Reactivity of 1,4-dihydropyridines toward SIN-1-derived peroxynitrite

Article abstract of DOI:10.1023/B:PHAM.0000045224.97323.8b

Purpose. To study the reactivity of C4-substituted 1,4-dihydropyri- dines (1,4-DHP), with either secondary or tertiary nitrogen in the dihydropyridine ring, toward SIN-1-derived peroxynitrite in aqueous media at pH 7.4. Methods. Reactivity was followed by

Full text of DOI:10.1023/B:PHAM.0000045224.97323.8b

Pharmaceutical Research, Vol. 21, No. 10, October 2004 (© 2004)
Research Paper
•
•_
peroxynitrite formation, v ס k[NO ][O ], reaching poten-
2
Reactivity of 1,4-Dihydropyridines
Toward SIN-1–Derived Peroxynitrite
tially cytotoxic levels.
Although the role of the peroxynitrite as a cytotoxic spe-
cies in vivo is still debated (3), its associated form (i.e., the
peroxynitrous acid) is a recognized oxidant and thereby it
may destroy crucial cellular targets. On the other hand, the
oxidizing capability of peroxynitrite toward a variety of bio-
molecules has widely been investigated and demonstrated in
vitro (4).
1
2
1
C. López-Alarcón, H. Speisky, J. A. Squella,
3
4
C. Olea-Azar, C. Camargo, and
1
,5
Luis J. Núñez-Vergara
The pathologic activity of peroxynitrite is presumably
based on its capability to oxidize protein and nonprotein sulf-
hydryls (4), membrane phospholipids (5), low-density lipo-
proteins (6), and tyrosine residues (7). Under in vivo condi-
tions, low amounts of peroxynitrite seem to be formed con-
tinuously (8). This process can be mimicked in experimental
Received March 30, 2004; accepted June 10, 2004
Purpose. To study the reactivity of C4-substituted 1,4-dihydropyri-
dines (1,4-DHP), with either secondary or tertiary nitrogen in the
dihydropyridine ring, toward SIN-1–derived peroxynitrite in aqueous
media at pH 7.4.
•
_
•
systems with the O
and NO releasing compound SIN-1
2
Methods. Reactivity was followed by changes in the absorptivity of (8). Peroxynitrite generated in situ from SIN-1 has been
the UV-Vis bands corresponding to 1,4-DHP. Gas Chromatography/ shown to attack many biological targets; for example, low-
Mass Spectrometer (GC-MS) and Electron Paramagnetic Resonance density lipoproteins in nearly the same manner as a bolus
(
EPR) spin trap techniques were used to characterize the final prod-
addition of authentic peroxynitrite (6).
uct and the intermediates of the reaction, respectively.
The mechanisms of peroxynitrite reactions, such as con-
version to nitrate, oxidation, and nitration, are controversial.
Thus, the oxidation reactions mediated by peroxynitrite are
expected to take place by complicated reaction pathways, and
despite extensive research, no unified model is available as
Results. 1,4-DHPs significantly reacted toward peroxynitrite at var-
ied rates, according to the calculated kinetic rate constants. By EPR
spectroscopy, a carbon-centered radical from the 1,4-DHP was inter-
cepted with N-tert-butylamine-␣–phenylnitrone (PBN), as the inter-
mediate for the reaction with peroxynitrite. Likewise, the oxidized
derivative (i.e., the pyridine) was identified as the final product of the yet. Both one- and two-electron oxidation have been found in
reaction by GC-MS. By using the technique of deuterium kinetic several reactions induced by peroxynitrite (9).
isotope effect, the participation of the hydrogen of the 1-position on
On the other hand, the 4-substituted Hantzsch 1,4-
the 1,4-DHP ring was shown not to be the rate-limiting step of the dihydropyridines (1,4-DHP), having at least a single hydrogen
reaction.
atom at the position 4, are interesting compounds not only
from the point of view of the heterocyclic chemistry but also
Conclusions. The direct participation of the 1,4-DHP derivatives in
the quenching of SIN-1–derived peroxynitrite has been demon-
as pharmacologically active substances, due to their potential
strated. Kinetic rate constant of tested 1,4-DHP toward peroxynitrite
to act as antioxidants (10,11) and as NADH coenzyme ana-
showed a direct relationship with the oxidation peak potential values;
logs that mediate hydrogen transfer reactions in biological
that is, compounds reacting faster were more easily oxidized.
systems (12–14). More recently (15–17), the oxidation of
Hantzsch 1,4-dihydropyridines has attracted much attention
from chemists because of its reactivity toward endobiotics
KEY WORDS: 1,4-dihydropyridines; EPR; GC-MS; kinetic rate
constants; peroxynitrite.
such as nitric oxide. The latter is coincident with the discovery
·
INTRODUCTION
of several active roles of NO in a wide range of human patho-
physiological processes (18).
−
Furthermore, because peroxynitrite can react with re-
duced NADH (19), the study of the ability of 1,4-dihydropyri-
dines to quench peroxynitrite seems also to be of interest.
Taking into account the above, in the current work a system-
atic study on the reactivity of a number of 1,4-DHP with
SIN-1–derived peroxynitrite was addressed. Kinetic rate con-
stant values, reaction mechanisms, and a detailed discussion
based on experimental results that relate the chemical struc-
ture and the oxidation potentials of the 1,4-DHP compounds
are presented.
Peroxynitrite, ONOO , is formed in vivo by the reaction
•
of nitric oxide (NO ) with superoxide (1). The biological rel-
evance of this species was first pointed out by Beckman et al.
2), who recognized that peroxynitrite may be formed in sig-
nificant quantities under pathologic conditions. Because both
NO and O are produced at high rates by phagocytic cells
such as macrophages, relatively small increases in the rates of
(
•
•_
2
•
•_
NO and O
production may greatly increase the rate of
2
1
Laboratory of Bioelectrochemistry, Faculty of Chemical and Phar- MATERIALS AND METHODS
maceutical Sciences, University of Chile, Santiago, Chile.
Nutritional Toxicology, INTA, Faculty of Chemical and Pharma-
ceutical Sciences, University of Chile, Santiago, Chile.
Department of Inorganic and Analytical Chemistry, Faculty of
Chemical and Pharmaceutical Sciences, University of Chile, San-
tiago, Chile.
Laboratory of Antidoping, Faculty of Chemical and Pharmaceutical
Sciences, University of Chile, Santiago, Chile.
2
3
Chemicals
All solvents were of high-pressure liquid chromatogra-
phy (HPLC) grade and all reagents were of analytical grade.
Compounds
4
5
The following 1,4-dihydropyridine derivatives (Fig. 1,
To whom correspondence should be addressed. (e-mail: lnunezv@ I–VI, VIII) were synthesized in our laboratory according to a
ciq.uchile.cl)
previously described procedures (20,21). The final products
0
724-8741/04/1000-1750/0 © 2004 Springer Science+Business Media, Inc.
1750

Reactivity of 1,4-Dihydropyridines with Peroxynitrite
1751
1
H NMR (300 MHz, CDCl ): ␦ 0.88 (d, 3H, J ס 6.6 Hz,
3
>
CHCH ), 1.16 (t, 3H, J ס 7.1 Hz N-CH CH ), 2.4 (s, 6H,
3 2 3
-
-
CH ), 3.7 (q, 2H, J ס 7.1 Hz N-CH CH ), 3.72 (s, 6H,
3
2
3
1
3
OCH ), 3.77 (q, 1H, J ס 6.6 Hz, >CHCH ). C NMR (75
3
3
MHz, CDCl ): 15.56, (16.15x2), 21.73, 28.12, 39.51, (51.12x2),
3
(
108.75x2), (148.01x2), (168.50x2). Anal. Calcd. for
C H NO : C, 62.84; H, 7.85; N, 5.23. Found: C, 63.05; H,
1
4
21
4
7.62; N, 5.13. m.p.: 78°C –80°C.
III. 4-(4-methoxyphenyl)-2,6-dimethyl-3,5-dimethoxy-
carbonyl-1,4-dihydropyridine.
IR (KBr): ␯ 3349, 2949, 1697, 1650, 1431, 1383, 1251,
max
−
1
1
213, 1027 cm .
1
H NMR (300 MHz, CDCl ): ␦ 2.33 (s, 6H, -CH ), 3.65 (s,
3
3
6
H, -O-CH ), 3.75 (s, 3H, Ar-O-CH ), 4.94 (s, 1H, Ar-CH<),
3
3
5
.76 (s, 1H, -NH-), 6.75 (d, 2H, J ס 8.6Hz, Ar-H), 7.2 (d, 2H,
1
3
J ס 8.6 Hz, Ar-H). C NMR (75 MHz, CDCl ): (19.51x2),
3
3
(
8.31, (50.94x2), 55.05, (104.00x2), (113.30x2),
128.53x2),139.86, (143.93x2), 157.85, (168.06x2). Anal. Calcd.
for C H O N: C, 65.24; H, 6.39; N, 4.23. Found: C, 65.00; H,
1
8
21
5
6.47; N, 4.36. m.p.:181°C–183°C.
IV. 4-(4-Methoxyphenyl)-2,6-dimethyl-3,5-dimethoxy-
carbonyl-N-ethyl-1,4-dihydropyridine.
IR (KBr): ␯ 2948, 1690, 1629, 1433, 1390, 1256, 1213,
max
−
1
1
152, 1035 cm .
1
H NMR (300 MHz, CDCl ): ␦ 1.06 (t, 3H, J ס 5.9 Hz
3
NCH -CH ), 2.47 (s, 6H, -CH ), 3.67 (q, 2H, J ס 5.9 -N-
2
3
3
CH CH ), 3.7 (s, 6H, -OCH ), 3.75 (s, 3H, Ar-OCH ), 5.04 (s,
2
3
3
3
1
H, Ar-CH<), 6.75 (d, 2H, J ס 8.7 Hz, Ar-H), 7.09 (d, 2H, J
1
3
ס 8.7 Hz, Ar-H). C NMR (75 MHz, CDCl ): 16.06,
3
(
(
16.42x2), 37.19, 40.42, (51.26x2), 55.17, (107.16x2),
113.32x2), (127.91x2), 138.53, (148.53x2), 157.90, (168.61x2).
Anal. Calcd. for C H NO : C, 66.77; H, 6.96; N, 3.9. Found:
2
0
25
5
C, 66.85; H, 7.08; N, 3.66. m.p.: 106°C –108°C.
V. 4-(4-Nitrophenyl)-2,6-dimethyl-3,5-dimethoxycar-
bonyl-1,4-dihydropyridine.
IR (KBr): ␯max 3343, 2948, 1703, 1655, 1518, 1434, 1384,
−
1
1
6
347, 1218, 1020 cm .
Fig. 1. Chemical structures of 1,4-dihydropyridines and NADH.
1
H NMR (300 MHz, CDCl ): ␦ 2.38 (s, 6H, -CH ), 3.66 (s,
H, -O-CH ), 5.12 (s, 1H, Ar-CH<), 5.85 (s, 1H, -NH-), 7.46
were subjected to IR, NMR, and elemental analyses obtaining (d, 2H, J ס 8.8Hz, Ar-H), 8.12 (d, 2H, J ס 8.8 Hz, Ar-H).
3
3
3
1
3
C
the following results:
NMR (75 MHz, CDCl ): (20.63x2), 40.73, (52.11x2),
3
(
(
8
103.92x2), (124.39x2), (129.53x2), (145.81x2), 147.29, 155.64,
I. 4-Methyl-2,6-dimethyl-3,5-dimethoxycarbonyl-1,4-
dihydropyridine.
168.38x2). Anal. Calcd. for C H O N : C, 58.96; H, 5.24; N.
1
7
18
6
2
.09. Found: C, 58.76; H, 5.03; N, 8.25. m.p.:165°C–168°C.
IR (KBr): ␯
056, cm .
3342, 2950, 1680, 1650, 1435, 1351, 1226,
max
VI. 4-(4-Nitrophenyl)-2,6-dimethyl-3,5-dimethoxy-
carbonyl-N-ethyl-1,4-dihydropyridine.
−
1
1
1
H NMR (300 MHz, CDCl ): ␦ 0.96 (d, 3H, J ס 6.5Hz,
3
>
CH-CH ), 2.29 (s, 6H, -CH ), 3.73 (s, 6H, -O-CH ), 3.83 (q,
3 3 3
IR (KBr): ␯ 2945, 1690, 1624, 1511, 1382, 1345, 1252,
max
13
−
1
1
H, J ס 6.5Hz, >CH-CH ), 5.73 (s, 1H, -NH-). C NMR (75
3
1154, 1025 cm .
1
MHz, CDCl ): (20.35x2), 23.20, 29.30, (51.92x2), (105.27x2),
3
H NMR (300 MHz, CDCl ): ␦ 1.04 (t, 3H, J ס 7.1 Hz,
3
(145.64x2), (169.19x2). Anal. Calcd. for C H O N: C, 60.25;
12 17 4
N-CH CH ), 2.5 (s, 6H, -CH ), 3.72 (s, 6H, -OCH ), 3.72 (q,
2
3
3
3
H, 7.13; N, 5.86. Found: C, 60.27; H, 7.23; N, 5.87. m.p.:147°C
149°C.
2
2
H, J ס 7.1 Hz, N-CH -CH ), 5.19 (s, 1H, Ar-CH<), 7.34 (d,
2 3
1
3
–
H, J ס 8.5 Hz, Ar-H) 8.07 (d, 2H, J ס 8.5 Hz, Ar-H). C
NMR (75 MHz, CDCl ): 16.06, (16.4x2), 38.25, 40.0,
3
II. 4-Methyl-2,6-dimethyl-3,5-dimethoxycarbonyl-N-
ethyl-1,4-dihydropyridine.
(
51.44x2), (105.70x2), (123.29x2), (127.74x2), (146.42x2),
49.32, 153.61, (167.94x2). Anal. Calcd. for C H N O : C,
1
1
9
22
2
6
IR (KBr): ␯
2954, 1696, 1631, 1434, 1389, 1212, 1163, 60.9; H, 5.88; N, 7.48. Found: C, 61.10; H, 5.99; N, 7.34. m.p.:
max
−
1
1
056 cm
.
156°C–157°C.

1
752
López-Alarcón et al.
VIII. 4-(3-Nitrosophenyl)-2,6–dimethyl-3,5-diethoxy- UNICAM UV-3 spectrophotometer (Cambridge, UK). Ac-
carbonyl-1,4-dihydropyridine.
quisition and data treatment were carried out with a 2.11
software (Vision software, Unicam Limited, Cambridge,
UK). UV-Vis spectra were recorded in the 220–500 nm range
at different intervals. For all compounds the absorption bands
at 330–370 nm were used. Drug concentrations in aqueous
buffer [KH PO 50 mM/acetonitrile, 70/30 (v/v) pH 7.4] were
IR (KBr): ␯max 3334.5, 29981.7, 1699.8, 1651.3, 1488.8,
−
1
1
371, 1300.2, 1213.3, 1102.1, 1020.2 cm .
1
H-NMR (300MHz, CDCl ): ␦1.2(t, 6H, J ס 7.2 Hz,
3
-
CH CH ), 2.36(s, 6H, R-CH ), 4.1(m, 4H, -CH CH ), 5.1(s,
2 3 3 2 3
1
3
2
4
1
H, >CH-), 5.7(s, 1H, >NH), 7.7(m, 4H, Ar-H) ppm.
C
determined from respective calibration curves (10–100 ␮M).
The rates of the reactions were calculated using the kinetic
rate constant between NADH and SIN-1–derived peroxyni-
NMR (75 MHz, CDCl ): (2x13.2302); (2x18.5819); 38.852;
3
5
1
8.9277, 102.533; 118.131; 119.819; 127.550; 128.035; 134.542;
43.681; (2x148.692); 165.457; 165.796; 166.248; 167.671 ppm.
4
−1 −1
trite reported in the literature, k ס 1 × 10 m
s
(19).
Calc. C H O N : C ס 63.67; H, 6.29; N, 7.62. Found: C,
1
9
22
5
2
Control solutions (in the absence of SIN-1) revealed no
changes either in their original UV-Vis absorption bands or
GC-MS mass fragmentation.
Also, a possible photodecomposition of 1,4-dihydro-
pyridines was assessed, but in the time-scale of the experi-
ments this was negligible.
6
3.52; H, 6.36; N, 7.66. mp: 118.2°C–119.1°C.
NMR spectroscopy: The NMR spectra were recorded on
a Bruker spectrometer Avance DRX 300 (Rheinstetten, Karls-
ruhe, Germany).
FT-IR: The IR spectra were recorded on a Bruker spec-
trometer IFS 55 Equinox (Rheinstetten, Karlsruhe, Germany).
Elemental analyses were performed in a Fisons Instru- GC-MS
ment equipment (Fisons-Carlo Erba modelo EA 1108 (Mi-
lano, Italy)).
A Gas Chromatograph/Mass Spectrometer Hewlett
Packard 5890/5972 Detector (Palo Alto, CA, USA) and Hew-
lett Packard 7673 Autosampler were used for the measure-
ments. A Hewlett Packard Pentium II Data System-Laser Jet
Drugs
Nisoldipine and nifedipine were supplied by Sanitas 4000 printer, controlled instrumentation, and data handling
Laboratories (Santiago, Chile). Both compounds were as- was also used.
sayed without previous purification. NADH was obtained
from Merck.
Chromatography Column
Hewlett-Packard Ultra-1 column, 25 m × 0.2 mm i.d. ×
Solutions
Care was taken to exclude possible contamination by
0.11 film thickness (Little Falls, Wilmington, Delaware,
USA).
bicarbonate/carbon dioxide. Milli-Q water (⍀ ס 18.6) was Chromatographic Conditions
bubbled with oxygen for 30 min. Potassium phosphate buffer
Detector temperature, 300°C; injector temperature,
50°C; split ratio, 1/10; pressure, 13 psi; purge flow, 40 ml
(
KH PO , 50 mM)/acetonitrile, 70/30 (v/v) solutions were
2 4
2
prepared on the day of the experiments and pH adjusted to
.4 at 37°C.
−1
−1
min ; purge time, 0.5 ml min
.
7
Temperature Program
Experimental Conditions
The oven temperature was programmed from 130°C to
SIN-1 (Aldrich Chemical Co.) was used as peroxynitrite 305°C (hold for 5 min) at 15°C min ; run time was 16.67 min.
generator (Fig. 2). SIN-1 (100 ␮M) was added to 3 ml of Helium was used as carrier gas with an inlet pressure of 35
−
1
1
,4-DHP or NADH (50 ␮M) solutions in potassium phos- kPa. The identification of the samples was based on the analy-
phate buffer/acetonitrile 70/30 (v/v) at pH 7.4. The final so- ses of the mass spectra (full scan).
lutions were incubated between 1 and 2.5 h at 37°C. The
After the reaction, the final solutions were diluted and
progress of the reaction was followed both via UV and Vis injected without any pretreatment. Consequently, no extrac-
spectroscopy. The final products were analyzed by gas chro- tion or filtration procedures were necessary.
matograph/mass spectrometer (GC-MS).
Deuterium Kinetic Isotope Effect (DKIE) Studies
UV-Vis
The progress of the reaction with SIN-1–derived peroxy-
1
Studies by H-NMR.Spectroscopy
In order to establish the exchange among the proton of
nitrite was followed by UV-Vis spectroscopy using an the position 1 with a deuterium atom, compound I was dis-
Fig. 2. Reaction scheme of peroxynitrite formation from SIN-1.

Reactivity of 1,4-Dihydropyridines with Peroxynitrite
1753
1
solved in DMSO-d carrying out a normal H-NMR. After
6
this, 20 ␮l D O was added to the sample in order to observe
2
the possible change in the typical –NH- signal.
DKIE Influence on the Kinetic Rate Constants of
Compound I
Both, control solutions [H O/DMSO 70/30 (v/v) in 50
2
mM phosphate buffer pH 7.4] and deutered solutions [D O/
2
DMSO-d6 70/30 (v/v) in 50 mM phosphate buffer pH 7.4]
containing 50 ␮M of compound I were incubated at 37°C for
2
h. Then, the same experimental procedure as was previously
described was followed (Experimental conditions and UV-
Vis sections).
Electron Paramagnetic Resonance Studies
The EPR spectra were recorded in situ on a Bruker spec-
trometer ECS 106 (Rheinstetten, Karlsruhe, Germany) with
1
00 kHz field modulation in X band (9.78 GHz) at room
temperature. The hyperfine splitting constants were esti- Fig. 3. Time-course of UV-Vis spectra corresponding to the reaction
mated to be accurate within 0.05 G.
between 50 ␮M compound IX and 100 ␮M SIN-1–derived peroxyni-
trite in 50 mM phosphate buffer/acetonitrile (70/30) at pH 7.4. a–j: 60
min. Inset: Time-course of the UV absorption at 370 nm.
Two types of studies were performed: a) controlled po-
tential electrolysis of 1 mM Compound I solutions in 50 mM
phosphate/acetonitrile (70/30, v/v) at pH 7.4. These studies
were performed in the EPR cell using an appropriate plati-
num mesh electrode at 1.0 V vs. Ag/AgCl in the presence of
following the addition of 100 ␮M SIN-1 to an aqueous solu-
tion (pH 7.4) containing the 1,4-DHP compounds. This effect
is illustrated in Fig. 3 for compound IX. To compare the
reactivity, kinetic rate constant values (k) were calculated as
described in “Materials and Methods” for the tested 1,4-DHP
derivatives (Table I). Also, we checked the rate of reaction of
NADH with peroxynitrite following the decrease in absor-
bance at 340 nm. However, for comparative purposes, a re-
1
00 mM spin trap, N-tert-butylamine-␣-phenylnitrone (PBN).
The spin trap was electrochemically tested for purity, as was
the magnitude of the so-called potential window (the poten-
tial range within which the trap is electrochemically inactive).
The oxidation peak potential of PBN in 50 mM phosphate/
acetonitrile (70/30) at a Pt electrode was + 1.42 V vs. Ag/
AgCl. EPR spectra were collected after 20 min of electrolysis.
b) Reaction of 1 mM Compound I solutions in the presence of
4
−1 −1
ported k value for NADH of 1 × 10 m
s
was used (19).
Table I shows that all the 1,4-DHP derivatives reacted
with peroxynitrite at varied rates; the C-4 unsubstituted 1,4-
1
00 mM PBN and 2 mM SIN-1 in 50 mM phosphate/
acetonitrile (70/30, v/v) at pH 7.4 were analyzed by EPR.
Spectra were collected up to 1 h of reaction.
Table I. Kinetic Rate Constants (k) for the Reaction Between SIN-
Also, two types of blank solutions containing 2 mM
SIN-1 + 100 mM PBN and 1 mM compound I + 100 mM PBN
were analyzed by EPR in the same experimental conditions.
Under the experimental conditions, none of these solutions
revealed any signal.
1
–Derived Peroxynitrite and 1,4-DHP and their Relationship with
NADH and the Oxidation Peak Potentials
−3
−1 −1a
b
c
Compound
k × 10 /M
s
k1,4-DHP/kNADH
Ep/mV
I
II
3.0 ± 0.1
1.0 ± 0.05
3.0 ± 0.2
0.8 ± 0.06
1.8 ± 0.12
0.5 ± 0.03
2.5 ± 0.12
1.8 ± 0.08
10.3 ± 0.4
1.1 ± 0.07
1.0 ± 0.08
10
0.3
0.1
0.3
660
748
680
772
768
830
660
742
352
744
681
492
Voltammetry
III
IV
V
VI
VII
VIII
IX
Differential pulse voltammetry (DPV) was performed
with a BAS CV50, (Bioanalytical Systems, West Layfayette,
USA) assembly. A glassy carbon stationary electrode was
used as working electrode. A platinum wire was used as a
counterelectrode, and all potentials were measured against an
Ag/AgCl electrode.
0.08
0.18
0.05
0.25
0.18
1.03
0.11
0.1
Operating conditions: pulse amplitude, 40 mV; potential X (nifedipine)
−
1
scan, 4 mVs ; voltage range, 0 to 1000 mV, current range, 5 XI (nisoldipine)
NADH
1.0
to 25 ␮A, temperature, 25°C. All the solutions were purged
with pure nitrogen for 10 min before the voltammetric runs.
a
Kinetic rate constants were calculated by using the kinetic rate con-
stant for the reaction between NADH and SIN-1–derived peroxy-
RESULTS AND DISCUSSION
4
−1 −1
nitrite (k ס 1 × 10 M
s , taken from Ref. 19).
b
c
Reactivity of 1,4-DHP Toward SIN-1–Derived
Peroxynitrite: UV-Vis Spectroscopic Studies
Ratio between kinetic rate constants of the tested 1,4-DHP deriva-
tives/kinetic rate constant of NADH in presence of SIN-1.
Oxidation peak potential values determined by differential pulse
voltammetry using a glassy carbon working electrode in 50 mM
phosphate buffer/acetonitrile (70/30, v/v) + 0.1 M KCl at pH 7.4.
Potentials are expressed vs. Ag/AgCl reference electrode. 1,4-DHP
concentration: 0.1 mM
The 1,4-DHP tested compounds displayed UV absorp-
tion bands that ranged typically between ␭ ס 330 and 430 nm
and were maximal between 330 nm and 370 nm (Fig. 3). A
sustained decrease of the maximal UV bands was observed

1
754
López-Alarcón et al.
DHP (compound IX) being the most reactive one, exhibiting Table III. GC-MS Characteristics of the Oxidized Derivatives Ob-
4
−1 −1
tained After the Reaction Between 1,4-DHP with SIN-1–Derived
a k value of 1.03 × 10
m s , which was similar to that
Peroxynitrite
described for NADH (19). Such a result could be explained
by the similarity of the chemical moiety involved in the reac-
tion; that is, 1,4-dihydropyridine ring vs. N-substituted nico-
tinamide (Fig. 1).
Oxidized derivatives
+
Compound
Bp
M
Rt/min.
Compound IX shows a range of reactivity between 20.6
and 3.4 times higher than that of the other tested C-4 substi-
tuted 1,4-DHP derivatives (Table I). Also, compound IX was
found to be more reactive than the two well-known 1,4-DHP,
nifedipine and nisoldipine.
I. Pyridine
II. Pyridinium salt
III. Pyridine
IV. Pyridinium salt
V. Pyridine
VI. Pyridinium salt
VII. Pyridine
206
—
266
—
313
—
236
342
206
298
284
237
—
329
—
344
—
299
356
251
344
386
4.8
—
8.7
—
9.6
—
7.3
9.9
5.7
9.1
10.2
The inclusion of an electron-donor group on the C-4 phe-
nyl moiety slightly increased (by 20%) the rate of reaction
(
compound III vs. compound VII). In contrast, the inclusion VIII. Pyridine
of an electron-withdrawing group on the C-4 phenyl moiety
gave rise to a slight decrease (of 28%) in the k value (com-
pound V vs. compound VII).
IX. Pyridine
X. Pyridine
XI. Pyridine
On the other hand, the N-alkylation of the 1-position
produced a significant decrease of the reaction rates (Table
I). Comparing compound V and compound VIII, no differ-
+
Bp: Base peak; M : Mass-charge ion; Rt/min: Retention time in min-
utes.
ences in their k values were found; the latter despite the well- ent 1,4-DHP and its subsequent reactivity with peroxynitrite
known spin-trapping properties of the nitroso group (22).
did not require derivatization. b) After the reaction with per-
oxinitrite, the 1,4-DHP suffered a dehydrogenation process
yielding the pyridine-derivative. The latter conclusion is sup-
ported by the respective retention times and mass fragmen-
tation pattern for each compound as displayed in Table III.
Noteworthy, in the scavenging of peroxynitrite by 1,4-DHP,
an electron transfer reaction is involved. c) Retention times of
the corresponding pyridine derivatives were lower than that
of the parent compounds (Fig. 4). d) The mass fragmentation
pattern of the aromatized derivatives shows different peak
bases, depending on C-4 substitution on the 1,4-dihydropyri-
dine ring. e) The reaction between N-ethyl-DHP derivatives
with peroxynitrite, as assessed by the GC-MS technique, did
not reveal any new signal. Thus, the lack of a new signal
observed by this technique could be explained on the basis of
our previous observation that the electrochemical oxidation
of N-substituted DHP gives rise to a pyridinium salt as a final
product (23). This final product is soluble in aqueous media
preventing its subsequent extraction to inject it into a chro-
matograph (24).
Reaction Mechanism
In order to investigate the possible reaction mechanism,
different techniques were used for this purpose. These in-
cluded GC-MS technique to identify final product (s), deute-
rium kinetic isotope effect (DKIE) studies to determine the
participation of the hydrogen of the 1-position in rate of re-
action, and EPR studies to assess the possible formation of
radical species as intermediates from the 1,4-DHP after the
reaction with SIN-1–derived peroxynitrite. Also, correlations
between oxidation potentials and kinetic rate constants are
reported.
GC-MS Technique
In the following section, the identification of the prod-
ucts obtained after the reaction between the 1,4-DHP deriva-
tives and peroxynitrite is shown. The chromatographic data
corresponding to the 1,4-DHP compounds and their corre-
sponding oxidized derivatives are shown in Tables II and III.
Some conclusions on these studies can be summarized as fol-
lows: a) The GC-MS procedure used to characterize the par-
Deuterium Kinetic Isotope Effect Studies
To assess the feasibility of participation of the hydrogen
in the 1-position, DKIE studies were carried out selecting
compound I. As noted from the NMR experiments, the ad-
Table II. GC-MS Characteristics of Parent 1,4-DHP
1
,4-DHP
dition of 20 ␮l D O to a solution of compound I (600 ␮l)
2
made the –NH- signal at 8.7 ppm disappear, thus demonstrat-
ing that the proton of the secondary amine group was isoto-
pically exhanged with deuterium. A detailed experimental
+
Compound
Bp
M
Rt/min.
I
II
224
252
224
300
224
315
224
252
224
329
371
239
267
331
359
346
374
301
358
253
346
388
6.2
6.9
1
description of the H-NMR signals is as follows:
III
IV
V
VI
VII
VIII
IX
X
10.5
11.0
11.9
12.3
9.2
11.3
7.1
10.3
11.4
a. Normal spectra corresponding to compound I:
1
H-NMR (300 MHz, DMSO-d ): ␦ 0.82 (d, 3H, J ס 6.4 Hz,
6
>
CH-CH ), 2.2 (s, 6H, -CH ), 3.6 (s, 6H, -O-CH ), 3.7 (q, 1H,
3 3 3
J ס 6.4Hz, >CH-CH ), 8.7 (s, 1H, -NH-).
3
b. Spectra of compound I with addition of deuterium
oxide:
XI
1
H-NMR (300 MHz, DMSO-d +D O): ␦ 0.82 (d, 3H, J ס 6.4
6
2
+
Bp: Base peak; M : Mass-charge ion; Rt/min: Retention time in min- Hz, >CH-CH ), 2.2 (s, 6H, -CH ), 3.6 (s, 6H, -O-CH ), 3.7 (q,
3
3
3
utes.
1H, J ס 6.4 Hz, >CH-CH3).

Reactivity of 1,4-Dihydropyridines with Peroxynitrite
1755
Fig. 4. Ion chromatogram (A) and mass spectrum corresponding to compound VII after the reaction
with peroxynitrite (B) and its corresponding pyridine derivative (C).
The kinetic constant corresponding to the deutereted EPR Spin-Trapping Studies
compound I was estimated as described in “Materials and
Methods.” The ratio kH/kD for the peroxynitrite/compound I
In order to gather evidence on the possible formation of
reaction was 1.3. Because this value falls below 2 (25), our a radical as an intermediate during the oxidation of 1,4-DHP
experimental value of 1.3 can be interpreted as an indication induced by peroxynitrite, EPR experiments using the spin
that the hydrogen abstraction would not be the rate-limiting trap N-tert-butylamine-␣-phenylnitrone (PBN) were carried
step of the reaction (25). However further studies should be out.
conducted to clarify this, mainly to explore the possibility that
a more complex mechanism of reaction with this oxidizing intermediates generated in the electrochemical oxidation of
agent (peroxynitrite) is taking place.
1,4-DHP by PBN were also followed.
For comparative purposes, the trapping of the radical

1
756
López-Alarcón et al.
Figure 5 displays the EPR spectra correspond to the radi-
cals intercepted with PBN during a controlled potential elec-
trolysis (CPE) of compound I (part 5 A), and during the
reaction between compound I and SIN-1–derived peroxyni-
trite (part 5 B). The spectrum C corresponds to the base line
(see “Materials and Methods”). Spectra characteristics corre-
sponding to a nitroxide spin adduct appeared in both, the
electrodic reaction and the reaction with peroxynitrite in the
presence of PBN. The EPR spectra show the triplet, due to
the nitrogen, and its splitting into a doublet due to the pres-
ence of the adjacent hydrogen. aN values around 14 G and aH
splitting values of 3 G were obtained for the 1,4-DHP spin
adduct. The splitting constants for the spin adduct of about 14
G are consistent with the fact that PBN interacted with car-
bon-centered radicals as reported for 1,4-DHP derivatives
(26) and for other structurally related compounds (27).
Previous work conducted by us (28) and others (29)
shows that the electro-oxidation of 1,4-DHP derivatives
yields different types of radical intermediates: primarily the
+
и
radical cation PyH , and after its deprotonation, the neutral
Fig. 6. Relationship between kinetic rate constants (k) for the reac-
radical Pyи. According to the current EPR spectra results, it tion with peroxynitrite and the oxidation peak potentials of the 1,4-
seems likely that the species added to the PBN spin trap is a DHP derivatives at pH 7.4.
pyridinium radical. From previous quantum chemical calcu-
lations (30), it follows that the unsubstituted pyridinium radi- may assume that the radicals generated in our experiments
cal, Pyи, has the unpaired electron possibly localized in the 2-, are preferentially added to the spin trap by this C-4 reactive
4
-, or 6- position; however, the 4-position seems to be the position.
preferred one (30). In addition, electronegative substituents
in 3- and 5-position increase the spin density in the vicinal Correlations Between Kinetic Rate Constants and Oxidation
positions. Such effects are additive in 4-position, leading to an Peak Potential Values
even greater preference for this position. Consequently, one
Because it is demonstrated that the metabolism of 1,4-
DHP used in the treatment of hypertension proceeds through
oxidation (i.e., aromatization) of 1,4-DHP, significant re-
search has been carried out to study the features of these
oxidations (31). Therefore, in this section, attempts to corre-
late oxidation peak potential values, determined in aqueous
media at pH 7.4, with kinetic rate constants for the reaction
with SIN-1–derived peroxynitrite are discussed.
The electrochemical oxidation of the 1,4-dihydropyri-
dines was studied in the same experimental conditions used
in the reactivity studies (aqueous media). Under these
conditions, all compounds exhibited only a single oxida-
tion peak, which is consistent with previous reports (28,29).
Figure 6 displays the relationship between the kinetic rate
constants and the oxidation peak potentials. Data obtained
Fig. 5. Experimental EPR spectra of adduct PBN-pyridinium radical.
(A) Radical electrochemically generated from 1 mM compound I
solution in 50 mM phosphate buffer/acetonitrile (70/30). (B) Radical
generated after the reaction between 1 mM compound I and 2 mM
SIN-1. (C) Base line [2 mM SIN-1 + 100 mM PBN in 50 mM phos- Fig. 7. Scheme for the reaction between 1,4-DHP and SIN-1–derived
phate buffer/acetonitrile (70/30) at pH 7.4].
peroxynitrite.

Reactivity of 1,4-Dihydropyridines with Peroxynitrite
1757
from these studies indicate that the kinetic rate constants for
the reaction between 1,4-DHP compounds and SIN-1–
derived peroxynitrite exhibited a fairly good linear correla-
tion with oxidation peak potential (kinetic rate constant ס
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1
1
7,000 – 21.04 Ep mV; c.c. ס 0.96018). Thus, it is concluded
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1
. In the current paper, we have demonstrated the direct
2
264 (1955).
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7
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1
4
. DKIE studies clearly demonstrate that the hydrogen
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. After the reaction between 1,4-DHP and peroxyni-
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. Kinetic rate constant of tested 1,4-DHP toward per-
5
1
6
oxynitrite showed a direct relationship with the oxidation 19. M. Kirsch and H. Groot. Reaction of peroxynitrite with reduced
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. Based on the experimental evidences obtained in the
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2
2
ACKNOWLEDGMENTS
This work was partially supported by grants from
FONDECYT 8000016. Also, the support of DID of Univer-
sity of Chile is acknowledged.
2
2
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