Development of N-substituted hydroxylamines as efficient nitroxyl (HNO) donors

Article abstract of DOI:10.1021/ja2103923

Due to its inherent reactivity, nitroxyl (HNO), must be generated in situ through the use of donor compounds, but very few physiologically useful HNO donors exist. Novel N-substituted hydroxylamines with carbon-based leaving groups have been synthesized, and their structures confirmed by X-ray crystallography. These compounds generate HNO under nonenzymatic, physiological conditions, with the rate and amount of HNO released being dependent mainly on the nature of the leaving group. A barbituric acid and a pyrazolone derivative have been developed as efficient HNO donors with half-lives at pH 7.4, 37 °C of 0.7 and 9.5 min, respectively.

Full text of DOI:10.1021/ja2103923

Communication
pubs.acs.org/JACS
Development of N-Substituted Hydroxylamines as Efficient Nitroxyl
(HNO) Donors
Daryl A. Guthrie, Nam Y. Kim, Maxime A. Siegler, Cathy D. Moore, and John P. Toscano*
Department of Chemistry, 3400 North Charles Street, Johns Hopkins University, Baltimore, Maryland 21218, United States
S
* Supporting Information
Scheme 1. General Strategy for HNO Release
ABSTRACT: Due to its inherent reactivity, nitroxyl
(HNO), must be generated in situ through the use of
donor compounds, but very few physiologically useful
HNO donors exist. Novel N-substituted hydroxylamines
substituted hydroxylamines where X is a good leaving group.
Piloty’s acid and its derivatives,24 with sulfinate leaving groups,
are classic examples of this strategy. We have employed good
carbon-based leaving groups such that HNO is released along
with a stable carbanion at neutral pH.
with carbon-based leaving groups have been synthesized,
and their structures confirmed by X-ray crystallography.
These compounds generate HNO under nonenzymatic,
physiological conditions, with the rate and amount of
HNO released being dependent mainly on the nature of
the leaving group. A barbituric acid and a pyrazolone
derivative have been developed as efficient HNO donors
with half-lives at pH 7.4, 37 °C of 0.7 and 9.5 min,
respectively.
N-Hydroxycyanamide (HONHCN) is an N-substituted
hydroxylamine with a carbon-based leaving group (cyanide)
and a proposed intermediate in the oxidative bioactivation of
cyanamide (H2NCN), an alcohol deterrent used clinically in
Europe, Canada, and Japan.25−27 Cyanamide has been shown to
generate HNO, which is a potent inhibitor of aldehyde
dehydrogenase, following metabolic activation. However, N-
hydroxycyanamide is unstable and has not yet been isolated to
confirm its reactivity. In addition, evidence exists that it can be
further oxidatively metabolized to a nitrosyl cyanide inter-
mediate (ONCN) that can also generate HNO following
hydrolysis of the nitrile group to form an acyl nitroso
compound.26 An N,O-dibenzoyl derivative of N-hydroxycyana-
mide has been reported, but this precursor releases HNO not
via N-hydroxycyanamide, but presumably via an acyl nitroso
intermediate, and only following treatment with esterase or
base.28
itroxyl (HNO) has been shown to have biological activity
N
distinct from that of its redox cousin, nitric oxide (NO),
and related nitrogen oxides.1−11 Much of the recent interest in
HNO has been catalyzed by research suggesting that it may be
a novel therapeutic for the treatment of heart failure.1−5 At
neutral pH in the absence of chemical traps, HNO efficiently
dimerizes (k = 8 × 106 M−1s−1) to hyponitrous acid (HON
NOH), which subsequently dehydrates to nitrous oxide
(N2O).12 Given this inherent reactivity, HNO cannot be used
directly; donor molecules are required for the generation of
HNO in situ.
The most commonly used HNO donor is Angeli’s salt (AS)
We have synthesized and examined alternative N-substituted
hydroxylamines with carbon-based leaving groups suitable for
HNO generation at neutral pH without enzymatic activation. In
addition, these derivatives importantly avoid the release of toxic
cyanide.
(Figure 1), a compound that has been known since 1896.13
We first considered Meldrum’s acid derivative 1a, which was
expected to generate HNO as shown in Scheme 2a. This
precursor was easily synthesized without the need for
chromatographic purification by formation of the correspond-
ing bromide followed by reaction with N,O-bis(tert-butox-
ycarbonyl) hydroxylamine and subsequent acid deprotection, as
shown in Scheme 3. The other precursors reported here were
synthesized analogously, and all structures were confirmed by
X-ray crystallography. (See Supporting Information (SI) for
experimental details concerning the synthesis and character-
ization of all precursors.)
Figure 1. Previously reported classes of HNO donors.
That same year, another HNO donor, Piloty’s acid (PA), was
also reported.14 In the intervening 115 years, very few other
classes of HNO donors suitable for use under physiological
conditions have been developed.15,16 These include primary
amine-based diazeniumdiolates such as IPA/NO,17,18 acyloxy
nitroso compounds (AcON),19,20 and precursors to the acyl
nitroso compounds (AcN),21−23 which themselves are unstable.
Indeed, several recent reviews of HNO chemistry and biology
have made pleas for new donors.6−8
Compound 1a was examined for HNO generation by gas
chromatographic (GC) headspace analysis to quantify the
Herein, we are pleased to report a new class of HNO donors
based on the general strategy shown in Scheme 1 for N-
Received: November 4, 2011
Published: January 9, 2012
© 2012 American Chemical Society
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Journal of the American Chemical Society
Communication
1). Following a large-scale decomposition, the major organic
byproduct was isolated and identified by X-ray crystallography,
revealing that barbiturate 3a primarily undergoes an intra-
molecular rearrangement to compound 5 in pH 7.4 buffer
solutions (Scheme 4).
Scheme 2. Potential HNO Release Pathways from (a)
Meldrum’s Acid Derivatives 1a and 2a and (b) Barbituric
Acid Derivatives 3a and 4a
Scheme 4. Major Reaction Pathway for Barbiturate 3a
Given the positive impact that an electron-deficient O-
methyloxime group had on HNO production from Meldrum’s
acid derivatives above, we analyzed an analogous substitution
on the barbituric acid ring system. Satisfyingly, exchanging the
ethyl group in 3a with an O-methyloxime in barbiturate 4a
strongly favors the generation of HNO, as reflected by the high
yield of N2O observed following decomposition (Table 1).
HNO was confirmed as the source of N2O for this and the
other precursors examined by quenching with glutathione, a
known efficient trap for HNO.31,32
Scheme 3. Synthesis of Meldrum’s Acid Derivative 1a
The decomposition of 4a was monitored by 1H NMR
spectroscopy in PBS (pH 7.4, room temperature), and the only
detectable organic byproduct was the expected carbanion 4b
(Figure 2a). With an estimated pKa of ca. 4 (Figure 3a), the
amount of its dimerization product, N2O, formed following
decomposition in pH 7.4 phosphate buffered saline (PBS) at 37
°C. (See SI for experimental details.) Unfortunately, only trace
amounts of N2O are observed (Table 1). Meldrum’s acid (pKa
Table 1. Decomposition of HNO Donors
a
b
c
donor
% HNO
% carbanion
t1/2 (min)
d
1a
2a
3a
4a
6a
2
4
f
d
25
2
34
0.9
f
e
6
110
110
>95
>95
0.7
9.5
a
Donor compounds (0.1 mM) were incubated at 37 °C in PBS, pH
7.4. HNO yields are reported relative to the standard HNO donor,
Angeli’s salt, as determined by N2O headspace analysis (SEM 5%; n
≥ 3). N2O production was completely quenched with added
b
glutathione (0.2 mM). Yields of carbanions 1b−4b, 6b were
c
determined by 1H NMR spectroscopy. Determined from UV−vis
d
kinetic experiments. Relative to the major byproduct, acetone.
e
f
Relative to rearrangement byproduct 5. Not determined.
= 4.8)29 is completely ionized at pH 7.4, and its 5,5-dimethyl
derivative has a hydrolysis half-life of about 12 h under
physiological conditions.30 Nonetheless, the major product
Figure 2. 1H NMR analysis of the decomposition of (a) 4a to 4b and
(b) 6a to 6b in 10% D2O, PBS, pH 7.4 at room temperature. In each
case, the red spectrum was collected at the start of the experiment, and
the blue spectrum after complete decomposition. In the insert of (a),
the kinetics of decomposition are shown. The red triangles represent
the N-CH3’s (6H) and the oxime C-CH3 (3H) of 4a, and the blue
circles represent the N-CH3’s (6H) and the oxime C-CH3 (3H) of
carbanion 4b. The solid curves are calculated best fits to a single
exponential function (k = 2.4 × 10−3 s−1 for each fit). In (b), the
asterisks (*) indicate signals due to the minor anti-6b isomer.
1
observed for 1a by H NMR spectroscopy is acetone (SI),
indicative of a dominant ring-opening reaction pathway.
We next examined Meldrum’s acid derivative 2a, equipped
with an electron-deficient O-methyloxime moiety. Relative to
1a, an enhanced HNO yield is observed (Table 1), attributed to
2b being a better leaving group. However, acetone is still the
major product, indicating that the non-HNO producing ring-
opening reaction pathway remains competitive with the desired
pathway. Evidently, a more robust ring system that disfavors
alternative decomposition pathways is necessary.
byproduct is completely ionized at neutral pH. Influenced by
the O-methyloxime group, this pKa is slightly lower than that of
N,N-dimethyl barbituric acid (pKa = 4.7).33 Although the
To explore the impact of a different ring system, we
evaluated barbituric acid derivatives 3a and 4a (Scheme 2b).
Barbiturate 3a unexpectedly produced very little HNO (Table
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Journal of the American Chemical Society
Communication
Figure 3. Plot of the concentration of (a) 4b, 4b-H+, and (b) 6b, 6b-
H+ as a function of pH. In (a), the green squares represent 4b-H+
(λmax = 298 nm), and blue circles represent carbanion 4b (λmax = 261
nm). In (b), the green squares represent 6b-H+ (λmax = 270 nm),
where the last three data points were omitted due to spectral overlap,
and the blue circles represent carbanion 6b (λmax = 253 nm). See SI for
representative UV−vis spectra of 4b and 6b at high and low pH.
pharmacology of 4b has not yet been evaluated, barbituric acids
and their C5-substituted derivatives in general have an
expansive history in medicinal chemistry including hypnotic,
sedative, antiepileptic, and immunomodulation applications.34
The kinetics of decomposition from 4a to 4b was easily
monitored by UV−vis spectroscopy given the distinctive
absorbance of anion 4b (λmax = 261 nm) (Figure 4a). Analysis
of the decomposition rate as a function of pH reveals a sharp
increase near pH 8 (Figure 4b). Barbiturate 4a has a half-life of
ca. 1 min at pH 7.4 and 37 °C (Figure 4a) but is relatively
stable at pH 4.0 and room temperature, with a half-life of
approximately 12 h under these conditions (SI).
Figure 4. (a) UV−vis analysis of the decomposition of (a) 4a (time
between traces = 30 s) and (b) 6a (time between traces = 240 s) at 37
°C in PBS, pH 7.4. (c) Plot of UV−vis determined decomposition rate
as a function of pH at 25 °C for 4a (monitored at 261 nm) and 6a
(monitored at 253 nm).
Scheme 5. HNO Release from Pyrazolone Derivative 6a
To demonstrate the generality of this approach for HNO
generation, we have also examined another N-substituted
hydroxylamine with a suitable carbon-based leaving group. Like
barbiturates 3a and 4a, pyrazolone 6a (synthesized analogously
to compounds 1a−4a) also takes advantage of the formation of
an aromatic byproduct (6b) (Scheme 5) and efficiently
produces HNO with a half-life of ca. 10 min at pH 7.4, 37
°C (Table 1). Another potentially practical benefit enjoyed by
precursor 6a is that byproduct 6b, formed along with HNO, is a
4-substituted derivative of edaravone (Scheme 5), a potent
antioxidant already in clinical use for the treatment of stroke
and cardiovascular disease.35,36 Moreover, a variety of 4-
substituted edaravone analogues have also shown strong
antioxidant activity.37
The decomposition of 6a was analyzed similarly to that of 4a
by 1H NMR spectroscopy in PBS (pH 7.4, room temperature)
(Figure 2b). The only organic byproducts observed by this
analysis are the syn (major) and anti (minor) isomers of 6b
(Scheme 5), and the relative abundance of these isomers is
unchanged at high and low pH. Donors 6a, 2a, and 4a are
observed to be all syn by NMR analysis and X-ray
crystallography.
The pKa of 6b/6b-H+ is estimated to be ca. 6 (Figure 3b),
shifted below that of edaravone (pKa = 7) by O-methyloxime
substitution, indicating that nearly all of this byproduct is
ionized at pH 7.4. As was the case for precursor 4a, the
decomposition rate of pyrazolone 6a is highly pH dependent,
here with a sharp increase near pH 10 (Figure 4c); 6a is much
more stable at pH 4.0, with a half-life of about 25 h at room
temperature (SI).
The data presented on compounds 1a−4a and 6a suggest
that the ability of these N-substituted hydroxylamines to
generate HNO is based mainly on the nature of the leaving
group. To produce HNO efficiently, the driving force for the
formation of a stabilized carbanion must overcome other non-
HNO producing reaction pathways. Barbiturate 4a and
pyrazolone 6a, easily synthesized without the need for
chromatographic purification, have been developed as effective
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Journal of the American Chemical Society
Communication
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ASSOCIATED CONTENT
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Experimental procedures; compound characterization data,
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Corresponding Author
jtoscano@jhu.edu
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■
J.P.T. gratefully acknowledges the National Science Foundation
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