Synthesis, characterization, electrochemical behavior and in vitro protein tyrosine kinase inhibitory activity of the cymene-halogenobenzohydroxamato [Ru(η6-cymene)(bha)Cl] complexes

Article abstract of DOI:10.1016/j.jorganchem.2012.12.013

The ruthenium(II)-cymene complexes [Ru(η⁶-cymene)(bha)Cl] with substituted halogenobenzohydroxamato (bha) ligands (substituents = 4-F, 4-Cl, 4-Br, 2,4-F₂, 3,4-F₂, 2,5-F₂, 2,6-F ₂) have been synthesize

Full text of DOI:10.1016/j.jorganchem.2012.12.013

Journal of Organometallic Chemistry 730 (2013) 137e143
Contents lists available at SciVerse ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis, characterization, electrochemical behavior and in vitro
protein tyrosine kinase inhibitory activity of the cymene-
halogenobenzohydroxamato [Ru(
h
6-cymene)(bha)Cl] complexes
,
,
Xianmei Shang a b, Telma F.S. Silva a, Luísa M.D.R.S. Martins a c, Qingshan Li d,
M. Fátima C. Guedes da Silva a e, Maxim L. Kuznetsov a, Armando J.L. Pombeiro a
,
,
*
a Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, Technical University of Lisbon, Av. Rovisco Pais, 1049-001 Lisbon, Portugal
b Tongji School of Pharmacy, Huazhong University of Science and Technology, 13 Hangkong Road, 430030 Wuhan, China
c Chemical Engineering Departmental Area, ISEL e Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro 1, 1959-007 Lisbon, Portugal
d School of Pharmaceutical Science, Shanxi Medical University, 86 South Xinjian Road, 030001 Taiyuan, China
e Universidade Lusófona de Humanidades e Tecnologias, ULHT Lisbon, Av. do Campo Grande, 376, 1749-024 Lisbon, Portugal
a r t i c l e i n f o
a b s t r a c t
The ruthenium(II)ecymene complexes [Ru(h
6-cymene)(bha)Cl] with substituted halogenobenzohy-
Article history:
Received 12 October 2012
Received in revised form
9 December 2012
droxamato (bha) ligands (substituents ¼ 4-F, 4-Cl, 4-Br, 2,4-F2, 3,4-F2, 2,5-F2, 2,6-F2) have been
synthesized and characterized by elemental analysis, IR, 1H NMR, 13C NMR, cyclic voltammetry and
controlled-potential electrolysis, and density functional theory (DFT) studies. The compositions of their
frontier molecular orbitals (MOs) were established by DFT calculations, and the oxidation and reduction
potentials are shown to follow the orders of the estimated vertical ionization potential and electron
affinity, respectively. The electrochemical EL Lever parameter is estimated for the first time for the
various bha ligands, which can thus be ordered according to their electron-donor character. All
complexes exhibit very strong protein tyrosine kinase (PTK) inhibitory activity, even much higher than
that of genistein, the clinically used PTK inhibitory drug. The complex containing the 2,4-
difluorobenzohydroxamato ligand is the most active one, and the dependences of the PTK activity of
the complexes and of their redox potentials on the ring substituents are discussed.
Accepted 10 December 2012
Keywords:
Ruthenium(II) complexes
Synthesis
Protein tyrosine kinase inhibitor
Electrochemistry
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
signal transduction, and are involved in the control of cell prolifer-
ation, differentiation and transformation [19]. Continuing activation
The development of metal-containing drugs with high anti-
tumor activity, low toxicity and pharmacological outline
of PTK is associated with proliferative disorders such as cancer, and
PTK inhibitors have been developed as molecular-targeting cancer
therapeutic agents. The discovery and development of PTK inhibi-
tors as newcancer therapeutic agents have attracted much attention
[20e27]. Many PTK inhibitors with potent activities have already
passed or are currently in clinical trials to investigate their applica-
bility as anti-cancer drugs [28].
a
different from that of the platinum compounds, is a challenge in
drug design [1e5]. Ruthenium complexes have shown very
promising results in preclinical and clinical studies, and attracted
a high attention in cancer research because of their comparative
antitumor activity and lower cytotoxicity [6e15]. Ruthenium
compounds can have a mechanism of action different from that
of Pt drugs and a different spectrum of activity and non-cross-
resistance [7,8,16e18].
Ru(II) complexes with cymene ligands possess significant anti-
tumor activity [8b,c,12e15]. Until now, the PTK inhibitory activity of
mixed-ligand ruthenium(II) complexes with cymene and halo-
genobenzohydroxamic acid had not been disclosed, although some
ruthenium(II) complexes with cymene and hydroxamic acid have
been reported [29]. In the current work, we describe the synthesis,
characterization, electrochemical behavior and in vitro PTK inhibi-
tory activity of seven new mixed ligand ruthenium(II) complexes
with cymene and substituted hydroxamato ligands of general
Protein tyrosine kinases (PTKs) are members of a large family of
oncoproteins and proto-oncoproteins, playa major role in mitogenic
* Corresponding author. Tel.: þ351 2184 19237; fax: þ351 218464455.
E-mail
addresses:
pombeiro@ist.utl.pt,
pombeiro@popsrv.ist.utl.pt
(A.J.L. Pombeiro).
formula [Ru(
h
6-cymene)(bha)Cl].
0022-328X/$ e see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jorganchem.2012.12.013

138
X. Shang et al. / Journal of Organometallic Chemistry 730 (2013) 137e143
2. Experimental section
30 mL) with the appropriate halogenobenzohydroxamic acid
(1 mmol) and NaOMe (0.054 g, 1 mmol), the resulting clear solution
was stirred for 4 h at room temperature, and the color changed
from red to orange. The solvent was removed in vacuum, and the
residue was dissolved in dichloromethane (10 mL), and the solution
was filtered to remove sodium chloride. The orange solution was
concentrated (2 mL) and addition of hexane gave an orange
precipitate of the complex, which was separated by filtration and
dried under vacuum to afford an orange-red solid. The compound is
soluble in alcohols, acetone, acetonitrile, dimethyl sulfoxide, and
chlorinated solvents.
2.1. Materials
[RuCl(h m-Cl)]2, methyl 4-fluorobenzoate, methyl
6-p-cymene)(
4-chlorobenzoate, methyl 4-bromobenzoate, methyl 2,4-
difluorobenzoate, methyl 2,5-difluorobenzoate and methyl 2,6-
difluorobenzoate were purchased from Aldrich or Alfa and used
as received. 4-fluoro-, 4-chloro-, 4-bromo-, 2,4-difluoro-, 3,4-
difluoro-, 2,5-difluoro- and 2,6-difluoro-benzohydroxamic acids
were prepared as previously reported [30]. The basic forms of
these acids (bha) are denoted by F4bha, Cl4bha, Br4bha, F24bha,
F34bha, F25bha and F26bha, respectively. The other reagents were
of analytical grade. C and H elemental analyses were performed on
2.4.1. Synthesis of [Ru(h
6-p-cymene)(F4bha)Cl] (1)
Yield: 63%. Anal. Calcd for C17H19NO2ClFRu$1/2H2O (433.87): C,
47.06; H, 4.65; N, 3.23. Found: C, 47.53; H, 4.64; N, 3.22. IR: 3442
(NeH), 3044 (CaromeH), 2959, 2916, 2869, 1606 (C]O), 1488, 1234,
858, 630, 564, 513 (RueO) cmꢀ1. 1H NMR (400 MHz, CDCl3): 7.92e
a
PE-2400-II elemental analyzer. Infrared spectra (4000e
400 cmꢀ1) were recorded with a Biorad FTS 3000MX instrument
in KBr pellets. The 1H and 13C (Me4Si internal standard) NMR
spectra were recorded on a Bruker Avance IIþ 400 MHz (Ultra-
Shield Magnet) spectrometer.
2
7.72 (Harom, F4bha), 7.13e7.00 (Harom, F4bha), 5.37 (d, JHH ¼ 8 Hz,
4H, cymene), 2.92 (m, 1H, eCH(CH3)2), 2.28 (s, 3H, eCH3), 1.31 (d,
JHH ¼ 7.2 Hz, 6H, (CH3)2CHe) ppm. 13C NMR (100 MHz, CDCl3):
165.3 (C]O), 163.5 (CeF), 129.7, 129.1, 126.2, 115.8, 101.3, 96.8, 81.3,
80.5, 30.9, 30.6, 22.1, 18.9 ppm.
2.2. Instrumentation and measurement
The electrochemical experiments were performed on an EG&G
PAR 273A potentiostat/galvanostat connected to personal
computer through a GPIB interface. Cyclic voltammograms (CV)
were obtained in 0.2 M [nBu4N][BF4]/CH2Cl2, at a platinum disc
working electrode (d ¼ 1 mm). Controlled-potential electrolyses
(CPE) were carried out in electrolyte solutions with the above
mentioned composition, in a three-electrode H-type cell. The
compartments were separated by a sintered glass frit and equipped
with platinum gauze working and counter electrodes. For both CV
and CPE experiments, a Luggin capillary connected to a silver wire
pseudo-reference electrode was used to control the working elec-
trode potential. The CPE experiments were monitored regularly by
cyclic voltammetry, thus assuring no significant potential drift
occurred along the electrolyses. Ferrocene was used as an internal
standard for the measurement of the oxidation potentials of the
complexes; the redox potential values are quoted relative to the SCE
2.4.2. Synthesis of [Ru(h
6-p-cymene)(Cl4bha)Cl] (2)
Yield: 75%. Anal. Calcd for C17H19NO2Cl2Ru (441.31): C, 46.27; H,
4.34; N, 3.17. Found: C, 46.24; H, 4.44; N, 3.08. IR: 3442 (NeH), 3056
(CaromeH), 2961, 2924, 1846, 1637 (C]O), 1467, 1389, 1091, 878, 567
(RueO) cmꢀ1
.
1H NMR (400 MHz, CDCl3): 7.79 (d, JHH ¼ 8.4 Hz,
Harom, Cl4bha), 7.46 (d, JHH ¼ 8.8 Hz, Harom, Cl4bha), 5.50 (d,
JHH ¼ 6 Hz, 4H, Harom, cymene), 5.37 (d, JHH ¼ 6 Hz), 2.95 (m, 1H, e
CH(CH3)2), 2.30 (s, 3H, eCH3), 1.31 (d, JHH ¼ 7.2 Hz, 6H, (CH3)2CHe
) ppm. 13C NMR (100 MHz, CDCl3): 163.3 (C]O), 159.8 (CeCl), 140.5,
121.7, 114.7, 102.9, 101.4, 96.5, 81.2, 80.6, 30.8, 30.6, 22.1, 19.0 ppm.
2.4.3. Synthesis of [Ru(h
6-p-cymene)(Br4bha)Cl] (3)
Yield: 66%. Anal. Calcd for C17H19NO2ClBrRu (485.77): C, 42.03;
H, 3.94; N, 2.88. Found: C, 42.10; H, 3.72; N, 2.83. IR: 3442 (NeH),
3050 (CaromeH), 2960, 2923, 1831, 1583 (C]O), 1384, 527 (Rue
by using as internal reference the ferrocene/ferricinium ([Fe(h5
-
O) cmꢀ1
.
1H NMR (400 MHz, CDCl3): 7.76 (d, JHH ¼ 8.4 Hz,
C5H5)2]0/þ) couple (Eox ¼ 0:525 V vs. SCE in CH2Cl2) [31].
PBrbha), 7.50 (d, JHH ¼ 8.8 Hz, Br4bha), 5.73 (d, JHH ¼ 6.0 Hz, 2H,
1=2
2
Harom, cymene), 5.50 (d, JHH ¼ 6.0 Hz, 2H), 5.37 (d, JHH ¼ 6.0 Hz,
2.3. Computational details
2H), 3.01e2.91 (m, 1H, eCH(CH3)2), 2.40 (s, 3H, eCH3), 1.44 (d,
JHH ¼ 7.2 Hz, (CH3)2CHe), 1.31 (d, JHH ¼ 6.8 Hz, (CH3)2CHe) ppm. 13
C
The full geometry optimization of the complexes has been
carried out in Cartesian coordinates at the DFT level of theory using
Becke’s three-parameter hybrid exchange functional in combina-
tion with the gradient-corrected correlation functional of Lee, Yang
and Parr (B3LYP) [32] with the help of the Gaussian-03 [33]
program package. Symmetry operations were not applied for all
structures. A quasi-relativistic Stuttgart pseudopotential described
28 core electrons and the appropriate contracted basis set
(8s7p6d)/[6s5p3d] [34] for the ruthenium atom and the 6-31G(d)
basis set for other atoms were used. The Hessian matrix was
calculated analytically to prove the location of correct minima (no
imaginary frequencies were found). Vertical ionization potentials
and electron affinities were calculated as differences of the total
energies Eox ꢀ Eneut and Eneut ꢀ Ered, where the index “neut”
corresponds to a neutral complex, and the indexes “ox” and “red”
correspond to oxidized and reduced complexes with unrelaxed
geometries.
NMR (100 MHz, CDCl3): 168.4 (C]O), 164.0 (CeBr), 131.3, 130.2,
111.9, 101.3, 96.8, 90.9, 81.3, 80.6, 30.9, 30.7, 22.4, 22.1, 18.9 ppm.
2.4.4. Synthesis of [Ru(h
6-p-cymene)(F24bha)Cl] (4)
Yield: 58%. Anal. Calcd for C17H18NO2ClF2Ru (442.85): C, 46.11;
H, 4.10; N, 3.16. Found: C, 46.21; H, 4.42; N, 3.28. IR: 3440 (NeH),
3048 (CaromeH), 2958, 2923, 2866, 1610 (C]O), 1484, 875, 627,
510 (RueO) cmꢀ1. 1H NMR (400 MHz, CDCl3): 9.58 (d, NeH), 8.17e
8.09 (m, Harom, F24bha), 7.02e6.80 (m, Harom, F24bha), 5.48 (dd,
JHH ¼ 6.0 Hz, 4H, Harom, cymene), 2.91 (m, eCH(CH3)2), 2.33 (s, 3H,
eCH3), 1.38 (d, JHH ¼ 6.9 Hz, 6H, (CH3)2CHe) ppm. 13C NMR
(100 MHz, CDCl3): 175.6 (C]O), 167.5 (CeF), 160.1 (CeF), 143.7,
139.9, 131.9, 128.7, 126.3, 112.1, 104.3, 101.2, 99.4, 96.7, 81.3, 80.5,
31.2, 30.9, 30.6, 22.1, 18.9, 18.5 ppm.
2.4.5. Synthesis of [Ru(h
6-p-cymene)(F34bha)Cl] (5)
Yield: 74%. Anal. Calcd for C17H18NO2ClF2Ru (442.85): C, 46.11;
H, 4.10; N, 3.16. Found: C, 46.02; H, 4.22; N, 3.23. IR: 3436 (NeH),
3061 (CaromeH), 2963, 2925, 1869(vs), 1617, 1600 (C]O), 1521,
2.4. General procedure for the synthesis of compounds
1468, 1386, 776, 552 (RueO) cmꢀ1 1H NMR (400 MHz, CDCl3):
.
The starting complex [RuCl(
h
6-p-cymene)(
m-Cl)]2 (0.306 g,
7.77e6.88 (m, 3H, Harom, F34bha), 5.49 (m, 4H, Harom, cymene), 2.34
0.5 mmol) was added to a mixture of methanol and CH2Cl2 (1:1, v/v,
(s, 3H, eCH3, cymene), 1.28 (m, CH(CH3)2) ppm. 13C NMR (100 MHz,

X. Shang et al. / Journal of Organometallic Chemistry 730 (2013) 137e143
139
CDCl3): 165.9 (C]O), 159.3 (CeF), 156.3 (CeF), 137.8, 128.9, 126.3,
118.8, 81.3, 80.5, 30.9, 30.6, 24.1, 22.1, 18.9 ppm.
The phosphorylation assays were performed at 37 ꢁC in a final
volume of 40 L tyrosine kinase. The concentrations of PTKs used to
m
construct calibration curves were as follows: 600, 500, 400, 300,
2.4.6. Synthesis of [Ru(
h
6-p-cymene)(F25bha)Cl](6)
200 and 100 ꢂ 10ꢀ7 U/mL for PTK. A concentration of 500 ꢂ 10ꢀ7
/mL
Yield: 71%. Anal. Calcd for C17H18NO2ClF2Ru (442.85): C, 46.11;
H, 4.10; N, 3.16. Found: C, 45.94; H, 4.26; N, 3.26. IR: 3436 (NeH),
3055 (CaromeH), 2965, 2925, 1602 (C]O), 1577 (strong), 1507,
was used for each inhibitor. Phosphorylation reactions were initi-
ated with the addition of 40 mM ATP (10 mL) into each vessel, and
the plate was incubated at 37 ꢁC for 30 min. After completion of
reaction, liquid was decanted and the vessels were washed four
1203, 1127, 859, 772, 540, 465 (RueO) cmꢀ1 1H NMR (400 MHz,
.
CDCl3): 9.73 (d, NeH), 7.85e7.79 (m, H6, F25bha), 7.12e7.04 (m, H3,
H4, F25bha), 5.56e5.32 (m, 4H, Harom, cymene), 2.94 (m, 2H, e
CH(CH3)2), 1.39e1.37 (d, JHH ¼ 6.9 Hz, 6H, (CH3)2CHe), 1.31e1.28
(d, JHH ¼ 6.9 Hz, 6H, (CH3)2CHe) ppm. 13C NMR (100 MHz,
CDCl3): 169.5 (C]O), 152 (CeF), 148.0 (CeF), 133.4, 128.9, 126.2,
113.9, 81.3, 80.5, 33.6, 30.9, 24.0, 22.1 ppm.
times with Tween-PBS. A volume of 100
mL of blocking solution was
added to the vessels and incubated at 37 ꢁC for 30 min. After
washing the plate with Tween-PBS, anti-phosphotyrosine (50 mL)
was added to the vessels and incubated at 37 ꢁC for 30 min. The
reaction liquid was decanted and the remaining solution was
removed by rinsing four times with Tween-PBS. One hundred mL of
HRP coloring agent was added and incubated at 37 ꢁC for 15 min.
The reaction was terminated by addition of 1 N sulfuric acid
2.4.7. Synthesis of [Ru(h
6-p-cymene)(F26bha)Cl] (7)
Yield: 50%. Anal. Calcd for C17H18NO2ClF2Ru (442.85): C, 46.11;
H, 4.10; N, 3.16. Found: C, 46.02; H, 4.24; N, 3.22. IR: 3440 (Ne
H), 3055 (CaromeH), 2961, 2924, 1626 (C]O), 1466, 1004, 791,
(100 mL/well). Absorbance was measured at 450 nm in a microplate
reader (SpectraMax M5) [22]. All experiments were performed in
triplicate.
525, 447 (RueO) cmꢀ1 1H NMR (400 MHz, CDCl3): 7.62e7.35
.
(Harom, F26bha), 7.14 (Harom, F26bha), 6.96e6.92 (Harom, F26bha),
3. Results and discussion
2
5.51, 5.49, 5.37, 5.35 (dd, JHH ¼ 6 Hz, 4H, Harom, cymene), 2.93
(m, 1H, eCH(CH3)2), 2.34 (s, 3H, cymene), 1.31e1.29 (d,
JHH ¼ 6.8 Hz, 6H, (CH3)2CHe), 1.27e1.25 (d, JHH ¼ 6.8 Hz, 6H,
(CH3)2CHe) ppm. 13C NMR (100 MHz, CDCl3): 168.1 (C]O), 164.1
(CeF), 153.1 (CeF), 135.6, 133.0, 124.9, 103.7, 96.8, 81.5, 80.4, 30.9,
30.6, 22.1 ppm.
3.1. Synthesis and characterization
The new ruthenium(II) complexes [Ru(
(1), [Ru(
6-cymene)(Cl4bha)Cl] (2), [Ru( 6-cymene)(Br4bha)Cl] (3),
6-cymene)(F24bha)Cl] (4), [Ru( 6-cymene)(F34bha)Cl] (5),
6-cymene)(F25bha)Cl] (6) and [Ru( 6-cymene)(F26bha)Cl] (7)
6-p-cymene)(
-Cl)]2
h
6-cymene)(F4bha)Cl]
h
h
h
h
[Ru(
[Ru(
h
h
2.5. PTK inhibitory activity assay
have been prepared by the reaction of [RuCl(
h
m
with the appropriate halogenobenzohydroxamic acid in a mixture
of CH3OH/CH2Cl2 (1:1, v/v) under basic conditions in the presence
of NaOMe (Scheme 1).
PTK activity was determined by the ELISA method. The tyrosine
kinase was extracted from brain tissue of rat, and microtiter plates
were coated using poly-Glu-Tyr (PGT) as substrates. If the tyrosine
residues of PGT were phosphorylated by PTKs, they bound to
phospho-specific monoclonal antibody that was labeled specifi-
cally with HRP. The absorbance was measured to reflect the
activity of PTK.
The elemental analysis data of the complexes are in good
agreement with the calculated values, and their IR spectra display
a sharp band at 1583e1637 cmꢀ1, not present in [RuCl( 6-p-cym-
h
ene)(m-Cl)]2, which is assigned to nCO of the coordinating hydrox-
amato. Compared to that of the free acid (nCO for substituted
Br
Cl
R =
F
2
5
3
1
F
F
F
F
F
F
F
F
7
4
6
Scheme 1. Synthesis of the [Ru(h
6-cymene)(bha)Cl] complexes (1e7).

140
X. Shang et al. / Journal of Organometallic Chemistry 730 (2013) 137e143
benzohydroxamic acids are in the 1619e1634 cmꢀ1 range), a shift of
ca. 17e45 cmꢀ1 is observed, what is consistent with previous
findings [35e38].
Table 2
Cyclic voltammetric dataa for [Ru( 6-p-cymene)(bha)Cl] halo-substituted
h
complexes 1e7 (substituents ¼ 4-F, 4-Cl, 4-Br, 2,4-F2, 3,4-F2, 2,5-F2, 2,6-F2), and
calculated vertical ionization potentials (I) and electron affinities (A) (eV) of the
models 10e70.
In the 1H NMR spectra of 1e7, the expected resonances are
observed for the cymene and the substituted benzohydroxamato.
As a result of the coordination of the substituted hydroxamato unit,
downfield shifts (0.10e0.20 ppm) of the ligand ring protons are
observed relative to the free acid. Similar downfield shifts were also
detected for the coordinated p-cymene in 1e7 as compared to the
Complex
Anodic waves
Cathodic waves
ox
red
E
I
E
A
p=2
p=2
[Ru(
[Ru(
[Ru(
[Ru(
[Ru(
[Ru(
[Ru(
h
h
h
h
h
h
h
6-p-cymene)(F4bha)Cl] (1)
6-p-cymene)(Cl4bha)Cl] (2)
6-p-cymene)(Br4bha)Cl] (3)
6-p-cymene)(F24bha)Cl] (4)
6-p-cymene)(F34bha)Cl] (5)
6-p-cymene)(F25bha)Cl] (6)
6-p-cymene)(F26bha)Cl] (7)
1.60
1.69
1.67
1.51
2.05
1.70
1.98
6.74
6.77
6.76
6.70
6.82
6.74
6.65
ꢀ1.33
ꢀ0.51
ꢀ0.55
ꢀ1.51
ꢀ0.87
ꢀ0.82
ꢀ0.77
0.09
0.25
0.26
0.11
0.22
0.21
0.13
arene ligand in [RuCl(h m
6-p-cymene)( -Cl)]2. The 13C NMR spectra
for 1e7 also show the expected resonance signals.
3.2. Biological evaluation
a
Potential values (half-peak) in Volt ꢃ 0.02 vs. SCE, in a 0.2 M [nBu4N][BF4]/CH2Cl2
solution, at a Pt disc working electrode determined by using the [Fe(
h
5-C5H5)2]0/þ
All of the synthesized compounds (1e7) and the precursor
ox
redox couple (E
¼ 0:525 V vs. SCE) as internal standard at
a scan rate of
[RuCl(h m-Cl)]2 were screened for preliminary in vitro
6-p-cymene)(
p=2
200 mV sꢀ1
.
PTK inhibitory activity by ELISA with genistein as a positive refer-
ence compound. As shown in Table 1, all of them exhibit very strong
activities with IC50 values below 3.11 mM, which are even much
inhibitory activity. However, no clear relation, along the series of the
complexes, appears to be observed between the redox potential and
such a bioactivity.
higher than that of genistein with an IC50 value of 13.65
same model [39]
mM in the
Among 1, 2 and 3 with different para-halogen atoms, 1 with the
para-fluoro substituent showed the best activity with an IC50 value
of 0.02 mM (the order of activity follows F > Cl > Br), what suggests
that the fluorine atom may play an important role for the interac-
tion of the active ruthenium complex with PTK.
In order to interpret the experimental redox potentials of the
complexes, quantum-chemical calculations of the model
compounds [Ru(h6-C6H6)(bha)Cl] (10e70) with a benzene ligand
instead of cymene, have been performed at the DFT level of theory
(Fig. 2). The calculated structural parameters of bha in 10 are in good
agreement with the experimental data for the Ru(III) complex
For the isomeric difluoro compounds 4e7, the order of activity is
[Ru(H2edta)(2-OMe-Pha)] [41] (H2edta
¼
(HOOC)(ꢀOOC)
2-
F24 > F26 > F34 > F25, indicating a higher importance of the ortho-
NCH2CH2N(COOH)(COOꢀ);
2-OMe-Pha
¼
and para-positions, in comparison with the meta-one, for PTK
activity. In fact, the most active di-fluoro substituted compounds (4
and 7) bear the fluoro substituents only at the ortho- and/or para-
positions, whereas those with a meta-F (5 and 6) are less active.
methoxyphenylhydroxamate), the only Ru species with hydrox-
amato ligand for which the X-ray structure is known. Among
comparable bonds, the maximum deviation was found for the C]O
0
ꢀ
bond (0.04 A). The analysis of the composition of frontier MOs of 1
D
and the oxidized or reduced species with unrelaxed geometry {10
}
3.3. Electrochemistry behavior and theoretical study
or {10L} indicates that, upon oxidation, the electron is removed
from the first HOMO of 10 whereas, upon reduction, the electron
goes to the first LUMO.
The redox properties of 1e7 have been investigated by cyclic
voltammetry, at a platinum electrode, in a 0.2 M [nBu4N][BF4]/
CH2Cl2 solution, at 25 ꢁC. They exhibit a single-electron irreversible
The main contribution to the HOMOs of 10e70 comes from the
metal and p orbitals of the ONCO fragment of the bha ligand as well
as from the Clꢀ ligand (Fig. 3). The full geometry optimization of the
oxidized complex 10D does not result in a noticeable change of the
structure. The RueCl, RueO(1), N(2)eO(1), RueO(4), and C(3)eC(5)
anodic process, assigned [40] to the Ru(II/III) oxidation, at the
ox
oxidation potential values (E
in the range of 1.51 ꢀ 2.05 V vs. SCE)
p=2
given in Table 2 (Fig. 1 for compound 5 as a typical case).
In the cathodic region, a single-electron irreversible wave is
ꢀ
red
bonds are shortened by 0.020e0.083 A while the N(2)eC(3) bond is
detected, assigned to the Ru(II/I) reduction (E
in the range
p=2
D
0
ꢀ
elongated by 0.027 A upon oxidation. The spin density in 1 is
localized mostly on the Ru and O(1) atoms (the contributions are
0.42 and 0.30, respectively). Thus, the oxidation of 10 affects both
the metal atom and the bha ligand.
of ꢀ1.51 to ꢀ0.51 V vs. SCE). The occurrence of a single-electron
oxidation (or reduction) has been confirmed by exhaustive
controlled potential electrolysis (CPE) at a potential slightly anodic
(or cathodic) to that of the corresponding peak potential.
The LUMOs of complexes 10e70 are strongly delocalized along
the Ru atom and bha and benzene ligands (Fig. 3). The geometry
Complex [Ru(
oxidize (lowest oxidation potential, E
most difficult to reduce (lowest reduction potential, E
vs. SCE) (Table 2), and, moreover, is that with the highest PTK
h
6-p-cymene)(F24bha)Cl] (4) is the easiest to
ox
¼ 1:51 V vs. SCE) and the
p=2
red
¼ ꢀ1:51 V
p=2
Table 1
PTK inhibitory activity.a
Compounds
[RuCl(
6-p-cymene)(
IC50 (mM)
h
m
-Cl)]2
0.18
0.02
1.52
3.11
< 0.02
0.11
[Ru(
[Ru(
[Ru(
[Ru(
[Ru(
[Ru(
[Ru(
h
h
h
h
h
h
h
6-p-cymene)(F4bha)Cl] (1)
6-p-cymene)(Cl4bha)Cl] (2)
6-p-cymene)(Br4bha)Cl] (3)
6-p-cymene)(F24bha)Cl] (4)
6-p-cymene)(F34bha)Cl] (5)
6-p-cymene)(F25bha)Cl] (6)
6-p-cymene)(F26bha)Cl] (7)
0.23
0.02
Genistein
13.65 [39]
Fig. 1. Cyclic voltammogram (anodic region) of [Ru(h
6-p-cymene)(F34bha)Cl] 5 at a Pt
a
The IC50 values were determined in triplicate.
disc electrode, in a 0.2 M [nBu4N][BF4]/CH2Cl2 solution (v ¼ 0.2 V sꢀ1).

X. Shang et al. / Journal of Organometallic Chemistry 730 (2013) 137e143
141
influence their electron-donor properties and thus the electro-
chemical Lever EL ligand parameter. On the basis of the Lever [42e
47] linear relationship (Eq. (1)), by assuming that it is also valid for
half-sandwich ruthenium(II) cymene type complexes [48e51], we
propose the estimate of the Lever EL parameter for the various
chelating halogenobenzohydroxamatos bha (substituents ¼ 4-F, 4-
Cl, 4-Br, 2,4-F2, 3,4-F2, 2,5-F2, 2,6-F2). However, one should be very
cautious with the estimated values since, each of them is based on
a single complex and, moreover, the oxidation potential is not the
thermodynamic one in view of the irreversibility of the oxidation
wave.
ꢀ
ꢁ
X
E ¼ SM
EL þ IM ðV vs: NHEÞ
(1)
ox
As an example, application of Eq. (1) to 1 (E
¼ 1:60 V vs.
p=2
SCE ¼ 1.84 V vs. NHE) with the known values of SM (0.97) and IM
(0.04 V vs. NHE) for the RuII/III redox center [42] and of EL for Clꢀ
(ꢀ0.24 V vs. NHE) [42] and cymene (1.63 V vs. NHE) [48], allows to
determine the EL ligand parameter for chelating F4bha as 0.47 V vs.
NHE. By following the same calculation methodology, we have
determined the EL parameter for the other bidentate halogen-
obenzohydroxamato ligands of the present study (Table 3).
The EL parameter is a measure of the electron-donor character of
a ligand, the lower its value the stronger such a character is [42]. For
the mono halogenobenzohydroxamato compounds 1e3, the esti-
mated EL values of their bha ligands follow the order: FeC6H4C(O)
L
Fig. 2. Equilibrium structures of 10 and 10
.
L
optimization of the reduced species 10 leads to significant struc-
tural changes, i.e., a cleavage of the RueO(4) bond occurs. As
a result, the bha ligand in 10L acquired a monodentate coordination
mode (Fig. 2). The general structure is stabilized by the intra-
molecular H-bond between the NH group and the Clꢀ ligand. The
L
spin density in 10 is localized on the Ru atom with the contribu-
tion of 0.93. Thus, despite the delocalized character of the LUMO of
10, the reduction of this complex accompanied by the geometry
relaxation is metal centered.
The trends of the calculated vertical ionization potentials and
electron affinities along the row 10e70 are in good agreement with
the corresponding trends of the experimental oxidation and
reduction potentials, except for complex 7 for which the calculated
values are underestimated (Table 2, Fig. 1TS).
Table 3
EL ligand parameter for bidentate substituted halogenobenzohydroxamatos.
Ligand
EL/V vs. NHE
O
C
O-
0.47
F
N
H
3.4. Lever’s EL electrochemical parameter
O
The introduction of functional groups in the ortho-, meta- or
para-positions of the aromatic ring of the bha ligands should
O-
O-
0.56
0.54
0.37
Cl
C
N
H
O
C
Br
F
N
H
O
O-
C
N
H
F
O
C
O-
F
0.93
0.57
N
H
F
F
O
C
O-
N
H
F
F
O
O-
0.86
C
N
H
F
Fig. 3. Plots of the HOMO and LUMO of 10.

142
X. Shang et al. / Journal of Organometallic Chemistry 730 (2013) 137e143
Scheme 2. Possible tautomeric equilibria for the bha ligand in 1e7.
NHOꢀ (F4bha) < CleC6H4C(O)NHOꢀ (Cl4bha) z BreC6H4C(O)NHOꢀ
(Br4bha), indicating that the fluoro-benzohydroxamato acts as
a stronger electron-donor than the analogous chloro- and bromo-
benzohydroxamatos. This can be rationalized on the basis of the
Hammett’s substituent constant. In fact, the Fꢀ substituent
The PTK inhibitory activity does not appear to follow the redox
potential of the complexes, and further studies deserve to be
undertaken in order to get an insight in the mechanism of action of
these complexes which may be helpful for the design of new metal-
based anticancer agents.
(
sp ¼ 0.06) is an overall stronger electron-donor than the other
halogens (which have a common sp value of 0.23). Correlations of
redox potentials with Hammett’s and related constants of ligand
substituents are well documented [52,53].
Acknowledgments
This work has been partially supported by the Foundation for
Science and Technology (FCT) (grant no. SFRH/BPD/44773/2008)
and its PEst-OE/QUI/UI0100/2011 project, Portugal, the state
‘Innovative Drugs Development’ key and technology major projects
of China (no. 2009ZX09103-104) and the National Natural Science
Foundation of China (No: 81102311). T.F.S.S. is grateful to FCT for her
PhD (SFRH/BD/48087/2008) fellowship. M.L.K. is grateful to the FCT
and IST for a research contract within the Ciência 2007 scientific
programme.
The estimated EL values for the bidentate mono substituted
halogeno-benzohydroxamato ligands (avg. 0.52 V) indicate an
electron-donor ability comparable to that found for the tridentate
tris(pyrazol-1-yl)borate (
bis(pyrazol-1-yl)acetic acid (
h
3-Tpꢀ; EL ¼ 0.52 V) [48] or the bidentate
h
2-bpac; EL ¼ 0.54 V) [48], but
considerably lower than those found for the bidentate acetylacet-
onate (acacꢀ; EL ¼ ꢀ0.16 V) [43] and fluoro-substituted derivatives,
such as 1,1,1-trifluoro-2,4-pentanedionate (tfacꢀ; EL ¼ 0.06 V) [43]
or 1,1,1,5,5,5-hexafluoro-2,4-pentanedionate (hfacꢀ; EL ¼ 0.34 V)
[43]. The difluoro-benzohydroxamato ligands usually display EL
values lower than that of the mono-fluorinated ligand, thus
behaving as weaker electron-donors.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.jorganchem.2012.
12.013.
Concerning 4e7, the presence of two fluoro substituents in
different positions of the aromatic ring can lead to different redox
potentials, but any analysis has to be taken rather cautiously in
view, e.g., of the possibility of (i) establishment of H-bonds between
F and the NH group, or (ii) ligand tautomerization (Scheme 2), (iii)
H-bond between F and the OH group, and (iv) the irreversible
character of the oxidation waves.
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