Study on the oxidation of C4-phenolic-1,4-dihydropyridines and its reactivity towards superoxide radical anion in dimethylsulfoxide

Article abstract of DOI:10.1016/j.electacta.2010.09.063

Electrochemical characterization on glassy carbon electrode (GCE) and reactivity with superoxide radical anion in aprotic medium of three new synthesized C4-phenolic-1, 4-dihydropyridines is reported. Voltammetry, coulometry, controlled-potential electrolysis (CPE), UV-vis spectroscopy, ¹H NMR techniques were employed for the characterization of title compounds. The oxidation mechanism involves initially an oxidation process on the phenol moiety with the formation of the corresponding quinone followed by a second one affecting the dihydropyridine ring to give the pyridine derivative. Both processes appeared irreversible in character. Cyclic voltammetry was used to generate O₂^- by reduction on GCE of molecular oxygen in DMSO. The reactivity of DHPs towards O₂^- was directly measured by the anodic current decay of the radical in the presence of increasing concentration of tested 1,4-dihydropyridines and compared with the reaction of the reference antioxidant, Trolox. The linear correlations obtained between the anodic current of O₂^- and compound concentrations in the range between 0.01 mM and 1.00 mM allowed the determination of both the DHP antioxidant index (AI) and the concentrations needed to consume 50% of O₂^-. Synthesized C4-phenolic 1,4-dihydropyridines exhibited significant scavenging capacity towards superoxide radical anion higher than Trolox and tested commercial 1,4-dihydropyridines.

Full text of DOI:10.1016/j.electacta.2010.09.063

Electrochimica Acta 56 (2010) 841–852
Contents lists available at ScienceDirect
Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta
Study on the oxidation of C4-phenolic-1,4-dihydropyridines and its reactivity
towards superoxide radical anion in dimethylsulfoxide
Ricardo Salazara, P.A. Navarrete-Encinab, J.A. Squellac, C. Barrientosb,
V. Pardo-Jiménezc, Luis J. Nún˜ez-Vergarac,∗
a Laboratorio de Electrocatálisis. Facultad de Química y Biología. Universidad de Santiago de Chile, Santiago, Chile
b Laboratorio de Sintesis Orgánica y Modelación Molecular, Santiago, Chile
c Laboratorio de Bioelectroquímica, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, PO Box 233, Santiago, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
Electrochemical characterization on glassy carbon electrode (GCE) and reactivity with superoxide radical
anion in aprotic medium of three new synthesized C4-phenolic-1, 4-dihydropyridines is reported.
Voltammetry, coulometry, controlled-potential electrolysis (CPE), UV–vis spectroscopy, 1H NMR tech-
niques were employed for the characterization of title compounds.
Received 24 May 2010
Received in revised form
16 September 2010
Accepted 18 September 2010
Available online 25 September 2010
The oxidation mechanism involves initially an oxidation process on the phenol moiety with the forma-
tion of the corresponding quinone followed by a second one affecting the dihydropyridine ring to give
the pyridine derivative. Both processes appeared irreversible in character.
Keywords:
Cyclic voltammetry was used to generate O2•− by reduction on GCE of molecular oxygen in DMSO. The
Polyhydroxyphenyl dihydropyridines
Electrochemical oxidation
Quinones
•−
reactivity of DHPs towards O2 was directly measured by the anodic current decay of the radical in the
presence of increasing concentration of tested 1,4-dihydropyridines and compared with the reaction of
•−
the reference antioxidant, Trolox. The linear correlations obtained between the anodic current of O2
Antioxidant index
Superoxide radical anion
and compound concentrations in the range between 0.01 mM and 1.00 mM allowed the determination of
both the DHP antioxidant index (AI) and the concentrations needed to consume 50% of O2•−. Synthesized
C4-phenolic 1,4-dihydropyridines exhibited significant scavenging capacity towards superoxide radical
anion higher than Trolox and tested commercial 1,4-dihydropyridines.
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
erable antioxidant activity in the stabilization of edible oils [14]
and exhibits synergistic properties with antioxidants, such as
1,4-Dihydropyridines (DHPs), an important class of drugs,
are potent blockers of calcium (Ca2+) currents through voltage-
dependent L-class Ca2+ channels. They induce relaxation of vascular
smooth muscle, preferentially in arterial beds, and display a nega-
tive inotropic effect on isolated cardiac muscle. In therapy, this class
of drugs is principally used in the treatment of cardiac arrhythmias,
peripheral vascular disorders, and hypertension [1–3].
Additionally, several in vitro findings reported that 1,4-DHP
calcium antagonists display antioxidant properties [4–9]. This
effect has not been clearly proved due to its Ca2+ channel
antagonist effect, but it would rather be related with the reac-
tivity of these structures towards different radical species [10].
Investigations developed in our laboratory [11–13] have been
conducted to clarify how 1,4-DHPs interact with or scavenge rad-
icals. In this same line of evidences, it has been reported that
the 2,6-dimethyl-3,5-diethoxycarbonyl-1,4-DHP possesses consid-
␣-tocopherol [15] and 2,6-bis(tert-butyl)-4-methylphenol (BHT)
[16].
Recently, synthesis, electrochemical characterization and reac-
tivity towards free radicals of new C4-monophenolic and
C4-methoxy-phenolic substituted 1,4-DHPs have been reported
by our laboratory [17,18]. Two redox centers coexisting in these
compounds show mutual significant interactions affecting both
the electrochemical responses and reactivity towards free radicals.
Based on these encouraging results, we decided to synthesize new
derivatives containing dihydroxyphenyl- and trihydroxyphenyl
groups at 4-position on the 1,4-dihydropyridine ring.
Phenols have been described to have greater antioxidant activity
than vitamins C and E. The mechanism of their action as antioxi-
dants seems to involve the ability of phenols to scavenge radicals
by an electron transfer process by which the phenol is converted
into a phenoxyl radical [19].
Electrochemical oxidation of 1,4-dihydropyridines in aprotic
medium have been extensively reported by us [20–24] and other
researchers [25–33]. Also, the electrochemical characterization of
compounds containing a phenolic moiety has been studied [32–36].
∗
Corresponding author. Tel.: +56 2 9782812; fax: +56 2 7371241.
E-mail address: lnunezv@ciq.uchile.cl (L.J. Nún˜ez-Vergara).
0013-4686/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.electacta.2010.09.063

842
R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852
HO
OH
O
O
O
O
O
O
O
O
N
H
4-Ph-DHP
N
H
3,5-OH-DHP
OH
OH
OH
HO
HO
O
O
O
O
O
O
O
O
N
H
N
H
3,4,5-OH-DHP
3,4-OH-DHP
OH
OH
HO
OH
HO
HO
OH
pyrocathecol
resorcin
pyrogallol
O
CH3
CH3
H3C
O
OH
HO
CH3
Trolox
Fig. 1. Chemical structures.
Furthermore, the electrochemical measurements have been used to
assess antioxidant abilities of these types of compounds [37,38].
This paper reports both the electrochemical oxidation of some
new C4-phenolic-1,4-dihydropyridines and its reactivity towards
electrogenerated superoxide radical anion (O2•−) in dimethylsu-
foxide (DMSO). Synthesized derivatives contain two aromatic ring
systems with a number of conjugated double bonds and redox reac-
tive NH and OH groups (Fig. 1). These features affect both the redox
behavior and its reactivity towards free radicals.
2,6-dimethyl-3,5-diethoxycarbonyl-4-phenyl-1,4-
dihydropyridine (4-Ph-DHP) was synthesized according to a
methodology previously reported [12,17].
3,4-OH-DHP, 3,5-OH-DHP and 3,4,5-OH-DHP were synthe-
sized by the following general procedure: 10 mmol ethyl
3-aminocrotonate and 5 mmol of the respective aldehyde
(3,4-dihydroxy-benzaldehyde for 3,4-OH-DHP derivative; 3,5-
dihydroxy-benzaldehyde for 3,5-OH-DHP derivative and 3,4,5-
trihydroxy-benzaldehyde for 3,4,5-OH-DHP derivative) were
dissolved in glacial acetic acid (30 mL) with vigorous stirring at
60 ◦C. After 6 h heating, water is added until precipitation of the
dihydropyridine compound. The respective solids were filtered off
and washed with ethanol/water (1:1). The compounds were recrys-
tallized twice from absolute ethanol.
2. Experimental
2.1. Compounds
2,6-dimethyl-3,5-diethoxycarbonyl-4-(3,4-
dihydroxyphenyl)-1,4-dihydropyridine (3,4-OH-DHP): Yield:
Chemical structures of synthesized compounds are shown in
Fig. 1.

R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852
843
32%. mp 195.0–197.0 ◦C. FT-IR ꢀmax (KBr) cm−1 3411.3, 1666.4,
1470.9, 1370.8, 1247.6, 1218.8, 1123.0, 1020.7 and 781.0. 1H NMR
ı (300 MHz; acetone-d6), 1.2 (6 H, t, J = 7.4, R–CO–O–CH2–CH3),
2.3 (6 H, s, R–CH3), 3.0 (1 H, s, R2–CH), 4.0 (4 H, q, J = 7.4,
R–CO–O–CH2–CH3), 4.9 (2 H, s, –OH), 6.6–7.6 (4H,m,Ar-H), 7.8
(1-H, s, N-H). 13C-NMR ı (75 MHz; acetone-d6) 13.8 (2 × s), 17.9
(2 × s), 38.6 (s), 58.9 (2 × s), 103.3 (2 × s), 114.4 (s), 115.1 (s), 119.1
(s), 140.5 (s), 144.3 (2 × s), 160.2 (s), 165.3 (s) and 205.5 (2 × s).
(from EtOH). Found: C, 62.95; H 6.40; N, 3.89. C19H23NO6 requires
C, 63.15; H, 6.41; N, 3.88%).
2.2.1. Electrolytic medium
DMSO containing 0.1 M tetrabutylammonium hexafluorophos-
phate (TBAFP6).
2.2.2. Controlled-potential electrolysis (CPE)
A
three electrode circuit with an (Ag/AgCl) as a refer-
ence electrode, platinum as counter-electrode and a reticulated
glassy carbon working electrode was used. Exhaustive electroly-
sis was carried out at the following potentials: Ep1 = 900 mV and
Ep2 = 1400 mV for 3,4-OH-DHP and 3,4,5-OH-DHP. The applied
potentials for 3,5-OH-DHP were Ep1 = 1050 mV and Ep2 = 1400 mV.
A BAS-CV 100 assembly was used to electrolyze the 1,4-DHPs
solutions. Net charge was calculated including correction for the
estimated background current.
2,6-dimethyl-3,5-diethoxycarbonyl-4-(3,5-
dihydroxyphenyl)-1,4-dihydropyridine (3,5-OH-DHP): Yield:
40%. mp 245.0–247.0 ◦C (from EtOH). FT-IR ꢀmax (KBr) cm−1 3403.7,
2979.6, 1672.2, 1599.0, 1473.1, 1373.3, 1221.2, 1156.5, 1004.2,
844.6, 800.3 and 619.5. 1H NMR ı (300 MHz; acetone-d6) 1.1 (6
H, t, J = 7.4, R–CO–O–CH2–CH3), 2.2 (6 H, s, R–CH3), 2.9 (1 H, s,
R2–CH), 3.9 (4 H, q, J = 7.4, R–CO–O–CH2–CH3), 4.8 (2 H, s, –OH),
6.0 (1 H, s, Ar-H), 6.2 (1 H, s, Ar-H), 7.7 (1 H, s, Ar-H), 7.8 (1-H, s,
N-H). 13C-NMR ı (75 MHz; acetone-d6) 13.8 (2 × s), 17.9 (2 × s),
39.1 (s), 59.0 (2 × s), 100.2 (2 × s), 102.8 (s), 106.2 (2 × s), 144.6
(s), 150.4 (2 × s), 158.0 (s), 167.2 (s) and 205.4 (2 × s). Found: C,
62.90; H 6.43; N, 3.89. C19H23NO6 requires C, 63.15; H, 6.41; N,
3.88%.
2.3. Characterization of oxidation products from 3,4-OH-DHP,
3,5-OH-DHP and 3,4,5-OH-DHP
All UV–vis spectra were recorded in the 200–1000 nm range by
using an Agilent spectrophotometer with diode array using 1 cm
quartz cell. A quartz crystal glass spectroelectrochemical cell (140A
CH Instrument, path length: 1 mm) of 0.1 mL with three electrodes,
a Pt gauze as a working electrode (CH Instrument 011547), a non
aqueous (Ag/AgCl) as reference electrode (CH Instrument 010249)
and a Pt as counter electrode (CH Instrument 011548) – were used.
2,6-dimethyl-3,5-diethoxycarbonyl-4-(3,4,5-
trihydroxyphenyl)-1,4-dihydropyridine (3,4,5-OH-DHP): (Yield:
35%. mp 245.0–247.0 ◦C (from EtOH). FT-IR ꢀmax (KBr)/cm−1
3400.5, 2960.2, 1682.2, 1660.2.0, 1654.5, 1449.9, 1374.5, 1228.1,
1159.4, 1054.5, 1033.5 and 783.7. 1H NMR ı (300 MHz; acetone-d6,
Me4Si) 1.1 (6 H, t, J = 7.3, R–CO–O–CH2–CH3), 2.2 (6 H, s, R–CH3),
4.3 (4 H, q, J = 7.3, R–CO–O–CH2–CH3), 4.7 (1 H, s, R2–CH), 6.2
(2 H, s, Ar-H), 7.1 (1 H, s, –OH), 7.6 (2 H, s, –OH), 7.9 (1-H, s,
N-H). 13C-NMR ı (75 MHz; acetone-d6) 17.0 (2 × s), 21.1 (2 × s),
41.7 (s), 62.0 (2 × s), 106.2 (2 × s), 109.9 (2 × s), 134.2 (s), 142.6
(2 × s), 146.5 (s), 148.4 (2 × s) and 170.56 (2 × s). Found: C,
60.28; H 6.15; N, 3.72. C19H23NO7 requires C, 60.47; H, 6.14; N,
3.71%)
(a) Spectroelectrochemical experiments of 0.1 mM and 0.05 mM
solutions of compounds were used to electrolyze the dihy-
dropyridine moiety and phenolic group and to characterize
the oxidation products produced during the electrolysis of
both electroactive groups. Applied potentials were the same as
described in Section 2.2.
(b) To assess the chemical nature of the oxidation products
corresponding to the phenolic moiety, 1 mM phenolic 1,4-
DHP solutions in the presence of 5 mM hydroxylamine were
electrolyzed. Reaction medium was a mixture of aqueous
0.1 M Britton–Robinson buffer pH 5.5/acetonitrile (70/30)
to assure the solubility of the compounds. Blank solutions
were prepared containing the phenolic 1,4-DHP derivatives at
1 mM concentration + 1 mM hydroxylamine in 0.1 M aqueous
Britton–Robinson buffer pH 5.5/acetonitrile (70/30). Time-
course of reactions was followed between 200 nm and 1000 nm
during 45 min for each compound. All experiments were con-
ducted at room temperature.
Commercial 1,4-dihydropyridines. Nifedipine: 1,4-dihydro-
2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic
acid dimethyl ester (Mintlab Laboratories, Santiago, Chile).
Nisoldipine:
(1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,
5-pyridine carboxylic acid methyl 2-methylpropyl ester) was
obtained from Sanitas Laboratories, Santiago, Chile. Nimodip-
ine:
(1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine
dicarboxylic acid 2-methoxyethyl-1-methylethyl ester) was
obtained from Saval Laboratories, Santiago, Chile. Amlodipine:
2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-
methyl-3,5-pyridine dicarboxylic 3-ethyl 5-methyl ester was
obtained from Chile Laboratories, Santiago, Chile.
Trolox, (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid) was purchased from Sigma–Aldrich and it was used as
received. A stock solution of Trolox was prepared in dimethylsul-
foxide (DMSO) at a concentration of 10 mM.
(c) Chemical oxidation of phenolic 1,4-DHP. 1 mM solution of each
phenolic 1,4-DHP was mixed with 0.1 M KIO4 solution in ace-
tonitrile/water (1:1) and further reaction with hydroxylamine.
Time-course of reactions was followed by UV–visible spec-
troscopy in a range of 200–1000 nm. Spectral characteristics
were compared with those obtained through the CPE experi-
ments.
2.4. Electrochemical experiments to assess the reactivity of
C4-substituted 1,4-DHPs with superoxide radical anion
2.2. Voltammetry
Cyclic voltammetric (CV) experiments were performed in DMSO
(Merck) with 0.1 M TBAPF6 purchased from Fluka. DMSO was dried
with 0.3 nm molecular sieves. Oxygen (99.8% pure) and nitrogen
(99.9% pure) were purchased from AGA (Santiago, Chile). All the
measurements were carried out in a BAS CV-100 W voltammetric
analyzer with a three electrode measuring cell. A glassy carbon disc
electrode with an area of 0.071 cm2, a platinum wire counter elec-
trode and an (Ag/AgCl/NaClsat) reference electrode were used for
the measurements. The glassy carbon disk working electrode was
polished using successively 0.3 ␮m and 0.05 ␮m alumina powder
on a polishing cloth before each measurement.
Differential pulse voltammetry (DPV) and cyclic voltammetry
(CV) were performed with a BAS-CV 100 assembly. All voltammet-
ric experiments were carried out with 1 mM solutions of 1,4-DHPs.
A stationary glassy carbon electrode (GCE) was used as working
electrode (0.07 cm2) for DPV and CV experiments. The surface of
the electrode was polished to a mirror finish with alumina pow-
der (0.3 ␮m and 0.05 ␮m) before use and after each measurement.
Platinum wire was used as auxiliary electrode and all poten-
tials were measured against an (Ag/AgCl) electrode in saturated
KCl.

844
R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852
Table 1
1212 mV. The oxidation peak potential values corresponding to
3,4,5-OH-DHP were 600 mV and 1200 mV (Fig. 2A, Table 1).
The electroactive groups responsible for these oxidation peaks
could be assigned by comparison with reference compounds based
on the following considerations: (a) 4-Ph-DHP derivative, having
no OH groups at all, experimented only the oxidation of the dihy-
dropyridine ring, which occurs at 1064 mV in the same electrolytic
media (Fig. 2B, Table 1) (b) pyrocatechol (1,2-dihydroxybenzene)
and resorcin (1,3-dihydroxybenzene) exhibited a single oxidation
signal at potentials of 792 mV and 1092 mV, respectively. (c) Pyro-
gallol (1,2,3-trihydroxybenzene) shows two oxidation signals at
692 mV and 928 mV. Considering that these compounds lack of
a dihydropyridine ring, the signals corresponded to the oxidation
of the OH groups (Fig. 2B, Table 1). Consequently, the oxidation
peaks in the Fig. 2A can be assigned as follows: peak I appear-
ing in the range 600–1000 mV belong to the oxidation of the OH
groups. Peak II correspond to the oxidation of the DHP ring. It can
be seen from Fig. 2A and B that the oxidation potential of 4-Ph-
DHP (Ep = 1064 mV) is shifted towards higher oxidation potential
values related to the hydroxyphenyl-substituted 1,4-DHP, which
can be explained considering the hydroxyphenyl moiety as an
electron-withdrawing group because its conversion to quinones
(3,4-OH-DHP and 3,4,5-OH-DHP).
The coexistence of two redox centers (i.e. phenolic and DHP
moieties) in the same molecule, produced significant changes in
the oxidation potential respect to the molecules wherein the cen-
ters are isolated (Fig. 2, Table 1). A shift toward lower oxidation
potential values for the phenolic groups in the C4-substituted 1,4-
DHPs was observed. In contrast, the oxidation of the DHP ring in
these compounds occurred at higher oxidation potential values.
The results above-summarized are consistent with the electron-
donating effect of the dihydropyridine ring on the OH groups
oxidation. In the case of 3,4-OH-DHP, the DHP ring increases the
electronic density of the OH group in p-position (4-position of
phenyl moiety), facilitating the oxidation of this group. This behav-
ior is consistent with previous results obtained by Michalkiewicz
and Skorupa [41] indicating that the first stage of the anodic oxi-
dation of 3,4-dihydroxyphenyl acetic acid (DOPAC), proceed on the
OH group at 4-position of the aromatic ring.
Oxidation peak potential values on glassy carbon electrode in aprotic medium
(DMSO + 0.1 M TBAPF6).
Compound
E vs. (Ag/AgCl) (mV)
Peak IOH
Peak IIOH
ꢁEp-OHa
PeakNH
ꢁEp-NHb
3,4-OH-DHP
3,5-OH-DHP
3,4,5-OH-DHP
4-Ph-DHP
Pyrocatechol
Resorcin
780
952
600
–
−12
−140
−92
–
–
–
1172
1212
1200
1064
–
+108
+148
+136
–
–
–
792
1092
692
–
–
Pyrogallol
928
–
–
a
Differences of oxidation peak potential values of OH group (peak I) with respect
to the phenol reference derivative (pyrocatechol, resorcin and pyrogallol).
b
Differences of oxidation peak potential values of 1,4-dihydropyridine ring with
respect to 4-Ph-DHP derivative.
In oxygen media, O2 gas was bubbled directly into the cell in
order to obtain a fixed concentration of oxygen, and during the
measurement, O2 gas was flushed over the cell solution. In order
to maintain fixed oxygen concentration in the measurement cell
an apparatus consisting of two flow-meters (Cole Palmer 316SS)
for oxygen and nitrogen, respectively, equipped with needle valves
were used. Knowing the oxygen solubility in DMSO [39] and by
establishing the oxygen and nitrogen flow rates, it was possi-
ble to determine the concentration of oxygen in the gas passing
through the measurement cell [40]. All cyclic voltammograms were
recorded at a constant temperature of 25 ◦C, the initial potential
was fixed at 0.0 V vs. (Ag/AgCl/NaClsat) and the scanning poten-
tial was reversed at −0.9 V vs. (Ag/AgCl/NaClsat). Concentration of
antioxidants varied between 0 and 50 mM.
3. Results and discussion
3.1. Electrochemical oxidation of 3,4-OH-DHP, 3,5-OH-DHP and
3,4,5-OH-DHP in DMSO + 0.1 M TBAFP6
3.1.1. Voltammetry
PV results revealed that compounds 3,5-OH-DHP and 3,4,5-OH-
DHP exhibited two well-defined anodic peaks. In Table 1 oxidation
peak potential values and ꢁEp of different derivatives in DMSO are
summarized.
The corresponding potential values for 3,4-OH-DHP were
780 mV and 1172 mV and for 3,5-OH-DHP were 952 mV and
This effect can be corroborated from Fig. 2A, where 3,4-OH-DHP
exhibits a lower oxidation potential of the OH group than 3,5-OH-
DHP derivative where the hydroxyl group are in meta-position with
respect to the DHP ring position (ꢁEp = 172 mV), in concordance
with studies that demonstrated that cathechol moiety is more eas-
II
A
B
I
II
4-Ph-DHP
3,4-OH-DHP
I
pyrocatechol
resorcin
II
3,5-OH-DHP
I
5μA
10μA
*
3,4,5-OH-DHP
pyrogallol
400
600
800
1000 1200 1400
400
600
800
1000 1200 1400
E vs (Ag / AgCl) / mV
E vs (Ag / AgCl)/ mV
Fig. 2. Dp voltammograms in DMSO + 0.1 M TBAFP6 of: (A) 1 mM solutions of 3,4-OH-DHP, 3,5-OH-DHP and 3,4,5-OH-DHP. (B) 1 mM 4-Ph-DHP, pyrocatechol, resorcin and
pyrogallol solutions.

R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852
845
II
A
I
3,4-OH-DHP
3,5-OH-DHP
10 μA
3,4,5-OH-DHP
0
200
400
600
800
1000
1200
1400
E vs (Ag / AgCl) / mV
B
I
I
3,4,5-OH-DHP
3,4-OH-DHP
10 μA
10 μA
800
-200
0
200
400
600
-200
0
200
400
600
800
E vs (Ag / AgCl)/mV
E vs (Ag / AgCl)/mV
Fig. 3. Cyclic voltammograms of 1 mM solutions of synthesized C4-phenolic 1,4-DHP in DMSO + 0.1 M TBAPF6 (A) Extended sweep (B) short sweep for peak I. Sweep rate:
0.1 V s−1
.
ily oxidizable than resorcin [34]. Moreover, the oxidation of the
OH group at 4-position on the 3,4,5-OH-DHP derivative is eas-
ier than that of 3,4-OH-DHP (ꢁEp = 180 mV), which is consistent
with the interaction between the hydroxyl in 4-position with both
hydroxyl groups at 3- and 5-position together with the electron-
donating effects of the DHP ring at 1-position. The interaction may
be hydrogen-bonding between the OH groups and also electronic
substituent effects on the hydroxyl group at 4-position. The final
result of these interactions may explain the oxidation easiness of
this derivative. In any case, the electrode oxidation at lower oxida-
tion potentials produces the substituted quinones as final products.
In conclusion, for 3,4-OH-DHP and 3,4,5-OH-DHP derivatives
the first step involves the oxidation of the OH group at 4-position of
aromatic ring generating a phenoxyl radical followed by the oxida-
tion of the second hydroxyl group at 3-position with the formation
of an ortho-quinone derivative [42,43], which could affect the sub-
sequent oxidation of the DHP ring. This fact produces a shift toward
higher oxidation potential values for the second anodic peak.
Cyclic voltammetric experiments were carried out at different
sweep rates ranging from 0.1 V s−1 up to 3 V s−1. Under these con-
ditions, two anodic signals were observed for three derivatives
(Fig. 3A). From cyclic voltammograms in the range of −1000 mV
to 1000 mV, reduction peaks at 185 mV for 3,4-OH-DHP and at
−85 mV for 3,4,5-OH-DHP, corresponding to the reduction of the
respective quinone (Fig. 3B) were found. Thus, these two deriva-
tives were oxidized through a quasi-reversible process. In the case
of 3,5-OH-DHP, no cathodic peak was found in the same range.
Therefore, this derivative is oxidized by an irreversible process.
Peak II corresponding to the oxidation of DHP ring (Fig. 3A) shows
no signal in the reverse scan for all compounds indicating that this
oxidation process is of irreversible character.
Plots of log (I/␮A) vs. log(v/V s−1) had slopes between 0.5 and 1.0,
indicating that the current is controlled by a mixed process (diffu-
sion and adsorption). On the other hand, oxidation potential values
were dependent on the sweep rates supporting the irreversible
character of the oxidation of the compounds in this medium.
To understand the mechanism of oxidation in more detail and
to determine the number of electrons transferred in the oxidation
process of synthesized 1,4-DHPs, controlled-potential electrolysis
(CPE) in DMSO + 0.1 M TBAFP6 were carried out. For all the syn-
thesized C4-phenolic-DHP, the first oxidation process an average
n value of 2.25 0.25 and an overall process with an average n
value of 4.55 0.30 were obtained. CPE of pyrocatechol, resorcinol,
pyrogallol and 4-Ph-DHP derivatives exhibited an average n value
of 2.15 0.15 electrons. In conclusion, the first oxidation process
comprises the transformation from the phenol to the respective
quinone, while the second value is compatible with the oxidation
of both the phenolic moiety and the dihydropyridine ring.
In conclusion, for 3,4-OH-DHP and 3,4,5-OH-DHP derivatives
the oxidation involves a first stage of anodic oxidation proceeding
on the OH group in position four of the aromatic ring and pro-
duces a phenoxyl radical. This radical is chemically unstable and
participates in the successive homogenous reaction. As a conse-
quence of this, only a small part of this product could takes part
in the second stage of the electrode reaction. This fast charge-
transfer step produces substituted ortho-quinone which undergoes
the cathodic reduction at the potential 185 mV for 3,4-OH-DHP
and −85 mV for 3,4,5-OH-DHP (Fig. 3B). This interpretation is

846
R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852
Fig. 4. (A) UV–visible spectra of the CPE of a 0.1 mM solution of 3,4-OH-DHP at an applied potential of 900 mV during 500 s. (B) UV–visible differential spectra of (A). (C)
UV–visible spectra of the CPE of electrolyzed solution (A) at an applied potential of 1200 mV during 500 s. (D) UV–visible differential spectra of the solution in (C).
consistent with previous reports on the oxidation of cathecols
[41,44–46].
(Fig. 5A). Clearly, these new maxima are related with the oxida-
tion product, i.e. the quinone derivative, which exhibits a similar
response to that shown in Fig. 4A and B for the electrolysis of 3,4-
OH-DHP at an applied potential of 900 mV.
3.2. Characterization of oxidation products from 3,4-OH-DHP,
3,5-OH-DHP and 3,4,5-OH-DHP
Furthermore, after the CPE of 4-Ph-DHP the most significant
changes on the UV–vis spectra were the decrease in the intensity of
the original UV–vis band at 354 nm (which is due to the oxidation
of the dihydropyridine nucleus) and the appearance of a new band
at 278 nm which corresponds to the oxidation product, i.e. the pyri-
dine derivative (Fig. 5C and D). The above UV assignments proving
the oxidation of phenolic and 1,4-DHP moieties were confirmed by
comparison with the spectroelectrochemical results obtained with
reference compounds having only one moiety in its structure.
To support quinone formation after the electrolysis at an
applied potential of 900 mV for 3,4-OH-DHP and 3,4,5-OH-DHP
and at applied potential of 1050 mV for 3,5-OH-DHP, we used the
well-known chemical characterization reaction of ketones with
hydroxylamine leading to oxime formation [49,50]. The idea behind
this characterization was to compare the chemically synthesized
DHP-oximes with the in situ electrochemically produced ones.
Therefore, we can compare the UV–vis spectra obtained after the
electrochemical oxidation of the derivatives in the presence of
5 mM hydroxylamine with the chemical oxidation of 1 mM phe-
nolic 1,4-DHP with 0.1 M KIO4 solution in acetonitrile/water (1:1)
in the presence of hydroxylamine to produce the oxime derivative.
In Fig. 6A, the time-course of CPE corresponding to 3,4,5-OH-
DHP after 15 min in the presence of hydroxylamine is shown. As
can be seen from this figure, two bands at 288 nm and 350 nm
3.2.1. Spectroelectrochemical experiments
3,4-OH-DHP shows two original UV–vis maxima absorption
bands near to 290 nm and 350 nm (Fig. 4A). CPE at 900 mV pro-
duce significant changes in the UV–vis differential spectra as are
shown in Fig. 4B. Three new bands at 272 nm, 312 nm and 406 nm
appeared. A decrease in the UV–vis zone at 365 nm was also found
which corresponds to the cathecol part of the molecule. This elec-
trolyzed solution was used for a second further electrolysis at a
controlled-potential of 1200 mV during 500 s (Fig. 4C). We can
observe the appearance of a new UV–vis band at 268 nm and the
significant decrease in the intensity of the original UV–vis band at
343 nm (Fig. 4D). From these spectral changes it can be concluded
that: (a) at an applied potential of 900 mV the maximum at 406 nm
supports the formation of the corresponding ortho-quinone deriva-
tive from 3,4-OH-DHP, which is consistent with a previous work of
Giacomelli et al. [47]. (b) At an applied potential of 1400 mV, the
new maximum at 268 nm accounts for pyridine formation accord-
ing to previous works of us [13,17,25] and others [48].
CPE of both pyrocatechol and 4-Ph-DHP were also carried out.
The original spectrum of pyrocatechol shows a single sharp UV–vis
band at 280 nm. The application of an oxidation potential at 900 mV
produced the appearance of maxima at 262 nm, 296 nm and 374 nm

R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852
847
Fig. 5. (A) UV–visible spectra of the CPE of a 0.5 mM pyrocatechol solution at an applied potential of 900 mV during 500 s. (B) UV–visible differential spectra of (A). (C)
UV–visible spectra of CPE of 0.1 mM 4-Ph-DHP solution at an applied potential of 1200 mV during 500 s. (D) UV–visible differential spectra of (C).
appeared supporting the formation of the corresponding oxime
as a consequence of the reaction between the quinone electro-
chemically generated and the hydroxylamine. Fig. 6B shows: (1)
the UV–vis spectrum for the electrochemical oxidation and (2) the
chemical oxidation with KIO4 for 3,4,5-OH-DHP compound, respec-
tively. As can be seen, both oxidation products exhibited a similar
UV–vis spectrum. A similar behavior was shown by 3,4-OH-DHP
and 3,5-OH-DHP derivatives. Two bands with maxima at 286 nm
and 356 nm were observed in the case of 3,4-OH-DHP derivative
either by electrochemical and chemical oxidation. For 3,5-OH-DHP
the absorbance maxima for the oxidation product were at 286 nm
and 355 nm. As expected, 4-Ph-DHP did not show any change in
the UV–vis spectrum at an applied potential of 900 mV either with
or without addition of hydroxylamine. Taking into account the
above results, in Scheme 1 the oxidation mechanism of C4-phenolic
synthesized compounds is depicted. Also, these results are in agree-
ment with previous observations [26,28,32–34].
3.3. Reactivity of 1,4-dihydropyridines towards superoxide
radical anion
3.3.1. NMR experiments. Assessment of the acid–base reaction
between DHPs derivatives and superoxide radical anion
In order to test the acid–base reactivity of protons belonging to
the –NH– part of the dihydropyridine ring and hydroxyl groups on
the phenyl ring of 1,4-DHP compounds towards superoxide anion,
their NMR spectra in DMSO-d6 in the absence and in the presence
of O2•− were recorded. The 1H NMR spectra of substituted DHPs in
A
3
2
1
0
B
6
2
5
4
3
2
1
1
2
1
0
250
300
350
400
450
500
550
250
300
350
400
450
500
550
λ / nm
λ / nm
Fig. 6. (A) Time-course of CPE 0.1 mM 3,4,5-OH-DHP solution at an applied potential of 900 mV in the presence of 1 mM of hydroxylamine in 0.1 M aqueous Britton–Robinson
buffer pH 5.5/acetonitrile (1/1). 1: 0 min, 2–6: 15–45 min. (B) 1: UV–visible spectrum of CPE of 0.1 mM 3,4,5-OH-DHP solution at an applied potential of 900 mV in the presence
of 1 mM of hydroxylamine at 15 min. 2: UV–vis spectrum for the reaction product between 1 mM 3,4,5-OH-DHP solution and 0.1 M KIO4 solution in a acetonitrile/water
solution (1:1) in the presence of 0.1 M of hydroxylamine after 15 min.

848
R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852
OH
O
OH
O
O
O
-2e, -2H+
900 mV
O
O
O
O
O
O
N
H
N
H
-2e, -2H+
1400 mV
1. KIO4, AcCN/H2O (1:1), 25 ºC, 20 min.
2. NH2OH
NH2OH
O
NOH
O
O
NOH
O
O
O
O
O
O
O
N
N
H
Scheme 1. Oxidation mechanism exemplified for 3,4-OH-DHP in DMSO and the trapping by hydroxylamine of the oxidized products corresponding to the phenolic moiety
either after the electrochemical or the chemical oxidations.
•−
DMSO-d6 show signals between 8.2 ppm and 8.6 ppm correspond-
ing to the hydroxyl and secondary amino groups of the molecules.
In Fig. 7A this behavior is represented for 3,4-OH-DHP derivative.
3.3.2. About the O2 scavenging by 1,4-dihydropyridines
As it was demonstratedin aprevious publication [13] the anionic
form of DHPs does not led to products and therefore O2•− scaveng-
ing is carried out by the DHP itself.
When we mix the C4-phenolic 1,4-DHP derivatives with an excess
•−
of O2
in DMSO-d6 an acid–base equilibrium is quickly estab-
Hydroxy phenyl DHPs have two redox centers (i.e. the hydrox-
yphenyl and DHP rings) that scavenge O2
mechanisms: (a) the interaction of O2
ring leading to quinone type of compounds (I) and (b) the oxida-
tion of the DHP ring to form pyridine derivatives (II) as is shown in
Scheme 2.
lished. After the addition of O2•−, the signals in the range (8.2–8.6)
ppm disappeared, evidencing anion formation (Fig. 7B). From these
results it can be concluded that the superoxide anion acts as a base
abstracting the proton at 1-position of the dihydropyridine ring and
also the hydroxyl group protons on the aromatic ring at C4-position.
by two different
•−
•−
and the hydroxyphenyl
C4-H
C4-H
A
B
Ar-OH
DHP-NH
9
8
7
ppm
6
5
9
8
7
ppm
6
5
•−
Fig. 7. 1H NMR for 3,4-OH-DHP derivative in the absence (A) and in the presence of O2 (B).

R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852
849
O
OH
O
OH
3 O2
+ 3 HOO
CO2Et
CO2Et
EtO2C
EtO2C
acid-base
equilibrium
N
N
H
scavenging by hydroxyphenyl
group.
2 O2
O
O
+ 2 HOO
CO2Et
(I)
EtO2C
N
H
scavenging by dihydropyridine
ring.
2 O2
O
O
(II)
CO2Et
EtO2C
N
Scheme 2. Mechanism involved in the scavenging activity of C4-phenolic 1,4-dihydropyridines.
30
20
30
A
O2-.
B
O2
20
10
0
Ipa
10
0
0
10 mA
-10
-20
-30
-40
-10
-20
-30
-40
O2-.
O2
-800
-700
-600
-500
-400
-300
-800 -700 -600 -500 -400 -300
E vs (Ag / AgCl)/mV
E vs (Ag / AgCl)/mV
Fig. 8. (A) Typical cyclic voltammogram of the electroreduction of O2 showing the electrode process and the parameters involved. (B) Cyclic voltamograms of the electrore-
duction of oxygen in absence (solid line) and in the presence (dashed line) of 1.0 mM of 3,4,5-OH-DHP.

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R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852
3,5-OH-DHP Trolox
3,4,5-OH-DHP
3,4-OH-DHP
Phenyl-DHP
Nimodipine
Nifedipine
Amlodipine
Nisoldipine
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
0.80
0.75
0.70
0.65
0.60
0.55
0.50
0.45
AI 50
AI 30
0.40
0.0
0.1
0.2
0.3
0.4
0.5
[1,4-DHP] / mM
0
10
20
30
40
50
0
5
10
15
20
25
30
[1,4-DHP] / mmol dm^-3
[1,4-DHP] / mmol dm^-3
Fig. 10. Dimensionless parameter related to the decrease of superoxide anodic cur-
rent, vs. the concentration of commercial 1,4-dihydropyridines.
Fig. 9. Dimensionless parameter related to the decrease of superoxide anodic cur-
rent, [Ip0a − IpDaHP]/Ip0a vs. the concentration of synthesized 1,4-dihydropyridines and
the reference antioxidant, Trolox. Inset: Typical linear dependence at a concentra-
tion level between 0.01 and 1 mM for 3,4,5-OH-DHP derivative.
ilar effect on the O2 reduction, i.e. a linear dependence between
the dimensionless parameter [Ip0a − IpDaHP]/Ip0a and the 1,4-DHP con-
centration range between 0.01 mM and 1.00 mM (inset in Fig. 9).
Exceeding this concentration level, linearity is lost. Calculation of
antioxidant index, AI50 (concentration of 1,4-DHP needed to con-
sume superoxide radical by a current decrease of 50% of the initial
anodic current) were carried out in the concentration linear range
(Fig. 10). The kinetics of the reaction between the 1,4-DHPs and
3.4. Reactivity of 1,4-DHPs towards electrogenerated superoxide
radical anion assessed by cyclic voltammetry
Cyclic voltammetry can be employed for the generation and
•−
detection of O2
oxygen. O2
by the electrochemical reduction of molecular
•−
is derived from the direct electro-reduction, in the
•−
O2 may be related to the slopes of straight lines obtained from
diffusion layer of glassy carbon electrodes, of molecular oxygen in
DMSO medium (Fig. 8A), without inclusion of any other compound.
The presence of the radical O2•− is easily detected by its anodic oxi-
dation current measured at the same electrode during the reverse
scan (Fig. 8A). It is known that O2•− radical is stable in aprotic media
and dismutation does not occur during the time scale of the cyclic
voltammetry in DMSO solution [51]. By using this procedure we
can instantly follow the reaction of O2•− with bioactive compounds
at the electrode surface in the oxygen reduction potential range.
In fact, several authors have•r−eported studies on the reactivity of
superoxide radical anion, O2 with 1,4-dihydropyridines [52,53],
flavonoids [37] and sulfur amino acids [54].
the graphics of [Ip0a − IpDaHP]/Ip0a vs. 1,4-DHP concentration (Inset,
Fig. 9, i.e. the biggest slope means the most active compound).
The corresponding slope values are shown in Table 2. As can be
seen, commercial 1,4-DHPs were significantly less reactive than
the C4-phenolic synthesized 1,4-DHPs and Trolox. 3,4,5-OH-DHP
and 3,4-OH-DHP were respectively, 2.45 and 2.10-fold more reac-
tive towards O2•− than Trolox. A similar trend for the AI50 values is
observed, i.e., 3,4,5-OH-DHP has an AI50 value that is 31-fold lower
than Trolox. In Table 2, it can be also observed that the 3,5-OH-DHP
is the less reactive hydroxyl derivative.
•−
The lower reactivity towards O2
exhibited by 3,5-OH-DHP
compared with the 3,4-OH-DHP derivative, is in agreement with
theoretical and experimental studies which document that 3,5-
dihydroxy phenyl group is significant less reactive than the
3,4-dihydroxy phenyl group due to differences in the phenolic O–H
bond dissociation enthalpies of the OH groups at position 3 and 5
in the phenyl with respect to the same OH at position 3 and 4. The
explanation given is related to the ability of losing the proton of the
hydroxyl group at 4-position of the phenyl group easier than the
proton of hydroxyl at 3-position [55].
In the present paper, the reactivity of some new synthesized
and commercial 1,4-DHPs towards superoxide radical anion, O2
•−
is assessed by a cyclic voltammetric methodology. From the exper-
imental point of view, single cyclic voltammograms in the absence
of 1,4-DHPs were recorded to determine the current value Ip0a, as the
•−
anodic peak current of O2 (Fig. 8A). This value is directly related
•−
to the O2 concentration at the electrode surface and depends on
the solubility of oxygen in DMSO and the experimental parameters
selected to record the cyclic voltammograms. The time-scale was
controlled by the scan rate at 0.1 V s−1 and the working potential
range was settled from 0 mV to −900 mV vs. (Ag/AgClsat). Cyclic
voltammograms of O2 reduction were recorded in the presence of
different concentrations of synthesized and commercial 1,4-DHPs
and the reference antioxidant, Trolox, to evaluate their antioxidant
index towards superoxide radical anion.
Table 2
Parameters for the regression equations for the synthesized and commercial 1,4-
dihydropyridines. .
Compound
Antioxidant index (AI)a
AI30 (mM)
AI50 (mM)
r2
The increase of 1,4-DHPs concentration leads to a decrease of
3,4,5-OH-DHP
3,4-OH-DHP
3,5-OH-DHP
4-Ph-DHP
Nifedipine
Nisoldipine
Nimodipine
Amlodipine
Trolox
0.689 0.076
0.583 0.024
0.114 0.019
0.112 0.017
0.213 0.022
0.188 0.020
0.222 0.020
0.209 0.019
0.281 0.036
–
0.11
0.81
3.78
50.00
5.13
5.58
4.63
5.35
3.41
0.995
0.993
0.997
0.996
0.996
0.994
0.997
0.998
0.999
O2 anodic peak current, IpDaHP, with no significant modification of
•−
0.49
2.00
0.30
1.92
1.86
2.04
2.04
0.78
the cathodic current (Fig. 8B).
So our observations suggest that the 1,4-DHP compounds
reacted with superoxide radical as H atom donors leading to OH-
DHP• radicaland theconjugatedbaseofhydrogen peroxide. Aseries
DHP
of I
values were determined from the cyclic voltammograms
pa
recorded at increasing concentrations of each tested compound. As
can be seen from Fig. 9, all the tested compounds exhibited a sim-
a
Average slopes obtained from five independent runs for each compound.

R. Salazar et al. / Electrochimica Acta 56 (2010) 841–852
851
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Trolox
3,5-OH-DHP
500
600
700
800
900
1000
1100
1200
1300
E vs (Ag/AgCl)/mV
Fig. 11. Relationship between antioxidant index and peak potential values for the oxidation of: OH group to quinone-type derivative (ꢀ) and 1,4-DHP ring to pyridine
derivative (ꢁ).
The examination of inhibitory concentration of 1,4-DHPs
(Table 2) reveals that 3,4-OH-DHP and 3,4,5-OH-DHP were the most
potent compounds exhibiting the lowest AI50 values, meaning that
they have significant antioxidant index towards superoxide radical.
In Fig. 11, the relationship between the antioxidant index (AI,
sponding dioxime derivative. The 1,4-dihydropyridine ring was
oxidized to the pyridine derivative, which exhibited the charac-
teristic UV–visible band with maxima at (270–278) nm.
All synthesized C4-polyphenolic 1,4-dihydropyridines were
more reactive than the tested commercial ones and even than the
reference antioxidant, Trolox, towards superoxide radical anion.
•−
measured as O2 consumption) and oxidation peak potential val-
•−
ues of phenolic DHPs, 1,4-DHP (with no substituents at phenyl ring)
and commercial DHPs are depicted. As can be seen, the synthesized
C4-phenolic DHPs and Trolox exhibit a nice dependence between AI
Scavenging of O2 by C4-phenolic 1,4-DHPs are related to the
existence of hydrogens at the N-position of the dihydropyridine
ring and hydroxyl groups on the phenyl ring according to NMR
experiments. It is well-known that the high reactivi•ty− of phenolic
compounds is based on the abstraction of H• by O2 [56] which
gives rise to the phenoxyl radical and the further formation of the
respective quinone plus HOO− [42]. On the other hand, the oxida-
tion of 1,4-dihydropyridine ring to the pyridine derivative support
the participation of this moiety in the scavenging of this type of
radicals.
•−
and Ep ox, i.e., the most active compound towards O2 has a lower
oxidation potential value. In contrast, commercial DHPs maintain a
constant relationship between AI and Ep ox. Low Ep ox values exhibit
comparatively high AI properties of the synthesized 3,4,5-OH and
3,4-OH-DHPs and are consistent with the oxidation easiness of the
phenolic part of the molecules to their respective quinones. 3,4,5-
OH-DHP may form the o-quinone easier than 3,4-OH-DHP while
the 3,5-OH-DHP does not form a quinonic compound demonstrat-
ing poor antioxidant index. Commercial DHPs and 4-Ph-DHP have
high Ep ox and low AI values and their antioxidant effects may be
attributed to the oxidation of DHP ring to the respective pyridine
derivative.
Acknowledgement
This work was supported by Grant from FONDECYT 1080132.
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