Mechanistic insights into the: In vitro metal-promoted oxidation of (di)azine hydroxamic acids: Evidence of HNO release and N, O -di(di)azinoyl hydroxylamine intermediate

Article abstract of DOI:10.1039/d0nj00753f

The oxidant-dependent ability of hydroxamic acids to release nitroxyl (HNO), a small inorganic molecule endowed with various biological properties, is addressed from a mechanistic standpoint. Indeed, the exact mechanism of the hydroxamic acid oxidation in physiological conditions and the direct or indirect characterization of the intermediates remain elusive. In this work, intermolecular oxidation of isonicotino-, nicotino- and pyrazino-hydroxamic acids with K3[FeIII(CN)6] at physiological pH (7.4), was monitored by 1H NMR, MS, EPR and UV-vis techniques. While nitrosocarbonyl (di)azine intermediates, (di)Az-C(O)-NO, could be a priori envisaged, it was in fact the corresponding N,O-di(di)azinoylhydroxylamines (AzC(O)NHOC(O)Az) and HNO that were identified, the first by 1H NMR and the second on the basis of EPR and UV-vis experiments using the [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] (cPTIO) spin trap. The decomposition of the unstable N,O-di(di)azinoylhydroxylamine intermediates in aqueous buffer media was shown to generate the corresponding carboxylic acids as final organic products, envisaged as possible in vivo metabolites. The same oxidation experiments performed in the presence of methylamine led to the corresponding N-methyl amides suggesting that, unlike hydroxamic acids, N,O-di(di)azinoylhydroxylamines act as acylating agents in physiological pH conditions.

Full text of DOI:10.1039/d0nj00753f

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Mechanistic insights into the in vitro metal-promoted oxidation of (di)azine
DOI: 10.1039/D0NJ00753F
hydroxamic acids: evidence of HNO release and N,O-di(di)azinoyl
hydroxylamine intermediate
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Edinilton Muniz Carvalho,a,b,c Lionel Rechignat,a,b Eduardo Henrique Silva Sousa,c Luiz Gonzaga França
Lopes,*c Remi Chauvin,a,b and Vania Bernardes-Génisson*a,b
a CNRS, Laboratoire de Chimie de Coordination, LCC, UPR 8241, 205 Route de Narbonne, BP 44099, F-
31077 Toulouse, Cedex 4, France
b
Université de Toulouse, Université Paul Sabatier, UPS, 118 Route de Narbonne, F-31062, Toulouse,
Cedex 9, France
c Laboratório de Bioinorgânica, Universidade Federal do Ceará, Departamento de Química Orgânica e
Inorgânica, Campus Pici, Fortaleza, CE 60455-760, Brazil.
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Abstract
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The oxidant-dependent ability of hydroxamic acids to release nitroxyl (HNO), a small inorganic
molecule endowed with various biological properties, is addressed from a mechanistic standpoint.
Indeed, the exact mechanism of the hydroxamic acid oxidation in physiological conditions and the
direct or indirect characterization of the intermediates remain elusive. In this work, intermolecular
oxidation of isonicotino-, nicotino- and pyrazino-hydroxamic acids with K3[FeIII(CN)6] at physiological
pH (7.4), was monitored by 1H NMR, MS, EPR and UV-vis techniques. While nitrosocarbonyl (di)azine
intermediates, (di)Az-C(O)–NO, could be a priori envisaged, it was in fact the corresponding N,O-
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di(di)azinoylhydroxylamines (AzC(O)NHOC(O)Az) and HNO that were identified, the first by NMR H
and the second on the basis of EPR and UV-vis experiments using the [2-(4-carboxyphenyl)-4,4,5,5-
tetramethylimidazoline-1-oxyl 3 oxide] (cPTIO) spin trap. The decomposition of the unstable N,O-
di(di)azinoylhydroxylamine intermediates in aqueous buffer media was shown to generate the
corresponding carboxylic acids as final organic products, envisaged as possible in vivo metabolites.
The same oxidation experiments performed in the presence of methylamine led to the formation of
the corresponding N-methyl amides suggesting that, unlike hydroxamic acids, N,O-
di(di)azinoylhydroxylamines act as acylating agents in physiological pH conditions.
Key-words: nitroxyl donors;(di)azine hydroxamic acids; Fe(III) oxidation; N,O-diaroylhydroxylamine
intermediate; physiological pH conditions.
Introduction:
Nitroxyl or azanone (HNO) is an unstable inorganic weak acid (pKa = 11.4),[1,2]
which can also be considered as the protonated form of the one-electron reduction product of the
.
nitric oxide radical (NO ). Nitroxyl displays several important biological activities,[3] remaining
however, poorly understood. One of the main pharmacological properties of HNO is its beneficial
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[4,5]
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influence on the cardiovascular system. HNO, which is indeed known to induce vasodilation,
DOI: 10.1039/D0NJ00753F
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exhibit positive inotropic/lusitropic effects[6,7] and inhibit platelet aggregation,[8] is thus envisaged to
play a key role for the treatment of patients with heart failure. HNO also exhibits an anti-angiogenic
activity, as opposed to its sibling NO that promotes angiogenesis. This HNO property has opened
many potential pharmacological applications including in cancer therapy.[9] Since HNO cannot be
directly employed in vivo due its high irreversible instability in aqueous media (dimerization and
dehydration giving nitrous oxide, N2O, Scheme 1)[1,2] and high reactivity towards nucleophilic
biomolecules (mainly with sulfur compounds),[3,10] all studies on biological properties of HNO or its
use as a therapeutic agent require the design of HNO donor compounds. Hence research into the
development of different classes of pro-drugs, with the ability to release HNO under physiological
conditions and in a tunable manner, attracts the attention of the scientific community. Among the
different classes of HNO donors,[11] one can mention the hydroxamic acid function -C(O)NHOH, which
is prone to generate HNO upon reaction with various oxidizing agents (NaIO4, metalloenzymes,
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.
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Na3[FeII(CN)5NH3]/oxidant, K3[FeIII(CN)6], radicals generated by radiolysis such as OH, N3, etc.).[12]
However, the exact mechanism of the hydroxamic acid oxidation in physiological conditions and the
direct or indirect characterization of the transient intermediates, generated by an oxidative process
in aqueous media, still constitute a matter of study.[12-14]
OH
N
O
N
O
N
N
N
N
N
2
H
O
HO
RSH
RSNHOH
Scheme 1. Reactions of HNO in aqueous or biological media.
Taking into account the potential of hydroxamic acids to act as HNO donors for therapeutic
treatments, the elucidation of their oxidation mechanism in physiological pH conditions is crucial to
improve their properties as possible pro-drugs able to release HNO in a controllable way. Recently,
we have developed a pentacyanoferrate(II) complex of pyrazinohydroxamic acid and have
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investigated in vitro its oxidation in the presence of H2O2 as well as its ability to release
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simultaneously HNO and the corresponding carboxylic acid (pyrazinoic acid) as the antituberculosis
active metabolites of the delamanid and pyrazinamide drugs, respectively.[15] It has been proposed
that oxidation of the pentacyanoferrate(II) complex of pyrazinohydroxamic acid proceeds via
intramolecular (FeIII)-mediated oxidation of the C(O)NHOH function after initial oxidation of the FeII
center into FeIII center by H2O2.
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In the present study, we examine if the direct intermolecular oxidation of isonicotino- (1),
nicotino- (2), and pyrazino- (3) hydroxamic acids promoted by potassium ferricyanide (K3[FeIII(CN)6])
at pH = 7.4 (Scheme 2) occurs and leads to the same final metabolites.
Scheme 2. Hydroxamic derivatives of isonicotinic hydrazine (isoniazid), nicotinamide, and
pyrazinamide.
2. Results and Discussion
The hexacyanoferrate(III) ion is a mild oxidizing species, which operates by abstracting one
electron from a substrate through an outer sphere (intermolecular) pathway. This reagent allows
oxidative processes[16] without atom transfer. Its use is relevant for a mechanistic study that could
corroborate our previous results on the oxidation of the pyrazinohydroxamic acid acting as a N-ligand
at a FeII(CN)53– center, for which an oxidation of the hydroxamic portion is proposed via an action of a
FeIII center generated by oxidation of the FeII center by H2O2.
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It has been reported that one-electron oxidation of hydroxamic acids leads to the unstable
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nitroxide radical RCONHO .[13,14,17] The fate of this radical is, however, uncertain: several alternative
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theoretical routes have been proposed,[14,16] and conceivable intermediates are a priori too unstable
to be fully characterized (Scheme 3). On the one hand, the nitroxide radical intermediate could
undergo a homolytic cleavage to form HNO and the aroyl radical (pathway a), or quickly dimerize,
then rearrange to a N,O-diacylhydroxylamine intermediate (pathway e and f, the latter proposed by
W. A. Waters).[12] On the other hand, it is proposed that the nitroxide radical readily undergoes a
second oxidation to give the nitrosocarbonyl derivative, for which, however, direct evidence and
complete spectral characterization in aqueous solution are lacking.[18,19] Such species are highly
reactive and can decompose by either nucleophilic addition/HNO elimination (pathway c and d), or
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homolytic cleavage releasing NO and an aroyl radical (pathway b) (Scheme 3). In the first issue, it
must be reminded that direct identification of HNO is still a real challenge. In the following are
reported mechanistic investigations of the oxidation of isonicotino- (1), nicotino- (2) and pyrazino- (3)
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hydroxamic acids by K3[Fe(CN)6] in aqueous buffer solution using H NMR, MS, EPR and UV-
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vis techniques.
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O
O
R
N
R
O
N
O
N
O
f
O
R
R
N
O
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O
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f
H2O
N-O
HN(OH)2
O
H2N2O2
O
dimerization
f
W.A.Waters, 1971
H
R
pH = 9
O
O
O
O
HNO
e
+
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R
R
O N
H
N-O
H
R
O
O
N
R
HO N
R
OH
H
H
R
NH-O
R
diacylhydroxylamine intermediate
NH-O
O
e
f
R
NH-OH
O
HNO
d
mesomerization
dimerization
R
N=O
O
O
OH
oxid
oxid
H2O
NH-OH
N-O
H
O
O
R
R
R
R
acylnitroso
1e-
+ H+
1e-
+ H+
HNO
c
nitroxide radical
intermediate
R = alkyl, aryl
.
HNO
homolytic
cleavage
a
homolytic
cleavage
NO
b
O
R
Scheme 3. Possible pathways and intermediates for the oxidation of hydroxamic acids (adapted from
ref. 13).
2.1 Monitoring of FeIII-mediated oxidation of heteroarylhydroxamic acids by 1H NMR.
Oxidation of isonicotino-, nicotino-, and pyrazino-hydroxamic acids (1 eq) by K3[Fe(CN)6] (2.5
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eq) carried out in a sodium phosphate D2O buffer solution (pH = 7.4), was monitored by H NMR
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spectroscopy for 312 h (Figure 1). H NMR spectroscopy was also used to determine the in situ
relative ratio of various products.
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NH-OH
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NH-OH
N
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N
Figure 1. 1H NMR monitoring of K3[FeIII(CN)6]-mediated oxidation of isonicotinohydroxamic acid 1 (24
mM, a) nicotinohydroxamic acid 2 (24 mM, b) and pyrazinohydroxamic acid 3 (24 mM, c) by
K3[FeIII(CN)6] (60 mM) in a sodium phosphate D2O buffer solution (pH = 7.4) at room temperature.
The spectrum at 0 h corresponds to the reaction medium before addition of K3[FeIII(CN)6].
After 1 h, 1H NMR analysis indicated a partial conversion of the starting material (50%, 56% and 38%
for 1, 2, and 3, respectively), mainly into a single primary product evidenced through a duplication of
all aromatic signals (Figure 1). Over time, the primary product tends to disappear (not completely),
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while giving another product characterized by new H NMR data: 7.61-8.70 and 7.91 ppm from 1,
8.42 and 8.68 ppm from 2, 8.70-9.04 and 9.15 ppm from 3 (Figure 1). Even though these data are
slightly shifted with respect to authentic samples of the corresponding carboxylates (Figure 2), signal
patterns remain the same, in particular showing an overlap for the two vicinal aromatic protons of
the pyrazine ring (Figure 1). These oxidative protocols were repeated using pure D2O as solvent: in
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these conditions, the reactions were found to proceed at slower rate, and the H NMR chemical
shifts measured at t = 226 h were in agreement with those of the corresponding carboxylate form
(Figure S1). These results support our proposal that the carboxylic acid is the ultimate oxidative
product - potential metabolite - of the hydroxamic acid (Figure 2).
a)
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b)
c)
Figure 2: 1H NMR spectra of the K3[FeIII(CN)6]-mediated oxidation of a) isonicotinohydroxamic acid b)
nicotinohydroxamic acid and c) pyrazinohydroxamic acid at 0.5 h and 226 h using K3[FeIII(CN)6] in D2O,
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at room temperature and authentic samples of a) isonicotinohydroxamic acid and sodium
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isonicotinoate, b) nicotinohydroxamic acid and sodium nicotinoate and c) pyrazinohydroxamic acid
and sodium pyrazinoate. Symbols: blue squares: starting hydroxamic acid, red stars: intermediate
product, pink triangle: final carboxylic acid under carboxylate salt form.
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Thus, we propose that the intermediate product, tentatively assigned to the
di(di)azinoylhydroxylamine structure of Scheme 4, is hydrolyzed to the corresponding carboxylic acid
and the starting hydroxamic acid after a nucleophilic attack at the most electrophilic carbonyl group
(Scheme 4). As indicated on the 1H NMR spectra, the amount of hydroxamic acid remains lower than
that of the carboxylic acid because the former is oxidized again by the four-fold excess of K3[Fe(CN)6],
yielding de novo the intermediate di(di)azinoylhydroxylamine product.
Scheme 4. Products suggested to appear during oxidation of (di)azine carbohydroxamic acids with a
five-fold excess of potassium ferricyanide.
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After 312 h in buffer solution (Figure 1), H NMR spectra showed essentially peaks according to the
carboxylate structure (79%, 80% and 76% yield for isonicotinoic, nicotinoic and pyrazinoic acid from
1, 2 and 3, respectively).
On the basis of these results, we postulate that oxidation of (di)azine hydroxamic acids under such
mild conditions occurs by first giving an unstable nitroxide radical, which is converted, after
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dimerization, to HNO and a N,O-di(di)azinoylhydroxylamine (Scheme 3, pathway e). The formation of
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the latter species could explain the duplication of the aromatic signals observed on 1H NMR spectra.
As already mentioned, it could also be envisaged that the nitroxide radical undergoes a second
oxidation leading to a transient nitrosocarbonyl (di)azine, which would readily react with any
available nucleophile occurring in the medium. Thereby, reaction of the nitrosocarbonyl (di)azine
with remaining hydroxamic acid would also lead to N,O-di(di)azinoylhydroxylamine and HNO
(pathway d). In these conditions, one would also expect the nitrosocarbonyl (di)azine to react with
H2O, breaking down into the corresponding carboxylic acid and HNO (pathway c). However, 1H NMR
analysis of the reaction after 1 h showed that the conversion of the starting material (see e.g. 1,
Figure 1 and 2) leads initially only to the putative N,O-di(di)azinoylhydroxylamine intermediate 4
whereas the corresponding carboxylic acid product had not yet begun to be produced. All together,
these results suggest that no nitrosocarbonyl (di)azine is involved in the formation of the N,O-
di(di)azinoylhydroxylamine and carboxylic acid. This scenario is also in total agreement with the work
carried out by A. Cerami et al. from benzene hydroxamic acid.[14]
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2.2 Identification of intermediates in FeIII-mediated oxidation of (di)azine hydroxamic acids
In an attempt to characterize the proposed intermediates, the reaction of isonicotinohydroxamic
acid was repeated at a preparative scale (0.72 mmol, using 2.5 eq FeIII(CN)6 ) and stopped after 24 h.
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The new product was isolated and then characterized by H NMR, 13C NMR and MS techniques. For
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the isonicotinic derivative, H NMR analysis showed a product exhibiting four doublets at 8.91 (d,
1H), 8.81 (d, 1H), 7.99 (d, 1H) and 7.79 (d, 1H) ppm, confirming the results of the 1H NMR monitoring
experiment. Moreover, the 13C NMR spectrum revealed indeed the presence of two different
carbonyl groups (167.7 and 166.0 ppm), four CH (149.7, 147.8, 123.4 and 122.7 ppm) and two Cq
(146.0 and 137.5 ppm) centers, which correlate with the dissymmetric N,O-
diisonicotinoylhydroxylamine structure (4). Analysis by mass spectrometry (MS, DCI-CH4) did not
allow the identification of the molecular ion peak but allowed the detection of three peaks at m/z =
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(Figure 3), respectively. The amide 13 would result from a decarboxylation of 4, while the acid 7a and
amide 10 would result from a cleavage of the hydroxamic N-O bond (Figure S2). By HRMS (ESI-
positive mode), a small peak at m/z = 244.0726 corresponding to 4 ([M+ + 1]) was detected. In the
nicotinic or pyrazinic series, a mixture of compounds was obtained, containing the intermediate 5 or
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6 along with the starting material 2 or 3 and the corresponding carboxylic acids. The H NMR
spectrum of the nicotinic intermediate showed signals clearly attributable to all the non-equivalent
aromatic CH groups of 5 (9.18, 9.00, 8.86, 8.68, 8.39, 8.18, 7.62, 7.49 ppm), while that of the
pyrazinic product 6 was slightly more speculative because of signal overlaps. The mass spectrum
(DCI-CH4) of the nicotinic products (from 2) showed, besides peaks relative to the nicotinic acid
impurity, peaks at m/z = 215, m/z = 200 and m/z = 123, attributable to the secondary amide 14 ([M +
+.
CH4 ], [M + H+]) and primary amide 11 ([M + H+]), respectively. As observed in the isonicotinic series
(from 1), in the nicotinic series (from 2) HRMS (ESI-positive mode) allowed detection of a peak
consistent with the structure 5 at m/z = 244.0726 ([M + H+]). In the pyrazinic series (from 3), MS (DCI-
+
CH4) revealed peaks at m/z = 230, 217 and 202 assignable to the secondary amide 15 ([M + C2H5 ], [M
+
+ CH3 ] and [M+ H+], respectively), and at m/z = 124 assignable to the primary amide 12 ([M + H+]).
Oxidation of 3 in the presence of K3[FeIII(CN)6] and H2O2 (conditions already used for oxidation of the
pyrazinohydroxamic acid-[FeII(CN)5 complex])15 allowed detection of the molecular ion peak of N,O-
dipyrazinoylhydroxylamine 6 by HRMS-ESI+ at m/z = 246.0619 ([M + H+]), and this in spite of the
harsher conditions. In summary, the presence of the molecular ion peaks for the
di(di)azinoylhydroxylamines 4-6 and secondary carboxamides 13-15 supports the proposed
mechanism.
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X
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13 W = CH, X = CH, Y = N, Z = CH: m/z = 199
14 W = CH, X = N, Y = CH, Z = CH : m/z = 199
15 W = N, X = CH, Y = CH, Z = N: m/z = 201
O
NH-OH
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NH-O
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O
K3[Fe(CN)6]
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NH2
O
OH
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1 W = CH, X = CH, Y = N, Z = CH: m/z = 138
2 W = CH, X = N, Y = CH, Z = CH : m/z = 138
3 W = N, X = CH, Y = CH, Z = N: m/z = 139
4 W = CH, X = CH, Y = N, Z = CH: m/z = 243
5 W = CH, X = N, Y = CH, Z = CH : m/z = 243
6 W = N, X = CH, Y = CH, Z = N: m/z = 245
W
X
W
X
Z
Y
Y
10 W = CH, X = CH, Y = N, Z = CH: m/z =122
11 W = CH, X = N, Y = CH, Z = CH : m/z = 122
12 W = N, X = CH, Y = CH, Z = N: m/z = 123
7a R = H; W = CH, X = CH, Y = N, Z = CH
7b R = Na+/K+; W = CH, X = CH, Y = N, Z = CH
8a R = H; W = CH, X = N, Y = CH, Z = CH
8b R = Na+/K+; W = CH, X = CH, Y = N, Z = CH
9a R = H; W = N, X = CH, Y = CH, Z = N
9b R = Na+/K+; W = CH, X = CH, Y = N, Z = CH
Figure 3. MS fragmentation of the proposed N,O-di(di)azinoylhydroxylamine intermediates in the
oxidation of (di)azine hydroxamic acids.
2.3 FeIII-mediated oxidation of (di)azine hydroxamic acids in the presence of methylamine.
With the view to controlling the apparent electrophilic trans-acylation reactivity of the
postulated N,O-di(di)azinoylhydroxylamine intermediates, the reaction with K3[FeIII(CN)6] was
performed in the presence of an excess of methylamine (10 eq) as a potential nucleophile. From the
three hydroxamic acids 1-3, a selective reaction was observed, allowing isolation of the N-methyl
carboxamides 16-18 with c.a. 70% yield, as confirmed by H NMR, 13C NMR, IR and HRMS analyses
1
(Scheme 5). It is noteworthy that control experiment of the hydroxamic acids 1-3 with methylamine
in the absence of K3[FeIII(CN)6] did not produce the corresponding carboxamides by elimination of
NH2OH. The requirement of K3[FeIII(CN)6] suggests the occurrence of the intermediates 4-6, as
mentioned above, which could react as an acyl donor towards methylamine.
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NH-OH
NHCH3
Z
O
O
X
NH-O
Z
O
NH-OH
Z
O
W
+
CH3NH2
HNO
K3[Fe(CN)6] (2.5 eq.)
W
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W
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O
W
X
W
X
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Y
Y
Y
Y
16 W = CH, X = CH, Y = N, Z = CH
17 W = CH, X = N, Y = CH, Z = CH
18 W = N, X = CH, Y = CH, Z = N
1 W = CH, X = CH, Y = N, Z = CH
2 W = CH, X = N, Y = CH, Z = CH
3 W = N, X = CH, Y = CH, Z = N
1 W = CH, X = CH, Y = N, Z = CH
2 W = CH, X = N, Y = CH, Z = CH
3 W = N, X = CH, Y = CH, Z = N
4 W = CH, X = CH, Y = N, Z = CH
5 W = CH, X = N, Y = CH, Z = CH
6 W = N, X = CH, Y = CH, Z = N
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HNO
CH3NH2
NHCH3
Z
O
W
X
Y
Scheme 5. Oxidation of (di)azine carbohydroxamic acids in the presence of methyl amine.
2.4 Monitoring of FeIII-mediated oxidation of (di)azine carbohydroxamic acids by EPR
In order to test whether the oxidation of hydroxamic acids promoted by K3[FeIII(CN)6], in
physiological pH condition, is accompanied by HNO release, the nitronyl nitroxide radical [2-(4-
carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3 oxide] (cPTIO) was employed. This spin trap
.
probe is currently used to discriminate between the NO radical and HNO using electronic
.
.
paramagnetic resonance (EPR).[20] The NO radical reacts with cPTIO by deoxygenation, giving NO2
and the cPTI radical (Figure S3), which exhibits a more complex EPR signal (septet pattern). In
contrast, HNO readily reduces cPTIO into the corresponding nitronyl hydroxylamine (cPTIO-H) that is
an EPR-silent diamagnetic species, inducing a decrease of the initial cPTIO EPR signal (Figure S3).
Reactions of the hydroxamic acids 1, 2 and 3 (5 mM) with K3[FeIII(CN)6] (15 mM), in
phosphate buffer (40 mM, pH = 7.4) and in the presence cPTIO (200 M), were carried out for 5.5 h
and analyzed by EPR. The stability of cPTIO in the reaction conditions was first checked by EPR: alone
or in the presence of hydroxamic acid, cPTIO was found to be stable without modification of the
signal (Figure S4a). In the presence of [FeIII(CN)6]3- or [FeII(CN)6]4-, two complex ions expected to
occur during the redox reaction, a decrease of the cPTIO EPR signal by 65% or 30%, respectively, was
observed (Figure S4a). In the reaction mixture, where hydroxamic acid, [FeIII(CN)6]3-/[FeII(CN)6]4- and
cPTIO co-exist, the EPR signal corresponding to the cPTIO radical was almost completely abolished
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(85% of reduction) after 5.5 h (Figure 4). This result disclosed that the cPTIO signal decrease is
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accelerated with the concomitant presence of hydroxamic acid and K3[FeIII(CN)6], and suggesting that
HNO is indeed released during oxidation of hydroxamic acids promoted by K3[FeIII(CN)6].
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Figure 4. EPR spectra of the reaction of a) isonicotinohydroxamic acid, b) nicotinohydroxamic acid
and c) pyrazinohydroxamic acid with K3[FeIII(CN)6] (15mM) in the presence of cPTIO (200 µM) for 5.5
h in 40 mM phosphate buffer (pH 7.4), at room temperature.
Monitoring of FeIII-mediated oxidation of (di)azine carbohydroxamic acids by UV-vis
The release of HNO during the oxidation of the (di)azine hydroxamic acids 1-3 promoted by
K3[FeIII(CN)6], in the presence of cPTIO was also studied by UV-vis spectroscopic technique. cPTIO
exhibits characteristic electronic absorptions at 360 and 560 nm. Since no other reaction component
absorbs around 560 nm (in contrast to the 360 nm region), this wavelength was chosen for
monitoring. The evolution of this band was first followed in the sole presence of K3[FeIII(CN)6] or
hydroxamic acids. No significant interaction was evidenced between [FeIII(CN)6]3-and cPTIO (Figure
S5), or hydroxamic acids and cPTIO (Figures 5D-F). In the oxidative reaction conditions (hydroxamic
acid + K3[FeIII(CN)6] + cPTIO), monitoring over 24 h showed a decline of the absorption band at 560
nm for the three substrates 1-3 (Figures 5 A-C). These observations confirm the consumption of
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cPTIO under the given conditions, while the disappearance of the EPR signal indicated that it is
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accompanied by the generation of HNO (see above section).
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Figure 5. Electronic absorption spectra of cPTIO in oxidative reaction mixtures or with hydroxamic
acids alone. Panels on the left show spectra of the reaction between isonicotino- (A), nicotino- (B)
and pyrazino- (C) hydroxamic acids (750 µM) with K3[FeIII(CN)6] (15 mM) in the presence of cPTIO
(150 µM). Panels on the right show the spectra of cPTIO (150 µM) in the presence of isonicotino- (D),
nicotino- (E) and pyrazino- (F) hydroxamic acids (750 µM). All reactions were monitored at 0 and 24 h
in 40 mM phosphate buffer (pH 7.4), at room temperature.
3. Conclusion
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In this work, it was shown that (di)azine carbohydroxamic acids generate HNO at physiological pH
5
6
7
8
upon oxidation by K3[FeIII(CN)6] through an intermolecular/outer-sphere process, as it was previously
shown upon oxidation by H2O2 through an intramolecular/inner-sphere process. However, in
contrast to what is often reported, HNO was not generated from the putative nitrosocarbonyl
(di)azine, which would be formed after two oxidation steps of hydroxamic acid. Instead, HNO is
released through a one-electron oxidation of the hydroxamic acid along with the N,O-
di(di)azinoylhydroxylamine intermediate. This intermediate was characterized by NMR, MS, UV
techniques and proved to be quite reactive, breaking down into the corresponding carboxylic acid
and hydroxamic acid, which is then re-oxidized. So, globally, one molecule of hydroxamic acid
generates one molecule of HNO. Since oxidation processes of arene hydroxamic acids going through
either nitrosocarbonyl or N,O-di(di)aroylhydroxylamine intermediates, can have similar features in
terms of (i) stoichiometry of HNO release, (ii) electrophilic reactivity of the intermediate and (iii)
chemical nature of the final carboxylic products, a particular attention should be addressed to such
mechanistic proposals. Computational studies would deserve to be considered to discriminate
between the intrinsically highly reactive nitrosocarbonyl and N,O-diaroylhydroxylamine
intermediates.
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Supplementary Information
Experimental synthesis protocols and 1H NMR, MS, UV-vis and EPR spectra of relevant species.
Conflicts of Interest
There are no conflicts to declare
Acknowledgments
We are thankful to CAPES (PROEX 23038.000936/2018-46), Capes/Cofecub (88887.130198/2017-01)
CNPq (L. G. F. Lopes 303355/2018-2; E. H. S. Sousa 308383/2018-4, Universal 403866/2016-2),
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FUNCAP (PRONEX PR2 0101-00030.01.00/15 SPU Nº: 3265612/2015) for financial support. This study
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was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -
Brasil (CAPES) - Finance Code 001.
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References
. F. Doctorovich, D. Bikiel, J. Pellegrino, S. A. Suárez, A. Larsen, M. A. Martí. Coord. Chem. Rev.
2011, 255, 2764-2784.
2. V. Shafirovich and S. V. Lymar. Proc. Natl. Acad. Sci. USA, 2002, 99, 7340-7345.
3. J. C. Irvine, R. H. Ritchie, J. L. Favaloro, K. L. Andrews, R. E. Widdop, B. K. Kemp-Harper. Trends
Pharmacol. Sci. 2008, 29, 601-608.
4. J. C. Irvine, J. L. Favaloro, B. K. Kemp-Harper. Hypertension 2003, 41, 1301-1307.
5. J. C. Irvine, J. L. Favaloro, R. E. Widdop, B. K. Kemp-Harper. Hypertension 2007, 49, 885-892.
6. N. Paolocci, T. Katori, H. C. Champion, M. E. St. John, K. M. Miranda, J. M. Fukuto, D. A. Wink, and
D. A. Kass. Proc. Natl. Acad. Sci. USA, 2003, 100, 5537-5542.
7. N. Paolocci, W. F. Saavedra, K. M. Miranda, C. Martignani, T. Isoda, J. M. Hare, M. G. Espey, J. M.
Fukuto, M. Feelisch, D. A. Wink and D. A. Kass. Proc. Natl. Acad. Sci. U S A, 2001, 98, 10463–10468.
8. E. Bermejo, D. A. Sáenz, Alberto, R. E. Rosenstein, S. E. Bari, M. A. Lazzari. Thromb. Haemost. 2005,
94, 578-584.
9. M. E. Shoman, O. M. Aly. Curr. Top. Med. Chem. 2016, 16, 2462-2470.
0. M. D. Bartberger, J. M. Fukuto, and K. N. Houk, Proc. Natl. Acad. Sci. USA 2001, 98, 2194-2198.
1 . a) A. Porcheddu, L. De Luca, G. Giacomelli. Synlett 2009, 13, 2149-2153. b) A. D. Sutton, M.
Williamson, H. Weismiller, J. P. Toscano. Org. Lett. 2012, 14, 472-475. c) X. Sha, T. S. Isbell, R. P. Patel,
C. S. Day, S. B. King. J. Am. Chem. Soc. 2006, 128, 9687-9692. d) M. E. Shoman, J. F. Du Mond, T. S.
Isbell, J. H. Crawford, A. Brandon, J. Honovar, D. A. Vitturi, C. R. White, R. P. Patel, S. B. King. J. Med.
Chem. 2011, 54, 1059-1070. e) D. A. Guthrie, A. Ho, C. G. Takahashi, A. Collins, M. Morris, J. P.
Toscano, J. Org. Chem. 2015, 80, 1338-1348. f) D. A. Guthrie, S. Nourian, C. G. Takahashi, J. P.
Toscano. J. Org. Chem. 2015, 80, 1349-1356. g) A. El-Armouche, A. Wahab, K. Wittköpper, T. Schulze,
F. Böttcher, L. Pohlmann, S. B. King, J. F. DuMond, C. Gerloff,R. H. Böger, T. Eschenhagen, L. Carrier, S.
Donzelli. Biochem. Biophys. Res. Commun. 2010, 402, 340-344.
2. T. R. Oliver and W. A. Waters. J. Chem. Soc. (B) : Phys. Org 1971, 677-681.
3. M. M. Gutiérrez, A. E. Almaraz, S.E. Bari, J. A. Olabe Iparraguirre, V. T. Amorebieta. J. Coord. Chem.
2015, 68, 3236-3246.
4. S. H. Blobstein, R. W. Grady, S. R. Meshnick and A. Cerami. Biochemical. Pharmacology 1978, 27,
2939-2945.
5. Results not yet published.
6. J. M. Leal, B. Garcia, P. L. Domingo. Coord Chem Rev. 1998. 173, 79-131.
7. U. Samuni, E. Mainon and S. Goldstein. J. Coord. Chem. 2018, 71, 1728-1737.
8. E. Maimon, A. Lerner, A. Samuni and S. Goldstein. J. Phys. Chem. 2018, 122, 7006-7013.
9. E. Maimon, A. Samuni and S. Goldstein. J. Phys. Chem. 2018, 122, 3747-3753.
20. A. A. Bobko, V. V. Khramtsov. Nitric Oxide. 2014, 40, 92-98.
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