Catch-and-release of HNO with pyrazolones

Article abstract of DOI:10.1021/jo502330w

A new and versatile class of HNO donors, the (hydroxylamino)pyrazolone (HAPY) series of HNO donors utilizing pyrazolone (PY) leaving groups, is described. HNO, the smallest N-based aldehyde equivalent, is used as a reagent along with a variety of PY compounds to synthesize the desired HAPY donors in what can be considered an N-selective HNO-aldol reaction in up to quantitative yields. The bimolecular rate constant of HNO with PY in pH 7.4 phosphate buffer at 37 °C can reach 8 × 10⁵ M^-1 s^-1. In ¹H NMR experiments, the HAPY compounds generate HNO quantitatively (trapped as a phosphine aza-ylide) with half-lives spanning 3 orders of magnitude (minutes to days) under physiologically relevant conditions. B3LYP/6-31G calculations confirm the energetically favorable reactions between HNO and the PY enol and enolate, whereas HNO release is expected to occur through the oxyanion (OHN-PY) of each HAPY compound. HNO has been shown to provide functional support to failing hearts.

Full text of DOI:10.1021/jo502330w

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pubs.acs.org/joc
“Catch-and-Release” of HNO with Pyrazolones
Daryl A. Guthrie, Anthony Ho, Cyrus G. Takahashi, Anthony Collins, Matthew Morris,
and John P. Toscano*
Department of Chemistry, 3400 North Charles Street, Johns Hopkins University, Baltimore, Maryland 21218, United States
S
* Supporting Information
ABSTRACT: A new and versatile class of HNO donors, the (hydroxylamino)-
pyrazolone (HAPY) series of HNO donors utilizing pyrazolone (PY) leaving
groups, is described. HNO, the smallest N-based aldehyde equivalent, is used as a
reagent along with a variety of PY compounds to synthesize the desired HAPY
donors in what can be considered an N-selective HNO-aldol reaction in up to
quantitative yields. The bimolecular rate constant of HNO with PY in pH 7.4
phosphate buffer at 37 °C can reach 8 × 105 M−1 s−1. In 1H NMR experiments, the
HAPY compounds generate HNO quantitatively (trapped as a phosphine aza-
ylide) with half-lives spanning 3 orders of magnitude (minutes to days) under
physiologically relevant conditions. B3LYP/6-31G* calculations confirm the
energetically favorable reactions between HNO and the PY enol and enolate, whereas HNO release is expected to occur
through the oxyanion (OHN-PY) of each HAPY compound. HNO has been shown to provide functional support to failing
hearts.
designed to optimize both physiochemical and physiological
properties are desired.
INTRODUCTION
■
Heart failure, defined as the inability of the heart to pump
enough blood to supply the metabolic demands of the body,
affects more than 23 million people worldwide with total
annual costs in 2013 of $32 billion in the US alone.1−3 Azanone
(HNO), commonly referred to as nitroxyl, is the one-electron
reduced and protonated form of nitric oxide (NO) and has
been shown to improve both vasorelaxation and myocardial
contractility, making it a promising alternative therapeutic in
the fight against congestive heart failure.4−8
The practical use of HNO as a therapeutic agent is
complicated due to its propensity to spontaneously dimerize
(k = 8 × 106 M−1 s−1) to hyponitrous acid (HONNOH),
which dehydrates to give nitrous oxide (N2O).9 Thus, HNO
donors, such as Angeli’s salt (Na2N2O3), have been utilized to
discern the biological activity of HNO.4−8 Angeli’s salt has a
relatively short half-life of ca. 2−3 min at pH 7.4, 37 °C10 and
produces HNO along with an equimolar amount of nitrite,
which possesses its own biological activity.11,12 Unfortunately,
Angeli’s salt is not ideal from a drug development perspective
since derivatives that are HNO donors have yet to be
successfully realized.
Identifying physiologically useful donor molecules that
evolve HNO in a controlled and tunable fashion is an
important and ongoing challenge for fundamental research
and biomedical applications. Furthermore, sustained release of
low concentrations of HNO might be an important step to
mimic hypothesized endogenous HNO production.13 In
comparison, the availability of NO donors was invaluable to
the elucidation of the biological properties and endogenous
production of NO. Thus, new robust HNO donor platforms
amenable to structure−activity relationship (SAR) studies
The most commonly used strategy for prolonging HNO
release is through the use of ester prodrugs of otherwise
efficient HNO donors. These include acyloxy nitroso
compounds,14,15 esters of primary amine-based diazeniumdio-
lates such as IPA/NO,16−19 and precursors to acyl nitroso
compounds20−22 such as N,O-bis-acylated hydroxylamine
derivatives.23 The tunability of this strategy is reliant on the
precursor ester hydrolysis rates, which can vary from seconds to
hours. However, the overall chemistry of these long-acting
HNO donors can be more complicated than is originally
anticipated. For example, direct and HNO-mediated reactions
of acyloxy nitroso compounds have been demonstrated with
thiol-containing proteins;24,25 dual mechanisms of HNO
generation by IPA/NO-AcOM (iPrHN-N(O)NO-
CH2OAc) and IPA/NO-aspirin can occur in the absence of
esterase,18,19 and optimization of HNO production from N,O-
bis-acylated hydroxylamine derivatives requires the use of urea-
based precursors with arenesulfonyl leaving groups in order to
avoid both amide hydrolysis and acyl migration pathways.23
Additionally, these precursors are all susceptible to esterase-
mediated hydrolysis, which complicates drug development
since esterase activity not only depends on the substrate but
also shows strong interspecies, interorgan, and interindividual
variability.26
Previously, we have reported the development of N-
substituted hydroxylamines as efficient HNO donors based
on the general formula (HOHN-X) where X is a good carbon-
based leaving group.27 The rate and amount of HNO released
Received: October 12, 2014
Published: January 16, 2015
© 2015 American Chemical Society
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The Journal of Organic Chemistry
Featured Article
from these compounds is dependent mainly on the nature of
the leaving group X. We identified examples from three
independent hydroxylamine-substituted classes shown in Figure
1: (1) the hydroxylamino-Meldrum’s acid (HAMA) class, (2)
the (hydroxylamino)barbituric acid (HABA) class, and (3) the
(hydroxylamino)pyrazolone (HAPY) class.
RESULTS AND DISCUSSION
Early Studies. We first considered HAPY-2 (R1 = phenyl,
■
R2 and R3 = methyl), which was expected to generate HNO as
shown in Scheme 1. This precursor was synthesized from the
expected organic byproduct of HNO release, PY-2, which is
structurally similar to edaravone and has comparable in vitro
inhibitory activity with edaravone against lipid peroxidation.31
PY-2 was readily prepared through the condensation of ethyl 2-
methylacetoacetate and phenylhydrazine. Installation of the
hydroxylamine group was accomplished analogously to the
previously reported synthesis of HAPY-1 by formation of the
corresponding bromide followed by reaction with N,O-bis(tert-
butoxycarbonyl)hydroxylamine and subsequent acid deprotec-
tion (Scheme 2, path A).27
Figure 1. Previous examples from the HAMA, HABA, and HAPY
classes of HNO donors.
Scheme 2. Possible Synthetic Strategies: Traditional (Path
A) vs Umpolung (Path B)
Each example shares in common an O-methyloxime moiety
attached to the carbon bearing the hydroxylamine group.
Replacement of the O-methyloxime with simple alkyl groups on
the HAMA and HABA scaffolds results in negligible HNO
production following incubation at pH 7.4, 37 °C due to either
competitive hydrolysis or rearrangement mechanisms of their
ring core systems, respectively.27 The hydrolysis pathway
remains dominant in HAMA-1 even with O-methyloxime
substitution, restricting further development of the HAMA class
to examples with fast HNO release profiles. Strategies to curtail
the non-HNO producing rearrangement pathway in the HABA
class to favor HNO generation is the subject of a companion
paper.28
Previously, we examined HAPY-1 for HNO production in
pH 7.4 phosphate-buffered saline at 37 °C by gas chromato-
graphic headspace analysis to quantify the amount of its
dimerization product, N2O, as well as 1H NMR spectroscopy to
quantify the amount of the expected organic byproduct PY-1.
HNO was confirmed as the source of N2O for HAPY-1 by
quenching with glutathione (GSH), a known efficient trap for
HNO.32,33
On the other hand, the HAPY class of HNO donors
(Scheme 1) offers several distinct advantages: (1) the
Upon examination of HAPY-2, however, only trace amounts
1
of N2O are detected following 24 h incubation, and H NMR
analysis reveals that HAPY-2 is essentially stable. Interestingly,
upon 24 h incubation with excess GSH (ca. 5 equiv), enhanced
conversion of HAPY-2 to byproduct PY-2 is observed by H
Scheme 1. General HNO Release from HAPY Class
1
NMR spectroscopy; the relative yield of PY-2 is 36%.
Importantly, no other products arising from HAPY-2 are
observed. Four conclusions can be drawn from these results:
(1) the pyrazolone ring is stable against hydrolysis and
rearrangement, (2) the decomposition rates of HAPY-1 and
HAPY-2 differ dramatically, (3) HAPY decomposition may be
reversible, and (4) an alternative means of monitoring HNO
release in an expanded HAPY class is necessary.
Synthesis. We tested the possibility that PY-2 may be an
efficient trap for HNO by monitoring the reaction of PY-2 and
Angeli’s salt in 10% D2O, pH 7.4 phosphate buffer with the
metal chelator, diethylenetriaminepentaacetic acid (DTPA), at
room temperature by 1H NMR spectroscopy (Figure 2).
Consistent with our hypothesis, we observe the complete
conversion of PY-2 to HAPY-2, which is the first example of
what can be considered an N-selective nitroso-aldol reac-
tion,34−36 utilizing the smallest nitroso compound, HNO, as the
reagent.
This new reactivity of HNO is not so unexpected given what
has been reported in the literature. The chemical similarity
between HNO and an aldehyde is greater than between HNO
and an oxyacid.9 In addition, HNO is a potent inhibitor of
aldehyde dehydrogenase,37,38 demonstrating its chemical
similarity to acetaldehyde, though HNO is isoelectronic with
formaldehyde. Pyrazolones, as well as other reactive enols and
enolates, readily form aldol condensation products with
pyrazolone ring is expected to be resistant to hydrolysis and
rearrangement, (2) multiple synthetic handles are available to
investigate SAR, and (3) the byproducts are related to
edaravone,29−31 a potent antioxidant marketed in Japan to
treat acute ischemic stroke, which has been reported to be
effective for myocardial ischemia as well.
Herein, we are pleased to report the first series extension of
the HAPY class of HNO donors by varying the groups at the
R1, R2, and R3 positions on the pyrazolone ring. In doing so, we
have discovered a synthetically advantageous umpolung process
in which the PY’s 3-position switches from electrophilic to
nucleophilic, thereby reducing the steps to the desired HAPY
product from three to one. This discovery is made possible by
recognizing that HNO can be viewed as an aldehyde equivalent.
The phrase “catch-and-release” is thus attributed to describe the
HAPY compounds’ ability to undergo reversible generation of
1
HNO. Because of this attribute, we have developed an H
NMR assay for the evaluation of half-life and HNO production
in the presence of a phosphine-based trap for HNO to gauge
how these donors might behave under physiological conditions
where numerous biological targets for HNO exist.
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(Table 1) were prepared (see the Experimental Section for
synthetic procedures).
Table 1. HNO−Aldol Reaction of Pyrazolones
HAPY
R1
Ph
R2
Me
R3
% yield
1
C(NOMe)Me
74
89
44
76
35
75
94
96
89
99
99
87
2
Ph
Me
Me
3
Me
Me
H
Me
C(NOMe)Me
4
Me
Me
5
Me
C(NOMe)Me
6
H
Me
Me
Me
Me
Me
Me
Me
Me
7
4-ClPh
2-ClPh
Me
Ph
Me
8
Me
9
Ph
10
11
12
Ph
Me
Me
4-ClPh
2-ClPh
We have been able to develop a general synthetic protocol at
room temperature that features the use of Angeli’s salt, which
has a suitable HNO generation rate for this procedure. An
added advantage is that the nitrite byproduct is readily
separated from the HAPY product. To initiate decomposition
of Angeli’s salt, DTPA was used as a proton source as well as to
maintain a buffered solution (pH ≈ 8.0−8.5) and to preclude
HNO reactivity with trace metals. A 50% v/v aqueous ethanol
solution allowed both the organic and salt reagents to be readily
soluble at typical reaction concentrations (0.1−0.4 M) (see the
Experimental Section for details). The use of ethanol also aided
in the preliminary drying steps by allowing the mild azeotropic
removal of the water. This procedure is applicable for each
HAPY example; the isolated synthetic yields are shown in Table
1. Further investigations are underway to render this process
asymmetric.
Surprisingly, even the relatively efficient HNO donor, HAPY-
1, can be isolated starting from PY-1 in better yield and in fewer
steps when compared to the traditional bromination−displace-
ment−deprotection strategy (one step, 74% vs three steps, 34%
overall). The caveat here is that conversion to HAPY-1, or the
other O-methyloxime derivatives HAPY-3 and HAPY-5, is not
complete and requires the use of chromatographic separation to
isolate. Fortunately, the respective isolated yields correspond
well with the conversion yields, and under these reaction
conditions, the HAPY products are relatively stable several
hours after HNO generation from Angeli’s salt has completed.
Therefore, we believe the lower yields for these compounds are
primarily due to less efficient trapping of HNO rather than the
reactivity of the HAPY product. Otherwise, complete
conversion was observed in the other HAPY examples, each
equipped with a methyl group in the R3 position, resulting in
excellent isolated yields.
Figure 2. (a) N-Selective HNO−aldol reaction between PY-2 and
1
Angeli’s salt-derived HNO. (b) H NMR spectra of the formation of
HAPY-2 from PY-2 and Angeli’s salt derived HNO in 10% D2O, pH
7.4 phosphate buffer (0.25 M) with DTPA (0.2 mM) at room
temperature. (c) Kinetics of the reaction using the integrated area of
the upfield methyl group of PY-2 relative to the upfield methyl group
of HAPY-2. The solid curves are calculated best fits to a single
exponential function (k = 4.0 × 10−4 s−1 for each fit).
aldehydes in aqueous media.39 Moreover, the nitroso-aldol
reaction between carbonyl compounds and nitrosoarene and
acyl nitroso compounds to produce enantioselective amines
and alcohols following N−O bond reduction has been recently
well documented.34−36
The reaction of HNO with PY to give HAPY is synthetically
attractive, as it is the one-step umpolung strategy (Scheme 2,
path B) to the traditional bromination−displacement−depro-
tection strategy (Scheme 2, path A). As such, we endeavored to
find a useful preparatory procedure for this HNO−aldol
reaction and apply it to an expanded HAPY class.
On the basis of the early studies comparing HAPY-1 and
HAPY-2, a large variation in effective half-life is observed when
the O-methyloxime group at the R3 position of the pyrazolone
ring is replaced with a methyl group. Moving forward, we
probed the R1 and R2 positions with simple hydrogen, methyl,
and substituted phenyl variations. Accordingly, PY-3−PY-12
Kinetic Evaluation. Given the high conversion yields,
particularly for the R3 = methyl examples, we have quantified
this reactivity of HNO with pyrazolones utilizing PY-2 as an
example. We employed a competitive trapping experiment
using the HNO donor, HABA-1, and tris(4,6-dimethyl-3-
sulfanatophenyl)phosphine trisodium salt (TXPTS), another
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efficient and selective trap for HNO at pH 7.4, 37 °C (kTXPTS
=
9 × 105 M−1 s−1) following Scheme 3.40,41 HNO reacts with 2
Scheme 3. Competitive HNO Trapping Experiment at pH
7.4, 37 °C
Figure 3. Competition for HABA-1-derived HNO at pH 7.4, 37 °C:
PY-2 vs TXPTS. (a) All competition reactions contained HABA-1 (5
mM) and PY-2 (5 mM) in 2.0 mL of 10% D2O, pH 7.4 phosphate
buffer (0.25 M) with DTPA (0.2 mM). Each reaction was incubated
under argon for 30 min at 37 °C to ensure complete decomposition of
HABA-1 before 1H NMR analysis of the reaction mixture. (b)
equiv of TXPTS to give the diagnostic aza-ylide product as well
as the corresponding phosphine oxide, where the rate-limiting
step is assumed to be the initial reaction between HNO and the
first equivalent of TXPTS.
To determine the rate constant for the reaction of PY-2 with
HNO (eq 1), we evaluated the concentration of TXPTS
required to achieve equal product distributions of HAPY-2
compared to the aza-ylide product of TXPTS reaction.41,42
Importantly, all the HNO produced under these conditions
must react with either PY-2 or TXPTS in order for eq 1 to be
valid; HNO dimerization cannot occur.
1
Representative H NMR spectrum of the reaction mixture, and each
designated signal was used in the postreaction analysis.
Scheme 4. Possible Reactions Involving HNO with
a
Pyrazolones
k
TXPTS[TXPTS]
kPY‐2
=
[PY‐2]
(1)
Accordingly, due to its diagnostic 1H NMR signals,
byproduct BA-1 (Scheme 3) served as an internal reference
such that all species involved could be quantified, including the
aza-ylide product of TXPTS. The results shown in Figure 3
indicate that all HNO is effectively consumed by the traps and
that the concentration of TXPTS required to affect equal
product distributions is ca. 8.5 mM, which gives an HNO
trapping rate constant for PY-2 of ca. 8 × 105 M−1 s−1 at pH
7.4, 37 °C, comparable to that of TXPTS.
These results indicate that PY-2 to HAPY-2 formation is
efficient and can be used diagnostically as an alternative means
of HNO quantification. Also, the “HNO transfer reaction”
between HABA-1 and PY-2 demonstrates that HNO donors
beyond Angeli’s salt can be used in the HNO−aldol reaction.
Computational Studies. We modeled the thermodynamic
driving forces for three possible reactions involving HNO with
the synthesized pyrazolone examples (Scheme 4). All
calculations were performed with Spartan ‘14 at the B3LYP/
6-31G* level with an SM8 solvation model for aqueous
solvation.43 Optimized geometries and vibrational frequencies
were calculated for each reactant and product independently
(Supporting Information). Reaction II was balanced using a
hydronium cation and a water molecule on the left and right
a
Reactions: (I) HNO with neutral PY in the enol tautomer to give
neutral HAPY, (II) HNO with the enolate of PY to give neutral
HAPY, and (III) HNO with the enolate of PY to give HAPY oxyanion.
side of the reaction, respectively. Table 2 shows the Gibbs free
energy differences for reactions I−III.
Consistent with the experimental results above, these
calculations indicate that both reaction I and, particularly,
reaction II are thermodynamically favorable for the forward PY
plus HNO to HAPY reaction for all examples. On the other
hand, reaction III is thermodynamically unfavorable, indicating
that HNO formation is dependent on HAPY oxyanion
formation. In addition, ground-state structures were not
found for the oxyanions of HAPY-1, HAPY-3, HAPY-5, and
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Table 2. Gibbs Free Energy Differences in Reactions I−III
entry
R1
R2
R3
ΔGI° (kcal/mol)
ΔGI°I (kcal/mol)
ΔGI°II (kcal/mol)
1
Ph
Me
C(NOMe)Me
−0.7
−13.0
−0.6
−13.1
−0.6
−12.5
−13.0
−12.2
−10.8
−11.0
−10.6
−8.0
−27.5
−34.4
−30.6
−38.7
−30.8
−38.1
−33.5
−36.3
−34.7
−31.5
−34.3
−32.8
a
13.7
a
2
Ph
Me
Me
3
Me
Me
H
Me
C(NOMe)Me
4
Me
Me
10.2
a
5
Me
C(NOMe)Me
6
H
Me
Me
Me
Me
Me
Me
Me
Me
10.9
14.0
12.0
13.2
a
7
4-ClPh
2-ClPh
Me
Ph
Me
8
Me
9
Ph
10
11
12
Ph
Me
Me
4-ClPh
2-ClPh
14.6
15.8
a
Ground-state structures were not found for these oxyanions since the calculated structures resemble dissociated HNO and the corresponding
organic byproduct. Free energies are given for 298.15 K.
HAPY-10 since the calculated structures resemble dissociated
HNO and the corresponding PY byproduct. Thus, HNO
generation from the expanded HAPY class is expected to be
dependent on the pKa of the HAPY donor, which in turn is
likely dependent on the pKa of the PY bydroduct.
favors HAPY-2 formation at low (0−4 equiv) [TXPTS], while
HNO generation is rendered irreversible at high (>4 equiv)
[TXPTS]. Conversely, the reaction of HNO with TXPTS (or
biologically relevant GSH, for example) is irreversible.
Importantly, these results also indicate no bimolecular, direct
reaction between TXPTS and HAPY-2, since HAPY-2
consumption is not linear with the change in initial [TXPTS].
The timecourse for the disappearance of HAPY-2 and
appearance of PY-2 and HNO (trapped as the TXPTS aza-
HNO Release from the Expanded HAPY Class. As
described above, the reaction of HNO with pyrazolones can be
taken advantage of synthetically and potentially used as an
alternative means of quantifying HNO production. However,
the original purpose of this investigation was to study the HNO
production from these newly prepared HAPY compounds.
With this reversible reactivity in mind, an additional trap for
HNO is required to drive the HAPY to PY reaction forward.
We had demonstrated this earlier by using GSH, a biologically
relevant trap for HNO in vivo. However, TXPTS is best suited
for these mechanistic studies since it has selective and
irreversible reactivity with HNO, and the diagnostic aza-ylide
singlet in the 1H NMR spectrum is distinct from the HAPY and
PY compounds.
1
ylide) with ca. 6 equiv of TXPTS as well as representative H
NMR spectra at t = 0, 240, and 480 h are shown in Figure 5.
With a half-life of 47 h and a quantitative TXPTS aza-ylide
yield, HAPY-2 is a very long-lived, yet pure, HNO donor.
We first examined the effect that increasing [TXPTS] has on
the half-life of HAPY-2, as an example. Following a 48 h
incubation of HAPY-2 with 0−20 equiv of TXPTS in pH 7.4
1
phosphate buffer at 37 °C, H NMR analysis indicates the
incremental consumption of HAPY-2 with TXPTS increasing
from 0 to ca. 4 equiv, whereas the relative percentage of HAPY-
2 remains effectively unchanged when TXPTS exceeded ca. 4
equiv (Figure 4). These results highlight the reversibility of
HNO generation from the HAPY series as the equilibrium
Figure 5. (a) Timecourse for the disappearance of HAPY-2 and
appearance of PY-2 and TXPTS aza-ylide with added TXPTS (6
equiv). The solid curves are calculated best fits to a single exponential
function (k = 4.1 × 10−6 s−1 for each fit). (b) 1H NMR spectra at t = 0,
240, and 480 h; see the Experimental Section for details.
Figure 4. Incubation of HAPY-2 at pH 7.4, 37 °C after 48 h as a
function of [TXPTS]0. Conditions: HAPY-2 (1 mM) in 10% D2O, pH
7.4 phosphate buffer (0.25 M) with DTPA (0.2 mM) at 37 °C under
argon.
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Table 3. Incubation of HAPY-1−HAPY-12 in pH 7.4 Phosphate Buffer at 37 °C under Argon with Added TXPTS
a
b
c
HAPY
R1
Ph
R2
Me
R3
t1/2 (min)
k (s−1
)
PY pKa
1
C(NOMe)Me
4
2793
11
3.0 × 10−3
4.1 × 10−6
1.0 × 10−3
2.0 × 10−6
8.2 × 10−4
2.5 × 10−6
7.8 × 10−6
6.1 × 10−6
1.5 × 10−5
2.5 × 10−5
1.4 × 10−5
1.6 × 10−4
6.0 (6.8)
7.6 (8.8)
6.7 (7.4)
8.5 (9.5)
7.3 (8.2)
9.0 (10.0)
(8.4)
2
Ph
Me
Me
3
Me
Me
H
Me
C(NOMe)Me
4
Me
Me
5674
14
5
Me
C(NOMe)Me
6
H
Me
Me
Me
Me
Me
Me
Me
Me
4585
1473
1887
753
460
799
71
7
4-ClPh
2-ClPh
Me
Ph
Me
8
Me
7.6 (8.7)
7.6 (8.5)
(8.1)
9
Ph
10
11
12
Ph
Me
Me
4-ClPh
2-ClPh
(8.0)
(8.0)
a
Incubation conditions: HAPY (1 mM) and TXPTS (5 mM) in 10% D2O, pH 7.4 phosphate buffer (0.25 M) with DTPA (0.2 mM) at 37 °C under
b
1
argon. HNO yields are quantitative for all examples. The rates are calculated best fits to a single exponential function of the integrated H NMR
data for HAPY disappearance/PY appearance. The pKa values were determined by titration in water; the values in parentheses were determined in
c
50% v/v aqueous ethanol.
Fortunately, under these anaerobic assay conditions, the
TXPTS aza-ylide product is stable. The TXPTS oxide product
slowly increases beyond the expected stoichiometric amount
due to oxidation of the excess TXPTS (Figure 5b), as
maintaining a rigorously anaerobic assay condition for this
extended time period is experimentally challenging.
the reverse reaction, PY-1 plus HNO to give HAPY-1 (Table
3).
Next, we explored the perhaps more subtle effects at the R1
and R2 positions with additional examples, specifically HAPY-
3−HAPY-12. With a variety of derivatives in hand, we followed
1
their decomposition according to the H NMR protocol used
for HAPY-1 and HAPY-2 described above. We also measured
the pKa values of their respective PY byproducts by titration in
water (solubility permitting). For comparison, the pKa values of
all PY examples were also determined in 50% v/v aqueous
ethanol. The results are shown in Table 3.
Likewise, an unknown oxidation product forms slowly from
the organic byproduct PY-2 (Figure 5). Following overnight
aerobic incubation of PY-2 alone in pH 7.4 phosphate buffer at
37 °C in the absence of metal chelator, the oxidized product
precipitated from solution, which was briefly reheated to affect
recrystallization. Single-crystal X-ray crystallography confirmed
the identity of the oxidized product as PY-2OH, the
corresponding alcohol of PY-2 (Supporting Information).
Recall that PY-2 is a derivative of edaravone, which has
beneficial effects attributed to its free radical scavenging ability
through a single electron transfer mechanism with reactive
oxygen species such as hydroxyl and peroxyl radicals.44 Also,
the relatively rapid aerobic oxidation of PY-2 alone versus PY-
2OH formation under the conditions outlined in Figure 5
suggests that PY-2 is effectively inert to oxidation via the
First, we changed the R1 position from phenyl in HAPY-1
and HAPY-2 to methyl (HAPY-3 and HAPY-4, respectively)
and hydrogen (HAPY-5 and HAPY-6, respectively). As the R1
substituent is changed from phenyl to methyl or hydrogen, we
observe a decrease in dissociation rate, which seems to correlate
well with the pKa values of the respective PY byproducts.
Although the R3 = O-methyloxime derivatives, HAPY-3 and
HAPY-5, have half-lives between those of HAPY-1 and HAPY-
2, the R3 = methyl derivatives, HAPY-4 and HAPY-6, have even
slower dissociation rates than HAPY-2. Importantly, the HNO
yield for all of these precursors remains quantitative, and as
such, HAPY-4 and HAPY-6 are the longest lived reported pure
HNO donors to date with half-lives measuring 95 and 76 h,
respectively.
1
TXPTS oxide product (R3PO). Therefore, this H NMR
assay for the evaluation of half-life and HNO production
requires the use of excess TXPTS (>5 equiv) under anaerobic
conditions in order to reduce complications from the reverse
reaction, PY plus HNO to give HAPY, as well as from TXPTS
and PY oxidation.
Next, we explored substitution on the phenyl ring of HAPY-2
with 4-chloro (HAPY-7) and 2-chloro (HAPY-8) groups.
These derivatives have slightly faster rates when compared to
HAPY-2 but are still over 2 orders of magnitude slower than
HAPY-1.
The half-life of HAPY-1 was previously reported to be 9.5
min in pH 7.4 phosphate buffer at 37 °C, which was
determined by UV−vis analysis in the absence of an additional
We then made changes to the R2 position first by
synthesizing HAPY-9, the isomer of HAPY-2. Interestingly,
the decomposition rate of HAPY-9 is over three times faster
than that of HAPY-2. The impact of R2 substitution is best
trap for HNO (such as TXPTS).27 Following the H NMR
1
assay described above, the observed half-life of HAPY-1 is
reduced in half to 4 min, presumably due to minimization of
1343
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illustrated by comparing HAPY-4 with HAPY-9, HAPY-11, and
HAPY-12, where the observed rate changes span 2 orders of
magnitude.
HAPY-1 (pKa > 10) and HABA-1 (pKa > 9),27 and (3) the
barrier to dissociation must be very low once HAPY
deprotonation occurs. Moreover, with such large approximate
pKa values, HNO evolution at neutral pH suggests a near
barrierless dissociation from the deprotonated donor.
All told, by varying groups at the R1, R2, and R3 positions on
the pyrazolone ring, the observed rate changes span 1, 2, and 3
orders of magnitude, respectively. The distance removed from
the PY’s 3-position carbon, formerly bearing the hydroxyl-
amine, is greatest for the R1 position and shortest for the R3
position, and inductive effects are expected to follow likewise.
However, only the R3 position allows for additional resonance
stabilization in the dissociated PY byproduct. Because of the
combined resonance and inductive stabilization effects, the
impact on the PY byproduct pKa is greatest when the R3
position is varied. Given the relatively large pKa difference of
two units for PY-1 and PY-2, this must be a contributing factor
in the large difference in decomposition rates of the
corresponding HAPY products (Table 3).
However, the PY pKa alone does not always correlate with
the HAPY pKa. For example, the pKa values of PY-5, PY-10 −
PY-12 are all ca. 8 in 50% v/v aqueous ethanol (Table 3), yet
HAPY-5 (pKa = 10.5) is more acidic than HAPY-12 (pKa >
11.5), which in turn is more acidic than HAPY-10 and HAPY-
11 (pKa > 12.0) (Figure 6b). This trend is mirrored in the half-
lives of these HAPY examples at neutral pH (Table 3); the half-
lives of HAPY-5, HAPY-10, HAPY-11, and HAPY-12 are 14,
460, 799, and 71 min, respectively. These results suggest that
the HAPY pKa, as opposed to the PY pKa, is a better predictor
of HNO generation rate under physiological conditions. The
fact that decomposition of HAPY-5 as a function of pH
approaches a limiting rate constant at a pH apparently much
lower than that for HAPY-10−HAPY-12 in this analysis (Figure
6b), suggests contribution from deprotonation at the R1
position proton (N-H) of the pyrazolone ring (Scheme 5).
As suggested from our calculations, HNO release is governed
by the HAPY pKa, which likely correlates with the PY pKa. This
correlation is confirmed by the UV−vis−determined decom-
position rates for HAPY-1 and HAPY-2 as a function of pH,
where the observed sharp increases reflect rapid PY formation
presumably as a result of HAPY deprotonation (Figure 6a).
Scheme 5. Proposed Mechanism of HNO Release
This may explain the greater acidity of HAPY-5. In comparison,
a pKa value of 11.5 is estimated for the R1 position proton of
HAPY-6, the R3 = methyl analog of HAPY-5 (Supporting
Information). For the HAPY class as a whole, HNO release
following HOHN-PY deprotonation remains the most plausible
mechanism in water (Scheme 5).
The pKa values of the PY byproducts are a reflection of the
weighted average of the various tautomers that are known to
exist in pyrazolones, such as edaravone.45−47 Presumably, the
pKa value related to deprotonation of the PY’s 3-position
carbon, formerly bearing the hydroxylamine, is the most
relevant to predicting SAR for HNO release; however, we can
assume from this first HAPY series extension study that PY
byproducts with measured pKa values ≤ ca. 8 in 50% v/v
aqueous ethanol are useful targets for further development of
the HAPY class of HNO donors.
Figure 6. Plot of UV−vis determined decomposition rates as a
function of pH in 0.1 M phosphate buffer at 25 °C (SEM 10%; n ≥
3) for (a) HAPY-1 and HAPY-2, and (b) HAPY-5 (red), HAPY-12
(green), HAPY-11 (blue), and HAPY-10 (purple). For HAPY-5, the
solid curve is a calculated best fit to a sigmoid function (pKa = 10.5).
CONCLUSION
■
HNO has been shown to enhance myocardial function,
demonstrating its potential usefulness in the treatment of
heart failure. The HAPY class of HNO donors represents a new
and robust platform capable of producing HNO quantitatively
over extended periods of time along with a PY byproduct that is
related to edaravone, a potent antioxidant already in clinical use
for the treatment of stroke and cardiovascular disease. The
HNO release rate is highly tunable by varying the groups at the
R1, R2, and R3 positions on the pyrazolone ring; the effect of the
R-position on half-life is R3 > R2 > R1. Experimental and
Decomposition rates reach the time resolution limit (ca. 0.15
s−1) of the UV−vis experiment, precluding a complete pKa
analysis for each donor as the magnitude of the sigmoidal curve
extends well beyond experimental parameters. However, these
results suggest that (1) the pKa difference of HAPY-1 and
HAPY-2 is also ca. two units, corresponding well to the PY-1/
PY-2 pKa difference, (2) the observed sharp increases in k
values as a function of pH is not likely related to HNO
deprotonation (pKa ∼11.4) since this behavior is observed with
1344
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The Journal of Organic Chemistry
Featured Article
General HNO−Aldol Procedure. To the PY substrate (0.20
mmol), Angeli’s salt (49 mg, 0.40 mmol), and powdered DTPA (39
mg, 0.10 mmol) under argon at room temperature was added a
degassed mixture of 50% v/v aqueous ethanol (1 mL) via syringe. The
reaction was allowed to stir for 1.5 h, diluted with ethanol (>5 mL),
and concentrated to dryness in vacuo with minimum heat (<30 °C).
The material was then taken up in minimum ethanol, eluted through a
short silica plug, and concentrated in vacuo to give the desired HAPY
product or to allow for further purification if complete conversion was
not observed (i.e., for the O-methyloxime derivatives HAPY-1, HAPY-
3, and HAPY-5). The crude reaction mixture was loaded with
methanol onto an analytical TLC plate (250 μm thickness) and
developed in either 50% v/v ethyl acetate/dichloromethane (HAPY-1)
or ethyl acetate (HAPY-3 and HAPY-5). The product band (Rf > 0.5)
was scraped off the plate into a flask using a metal spatula. The product
was eluted with methanol (10 mL), filtered through cotton, and
concentrated in vacuo to yield the desired HAPY product typically as
off-white/white solids. Isolated yields are given in Table 1.
4-(N-Hydroxylamino)-4-methyl-N-phenyl-3-methylpyrazolone
(HAPY-2). Mp: 152−154 °C. 1H NMR (400 MHz, CDCl3) δ: 7.93 (d,
2H, J = 8.8 Hz), 7.40 (dd, 2H, J = 7.4 Hz), 7.18 (t, 1H, J = 7.4 Hz),
5.70 (bs, 1H), 4.77 (s, 1H), 2.21 (s, 3H), 1.28 (s, 3H). 13C NMR (100
MHz, CDCl3) δ: 174.1, 162.1, 138.1, 129.1, 125.3, 118.9, 71.2, 17.0,
13.3. HR-MS (FAB): found m/z = 220.10932 (MH+), calcd for
C11H14N3O2 220.10860 (MH+).
4-(N-Hydroxylamino)-4-(acetyl-O-methoxyoxime)-N-methyl-3-
methylpyrazolone (HAPY-3). Mp: 105−108 °C. 1H NMR (400 MHz,
CDCl3) δ: 6.12 (s, 1H), 4.67 (s, 1H), 3.90 (s, 3H), 3.33 (s, 3H), 2.12
(s, 3H), 1.74 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 171.7, 159.0,
76.6, 62.5, 31.8, 14.4, 11.0. HR-MS (FAB): found m/z = 215.11454
(MH+), calcd for C8H15N4O3 215.11442 (MH+).
4-(N-Hydroxylamino)-4-methyl-N-methyl-3-methylpyrazolone
(HAPY-4). Mp: 135−137 °C. 1H NMR (400 MHz, DMSO-d6) δ: 7.52
(d, 1H, J = 2.8 Hz), 6.25 (d, 1H, J = 2.8 Hz), 3.12 (s, 3H), 1.96 (s,
3H), 1.00 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 175.3, 161.9,
69.0, 30.7, 16.0, 12.7. HR-MS (FAB): found m/z = 158.09320 (MH+),
calcd for C6H12N3O2 158.09295 (MH+).
4-(N-Hydroxylamino)-4-(acetyl-O-methoxyoxime)-3-methylpyra-
zolone (HAPY-5). Mp: 148−150 °C. 1H NMR (400 MHz, DMSO-d6)
δ: 10.96 (s, 1H), 7.76, (d, 1H, J = 2.7 Hz), 6.60 (d, 1H, J = 2.7 Hz),
3.76 (s, 3H), 1.98 (s, 3H), 1.79 (s, 3H). 13C NMR (100 MHz, DMSO-
d6) δ: 173.9, 159.5, 151.8, 74.2, 61.6, 14.9, 10.6. HR-MS (FAB): found
m/z = 201.09920 (MH+), calcd for C7H13N4O3 201.09817 (MH+).
4-(N-Hydroxylamino)-4-methyl-3-methylpyrazolone (HAPY-6).
Mp: 185−187 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.77 (s,
1H), 7.50 (d, 1H, J = 2.9 Hz), 6.16 (d, 1H, J = 2.9 Hz), 1.93 (s, 3H),
0.97 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 178.3, 161.9, 68.0,
16.0, 13.0. HR-MS (FAB): found m/z = 144.07683 (MH+), calcd for
C5H10N3O2 144.07730 (MH+)..
4-(N-Hydroxylamino)-4-methyl-N-(4-chlorophenyl)-3-methylpyr-
azolone (HAPY-7). Mp: 157−159 °C. 1H NMR (400 MHz, CDCl3) δ:
7.90 (d, 2H, J = 9.0 Hz), 7.35 (d, 2H, J = 9.0 Hz), 5.66 (d, 1H, J = 2.8
Hz), 4.45 (d, 1H, J = 2.8 Hz), 2.20 (s, 3H), 1.28 (s, 3H). 13C NMR
(100 MHz, CDCl3) δ: 174.0, 162.2, 136.8, 130.4, 129.1, 119.9, 71.2,
16.9, 13.3. HR-MS (FAB): found m/z = 254.06968 (MH+,35Cl),
256.06703 (MH+,37Cl), calcd for C11H13ClN3O2 254.06963
(MH+,35Cl), 256.06668 (MH+,37Cl).
computational investigations suggest that the HNO release rate
correlates with the HAPY and PY pKa values. The catch is a
strong affinity that is shared between HNO and the released PY
byproduct since the bimolecular rate constant in pH 7.4
phosphate buffer at 37 °C can reach 8 × 105 M−1 s−1. Given
that biological targets for HNO are in excess of HNO in vivo,
the reverse reaction of PY plus HNO to give HAPY is expected
to be negligible under these conditions; thus, we believe these
donors will be useful in in vivo studies. This is also the case in
1
the reported H NMR assay for the evaluation of half-life and
HNO production utilizing the selective, distinctive, and
irreversible trap for HNO, TXPTS. Yet, this reverse reaction,
described herein for the first time as the HNO-aldol reaction
with pyrazolones, is an efficient, alternative synthetic strategy to
the cumbersome, three-step bromination-displacement-depro-
tection strategy, thus illustrating a synthetically useful route to
HAPY donors. We anticipate utilizing these methodologies for
the development of new N-substituted hydroxylamines with
other suitable carbon-based leaving groups.
EXPERIMENTAL SECTION
■
Materials. All starting materials were of reagent grade and used
without further purification. Angeli’s salt (Na2N2O3);48 compounds
PY-1 and HAPY-1;27 PY-2, PY-4, PY-9, and PY-10;49 PY-6;50 PY-11
and N-methyl-3-(2-chlorophenyl)pyrazolone;51 and 4-acetyl-3-meth-
ylpyrazolone52 are all known compounds and were prepared according
to literature procedures. Tris(4,6-dimethyl-3-sulfanatophenyl)-
phosphine trisodium salt (TXPTS) was of reagent grade and used
without further purification. Synthetic TXPTS aza-ylide was obtained
through the amidation of TXPTS using hydroxylamine O-sulfonic acid
in water.53 Melting point measurements are uncorrected. NMR spectra
were obtained on a 400 MHz FT-NMR spectrometer. All chemical
shifts are reported in parts per million (ppm) relative to residual
1
CHCl3 (7.26 ppm for H, 77.2 ppm for 13C), residual DMSO (2.50
1
1
ppm for H, 39.5 for 13C), or residual MeOH (3.31 ppm for H, 49.2
ppm for 13C). High-resolution mass spectra were obtained on a
magnetic sector mass spectrometer operating in fast atom bombard-
ment (FAB) mode. Ultraviolet-visible (UV-vis) absorption spectra
were obtained using a diode array spectrometer. Buffered solutions
(0.1 M) for UV-vis experiments were prepared from Na2PO3H/
Na3PO4 (pH 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.2).
1H NMR Incubation Procedure of HAPY Donors at pH 7.4, 37
°C with TXPTS. General Methods. All 1H NMR spectra were
obtained in pH 7.4 solution containing 0.25 M phosphate buffer, 0.2
mM of the metal chelator diethylenetriaminepentaacetic acid (DTPA),
and 10% D2O on a 400 MHz FT-NMR spectrometer using a 1 s
presaturation pulse to suppress the water signal. Each free induction
decay was Fourier transformed, phased, and baseline-corrected, and
integral areas were measured for the methyl groups of compounds
HAPY-1−HAPY-12 and PY-1−PY-2 and the downfield methyl group
1
of TXPTS aza-ylide. The H NMR spectrum of the HNO-derived
TXPTS aza-ylide product matched that of synthetic TXPTS aza-ylide.
The HNO yield from compounds HAPY-1−HAPY-12 was determined
from the final TXPTS aza-ylide yield.
4-(N-Hydroxylamino)-4-methyl-N-(2-chlorophenyl)-3-methylpyr-
1
1
Procedure. The H NMR procedure used was based on an HPLC
azolone (HAPY-8). Mp: 150−152 °C. H NMR (400 MHz, DMSO-
protocol developed by King and co-workers.40,41 To an argon-purged
NMR solution (1.00 mL) containing TXPTS (3.3 mg, 5 mM) was
added HAPY (10 μL of 100 mM in methanol-d4) to give 1 mM as the
initial concentration of HAPY. The solution was briefly mixed, ca. 0.5
d6) δ: 7.73 (d, 1H, J = 2.8 Hz), 7.60 (m, 1H), 7.43 (m, 3H), 6.50 (d,
1H, J = 2.8 Hz), 2.07 (s, 3H), 1.17 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ: 174.8, 163.3, 134.8, 130.8, 130.1, 129.9, 129.1, 128.0,
69.4, 16.2, 12.9. HR-MS (FAB): found m/z = 254.06967 (MH+,35Cl),
256.06718 (MH+,37Cl), calcd for C11H13ClN3O2 254.06963
(MH+,35Cl), 256.06668 (MH+,37Cl).
1
mL was transferred to an argon-purged NMR tube, and an initial H
NMR spectrum was obtained. The sample was then either (1)
1
externally incubated at 37 °C and H NMR spectra were collected at
4-(N-Hydroxylamino)-4-methyl-N-methyl-3-phenylpyrazolone
(HAPY-9). Mp: 146−148 °C. 1H NMR (400 MHz, DMSO-d6) δ: 8.06
(m, 2H), 7.64 (d, 2H, J = 2.5 Hz), 7.45 (m, 3H), 6.48 (d, 2H, J = 2.5
Hz), 3.29 (s, 3H), 1.21 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ:
176.1, 157.4, 130.4, 129.9, 128.6, 126.2, 69.3, 31.2, 17.9. HR-MS
1
regular time intervals or (2) internally incubated at 37 °C and H
NMR spectra were collected using a canned pulse sequence, zg2d,
modified to include a 1 s presaturation pulse during the relaxation
delay.
1345
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Featured Article
(FAB): found m/z = 220.10897 (MH+), calcd for C11H14N3O2
220.10860 (MH+).
4-(Acetyl-O-methoxyoxime)-3-methylpyrazolone (PY-5). 4-Acetyl-
3-methylpyrazolone (2.935 g, 20.9 mmol), O-methylhydroxylamine
hydrochloride (1.921 g, 23 mmol), and sodium bicarbonate (1.932 g,
23 mmol) were taken up in methanol (125 mL), and the reaction was
refluxed with stirring for 21 h followed by concentration in vacuo. The
resultant material was taken up in ethyl acetated (2 × 125 mL), filtered
through cotton, and concentrated in vacuo to give the title compound
as a pale yellow solid (2.487 g, 70%). Mp: 189−191 °C. 1H NMR (400
MHz, DMSO-d6) δ: 3.78 (s, 3H), 2.23 (s, 3H), 2.08 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ: 159.9, 151.5, 138.2, 98.5, 61.0, 13.5,
12.2. HR-MS (FAB): found m/z = 170.09335 (MH+), calcd for
C7H12N3O2 170.09295 (MH+).
4-(N-Hydroxylamino)-4-methyl-N-phenyl-3-phenylpyrazolone
1
(HAPY-10). Mp: 148−150 °C. H NMR (400 MHz, CDCl3) δ: 8.21
(m, 2H), 8.05 (m, 2H), 7.45 (m, 5H), 7.24 (m, 1H), 5.95 (d, 1H, J =
3.2 Hz), 4.50 (d, 1H, J = 3.3 Hz), 1.48 (s, 3H). 13C NMR (100 MHz,
CDCl3) δ: 175.0, 157.7, 138.2, 131.0, 130.3, 129.1, 129.1, 126.8, 125.5,
119.0, 71.5, 18.9. HR-MS (FAB): found m/z = 282.12461 (MH+),
calcd for C16H16N3O2 282.12425 (MH+).
4-(N-Hydroxylamino)-4-methyl-N-methyl-3-(4-chlorophenyl)-
pyrazolone (HAPY-11). Mp: 182−184 °C. 1H NMR (400 MHz,
DMSO-d6) δ: 8.06 (d, 2H, J = 8.7 Hz), 7.66 (d, 1H, J = 2.6 Hz), 7.53
(d, 2H, J = 8.7 Hz), 6.54 (d, 1H, J = 2.6 Hz), 3.29 (s, 3H), 1.19 (s,
3H). 13C NMR (100 MHz, DMSO-d6) δ: 176.1, 156.4, 134.5, 129.2,
128.7, 128.0, 62.3, 31.2, 17.7. HR-MS (FAB): found m/z = 254.06941
(MH+, 35Cl), 256.06193 (MH+, 37Cl), calcd for C11H13ClN3O2:
254.06963 (MH+, 35Cl), 256.06668 (MH+, 37Cl).
4-(N-Hydroxylamino)-4-methyl-N-methyl-3-(2-chlorophenyl)-
pyrazolone (HAPY-12). Mp: 126−128 °C. 1H NMR (400 MHz,
CDCl3) δ: 7.71 (m, 1H), 7.50 (m, 1H), 7.38 (m, 2H), 5.90 (br, 2H),
3.45 (s, 3H), 1.26 (s, 3H). 13C NMR (100 MHz, CDCl3) δ: 174.9,
158.6, 133.5, 131.1, 131.1, 130.7, 129.3, 127.1, 71.6, 32.0, 16.9. HR-MS
(FAB): found m/z = 254.06981(MH+, 35Cl), 256.06755 (MH+, 37Cl),
calcd for C11H13ClN3O2 254.06963 (MH+, 35Cl), 256.06668 (MH+,
37Cl).
4-Methyl-N-(4-chlorophenyl)-3-methylpyrazolone (PY-7). Ethyl 2-
methylacetoacetate (0.71 mL, 5 mmol), 4-chlorophenylhydrazine
hydrochloride (0.895 g, 5 mmol), and sodium acetate (0.410 g, 5
mmol) were taken up in ethanol (50 mL), and the reaction was
refluxed with stirring for 1 d followed by filtration through cotton and
concentration in vacuo. The resultant material was recrystallized from
hot ethanol and 1 N HCl to give the title compound as a white solid
(0.867 g, 78%). Mp: 173−175 °C. 1H NMR (400 MHz, DMSO-d6) δ:
7.75 (d, 2H, J = 8.9 Hz), 7.50 (d, 2H, J = 8.9 Hz), 2.13 (s, 3H), 1.80
(s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 157.0, 147.7, 135.7,
129.7, 129.0, 122.0, 99.3, 11.3, 6.6. HR-MS (FAB): found m/z =
223.06396 (MH+,35Cl), 225.06151 (MH+,37Cl), calcd for
C11H12ClN2O: 223.06382 (MH+,35Cl), 225.06087 (MH+,37Cl).
4-Methyl-N-(2-chlorophenyl)-3-methylpyrazolone (PY-8). Ethyl 2-
methylacetoacetate (0.71 mL, 5 mmol), 2-chlorophenylhydrazine
hydrochloride (0.895 g, 5 mmol), and sodium acetate (0.410 g, 5
mmol) were taken up in acetic acid (50 mL), and the reaction was
refluxed with stirring for 18 h followed by filtration through cotton and
concentration in vacuo. The resultant material was triturated with
diethyl ether to remove the colored impurities, filtered, extracted into
ethyl acetate (2 × 100 mL), washed with water and brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo to give the title
compound as an off-white solid (0.696 g, 63%). Mp: 166−168 °C. 1H
NMR (400 MHz, MeOD-d4) δ: 7.58 (m, 1H), 7.45 (m, 3H), 2.17 (s,
3H), 1.85 (s, 3H). 13C NMR (100 MHz, MeOD-d4) δ: 164.8, 148.6,
135.1, 134.3, 132.1, 131.7, 131.5, 129.1. HR-MS (FAB): found m/z =
223.06348 (MH+,35Cl), 225.06092 (MH+,37Cl), calcd for
C11H12ClN2O 223.06382 (MH+,35Cl), 225.06087 (MH+,37Cl).
4-Methyl-N-methyl-3-(2-chlorophenyl)pyrazolone (PY-12). To a
solution of compound 2 (0.370 g, 1.56 mmol) in acetic acid (20 mL)
was added sodium cyanoborohydride (0.211 g, 3.2 mmol), and the
reaction was heated to 65 °C and stirred for 2 h. The reaction was then
concentrated in vacuo, taken up in 1 N HCl, extracted into ethyl
acetate (2 × 50 mL), washed with brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resultant viscous oil was
triturated with dichloromethane and hexanes and filtered to give the
title compound as a white solid (0.070 g, 20%). Mp: 168−170 °C. 1H
NMR (400 MHz, DMSO-d6) δ: 7.51 (d, 1H, J = 6.8 Hz), 7.38 (m,
3H), 3.57 (s, 3H), 1.76 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ:
150.5, 145.5, 133.0, 132.7, 132.1, 129.8, 129.4, 127.0, 95.7, 33.2, 7.7.
HR-MS (FAB): found m/z = 225.06188 (MH+,37Cl), calcd for
C11H12ClN2O 225.06087 (MH+,37Cl).
4-Acetyl-1,3-dimethylpyrazolone (1). To methyl acetoacetate
(2.32 g, 20 mmol) on ice was added methylhydrazine (1.05 mL, 20
mmol) over 2−3 min with stirring. The reaction was then stirred for 5
min, whereupon 1,3-dimethylpyrazolone precipitated from solution.
The reaction was diluted with ethanol (20 mL) and concentrated in
vacuo to give dry 1,3-dimethylpyrazolone as an off-white solid. This
was then taken up with trimethyl orthoacetate (10 mL) under nitrogen
and heated to 65 °C for 2 h. The clear, red-orange solution was
concentrated in vacuo, the solid was dissolved in water (10 mL), and
the solution was allowed to stand at 4 °C overnight. Filtration of the
resulting precipitate gave the title compound as an orange solid (1.36
1
g, 44%). Mp: 118−120 °C. H NMR (400 MHz, CDCl3) δ: 3.55 (s,
3H), 2.38 (s, 3H), 2.36 (s, 3H). 13C NMR (100 MHz, CDCl3) δ:
195.3, 159.6, 146.8, 103.2, 32.5, 27.5, 15.5. HR-MS (FAB): found m/z
= 155.08184 (MH+), calcd for C7H11N2O2 155.08205 (MH+).
3-(2-Chlorophenyl)-4-formyl-1-methylpyrazolone (2). To ethyl
(2-chlorobenzoyl)acetate (0.937 g, 4.13 mmol) at room temperature
was added methylhydrazine (0.22 mL, 4.1 mmol) over two to 3 min
with stirring. The reaction was then heated to 90 °C for 15 min and
allowed to cool to room temperature where white solids formed upon
standing. This material was recrystallized from hot ethanol and water
to give pure N-methyl-3-(2-chlorophenyl)-pyrazolone (0.410 g, 48%).
To this product dissolved in dimethylformamide (1 mL) was added
phosphoryl chloride (0.13 mL, 1.4 mmol), and the reaction was heated
to 65 °C with stirring for 2 h followed by addition of water (5 mL).
This solution was allowed to stand at room temperature for 1 d, and
the resultant yellow precipitate was collected by vacuum filtration to
1
give the title compound (0.370 g, 79%). Mp: 163−165 °C. H NMR
(400 MHz, DMSO-d6) δ: 9.51 (s, 1H), 7.44 (m, 4H), 3.60 (s, 3H).
13C NMR (100 MHz, DMSO-d6) δ: 182.0, 155.6, 132.9, 132.2, 131.7,
130.2, 129.3, 126.9, 104.4, 33.1. HR-MS (FAB): found m/z =
237.04354 (MH+,35Cl), 239.04114 (MH+,37Cl); calcd for
C11H10ClN2O2: 237.04308 (MH+,35Cl), 239.04013 (MH+,37Cl).
4-(Acetyl-O-methoxyoxime)-1,3-dimethylpyrazolone (PY-3).
Compound 1 (0.771 g, 5 mmol), O-methylhydroxylamine hydro-
chloride (0.459 g, 5.5 mmol), and sodium bicarbonate (0.462 g, 5.5
mmol) were taken up in methanol (50 mL), and the reaction was
refluxed with stirring for 2 h followed by concentration in vacuo. The
resultant material was taken up in dichloromethane, filtered through
cotton, and concentrated in vacuo to give the title compound as an off-
ASSOCIATED CONTENT
■
S
* Supporting Information
Computational results including optimized geometries, ener-
gies, and vibrational frequencies and intensities; synthetic
schemes; single-crystal X-ray crystallographic data; NMR
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
1
white solid (0.731 g, 80%). Mp: 105−107 °C. H NMR (400 MHz,
CDCl3) δ: 3.85 (s, 3H), 3.56 (s, 3H), 2.29 (s, 3H), 2.19 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ: 155.2, 153.8, 144.7, 95.2, 62.0, 32.8, 15.6,
13.1. HR-MS (FAB): found m/z = 184.10819 (MH+), calcd for
C8H14N3O2 184.10860 (MH+).
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: jtoscano@jhu.edu.
1346
DOI: 10.1021/jo502330w
J. Org. Chem. 2015, 80, 1338−1348

The Journal of Organic Chemistry
Featured Article
(16) Miranda, K. M.; Katori, T.; Torres de Holding, C. L.; Thomas,
L.; Ridnour, L. A.; McLendon, W. J.; Cologna, S. M.; Dutton, A. S.;
Champion, H. C.; Mancardi, D.; Tocchetti, C. G.; Saavedra, J. E.;
Keefer, L. K.; Houk, K. N.; Fukuto, J. M.; Kass, D. A.; Paolocci, N.;
Wink, D. A. J. Med. Chem. 2005, 48, 8220−8228.
Notes
The authors declare the following competing financial
interest(s): J.P.T. is a cofounder and stockholder and serves
on the Scientific Advisory Board of Cardioxyl Pharmaceuticals.
(17) Salmon, D. J.; Torres de Holding, C. L.; Thomas, L.; Peterson,
K. V.; Goodman, G. P.; Saavedra, J. E.; Srinivasan, A.; Davies, K. M.;
Keefer, L. K.; Miranda, K. M. Inorg. Chem. 2011, 50, 3262−3270.
(18) Andrei, D.; Salmon, D. J.; Donzelli, S.; Wahab, A.; Klose, J. R.;
Citro, M. L.; Saavedra, J. E.; Wink, D. A.; Miranda, K. M.; Keefer, L. K.
J. Am. Chem. Soc. 2010, 132, 16526−16532.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the National Science Foundation
(CHE-1213438) and Cardioxyl Pharmaceuticals for generous
support of this research. We also thank the Johns Hopkins
University, Department of Chemistry Facility Managers, Dr.
Cathy D. Moore (NMR spectroscopy), Dr. Maxime A. Siegler
(single-crystal X-ray crystallography), and Dr. I. Phil Mortimer
(HR-MS) for assistance with compound identification and
characterization.
(19) Basudhar, D.; Bharadwaj, G.; Cheng, R. Y.; Jain, S.; Shi, S.;
Heinecke, J. L.; Holland, R. J.; Ridnour, L. A.; Caceres, V. M.; Spadari-
́
Bratfisch, R. C.; Paolocci, N.; Velazquez-Martínez, C. A.; Wink, D. A.;
Miranda, K. M. J. Med. Chem. 2013, 56, 7804−7820.
(20) Corrie, J. E. T.; Kirby, G. W.; Mackinnon, J. W. M. J. Chem. Soc.,
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(21) Cohen, A. D.; Zeng, B.-B.; King, S. B.; Toscano, J. P. J. Am.
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