Mendeleev
Communications
Mendeleev Commun., 2020, 30, 482–484
Mechanism of the 2-ethyl-3-hydroxy-6-methylpyridinium
2-nitroxysuccinate reduction in nitrite-generating systems
Olesya V. Pokidova,*a Boris L. Psikha,a Alexandra Yu. Kormukhina,b
Alexander I. Kotel’nikova,b and Boris S. Fedorova
a Institute of Problems of Chemical Physics, Russian Academy of Sciences, 142432 Chernogolovka,
Moscow Region, Russian Federation. E-mail: pov@icp.ac.ru
b Department of Fundamental Physical and Chemical Engineering, M. V. Lomonosov Moscow State
University, 119991 Moscow, Russian Federation
DOI: 10.1016/j.mencom.2020.07.025
O
OH
Et
k1
The nitrite-generating activity of 2-ethyl-3-hydroxy-6-methyl-
pyridinium 2-nitroxysuccinate as a promising NO-donor has
been investigated in reactions with various reducing agents.
Reduction of the NO-donor with cysteine was analyzed using
a kinetic modeling method. The calculated rate constants
satisfactorily describe the experimental data, thereby
confirming the proposed reaction mechanism.
+
HS
OH
NH2
Me
N
H
HOOCCH2(ONO2)CHCOO–
O
k1 = (9.7±2)×10–2 dm3 mol–1 s–1
k2 = (2.1±1.5)×10–2 dm3 mol–1 s–1
k2
–
NO2
O2NS
OH
NH2
Keywords: organic nitrate, thionitrate, cysteine, Griess method, nitrite ion.
Organic nitrates as exogenous donors of nitric oxide (NO),
which represent a signaling molecule involving in regulation of
vascular tone, are widely used in clinical practice for the complex
treatment of cardiovascular diseases.1–3 One of the promising
examples of this class of compounds is 2-ethyl-3-hydroxy-6-methyl-
pyridinium 2-nitroxysuccinate 1, a multifunctional substance4 with
combined properties of organic nitrate and effective antioxidant.5
Compound 1 is a nitroxy derivative of the known antioxidant
Mexidol or 2-ethyl-3-hydroxy-6-methylpyridinium succinate,
widely used as pharmaceutical in Russia. The nitroxy derivative
demonstrates effective NO-donor and nitrite-generating activities
in vitro compared with Nitroglycerin and Nicorandil.6 Besides,
compound 1 has proved to be an effective inhibitor of phospho-
diesterases,7 which along with guanylate cyclase represent key
enzymes in regulation of the cyclic nucleotides level.
easily converted to NO in vivo by deoxyhemoglobin and other
heme-containing proteins as well as by xanthine oxidoreductase
and various thiol-containing enzymes.15,16 Thus, thiols play an
important role in the initial stage of the transformation of organic
nitrates into NO, and depletion of the thiol pool can result in tolerance
for the corresponding group of drugs.17
Based on the foregoing, the aim of this work was a detailed
investigation of the compound 1 reduction into nitrite ion under
the action of Cys. Quantitative determination of the nitrite ion content
in solution was performed using a convenient and widely used
Griess method.† To avoid nitrite oxidation by atmospheric
oxygen, the experiments were carried out under anaerobic
conditions.‡
First, the reactivity of compound 1 was explored in model
systemswithvariousreducingagents. Fromthekineticdependence
for the accumulation of nitrite ions [Figure 1(a)] formed during
the interaction of compound 1 with Cys, sodium ascorbate and
OH
†
Me
N
Et
The experimental technique consisted in the measurement of optical
H
density for an azo dye formed during the interaction of nitrite ions with
sulfanilamide (SA) (Sigma, USA) and N-(1-naphthyl)ethylenediamine
(NEDA) (MP Biomedicals, Germany). The reaction was initiated by
addition of a freshly prepared solution of compound 1 to the solution of
reducing agent, namely Cys, sodium ascorbate, glutathione (GSH) or
FeSO4 (Sigma, USA), in buffer at pH 7.0.Aliquots of the reaction mixture
were taken at certain time intervals, introduced into vessels with a
solution of 0.5% SA in 0.25 m HCl (1.5 ml) and incubated for 5 min, then
a solution of 0.02% NEDA in 0.5 m HCl (1 ml) was added to the mixture.
Absorbance was measured after 10 min at 540 nm using anAgilent Cary 60
spectrophotometer (USA). The concentration of nitrite ion was calculated
from the calibration curve plotted for sodium nitrite as a reference.
HOOCCH2(ONO2)CHCOO–
1
For further employment of compound 1 as a drug, it is critical
to investigate its biotransformation in the presence of biological
substrates in vitro and in vivo. From this point of view, an
important step is to explore the interaction of compound 1 with
thiols, in particular with cysteine (Cys), which is a component of
the catalytic centers of enzymes involved in metabolism of organic
nitrates, such as the aldehyde dehydrogenase enzyme family.8,9
Non-protein thiols are also able to reduce organic nitrates.10,11
Moreover, for all thiols including the protein ones, it has been
experimentally confirmed that during the interaction with organic
nitrates a thionitrate is formed as unstable intermediate.8,12
Its further reduction leads to release of nitrite ions,13,14 which are
‡
Kinetic experiments on the interaction of compound 1 with reducing
agents were carried out in an argon atmosphere. All vessels and quartz
cuvettes used were sealed with Rubber Septa seals (Sigma, USA). The
vessels containing phosphate buffer and weighed samples were preliminarily
purged with argon for 30 min.All the solutions employed were transferred
using syringes with soldered needles.
© 2020 Mendeleev Communications. Published by ELSEVIER B.V.
on behalf of the N. D. Zelinsky Institute of Organic Chemistry of the
Russian Academy of Sciences.
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