C, C^5′-py), 108.54 (s, 1C, C¹-cym), 99.36 (s, 1C, C⁴-cym), 95.33 (s, 1C,

C^a-dbzm), 88.95 (d, J_PC=3.6 Hz, 2C, C^3,5-cym), 88.82 (d, J_PC=4.2 Hz,

2C, C^2,6-cym), 30.69 (s, 1C, C⁷-cym), 22.18 (s, 2C, C^8,9-cym), 17.67 (s, 1C,

C¹⁰-cym) ppm. ³¹P NMR (162 MHz, CDCl₃) δ=28.92 (s, 1P, κ¹-PPh₂py)

ppm. ¹⁹F NMR (376 MHz, CDCl₃) δ=−154.44 (s, ¹⁰B-F, BF₄⁻), −154.49

(s, ¹¹B-F, BF₄⁻) ppm. FT-IR (KBr, cm⁻¹): 3059 (ν_C²_{H, sp}), 2964 (ν_C³_{H, sp}),

1589 (m, ν_{C_O+C_C}), 1571 (ν_C=N), 1538, (vs, ν_{C_O+C_C}), 1520 (vs,

4

ν_{C_C+C_O}), 1484 (s, ν_{C_C+C_O}), 1452, 1437 (ν_P-C), 1374 (s, δ_s,CH₃),

1315, 1059 (s, ν_{d B-F}), 756 (δ_CH,oop), 724, 696 (m, δ_{C_C, oop}), 529 (m,

PPh₂py), 514 (PPh₂py). MS (FAB+, CHCl₃): m/z (%)=722 (22)

[M-BF₄]⁺, 588 (14) [M-BF₄-cym]⁺, 459 (100) [M-BF₄-PPh₂py]⁺

.

Molar conductivity (CH₃CN): 137.1 S·cm²·mol⁻¹. Solubility: solu-

ble in acetone, methanol, ethanol, dichloromethane and chloroform,

and insoluble in water.

3. Results and discussion

2.11.12. [(η⁶-p-cymene)Ru(dbzm)(PPh₂py)]BPh₄(3b·BPh₄)

[(η⁶-p-cym)RuCl(dbzm)] (80.0 mg, 0.160 mmol); NaBPh₄(56.1 mg,

0.164 mmol), PPh₂py (45.2 mg, 0.172 mmol).Yield: 130.5 mg

(0.125 mmol, 78.1%). FW (C₆₆H₅₉NPO₂RuB)=1041.05 g/mol. Anal.

calcd for C₆₆H₅₉NPO₂RuB·3.5H₂O: C, 71.8; H, 6.03; N, 1.27. Found: C,

71.74; H, 5.83; N, 1.40. ¹H NMR (400 MHz, CDCl₃) δ=8.72 (m, 1 H,

H^6′-py), 7.56 (m, 4 H, H^o-Ph, dbzm), 7.50 (m, 2 H, H^p-Ph, dbzm), 7.46

(m, 1 H, H^4′-py), 7.42–7.32 (m, 18 H, H^o,p-PPh, H^o-BPh, H^m-CPh-dbzm),

7.26 (m, 1 H, H^5′-py), 7.21 (m , 4 H, H^o,p-PPh), 7.05 (m, 1 H, H^3′-py), 6.94

3.1. Synthesis and general characterization

Precursors of formula [(η⁶-p-cym)Ru(κ²-O,O′-X)Cl] 1–4 (X=acac,

bzac, dbzm, hfac) were prepared according to the method described

by Dyson et al. [23] in a one-pot reaction by treating a solution of

[(η⁶-p-cymene)Ru(μ-Cl)Cl]₂[26] in acetone with the appropriate

1,3-diketone and an excess of Na₂CO₃at room temperature for

approximately 3 h (see Scheme 2 and Eq. (1) in Scheme 3).

(t, J_HH=7.4 Hz, 8 H, H^m-Ph, BPh₄⁻), 6.79 (t, J_HH=7.1 Hz, 4 H, H^p-Ph,

Novel complexes of general formula [(η⁶-p-cym)Ru(κ²-O,O′-X)

(κ¹-P-L)]Y (X=acac, bzac, dbzm), 1a·Y-3a·Y and 1b·Y-3b·Y, were pre-

pared in acetone as BF₄⁻or BPh₄⁻salts by treatment of the corresponding

precursors 1–3 with an excess of NaBF₄or slight excess of NaBPh₄and

the corresponding phosphine: L=dpim for series a (1a·Y–3a·Y) and

L=PPh₂py, for series b (1b·Y–3b·Y) (see Scheme 2 and Eq. (2) in

Scheme 3). The complexes with bzac were obtained as racemic mixtures

due to the chirality of the Ru ion. Attempts to prepare the corresponding

3

BPh₄⁻), 6.08 (s, 1 H, H^a-dbzm), 5.43 (d, J_HH=6.2 Hz, 2 H, H^2,6-cym),

3

5.23 (d, J_HH=6.2 Hz, 2 H, H^3,5-cym), 2.40 (sept, J_HH=6.9 Hz, 1 H,

H⁷-cym), 1.74 (s, 3 H, H¹⁰-cym), 1.11 (d, J_HH=6.9 Hz, 6 H, H^8,9-cym)

3

ppm. ¹³C{¹H} NMR (101 MHz, CDCl₃) δ=182.74 (s, 2C, C^b-dbzm),

164.36 (q, J_BC=49.2 Hz, 4C, Cⁱ-BPh, BPh₄⁻), 156.56 (d, J_PC=72.03 Hz,

1

1C, C^2′-py), 150.62 (d, J_PC=17.6 Hz, 1C, C^6′-py), 137.70 (s, 2C,

3

Cⁱ-Ph-dbzm), 136.66 (d, J_PC=6.3 Hz, 1C, C^4′-py), 136.47 (s, 8C,

3

C^o-BPh, BPh₄⁻), 134.38 (d, J_PC=9.7 Hz, 4C, C^o-Ph₂P), 132.03 (s, 2C,

analogues from 4

were unsuccessful. Complexes [(η⁶-p-cymene)

2

C^p-Ph-dbzm), 131.61 (d, J_PC=2.5 Hz, 2C, C^p-Ph₂P), 129.24 (d, J_PC

=

RuCl(κ²-P,N-L)]BF₄(5a·BF₄and 5b·BF₄) were obtained instead, thus dem-

onstrating that hfac behaves as a better leaving group than Cl⁻under the

conditions employed (see Eq. (3) in Scheme 3). Indeed, the weakness of

the Ru\O bond in complex 4 has been reported previously [24].

4

2

17.1 Hz, 1C, C^3′-py), 128.89 (d, J_PC=9.8 Hz, 4C, C^m-Ph₂P), 128.72

3

(s, 4C, C^m-Ph-dbzm), 128.58 (d, J_PC=44.7 Hz, 2C, Cⁱ-Ph₂P), 126.96

1

(s, 4C, C^o-Ph-dbzm), 125.67 (q, J_BC=2.7 Hz, 8C, C^m-BPh, BPh₄^-),

3

125.20 (d, J_PC=2.3 Hz, 1C, C^5′-py), 109.51 (s, 1C, C¹-cym), 98.11

All of the ionic complexes were isolated in moderate to good

yields (from 47 to 88%) as yellow, orange or brown solids that were

4

(s, 1C, C^a-dbzm), 95.41 (s, 1C, C⁴-cym), 89.41 (d, J_PC=3.8 Hz, 2C,

178

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

air and moisture resistant — with the exception of 1b·BF₄, which

decomposed to give a dark solid in a few days under an air atmo-

sphere. Qualitative tests indicated that the BF₄⁻salts are soluble in

common organic solvents such as acetone, methanol, ethanol, chloro-

form and dichloromethane, whereas the BPh₄⁻derivatives are only

soluble in dichloromethane, chloroform and acetone. Furthermore,

neither of the salts is soluble in pure water despite being ionic,

although BF₄⁻salts are soluble in H₂O/DMSO (98:2) mixtures. All of

the compounds were comprehensively characterized on the basis of

¹H, ¹³C{¹H}, ¹⁹F (BF₄⁻salts) and ³¹P{¹H} NMR in CDCl₃, as well as by IR

spectroscopy, positive ion FAB mass and elemental analysis. In addi-

tion, the solid state structures of 1a·BF₄, 1a·BPh₄, 3a·BF₄, 1b·BPh₄

and 3b·BPh₄were determined by X-ray diffraction.

FAB+mass spectra were acquired for all the new ionic complexes

apart from 1b·BF₄, which decomposed. Every spectrum contained a

peak for the expected complex cation, [M−Y]⁺(Y=BF₄⁻or BPh₄⁻),

and two characteristic fragmentation peaks: (i) one for the loss of the

p-cymene ring [M−Y−cym]⁺and (ii) the base peak for the loss of

the respective aminophosphine, [M−Y−L]⁺(L=dpim or PPh₂py).

FT-IR spectra were recorded for all the complexes and are fully consis-

tent with the aforementioned formulations. Complexes 1–4 show charac-

teristic bands for the Ru–Cl stretching bands at around 280 cm⁻¹and

these are not present in the cases of the cationic derivatives 1a·Y–3a·Y

and 1b·Y–3b·Y. All of these spectra show the expected strong peaks for

the BF₄⁻anion at around 1058 cm⁻¹and for the BPh₄⁻at around

704 cm⁻¹[21,44]. Moreover, the coupled ν_{C_O+C_C}stretching peaks

appear at characteristic values for each β-diketonate, i.e. around 1573

and 1515 cm⁻¹for acac, 1588, 1555,1516 and 1488 cm⁻¹for bzac and

ﬁnally 1589, 1538, 1520 and 1484 cm⁻¹for dbzm [28,29,45,46].

All of the BF₄⁻and BPh₄⁻salts behave as 1:1 electrolytes in acetoni-

trile solutions (10⁻³M) according to the molar conductivity measure-

ments (Λ_M) [47]. The values are clearly higher for the BF₄⁻salts (from

121.3 to 145.4) than for the BPh₄⁻salts (from 92.3 to 119.7) (see

Experimental section and Table S6 of Supporting Information).

The ³¹P{¹H} NMR spectra of complexes 1a·Y–3a·Y and 1b·Y–3b·Y

in CDCl₃each contain one sharp singlet that is strongly shifted to higher

frequency with respect to that for the corresponding free ligand. This

shift is a consequence of coordination to the Ru ion (see Table 1). In-

deed, the signals appear in a very narrow range, i.e. between 10.82

and 11.51 ppm for series a and from 28.41 to 29.02 ppm for series b. In-

terestingly, the ‘β-diketonate’ effect on δ (³¹P) is negligible in both se-

ries. By contrast, a weak but characteristic displacement to higher

frequencies is observed for all of the BF₄⁻salts when compared to

their BPh₄⁻counterparts, Δδ (³¹P)=0.33–0.66, indicating that the do-

nation of electron density from the phosphorus to the metal is slightly

higher for BF₄⁻salts (Table 1). This counter-anion effect has been

explained as being a consequence of a reduced bonding interaction be-

tween the arene ring and the Ru in the BF₄derivatives as compared to

the BPh₄salts [23], and suggests that the counter-anions play a

non-innocent role in the electronic system of the cationic complex. In

any event, the recorded δ values compare well with those observed

for the analogues [(η⁶-p-cym)RuCl₂(κ¹-P-L)] (L=dpim, A; or PPh₂py,

B in Scheme 1) and they prove the κ¹-P coordination mode of the re-

spective ligands [21,48,49]. Nevertheless, the resonances of the neutral

complexes A and B appear at lower frequencies (meaning that the P

atoms are more shielded) than those of their cationic β-diketonato rel-

atives, 1a·Y–3a·Y and 1b·Y–3b·Y, and this ﬁnding could be due to the

higher electronic density at the metal center in the former. The spectra

of products 5a·BF₄and 5b·BF₄exhibit a singlet at −8.29 ppm and

−16.54 ppm, respectively, and these are consistent with a κ²-P,N coor-

dination mode for the corresponding ligands [21].

Complete assignment of the resonances in the ¹H and ¹³C{¹H}

NMR spectra of all complexes for the corresponding arene, phosphine

and β-diketonate was performed using 2D NMR experiments such as

Scheme 2. Structures and numbering of aminophospine ligands, Ru(II) precursors of general formula [(η⁶-p-cymene)Ru(κ²-O,O′-X)Cl] and novel Ru(II) arene derivatives of general

formula [(η⁶-p-cymene)Ru(κ²-O,O′-X)(κ¹-P-L)]Y reported in this work.

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

179

Scheme 3. Synthesis of the precursors 1–4 and the cationic complexes 1a·Y-3a·Y, 1b·Y-3b·Y (Y=BF₄⁻, BPh₄⁻), 4a·BF₄and 4b·BF₄.

gCOSY, NOESY, gHSQC and gHMBC (see Experimental section and

Table S7 of Supporting Information). The ¹H NMR spectra of com-

plexes 1a·Y, 3a·Y, 1b·Y and 3b·Y reveal the local C_ssymmetry of

the metallic center (non-stereogenic and achirotopic in nature),

with the characteristic AA′BB′ spin system for the cymene aromatic

protons and homotopic methyl groups for the isopropyl moiety

[50]. On the other hand, the ¹H NMR spectra for complexes 2a·Y

and 2b·Y display an ABCD spin system for the aromatic protons of

the cymene ring and diastereotopic methyl groups in the isopropyl

entity, which could indicate a C₁symmetry environment for the

stereogenic and chirotopic Ru ion [51]. The ¹³C{¹H} NMR spectra of

all the complexes show symmetry patterns that are fully consistent

with those established by ¹H NMR spectroscopy (see Experimental

section).

The p-cymene aromatic protons in complexes 1a·Y–3a·Y and

1b·Y–3b·Y exhibit higher frequencies than those of the stable com-

plexes [(η⁶-p-cym)RuCl₂(dpim)], A, and [(η⁶-p-cym)RuCl₂(PPh₂py)],

B, respectively [21,48,49]. Such deshielding is similar to that of free

p-cymene (7.13 ppm, multiplet, in CDCl₃) and could be interpreted

as a symptom of weak coordination to the metallic center. In fact,

free p-cymene was detected by ¹H and ¹³C{¹H} NMR spectroscopy

in the corresponding samples (CDCl₃) of all the cationic complexes

several hours after their preparation. The extent of decomposition

in solution was estimated by the integration of ¹H NMR signals,

which indicated less than 10% after 20 h in most cases. Exceptions

to this behavior are 1a·BPh₄and 3a·BPh₄, which also decompose

but at a slower rate. In particular, 1a·BPh₄, as a model complex,

was monitored over ﬁve days by ¹H and ³¹P{¹H} NMR spectroscopy

in CDCl₃and it was concluded that this compound undergoes a slow

and intricate evolution. Several emerging signals appeared in the

³¹P{¹H} NMR spectrum after 5 days (see Fig. 1). The ¹H NMR spec-

trum shows that 58% of 1a·BPh₄remained unchanged along with

23% of free p-cymene and another unknown Ru-arene product. It is

worth mentioning that both ring slippage and ring substitution

processes have been alleged to operate as activation mechanisms in

catalysis and also in reactions with potential biomolecular targets,

including DNA [52–54].

Interestingly, the ¹H NMR spectra of the BPh₄⁻salts in CDCl₃

showed lower frequencies for all the resonances of the p-cymene

and phosphine ligands in comparison to those recorded in the spectra

of the BF₄^-analogues. The effect is particularly pronounced for the

aromatic p-cymene signals (e.g. 5.88 and 5.69 ppm for 1a·BF₄and

5.65 and 5.40 ppm for 1a·BPh₄in CDCl₃). This anion effect has been

interpreted in similar complexes as evidence of stronger coordination

of the p-cymene ring in the case of BPh₄⁻salts [23]. In our case, we as-

cribe this observation to the formation of speciﬁc ion-pairs, favored in

solvents of low polarity such as CDCl₃, without detriment to the pre-

ceding argument. In particular, we propose that the p-cymene ring is

involved in the formation of ion-pairs that differ in nature depending

on the anion: (i) CH–π interactions involving aromatic CH groups of

p-cymene and phenyl rings of the BPh₄⁻anion, with a shielding effect

on the p-cymene NMR signals, and (ii) C\H⋯F hydrogen bonds, with

a deshielding effect on the p-cymene NMR peaks, in the case of BF₄^-

salts. Such an interpretation is supported by the interionic contacts

observed in the crystal structures determined by X-ray diffraction

(see below).

In an effort to ﬁnd evidence for the existence of ion-pairing we

recorded the ¹H and ³¹P{¹H} NMR spectra for a mixture of 1a·BF₄/

1a·BPh₄(1:1) in CDCl₃. The proton resonances of this mixture

showed average values between those of pure 1a·BF₄and pure

1a·BPh₄(see Fig. 2). This result is in accordance with a fast ionic

exchange at room temperature and conﬁrms the labile nature of

these weak interactions. The ³¹P{¹H} NMR spectrum of the mixture

produced a singlet at δ_P=11.11 ppm, i.e. between those of pure

1a·BPh₄(10.89 ppm) and pure 1a·BF₄(11.43 ppm).

Table 1

³¹P{¹H} NMR chemical shifts (ppm) for phosphines dpim and PPh₂py and complexes

[(p-cym)Ru(X)(κ¹-P-L)] (L=dpim, A, 1a·Y–3a·Y; L=PPh₂py, B, 1b·Y–3b·Y) in

CDCl₃at 25 °C. All resonances were observed as singlets.

Comparison of the NMR data for the β-diketonate CH^αgroups

within the two sets of compounds (series a and series b) reveals

some interesting trends. On the one hand, the ¹H NMR chemical shifts

increase in the order acacbbzacbdbzm. On the other hand, the ¹³C

NMR chemical shifts show the opposite trend and increase in the

order dbzmbbzacbacac. It seems that the electron-donating Me

group shields the H nucleus and deshields the C nucleus. In contrast,

the electron-withdrawing Ph group deshields the proton and shields

the carbon. These ﬁndings are consistent with the higher polarization

of the C\H bond expected for the β-diketonates with electron-

withdrawing groups.

Series a

δ (ppm)

Δδ^a

Series b

δ (ppm)

Δδ^a

dpim

A

−29.47

7.58

PPh₂py

B

−7.63

23.54

28.94

28.61

29.02

28.58

28.92

28.41

1a·BF₄

1a·BPh₄

2a·BF₄

2a·BPh₄

3a·BF₄

3a·BPh₄

11.43

10.89

11.51

10.85

11.44

10.82

0.54

0.66

0.62

1b·BF₄

1b·BPh₄

2b·BF₄

2b·BPh₄

3b·BF₄

3b·BPh₄

0.33

0.44

0.51

a

Δδ is the chemical shift difference for pairs of derivatives with identical cations and

BF₄^-or BPh₄^-counter-ions.

180

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

Fig. 1. Evolution with time of the ³¹P{¹H} NMR spectrum for 1a·BPh₄(ﬁlled red triangle) in CDCl₃. Evolution products are labelled as (ﬁlled green circle).

[21,23]. The coordination sphere consists of a η⁶-p-cymene ring that oc-

3.2. Solid state structures of 1a·BF₄, 1a·BPh₄, 3a·BF₄, 1b·BPh₄,

3b·BPh₄and 2

cupies three facial coordination positions, plus the κ¹-P-phosphine (chlo-

ride in the case of 2) and a κ²-O,O′-β-diketonate. Ru\C(arene) bond

distances are slightly different for each speciﬁc complex, with the

shortest distances for those carbon atoms in a cis orientation to the P

and the longest distances for those carbon atoms trans to the P, as one

would expect according to the strongest trans inﬂuence of the phos-

phines in comparison to the O-donor ligands. For the aminophosphine

derivatives with acac the range found for the Ru\C distances is 2.16–

2.23 Å while for those containing dbzm it is 2.20–2.24 Å, i.e. a higher

range is observed for complexes with acac than for those with dbzm.

The Ru-arene centroid distance is slightly higher for the dbzm deriva-

tives (1.70–1.71 Å) than for the acac complexes (1.69 Å). The values

are very similar to those of the stable complexes [(p-cym)RuCl₂(dpim)],

A, [1.700 Å] and [(p-cym)RuCl₂(PPh₂py)], B, [1.704 Å], for which loss of

the p-cymene ring in solution has not been observed. Therefore, the pos-

sible weakness of the bond between the metal and the p-cymene ob-

served for the novel β-diketonato complexes in solution (partial loss of

p-cymene in CDCl₃) does not seem to persist in the solid state. The

Ru-arene centroid distance is smaller for 2 (1.655 Å), which is the only

neutral derivative. The Ru\P bond lengths are in the range 2.34–

Single crystals suitable for X-ray analysis were obtained for 1a·BF₄,

1a·BPh₄, 3a·BF₄, 1b·BPh₄, 3b·BPh₄and 2 by either liquid–liquid diffu-

sion techniques from CH₂Cl₂/n-hexane (1a·BF₄), CH₂Cl₂/n-hexane

(3b·BPh₄), EtOH/n-hexane (3a·BF₄) or by slow evaporation experi-

ments from MeOH (1a·BPh₄) and acetonitrile (1b·BPh₄). Although

complex 2 is not described in this paper for the ﬁrst time, the determi-

nation of the structure by X-ray diffraction has not been reported and

thus it is included in this section. Crystallization conditions are de-

scribed in the Experimental section. The ORTEP diagrams are shown

in Fig. 3. Unfortunately, the X-ray data obtained for 1b·BPh₄were of

low quality. Nevertheless, the atom connectivity was deﬁned sufﬁcient-

ly well to determine the important features of the structure. A ball and

stick representation is depicted in Fig. 3. Relevant crystallographic pa-

rameters are listed in Table 2 and selected bond lengths and angles

are given in Table 3.

The molecular structures ﬁt the classical three-legged piano-stool ar-

rangement with a pseudo-octahedral geometry and display similar mo-

lecular features to those of structurally related ruthenium complexes

Fig. 2. (Left) Region of the aromatic p-cymene protons in the ¹H NMR spectra of (a) 1a·BF₄, (b) an equimolar mixture of 1a·BF₄and 1a·BPh₄and (c) 1a·BPh₄. (Right) ³¹P{¹H} NMR

spectra of (a) 1a·BF₄, (b) an equimolar mixture of 1a·BF₄and 1a·BPh₄and (c) 1a·BPh₄. CDCl₃was used as solvent in all the experiments.

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

181

Fig. 3. ORTEP or ball and stick drawing of the complex cations: (a) [(p-cym)Ru(acac)(dpim)]BF₄(1a·BF₄), (b) [(p-cym)Ru(acac)(dpim)]BPh₄(1a·BPh₄), (c) [(p-cym)

Ru(dbzm)(dpim)]BF₄(3a·BF₄), (d) [(p-cym)Ru(acac) (PPh₂py)]BPh₄(1b·BPh₄), (e) [(p-cym)Ru(dbzm)(Ph₂Ppy)]BPh₄(3b·BPh₄), (f) 2. Hydrogens and counterions have been

omitted for clarity.

182

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

Table 2

Crystal data and structure reﬁnement for 1a·BF₄, 1a·BPh₄, 1b·BPh₄, 2, 3a·BF₄and 3b·BPh_4.

1a·BF₄

1a·BPh₄

1b·BPh₄

2

3a·BF₄

3b·BPh₄

Empirical formula

Formula weight

C₃₁H₃₆BF₄N₂O₂PRu

687.47

C₅₅H₅₆BN₂O₂PRu

919.87

C₅₆H₅₅BNO₂PRu

916.86

C₂₀H₂₃ClO₂Ru

431.90

C₄₁H₄₀BF₄N₂O₂PRu

811.60

C₆₆H₅₉BNO₂PRu

1040.99

Temperature (K)

173(2)

100(2)

298(2)

173(2)

100(2)

Wavelength (Å)

0.71073

Crystal system

Space group

Monoclinic

C2/c

Monoclinic

P2₁

Monoclinic

P2₁/n

Triclinic

Pī

Orthorhombic

P2₁2₁2₁

Triclinic

Pī

a(Å)

b(Å)

c(Å)

α(°)

38.455(4)

9.384(1)

18.523(2)

10.260(2)

21.274(3)

11.662(2)

20.368(6)

11.325(3)

20.480(5)

9.6072(9)

12.138(1)

16.762(1)

92.881(2)

103.513(2)

96.531(2)

1882.2(3)

4

1.524

0.983

880

0.10×0.10×0.03

−11≤h≤11

−14≤k≤14

−19≤l≤19

18735

10.043(2)

18.874(4)

19.405(4)

9.5301(3)

15.9563(5)

17.3447(6)

91.628(2)

100.313(2)

96.294(2)

2576.2(1)

2

1.342

0.383

1084

0.29×0.21×0.08

−11≤h≤11

−19≤k≤19

0≤l≤21

10045

β(°)

112.962(2)

113.09(2)

105.138(7)

γ(°)

Volume (Å³)

6155(1)

8

1.484

0.617

2341.6(6)

2

1.305

0.412

960

0.60×0.60×0.10

−12≤h≤12

−25≤k≤25

−13≤l≤13

23083

4560(2)

4

1.335

0.423

3678(1)

4

1.465

0.529

Z

Density (calculated) (g/cm³)

Absorption coefﬁcient (mm⁻¹

F(000)

)

2816

1912

1664

Crystal size (mm³)

Index ranges

0.45×0.30×0.11

−45≤h≤45

−11≤k≤11

−22≤l≤22

28995

0.11×0.10×0.03

−20≤h≤19

−11≤k≤11

−16≤l≤20

20851

0.50×0.10×0.05

−11≤h≤11

−22≤k≤22

−23≤l≤23

36333

Reﬂections collected

Independent reﬂections

5354

8235

4739

6619

6468

10045

[R(int)=0.1262]

5354/0/417

0.990

[R(int)=0.1045]

8235/1/565

1.018

[R(int)=0.2256]

4739/0/259

0.853

[R(int)=0.0471]

6619/0/447

0.923

[R(int)=0.1170]

6468/0/470

1.035

[R(int)=0.0000]

10045/0/670

1.017

Data/restraints/parameters

Goodness-of-ﬁt on F²

Final R indices [I>2α(I)]

R1=0.0640

wR2=0.1375

R1=0.0380

wR2=0.0772

−0.05(2)

0.366 and −0.470

R1=0.0752

wR2=0.1589

R1=0.0353

wR2=0.0732

R1=0.0872

wR2=0.2008

−0.05(6)

1.145 and −0.922

R1=0.0332

wR2=0.0697

Absolute structure parameter

Largest diff. peak and hole

1.627 and −1.032

0.522 and −0.569

0.712 and −0.448

0.444 and −0.638

2.37 Å and this compares well with those of A [21] [2.352 Å] and B [49]

[2.364 Å].

complexes. This insinuates feasible C\H^⋯F contacts between the

BF₄⁻counterions and the p-cymene ring, a situation consistent with

the NMR deshielding of the cymene signals.

As stated, the β-diketonate groups adopt a bidentate coordination

mode through the two O atoms to form a six-membered chelate ring.

The O(2)-Ru(1)-O(1) bite angles are between 86.8° and 89.61° and

this reveals the absence of strain. The Ru\O distances are quite similar

for all the structures regardless of the nature of the β-diketonate (see

Table 3). There is a difference between 1a·BF₄and 1a·BPh₄, suggesting

that the counter-ion could affect the electronic distribution throughout

the cationic complex. The C\O bond distances are slightly shorter for

the acac complexes (about 1.27 Å with one value of 1.315 Å) as com-

pared to the dbzm derivatives (about 1.29 Å), a situation in agreement

with the higher electron-withdrawing ability of phenyl groups in com-

parison to the methyl groups and described for similar compounds [23].

In the case of 2, which contains bzac (with a methyl and a phenyl

group), the value found is intermediate (1.28 Å). The chelate rings are

not completely planar and have an envelope conformation in all cases,

with rather small dihedral angles between the diketonate backbone

and the respective O\Ru\O planes (in the range 8.35–15.70°).

Unfortunately, interionic contacts cannot be accurately studied for

the available structures of 1a·BF₄and 3a·BF₄because BF₄⁻anions

show both positional and rotational disorder. Nevertheless, a rough

analysis allowed us to conﬁrm the presence of the BF₄^-anions in the

vicinity of the p-cymene ﬂanks in the crystal network of both

In contrast to the above, the bigger BPh₄⁻anion does not show dis-

order in the crystal structures of 1a·BPh₄and 3b·BPh₄, and thus the

interactions between the BPh₄⁻and the cationic complexes could be

analyzed. In both cases, one or two BPh₄⁻anions seem to wrap the

p-cymene ring of the Ru cation and at least one phenyl group estab-

lishes a CH–π interaction with an H atom of the coordinated

p-cymene, as previously observed in related systems [55]. In fact,

these contacts seem to be strong enough to remain in

chloroform-d₆solutions and they shield the aromatic protons of

p-cymene (see NMR discussion). This CH–π interaction is depicted

in Fig. 4 for derivative 1a·BPh₄and the corresponding parameters

for these anion-cation interactions for 1a·BPh₄and 3b·BPh₄are

summarized in Table 4 and represented in Fig. S1 [56–58].

Similar complexes of formula [(η⁶-p-cymene)Ru(β-diketonate)

(PTA)]BPh₄(D type in Scheme 1) also show weak π interactions

between the p-cymene entity and two different BPh₄⁻anions, as de-

duced by the Mercury program. In this case, each anion uses two phe-

nyl groups to wrap both sides of the coordinated arene. Diversely, the

corresponding BF₄⁻complexes, [(η⁶-p-cymene)Ru(β-diketonate)

(PTA)]BF₄, show hydrogen bonds between the p-cymene ring and

the BF₄⁻anions [23].

Table 3

Selected bond lengths [Å] and angles (°) for 1a·BF₄, 1a·BPh₄, 3a·BF₄, 1b·BPh₄, 3b·BPh₄and 2.

Bond length/angle

1a·BF₄

1a·BPh₄

1b·BPh₄

3a·BF₄

3b·BPh₄

Bond length/angle

2

Ru-arene (centroid)

Ru(1)-P(1)

Ru(1)-O(1)

1.693

1.691

1.693

1.711

1.702

Ru-arene (centroid)

Ru(1)-Cl(1)

Ru(1)-O(1)

1.655

2.358(1)

2.074(2)

2.073(2)

87.9(1)

89.31(8)

83.29(8)

2.341(1)

2.061(3)

2.070(2)

88.4(1)

89.2(1)

83.8(1)

2.352(3)

2.053(7)

2.075(8)

89.7(3)

84.2(2)

88.7(2)

2.352(2)

2.080(6)

2.076(6)

88.4(2)

84.7(2)

88.2(2)

2.377(6)

2.063(1)

2.062(1)

88.51(6)

84.78(4)

88.03(4)

2.410(1)

2.079(3)

2.074(3)

86.8(1)

84.84(9)

85.02(9)

Ru(1)-O(2)

O(1)-Ru(1)-O(2)

O(1)-Ru(1)-P(1)

O(2)-Ru(1)-P(1)

O(1)-Ru(1)-O(2)

O(1)-Ru(1)-Cl(1)

O(2)-Ru(1)-Cl(1)

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

183

and 1a·BPh₄as well as with the evidence of ion-pairing observed in

solution by ¹H NMR spectroscopy.

Theoretical studies were carried out in order to provide more infor-

mation concerning the ion pairing nature in non-polar solvents. Initial-

ly, gas phase geometrical optimization of the ion pairs [(η⁶-p-cymene)

Ru(acac)(dpim)]BF₄(1a·BF₄) [(η⁶-p-cymene)Ru(acac)(dpim)]BPh₄

(1a·BPh₄) was carried out at the B3LYP level (see Computational

studies section). The relative anion–cation orientations are very similar

to the ones found in the solid state. In particular, in 1a·BF₄there is a dis-

tance of 1.947 Å between a ﬂuorine atom of the BF₄⁻anion and a hydro-

gen atom of the p-cymene located in the ortho position to the ⁱPr

substituent, with a C(H)⋯F distance of 3.028 Å and a C\H⋯F angle of

173.16°. These values are consistent with a strong hydrogen bonding

interaction [61]. There is also a short distance between a ﬂuorine

atom of the anion and a hydrogen atom of a methyl group of the coor-

dinated acetylacetonate (2.036 Å). In this case, the C(H) ⋯F distance is

3.120 Å and the C\H⋯F angle is 167.73°. These values account for

another hydrogen bond.

In 1a·BPh₄there is a distance of 2.604 Å between a hydrogen

atom of the p-cymene and the centroid of a phenyl ring of the anion

BPh₄⁻. In addition, the shortest H(p-cymene)⋯C(phenyl) distance is

2.714 Å and the C\H-centroid angle is 177.43°. These values ﬁt well

with the reference parameters for a CH–π interaction [62,63].

In an effort to explain the formation of the ionic pairs in solution

with solvents of low polarity, free energies of solvatation were calcu-

lated for 1a·BF₄and 1a·BPh₄in chloroform and water (see

Computational studies section). The stabilization of the ionic pairs

with respect to the gas phase is higher for chloroform than for

water. In the case of 1a·BF₄the difference is 6.2 kcal/mol in favor of

chloroform and in the case of 1a·BPh₄this difference is 10.3 kcal/

mol, again in favor of chloroform. All in all, these results support the

stabilization of the ionic pairs 1a·BF₄and 1a·BPh₄in solution when

solvents of low polarity are used, a situation in good agreement

with the aforementioned NMR evidence, i.e. the differences in the

chemical shifts for the two different salts.

In addition, NBO [64] calculations were performed in order to de-

termine the charges on the phosphorus atom in the ion pairs 1a·BF₄

and 1a·BPh₄. The calculated charges are 1.055 for 1a·BF₄and 1.054

for 1a·BPh₄. As can be seen, the charge on the phosphorus atom for

1a·BF₄is slightly more positive than the charge for the same atom

in 1a·BPh₄. This is in accordance with the observed differences in

the ³¹P NMR resonances, since the more positive phosphorus in

1a·BF₄appears at higher frequencies (less shielded) and the less pos-

itive phosphorus atom (in 1a·BPh₄) appears at lower frequencies.

This conﬁrms that the donation of electron density from the P to

the Ru ion is slightly lower in 1a·BPh₄as a consequence of the inter-

action between the counter-ion and the arene, which would affect the

electronic distribution throughout the complex.

Fig. 4. CH–π interaction in complex 1a·BPh₄, where p-cym is the C\H donor and B-Ph

the acceptor ring.

3.3. Calculations of electrostatic potential surfaces and optimization of

ionic pairs

Electrostatic potential maps were obtained for the complex cation

1a⁺and for anions BF₄⁻and BPh₄⁻(Fig. 5) through DFT energy calcu-

lations in order to visualize charge distributions and locate the prob-

able areas involved in electrostatic supramolecular (interionic)

interactions. Calculations were performed using experimental struc-

tural parameters for 1a⁺in 1a·BF₄, whereas the structures of both

anions were optimized in gas phase. Major positive charge regions

were found on the hydrogen atoms of the p-cymene ring. On the

other hand, the BPh₄⁻diagram shows that the negative charge is lo-

cated on the π-electronic cloud of the electron-rich aromatic rings,

while the hydrogens of the Ph groups have less electron density.

Thus, it seems reasonable to ﬁnd this anion involved in noncovalent

CH–π interactions with the Ru-cymene cationic fragment, which

acts as the hydrogen donor. As a matter of fact, a recent study empha-

sized the enhanced CH–π donor ability of Ru-coordinated arenes on

the basis of DFT calculations [59]. The BF₄⁻map, as expected, reveals

that the F atoms are highly electronegative and are consequently

prone to form hydrogen bonds. Hence, speciﬁc CH⋯F-BF₃⁻interactions

with the p-cymene electropositive protons seem credible. Similar

noncovalent contacts have been found for other Ru arene complexes

with the same anions [55,60]. These predictions are in good agree-

ment with the contacts observed in the crystal structures of 1a·BF₄

3.4. DNA–ruthenium complex interaction studies

DNA is the main biological target for most of the anticancer com-

pounds. In order to evaluate the ability of our ruthenium complexes

to bind to DNA, agarose gel electrophoresis and atomic force micros-

copy studies were carried out. For comparison purposes, cisplatin was

considered as the reference drug and it was also evaluated under the

same experimental conditions.

Table 4

Parameters for the CH–π interactions in 1a·BPh₄and 3b·BPh₄, where p-cym is the

C\H donor and B-Ph the acceptor ring (see Fig. S1 in Supporting Information).

Complex 1b·BF₄was excluded from these experiments because it

is very unstable and the BPh₄derivatives were also discarded because

they were insoluble in water. The same applies for the cytotoxicity

studies.

Compound

d_C-Ct(Å)

d_H-Ct(Å)

d_H-Atm(Å)

α (º)

1a·BPh₄

3b·BPh₄

3.57

3.50

2.64

2.61

2.87

2.80

163.95

160.70

3.4.1. Agarose gel electrophoresis

d_C–Ct=distance between the C atom in the C\H donor and the centroid of the acceptor

The inﬂuence of the compounds on the tertiary structure of DNA

was determined by their ability to modify the electrophoretic mobil-

ity of the relaxed open circular form (OC) and/or supercoiled

ring; d_H–Ct=distance between the H atom in the C\H donor and the centroid of the

acceptor ring; d_H–Atm=distance between the H atom in the C\H donor and the closest

C atom in the acceptor ring; α=C\H⋯centroid angle (see Fig. S1).

184

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

Fig. 5. Electrostatic potential maps for: the complex cation 1a⁺(a), and anions BPh₄⁻(b) and BF₄⁻(c). The most electronegative areas are in red and the least electronegative areas

in blue.

covalently closed circular form (CCC) of the pBR322 plasmid DNA.

The electrophoretic mobility of these two forms is very different

since it depends on the degree of DNA folding. The BF₄complexes

1a·BF₄, 2a·BF₄, 3a·BF₄, 2b·BF₄and 3b·BF₄were tested after 48 h

of incubation with the plasmid DNA at the molar ratio r_i=0.50. As

shown in Fig. 6, complexes 1a·BF₄, 2a·BF₄and 3a·BF₄(lanes 5, 6,

and 7) did not give rise to observable interactions with the DNA as

the degree of folding of the OC and/or CCC forms of the plasmid was

unaltered by the presence of the ruthenium complexes. In sharp con-

trast, under similar conditions, cisplatin and complexes 2b·BF₄and

3b·BF₄induced signiﬁcant changes in the electrophoretic mobility

of these plasmid forms. While cisplatin induced a co-migration of

the two DNA bands, next to the coalescence point (lanes 3 and 4)

[65], compounds 2b·BF₄(lane 8) and 3b·BF₄(lane 9) produced an

appreciable progressive conversion of the CCC bands into the OC

bands, indicating that the supercoiled plasmid DNA forms were re-

laxed by the interaction with the ruthenium complexes. Similar re-

sults were obtained when the incubation time of the samples was

reduced to 24 h (see Fig. S3 in Supplementary material).

modiﬁcations caused in these conformers, after being incubated in

the presence of the ruthenium compounds, can be graphically evalu-

ated. Based on the DNA-interacting behavior observed in the electro-

phoretic mobility experiments, complexes 2b·BF₄and 3b·BF₄were

selected for further analysis by AFM. The AFM images in Fig. 7 are of

free pBR322 plasmid DNA without any treatment (A), pBR322 incu-

bated for 48 h at 37 °C (r_i=0.5) with cisplatin as positive control

(B), and with BF₄complexes 2b·BF₄and 3b·BF₄(C and D, respective-

ly). The typical AFM image of native pBR322 can be observed in

Fig. 7A. In Fig. 7B it can be seen that DNA structure is distorted by cis-

platin through the development of numerous kinks in the DNA

chains, which is very characteristic of this compound as it covalently

binds to DNA. In addition, the ability of cisplatin to crosslink DNA led

to the formation of some plasmid aggregates [66,68,69], a situation in

accordance with the signiﬁcant decrease in electrophoretic mobility

of the conformer forms. It can be seen in Fig. 7C and D that the inter-

action of both 2b·BF₄and 3b·BF₄with DNA produced similar

changes in structure, with a general tendency to induce relaxed circu-

lar forms and some cross-links between plasmids. However, a re-

duced number of kinked forms were present in the DNA chains

compared with cisplatin-treated samples. The conformational

changes in the plasmid DNA structure induced by these compounds

may presumably be attributed to covalent interactions between ru-

thenium and DNA. These interactions are similar to those induced

by cisplatin or cisplatinum(II) analogues [70–73] and very different

3.4.2. AFM studies

Tapping mode atomic force microscopy (TMAFM) is an excellent

tool to study the surface relief, morphology and topology of DNA mol-

ecules [66,67]. With this technique, direct visualization of three con-

formers of pBR322 plasmid DNA can be achieved and therefore

Fig. 6. Electrophoretic mobility pattern in agarose gel of pBR322 plasmid DNA treated with 1a⋅BF₄(lane 5), 2a⋅BF₄(lane 6), 3a⋅BF₄(lane 7), 2b⋅BF₄(lane 8) and 3b⋅BF₄(lane 9) for

48 h at 37 °C (r_i=0.5). Control plasmid DNA samples treated with TE buffer and TE buffer+vehicle (milliQ water+5% DMSO) are represented in lanes 1 and 2, respectively. Lanes

3 and 4 show the mobility pattern of the plasmid DNA incubated with cisplatin (dissolved in milliQ water alone and milliQ water+5% DMSO). OC, open circular form; CCC, cova-

lently closed circular form.

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

185

Fig. 7. AFM images of pBR322 plasmid DNA free (A) and incubated with cisplatin (B), 2b⋅BF₄(C) and 3b⋅BF₄(D) for 48 h at 37 °C (ri=0.5).

to those observed for classical intercalators such as ethydium bro-

mide and other planar heterocycles [74,75]. Finally, it is important

to highlight that both compounds were able to produce some frag-

mentation of DNA chains during the reaction, even in the absence of

an activator. In conclusion, AFM results conﬁrm that the new com-

plexes 2b·BF₄and 3b·BF₄interact with DNA and modify its tertiary

structure, as we previously observed in the electrophoretic pattern.

cell lines (Table 5). Remarkably, complexes 2b·BF₄and 3b·BF₄

were the most efﬁcient at inhibiting the cell proliferation, with IC₅₀

values equivalent to those established for cisplatin in these cell lines

(IC₅₀=4 μM in MCF-7 cells and 2.5 μM in CAPAN-1 cells). The signif-

icant antiproliferative activity of these two complexes may be closely

related to their ability to interact with DNA. However, we cannot rule

out other biological targets for these complexes, most likely proteins,

as complexes 1a·BF₄, 2a·BF₄and 3a·BF₄, which did not interact with

DNA in the in vitro experiments, have demonstrated relevant cytotox-

ic activity. On the other hand, the low solubility of these derivatives in

water could limit their potential in vivo antitumor activity. Never-

theless, strategies such as the use of polymeric micelles as carrier sys-

tems for water-insoluble anticancer drugs could be helpful to

improve the in vivo activity [76].

3.5. Cytotoxicity

The cytotoxic activity levels of the BF₄⁻salts 1a·BF₄, 2a·BF₄,

3a·BF₄, 2b·BF₄and 3b·BF₄were established in a comparative in

vitro MTT cell viability assay with both human breast cancer cells

(MCF-7) and human pancreatic cancer cells (CAPAN-1). The different

ruthenium compounds were freshly dissolved in DMSO and milli-Q

water and then serially diluted in complete culture medium at con-

centrations ranging from 0 μM to 100 μM (DMSO ﬁnal concentration

in the culture mediumb1%). The activity values are expressed as the

concentration of each complex required to decrease the cell viability

by 50% (IC₅₀). The ﬁve complexes displayed IC₅₀values below

50 μM in both MCF-7 (except complex 1a·BF₄) and CAPAN-1 cancer

3.6. Stability studies

In order to obtain more information concerning the reasons for the

different behavior of the dpim or PPh₂py Ru complexes in the interac-

tion with DNA or in the cytotoxicity experiments, complementary stud-

ies on the stability of the Ru derivatives in the solvent mixture used for

186

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

Table 5

aquation would be enhanced for complexes belonging to series b. This

ﬁnding is in agreement with the higher hydrolysis rate observed for

3b·BF₄relative to that of 3a·BF₄and can explain the different

antiproliferative activities of the two series.

IC₅₀(μM) values for the compounds 1a·BF₄, 2a·BF₄, 3a·BF₄, 2b·BF₄and 3b·BF₄.

Compound

MCF-7

CAPAN-1

1a·BF₄

2a·BF₄

3a·BF₄

2b·BF₄

3b·BF₄

Cisplatin

62

23.67

35.33

25.5

6.6

6.94

2.5

19.67

22.95

3.77

3.3

3.8. Pro-apoptotic activity studies

To further characterize the cytotoxicity of the BF₄complexes, their

pro-apoptotic activity was also determined. The disruption of the reg-

ulation of apoptosis is a key event in the transformation of normal

cells into tumor cells and many traditional anticancer drugs exert

their cytotoxic effects by inducing apoptosis, most often as a result

of DNA damage [77,78]. Thus, it is important to evaluate the suitabil-

ity of the complexes as possible anticancer agents in terms of their

ability to induce apoptosis, as the cellular viability assays, like the

MTT test, measure cytotoxicity but do not distinguish between apo-

ptosis and necrosis. The pro-apoptotic activity of the complexes was

quantiﬁed by annexin V–propidium iodide ﬂow cytometry after

treating CAPAN-1 cells for 24 h with two different concentrations

(10 and 20 mM) of complex 2b·BF₄, which demonstrated a high cy-

totoxic activity, or medium alone as a control. Annexin V binds phos-

phatidyl serine residues, which are asymmetrically distributed

towards the inner plasma membrane but migrate to the outer plasma

membrane during apoptosis. Propidium iodide is impermeant to live

cells and apoptotic cells but stains nucleic acids in dead cells. Hence,

the percentage of annexin V positive cells indicates the proportion

of apoptotic cells, while double positive staining for annexin V and

propidium iodide indicates both late apoptotic and necrotic cells

(dead cells). As represented in Table 7, both 2b·BF₄concentrations

markedly increased the percentage of apoptotic cells compared to

the basal level of apoptosis seen in the control cells. The treatments

also led to a relevant percentage of dead cells, which may be the re-

sult of either late apoptotic or necrotic pathways, as apoptosis de-

velops rapidly after the treatment.

4

the AFM studies (DMSO/H₂O) and cyclic voltammetry of the new deriv-

atives were undertaken.

Hydrolysis processes are of great interest to gain a more precise

outlook about the stability of the potential drugs under pseudo-

pharmacological conditions. However, all of our complexes are insol-

uble in water, so we monitored complexes 3a·BF₄and 3b·BF₄by ³¹P

{¹H} NMR spectroscopy in a mixture of DMSO-d₆/D₂O (50:50) for

several days. The dpim complex, 3a·BF₄, showed high stability,

since evolution was not observed after 48 h and only traces of evolu-

tion products were detected after 4 days (Fig. 8). By contrast, the

PPh₂py derivative, 3b·BF₄, displayed signs of hydrolytic evolution

after 1 h and several emerging peaks were conﬁrmed over the next

7 days (Fig. 9).

3.7. Cyclic voltammetry experiments

Cyclic voltammetry experiments were carried out on all the BF₄salts –

except for the unstable 1b·BF₄– in order to determine their redox be-

havior. The measurements were made on solutions of the respective

complexes (10⁻³M) and tetrabutylammonium hexaﬂuorophosphate

(TBAPF₆, 0.1 M) as the supporting salt, in dry CH₂Cl₂, using a conven-

tional three-electrode cell with a graphite working electrode. In partic-

ular, the abovementioned BF₄salts showed a Ru-based one-electron

quasi-reversible oxidation (Ru^III/Ru^II) with E_1/2values between +0.82

and +0.76 V for complexes of series a, and +0.58 and +0.57 V for

complexes of series b, with respect to a calomel reference electrode at

a scan rate 100 mV·s⁻¹(see Table 6 and Fig. S2). The higher oxidation

potentials observed for series a relative to series b suggest that dpim has

a worse σ-donor character and/or a better π-acceptor character than

PPh₂py, so that the Ru(II) ion becomes poorer in electron density in

complexes of series a compared to their analogues in series b. As a con-

sequence, the β-diketonate dissociation in water and subsequent

These results demonstrate that, according to their ability to inter-

act with DNA, the complex induces cell death largely through the ac-

tivation of apoptotic pathways.

4. Concluding remarks

A

collection of new Ru(II) compounds of general formula

[(η⁶-p-cymene)Ru(κ²-O,O′-X)(κ¹-P-L)]Y has been prepared through

Fig. 8. Evolution with time of the ³¹P{¹H} NMR spectrum for 3a·BF₄(ﬁlled red triangle) in a mixture of DMSO-d₆/D₂O (50:50). Evolution products are labelled as (ﬁlled green circle).

C. Aliende et al. / Journal of Inorganic Biochemistry 117 (2012) 171–188

187

Fig. 9. Evolution with time of the ³¹P{¹H} NMR spectrum for 3b·BF₄(ﬁlled red triangle) in a mixture of DMSO-d₆/D₂O (50:50). Evolution products are labelled as (ﬁlled green circle).

the combination of two aminophosphines (L=dpim and PPh₂py) as

neutral ligands, three β-diketonates (X=acac, bzac and dbzm) as

monoanionic leaving groups, and two borate anions (Y=BF₄⁻and

BPh₄⁻) as counter-ions. NMR, structural and computational studies

provide evidence of ion-pairing for these complexes with both BF₄⁻

and BPh₄⁻in CDCl₃. For the BF₄⁻salts the ion-pairs are likely to be

held together by interionic F⋯H\C hydrogen bonds, whereas for the

BPh₄⁻salts, CH–π interactions are thought to be the driving force for

the ion pair formation. In addition, most of the complexes show a

general tendency to lose the p-cymene ring slowly in apolar solvents

such as CDCl₃. On the other hand, the more soluble BF₄⁻salts have

shown cytotoxic activity in MTT assays with human breast cancer

cells (MCF-7) and human pancreatic cancer cells (CAPAN-1). Interest-

ingly, the most active complexes in both cell lines are those with

PPh₂py, namely 2b·BF₄and 3b·BF₄, which also exhibit a remarkable

interaction with DNA — as established by electrophoretic and AFM

studies. The corresponding analogs with dpim, 1a·BF₄, 2a·BF₄and

3a·BF₄, are less active and are reluctant to take part in any interaction

with DNA. Moreover, evolution experiments suggest a correlation be-

tween these trends and the hydrolysis rate in DMSO/H₂O solutions, so

that the higher reactivity of 3b·BF₄as compared to 3a·BF₄could ex-

plain the enhanced biological performance of the former. This ligand

effect is rationalized in terms of the electronic features of the two

phosphines, as established by cyclic voltammetry measurements. In

summary, we are inclined to believe that activation by β-diketonate

dissociation and aquation might be favored in complexes that belong

to series b due to the higher σ-donor ability of PPh₂py compared to

dpim. In addition, we believe that two attributes of our complexes

contribute to their cytotoxic properties by avoiding premature reac-

tions on the metallic center: (i) the strong binding of the

β-diketonate anions to the Ru(II) ion as compared with other

monodentate ligands such as chloro [79,80] and (ii) protection of

the metal by the pendant groups in the arene entity and the bulky

substituents on the β-diketonate ligands [24].

Acknowledgments

This work was supported by the MINECO of Spain (project

CTQ-2011-24434, FEDER funds). We would like to acknowledge

Emilie Goossens for technical support.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://

dx.doi.org/10.1016/j.jinorgbio.2012.07.022.

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Cyclic voltammetric data for BF₄salts of series a and b.

Compound

E_ox(V)

E_red(V)

E_1/2(V)

1a·BF₄

2a·BF₄

0.8

0.71

0.27

0.77

0.79

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0.80

0.82

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0.58

0.34

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0.85

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Table 7

Percentage of live, apoptotic and dead cells after treating CAPAN-1 cells with the com-

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