Nitrite anion: The key intermediate in alkyl nitrates degradative mechanism

Article abstract of DOI:10.1021/jm701390c

Alkyl nitrates are metabolized in vitro to yield nitric oxide, and thiol groups have long been considered necessary cofactors. Here, we report evidence that no reaction between thiols and alkyl nitrates takes place in vitro, but stronger reducing agents, such as iron(II) derivatives, are necessary; alkoxy radicals and nitrite anions are the reaction intermediates. The latter, in slightly acidic conditions, can nitrosate thiols to the corresponding S-nitrosothiols, the real NO releasers.

Full text of DOI:10.1021/jm701390c

3318
J. Med. Chem. 2008, 51, 3318–3321
Nitrite Anion: The Key Intermediate in Alkyl Nitrates Degradative Mechanism
Loris Grossi†
Dipartimento di Chimica Organica “A. Mangini”, UniVersita` di Bologna, I-40136 Bologna, Italy
ReceiVed NoVember 6, 2007
Alkyl nitrates are metabolized in vitro to yield nitric oxide, and thiol groups have long been considered
necessary cofactors. Here, we report evidence that no reaction between thiols and alkyl nitrates takes place
in vitro, but stronger reducing agents, such as iron(II) derivatives, are necessary; alkoxy radicals and nitrite
anions are the reaction intermediates. The latter, in slightly acidic conditions, can nitrosate thiols to the
corresponding S-nitrosothiols, the real NO releasers.
Introduction
role of thiols mediated by the corresponding S-nitrosothiols;9
i.e., the degradative process conducted in the presence of thiols
leads to the formation of the corresponding S-nitrosothiols.
However, assuming a two-electron mechanism, NO should be
formed immediately, and to perform the thiols’ nitrosation, it
should be oxidized to nitrous anhydride (N2O3).12 But this
oxygen-induced process has been shown to be irrelevant in vivo
because it is negligibly slow.13,14 In contrast, if a one-electron
mechanism is considered, the intermediate nitrite anion itself
could carry out the thiol nitrosation, a process that can take
place also in very light acidic conditions (Scheme 2).15,16
To discriminate between these hypotheses, the 5-phenyl-4-
penten-1-nitrate was first reacted with FeCl2 in the presence of
GSH in a neutral and aerated solution directly in the cell of a
UV spectrometer. In this way the formation of the S-nitroso-
glutathione could be directly detected through its UV absorption
spectrum. But no GSNO was indicated. This result indicated
against a two-electron mechanism, i.e., the direct formation of
NO, and underlined the low probability of its oxidation to N2O3.
In contrast, when the same experiment was repeated in anaerobic
conditions and at pH 6.5, the formation of GSNO was clearly
evidenced. In fact, the nitrite anion through its acidic equilibrium
is in equilibrium with the nitrous acid and the nitrous anhydride,
a well-known nitrosating species.12 This result definitely
confirmed the possibility of iron(II) derivatives to induce a
nitrate reductive process via a monoelectron transfer, leading
to alkoxy radicals and nitrite anions as intermediates.
Several troublesome aspects of organic nitrate ester therapy
are known in vivo, and one of the most serious is that patients
become refractory to its effect: tolerance. This decrease in NO
production, concomitant to the decrease of thiols’ level in tissue,
depletion,17 has been hypothesized to be affected by the enzyme
activity.3 In particular, tolerance seems to increase with the
oxidation of the enzyme, as supported by the counteraction of
supplied reducing agents.10,18–23 Furthermore, the role of the
enzyme is indirectly underlined by the absence of tolerance in
NO releasing substrates that do not require enzymes.24 To prove
if an analogous behavior could be hypothesized also in vitro,
experiments with nitrates and FeCl3 in both the absence and in
the presence of thiol derivatives were conducted. When experi-
ments were carried out in the absence of thiols, no reaction took
place. That stressed the impossibility for iron(III) derivatives
(oxidized state) to interact directly with nitrates. In contrast,
when the experiments were repeated in the presence of GSH,
cysteine, or benzylthiol, the nitrate degradative reaction did
occur.25,26 In particular, from the reaction of 5-phenyl-4-penten-
1-nitrate with FeCl3 in the presence of GSH, products 1 and 6
The role of sulfhydryl derivatives has always been underlined
by the effects of compounds such as N-acetylcysteine and
glutathione on the hemodynamic responsiveness to nitrates,
inducing a decrease in tone paralleling an increase in sulfhydryl
derivatives concentration. In fact, the direct interaction of thiols
and nitrates has usually been considered the source of NO, and
only more recently an enzymatic pathway has been taken into
account.1–5 In particular, it has been hypothesized that P450,
the enzyme most commonly involved in drug metabolism,1 takes
part in the biotransformation of organic nitrates, leading to the
formation of NO3,6 via a multi-electron mechanism. However,
the latter hypothesis contrasts with other reports7,8 that highlight
the role of thiols mediated by the formation of the corresponding
S-nitrosothiols.9,10
Results and Discussion
A definitive study on the role of thiols, i.e., if they act as
direct reducing agents or if other reductants are responsible for
the nitrate degradation, has never been reported. Thus, to
investigate this topic, experiments between glutathione, cysteine
or benzylthiol, and alkyl nitrates such as the 5-phenyl-4-pentene-
1-nitrate and the 1-penten-5-nitrate were conducted. These
experiments led to the discovery that the thiol and the nitrate
were unchanged; i.e., no reaction took place. This result,
definitely in contrast with those reported in the literature, seemed
to indicate the inability of thiols to induce directly the nitrate
reductive process. On the contrary, it suggested that a stronger
reducing agent was necessary. In particular, the behavior of P450
in vivo6 supported the hypothesis that iron(II) derivatives could
succeed. Thus, experiments with FeCl2 and the 5-phenyl-4-
penten-1-nitrate or the 5-nitrate-1-penten, in anhydrous solvents,
were conducted. As a matter of fact, the formation of 1-5 can
be accounted for only through a radical mechanism (Scheme
1). In particular, the intermediate alkoxy radicals, via a 1,2- or
a 1,5-exo ring closure process, led to the formation of carbon
centered radicals, which are precursors of these species, are well
known markers of radical mechanisms (Table 1).11 These results
proved the capability of iron(II) derivatives to induce the
degradative nitrate reduction via an electron transfer but not
which mechanism is involved (Scheme 2).
One of the most accredited hypotheses for the NO production,
contrasting with that usually acknowledged,7,8 highlights the
†
Contact information. Phone: +390512093627. Fax: +390512093654.
E-mail: loris.grossi@unibo.it.
10.1021/jm701390c CCC: $40.75
2008 American Chemical Society
Published on Web 04/29/2008

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3319
Scheme 1. Electron Transfer Process: Radical Intermediates
Table 1. Reaction Products: Markers of the Radical Process
were detected, species corresponding to those obtained when
the 5-phenyl-4-penten-1-nitrate was reacted with FeCl2 alone
(Table 1). This confirmed the role of iron(II) in the nitrate
reductive process, and its formation is accounted for via the
reduction of iron(III) carried out by reducing species such as
thiols. The role of antioxidants (thiols) was also stressed by
results obtained in experiments conducted at steady concentra-
tion of nitrate and variable thiol/iron(III) molar ratios; these
showed the highest nitrate degradation yield when a 2.5:1 thiol/
iron(III) molar ratio was used, i.e., conditions in which the
conversion of iron(III) into iron(II) is favored (Scheme 3).27,28
These in vitro results led to the hypothesis of a parallel behavior
of P450 in vivo. In fact, in the cell, the P450-iron(II), acting as
nitrate reducing agent, turns into P450-iron(III), but in order for
the enzyme to continue its function, the P450-iron(III) must be
reconverted into P450-iron(II), and this can be performed by thiols.
In fact, cysteine and GSH present in the cell in high concentration
could succeed.
Conclusion
The degradative NO release from alkyl nitrates starts from a
one-electron transfer process between the nitrate, the oxidant,

3320 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Scheme 2. Mechanism of Degradation of Nitrates
Brief Articles
Scheme 3. Reduction of Nitrates: Role of Thiols
106, 105, 77, 51, 28], 2 [GC-MS ) (M+) 182, 105, 77, 51, 28]
and 3a (H-derivative) [GC-MS ) (M+) 161, 91, 71]. When the
experiment was conducted in the presence of a Br atom donor
(CBr3Cl or N-bromosuccinimide), 1, 2, 3b (Br derivative) [GC-MS
) (M+) 240, 171, 160, 91, 71] and 4a (Br derivative) [GC-MS )
(M+) 240, 160, 133, 117, 105, 91, 77] were detected. When a Cl
atom donor (SO2Cl2) was used, 1 and 4b (Cl derivative) [GC-MS
(M+) 196, 160, 131, 115, 105, 91, 77] were detected. When the
5-nitrate-1-pentene (0.15 mmol) was reacted with ferrous chloride
(0.3 mmol) in anhydrous acetonitrile (5.0 mL), besides unreacted
nitrate and the corresponding alcohol, the compound 5 [GC-MS
) (M+) 86, 71, 56] was detected.
When a cis/trans (90:10) mixture of the 5-phenyl-4-penten-1-
nitrate (1.0 mmol), ferric chloride (1.6 mmol), and GSH (3.0 mmol)
was dissolved in a 50:50 anhydrous acetonitrile/methanol solution
(10.0 mL), carefully deoxygenated under N2 atmosphere, and stirred
at room temperature for 72, besides the corresponding alcohol and
the oxidized glutathione, 1 and 6 [ES-MS 199 [M + Na]+; GC-MS
) (M+) 176, 148, 105,77, 71] were detected.
and an iron(II) derivative, the reductant. This mechanism is
supported by experiments that led to the detection of products
that unquestionably sustained a radical mechanism, i.e., that can
be accounted for only via an electron transfer process. The
nitrate degradative process, besides alkoxy radicals, led to the
formation of nitrite anions, an inactive NO derivative, which
in slightly acidic conditions, for example, like in ischemic
conditions, is able to induce the nitrosation of thiols to the
corresponding S-nitrothiols, the real NO suppliers. These results
led to a hypothesis of a parallel behavior with P450, i.e., the
role of the iron(II)/iron(III) redox couple and thiols inside the
cell, in support of the intermediacy of S-nitrosthiols. It follows
that intracellular thiols, in particular cysteine and GSH, being
involved in the fundamental processes, will decrease in con-
centration over time. This accounts for depletion and for less
efficiency in re-establishing the enzyme reducing capability,
tolerance.
Acknowledgment. This work was financially supported by
the Ministero dell’Istruzione, dell’Universita` e della Ricerca
(MIUR), Rome, Funds PRIN 2006. I thank L. A. Winter for
helpful discussions.
Experimental Section
The 5-phenyl-4-penten-1-nitrate was synthesized from the parent
5-phenyl-4-penten-1-ol as reported29 and reacted with N-bromo-
succinimide to lead to the 5-phenyl-4-penten-1-bromo, which under
the action of silver nitrate leads to the nitrate. The 5-nitrate-1-
pentene was obtained by reacting the 5-bromo-1-pentene, com-
mercial grade, with silver nitrate. All the other reagents were
commercial products and used as received. In particular, solvents
such as acetonitrile, methanol, and methylene chloride, and ferrous
and ferric chloride were carefully kept anhydrous. A buffer
phosphate solution at pH 6.5 was used for some UV experiments.
Nitrates Degradative Reactions. All the reactions were con-
ducted under nitrogen atmosphere and at thermostatted temperature
(ranging between 37 and 60 °C). After the workup, the products
were identified using standard techniques (NMR, GC-MSa) and
compared with data reported in the literature. The formation of
S-nitrosothiols was from two solutions containing the nitrate/thiol
mixture and the iron(II) derivative and from reaction directly in a
flat cell inside the spectrometer by a mixing flow system. The
presence of the characteristic S-NO absorption band at λ ) 540
nm (ꢀmax ) 16.34 M-1 cm-1) was followed. The reaction yield
was more then 75% in comparison to the reacted nitrate for all the
experiments. In particular, a cis/trans (90:10) mixture of the
5-phenyl-4-penten-1-nitrate (0.35 mmol) was dissolved in anhydrous
CH3CN or CH2Cl2, (4.0 mL) and was deoxygenated by bubbling
of N2 for 1 h. To this solution ferrous chloride (0.35 mmol) was
added, and the solution was stirred at 60 °C for 60 min under N2
atmosphere. The workup of the reaction allowed us to identify,
besides the corresponding alcohol, the products 1 [GC-MS ) (M+)
Supporting Information Available: UV-vis spectra of iron(II)
and iron(III) in the presence and in the absence of the nitrite anion.
This material is available free of charge via the Internet at http://
pubs.acs.org.
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JM701390C