Reactivity of C4-indolyl substituted 1,4-dihydropyridines toward superoxide anion (O2O) in dimethylsulfoxide

Article abstract of DOI:10.1002/poc.1453

Reactivity of two new C4-indolyl substituted 1,4-dihydropyridines (1,4-DHPs) toward superoxide anion (O2-) in dimethylsulfoxide (DMSO) is reported. Reactivity was followed by electrochemical and spectroscopic techniques. Gas chromatography-mass spectrometry (GC-MS) was used to identify the final products of the reaction. C4 indolyl-substituted-1,4-DHPs reacted toward O₂O at significant rates, according to the calculated kinetic rate constants. Results are compared with 4-phenyl-DHP and the commercial 1,4-DHPs, nimodipine, nisoldipine, and amlodipine. Indolyl-substituted 1,4-DHPs were more reactive than the commercial derivatives. The direct participation of proton of the 1-position of the secondary amine in the quenching of O ₂ was demonstrated.

Full text of DOI:10.1002/poc.1453

Research Article
Received: 6 June 2008,
Revised: 11 August 2008,
Accepted: 30 August 2008,
Published online in Wiley InterScience: 4 November 2008
(
www.interscience.wiley.com) DOI 10.1002/poc.1453
Reactivity of C4-indolyl substituted
1,4-dihydropyridines toward superoxide
ꢀ
anion (O ) in dimethylsulfoxide
2
a
b
a
c
Ricardo Salazar , P. A. Navarrete-Encina , J. A. Squella , C. Camargo
a
*
and Luis J. N u´ n˜ ez-Vergara
ꢀ
Reactivity of two new C4-indolyl substituted 1,4-dihydropyridines (1,4-DHPs) toward superoxide anion (O2 ) in
dimethylsulfoxide (DMSO) is reported. Reactivity was followed by electrochemical and spectroscopic techniques. Gas
chromatography-mass spectrometry (GC–MS) was used to identify the final products of the reaction. C4 indolyl-
ꢀ
substituted-1,4-DHPs reacted toward O2 at significant rates, according to the calculated kinetic rate constants.
Results are compared with 4-phenyl-DHP and the commercial 1,4-DHPs, nimodipine, nisoldipine, and amlodipine.
Indolyl-substituted 1,4-DHPs were more reactive than the commercial derivatives. The direct participation of proton
ꢀ
of the 1-position of the secondary amine in the quenching of O2 was demonstrated. Copyright ß 2008 John Wiley &
Sons, Ltd.
Keywords: C4-indolyl substituted 1,4-dihydropyridines; superoxide radical anion; reactivity cyclic voltammetry; UV–Visible
spectroscopy; first-order kinetic rate constants
inclusion of the indole moiety as good choice for a second redox
INTRODUCTION
center in a 1,4-DHP molecule. The indole nucleus it has been
reported to possess a wide variety of important biological
Interest has recently grown in studying antioxidants, an
important class of biologically active compounds essential for
maintaining the so-called antioxidant status of the human body.
Their main function consists in diminishing the concentrations of
reactive oxygen species in the cells of living organisms. Oxidative
stress may arise in the human body as a result of the formation of
active intermediates of radical nature in the course of oxygen
reduction. Reactive oxygen species are the by-products of the
most important metabolic reactions involving molecular oxygen.
A variety of enzymatic and non-enzymatic biomolecules have
been reported to reduce molecular oxygen to superoxide
[
12]
[13]
properties such as anti-inflammatory,
antibacterial,
antic-
[
14]
[15]
onvulsant,
and antioxidant.
[
16]
Lavilla et al. have previously reported the design, synthesis,
and pharmacological evaluation of a series of 4-(3-indolyl) DHPs.
Also, these authors demonstrated that these derivatives show a
similar activity to that of nifedipine on the inhibition of radical
oxygen-derived species production. The antioxidant properties of
such DHPs was displayed regardless of their substitution pattern.
[
17–19]
Several authors
have recognized that 1,4-DHPs offer an
antioxidant protective effect that may contribute to their
pharmacological activity. Moreover, a direct reactivity between
some commercial 1,4-DHPs and superoxide radical anion based
on electrochemical methods was also previously reported by our
ꢀ
[1–3]
radicals, O2 .
This radical can be induced by electron transfer
reaction in vivo to generate other reactive oxygen species such as
hydroxyl free radical and hydrogen peroxide. The O2 is reactive
and toxic, causing oxidation of bio-macromolecules as well as
initiate radical-chain oxidation in tissues. In consequence,
compounds maintaining their original pharmacological activities
ꢀ
[
20,21]
laboratory.
ꢀ
but with another added ability, such as its reactivity toward O2 ,
are of major interest.
*
Correspondence to: L. J. N u´ n˜ ez-Vergara, Faculty of Chemical and Pharma-
ceutical Sciences, University of Chile, P.O. Box 233, Santiago, Chile.
E-mail: lnunezv@ciq.uchile.cl
1,4-DHP analogs of nifedipine, are of major significance in the
treatment of a number of cardiovascular diseases, including
angina, hypertension, peripheral vascular disorders, and a
number of arrhythmic conditions. The synthesis, the properties
of these drugs and the mechanisms by which they function have
a
R. Salazar, J. A. Squella, L. J. N u´ n˜ ez-Vergara
Laboratory of Bioelectrochemistry, University of Chile, Santiago, Chile
b
P. A. Navarrete-Encina
[
4–7]
been extensively reviewed during the past years.
In the last years our effort has been focused in the synthesis of
Laboratory of Organic Synthesis and Molecular Modeling, University of Chile,
Santiago, Chile
[
8–11]
new C4-substituted-1,4-DHPs
with strengthened antioxidant
c
C. Camargo
properties and the study of the interactions of different redox
centers coexisting in the DHP molecule. An example of this is the
Laboratory of Antidoping, Faculty of Chemical and Pharmaceutical Sciences,
University of Chile, Santiago, Chile
J. Phys. Org. Chem. 2009, 22 569–577
Copyright ß 2008 John Wiley & Sons, Ltd.

R. SALAZAR ET AL.
In this paper, a study on the reactivity of synthesized C4-indolyl
ꢀ
2ꢁ —CH ); 4.04 (q, 4H, 2ꢁ —O—CH —CH ); 4.99 (s, 1H,
3
2
3
substituted 1,4-DHP derivatives with superoxide anion (O2 ) in
Ar—CH<); 6.37 (s, 1H, Ar—H); 7.00 (d, 1H, J ¼ 7.92 Hz, 1ꢁ Ar—H);
7.25 (m, 3H, J ¼ 7,56 Hz, 3ꢁ Ar—H); 8.76 (s, 1H, DHP-N—H); 10.94
dimethylsulfoxide (DMSO) is reported. To account the reactivity,
spectroscopic, electrochemical, and chromatographic techniques
were used. Apparent first-order kinetic rate constant values were
calculated.
1
3
(s, 1H, Indole-N—H). C-NMR (75 MHz, DMSO d ): (2ꢁ)13.74;
6
(2ꢁ)17.96: 48.802; (2ꢁ)58.37; (2ꢁ)100.20; 102.53; 110.01; 117.72;
120.76; 124.49; 127.07; 134.08; 138.52; (2ꢁ)143.88; (2ꢁ)166.71.
Anal. Calcd for C21
H 6.85; N 7.38.
24 2 4
H N O : C 68.46; H 6.57; N 7.60. Found: C 67.00;
EXPERIMENTAL METHODS
2,6-Dimethyl-3,5-diethoxycarbonyl-4-phenyl-1,4-dihydropyridine
[4-phenyl-DHP]
C4-indolyl substituted 1,4-dihydropyridines (Fig. 1)
Ethylacetoacetate (15.0 mmol) and concentrated ammonia
hydroxide (10.0 mmol) were added to a mixture of 6 mmol of
indole 3-carboxaldehyde or indole 5-carboxyaldehyde in 20 ml of
ethyl alcohol.
2,6-Dimethyl-3,5-diethoxycarbonyl-4-phenyl-1,4-dihydropyridine
[4-phenyl-DHP] was synthesized according to a previous
paper.
[
6,7]
This mixture was heated under reflux for 30 h under nitrogen
atmosphere. The crude solid is filtered and recrystallized in ethyl
alcohol/water (50/50). Synthesized compounds had the following
characteristics.
Commercial 1,4-dihydropyridine
Amlodipine (99%): (2-[(2-aminoethoxy)methyl]-(2-chlorophenyl)-
1
,4-dihydro-6-methyl-3,5-pyridinedicarboxylic acid 3-ethyl 5-
methyl ester) was purchased from Sigma-Aldrich. Nisoldipine
97.8%): (1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine
(
carboxylic acid methyl 2-methylpropyl ester) was obtained from
Laboratorio Chile, Santiago, Chile. Nimodipine: 1,4-dihydro-2,
2
,6-Dimethyl-3,5-dietoxycarbonyl-4-(3-Indolyl)-1,4-dihydropyridine
(
4-(3-indolyl)-DHP)
6
-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid, 2-
Yield: 73%. m.p.:183–184 8C. IR (KBr): dmax 3344.4; 2978.1; 1676.7;
487.1; 1367.9; 1305.6; 1215.4; 1100.3; 1020.4; 807.2; 744.7.
methoxyethyl 1-methylethyl ester (Laboratorio Saval, Santiago,
Chile).
1
1
H-NMR (300 MHz, DMSO d ): d
1.18 (t, 6H, 2ꢁ —CH —CH );
2 3
6
max
2
3
2
.31 (s, 6H, 2ꢁ —CH ); 4.03 (q, 4H, 2ꢁ —O—CH —CH ); 5.23 (s,
3
UV–Vis spectrophotometry
1
H, Ar—CH<); 6.975 (m, 3H, J ¼ 7,83 Hz, 3ꢁ Ar—H); 7.33 (d, 2H,
J ¼ 7.92 Hz, 2ꢁ Ar—H); 7.92 (d, 2H, J ¼ 7.92 Hz, 2ꢁ Ar—H); 8.89 (s,
UV–Vis spectra were recorded in the 200–1000 nm range by
using a diode array detector Agilent spectrophotometer.
1
3
1
H, DHP-N—H); 10.74 (s, 1H, Indole-N—H). C-NMR (75 MHz,
DMSO d
): (2ꢁ)13.70; (2ꢁ)17.64: 29.98; (2ꢁ)58.25; (2ꢁ)101.43;
10.85; 117.54; 118.91; 119.77; 121.31; 122.17; 125.25; 135.54;
6
1
Molar absorptivity
(
6
2ꢁ)143.58; (2ꢁ)166.71. Anal. Calcd. for C H N O : C 68.46; H
2
1 24 2 4
.57; N 7.60. Found: C 67.89; H 6.71; N 7.61.
This parameter was calculated from 30–200 mM 1,4-DHP
solutions in the absence and with an excess of tetrabutylammo-
nium hydroxide (TBA-OH) in DMSO.
2
,6-Dimethyl-3,5-dietoxycarbonyl-4-(5-Indolyl)-1,4-dihydropyridine
Stock superoxide anion solution. It was prepared by weighing
(
4-(5-indolyl)-DHP)
0
.071 g (1 mmol) of potassium superoxide (Sigma-Aldrich, USA),
Yield: 61%. m.p.:178–180 8C. IR (KBr): dmax 3351.2; 2978.5; 1657.6;
dissolving it in DMSO (spectroscopic grade) and sonicating
during 0.5–3 h. From the stock solution stored at ꢂ40 8C were
prepared individual solutions to study the reactivity with selected
1
1481.6; 1371.4; 1303.5; 1208.2; 1100.5; 1018.2; 727.0. H-NMR
(
300 MHz, DMSO d ): d
1.20 (t, 6H, 2ꢁ —CH —CH ); 2.32 (s, 6H,
6
max
2
3
Figure 1. Chemical Structure of derivatives.
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Copyright ß 2008 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2009, 22 569–577

REACTIVITY OF C4-INDOLYL SUBSTITUTED 1,4-DHP
1,4-DHPs. Stability studies revealed that the stock solution
Equipment and operation conditions
remains unalterable for at least 2 weeks.
Kinetic experiments were conducted in a pseudo-first order
kinetic condition by using a 0.1 mM 1,4-DHP solution and a
HPLC measurement were carried out by using a Waters assembly
equipped with a model 600 controller pump and a model 996
photodiode array (PDA) detector. The acquisition and treatment
of data were made by means of the Millennium version 2.1
20 mM potassium superoxide salt. Measurements were pro-
grammed at intervals of 10 min during 5 h.
software. As chromatographic column a Kromasil C-18 column
2
(
4.6 ꢁ 150 mm ) was used. As precolumn a C-18 Bondapak
2
NMR spectroscopy
precolumn (30 ꢁ 4.6 mm ) was employed. The injector was a
2
0 ml Rheodyne valve. An aliquot of the electrolyzed solution and
The H-NMR spectra were recorded on a Bruker spectrometer
advance DRX 300.
the result solutions of 5 h of reaction toward superoxide radical
anion of each 1,4-DHPs was taken and a 20 ml volume of these
solutions were injected into the chromatographic system. A
mixture of acetonitrile/0.05 M phosphate buffer (55/45) at pH 4.3
was used as mobile phase. The PDA detector operated at 250 nm
Electrochemical experiments
Electrolytic medium was DMSO spectroscopic grade purchased
from Merck containing 0.1 M tetrabutylammonium hexafluoro
phosphate (TBAHFP, Sigma-Aldrich).
(
product) and 360 nm (1,4-DHPs) for quantification. The flow of
mobile phase was maintained at 1 ml/min and a helium bubbling
of 30 ml/min was applied to remove dissolved gases.
2
A stationary glassy carbon electrode (0.071 cm ) was used as
working electrode for cyclic voltammetry (CV) and differential
pulse voltammetry (DPV) techniques. The surface of the
electrode was polished to a mirror finish with alumina powder
RESULTS AND DISCUSSION
(
0.3–0.05 mm) before use and after each measurement. Platinum
wire was used as auxiliary electrode and all potentials were
measured against an Ag/AgCl electrode in saturated KCl. All cyclic
voltammograms were carried out at a constant temperature of
Reactivity of C4-substituted 1,4-dihydropyridines with
superoxide radical anion assessed by voltammetric
techniques
2
5 8C. The return-to-forward peak current ratio, Ipa/Ipc, for the
Cyclic voltammetry
oxygen/superoxide couple was measured for each cyclic
voltammogram at 0.1 Vs according to the procedure described
ꢂ1
[23]
In a previous paper the optimal conditions for generating and
[
22]
by Nicholson
0–50 mM).
CV and DPV were performed with a BAS-CV 100 assembly and
the routine 1,4-DHP concentration was 1 mM. For measurements
in oxygen media, O gas was bubbled directly into the cell in
order to obtain solutions 1.0 mM O2 in DMSO, and during
the measurement, O gas was flushed over the cell solution using
by using different concentrations of 1,4-DHPs
studying the superoxide redox couple were reported. Moreover,
this couple was used to reveal an interaction between superoxide
with a commercial 1,4-DHP. In the present paper, we have used
such methodology to assess the reactivity of synthesized
1,4-DHPs with superoxide. Thus, we have examined the cyclic
voltammetric response of O /O redox couple on a glassy
carbon electrode in the absence and in the presence of 1,4-DHP
derivatives as shown in Fig. 2. In this Figure the cyclic
(
2
ꢀ
2
2
2
an apparatus consisting of two flow-meters (Cole Palmer 316SS)
for oxygen and nitrogen, respectively, equipped with needle
valves.
Controlled-potential electrolysis (CPE)
Studies on exhaustive electrolysis were carried out at constant
electrode potential (1100 mV) on glassy carbon mesh electrode
during 2 h continuously stirred in a 30 ml cell capacity at 1 mM
solutions. A three-electrode circuit with a reference electrode Ag/
AgCl and platinum wire as counter electrode were used. A BAS-CV
100 assembly was also used to electrolyze the 1,4-DHPs solutions.
GC–MS
A gas chromatograph/mass selective detector (5890/5972)
combination (Hewlett-Packard, Palo Alto, CA, USA) and a
Hewlett-Packard 7673 autosampler were used for the analyses.
The m/z range monitored was 45–550 with a scan rate of 1 scan/s;
the normal energy electron was set at 70 eV. A Hewlett-Packard
Ultra-1 column, 25 cm, ꢂ0.2 mm i.d., ꢂ0.11 mm film thickness
(
Little Falls, Wilmington, DE, USA), was used.
:
Figure 2. Cyclic voltammograms corresponding to the O
couple: (A)
þ30 mM; and (E) þ50 mM of 4-(5-indolyl)-DHP. Sweep rate: 0.1 Vs
Electrolytic media: DMSO þ 0.1 M de TBAHFP. Insert: Ipa/Ipc ratio of
2
/O :redox
2
HPLC
O
2
saturated solution; (B) þ1 mM; (C) þ10 mM; (D)
ꢂ1
.
This technique was used to analyze 1,4-DHP solutions and
products generated after the reaction with superoxide anion and
the electrolysis (CPE).
ꢀ
2
O /O redox couple in the presence of different concentrations of
2
4-(3-indolyl)-DHP
J. Phys. Org. Chem. 2009, 22 569–577
Copyright ß 2008 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/poc

R. SALAZAR ET AL.
voltammetric response at four concentrations of 4-(5-indolyl)-
DHP in oxygenated DMSO þ 0.1 M TBAHFP solutions is displayed.
As can be seen, after the addition of 4-(5-indolyl)-DHP solution,
ꢀ
the oxidation peak current of O2 (anodic current (Ipa), oxygen
regeneration) decreases whereas the reduction current (cathodic
ꢀ
current (Ipc), O2 formation) increases. On the other hand, in the
insert of Fig. 2 the effect of increasing concentrations of
4
-(5-indolyl)-DHP derivative on the Ipa/Ipc ratio is displayed.
ꢀ
These data support that 4-(5-indolyl)-DHP reacts with O2 , that is,
it scavenges O2 in DMSO, in a concentration-dependent way.
ꢀ
Considering that the direct interaction between the in situ
generated superoxide and the tested compounds is connected
with the decrease of Ipa/Ipc ratio, we have used this ratio as a
reactivity criterion. Thus, we have extended the study to other
molecules with comparative purposes. Figure 3 shows the
variation of Ipa/Ipc ratio as a function of the concentration of the
synthesized 1,4-DHPs and commercial 1,4-DHPs (nisoldipine,
amlodipine, and nisoldipine). In quantitative terms, 4-(5-indolyl)-
DHP produced a decrease of 20% on the current ratio at the same
concentration compared with the commercial ones.
Figure 4. Differential pulse voltammograms of: (A) 1 mM 4-(5-indolyl)-
DHP (B) 1 mM 4-(5-indolyl)-DHP þ 1.5 mM de KO . DMSO þ 0.1 M TBAHFP
2
Consistent with our results, other authors have reported that
[
24]
the hydrogen at the N-position of indole compounds also acts
ꢀ
as a proton donor to O2 .
These results substantiate that both indole and DHP moieties
would contribute to the reactivity toward superoxide anion. Also,
no significant differences in the reactivity between indolyl-
substituted 1,4-DHPs were found. In all the cases, the results also
support that superoxide radical anion reacted with the
derivatives in a concentration-dependent trend.
would correspond to the electrochemical oxidation of the DHP
anion (R—N ) generated by the action of superoxide radical
S
anion, which abstracted the proton of the 1-position. On the
other hand, peak II suffered a little shift toward more anodic
potential values, that is, 1300 mV (Fig. 4B). Current intensity of this
peak II did not change under these experimental conditions.
Similar experiments but using an organic base such as, TBA-OH
instead of superoxide anion were performed. Results revealed
that changes affected the intensity of the anodic signal of the
DHP ring and the appearance of the signal corresponding to the
DHP anion.
Differential pulse voltammetry (DPV)
In these experiments the voltammetric response of 1,4-DHPs on a
glassy carbon electrode both in the absence and in the presence
of a 1.5 mM potassium superoxide salt solution was observed.
C4-indolyl-substituted 1,4-DHPs exhibited two anodic voltam-
metric signals. As can be seen in Fig. 4A, 4-(5-indolyl)-DHP
derivative exhibited a peak I at 992 mV which correspond to the
oxidation of the DHP ring. The second peak II due to the oxidation
of the indole moiety appeared at 1288 mV. After the addition of
the superoxide, peak I completely disappeared (Fig. 4B), but a
new peak III at approximately 600 mV appeared. This peak III
In conclusion from different voltammetric experiments the
interaction between the 1,4-DHP derivatives and superoxide
anion has been documented. CV and DPV experiments required
quite different conditions and therefore this could explain the
apparent discrepancy in terms of the concentrations at which the
ꢀ
effects were achieved. Thus, in the CV experiments, the O2 is
electrochemically generated in situ on the electrode surface from
oxygen-saturated solutions, therefore only the DHP molecules
reaching the electrode surface will be able to modify the anodic
current corresponding to the superoxide anion. This could
explain the high concentration required to observe changes in
2
the current. In the VPD experiments, the superoxide anion (KO ) is
added to the solution together with the 1,4-DHP derivative.
Accordingly, DHP-anion is the species that reaches the electrode
surface and it is subsequently oxidized to pyridine. Consequently,
a lower concentration is required to observe changes in the
voltammograms.
NMR studies
6
NMR spectra of 1,4-DHPs in d -DMSO in the absence and in the
presence of KO were recorded. In Fig. 5A, the extended NMR
2
spectrum corresponding to 4-(3-indolyl)-DHP derivative is shown.
It can be observed the signal at 8.8 ppm accounting for the
proton of the N1-position of the secondary amine of DHP ring
and the signal at 10.7 ppm corresponding to the proton of the
indole moiety. These signals disappeared in the presence of an
ꢀ
Figure 3. Ipa/Ipc ratio of O
2
/O2 redox couple in the presence of
different concentrations of: 4-(3-indolyl)-DHP, 4-(5-indolyl)-DHP,
4
-phenyl-DHP, nimodipine, nisoldipine, and amlodipine. Sweep rate:
.1 Vsꢂ1. Electrolytic media: DMSO þ 0.1 M de TBAHFP
0
2
excess of KO (Fig. 5B). On the other hand, the signal at 5.2 ppm
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Copyright ß 2008 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2009, 22 569–577

REACTIVITY OF C4-INDOLYL SUBSTITUTED 1,4-DHP
Figure 6. UV–Vis spectra of (A) 0.1 mM 4-(5-indolyl)-DHP; and (B) 0.1 mM
-(5-indolyl)-DHP þ 20 mM KO solution in DMSO
4
2
1
,4-DHP solutions in DMSO at 37 8C). Time-course of the reaction
1
Figure 5. Extended HNMR spectrum in d
6
DMSO of 5 mM 4-(3-indolyl)-
revealed that the intensity of the UV–Vis band corresponding to
the DHP anion (l ¼ 440–450 nm) significantly decreased. This
effect is illustrated for 4-(5-indolyl)-DHP in Fig. 7A. UV–Vis spectra
exhibited two isosbestic points at l ¼ 279 and 396 nm,
respectively. This characteristic supports that there are two main
2 2
DHP: (A) Absence KO (B) 20 mM KO
corresponding to the proton of C-4 position of the DHP ring
remains unaltered. So, we can conclude that the superoxide acts
as a base abstracting the protons at 1-positions of both DHP ring
and the indole moiety. This was fully proved by H-NMR spectra
where the signals corresponding to the N—H group protons
disappeared and a new signal corresponding to the formation of
[
27]
species during the reaction.
As can be seen in Fig. 7B for
4-(5-indolyl)-DHP, the absorption at 446 nm clearly decreased
while the absorption at 308 nm increased. Analyses of the
reaction products by gas chromatography-mass spectrometry
(GC–MS) support the oxidation of the 1,4-DHP ring. Fragments
with m/z 366, m/z 275, m/z 245, and m/z 321 containing the
pyridine ring sustain the formation of this derivative as the final
product of the reaction (Fig. 8). This main product of the reaction
between superoxide and 1.4-DHPs absorbs in the region of
1
:
2
the HO moiety appeared at 6.8 ppm in all of the spectra of the
,4-DHP derivatives. pKa values of 1,4-DHPs and indole are 19.0
[
25]
1
[
26]
and 21.0,
respectively. In consequence, the first step of the
interaction between superoxide and the derivatives correspond
to the abstraction of both protons at 1-position on the DHP ring
and indole in the experimental conditions here used.
[
6]
310 nm. In this zone, superoxide also exhibits a wide absorption
band close to 300 nm, which interferes with the absorption of
pyridine derivative.
Taking into account the above-summarized experimental facts,
we decide the use of a methodology which was based in the
absorptivity changes in the region l ¼ 440–450 nm to determine
the apparent kinetic rate constants for the reaction between
1,4-DHPs and superoxide.
Reactivity of C4-indolyl substituted 1,4-DHP toward
superoxide anion assessed by UV–Visible spectroscopy
Indolyl-substituted 1,4-DHPs exhibited a main UV–Vis band in the
zone at l ¼ 360 nm, which in the presence of superoxide anion
potassium salt disappeared together with the appearance of a
new intense UV–Vis band close to 450 nm (Fig. 6). This latter band
is due to UV–Vis. absorption of the anionic species generated by
the addition of the superoxide anion. To test the above results, we
also use TBA-OH as a base. Results of these experiments were
similar.
Table 1. Molar absorptivities corresponding to parent
1,4-DHP derivatives and their anionic species
Molar absorptivity coefficients/
Considering the above results, anion molar absorptivities
ꢂ1
ꢂ1
M
cm
(
eAnion) corresponding to all 1,4-DHP derivatives were determined
in the region of l ¼ 450 nm by using an excess of TBA-OH. Then,
Anion values were used to calculate the DHP anion concentration
Derivative
l ¼ 350–360 nm
l ¼ 445–460 nm
e
involved in the reaction through the calibration curve method as
is described in Section ‘‘Experimental Method.’’ Results from these
experiments are summarized in Table 1. As expected molar
absorptivities corresponding to the DHP anions were significantly
higher than that of parent derivatives.
4
4
4
-(3-indolyl)-DHP
-(5-indolyl)-DHP
-phenyl-DHP
10 161 ꢃ 16
8 966 ꢃ 20
6 900 ꢃ 14
5 992 ꢃ 8
3 332 ꢃ 5
6 612 ꢃ 9
16 674 ꢃ 22
15 968 ꢃ 28
12 625 ꢃ 16
11 243 ꢃ 10
9 976 ꢃ 10
9 983 ꢃ 9
Amlodipine
Nisoldipine
Nimodipine
Now, kinetic studies were carried out under a pseudo
first-order kinetic (20 mM potassium superoxide and 0.1 mM
J. Phys. Org. Chem. 2009, 22 569–577
Copyright ß 2008 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/poc

R. SALAZAR ET AL.
Figure 8. Ion chromatogram and mass spectrum of 0.1 mM
2
4-(3-indolyl)-DHP in the presence 20 mM KO solution after 5 h reaction
ꢀ
the [O2 ] is in very large excess over (DHP) then we may consider
it as nearly constant.
d½pyiridineꢄ
0
0
ꢀ
m
Therefore,
assuming first-order condition.
=
¼ k ½DHPꢄ, where k ¼ k
3
[O2 ]
dt
However, if the DHP concentration is low, the steady-state
approximation will be useful.
d½DHPꢄ
ꢂ
0
¼
k1 ½DHP ꢄ ꢂ k2 ½DHPꢄ ꢂ k ½DHPꢄ ¼ 0
dt
therefore
and
ꢂ
k1½DHP ꢄ
½
DHPꢄ ¼
0
ðk2 þ k Þ
Figure 7. (A) Time-course of the reaction between 0.1 mM
-(5-indolyl)-DHP and 20 mM KO solutions in DMSO at 37 8C followed
by UV–Vis spectroscopy. (B) Differential UV–Vis spectra
4
2
ꢂ
0
ꢂ
ꢂd½DHP ꢄ d½pyridineꢄ ꢂk k1 ½DHP ꢄ
¼
¼
0
dt
dt
ðk2 þ k Þ
0
k k1
0
0
k ¼
ꢀ
ðk2 þ k0Þ
In this reaction, the O2 acts as a base as well as an oxidant.
ꢀ
When we add O2 to DHP solution a first fast acid–base
ꢂ
ꢂd½DHP ꢄ
0
0
ꢂ
equilibrium is established, leaving small quantity of DHP in its free
form. When we add a strong base such as TBA-OH to a DHP
solution in at least equimolar quantitites we convert DHP to
¼ ꢂk ½DHP ꢄ
dt
ꢂ
d½DHP ꢄ
00
k t
¼
ꢂ
ꢀ
ꢂ
DHP . However, if O2 is added to this solution, no conversion to
pyridine occurred. This experiment demonstrates that the anionic
½DHP ꢄ
Integrating:
ꢀ
form of DHP does not react with O2 in an oxidative process that
ꢂ
00
ꢂ 0
would lead to pyridine. Therefore, it is the DHP moiety that
experiments the oxidation process. Therefore, the following
kinetic treatment is proposed for the overall reaction when a
ln½DHP ꢄ ¼ ꢂk t þ ln½DHP ꢄ
ꢂ
As can we seen in Fig. 9, a plot of ln [DHP ] versus time is linear
00
with a slope equal to k . The linearity of the plot (r ¼ 0.9999)
ꢀ
large amount of O2 (at least 200-fold greater than (DHP)) is
supports the original assumption of pseudo first-order kinetic for
added.
ꢂ
00
ꢂ
the reaction. Also, from equation ln [DHP ] ¼ ꢂk t þ ln [DHP ]
0
From the following expressions is possible to determine the
apparent kinetic rate constants:
00
the apparent pseudo first-order kinetic rate constants, k can be
calculated.
acid ꢂ base equilibrium : DHP ꢂ DHP
oxidative process : DHP þ O2ꢀ ꢂ! pyridine
Kinetic experiments
k3
ꢂ
In Fig. 9, a typical linear plot between ln [DHP ] versus time for
d½pyiridineꢄ
4-(5-indolyl)-DHP is shown. All the derivatives exhibited a linear
dependence. Calculated apparent kinetic rate constant values are
n
ꢀ m
¼
k3 ½DHPꢄ ½O2 ꢄ
dt
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Copyright ß 2008 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2009, 22 569–577

REACTIVITY OF C4-INDOLYL SUBSTITUTED 1,4-DHP
ꢂ
Figure 9. Typical plot of ln [DHP ] versus time for the reaction between
2
0.1 mM 4-(5-indolyl)-DHP and 20 mM KO solutions
Scheme 1. Proposed mechanism of reaction between C4-indolyl
,4-dihydropyridines with superoxide anion
1
presented in Table 2. As can be seen C4-indolyl-substituted
,4-DHPs were the most reactive derivatives. Thus, 4-(3-indolyl)-
1
DHP exhibited a kinetic rate constant that is 16.1- and 15-fold
higher than those commercial 1,4-DHPs, nisoldipine, and
amlodipine, respectively. Also, indolyl-substituted 1,4-DHPs were
more reactive than the 4-phenyl-DHP derivative. From these
results it can be concluded that the inclusion of an indole moiety
substituting C-4 position of the DHP ring produced a significant
increase in the reactivity of the 1,4-DHP. The bigger reactivity of
HPLC
Time-course of the reaction between 1,4-DHP derivatives
and KO was also followed by HPLC technique. As can be seen
2
in the Fig. 10, the parent 4-(5-indolyl)-DHP at 0.1 mM concen-
tration exhibited a single chromatographic signal (Fig. 10a) with a
retention time of 5.48 min in the experimental conditions. The
2
addition of the free radical KO , at a 20 mM concentration
4-(3-indolyl)-DHP derivative may be is due to the instability of the
produced the appearance of a new signal at 5.83 min, which
corresponds to the pyridine formation. Time-course of reaction
was followed during 5 h with 1 hour intervals (Fig. 10a–f).
Oxidation of 4-(5-indolyl)-DHP by superoxide to pyridine was
finished at 5 h (Fig. 10f). To compare the chromatographic
characteristics of the oxidized products, reaction solutions
with KO and those electrolyzed at 1100 mV, were analyzed.
2
Analysis by HPLC of these solutions revealed similar chromato-
graphic characteristics (Table 3). As can be seen from this Table,
radical generated at the DHP ring (dihydropyridyl radical)
compared with the radical formed in the 4-(5-indolyl)-DHP.
In the following Scheme 1 we propose a logical mechanism for
the oxidation of 1,4-DHP free form to pyridine, which was
identified by GC–MS as the product of the reaction. Also in
[
8]
previous paper,
we have identified a C-centered radical
(
dihydropyiridyl radical) by electron spin resonance (ESR) after
exhaustive oxidation by means of controlled-potential electro-
lysis (CPE). This radical was trapped with N-tert-butylamine-a-
phenylnitrone (PBN) as intermediate of the electrochemical
oxidation.
Table 2. Apparent first-order kinetic rate constant values for
the reaction between C-4 substituted 1,4-DHP derivatives and
superoxide anion (KO ) and their relationship
2
Pseudo first-order kinetic rate
ꢂ1
constant Kpy/s
0
0
a
(sꢂ1) ꢁ 10ꢂ4
b
Derivative
k
Ratio
4
4
4
-(3-indolyl)-DHP
-(5-indolyl)-DHP
-phenyl-DHP
2.25 ꢃ 0.04
1.79 ꢃ 0.06
0.38 ꢃ 0.03
0.15 ꢃ 0.02
0.14 ꢃ 0.01
0.03 ꢃ 0.01
16.1
12.8
2.7
3.6
1.0
Amlodipine
Nisoldipine
Nimodipine
0.2
Figure 10. Time-course
of
the
reaction
between
0.1 mM
a
Calculated apparent first-order kinetic rate constants.
Ratio kinetic rate constant derivative/kinetic rate constant
4-(5-indolyl)-DHP and 20 mM KO2 in DMSO at 37 8C, followed by HPLC
with diode array detector (l ¼ 250 nm). Parent 4-(5-indolyl)-DHP. Reaction
mixture at: (a) t ¼ 0, (b) t ¼ 1 hour, (c) t ¼ 2 hours, (d) 3 hours, (e) 4 hours,
b
nisoldipine.
(f) 5 hours.
J. Phys. Org. Chem. 2009, 22 569–577
Copyright ß 2008 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/poc

R. SALAZAR ET AL.
Table 3. Retention times (Rt) of oxidized products obtained by controlled-potential electrolysis (CPE) and the reaction of
1,4-dihydropyridines with superoxide anion obtained from HPLC with diode array detector (l ¼ 250 nm)
a
b
c
Derivative
Rt 1,4-DHP /min
Rt reaction solution /min
Rt electrolyzed derivative /min
4-(3-indolyl)-DHP
4-(5-indolyl)-DHP
4-phenyl-DHP
5.9
5.5
8.5
6.2
5.9
9.6
6.2 (
Eapp ¼ 1100 mV)
5.9 (Eapp ¼ 1100 mV)
9.5 (Eapp ¼ 1200 Mv)
a
b
c
Average of three independent measurements of parent 1,4-DHP.
Products of the reaction with superoxide radical anion of the derivatives after 5 h at 37 8C.
Controlled-potential electrolysis (CPE) in DMSO þ 0.1 M de TBAHFP.
ꢀ
the chromatographic signals and retention times support that
products are quite similar, corresponding to the pyridine
derivative.
DHP-anion was not possible its identification by HPLC
technique because the pH of the mobile phase was pH 4.3.
Kinetic experiments were carried out in DMSO (UV–Vis), and
aliquots of such reaction solutions were injected into the
chromatograph. In consequence, the DHP-anion returns to its
protonated-DHP form due to the acidity of the mobile phase,
precluding its determination.
4. The anionic form of DHP does not react with O2 to generate
pyridine.
5. Analyses of reaction solutions by GC–MS permit us the identi-
fication of the pyridine derivative as the final product.
6. Results from voltammetric techniques (CV and DPV) were
consistent with the spectroscopic ones, supporting the for-
ꢂ
mation of the DHP anion (R—N ) and the consumption of the
parent 1,4-DHPs in the reaction.
Acknowledgements
GC/MS
This work was partially supported by Grant 1050761 (L.J. N u´ n˜ ez-
Vergara) from FONDECYT and a Grant AT-23070093 (R. Salazar)
from CONICYT.
To identify the final products after the reaction between
superoxide and the 1,4-DHP derivatives, a GC–MS method was
used. Figure 8 shows typical extracted ion chromatograms and
mass spectra corresponding to a solution resulting after 5 h
reaction between an initial 0.1 mM 4-(3-indolyl)-DHP) solution
and 20 mM superoxide in DMSO at 37 8C. The chromatogram
shows a peak at 10.4 min corresponding to the pyridine
derivative. The other 1,4-DHP derivatives exhibited similar
characteristics, varying only the product’s abundance. In all the
compounds, the retention times of the oxidized derivatives were
lower than those of the parent derivatives, and the mass spectral
fragmentation pattern were different from that of the original
REFERENCES
[
[
1] E. W. Kellog, I. J. Fridovich, Biol. Chem. 1977, 252, 6721–6728.
2] S. Cuzzocrea, D. P. Riley, A. P. Caputi, D. Salvemini, Pharmacol. Rev.
2001, 53, 135–159.
[3] Y. Moroka, C. S. Foote, J. Am. Chem. Soc. 1975, 98, 1510–1516.
[
[
[
[
4] D. M. Stout, A. I. Meyers, Chem. Rev. 1982, 82, 223–243.
5] R. Lavilla, J. Chem. Soc. Perkin Trans. 1 2002, 1141–1156.
6] D. J. Triggle, Drug Dev. Res. 2003, 58, 5–17.
[
28]
derivatives.
We can conclude that the only product of the
7] D. J. Triggle, Biochem. Pharmacol. 2007, 74(1), 1–9.
reaction was the pyridine derivative. Analyses of electrolyzed
solutions of 1,4-DHPs at 1100 mV by this technique revealed
the similarity of the chromatographic characteristics of these
products with those obtained by the reaction with the free
radical. Average retention time of the oxidized derivatives was
[8] L. J. N u´ n˜ ez-Vergara, C. Lopez-Alarc o´ n, P. A. Navarrete-Encina, A. M.
Atria, C. Camargo, J. A. Squella, Free Rad. Res. 2003, 37(1), 109–120.
[
9] C. L o´ pez-Alarc o´ n, H. Speisky, J. A. Squella, C. Olea-Azar, C. Camargo,
L. J. N u´ n˜ ez-Vergara, Pharm. Res. 2004, 21, 1750–1757.
[
10] L. J. N u´ n˜ ez-Vergara, R. Salazar, C. Camargo, J. Carbajo, B. Conde, P. A.
Navarrete-Encina, J. A. Squella, Bioorg. Med. Chem. 2007, 15, 4318–
4326.
10.4 min and m/z of 366 was the most abundant fragment,
[
[
[
[
[
11] P. A. Navarrete-Encina, J. A. Squella, J. Carbajo, B. Conde, L. J.
N u´ n˜ ez-Vergara, Synth. Commun. 2007, 37, 2051–2060.
12] P. Rani, V. K. Srivastava, A. Kumar, Eur. J. Med. Chem. 2004, 39, 449–
supporting that both types of products are the same,
corresponding to the pyridine derivative.
452.
13] F. Palluotto, F. Campagna, A. Carotti, M. Ferappi, A. Rosato, C. Vitali,
Farmaco 2002, 57, 63–69.
14] M. Mugnaini, M. Antolini, M. Corsi, F. T. Vanamsterdam, J. Recept.
Signal Transduct Res. 1998, 18, 91–112.
15] M. Mor, C. Silva, F. Vacondio, P. V. Plazzi, S. Bertoni, G. Spadoni, G.
Diamantini, A. Bedini, G. Tarzia, M. Zusso, D. Franceschini, P. Giusti,
J. Pineal Res. 2004, 36, 95–102.
CONCLUSIONS
1
2
3
. C4-indolyl substituted 1,4-DHPs significantly reacted with
superoxide either electrochemically generated or as the pot-
assium salt.
. Apparent first-order kinetic rate constant values of synthesized
compounds were higher than that of commercial 1,4-DHPs,
nifedipine, nisoldipine, and amlodipine.
. Extended NMR spectra demonstrated an acid–base equi-
librium reaction involving the protons at 1-position of both
heterocycles.
[16] R. Lavilla, T. Gotsens, M. C. Santano, J. Bosch, A. Camins, N. Arnau, E.
Escubedo, J. Camarasa, M. Pallas, Bioorg. Chem. 1997, 25, 169–
1
78.
17] R. P. Mason, I. T. Mak, M. W. Trumbore, P. E. Mason, J. Cardiol. 1999, 84, 161–
21.
[
[
2
18] G. D ´ı az-Araya, L. Godoy, L. Naranjo, J. A. Squella, M. E. Letelier, L. J.
Nu n˜ ez-Vergara, Gen. Pharmacol. 1998, 31, 385–391.
www.interscience.wiley.com/journal/poc
Copyright ß 2008 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2009, 22 569–577

REACTIVITY OF C4-INDOLYL SUBSTITUTED 1,4-DHP
[
[
[
19] R. Toniolo, F. di Narda, G. Bontempelli, F. Ursini, Bielectrochemistry
000, 51, 193–200.
20] M. E. Ortiz, L. J. N u´ n˜ ez-Vergara, J. A. Squella, Pharmceut. Res. 2003, 20,
92–296.
21] M. E. Ortiz, L. J. Nu n˜ ez-Vergara, C. Camargo, J. A. Squella, Pharmaceut.
Res. 2004, 21, 428–435.
[24] T. Okajima, T. Ohsaka, Electrochimic. Acta 2002, 47, 1561–1565.
[25] J.-P. Cheng, Y. Lu, X.-Q. Zhu, Y. Sun, F. Bi, J. He, J. Org. Chem. 2000, 65,
3853–3857.
[26] F. G. Bordwell, Acc. Chem. Res. 1988, 21, 456–463.
[27] H. H. Perkampus, UV-Vis. Spectroscopy and its Applications. Springer
Laboratory Berlin, 1992, 182–183.
2
2
[
[
22] R. S. Nicholson, Anal. Chem. 1966, 38, 1406.
23] M. E. Ortiz, L. J. N u´ n˜ ez-Vergara, J. A. Squella, J. Electroanal. Chem.
[28] L. J. N u´ n˜ ez-Vergara, P. A. Navarrete-Encina, S. Salas, B. Conde, J.
Carbajo, J. A. Squella, C. Camargo, J. Pharm. Biomed. Anal. 2007, 44,
236–242.
2
002, 519, 46–52.
J. Phys. Org. Chem. 2009, 22 569–577
Copyright ß 2008 John Wiley & Sons, Ltd.
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