Influence of ancillary ligand L in the nitric oxide photorelease by the [Ru(L)(tpy)NO]3+ complex and its vasodilator activity based on visible light irradiation

Article abstract of DOI:10.1016/j.ica.2006.02.020

The photochemical and pharmacological studies of the novel [Ru(L)(tpy)NO]³⁺ L = bpy (2,2′-bipyridine), NH · NHq (quinonediimine) and NH₂.NH₂cat (o-phenylenediamine) were investigated in aqueous medium. The synthesized nitrosyl ruthenium complexes showed nitric oxide (NO) release under light irradiation at 355 nm for [Ru(L)(tpy)NO]³⁺ complex with quantum yield of 0.14 ± 0.02, 0.47 ± 0.03 and 0.46 ± 0.02 mol Einstein^-1 for L = bpy, NH · NHq and NH₂ · NH₂cat, respectively, and 0.0065 ± 0.001 mol Einstein^-1 for light irradiation at 532 nm for [Ru(NH · NHq)(tpy)NO]³⁺ complex. The photochemical pathway at 355 nm light irradiation was described as a multi-step mechanism, although at 532 nm it was better attributed to a photo-induced electron transfer. The vasorelaxation induced by NO release produced by light irradiation in visible region from physiological solution of [Ru(NH · NHq)(tpy)NO]³⁺ complex was evaluated and compared with sodium nitroprusside (SNP). The results showed very similar vasodilator power between both species.

Full text of DOI:10.1016/j.ica.2006.02.020

Inorganica Chimica Acta 359 (2006) 2543–2549
www.elsevier.com/locate/ica
Influence of ancillary ligand L in the nitric oxide photorelease by
the [Ru(L)(tpy)NO]3+ complex and its vasodilator activity based
on visible light irradiation
a
a
b
Renata Galvao de Lima , Marilia Gama Sauaia , Daniella Bonaventura ,
˜
Antonio Claudio Tedesco a,b, Lusiane Maria Bendhack , Roberto Santana da Silva
b
a,b,
*
a
ˆ
Departamento de Quı´mica da Faculdade de Filosofia Ciencias e Letras de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Av. dos Bandeirantes 3900,
14040-901 Ribeira˜o Preto, SP, Brazil
Faculdade de Ciencias Farmaceuticas de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Av. do Cafe s/n, 14040-903 Ribeira˜o Preto, SP, Brazil
b
ˆ
ˆ
´
Received 7 November 2005; received in revised form 2 February 2006; accepted 7 February 2006
Available online 6 March 2006
Abstract
The photochemical and pharmacological studies of the novel [Ru(L)(tpy)NO]3+ L = bpy (2,20-bipyridine), NH Æ NHq (quinone-
diimine) and NH2.NH2cat (o-phenylenediamine) were investigated in aqueous medium. The synthesized nitrosyl ruthenium complexes
showed nitric oxide (NO) release under light irradiation at 355 nm for [Ru(L)(tpy)NO]3+ complex with quantum yield of 0.14 0.02,
0.47 0.03 and 0.46 0.02 mol Einsteinꢀ1 for L = bpy, NH Æ NHq and NH2 Æ NH2cat, respectively, and 0.0065 0.001 mol Einsteinꢀ1
for light irradiation at 532 nm for [Ru(NH Æ NHq)(tpy)NO]3+ complex. The photochemical pathway at 355 nm light irradiation was
described as a multi-step mechanism, although at 532 nm it was better attributed to a photo-induced electron transfer. The vasorelax-
ation induced by NO release produced by light irradiation in visible region from physiological solution of [Ru(NH Æ NHq)(tpy)NO]3+
complex was evaluated and compared with sodium nitroprusside (SNP). The results showed very similar vasodilator power between both
species.
ꢀ 2006 Elsevier B.V. All rights reserved.
Keywords: Nitrosyl ruthenium complexes; Vasodilation; Nitric oxide
1. Introduction
oxide, several species have been synthesized with the aim
of developing nitric oxide-donor agents [8–14]. It might
benefit from nitric oxide-enhancing medicines [15–17].
Among them, coordination compounds containing {M–
NO+} bond have been successfully employed as NO deliv-
ery agents by accessing the reduction potential of nitrosyl
ligand [18–20] or by light stimulation [8–13,21,22]. Perhaps,
a class of those nitrosyl species that have been most inves-
tigated is that concerning to the use of ruthenium(II) as
metal center [21–24]. The ruthenium ion configurations
are very useful due to their rich and versatile redox chem-
istry, in addition to their favorable thermodynamic and
kinetic properties. Nitrosyl ruthenium complexes have usu-
ally been described as containing {RuII–NO+} bond,
whose stability is pH-dependent, since it can react with
Nitric oxide (NO) is a radical molecule produced by
many cells in the human body and has become recognized
as a major effector molecule in a diverse array of physiolog-
ical and pathological processes [1–5]. NO action has also
been found in different cells and tissues in which it exhibits
several different functions, such as neurotransmission,
blood pressure control, inhibition of platelet aggregation,
and immunological responses [6,7]. Due to the great bio-
logical importance of NO and also considering that many
disease states are associated with a deficiency in nitric
*
Corresponding author.
E-mail address: silva@usp.br (R.S. da Silva).
0020-1693/$ - see front matter ꢀ 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2006.02.020

2544
R.G. de Lima et al. / Inorganica Chimica Acta 359 (2006) 2543–2549
hydroxide ion to generate nitroruthenium(II) species [8–
14,22,25]. The degree of electrophilicity of coordinated
NO+ can be systematically tuned through the modulation
of ancillary ligand ‘‘L’’ in [RuL5NO]n+ type complex [26–
28]. Two classes of nitrosyl ruthenium complexes, cis-
2. Experimental
2.1. Apparatus
The ultraviolet–visible (UV–Vis) spectra were recorded
on a Hitachi U-3501 and Genesys-2 apparatus from Spec-
[RuII(bpy)2L(NO+)]n+ and trans-[RuIIL(NH3)4(NO+)]3+
,
´
have been thoroughly studied because of their capability
of releasing NO under light stimulation [8,10,23], which
is one of our current interests [8–14]. The photochemical
pathway for those complexes is produced by light irradia-
tion on metal–ligand charge transfer band (MLCT),
characterized as (dpRuII)–p*(NO+) transition or by
photo-induced electron transfer [8–14,22,25].
In an attempt to determine the controlling factors that
allow managing the quantum yield of nitric oxide release,
we have studied the photoreactivity of [Ru(bpy)(tpy)-
NO](PF6)3, [Ru(NH Æ NHq)(tpy)NO](PF6)3 and [Ru(NH2 Æ
NH2cat)(tpy)NO](PF6)3 complexes, in which tpy is
2,20:60,200-terpyridine, bpy is 2,20-bipyridine, NH Æ NHq is
quinonediimine and NH2 Æ NH2cat is o-phenylenediamine
(Fig. 1).
Some spectroscopic properties of [RuL4(benzoquinone-
diimine)]2+ complex have been described [31–33]. The spe-
cies have shown great electron delocalization between the
metal and the ligand that seems to be dependent on the
energy, symmetries and overlap of valence metal and
ligand orbital [34]. The non-innocent character of those
quinone-related ligands permits to illustrate it in three dif-
ferent oxidation states, which are characterized as quinone-
diimine (NH Æ NHq), semiquinonediimine(NH Æ NHsq)
and o-phenylenediamine (NH2.NH2cat) [35]. Based on this,
our goal in this work was to describe the effect of this
possible electron delocalization in some chemical and
photochemical properties of [Ru(bpy)(tpy)NO](PF6)3,
[Ru(tpy)(NH Æ NHq)NO](PF6)3 and [Ru(NH2.NH2cat)-
(tpy)NO](PF6)3 complexes. In principle, the photolysis of
both classes of complexes shows NO release by similar
photochemical pathway. The possibility of using this type
of complex as a NO delivery agent by light stimulation
was also evaluated and interpreted by the pharmacological
viewpoint.
tronic. Infrared (IR) spectra were recorded on a protege
460 series FT-IR spectrometer, using solid samples pressed
in KBr pellets. The pH measurements were made using a
430 pH meter from Corning.
The photolysis of the complexes in a trifluoroacetate
buffer solution at pH 2.01 and ionic strength of 0.1 M
adjusted with NaBF4 was performed using a laser flash
photolysis apparatus consisting of a Continuum Q-
switched Nd:YAG laser (Continuum, Santa Clara, CA)
with excitation provided by a third (355 nm) and second
(532 nm) harmonic. The pulse length was 8 ns, the beam
diameter incident on the sample was 6 mm, and the repeti-
tion rate was 10 Hz. The pulse energy was typically 10 mJ/
pulse measured with a Field Master power-meter with an
L-30 V head. NO releasing was detected and measured
with an ISO-NOP NO meter from World Precision Instru-
ments, which directly detects NO concentration by an
amperometric technique. The sensitivity of this apparatus
ranges from 1 nM to 20 lM, with a 2-mm sensor, which
directly detects NO concentration by an amperometric
technique. The sensor output was recorded with an IBM-
PC computer linked to a DUO-18 acquisition board from
WPI.
2.2. Chemicals and reagents
RuCl3 Æ nH2O, 2,20:60,200-tpyridine, 2,20-bipyridine, 3,4-
benzoquinonediiminebenzoic acid, and 1,100-phenanthro-
line, and Reinecke’s salt {(NH4)Cr(NH3)2(NCS)4 H2O}
were purchased as high purity reagents from Aldrich
Chemicals and were used as supplied. Potassium ferrioxa-
late, {K3[Fe(C2O4)3] Æ 3H2O}, was purchased from Fisher
Scientific Co. Doubly distilled H2O was used for all
experiments.
The recrystallized complex salts [RuCl(bpy)(tpy)]Cl and
[Ru(bpy)(tpy)NO](PF6)3 were prepared as previously pub-
lished [36,37].
The [RuCl(NH Æ NHq)(tpy)]Cl complex was synthesized
by a similar procedure described for [RuCl(bpy)(tpy)]Cl
complex, however using 3,4-benzoquinonediiminebenzoic
acid as ligand instead of 2,20-bipyridine. The obtained ruthe-
nium species was purified through a silica-gel column (70–
230 mesh/Merck) by elution with methanol. A red solution
fraction was separated and the solid was recovered by rotary
evaporation and used in the synthesis of [Ru(NH Æ NHq)-
(tpy)NO](PF6)3 complex. Yield = 20.0%.
The [Ru(NH Æ NHq)(tpy)NO](PF6)3 complex was pre-
pared by mixing 0.038 g of [RuCl(NH Æ NHq)(tpy)]Cl salt
(0.069 mmol) previously dissolved in water (30 ml) under
argon atmosphere with 0.024 g of NaNO2 (0.34 mmol)
and refluxed for 1 h. Two milliliters of HPF6 was added
N
N
N
N
N
bpy
RuII
L=
L
HN
COOH
COOH
NO+
HN
L
NH.NHq
H2N
H2N
[Ru(tpy)(L)NO+]3+
NH2.NH2.cat
Fig. 1. Structure of nitrosyl ruthenium species.

R.G. de Lima et al. / Inorganica Chimica Acta 359 (2006) 2543–2549
2545
to the stirred solution. The resulting orange precipitate was
collected by filtration, washed with diethyl ether and stored
under vacuum in the dark. Typical yield for [Ru(NH Æ
NHq)(tpy)NO](PF6)3 was 87.0 %. Anal. Calc. for
C22H17N6O3P3F18Ru: C, 27.82; H, 1.89; N, 8.83. Found:
C, 27.90; H, 1.99; N, 8.99%.
The [Ru(NH2.NH2cat)(tpy)NO](PF6)3 complex was pre-
pared by mixing 0.023 g of [RuCl(NH Æ NHq)(tpy)]Cl salt
(0.041 mmol) previously dissolved in water (50 ml) under
argon atmosphere with 0.014 g of NaNO2 (0.21 mmol).
The mixture was put in a microwave with 30% potency
during 2 min. Two milliliters of HPF6 was added to the
stirred solution. The resulting orange precipitate was col-
lected by filtration, washed with diethyl ether and stored
under vacuum in the dark. Typical yield for [Ru(NH2 Æ
NH2cat)(tpy)NO](PF6)3 was 75.0%. Anal. Calc. for
C22H17N6O3P3F18Ru: C, 27.76; H, 2.00; N, 8.83. Found:
C, 27.51; H, 2.00; N, 8.82%.
was obtained from the numeric values of light intensity,
which were calculated according to the literature [41].
NO quantum yields (/t) were calculated based on NO
concentrations, obtained by NO meter measurement. The
calculated values were plotted versus t. These plots were
linear, with a negative slope, for the first 40% of the reac-
tion. The extrapolated quantum yield at t = 0 (y intercept)
was taken as /NO for the photolabilization of NO from
[Ru(L)(tpy)NO]3+compounds. Evaluation of /NO at t = 0
eliminates possible complications, resulting from primary
products photoreactions [41,42]. All the reported data are
the average of three independent experiments, and the
error is the standard deviation.
2.6. Pharmacological experimental protocols
Vessel preparation was done as previously described
[12].
2.3. Laser flash photolysis experiments
2.6.1. Experimental protocols
2.6.1.1. Relaxant effect of [Ru(NH Æ NHq)(tpy)NO]3+
after pre-contraction with norepinephrine, phenylephrine
and prostaglandin F2a. To examine whether [Ru(NH Æ
NHq)(tpy)NO]3+ induces smooth muscle cell relaxation,
aortic rings were pre-contracted with 0.1 lM norepineph-
rine (NOR), 0.1 lM phenylephrine (Phe) and 3 lM prosta-
glandin F2a (PGF2a). When the contraction had reached, a
plateau [Ru(NH Æ NHq)(tpy)NO]3+ (1.0 · 10ꢀ10–3.0 ·
10ꢀ4 M) was cumulatively added.
Photolysis of the nitrosyl ruthenium species (1.0 · 10ꢀ4
–
1.0 · 10ꢀ5 M) was performed in trifluoroacetate buffer solu-
tion at pH 2.01 and the ionic strength of 0.1 M was adjusted
with NaBF4. A sample (3.0 ml) in a quartz 1-cm square cuv-
ette with a magnetic stirring bar was routinely thermostated
at 25.0 0.1 ꢁC. The sample solution was stirred continu-
ously and was irradiated for a defined time period.
Two types of analyses were carried out during the flash
photolysis experiments. First, the UV–Vis spectrum of the
sample was recorded after each irradiated period. Second,
the NO measurements were made with the selective elec-
trode (NOmeter) into the cuvette, but positioned outside
the light path to avoid any photoelectric interference.
2.6.1.2. Time-course for the relaxation induced by [Ru(NH Æ
NHq)(tpy)NO]3+
.
[Ru(NH Æ NHq)(tpy)NO+]3+ (1.0 ·
10ꢀ4 M) was added to the organ chamber when a stable
contraction in response to 1 · 10ꢀ5 M phenylephrine was
achieved. Time-course for relaxation induced by this com-
pound was evaluated and a similar protocol was followed
for sodium nitroprusside (SNP) (3.0 · 10ꢀ5 M).
2.4. NOmeter calibration
The calibration curve of the selective electrode was con-
structed by several dilutions of a known volume of a satu-
rated nitric oxide solution in 10.0 ml of a previously
degassed trifluoroacetate buffer solution (pH = 2.01), to
which the NOmeter electrode was adapted. The current
value in nA was recorded for each added volume. NO con-
centration was calculated according to the reported NO
molar fraction solubility (2.1 · 10ꢀ3 M at 25 0.1 ꢁC) [38].
Nitric oxide gas was produced by Cu0 dyed in HNO3
solution (50%) and was passed through a 5 M KOH solu-
tion to remove any trace of nitrite species. Nitric oxide
stock solution was prepared by degassing 10 ml of distilled
water in which the NO gas was bubbled during 30 min.
2.6.1.3. Data analysis. Data are expressed as mean-
s
SEM. In each set of experiments, n indicates the num-
ber of rats studied. The maximum relaxant effect (Emax
)
was considered as the maximal amplitude response reached
in the concentration-effect curves for the compounds. The
concentrations of the agents that produced the half-maxi-
mal relaxation amplitude (EC50) were determined after
logit transformation of the normalized concentration-
response curves and were reported as the negative loga-
rithm (pEC50) of the mean of individual values for each
tissue using the GraphPad Prism version 3.0 (GraphPad
Software Corporation San Diego, CA).
2.5. Quantum yield measurements
3. Results and discussion
Light intensities were determined before each photolysis
experiments by chemical actinometry procedure. The acti-
nometers used were potassium ferrioxalate to kirr = 355 nm
and Reinecke’s salt to kirr = 532 nm [39,40]. An average
The [Ru(NH Æ NHq)(tpy)NO](PF6)3 complex described
in this work was prepared by a similar procedure previ-
ously published for [Ru(bpy)(tpy)NO](PF6)3 [36]. The
[Ru(NH2 Æ NH2q)(tpy)NO](PF6)3 was achieved using

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R.G. de Lima et al. / Inorganica Chimica Acta 359 (2006) 2543–2549
microwave as a synthetic method. It is likely that the short
time used in that reaction was responsible for the isolation
of the quinoneammine ruthenium complex. Those nitrosyl
ruthenium complexes were characterized by elemental
analysis and conventional spectroscopic techniques. Some
data concerning to UV–Vis and IR spectra results of the
nitrosyl ruthenium complexes and related species are
shown in Table 1.
4x104
3x104
2x104
1x104
0
The [Ru(L)(tpy)NO]3+ and [Ru(X)(L)(tpy)]n+ com-
plexes, in which L = bpy or benzoquinonediimine ligands
and X = Cl, show intraligand bands in the UV region that
are attributed to p ! p* transitions of the unsaturated
ligands in comparison to the electronic spectrum of free
ligand. The [Ru(H2O)(NH Æ NHq)(tpy)]2+ was obtained
in situ by the reduction of [Ru(NH Æ NHq)(tpy)NO]3+ with
zinc amalgam, as previously described for similar nitrosyl
ruthenium complexes [8–13]. All the studied nitrosyl ruthe-
nium species also showed a shoulder on the 300–400 nm
region (Table 1) that is not observed on the electronic spec-
250 300 350 400 450 500 550 600 650 700 750 800
wavelength (nm)
Fig. 2. Electronic spectra of [Ru(NH Æ NHq)(tpy)NO]3+ complex at (solid
line), [Ru(H2O)(NH Æ NHq)(tpy)]2+ (dash line) and [RuCl(NH Æ NHq)-
(tpy)]+ (dot line) complexes in HCl 0.1 M.
The photolysis at 355 nm of an aqueous solution con-
taining [Ru(bpy)(tpy)NO]3+, [Ru(NH Æ NHq)(tpy)NO]3+
and [Ru(NH2 Æ NH2cat)(tpy)NO]3+ was monitored by
UV–Vis spectra. The UV–Vis absorption spectral change
trum of [RuCl(bpy)(tpy)]+ and [Ru(X)(NH Æ NHq)(tpy)]n+
.
Based on this and by comparison to the described electronic
spectrum of similar nitrosyl ruthenium species [8,10,26–28],
we tentatively assigned this band as metal ligand charge
transfer (MLCT) due to dp(RuII) ! p*(NO) transition.
For the [Ru(NH Æ NHq)(tpy)NO]3+ complex, we found,
on its electronic spectrum, a weak band at 510 nm that
was attributed to the dp(RuII) ! p*(NH Æ NHq) transition
(Fig. 2) by comparison to the electronic spectrum of
[Ru(X)(NH Æ NHq)(tpy)]n+ (X = H2O or Clꢀ).
containing [Ru(bpy)(tpy)NO]3+
, during irradiation is
shown in Fig. 3. The electronic spectrum of the photoprod-
uct is very similar to the synthesized [RuII(H2O)(bpy)-
(tpy)]2+ complex [36]. Assuming that the metal ligand
0.30
2.5
0.25
0.20
The IR spectrum exhibits a strong vibrational peak at
1850–1950 cmꢀ1 region ascribed to mNO stretching. The cat-
ionic character of NO+ of coordinated nitric oxide is
related to the extent of the dp(RuII)–NO+ interaction,
which is affected by the binding metal center with ancillary
ligands in the nitrosyl ruthenium species. Thus, we expect
that a change of the L ligand on [Ru(L)(tpy)NO]3+ com-
plex should modify the ruthenium nitrosyl interaction
and consequently the mNO stretching, as observed for sev-
eral different classes of nitrosyl ruthenium complexes
[10,26–30,43].
The observed lowest mNO stretching energy for [Ru
(tpy)(NH2 Æ NH2cat)(NO)]3+ complex, in comparison to
the other described nitrosyl terpyridine ruthenium com-
plex, can roughly be used to the attribution of strengthen-
ing {RuII–NO+} interaction or may be due to the effect
occasioned by the co-ligand trans to NO+, as observed
for [RuCl2(tpy)NO]Cl [18].
0.15
2.0
0.10
0.05
1.5
0.00
0
20
40
60
80
100
120
140
160
time (s)
1.0
0.5
0.0
240
300
360
420
480
540
600
660
720
780
wavelength (nm)
Fig. 3. Electronic spectra of [Ru(bpy)(tpy)NO]3+ complex, pH 2.01,
during flash-photolysis at 355 nm. [Complex] = 5.87 · 10ꢀ5 M. Inset:
Chronoamperogram of NO release by photolysis of [Ru(bpy)(tpy)NO]3+
complex. [Complex] = 1.00 · 10ꢀ4 M.
Table 1
UV–Vis and infrared data of [Ru(L)(tpy)NO]3+ complex, with L = bipyridine (bpy), quinonediimine (NH Æ NHq) and o-phenylenediamine
(NH2 Æ NH2cat) and related species
Complex
k (nm) (log e)a
mNO (cmꢀ1 b
)
[Ru(bpy)(tpy)NO](PF6)3
230 (4.63); 277 (4.25); 288 (4.32); 305 (4.24); 332 (4.02); 358 (3.92); 480 (2.84)
288 (4.21); 325 (4.11); 355 (3.98)
285 (4.39); 324 (4.27); 358 (4.15); 510 (3.65)
1944
1874
1888
[Ru(NH2 Æ NH2cat)(tpy)NO](PF6)3
[Ru(NH Æ NHq)(tpy)NO](PF6)3
[RuCl(tpy)(NH Æ NHq)]Cl
266 (4.07); 278 (4.05); 312 (4.02); 328 (3.89); 502 (3.98)
a
HCl 0.1 M.
KBr pellets.
b

R.G. de Lima et al. / Inorganica Chimica Acta 359 (2006) 2543–2549
2547
charge transfer between Ru(II) and NO+ occurs at 350 nm
for [RuII(tpy)(L)NO+]3+ complex, the irradiation in this
region could easily generate [RuIII(bpy)(tpy)NO0]3+ com-
shows a band in the visible region. The electronic spectrum
during light irradiation shows little decreasing of the inten-
sity of MLCT band attributed to dp(RuII) ! p*(N-
H Æ NHq) transition. The observed NO quantum yield
obtained by the NO-selective sensor measurement was
plex at excited state. Since the transient is formally RuIII
–
NO0, the substitution reaction is expected to occur consid-
ering many of the reports that present the NO0 species as a
labile ligand for ruthenium(III) complex [8–10,14]. How-
ever, the formation of an aqueous ruthenium(II) species
should be possible presumably by accessing a lowest energy
ligand field state, during the TCML state deactivation.
The photoreactivity of the nitrosyl complexes was also
accompanied by in situ NO detection. The signal recorded
by the NO sensor rose quickly when photolysis was initi-
ated, then decreased when the light was turned off owing
to NO consumption via various pathways, especially
auto-oxidation [44] (inset, Fig. 3). Considering that the
photolysis at 355 nm is due to light irradiation on the
MLCT band occasioned by dp(RuII) ! p*(NO) transition,
the [Ru(H2O)(bpy)(tpy)]3+ complex should be expected as
one of the photoproducts. Similar behavior was also
observed for all the studied complexes.
0.0065
0.001 mol Einsteinꢀ1. This value is consistent
with the expected quantum yield for NO release obtained
by photo-induced electron transfer for some nitrosyl ruthe-
nium species [11,14]. Assuming that the light irradiation at
532 nm is mainly attributed to the photochemical process
centered on the dp(RuII) ! p*(NH Æ NHq) transition, it is
licit to suppose that the photochemical pathway of this
process involves a photo-induced electron transfer by the
former NH Æ NHsemiquinone from {RuIII–(NH Æ NHsemi-
quinone)}* to the nitrosyl ligand, as described on Scheme 2.
Considering that semiquinone is a powerful reducing agent,
the proposed photochemical mechanism should involve the
production of {RuIII–(NH Æ NHq)} fragment.
3.1. Pharmacological experiment
Based on the spectroscopic results and by the NO mea-
surement, we could infer the photochemical pathway as a
multiple photochemical process as described on Scheme 1.
The quantum yields observed for NO release from
[Ru(L)(tpy)NO]3+ complexes, L = bpy, NH Æ NHq and
NH2 Æ NH2cat, obtained by 355 nm light irradiation were
The [Ru(NH Æ NHq)(tpy)NO]3+ complex shows kinetic
stability on physiological solution, which allowed us to
use this compound on vasorelaxation studies. The relaxa-
tion induced by this compound was concentration-depen-
dent in denuded rat aortas pre-contracted with
1.0 · 10ꢀ7 M norepinephrine or 1.0 · 10ꢀ7 M phenyleph-
rine or 3.0 · 10ꢀ6 M prostaglandin F2a (PGF2a). The
results showed that the relaxation induced by [Ru(NH Æ
NHq)(tpy)NO]3+ complex was the same for all contractive
0.14 0.02, 0.47 0.03 and 0.46 0.02 mol Einsteinꢀ1
,
respectively. The highest NO quantum yield found for
the derivative quinoneammine complexes can be taken as
indication for the formation of a large amount of {RuIII–
NO0} fragment at excited state in comparison to the bipyri-
dine ruthenium complex. Perhaps, the delocalized electron
density between the quinoneammine, nitrosyl and the
metal ion facilitates the incidence of {[RuIII(NH Æ NHq)-
(tpy)-NO0]3+}*, which is accentuated when the derivative
quinoneammine ligand is present. The [Ru(NH Æ NHq)-
(tpy)- NO]3+complex was also submitted to light irradia-
tion at 532 nm, since the aqueous solution of this species
agents used (NOR: Emax = 102.38 0.38%, pEC50
6.47 0.13, n = 6; Phe: Emax = 103.48 1.03%, pEC50
=
=
6.61 0.09, n = 8; PGF2a: Emax = 102.24 0.42%,
pEC50 = 6.48 0.08, n = 6).
In fact, the production of NO has been taken as a con-
sequence for the observed vasodilatation [45]. We found
that, in physiological medium, NO is produced by an exter-
nal stimulation of a nitrosyl ruthenium complex [9,10,12]
mainly by the action of an aqueous solution containing
*
H2O
H2O
{ [RuIII (L)(tpy)(NO+)]3+
}
[RuIII (L)(tpy)(H2O)]3+ + NO0
[RuII(L)(tpy)(H2O)]2+ + NO+
hν
[RuII(L)(tpy)(NO+)]3+
355 nm
*
{ [RuII(L)(tpy)(NO+)]3+
}
Scheme 1.
e
[RuII (NH.NHq)(tpy)(NO+)]3+
{[RuIII(NH.NHsemiquinone)(tpy)(NO+)] 3+
}
hν
*
532 nm
0
[RuIII (NH.NHq)(tpy)(H2O)]3+
Scheme 2.
+
NO

2548
R.G. de Lima et al. / Inorganica Chimica Acta 359 (2006) 2543–2549
noradrenaline, a known reducing agent. Considering that
the PGF2a is not a reducing agent, as established for nor-
adrenaline [20] or phenylephrine, and taking into account
that the vasorelaxation curve between the plots is very sim-
ilar (Fig. 4), we can infer that [Ru(NH Æ NHq)(tpy)NO]3+
complex induced vasorelaxation by an intracellular mecha-
nism, as previously attributed for SNP [45].
The study of time-course for the relaxation induced by
[Ru(NH Æ NHq)(tpy)NO]3+ showed that the time to reach
maximum relaxation under dark is around 3500 s
(Fig. 5), although under visible light irradiation it took
255 s, which is comparable to SNP (242 s, n = 5).
4. Conclusion
The photolysis of [Ru(L)(tpy)NO]3+ produced NO with
quantum yield dependent on the L ligand. Apparently, the
electron delocalization existent between ruthenium and
benzoquinonediimine ligand induces an increase of back
bonding in {RuII–NO+} interaction, thus producing high
NO quantum yield values when submitted to light irradia-
tion in ultraviolet region, in comparison to the complex
containing bipyridine ligand.
The observed vasorelaxation for the physiological
[Ru(NH Æ NHq)(tpy)NO]3+ complex solution showed that
this complex can be a powerful NO delivery agent and
may be a good candidate to be used in clinical therapy.
0
25
50
Acknowledgments
This work was supported by grants from FAPESP,
CNPq and Pronex.
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