Bioelectrochemistry 51 Ž2000.

193–200
www.elsevier.comrlocaterbioelechem

An electroanalytical investigation on the redox properties of lacidipine

supporting its anti-oxidant effect
Rosanna Toniolo a , Francesca Di Narda a , Gino Bontempelli a,) , Fulvio Ursini b
a
Department of Chemical Sciences and Technology, UniÕersity of Udine, Õia Cotonificio 108, 33100 Udine, Italy
b
Department of Biological Chemistry, UniÕersity of PadoÕa, Õia G. Colombo 3, 35121 Padua, Italy
Received 4 October 1999; received in revised form 14 March 2000; accepted 20 March 2000

Abstract

The redox properties of lacidipine ŽPyH 2 ., one of the most pharmacologically active N-unsubstituted 1,4-dihydropyridines, have been
studied by cyclic voltammetry and controlled potential electrolysis in acetonitrile, an aprotic solvent that is, at best, a mimic of the
lipofilic layer of biological membranes. PyH 2 undergoes a two-electron oxidation process involving two consecutive one-electron
releases, the latter requiring potentials much less positive than the former. The overall process occurs through a primary one-electron step
accompanied by a fast proton release, with the formation of a neutral radical ŽPyH P., which undergoes a further and quite easier
one-electron step, thus providing the main ultimate product ŽPyHq. consisting in the protonated form of the parent pyridine derivative.
This appears relevant for the anti-oxidant effect since the radical intermediate is much more prone to be oxidized than to be reduced, thus
preventing the propagation of the oxidative chain reaction. The mentioned release of protons in the primary electrode step causes the
overall process to be complicated by a parassite side reaction involving the coupling between one of the electrode products ŽHq. and the
starting species. The protonation of PyH 2 subtracts part of the original species from the electrode process because the parent cationic
species ŽPyHq 3 is no longer electroactive. This parassite reaction occurs rather slowly in the timescale of electroanalytical measurements
.
Žthe relevant kinetic constant has been estimated to be 6.4 l moly1 sy1 ., thus markedly affecting the process only in the presence of
relatively high PyH 2 concentrations and progressively decreasing with the starting PyH 2 concentration. All the products formed in the
oxidation process ŽPyHq, Hq and PyHq 3 have been identified by voltammetric evidences based on deep investigations on their cathodic
.
behaviour. The advantageous anti-oxidant properties displayed by PyH 2 with respect to those exhibited by phenolic anti-oxidants such as
vitamin E are also discussed. q 2000 Elsevier Science S.A. All rights reserved.

Keywords: Lacidipine, anti-oxidant properties; Lacidipine, electroanalysis; Lacidipine, redox properties; 1,4-Dihydropyridine, anti-oxidant properties;
1,4-Dihydropyridine, electroanalysis; 1,4-Dihydropyridine, redox properties

1. Introduction accepted theory of atherogenesis, oxidized low density

The most known pharmacological effect of 1,4-dihydro-
pyridines ŽDHPs. Ca2q channel modulators is their high
affinity interaction with a-1 subunit of the voltage depen-
dent L-type Ca2q channels, which supports their use in
treatment of hypertension w1x. Moreover, in several models
of atherosclerosis, all phases of the experimental disease
appear to be influenced by DHPs w2,3x, which directly
prevent the atherosclerotic plaque development, indepen-
dently of lowering blood pressure w4x. In this connection, it
must be emphasized that, according to the most widely
)
Corresponding author. Tel.: q39-0432-558842; fax: q39-0432-
558803.
E-mail address: gino.bontempelli@dstc.uniud.it ŽG. Bontempelli..
lipoprotein sparks the cascade of events, ultimately leading
to plaque formation w5x and several aspects of atherogene-
sis Že.g., secretion of cytokines, proliferation, migration
and margination of specific cell w6–8x. can be mimicked
by an overwhelming of cellular pro-oxidant reactions vs.
an anti-oxidant defence, usually referred to as ‘‘oxidative
stress’’. Eventually, epidemiological and experimental
studies support the concept that anti-oxidants protect
against spontaneous and experimental disease w9,10x. In
addition, it has been suggested recently that redox reac-
tions Žsuch as those produced by an oxidative stress.
behind the regulation of intracellular calcium homeostasis
are also deeply involved in cell signaling pathways and
control of gene expression w11,12x. Thus, the protective
effect of DPHs might be the consequence of their involve-

0302-4598r00r$ - see front matter q 2000 Elsevier Science S.A. All rights reserved.
PII: S 0 3 0 2 - 4 5 9 8 Ž 0 0 . 0 0 0 7 3 - 8
194 R. Toniolo et al.r Bioelectrochemistry 51 (2000) 193–200
ment in the complex network of cellular events, including
redox reactions, encompassing calcium fluxes. The basic
structure of DHPs actually suggests that these molecules
can undergo redox processes, the possible physiological
relevance of which was the aim of experiments specifically
addressed to measure their anti-oxidant capacity and to
evaluate the effect of DHPs on oxidative reactions promot-
ing a transmembrane calcium flux w13,14x.
The specific anti-oxidant capacity of lacidipine ŽPyH 2 ;
among other DHPs. has been analyzed using a kinetic
procedure, which showed that it can undergo redox pro-
cesses compatible with an anti-oxidant effect, although the
rate constant for the interaction with peroxil radicals is
three orders of magnitude lower than that of vitamin E
derivative w13x.
The calcium influx in smooth muscle cells induced by
hydrogen peroxide was prevented by both dihydropy-
ridines and typical anti-oxidants w14x. The calcium home-
ostasis impairment was sustained after the removal of the
oxidant and was repaired only by disulfide reducing agents,
thus suggesting that hydrogen peroxide produces the oxi-
dation of thiols in the membrane Žpossibly in the voltage
dependent channel., which is prevented by both anti-oxi-
dants and calcium channel blockers. Remarkably, the most
active for this effect among DHPs and typical anti-oxi-
dants such as vitamin E is the N-unsubstituted DHP named
PyH 2 .
Taken together, these evidence highlights the relevance
of redox transitions of DHPs as a possible molecular
element of the pharmacological effect. Thus, in this study,
the specific issue of the redox behaviour of PyH 2 and the
underlying thermodynamic constraints has been addressed
using an electroanalytical approach. Electroanalytical
methods are, in fact, particularly well suited for tackling
problems relative to redox processes, in that they are able
to provide straightforward information not only on the
electron transfers involved, but also on possible coupled
chemical reactions.
Investigations dealing with the electrochemical oxida-
tion of some DHPs have previously been performed in
both non-aqueous w15–19x and aqueous w15,20–22x media.
In water containing solvents, DHPs are reported to un-
dergo a simple two-electron anodic process affording the
corresponding pyridine derivative and the concomitant re-
lease of protons, but deep insight on the mechanism in-
volved is precluded just by the marked nucleophilic char-
acter displayed by H 2 O molecules. More detailed mecha-
nistic information has been obtained in non-aqueous sol-
vents, even though Žespecially when dihidropyridine nu-
cleotides have been considered w15,17,19x. major attention
has been almost always focused on the kinetics of deproto-
nation of the cation radical formed as the primary oxida-
tion product. Conversely, some features of the overall
oxidation process have led to different interpretations, so
that they have remained without reliable explanation as
yet. Thus, to account for controlled potential coulometric
results, yielding a number of electrons involved per mol of
oxidized DHP ranging from about 1.3 to 1.8, three alterna-
tive possibilities have been considered, all of them not
supported by experimental evidences: Ži. simultaneous oc-
currence of a two-electron and a single-electron process
w18x; Žii. occurrence of a two-electron process accompa-
nied by a side reaction consisting in the dimerization of the
radicals obtained by deprotonation of the cation radicals
formed in the primary anodic step w17x; Žiii. occurrence of
a two-electron process accompanied by a side reaction
consisting of the protonation of the starting species, which
is thus partially consumed w15,18x. In particular, in this last
hypothesis, a more-or-less quick decomposition of proto-
nated DHPs is advanced w15x.
It must also be emphasized that, despite the rather
abundant work in the field, some other important aspects
concerning the nature and molar ratio of the oxidation
products, as well as their acid–base properties, have not
been thoroughly clarified. Since these aspects might be
significant to account for the mentioned protective effect
of DHPs, we have carried out a detailed electroanalytical
study on the anodic behaviour of PyH 2 in acetonitrile, i.e.
in a non-aqueous medium that is, at best, a mimic of the
cellular-membrane environment.
In particular, special attention has been directed to the
protonation of the reactant Žoccurring in a timescale quite
different from that for the decay of the primary electrogen-
erated cation radical. in view of the possible implications
of the resulting protonated species on the biological activ-
ity of PyH 2 . This paper reports the results obtained, which
integrate the information gained as yet on DHPs, thus
giving further insight on their anti-oxidant properties.
2. Experimental
2.1. Chemicals
All the chemicals used were of reagent-grade quality
and they were employed without further purification, un-
less otherwise stated. Reagent-grade acetonitrile was puri-
fied further by the standard procedure reported in the
literature w23x and was stored over 0.4-nm molecular sieves
under a nitrogen atmosphere.
The supporting electrolyte, tetrabutylammonium per-
chlorate ŽTBAP., was recrystallized from methanol and
dried under vacuum at 508C.
Pure PyH 2 Žsee Fig. 1. and the parent pyridine species
ŽPy in Fig. 1. were synthesized and generously supplied by
Glaxo ŽVerona, Italy., while the related tetrahydropyridine
ŽPyH 4 in Fig. 1. was a commercial product ŽAldrich.. The
corresponding protonated cations ŽPyHq q q
3 , PyH and PyH 5 ,
respectively; see Fig. 1 were directly generated in aceto-
.
nitrile by electrochemical oxidation of pure hydrogen gas
continuously bubbled through the solvent containing the
supporting electrolyte and appropriate amounts of the men-
 R. Toniolo et al.r Bioelectrochemistry 51 (2000) 193–200 195

The voltammetric unit consisted of an EG & G PARC

model 273 potentiostat driven by an EG & G PARC model
270 software installed on an IBM 486 computer. In the
coulometric and preparative experiments, an AMEL mod.
552 potentiostat was used and the associated coulometer
was an AMEL integrator mod. 731. Unless stated other-
wise, all tests were carried out at 20 " 0.18C on 30-ml
aliquots of oxygen-free acetonitrile solutions containing
the desired species. Typically, voltammetric tests were run
at a scan rate of 0.1 V sy1 .

3. Results and discussion

3.1. Electrochemical oxidation of PyH2

The cyclic voltammetric picture typically recorded at a

Pt electrode on PyH 2 solutions in acetonitrile containing
0.1 M TBAP as the supporting electrolyte is reported in
Fig. 2a. A single well-defined anodic peak Ža. at ca. 1.2 V
was observed, with which no cathodic peak located in the
same potential region was associated even at quite fast

Fig. 1. Formulae of PyH 2 and its derivatives employed in this investiga-

tion.

tioned basic species. Stock solutions of free protons in

acetonitrile were prepared by the same electrochemical
approach, following the procedure reported elsewhere w24x.
In all cases, the resulting proton content was carefully
controlled by coulometric measurements.
Hydrogen and nitrogen Žwhich was used to remove
dissolved oxygen from the working solutions. were previ-
ously passed through sulphuric acid to remove traces of
water and then equilibrated to the vapour pressure of the
solvent.

2.2. Apparatus and procedure

Voltammetric, coulometric and preparative experiments

were conducted at 208C in an H-shaped three-electrode
cell with cathodic and anodic compartments separated by a
sintered glass disc. In voltammetric tests, the working
electrode was a platinum disc with an apparent geometric
area of 2.8 = 10y3 cm2 , mirror-polished with graded alu-
mina powder prior to each set of experiments. On the
contrary, a platinum gauze was used as the working elec-
trode in coulometric and preparative tests. In all cases, the
counter electrode was a platinum sheet, while the reference
Fig. 2. Cyclic voltammetric curves recorded at a scan rate of 0.1 V sy1
electrode was an aqueous AgrAgCl,Cly Žsat. connected to with a platinum electrode in a CH 3 CN solution containing 0.1 M TBAP
the cell by a salt bridge containing the medium employed and 10y3 M PyH 2 : Ža. before electrolysis; Žb. after exhaustive electroly-
also in the test solution. sis at 1.35 V.
196 R. Toniolo et al.r Bioelectrochemistry 51 (2000) 193–200

scan rates Žup to 10 V sy1 ., thus indicating that the

primary oxidation product undergoes a rapid decay. On the
contrary, two cathodic peaks at least Žc 1 and c 2 . located at
ca. 0.1 and y0.3 V, respectively, could be detected in the
reverse scan by suitable widening of the range of the
potential scan. When the Pt working electrode was re-
placed by a glassy carbon electrode, peaks Žc 1 . and Žc 2 .
were markedly shifted towards cathodic potentials and
concomitantly merged, thus suggesting that they were due
to protonated species produced in the anodic process. In
fact, it is well known that the reduction of both free and
bonded protons is affected by an overvoltage, which is
fairly slight at Pt surfaces, but becomes remarkable at
glassy carbon electrodes.
The involvement of acid species as the oxidation prod-
ucts is also suggested by the changes caused in peak Ža. by
the addition of small amounts of basic organic solvents Žin
a molar ratio of 20:1 with respect to the PyH 2 content.
such as dimethylsulphoxide ŽDMSO. and pyridine, i.e. two
species unable to interfere directly with the PyH 2 anodic
process since their oxidation occurs beyond 1.6 V under
the experimental conditions adopted, as preliminarily
checked by us. In fact, when voltammograms were run on
PyH 2 solutions in CH 3 CN added with DMSO, the anodic
peak Ža. increased of about 20–25% and was concomi-
tantly shifted towards less positive potentials Žof about 0.1
V.. As a matter of fact, this last result does not only
substantiate the formation of indefinite protonated species
as the oxidation products but it is also, in particular,
strongly suggestive of the protonation by hydrogen ions,
released just in the anodic reaction, of an aliquot of the
starting PyH 2 , which is thus subtracted from the electrode
process because the corresponding cationic species PyHq
is no longer electroactive.
Controlled potential coulometric experiments carried
out at 1.35 V Žoxidation of PyH 2 at peak ‘‘a’’. showed
that the apparent number of electrons involved Ž n app .
ranged from about 1.1 to 1.8, depending on the PyH 2
initial concentration, the revolution rate and the ratio be-
tween the working electrode area and the sample volume.
In particular, by keeping constant these two last experi-
mental parameters, its value decreased with increasing
PyH 2 concentrations Žin the range 3 = 10y4 –5 = 10y3 M.
as shown in Fig. 3, thus confirming the mentioned occur-
rence of a coupling reaction between one of the electrode
products and the starting species w25x.
The couples of values reported in this figure rise in
evidence that the reaction rate constant is too small to be
conveniently determined by voltammetric measurements.
Consequently, the rate constant k characterizing this cou-
pling reaction was estimated by resorting to the working
curves reported in the literature for this type of electro-
chemical process when studied by controlled potential
electrolysis experiments w26x. With this purpose, the exper-
imental dependence of n app on the logarithm of the PyH 2
concentration was compared with the theoretical working
Fig. 3. Comparison between the experimental trend of n app vs. the
logarithm of PyH 2 concentration Žfull points. and the corresponding
theoretical working curve derived for g s1034 Ždashed line..
curves calculated for different values of the parameter
g s krp, where p s mArV Ž m s mass transfer coeffi-
cient; A s electrode area; V s total solution volume.. A
satisfactory fit was obtained for g s 1034 l moly1 Žsee
dashed line in Fig. 3., which allowed a value of ca. 6.4 l
moly1 sy1 to be found for k, by using a value of
6.2 = 10y3 sy1 for p. This last quantity was estimated as
3 the slope of the linear plot of the logarithm of the current
decaying exponentially with time during a controlled po-
tential electrolysis w27x carried out in a solution containing
PyH 2 concentrated enough Ž5 = 10y3 M. so as to make
the current–time profile closely similar to that typical for a
simple one-electron charge transfer process, because the
coupling chemical reaction occurred nearly completely at
least in the early part of the electrolysis. Of course, this
electrolysis was conducted with the same electrode area
and under the same stirring and volume conditions adopted
in the electrolysis tests whose results are collected in Fig.
3.
Cyclic voltammetric tests carried out on PyH 2 solutions
at the end of these electrolyses showed the presence of
four peaks Žc 1 and c 2 already mentioned above; c 3 at ca.
y0.8 V and c 4 at ca.y 1.2 V. as reported in Fig. 2b, and
pointed out that peak Ža. was restored to some extent in the
reverse scan only after traversing peak Žc 2 ..
When the mentioned controlled potential electrolyses
were repeated in the presence of either DMSO or pyridine,
added to acetonitrile solutions in a molar ratio of 20:1 with
respect to PyH 2 , an n app value equal to 2.0 could be
obtained, but information concerning the oxidation prod-
ucts were precluded since the reduction of protonated
 R. Toniolo et al.r Bioelectrochemistry 51 (2000) 193–200
DMSO and pyridine occurs at potentials intermediate be-
tween peaks Žc 1 . and Žc 2 . w24x, thus preventing their
observation.
Such an increase of the n app value observed in the
presence of the employed basic species substantiates the
hypothesis above that a fraction of the initial PyH 2 species
undergoes the uptake of protons released in the anodic
reaction, thus becoming no longer electroactive.
The whole of these evidences could be accounted for by
advancing, fairly in agreement with previous reports on
DHPs w15,18x, the following reaction pathway for the
overall electrochemical oxidation process :
™ PyH q e Ž E .
PyH 2 Pq y o
PyH ™ PyH q H
2 1
Pq P q
Ž fast.
PyH ™ PyH q e
2 tive in the accessible anodic potential range., but lowered
P q y
Ž E -E . o o
PyH q H ™ PyH
2 1
q q
Ž parassite side reaction.
2PyH ™ PyH q PyH q 2e
2 3
2
q q
3
y these experiments for 3 = 10y4 M starting solutions of
Ž overall electrochemical reaction.
The fast occurrence of chemical decay Ž2. was inferred
from the absence of a backward cathodic peak at potentials
reasonably close to anodic peak Ža., as already reported
above, while chemical step Ž4. is advanced as the parassite
coupling reaction whose fairly small rate constant was
estimated above. Finally, the easier occurrence of the
second electrode step Ž3. with respect to the first Ž1. is
inferred from the good agreement of the experimental
voltammetric response with that expected for a two-elec-
tron ECE process characterized by E1o y E2o G 180 mV
w27,28x.
In order to gain further information that would enable
this reaction pathway to be substantiated, a careful investi-
gation was also carried out on the reduction peaks associ-
ated to the oxidation process. The relevant results are
reported in Section 4.
4. Reduction peaks
By comparison with the voltammetric picture displayed
by electrogenerated free hydrogen ions, the first cathodic
peak Žc 1 . was attributed to the reduction of free protons.
The relative height of this peak was not the same in all
experiments, but depended on the initial concentration of
PyH 2 . In particular, peak Žc 1 . was comparatively higher
after electrolysis of the most diluted PyH 2 solutions Ž3 =
10y4 M. and lowered progressively with increasing start-
ing concentrations of PyH 2 . This observation was con-
firmed by the results obtained in controlled potential ex-
haustive reductions carried out at potentials corresponding
to peak Žc 1 .. They pointed out in fact that the number of
mol of electrons per mol of PyH 2 initially present de-
creased from 0.8 to 0.1 as the concentration of the starting
species was increased from 3 = 10y4 to 5 = 10y3 M.
197
The presence in the solutions of free protons at the end
of PyH 2 oxidations is not in contrast with the reaction
pathway above, since the parassite side reaction Ž4. occurs
so slowly as to prevent a fraction of the hydrogen ions
released in the fast chemical step Ž2. from reacting with
the electroactive PyH 2 itself, this last species being more
rapidly subtracted by the fast electrode reaction Ž1.. On the
other hand, the slow occurrence of chemical step Ž4. was
also substantiated by suitable experiments performed on
purpose, based on the direct reaction of PyH 2 with electro-
generated hydrogen ions. In fact, when diluted enough
PyH 2 solutions were reacted with an equimolar amount of
Ž 1. protons, the corresponding anodic peak Ža. did not disap-
pear suddenly Žprotonated PyH 2 being no longer electroac-
Ž 2.
Ž 3. progressively and a completely similar behaviour was con-
comitantly displayed by peak Žc 1 .. In particular, a half-life
Ž 4.
Ž t 1r2 s 1rk Ž C0 . PyH . of about 10.2 min was inferred from
2
PyH 2 , in fairly good agreement with the value of the
Ž 5. kinetic constant k estimated above.
The lowering of peak Ža. observed after addition of
protons was accompanied by the appearance and concomi-
tant increase of peaks Žc 2 ., Žc 3 . and Žc 4 . irrespective of the
initial PyH 2 concentration, thus pointing out that they are
due to the protonated lacidipine ŽPyHq 3 formed as one of
.
the products of the overall electrochemical oxidation pro-
cess Ž5.. However, peak Žc 2 . was apparently due not only
to PyHq 3 , but also to the reduction of the other oxidation
product PyHq. This was checked by comparison with the
cathodic peak displayed by authentic samples of this
cationic species prepared from acetonitrile solutions of the
parent pyridine species Py in which equimolar amounts of
protons were electrogenerated. Such an apparent overlap-
ping is not surprising since PyHq 3 and PyH
q
are expected
to be both fairly weak acids characterized in acetonitrile by
comparable strength. Consequently, with the aim of gain-
ing further insight on the overall reduction occurring at
Žc 2 ., the cathodic process proper for both PyHq and PyHq 3
was investigated in detail by resorting to solutions contain-
ing separately authentic samples of each of these oxidation
products.
Cyclic voltammograms run on PyHq solutions showed
that this species was only responsible for peak Žc 2 ., with
which an anodic peak was associated in the reverse scan,
as shown in Fig. 4a. This return peak gave rise apparently
with peak Žc 2 . to a cathodic–anodic system involving a
reversible enough charge transfer since it was character-
ized by acceptably narrow peak separation Epa y Epc and
peak width Ep r2 y E p Žabout 140 mV.. Moreover, peak
potentials were scarcely affected by the potential sweep
rate.
Such a fairly reversible behaviour is strongly suggestive
of the occurrence at the cathodic peak of a simple charge
transfer involving merely the reduction to free hydrogen of
the protons trapped by the basic Py species Žand of the
198 R. Toniolo et al.r Bioelectrochemistry 51 (2000) 193–200
Fig. 4. Cyclic voltammetric curves recorded at a scan rate of 0.1 V sy1
with a platinum electrode in 0.1 M TBAP acetonitrile solutions contain-
ing: Ža. 10y3 M PyHq; Žb. 10y3 M PyH 3q.
opposite oxidation in the backward peak., in agreement
with the following equilibrium:
PyHqq ey ° Py q 1r2H 2 Ž 6.
The occurrence of this process was confirmed by con-
trolled potential electrolysis experiments which showed
that one mol of electrons per mol of PyHq was required in
the reduction process and that the electrolyzed solutions
displayed a ill-defined anodic wave at about 1.7 V, par-
tially mingled with the solvent discharge Žsee Fig. 4a., also
exhibited by solutions of authentic Py samples. Moreover,
the starting voltammetric picture proper for PyHq was
completely restored by electrogenerating again protons in
these electrolyzed solutions in a molar ratio 1:1 in respect
to the initial concentration of PyHq.
As to PyHq 3 , which was prepared directly in acetonitrile
from equimolar amounts of PyH 2 and electrogenerated
protons, its voltammetric profile showed the cathodic peak
Žc 2 . accompanied by peaks Žc 3 . and Žc 4 ., as already
mentioned above Žsee Fig. 4b.. Peak Žc 2 . for PyHq 3,
however, exhibited a markedly irreversible bahaviour in
that it was not associated with an anodic peak located at
quite close potential values, but only with the anodic peak
Ža. proper for the oxidation of PyH 2 .
A satisfactory coincidence of peaks Žc 3 . and Žc 4 . was
found with those displayed by the parent protonated tetra-
hydropyridine PyHq 5 , so as to enable these peaks to be
attributed to this reduced species. As a matter of fact, this
comparison was made by using the protonated form of
commercially available tetrahydropyridine not bearing the
substituents in positions 2, 3, 5 and 6 Žsee Fig. 1., owing to
the unavailability of the substituted compound. However,
we feel that this comparison is reliable since the effect of
the specific alkyl substituents on peak potentials is ex-
pected to be nearly negligible.
The whole of these findings indicated unambiguously
on one hand that the reduction of the acid PyHq 3 leads to
the parent base PyH 2 , but on the other hand that this
reaction does not occur through the simple cathodic con-
version to free hydrogen of protons bound to PyH 2 . Con-
versely, the data presented so far can be rationalized by
admitting that the cathodic process involving PyHq 3 leads
to the partial hydrogenation of its organic ring at the
expense of protons released from other PyHq 3 moieties,
according to the following reaction Žnote that PyH 4 turned
out to be a stronger base than PyH 2 . :
3PyHq
3 q 2e
y
™ PyH q 2PyHq
5 2 Ž 7.
A rather similar reaction pathway for the reduction of
PyHq 3 was advanced previously even though it was based
on relatively poor experimental evidences w18x.
A confirmation for the occurrence of reaction Ž7. was
gained by controlled potential electrolysis carried out on
PyHq 3 solutions at potentials corresponding to peak c 2 .
Ž .
Only about 0.66 mol of electrons per mol of starting PyHq 3
were, in fact, required and voltammetric tests performed
on the electrolyzed solutions rose in evidence peaks Žc 3 .
and Žc 4 ., together with peak Ža., this last with a height
equal to about 2r3 of that measured for the starting PyH 2
before the electrogeneration of equimolar amounts of pro-
tons. When these electrolyses were repeated in the pres-
ence of electrogenerated free protons, again directly at
potentials corresponding to Žc 2 ., a progressive increase of
peaks Žc 3 . and Žc 4 . with the excess of protons, accompa-
nied by a concomitant progressive decrease of peak Ža.,
was observed in the electrolyzed solutions. Attempts of
gaining quantitative information from these experiments
failed due to their poor reproducibility. This fact can be
conceivably accounted for by considering that the poten-
tials at which free protons are reduced is less negative Žof
about 0.4 V. than that required to reduce PyHq 3 , so that
they are reduced at the working potential much more
rapidly than PyHq 3 . Consequently, an appreciable fraction
of free protons are expected to be wasted in their direct
reduction to hydrogen gas, instead of supporting the hydro-
genation of the DHP ring.
All these statements are also in full agreement with the
results obtained by direct electroreduction at Žc 2 . of the
solutions obtained at the end of exhaustive electrooxida-
 R. Toniolo et al.r Bioelectrochemistry 51 (2000) 193–200
tion of PyH 2 . In fact, these electroreductions required
about 1.7 mol of electrons per mol of starting PyH 2 when
performed on solutions resulting from electrochemical oxi-
dation of the most diluted PyH 2 samples Ž3 = 10y4 M..
At the same time, very low amounts of PyHq 5
Žpeaks c 3
and c 4 . and only traces of PyH 2 Žpeak a. were found at the
end of these electrolyses. This was because the electrooxi-
dation products prevailing in these diluted PyH 2 solutions
are Hq and PyHq Žnearly 2 mol per mol of starting PyH 2 ,
on the whole, see reaction pathways 1–3. while only very
moderate amounts of PyHq 3 are formed see above , thus
Ž .
accounting for the number of electrons involved approach-
ing a value of two.
Conversely, when these electroreductions, following
electrooxidations, were performed on solutions containing
increasing concentrations of PyH 2 , the number of elec-
trons required decreased progressively attaining a value of
about 0.8 for the most concentrated PyH 2 solutions inves-
tigated Ž5 = 10y3 M.. In particular, the electroreduced
solutions obtained in this last case displayed the presence
of quite high amounts of both PyHq 5 peaks c 3 and c 4 and
Ž .
PyH 2 Žpeak a, whose height was about 30% of that
exhibited by the starting PyH 2 .. This result confirms once
again the occurrence of reaction Ž7. because the electrooxi-
dation of concentrated PyH 2 is expected to lead to large
amounts of PyHq 3
Žabout 45% of the starting PyH 2 , see
reaction pathways 1–4. accompanied by PyHq Žabout
55%. and small amounts of free Hq. Consequently, the
protons required to enable reductive hydrogenations of the
DHP ring at peak Žc 2 . must be supplied extensively by the
electroactive PyHq 3 itself, thus leading to a marked de-
crease of the electrons spent in the overall reduction
process.
5. Conclusions
The results obtained in this investigation indicate unam-
biguously that the oxidation of PyH 2 occurs through two
consecutive one-electron releases, the latter requiring po-
tentials less positive than the former. Even though our data
do not permit a detailed quantification of the relevant
potential separation Žonly the inferior limit E1o y E2o G 180
mV could be inferred., this feature makes the DHP investi-
gated an anti-oxidant which differs markedly from anti-
oxidants such as vitamin E. In fact, the reaction of these
with an initiating strong oxidant leads to one-electron
oxidized products, which, although weaker oxidants, are
still able to react with some target Že.g., unsaturated lipids.,
thus providing new radicals in turn able to propagate the
chain reaction. Such a mechanism accounts for the
‘‘tocopherol mediated peroxydation’’ w29x, which, in these
terms, is not so paradoxical as it could appear. In other
words, anti-oxidants such as vitamin E are only able to
convert strong initiating oxidants Že.g., peroxy radicals.
into progressively weaker oxidants, whose oxidative ability
199
has to be discharged by further anti-oxidants characterized
by lower oxidation potentials Že.g., the ascorbate ion,
whose parent radical terminates the chain reaction by
dismutation w30x..
The oxidation of PyH 2 Žas well as conceivably of other
similar DHP’s. promoted by a strong oxidant, instead,
leads to the formation of a radical species ŽPyH P., which is
easily removed by further oxidation, being even a better
one-electron donor than the original precursor. Moreover,
the PyH 2 oxidation products ŽPyHq, Hq and PyHq 3
. are
almost unable to promote in turn the oxidation of some
species present in a biological system, they being all
protonated species, i.e. effectively inactivated towards oxi-
dation. In addition, just their protonation, implying a high
solubility in water, could make possible their easy removal
through the usual elimination mechanisms activated by
biological systems.
Finally, it is worth to note that the inactivation of PyH 2
towards oxidation, caused by its protonation, could be
pharmacologically relevant since the compound is some-
how protected when in the water phase, i.e. in the biologi-
cal liquids acting as carriers towards cellular membranes
where the drug is designed to display its anti-oxidant
activity. Conversely, it becomes fully active only in the
deeper lipofilic layer of membranes, where it concentrates
in the deprotonated form thanks to the high partitioning
coefficient w1x. On the other hand, this inactivation by
protonation could also account for the unexpectedly low
rate constant found in water for the reaction of PyH 2 with
peroxy radicals w13x.
Acknowledgements
The authors are indebted to Glaxo ŽVerona, Italy. for
financial support and supply of some chemicals. Also the
financial aid from the Italian National Research Council
ŽCNR. and from the Ministry of the University and of
Scientific and Technological Research ŽMURST. is grate-
fully acknowledged.
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