Nitrosocarbonyl release from O-substituted hydroxamic acids with pyrazolone leaving groups

Article abstract of DOI:10.1016/j.tet.2016.08.016

A new class of nitrosocarbonyl precursors, O-substituted hydroxamic acids with pyrazolone leaving groups (OHPY), is described. These compounds generate nitrosocarbonyl intermediates, which upon hydrolysis release nitroxyl (azanone, HNO) under physiologically relevant conditions. Pyrazolones have been used to confirm the generation of nitrosocarbonyls by competitive trapping to form isomeric N-substituted hydroxamic acids (NHPY) via an N-selective nitrosocarbonyl aldol reaction. The rate of nitrosocarbonyl release from OHPY donors is impacted by donor substituents, including the pyrazolone leaving group.

Full text of DOI:10.1016/j.tet.2016.08.016

Tetrahedron 72 (2016) 6037e6042
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Nitrosocarbonyl release from O-substituted hydroxamic acids with
pyrazolone leaving groups
*
Saghar Nourian, Robert P. Lesko, Daryl A. Guthrie, John P. Toscano
Department of Chemistry, 3400 North Charles Street, Johns Hopkins University, Baltimore, MD 21218, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 June 2016
Received in revised form 31 July 2016
Accepted 4 August 2016
Available online 5 August 2016
A new class of nitrosocarbonyl precursors, O-substituted hydroxamic acids with pyrazolone leaving
groups (OHPY), is described. These compounds generate nitrosocarbonyl intermediates, which upon
hydrolysis release nitroxyl (azanone, HNO) under physiologically relevant conditions. Pyrazolones have
been used to confirm the generation of nitrosocarbonyls by competitive trapping to form isomeric N-
substituted hydroxamic acids (NHPY) via an N-selective nitrosocarbonyl aldol reaction. The rate of ni-
trosocarbonyl release from OHPY donors is impacted by donor substituents, including the pyrazolone
leaving group.
This article is dedicated to Professor Gary H.
Posner in appreciation of his career
contributions to organic chemistry
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Nitrosocarbonyl
Nitroxyl
HNO
Pyrazolone
1. Introduction
life.11 Derivatization of this inorganic salt has been unsuccessful to
date, thus preventing modification for tunable HNO release. Piloty’s
Nitroxyl (azanone, HNO), the one-electron reduced and pro-
tonated form of nitric oxide, has been shown to improve both
vasorelaxation and myocardial contractility, making HNO donors
ideal candidates for drug development.1e9 HNO is a reactive mol-
ecule that spontaneously dimerizes to produce hyponitrous acid
(HON]NOH), which then dehydrates to give nitrous oxide (N2O).10
Due to this inherent reactivity, HNO must be generated in situ
through the use of donor compounds.
acid (PA) derivatives and acyloxy nitroso compounds (AcON) rep-
resent other types of HNO donors that have been developed with
tunable half-lives.12e17 Our group has recently reported two new
series of HNO donors with half-lives that can be varied from min-
utes to hours under physiological conditions: (hydroxylamino)
pyrazolone (HAPY) and (hydroxylamino)barbituric acid (HABA)
derivatives.18e20 In addition to these examples, the continued de-
velopment of efficient HNO donors is important to expand the re-
search tools available to understand the potential role of HNO in
biological processes.
Angeli’s salt (Na2N2O3, AS, Fig. 1) is a well-known donor that
generates HNO under physiological conditions with a short half-
Fig. 1. Some previously reported HNO donors.
Another strategy to release HNO is based on the hydrolysis of
nitrosocarbonyl intermediates.21,22 Nitrosocarbonyls are highly
* Corresponding author. Fax: þ1 410 516 8420; e-mail address: jtoscano@jhu.edu
(J.P. Toscano).
http://dx.doi.org/10.1016/j.tet.2016.08.016
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.

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S. Nourian et al. / Tetrahedron 72 (2016) 6037e6042
reactive species that can react with nucleophiles including water to
generate HNO. Oxidation of hydroxamic acids and thermal de-
composition of 9,10-dimethylanthracene adducts represent com-
mon approaches to nitrosocarbonyl generation.23e25 The photolysis
of nitrodiazo compounds, nitronates with alpha leaving groups,
and 1,2,4-oxadiazole-4-oxides have also been shown to generate
nitrosocarbonyls efficiently.26e28 Recently, the aerobic oxidation of
hydroxamic acids by metal catalysts under mild conditions has
been developed as an efficient strategy for nitrosocarbonyl gen-
eration.29e46 In general, however, the above methods are not
suitable for HNO generation under physiological conditions.
Herein, we report a novel class of nitrosocarbonyl donors that
upon deprotonation and loss of the leaving group (Scheme 1,
HX¼pyrazolone) generate nitrosocarbonyl intermediates that can
hydrolyze to release HNO under physiological conditions. As has
been demonstrated in recent reports,33,35,36,40e42,45 nitro-
socarbonyls can react with nucleophiles through an N-selective
nitrosocarbonyl aldol reaction to produce N-substituted hydroxa-
mic acid adducts. We have recently found that pyrazolones are
efficient traps for nitrosocarbonyl intermediates to generate N-
substituted hydroxamic acid derivatives with pyrazolone leaving
groups (NHPY) in a reversible manner.47 In the current work, we
observe OHPY decomposition to generate nitrosocarbonyls which
further react with pyrazolones to produce isomeric NHPY com-
pounds (Scheme 1). We have synthesized and studied NHPY
compounds independently, and have recently demonstrated the
efficient formation of nitrosocarbonyl intermediates upon de-
composition of these compounds.47
structure was confirmed by X-ray crystallography (Supplementary
data). The other precursors were purified using column
chromatography.
The OHPY and NHPY isomers can obviously be distinguished by
X-ray crystallography. An analysis of 13C NMR data and available
crystal structures (Supplementary data), reveal that OHPY and
NHPY compounds have distinctive chemical shifts for their qua-
ternary carbons (
d
¼87.6e90.2 ppm for OHPY vs ¼68.4e76.5 ppm
d
for NHPY). Thus, 13C NMR spectroscopy conveniently allows the
two isomers to be distinguished if crystal structures cannot be
obtained.
2.2. Decomposition of OHPY compounds
Nitrosocarbonyl formation following OHPY decomposition un-
der physiological conditions was studied. As described above, the
reaction of nitrosocarbonyls and pyrazolones to produce NHPY
compounds can be efficient. Upon nitrosocarbonyl generation from
OHPY precursors 1, therefore, a competition exits between hydro-
lysis to generate HNO and carboxylic acid 4 (Scheme 3, Path A) and
trapping by the pyrazolone byproduct 2 to produce NHPY com-
pounds 3 (Scheme 3, Path B). NHPY compounds subsequently can
also release nitrosocarbonyl intermediates with half-lives that de-
pend on R1, R2, and R3 (Scheme 3, Path C).47
1H NMR spectroscopy was used to examine the decomposition
of OHPY donors and measure relative product yields in aqueous
solution (Table 1).19,20 Pyrazolone 2, NHPY 3, and carboxylic acid 4
are cleanly formed as the only observable organic products. Be-
Scheme 1. Reactivity of OHPY nitrosocarbonyl precursors.
2. Results and discussion
2.1. Synthesis
cause NHPY compounds 3a, 3c, and 3d all have half-lives on the
order of days,47 Path C in Scheme 3 does not contribute to the ob-
served chemistry for OHPY donors 1a, 1c, and 1d. Thus, the relative
yields of pyrazolone 2 and carboxylic acid 4 compared with that for
NHPY compound 3 in Table 1 reflect the competition between Path
A and Path B for these donors.
OHPY decomposition was also monitored by UVevis spectros-
copy to measure donor half-lives at pH 7.4 and 37 ꢀC (Table 1). Based
on the chemistry of related HNO donors,19,20 we propose that the
first step of decomposition is deprotonation which leads to release
of nitrosocarbonyl plus pyrazolone. If the barrier to dissociation
from anionic OHPY is very small, observed half-lives should corre-
late with donor pKa and the pyrazolone leaving group.19,20 Pyr-
azolone 2a (R1¼Ph, R2¼(C(¼NOMe)Me, pKa¼6) is a better leaving
group than pyrazolone 2b (R1¼Ph, R2¼Me, pKa¼7.6),19 consistent
with the much shorter half-life for OHPY 1a (t1/2¼25 min) compared
with 1b (stable). Exchanging the R1 group from phenyl to methyl
(OHPY 1c vs 1d) increases the half-life by a factor of two, consistent
with that previously reported for analogous HAPY and NHPY do-
nors.19,47 A comparison of the known pKa values of the related
OHPY compounds 1 have been synthesized by formation of the
corresponding bromide (Br-PY) followed by reaction with
hydroxamic acids (Scheme 2). Initially, OHPY 1a was synthesized
without the need for chromatographic purification, and its
Scheme 2. Synthesis of OHPY derivatives.

S. Nourian et al. / Tetrahedron 72 (2016) 6037e6042
6039
Scheme 3. Reactivity of OHPY donors 1.
Table 1
consistent with the corresponding yields of pyrazolone 2 and car-
boxylic acid 4 observed under the same conditions.
Product yields and half-lives for OHPY donors
OHPY
R1
R2
R3
%2a
74
%3a
26
%4a
73
t1/2 (min)b
The competition between nitrosocarbonyl hydrolysis to HNO
(Path A) and pyrazolone trapping (Path B) depends on the relative
concentration of pyrazolone. Lower concentrations of pyrazolone 2
following decomposition of lower concentrations of OHPY 1 will
disfavor NHPY formation and correspondingly increase the yield of
HNO. Determination of HNO yields at different concentrations of
donors (Table 2), confirms that the yield of HNO is impacted as
expected.
1a
1b
1c
1d
1e
Ph
Ph
Ph
Me
Ph
C(¼NOMe)Me
Me
C(¼NOMe)Me
C(¼NOMe)Me
C(¼NOMe)Me
Me
Me
MeO
MeO
t-BuO
25
Stablec
0.6
28
72
27
14
86
14
1.2
d
d
d
d
a
Relative yields determined from 1H NMR analysis of the complete de-
composition of 0.5 mM of the OHPY donor in 10% DMSO-d6, 10% D2O, and 80% H2O,
phosphate buffer (0.25 M) containing 0.2 mM of the metal chelator, diethylene-
triaminepentaacetic acid (DTPA), pH 7.4 at 37 ꢀC under argon.
b
Determined by UVevis spectroscopy.
Less than 5% decomposition after 2 days.
Not Determined due to low solubility.
2.3. Mechanistic studies
c
d
Our proposed mechanism for OHPY decomposition involves
deprotonation followed by initial formation of a nitrosocarbonyl
intermediate. Pyrazolone 2b has recently been demonstrated to be
an efficient trap for nitrosocarbonyls under physiological condi-
tions.47 To confirm the formation of nitrosocarbonyls upon OHPY
decomposition, we incubated OHPY 1a (0.5 mM) in presence of
pyrazolone 2b (0.5 mM) in pH 7.4 phosphate buffer at 37 ꢀC (Scheme
4). Upon decomposition of donor 1a under these conditions, we
observe quantitative formation of NHPY 3b (Supplementary data),
strong evidence for nitrosocarbonyl formation.
compounds, PhC(O)NHOH (8.8) and PhOC(O)NHOH (10.0),48 in-
dicates that the pKa of OHPY 1a is likely lower than that of OHPY 1c.
Although these two donors have the same pyrazolone leaving group,
the longer half-life for OHPY 1a (t1/2¼25 min) compared with 1c (t1/
¼0.6 min) suggests that the barrier to dissociation may also affect
2
the observed half-lives. In the case of NHYP donors, the dissociation
barrier was found to be strongly dependent on the R3 group.47 The
origin and impact of the R3 group on OHPY dissociation requires
further investigation.
In the absence of pyrazolone 2b, the decomposition of OHPY 1a
provides NHPY 3a (26%) (which has a half-life of 5 days), and HNO
(70%) and acetate (73%) (Scheme 4 and Tables 1 and 2). The stability
of NHPY 3a, along with the observation that it is not formed in the
presence of pyrazolone 2b, also argues against the possibility of
a direct intramolecular rearrangement from anionic OHPY 1a to
NHPY 3a without involvement of a nitrosocarbonyl intermediate.
Based on reactivity studies of bis-heteroatom-substituted am-
ides,49 the formation of NHPY 3b may be possible through the di-
rect attack of pyrazolone 2b on the amide nitrogen of the
hydroxamic acid moiety of NHPY 3a (Scheme 5). To examine this
possibility, NHPY 3a was incubated with pyrazolone 2b and its
stability was confirmed by 1H NMR spectroscopy at pH 7.4 and 10.
The pKa of NHPY 3a has recently been measured to be 10.0 and its
stability at or above pH 10 suggests a relatively high barrier for
nitrosocarbonyl formation from this NHPY donor even when
deprotonated.47
HNO generation was examined by gas chromatographic (GC)
headspace analysis to quantify the amount of its dimerization
product, N2O, formed following decomposition of OHPY donors in
pH 7.4 phosphate buffer solutions at 37 ꢀC (Table 2). The amount of
HNO release was measured relative to standard HNO donor,
Angeli’s salt. As expected, HNO yields from OHPY donors are
Table 2
HNO yields for OHPY donors at different concentrations
OHPY
% HNOa
500
m
M
100
m
M
20 mM
1a
1b
1c
1d
1e
70
75
91
b
b
b
20
29
17
26
41
29
8
c
35d
a
HNO yields are measured after complete decomposition of the donor and are
reported relative to the standard HNO donor, Angeli’s salt, as determined by N2O
headspace analysis (SEMꢁ5%; n¼3).
3. Conclusions
b
Stable.
c
The OHPY class of nitrosocarbonyl precursors efficiently gener-
ates these intermediates under physiological conditions following
Not determined due to low solubility.
t1/2¼3.2 min (measured using UVevis spectroscopy).
d

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S. Nourian et al. / Tetrahedron 72 (2016) 6037e6042
Scheme 4. Reactivity of OHPY 1a in the presence of pyrazolone 2b.
O-methoxyoxime)-4-bromo-3-methyl-1-phenyl-pyrazolone,
4-
bromo-3,4-dimethyl-1-phenyl-pyrazolone, 4-(acetyl-O-methox-
yoxime)-4-bromo-1,3-dimethyl-pyrazolone,18 C-methoxycarbohy-
droxamic acid, and tert-butylhydroxycarbamate50 were prepared
according to literature procedures. Acetohydroxamic acid was
purchased and used without further purification. NMR spectra
were obtained on a 400 MHz FT-NMR spectrometer. All chemical
shifts are reported in parts per million (ppm) relative to residual
CHCl3 (7.26 ppm for 1H, 77.23 ppm for 13C). High-resolution mass
spectra were collected on a magnetic sector mass spectrometer
working in fast atom bombardment (FAB) mode. Gas chromatog-
raphy (GC) headspace analysis was performed on the instrument
equipped with ECD detection and a molecular sieve packed column.
Ultravioletevisible (UVevis) absorption spectra were collected
using a diode array spectrophotometer.
4.2. Synthesis
Scheme 5. The possible reaction of NHPY 3a with pyrazolone 2b.
4.2.1. General procedure for the synthesis of N-((4-(acetyl-O-me-
thoxyoxime)-3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-
yl)oxy)-acetamide (OHPY 1a) and N-((3,4-dimethyl-5-oxo-1-phenyl-
4,5-dihydro-1H-pyrazol-4-yl)oxy)-acetamide (OHPY 1b). To a solu-
tion of acetohydroxamic acid (1 mmol) in dimethylformamide
(3 mL) at room temperature was added sodium hydride, 60%
(1.1 mmol), and the reaction stirred for one hour. This solution was
added to a solution of 1 mmol of brominated pyrazolone (4-(acetyl-
deprotonation and loss of pyrazolone. The nitrosocarbonyl pro-
duced is subsequently hydrolyzed to HNO in competition with
trapping by the pyrazolone byproduct via an N-selective nitro-
socarbonyl aldol reaction to form an isomeric NHPY compound. The
rate of nitrosocarbonyl release from OHPY compounds in aqueous
solution is dependent on the pyrazolone leaving group. This rate is
also influenced by the R3 substituent, an effect that is under current
investigation.
O-methoxyoxime)-4-bromo-3-methyl-1-phenyl-pyrazolone
for
the synthesis of OHPY 1a and 4-bromo-3,4-dimethyl-1-phenyl-
pyrazolone for the synthesis of OHPY 1b) in dimethylformamide
(2 mL), and the reaction proceeded at room temperature for 3 h.
The reaction was diluted with ether (20 mL) and washed with
ammonium chloride, water, and brine. The organic phase was
collected, dried over magnesium sulfate (MgSO4), and concentrated
in vacuo. Recrystallization from dichloromethane and hexane gave
the product OHPY 1a (25% yield) and, following purification by
flash chromatography (30% ethylacetate/hexane) on silica gel,
OHPY 1b (28% yield).
4. Experimental section
4.1. Method and materials
All starting materials were of reagent grade and used without
further purification. N-hydroxy-N-(4-(acetyl-O-methoxyoxime)-3-
methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)-N-acet-
amide (NHPY 3a), N-hydroxy-N-(4-(acetyl-O-methoxyoxime)-3-
methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)-N-methyl-
carbamate (NHPY 3c), N-hydroxy-N-(4-(acetyl-O-methoxyoxime)-
1,3-dimethyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)-N-methyl-
carbamate (NHPY 3d),47 4-(acetyl-O-methoxyoxime)-3-methyl-1-
phenyl-pyrazolone 2a, 3,4-dimethyl-1-phenyl-pyrazolone 2b, 4-
(acetyl-O-methoxyoxime)-1,3-dimethylpyrazolone 2c,19 4-(acetyl-
4.2.2. General procedure for the synthesis of N-((4-(acetyl-O-me-
thoxyoxime)-3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-
yl)oxy)-methylcarbamate (OHPY 1c), N-((4-(acetyl-O-methoxyox-
ime)-1,3-dimethyl-5-oxo-4,5-dihydro-1H-pyrazol-4-yl)oxy)-methyl-
carbamate (OHPY 1d), and N-(4-(acetyl-O-methoxyoxime)-3-methyl-
5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)oxy)-tert-

S. Nourian et al. / Tetrahedron 72 (2016) 6037e6042
6041
butylcarbamate (OHPY 1e). To a solution of 1 mmol of hydroxamic
acid (C-methoxycarbohydroxamic acid for the synthesis OHPY 1c
and 1d, and tert-butylhydroxycarbamate for the synthesis of OHPY
1e) in acetonitrile (3 mL) at room temperature was added trie-
thylamine (1 mmol), and the reaction stirred for one hour. This
solution was added to a solution of 1 mmol of brominated pyr-
solid; 1H NMR (400 MHz, CDCl3)
d
: 7.52 (1H, s, OH), 3.89 (3H, s, Me),
3.79 (3H, s, Me), 3.28 (3H, s, Me), 2.27 (3H, s, Me), 2.03 (3H, s, Me).
13C NMR (100 MHz, CDCl3)
d: 168.6, 158.3, 157.6, 151.1, 88.9, 62.5,
53.3, 31.4, 15.0, 10.3. HRMS (FAB): found m/z¼273.12013 (MHþ);
calcd for C10H17N4O5: 273.11989.
azolone
4-(acetyl-O-methoxyoxime)-4-bromo-3-methyl-1-
4.3.5. N-((4-(Acetyl-O-methoxyoxime)-3-methyl-5-oxo-1-phenyl-
phenyl-pyrazolone for the synthesis of OHPY 1c and 1e, and 4-
(acetyl-O-methoxyoxime)-4-bromo-1,3-dimethyl-pyrazolone for
the synthesis of OHPY 1d) in acetonitrile (2 mL), and the reaction
proceeded at room temperature for 3 h. The reaction mixture was
concentrated via rotary evaporation, redissolved in dichloro-
methane, and washed with water and brine. The organic phase was
collected, dried over MgSO4, and concentrated in vacuo. The com-
pound was purified by flash chromatography (20% ethylacetate/
hexane) on silica gel to give OHPY 1c, 1d, and 1e with yields of 34%,
28%, and 37%, respectively.
4,5-dihydro-1H-pyrazol-4-yl)oxy)-tert-butylcarbamate
(OHPY
1e). White solid, mp 102e104 ꢀC; 1H NMR (400 MHz, CDCl3)
d
:
7.94e7.92 (2H, m, arom), 7.43e7.40 (2H, m, arom), 7.31 (1H, s, OH),
7.23e7.21 (1H, m, arom), 3.91 (3H, s, Me), 2.36 (3H, s, Me), 2.06 (3H,
s, Me), 1.46 (1H, s, C(Me)3). 13C NMR (100 MHz, CDCl3)
d: 167.3,
159.4, 156.1, 151.2, 137.8, 129.2, 125.3, 118.4, 89.8, 82.9, 62.4, 28.0,
15.1, 10.2. HRMS (FAB): found m/z¼377.18180 (MHþ); calcd for
C18H25N4O5: 377.18250.
4.3.6. N-Hydroxy-N-(3,4-dimethyl-5-oxo-1-phenyl-4,5-dihydro-1H-
pyrazol-4-yl)-N-acetamide (NHPY 3b). White solid; 1H NMR
4.2.3. Synthesis of N-hydroxy-N-(3,4-dimethyl-5-oxo-1-phenyl-4,5-
dihydro-1H-pyrazol-4-yl)-N-acetamide (NHPY 3b). To a solution of
acetohydroxamic acid (210 mg, 2.80 mmol) and pyrazolone 2b
(105 mg, 0.56 mmol) in 50% aqueous ethanol (7 mL), was added
potassium carbonate (12 mg, 0.09 mmol) to adjust the pH to 7e8.
Sodium periodate (599 mg, 2.80 mmol) was added to the reaction
mixture, which was sonicated for 10 min, and then stirred for 3 h at
room temperature. The reaction mixture was diluted with ethanol
(12 mL) and the solid was filtered. The filtrate was concentrated via
rotary evaporation and the resulting solid was redissolved in eth-
ylacetate (50 mL) and washed three times with a saturated solution
of ammonium chloride (30 mL). The organic phase was collected,
dried over MgSO4, and concentrated in vacuo. Recrystallization from
dichloromethane and hexane gave the title compound (73% yield).
(400 MHz, CDCl3)
7.45e7.43 (2H, m, arom), 7.24e7.22 (1H, m, arom), 2.18 (3H, s, Me),
2.10 (3H, s, Me), 1.68 (3H, s, Me). 13C NMR (100 MHz, CDCl3)
: 167.8,
159.0, 157.2, 137.5, 127.9, 125.8, 119.5, 72.3, 22.5, 19.2, 12.4. HRMS
(FAB): found m/z¼262.11917 (MHþ); calcd for C13H15N3O3:
262.11981.
d: 9.08 (1H, s, OH), 7.87e7.85 (2H, m, arom),
d
Acknowledgements
We gratefully acknowledge the National Science Foundation
(CHE-1213438) and Cardioxyl Pharmaceuticals for generous sup-
port of this research. We also thank the Johns Hopkins Department
of Chemistry Facility Managers, Dr. Maxime A. Siegler (X-ray
Crystallography), and Dr. I. Phil Mortimer (Mass Spectrometry) for
assistance with compound identification and characterization.
4.3. Compound characterization
Supplementary data
4.3.1. N-((4-(Acetyl-O-methoxyoxime)-3-methyl-5-oxo-1-phenyl-
4,5-dihydro-1H-pyrazol-4-yl)oxy)-acetamide (OHPY 1a). Colorless
Supplementary data (Crystallographic data of OHPY 1a (CCDC
1495947) and NHPY 3b (CCDC 1495948) have been deposited at the
Cambridge Crystallographic Database Centre. These data can be
obtained for free via https://www.ccdc.cam.ac.uk/) related to this
article can be found in the online version, at http://dx.doi.org/
10.1016/j.tet.2016.08.016.
crystals, mp 143e145 ꢀC; 1H NMR (400 MHz, CDCl3)
d: 8.96 (1H, s,
OH), 7.85e7.83 (2H, m, arom), 7.41e7.38 (2H, m, arom), 7.22e7.20
(1H, m, arom), 3.90 (3H, s, Me), 2.34 (3H, s, Me), 2.06 (3H, s, Me),
1.92 (3H, s, Me). 13C NMR (100 MHz, CDCl3)
d: 167.5, 158.1, 156.7,
150.8, 137.8, 129.1, 125.7, 119.1, 90.2, 62.5, 20.0, 16.4, 10.2. HRMS
(FAB): found m/z¼319.14094 (MHþ); calcd for C15H19N4O4:
319.14063.
References and notes
4.3.2. N-((3,4-Dimethyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-
1. Paolocci, N.; Saavedra, W. F.; Miranda, K. M.; Martignani, C.; Isoda, T.; Hare, J.
M.; Espey, M. G.; Fukuto, J. M.; Feelisch, M.; Wink, D. A.; Kass, D. A. Proc. Natl.
Acad. Sci. U.S.A. 2001, 98, 10463e10468.
2. Paolocci, N.; Katori, T.; Champion, H. C.; St. John, M. E.; Miranda, K. M.; Fukuto,
J. M.; Wink, D. A.; Kass, D. A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5537e5542.
3. Dai, T.; Tian, Y.; Tocchetti, C. G.; Katori, T.; Murphy, A. M.; Kass, D. A.; Paolocci,
N.; Gao, W. D. J. Physiol. 2007, 580, 951e960.
4. Tocchetti, C. G.; Wang, W.; Froehlich, J. P.; Huke, S.; Aon, M. A.; Wilson, G. M.; Di
Benedetto, G.; O’Rourke, B.; Gao, W. D.; Wink, D. A.; Toscano, J. P.; Zaccolo, M.;
Bers, D. M.; Valdivia, H. H.; Cheng, H.; Kass, D. A.; Paolocci, N. Circ. Res. 2007,
100, 96e104.
yl)oxy)-acetamide (OHPY 1b). White solid, mp 142e144 ꢀC; 1H NMR
(400 MHz, CDCl3)
7.44e7.41 (2H, m, arom), 7.23e7.25 (1H, m, arom), 2.34 (3H, s, Me),
1.92 (3H, s, Me),1.56 (3H, s, Me). 13C NMR (100 MHz, CDCl3)
: 170.6,
d: 9.07 (1H, s, OH), 7.90e7.88 (2H, m, arom),
d
161.3, 158.1, 137.5, 129.1, 125.6, 118.4, 87.6, 20.4, 18.5, 13.2. HRMS
(FAB): found m/z¼262.11949 (MHþ); calcd for C13H15N3O3:
262.11917.
5. Tocchetti, C. G.; Stanley, B. A.; Murray, C. I.; Sivakumaran, V.; Donzelli, S.;
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White solid, mp 128e130 ꢀC; 1H NMR (400 MHz, CDCl3)
d:
7.92e7.90 (2H, m, arom), 7.50 (1H, s, OH), 7.41e7.44 (2H, m, arom),
7.20e7.22 (1H, m, arom), 3.90 (3H, s, Me), 3.77 (3H, s, Me), 2.38 (3H,
s, Me), 2.08 (3H, s, Me). 13C NMR (100 MHz, CDCl3)
d: 166.8, 158.8,
157.3, 151.3, 137.4, 129.2, 125.3, 118.7, 89.8, 62.8, 53.8, 14.9, 10.0.
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