O2-sulfonylethyl protected isopropylamine diazen-1-ium-1,2- diolates as nitroxyl (HNO) donors: Synthesis, β-elimination fragmentation, HNO release, positive inotropic properties, and blood pressure lowering studies

Article abstract of DOI:10.1021/jm301303p

New types of nonexplosive O²-sulfonylethyl protected (-CH ₂CH₂SO₂R; R = OMe, NHOMe, NHOBn, Me) derivatives of isopropylamine diazen-1-ium-1,2-diolate (IPA/NO) (2-5) were developed that are designed to act as novel HNO donors. These compounds, with suitable half-lives (6.6-17.1 h) at pH 7.4, undergo a base-induced β-elimination reaction that releases a methyl vinyl sulfone product and the parent IPA/NO anion which subsequently preferentially releases HNO (46-61% range). Importantly, the O²-methylsulfonylethyl compound 5 exhibited a significant in vitro inotropic effect up to 283% of the baseline value and increased the rates of contraction and relaxation but did not induce a chronotropic effect. Furthermore, compound 5 (22.5 mg/kg po dose) provided a significant reduction in blood pressure up to 6 h after drug administration. All these data suggest that O²-sulfonylethyl protected derivatives of IPA/NO, which are efficient HNO donors, could have potential applications to treat cardiovascular disease(s) such as congestive heart failure.

Full text of DOI:10.1021/jm301303p

Article
pubs.acs.org/jmc
O2‑Sulfonylethyl Protected Isopropylamine Diazen-1-ium-1,2-
diolates as Nitroxyl (HNO) Donors: Synthesis, β‑Elimination
Fragmentation, HNO Release, Positive Inotropic Properties, and
Blood Pressure Lowering Studies
Zhangjian Huang,‡,† Jatinder Kaur,† Atul Bhardwaj,† Nasser Alsaleh,† Julie A. Reisz,§ Jenna F. DuMond,§
S. Bruce King,§ John M. Seubert,† Yihua Zhang,‡ and Edward E. Knaus*,†
†Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2E1, Canada
‡Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, China
§Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina 27109, United States
S
* Supporting Information
ABSTRACT: New types of nonexplosive O2-sulfonylethyl
protected (-CH2CH2SO2R; R = OMe, NHOMe, NHOBn,
Me) derivatives of isopropylamine diazen-1-ium-1,2-diolate
(IPA/NO) (2−5) were developed that are designed to act as
novel HNO donors. These compounds, with suitable half-lives
(6.6−17.1 h) at pH 7.4, undergo a base-induced β-elimination
reaction that releases a methyl vinyl sulfone product and the
parent IPA/NO anion which subsequently preferentially releases HNO (46−61% range). Importantly, the O2-
methylsulfonylethyl compound 5 exhibited a significant in vitro inotropic effect up to 283% of the baseline value and increased
the rates of contraction and relaxation but did not induce a chronotropic effect. Furthermore, compound 5 (22.5 mg/kg po dose)
provided a significant reduction in blood pressure up to 6 h after drug administration. All these data suggest that O2-sulfonylethyl
protected derivatives of IPA/NO, which are efficient HNO donors, could have potential applications to treat cardiovascular
disease(s) such as congestive heart failure.
interest in HNO as a therapeutic agent continues to grow, new
and mechanistically unique HNO donors are urgently needed
in this challenging research area.
INTRODUCTION
■
Nitroxyl (HNO), the one-electron reduced and protonated
congener of nitric oxide (NO), exerts some biological activities
similar to that of NO. For example, HNO and NO are effective
vasodilators and inhibitors of platelet aggregation and
adhesion1 and show in vitro and in vivo anticancer activity.2
HNO has attracted recent attention because of some of its
unique biological and pharmacological properties that differ
from NO. In this regard, HNO exerts a positive inotropic
action on heart and it also shows lusitropic properties. Both
properties contribute to increased cardiac output.3−5 HNO also
induces beneficial effects in the treatment of ischemia−
reperfusion injury.6 These distinct biological actions indicate
that HNO release from HNO donors constitutes a drug design
strategy applicable to the treatment of heart failure.
Several HNO donor compounds have been discovered that
have been subjected to extensive studies. Angeli’s salt (AS,
Na2N2O3) is a well-known HNO donor that releases HNO in
the pH 4−8 range.7 The structural simplicity of Angeli’s salt
limits its extensive modification for medicinal chemistry utility.7
In our ongoing studies to design novel anti-inflammatory drugs
that act as HNO donors and are devoid of cardiovascular side
effects, we synthesized phenyl-8 and alkylsulfohydroxamic acid9
derivatives. Unfortunately, these sulfohydroxamic acids re-
quired a nonphysiological alkaline pH to release HNO. As
Diazeniumdiolates derived from secondary amines usually act
as NO donors under physiological conditions because of the
ease of protonation at the amine nitrogen (N3) and then
fragmentation to furnish the amine and 2 equiv of NO (Figure
1). In contrast, diazeniumdiolates of primary amines such as
isopropylamine diazeniumdiolate (IPA/NO), which is a HNO
donor, undergo preferential protonation at N2 prior to
decomposition to HNO and a nitrosamine in the pH range
5−13 (Figure 1).10,11 Recently a novel O2-acetyloxymethyl
IPA/NO (AcOM-IPA/NO) was reported by the Keefer group
that is a “pure” HNO donor with an ability to strengthen
contraction of beating cardiac myocytes.12 In a previous study,
we reported new types of O2-(N-methoxy-2-ethanesulfonyla-
mide) protected secondary amine diazeniumdiolates (repre-
sentative compound 1, Figure 1).13 NO release studies showed
that these compounds are able to release NO in the presence of
bases such as the basic natural amino acids arginine and
histidine or the non-nucleophilic organic base 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) in phosphate buffered
Received: September 10, 2012
Published: October 16, 2012
© 2012 American Chemical Society
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formation of the bromo compound 7, upon reaction with
potassium thioacetate in dry THF at reflux, afforded the desired
acetylthioethyl intermediate 8 in 85% yield. The thioacetate
moiety in 8 was converted to the sodium sulfonate analogue 9
in the presence of H2O2 in a solution of acetic acid. Reaction of
the sodium sulfonate 9 with thionyl chloride in DMF afforded
the corresponding relatively unstable sulfonyl chloride product
which was used immediately in the subsequent reaction without
further purification. Reaction of this sulfonyl chloride with
magnesium oxide in dry MeOH afforded the target methyl
sulfonate 2. Similar reactions of this sulfonyl chloride with
methoxylamine hydrochloride, or O-benzylhydroxylamine
hydrochloride, in the presence of sodium bicarbonate or
potassium carbonate in dry THF furnished the respective N-
methoxysulfonylamide 3 or N-benzyloxysulfonylamide 4 target
product.
Figure 1. Rationale for the design of novel base-sensitive HNO donors
2−5.
The synthetic reaction sequence used to prepare O2-(2-
methylsulfonylethyl) IPA/NO 5 is illustrated in Scheme 2.
Since the previously unknown 2-methylthio-1-
(trifluoromethanesulfonyloxy)ethane (structure not shown) is
a rather unstable compound, 2-(methylthio)ethyl iodide 1015
was found to be a useful alternative alkylating agent.
Accordingly, this key O2-alkylation reaction was performed
using the iodo compound 10 with isopropylamine diazenium-
diolate ammonium salt 6 rather than the corresponding IPA/
NO sodium salt in dry MeOH to yield the O2-alkylated product
11. The improved yield in this O2-alkylation reaction (28%
yield using the ammonium salt 6 relative to 8% for the sodium
salt) is likely due to the superior solubility in organic solvent
and higher reactivity of the ammonium salt 6.16 Oxidation of
the methylthio compound 7 using the mild oxidant potassium
peroxymonosulfate (KHSO5) afforded the target methyl
sulfone compound 5. The N-Boc compound 13 and the one-
carbon homologue of compound 5 (compound 15), required
for mechanistic NO and HNO release studies, were prepared
using the procedures illustrated in Scheme 2. In this regard,
reaction of the amine 11 with (Boc)2O and t-BuOLi afforded
the tert-butyl carbamate 12 (65%) that was oxidized using
KHSO5 to give the target compound 13 (56%). Condensation
of the IPA/NO salt 6 with chloromethyl methylsulfide in the
presence of Na2CO3 gave the O2-methylthiomethyl compound
14 (10%) which was subsequently oxidized using KHSO5 to
furnish the target product 15.
solution (PBS) at pH 7.4 via a β-elimination cleavage reaction.
Accordingly, it was of interest to investigate whether protecting
the primary amine IPA/NO using an O2-sufonylethyl group
would provide a hitherto-unknown class of stable (nonexplo-
sive) and readily isolated O2-protected IPA/NO that acts as an
HNO donor via a β-elimination reaction at physiological
relevant conditions. Herein, we now describe the synthesis of a
group of O2-sulfonylethyl IPA/NO compounds 2−5, half-lives,
fragmentation mechanism, HNO/NO release studies, positive
inotropic properties, and antihypertensive activities.
RESULTS AND DISCUSSION
■
Chemistry. The target O2-sulfonylethyl IPA/NO com-
pounds 2−4 were synthesized as depicted in Scheme 1,
Scheme 1. Synthesis of O2-Protected IPA/NO Analogues 2−
4
Mechanistic Decomposition of 5 Using 1H NMR
1
Spectrometry. H NMR has been used for the first time to
provide confirmation for the proposed base-induced β-
elimination for O2-sulfonylethyl NONOates. In this regard,
compound 5 (20 mM) was mixed with 1 equiv of DBU in
CD3OD in a NMR tube. This mixture was shaken at 25 °C for
exposure times of 0, 0.5, 1, and 4 h, respectively. The 1H NMR
spectrum was recorded at these 4 times, and the spectra are
presented in parts b, c, d, e of Figure 2, respectively. A
comparison of the spectra in Figure 2a and Figure 2b indicates
that addition of DBU to the solution of 5 in CD3OD results in
a rapid base-induced β-elimination reaction to furnish H2C
CHSO2Me (CH, δ 6.93; Htrans, δ 6.34; Hcis, δ 6.16, and Jtrans
= 17 Hz, Jcis = 10 Hz). The H2CCHSO2Me intermediate
product is gradually consumed, as illustrated by the NMR
spectral changes that occur proceeding from (b) to (c) and
then to (d). These spectral changes indicate that a Michael
reaction of methyl vinyl sulfone with deuterium methoxide
anion produces compound 16 (see structure in Figure 2). The
two geminal methylene protons on carbon adjacent to oxygen
employing a more efficient O2-alkylation reaction of IPA/NO.
The yield for the reaction of IPA/NO with 2-iodoethyl
thioacetate, which was initially used to prepare O2-(2-
acetylthioethyl) IPA/NO 8 according to a previous report,13
is very low (<2%). Fortunately, the reaction of 2-bromo-1-
(trifluoromethanesulfonyloxy)ethane with IPA/NO furnished
O2-(2-bromoethyl) IPA/NO 7 in a 65% yield, since the
trifluoromethanesulfonate moiety is a good leaving group for
the O2-alkylation of diazeniumdiolates.14 Subsequent trans-
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Scheme 2. Synthesis of the O2-Protected IPA/NO Analogue 5, the N3-Carbamoyl Compound 13 and the One-Carbon
Homologue 15
1
Figure 2. Partial H NMR spectrum of compound 5 (20 mM) without/with DBU (20 mM) in CD3OD. The spectrum of compound 5 (20 mM)
only is shown in (a). The spectra of a mixture of 5 (20 mM) with DBU (20 mM) in CD3OD after shaking at 25 °C for 0, 0.5, 1, and 4 h are shown in
(b), (c), (d), and (e), respectively.
in 16 appear as a doublet (Jvic = 5.5 Hz) at δ 3.77. The chemical
mechanism for the fragmentation of 5, which releases methyl
vinyl sulfone as a short-lived intermediate, is shown in Figure 2.
Methyl vinyl sulfone, a potentially toxic product from the β-
fragmentation reaction, undergoes a facile Michael reaction
with nucleophiles such as methoxide indicated above and with
glutathione (GSH) to furnish GS-CH2CH2SO2CH3 (see data
in Supporting Information). This provides strong credence that
the short-lived methyl vinyl sulfone should not induce adverse
in vivo toxicity.
Half-Life Determination of 2−5. The half-lives of target
compounds 2−5 were measured using a previously reported
spectrophotometric method17 via monitoring of the decrease in
the absorbance of the diazeniumdiolate chromophore at about
245 nm wavelength at pH 7.4 phosphate buffer at 37 °C.
Examples of the spectral changes occurring with time are shown
in Figure S1 in Supporting Information. A linear regression of
the ln(At − A∞) versus time plots generally showed good
linearity (R2, 0.97−0.99). The observed half-lives (t1/2) for
compounds 2, 3, 4, and 5 in pH 7.4 buffer solution containing
5% DMSO at 37 °C were 17.1, 8.9, 6.6, and 15.3 h, respectively,
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−
Figure 3. Plausible reactions of HNO and NO resulting in the formation of NO2 , N2O, and/or HNO during in vitro incubation studies.
−
Table 1. Percent (%) NO2 , N2O, and HNO Release from 2−5, 13, 15, IPA/NO, and AS in Buffer Solution at Different pH
Values
conditions
2
3
4
5
13
15
IPA/NO
AS
a
−
NO2
b
pH 7.4
10.6
42.0
0.8
6.0
1.8
28.6
0.4
4.6
62.0
36.0
c
pH 10.0
10.4
24.0
26.0
25.4
d
N2O
e
MeOH/TBS 7.4
0.5 h
2 h
1.4
1.9
1.9
0.2
0.4
0.2
16.7
15.1
1.2
1.3
5.9
3.8
0.5
9.4
8.3
7.0
6.1
7.4
6.8
5.9
5.7
8.5
9.5
12.9
33.5
0.4
9.7
1.6
48 h
0.5 h
2 h
27.5
0.3
10.4
1.3
f
MeOH/TBS 7.4/GSH
0.5
0.4
0.9
48 h
0.5 h
2 h
1.8
0.8
0.1
g
MeOH/TBS 10.0
52.6
62.9
17.3
29.0
56.8
61.3
24.2
30.3
52.9
62.2
0.4
h
MeOH/TBS 10.0/GSH
0.5 h
2 h
2.2
i
HNO
2 h
46.2 1.6
51.3 2.0
47.3 1.2
55.4 1.8
51.2 2.8
61.4 1.5
5.1 0.8
7.2 0.5
0.31 0.1
4.0 0.1
5.4 0.06
5.1 1.3
85.9 1.3
85.7 3.6
48 h
a
Percent NO2− is equal to (the concentration of NO2− detected)/(the initial concentration of test compound). The result is the mean value of three
b
measurements (n = 3) where variation from the mean % value was ≤0.2%. A solution of the test compound (2.4 mL of 50 μM) in phosphate buffer
at pH 7.4 containing 5% DMSO was incubated at 37 °C for 1.5 h. A solution of the test compound (2.4 mL of 50 μM) in Tris buffer containing 5%
DMSO at pH 10.0 was incubated at 37 °C for 1.5 h. Percent of nitrous oxide (N2O) released is based on the condensation of 2 mol of HNO → 1
c
d
mol of N2O + H2O. The result is the mean value of three measurements (n = 3). The HNO donor test compound (2−5 and IPA/NO)
e
concentration is 25 mM in each experiment and all samples were incubated at 37 °C. MeOH/TBS 7.4 solvent comprises 0.4 mL of MeOH and 0.4
f
mL of 500 mM Tris buffer solution (TBS) that contained 0.2 mM EDTA at pH 7.4. MeOH/TBS 7.4/GSH experiments are 50 mM in glutathione
g
(GSH). MeOH/TBS 10.0 solvent comprises 0.4 mL of MeOH and 0.4 mL of 500 mM TBS that also contained 0.2 mM EDTA at pH 10.0. The
h
solution pH was 9 at the conclusion of the experiment. MeOH/TBS 10.0/GSH experiments are 50 mM in glutathione (GSH). The solution pH
i
was 9 at the conclusion of the experiment. Percent of HNO released, based on a theoretical maximum release of 1 mol of HNO/mol of test
compounds 3−5, 13, 15, IPA/NO, and AS, and HNO was selectively trapped by phosphine 17 to produce an equivalent amount of HNO-derived
amide 19. The HNO donor test compound (3−5, 13, 15, IPA/NO, and AS) concentration is 200 μM. HNO trapping phosphine 17 concentration is
1 mM. The internal standard 4-hydroxybenzophenone concentration is 5.05 μM in each experiment, and all the samples were incubated at 37 °C.
The buffer solution contains 7.5% DMSO and 92.5% phosphate buffer (50 μM) at pH 7.4. The incubation mixture was quenched by addition of
H2O2 after 2 or 48 h. Quantification of the HNO-derived amide 19 was performed using a LC−MS assay.
which are much longer than that of IPA/NO (t1/2 = 2.3 min)18
and that of AcOM-IPA/NO (41 min).12 These data indicate
that 2−5 possess improved chemical stability at physiological
conditions relative to IPA/NO.
amine diazeniumdiolates to release NO rather than HNO, since
it does not undergo protonation at the N3-isopropylamino
nitrogen atom.19 Accordingly, compound 13, which is a N-Boc
derivative of compound 5, is expected to release NO rather
than HNO. Compound 15, a one-carbon homologue of the
two-carbon homologue 5, is not able to undergo a β-
elimination cleavage to release the IPA/NO anion. In this
regard, compound 15, unlike IPA/NO, is expected to release a
very low, or negligible, amount of HNO.
−
Nitrite (NO2 ), Nitrous Oxide (N2O), and HNO Release
Studies. A number of quantitative assays were performed to
determine the amount of HNO and NO released from the O2-
sulfonylethyl IPA/NO compounds 2−5 that takes place
following β-elimination cleavage. Prior to initiating these assays,
compounds 13 and 15 were prepared for use as controls (see
structures in Scheme 2). It has been reported that N3-
carbamoyl isopropylaminodiazeniumdiolate acts like secondary
Quantification of the amount of HNO and NO released from
donor compounds requires the use of several assays. Four
reactions that NO and/or HNO are expected to undergo
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during in vitro incubation studies in buffer at physiological pH
are illustrated in Figure 3. IPA/NO functions as a dual donor of
HNO and NO at pH 5−8.10,11 Consequently, compounds 2−5
are expected to release both NO and HNO following the β-
elimination and O2-cleavage reaction. Once produced, NO
could be rapidly oxidized to nitrite in the presence of oxygen in
aqueous PBS solution (reaction A).20 In contrast, HNO can
either dimerize to form N2O (8 × 106 M−1 s−1, reaction B)21,22
or react with NO to form a hyponitrite radical (with a rate
constant of 5.6 × 106 M−1 s−1) which subsequently reacts with
one more NO to ultimately furnish N2O and nitrite (reaction
C).23,24 Given this complexity, simply using the amount of
under physiological relevant conditions, 3−5 undergo a β-
elimination reaction with expulsion of the free isopropylamine
diazeniumdiolate anion that subsequently releases HNO.
Interestingly, compound 2 showed low percentages of N2O
production in MeOH/TBS at pH 7.4 even upon incubation for
48 h (1.9%), demonstrating that the O2-CH2CH2SO2OMe
moiety must be relatively stable under physiological conditions.
In contrast, in MeOH/TBS at pH 10, the % N2O produced
from compounds 2−5 was much higher as expected. For
instance, compound 5 liberated 1.6% of N2O in MeOH/TBS at
pH 7.4 relative to 62.2% N2O in MeOH/TBS at pH 10 for a 2
h incubation. The amount of N2O released from the
SO2NHOMe 3 (29.0%) and SO2NHOBn 4 (30.3%)
compounds in the MeOH/TBS 10/GSH system was much
greater than expected for a 2 h incubation. One plausible
explanation for this increased amount of N2O is attributed to a
competition between the reaction of GSH with the vinyl
sulfone arising from the β-elimination reaction and the reaction
(trapping) of HNO with GSH. In this regard, the vinyl sulfone
acts as a Michael acceptor to react with GSH, thereby
decreasing the amount of GSH available to react with HNO.
Credence for this explanation is based on the observation that
GSH reacts readily with methyl vinyl sulfone to furnish GS-
CH2CH2SO2CH3 (see data provided as Supporting Informa-
tion). A second plausible explanation for this increased amount
of N2O is attributed to the reaction C pathway shown in Figure
3. The reaction of HNO with NO to form a hyponitrite radical
(5.6 × 106 M−1 s−1) is faster than the trapping reaction of HNO
by GSH (2 × 106 M−1 s−1) such that GSH is not able to stop
the generation of N2O from the hyponitrite radical.
−
NO2 to quantify the percentage of NO and using N2O to
quantify the percentage of HNO may lack accuracy. It has been
reported that the reaction of the water-soluble phosphine 17
with HNO occurs at a rate comparable to the reaction of HNO
with glutathione (2 × 106 M−1 s−1), suggesting the utility of 17
as a selective and efficient agent for the detection (trapping)
and quantization of HNO.25,26 On the basis of these data, we
proceeded to use three independent assays to measure the
release of HNO and/or NO from the test and reference
compounds: (a) the colorimetric Griess reaction assay was used
−
to determine the amount of NO2 ; (b) a gas chromatographic
(GC) assay was used to determine the amount of N2O that was
derived from dimerization of HNO or the reaction of HNO
and NO; (c) a selective LC−MS assay was developed to
determine the percentage of HNO released by quantification of
HNO-derived amide 19 produced during a Staudinger reaction.
The data from these studies are presented in Table 1.
−
−
The % NO2 (% NO2 is equal to the concentration of
NO2− measured in the Griess assay/the initial concentration of
test compound) released from 2−5 in PBS at pH 7.4 and 37 °C
varied within a 0.8−10.6% range, which is indicative of slow
NO release (Table 1). In contrast, the % NO2− released in Tris
buffer solution at pH 10 and 37 °C was increased (10.4−42.0%
range), which is attributed to a more extensive β-elimination
The percentages of HNO released from compounds 3−5,
13, 15, IPA/NO, and AS were determined by quantification of
HNO-derived amide 19 produced in the reaction of phosphine
17 in the presence of the test compound using a modified LC−
MS assay (Table 1).26 Compounds 3−5 showed high HNO
release (46.2−61.4%) in PBS at pH 7.4 for 2 or 48 h
incubations. The amount of N2O released (8.3% at 2 h) from
IPA/NO in MeOH/TBS 7.4 is in good agreement with the
5.4% release of HNO determined in the LC−MS assay. Two
analogues of compound 5 (the carbamoyl compound 13 and
compound 15 having a one-carbon spacer) release a much
lower percentage of HNO (0.31−7.17%), indicating that a
primary amine moiety and a two-carbon spacer are two
important structural requirements that are highly relevant to
the concept of designing HNO donor compounds where HNO
release is triggered following a β-elimination reaction.
Collectively, the three independent assays employed to
−
cleavage at alkaline pH that furnishes IPA/NO. The % NO2
arising from compound 13 was expectedly higher than that
from parent compound 5 in PBS at pH 7.4 (28.6% for 13; 1.8%
for 5), since the carbamated primary amine diazeniumdiolate
acts like a secondary amine to predominately release NO.19
The one carbon linkage analogue 15 released a very low
amount of NO2− at both pH 7.4 and 10.0, revealing that the β-
elimination-induced O2-cleavage reaction is not favorable. The
observation that IPA/NO releases more NO2− at pH 7.4 (62%)
than at pH 10 (36%) is consistent with reports that IPA/NO
preferentially decomposes to release HNO rather than NO at
highly alkaline pH values.10,11 N2O release from 2−5 and IPA/
NO was quantified by gas chromatography (GC) using four
MeOH-based solvent mixtures at 37 °C (Table 1). Incubations
were also carried out at pH 10, since a highly alkaline pH will
accelerate the β-elimination reaction to furnish the IPA/NO
anion. Competition incubations experiments containing gluta-
thione (GSH) were also performed, since HNO reacts rapidly
with thiols to form disulfides and hydroxylamine or
sulfinamides.27,28 In this regard, the reaction of HNO with
GSH competes with the spontaneous dimerization of HNO to
N2O, thereby reducing the amount of N2O produced. The
percentages of N2O (based on the condensation of 2 mol of
HNO → 1 mol of N2O + H2O) arising from 3−5 in MeOH/
TBS at pH 7.4 increased with time. For compound 3, the
percentages of N2O after incubation for 0.5, 2, and 48 h were
5.9%, 12.9%, and 33.5%, respectively. These data indicate that
−
measure the percentages of NO2 , N2O, and HNO release
provide a relatively complete profile of HNO and/or NO
release properties of the test compounds. These data acquired
revealed that O2-sulfonylethyl compounds 3−5 are good HNO
donors compared to the parent IPA/NO at physiological
conditions.
Positive Inotropic, Rates of Contraction and Relaxa-
tion, and Heart Rate (Cardiac Hemodynamic) Studies.
The positive inotropic effect induced by HNO is considered to
be a unique biological and pharmacological action that differs
from that of NO. It was therefore of interest to carry out the
title studies for compound 5 which is an effective HNO donor.
To determine whether compound 5 possessed positive
inotropic properties, hearts from C57BL/6 mice were perfused
with Krebs−Henseleit buffer for 20 min. All hearts had normal
baseline contractile function, measured as LVDP (inotropic
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Table 2. Cardiac Hemodynamic Parameters Were Measured Using Isolated Perfused Hearts
a
a
baseline
6
5
isoproterenol
LVDP (cmH2O)
66
187 34*
157
5330
rate of contraction, dP/dtmax (cmH2O/ms)
2619 470
−2389 417
311 48
5698 679*
−5330 711*
269 22
rate of relaxation, −dP/dtmin (cmH2O/ms)
HR, perfused (beats/min)
−5882
334
a
*
Values are mean SEM: p < 0.05 vs baseline, n = 4 (compound 5), n = 1 (isoproterenol).
Figure 4. Systolic blood pressure (BPsys), diastolic blood pressure (BPdia), mean blood pressure (BPmean), and heart rate (HR) in conscious mice
following oral administration of either vehicle alone (control group, mean SD, n = 6) or test compound 5 (60.345 μmol/kg or 22.5 mg/kg po; n =
4 at 1 h, n = 5 at 3 h, and n = 6 at 6 h). Data are presented as group mean SD. Statistical differences between groups are denoted as ∗, p < 0.05.
effect), HR, and diastolic (rate of relaxation, −dP/dtmin) and
systolic (rate of contraction, dP/dtmax) contractions (Table 2).
Hearts perfused with compound 5 (100 μM) demonstrated a
significant increase in LVDP (positive inotropic effect) and
relaxation and contraction rates (Table 2) when compared to
baseline levels (290% above baseline). Similar inotropic effects
were observed with hearts perfused with isoproterenol (Table
2). However, no significant changes to heart rate were observed
with compound 5 at the concentration examined. Thus,
compound 5 possessed significant inotropic effects where the
force of contraction and degree of relaxation were increased
without inducing a chronotropic effect.
Blood Pressure Studies. Systolic blood pressure (BPsys,
mmHg), diastolic blood pressure (BPdia, mmHg), and heart rate
(HR, beats min−1) were measured at 1, 3, and 6 h time intervals
following oral administration of either the vehicle alone
(control group, n = 6) or the HNO donor compound 5
(60.345 μmol/kg or 22.5 mg/kg po, n = 4 at 1 h; n = 5 at 3 h; n
= 6 at 6 h) to C57 black mice. A minimum of three consecutive
measurements were made for each mouse at each time interval,
and the mean value is the average of these three measurements.
The data obtained for BPsys, BPdias, BPmean (average of BPsys and
BPdia), and HR are illustrated in panels a−d of Figure 4 (Table
S1 listing numerical data standard deviation is provided as
Supporting Information).
The O2-(2-methylsulfonylethyl) compound 5 showed a
significant reduction (∗, p < 0.05) in BPsys (mmHg) at 1
(120.2), 3 (103.4), and 6 (114.4) hours after drug
administration relative to the control group (1 h, 126.5; 3 h,
118.3; 6 h, 126.2; see data in Figure 4a). Significant reductions
in BPdia were also observed at 3 and 6 h, but not at 1 h, after
drug administration (see data in Figure 4b). The BPmean profile
(Figure 4c) shows that there was a significant reduction in
BPmean at all three times (1 h, 95.4; 3 h, 83.6; 6 h, 87.5) relative
to control values (1 h, 106.0; 3 h, 97.5; 6 h, 105.7).
The HR of mice was determined, and the data are shown in
Figure 4d (complete data are provided in Table S1 as
Supporting Information). The HR at 1 h (531) and 3 h
(488) was higher than, but nearly identical at 6 h (442), that for
control mice (449, 448, and 439 beats min−1 at 1, 3, and 6 h
after drug administration, respectivcly). This in vivo increase in
HR is attributed to neuroeffects that increase HR to
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subsequently isolated (purified) on a Combiflash Rf system using a
C18 column (complete details are provided as Supporting
Information). All reagents, purchased from the Sigma-Aldrich, were
used without further purification. In vitro cardiac hemodynamic
studies and in vivo blood pressure and heart rate measurements were
carried out using protocols approved by the Health Sciences Animal
Welfare Committee at the University of Alberta, Canada.
compensate for the significant reduction in BP observed. In
comparison, the in vitro perfused heart study described
previously showed that compound 5 did not induce a
chronotropic effect, since there are no neurocompensatory
effects to change HR in the in vitro (ex vivo) isolated heart.
CONCLUSIONS
■
O2-(2-Methoxysulfonylethyl) 1-(Isopropylamino)diazen-1-
ium-1,2-diolate (2). O2-(2-Oxysulfonylethylsodium salt) 1-
(isopropylamino)diazen-1-ium-1,2-diolate (9, 250 mg, 1.0 mmol)
was dissolved in DMF (2.5 mL), and SOCl2 (0.22 mL, 3.0 mmol) was
added dropwise. The reaction mixture was allowed to stir at 25 °C for
1.5 h, poured into cold water (30 mL), and extracted with ethyl ether
(3 × 30 mL). The combined organic fractions were washed with 2 N
HCl solution and brine, and the organic fraction was dried (MgSO4).
After concentration in vacuo at 25 °C, the resulting sulfonyl chloride
product obtained as a brown syrup was dissolved in dry MeOH (5
mL), and magnesium oxide (200 mg, 5.0 mmol) was added. The
reaction mixture was vigorously stirred at 25 °C until the sulfonyl
chloride had completely disappeared (TLC; EtOAc−hexane, 1:2, v/v)
in about 6 h. The reaction mixture was filtered through a pad of Celite
that provided a clear filtrate which was added to ethyl acetate (20 mL).
This mixture was washed with water (20 mL) and brine (20 mL), and
the organic fraction was dried (MgSO4). Removal of the solvent from
the organic fraction in vacuo gave a residue that was purified by flash
silica gel column chromatography using n-hexane−EtOAc (2:1, v/v) as
eluent to afford the title compound 2 (67 mg, 28%, two steps) as a
colorless oil. IR (film): 3275, 2986, 2934, 1742, 1362, 1165 cm−1. ESI-
MS: 242 [M + H]+, 264 [M + Na]+. 1H NMR (CD3OD): δ 1.22 (d, J
= 6.7 Hz, 6H, (CH3)2CH), 3.66 (t, J = 6.1 Hz, 2H, OCH2CH2S),
3.83−3.95 (m, 4H, (CH3)2CH and SO3CH3), 4.53 (t, J = 6.1 Hz, 2H,
OCH2CH2S), 8.09 (s, 1H, NH). 13C NMR (CD3OD): δ 19.5, 50.2,
57.1, 62.0, 67.9. Anal. Calcd for C6H15N3O5S: C, 29.87; H, 6.27.
Found: C, 29.90; H, 6.09.
The primary amine O2-sulfonylethyl IPA/NO compounds 2−5
described in this study possess the following distinctive
chemical and/or biological advantages compared to the
previously reported O2-sulfonylethyl secondary amine diaze-
niumdiolates10 and the parent IPA/NO. (i) O2-protected IPA/
NO analogues 2−5, unlike the unstable and highly explosive
parent IPA/NO,10,11 are stable, readily isolated compounds. (ii)
O2-protected IPA/NO analogues 2−5 have longer improved
half-lives (6.6−17.1 h) under physiological conditions relative
to IPA/NO which has a very short half-life (2.3 min).18 (iii) 1H
NMR spectroscopy, used to investigate the mechanistic
fragmentation of 5 in the presence of DBU in CD3OD
solution, provided validation for a previously proposed base-
induced β-elimination mechanism.13 Following the β-elimi-
nation reaction to furnish the IPA/NO anion and methyl vinyl
sulfone, the latter short-lived vinyl product undergoes
subsequent reaction with methoxide to form a Michael addition
product. (iv) It is highly probable that compounds 3−5
undergo preferential protonation at the primary amino N3-
position prior to decomposition, since a high amount of HNO
(46.2−61.4%), as quantified by selective formation of the
HNO-derived amide 19 using an efficient LC−MS assay, was
released. (v) Compound 5 showed a significant in vitro
inotropic effect, increasing the force of contraction and the rate
of relaxation without any chronotropic effect; and (vi)
compound 5 exhibited an antihypertensive effect, since BPmean
was lowered significantly up to 6 h after drug administration.
These results reveal that O2-sulfonylethyl IPA/NO compounds
3−5 represent a novel class of chemically stable HNO donor
compounds with excellent HNO release properties at
physiological pH. The IPA/NO donor compounds described
constitute useful lead compounds with relevant pharmaceutical
properties for the design of nitroxyl donors with potential
applications to treat cardiovascular disease(s) including
congestive heart failure.
O2-(2-Methoxyaminosulfonylethyl) 1-(Isopropylamino)-
diazen-1-ium-1,2-diolate (3). The same method was used to
prepare the intermediate sulfonyl chloride compound as described for
the preparation of compound 2. The resulting brown syrup of the
sulfonyl chloride product was dissolved in dry THF (5 mL), and then
methoxylamine hydrochloride (167 mg, 2.0 mmol) and NaHCO3 (250
mg, 3.0 mmol) were added. The reaction mixture was vigorously
stirred at 25 °C until the sulfonyl chloride had completely disappeared
(TLC; EtOAc−hexane, 1:2, v/v) in about 3 h. The reaction mixture
was filtered through a pad of Celite that provided a clear filtrate which
was added to ethyl acetate (20 mL). This mixture was washed with
water (20 mL) and brine (20 mL), and the organic fraction was dried
(MgSO4). Removal of the solvent from the organic fraction in vacuo
gave a residue that was purified by flash silica gel column
chromatography using n-hexane−EtOAc (2:1, v/v) as eluent to afford
the title compound 3 (82 mg, 32%, two steps) as a colorless oil. IR
(film): 3223, 2994, 2945, 1704, 1348, 1163 cm−1. ESI-MS: 257 [M +
H]+, 279 [M + Na]+. 1H NMR (DMSO-d6): δ 1.04 (d, J = 6.8 Hz, 6H,
(CH3)2CH), 3.56 (t, J = 6.1 Hz, 2H, OCH2CH2S), 3.67 (s, 3H,
NHOCH3), 3.72−3.77 (m, 1H, (CH3)2CH), 4.54 (t, J = 6.1 Hz, 2H,
OCH2CH2S), 8.70 (d, J = 9.2 Hz, 1H, (CH3)2CHNH), 10.2 (s, 1H,
NHOCH3). 13C NMR (DMSO-d6): δ 19.6, 47.3, 47.4, 64.4, 65.6. Anal.
Calcd for C6H16N4O5S: C, 28.12; H, 6.29. Found: C, 27.82; H, 6.07.
O2-(2-Benzyloxyaminosulfonylethyl) 1-(Isopropylamino)-
diazen-1-ium-1,2-diolate (4). The same method was used to
prepare intermediate sulfonyl chloride compound as described for the
preparation of compound 2. The resulting brown syrup of the sulfonyl
chloride product was dissolved in dry THF (5 mL), and then O-
benzyhydroxylamine hydrochloride (319 mg, 2.0 mmol) and K2CO3
(276 mg, 2.0 mmol) were added. The reaction mixture was vigorously
stirred at 25 °C until the sulfonyl chloride had completely disappeared
(TLC; EtOAc−hexane, 1:2, v/v) in about 4 h. The reaction mixture
was filtered through a pad of Celite that provided a clear filtrate which
was added to ethyl acetate (20 mL). This mixture was washed with
water (20 mL) and brine (20 mL), and the organic fraction was dried
(MgSO4). Removal of the solvent from the organic fraction in vacuo
EXPERIMENTAL SECTION
■
General. Melting points were determined on a Thomas-Hoover
capillary apparatus and are uncorrected. Infrared (IR) spectra were
recorded as films on NaCl plates using a Nicolet 550 series II Magna
1
FT-IR spectrometer. H NMR (300 MHz) and 13C NMR (75 MHz)
spectra were measured on a Bruker AM-300 spectrometer with TMS
as the internal standard, where J (coupling constant) values are
estimated in hertz (Hz). Liquid chromatography−mass spectrometry
(LC−MS) data were recorded on a Water’s Micromass ZQ 4000 mass
spectrometer using the ESI negative ionization mode and a reverse
phase C18 column. Microanalyses were performed for C, H, N by the
Microanalytical Service Laboratory, Department of Chemistry,
University of Alberta, Canada. Unknown compounds 2−5, 7−9, and
11−15 showed a single spot on Macherey-NagelPolygram Sil G/UV254
silica gel plates (0.2 mm) using a low, medium, and highly polar
solvent system, and no residue remained after combustion, indicating a
purity >95%. Column chromatography was performed on a
Combiflash Rf system using a gold silica (2−5, 7−9, and 11−15)
column. Although a method has been reported26,29 for the preparation
of compound 19, the amide compound 19 required as a reference
sample in the LC−MS assay was more conveniently synthesized via a
reaction of phosphine 17 with HNO released from AS and
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gave a residue that was purified by flash silica gel column
chromatography using n-hexane−EtOAc (2:1, v/v) as eluent to afford
the title compound 4 (83 mg, 25%, two steps) as a colorless oil. IR
(film): 3229, 2981, 2939, 1346, 1160 cm−1. ESI-MS: 355 [M + Na]+.
1H NMR (DMSO-d6): δ 1.02 (d, J = 6.7 Hz, 6H, (CH3)2CH), 3.59 (t,
J = 6.1 Hz, 2H, OCH2CH2S), 3.70−3.77 (m, 1H, (CH3)2CH), 4.42 (t,
J = 6.1 Hz, 2H, OCH2CH2S), 4.89 (s, 2H, CH2Ar), 7.31−7.38 (m, 5H,
ArH), 8.70 (d, J = 8.5 Hz, 1H, (CH3)2CHNH), 10.2 (s, 1H,
NHOCH2Ar). 13C NMR (DMSO-d6): δ 19.6, 47.4, 47.5, 65.6, 78.2,
128.3, 128.8, 135.7. Anal. Calcd for C12H20N4O5S: C, 43.36; H, 6.07.
Found: C, 43.43; H, 5.73.
1H, (CH3)2CH), 4.94 (s, 2H, OCH2S). 13C NMR (DMSO-d6): δ 20.3,
38.8, 49.4, 84.1. Anal. Calcd for C5H13N3O4S: C, 28.43; H, 6.20; N,
19.89. Found: C, 28.68; H, 6.24; N, 19.72.
Nitric Oxide Release Assay. In vitro nitric oxide release, upon
incubation of the test compound (2.4 mL of 5.0 × 10−2 mM) with
either (i) phosphate buffer solution (PBS) at pH 7.4 or (ii) TBS at pH
10 at 37 °C for 1.5 h was determined by quantification of nitrite
produced by the reaction of nitric oxide with oxygen and water using
the Griess reaction. Nitric oxide release data were acquired for test
compounds (2−5) and the reference compound IPA/NO using the
reported procedure.30
O2-(2-Methylsulfonylethyl) 1-(Isopropylamino)diazen-1-
ium-1,2-diolate (5). O2-(2-Methylthioethyl) 1-(isopropylamino)-
diazen-1-ium-1,2-diolate (11, 260 mg, 1.35 mmol) was dissolved in
THF−MeOH (1:1, v/v; 8 mL). A solution of potassium
peroxymonosulfate (1.66 g, 2.7 mmol) in water (8 mL) was added
dropwise at 0 °C, and the reaction was allowed to proceed for 4 h at
25 °C with stirring. Water (20 mL) was added to the reaction mixture.
This solution was extracted with EtOAc (3 × 30 mL). The combined
organic fractions were washed with brine (20 mL), and the organic
fraction was dried (MgSO4). Removal of the solvent in vacuo afforded
a residue which was purified by flash column chromatography using
EtOAc−hexane (1:1, v/v) as eluent to furnish the title compound 5
(167 mg, 55%) as a colorless oil, which solidified upon storage in a
refrigerator to give 5 as a yellow powder; mp 50−52 °C. IR (film):
3244, 2975, 2934, 1294, 1134 cm−1. ESI-MS: 226 [M + H]+, 248 [M +
Na]+, 473 [2M + Na]+. 1H NMR (CD3OD): δ 1.12 (d, J = 6.1 Hz, 6H,
(CH3)2CH), 3.03 (s, 3H, SO2CH3), 3.51 (t, J = 5.5 Hz, 2H,
OCH2CH2S), 3.85−3.91 (m, 1H, (CH3)2CH), 4.55 (t, J = 5.5 Hz, 2H,
OCH2CH2S). 13C NMR (DMSO-d6): δ 20.4, 42.9, 49.2, 53.8, 66.8.
Anal. Calcd for C6H15N3O4S: C, 31.99; H, 6.71; N, 18.65. Found: C,
32.08; H, 6.45; N, 18.32.
Gas Chromatographic N2O Analysis. For headspace analysis,
substrate (0.04 mmol) was placed in a 10 mL round-bottom flask,
which was sealed with a rubber septum and flushed with inert gas.
Solvent (0.8 mL) was added and the sample incubated at 37 °C, and at
desired time-points headspace aliquots (25 μL) were injected via a
gastight syringe onto a 7890A Agilent Technologies gas chromato-
graph equipped with a microelectron capture detector and a 30 m ×
0.32 m (25 μm) HP-MOLSIV capillary column. The oven was
operated at 200 °C for the duration of the run (4.5 min). The inlet was
held at 250 °C and run in split mode (split ratio 1:1) with a total flow
(N2 as carrier gas) of 4 mL/min and a pressure of 37.9 psi. The μECD
was held at 325 °C with a makeup flow (N2) of 5 mL/min. The
retention time of nitrous oxide was 3.4 min, and yields were calculated
based on a standard curve for nitrous oxide (Matheson Tri-Gas).
LC−MS Quantification of Phosphine-Mediated HNO Trap-
ping. A solution of phosphine 17 in DMSO (25 μL), the test
compound (2−5, 13, 15, IPA/NO, or AS) in DMSO (25 μL), and
internal standard compound 4-hydroxybenzophenone in DMSO (25
μL) were added to a PBS solution, pH 7.4 (925 μL), providing 17 (1
mM), test compound (200 μM), and 4-hydroxybenzophenone (5.05
μM). The incubation mixture was stirred at 37 °C in a sealed vial for 2
or 48 h. Hydrogen peroxide solution (30% w/v, 5 μL) was added into
the incubation mixture to quench the reaction of 17 and HNO. An
aliquot (500 μL) was removed and the solution was evaporated to
dryness under vacuum (using a Thermo Scientific Savant DNA 120
SpeedVac Concentrator) at about 60 °C. Methanol (500 μL) was
added to dissolve the residue. After centrifugation, an aliquot of the
clear methanol solution (250 μL) was analyzed by LC−MS (Water’s
Micromass ZQTM 4000 LC−MS instrument, operating in the ESI
negative mode, equipped with a Water’s 2795 separation module).
Separations were performed in triplicate using a Kromasil 100-5-C18
(100 μm pore size, 5 μm particle size) reverse phase column (2.1 mm
diameter × 50 mm length), preceded by a Kromasil 100-5-C18 2.1×
guard column. Separations were effected using a gradient going from
MeCN/1% aqueous formic acid (40:60, v/v) to MeCN/1% aqueous
formic acid (60:40, v/v) over a 12 min period at a flow rate of 0.25
mL/min. Operating parameters were as follows: capillary voltage = 3.5
kV; cone voltage = 20 V; source temperature = 140 °C; sesolvation
temperature = 250 °C; cone nitrogen gas flow = 100 L/h; desolvation
nitrogen gas flow = 550 L/h. The identities of products 18 (retention
time of 6.62 min), 19 (retention time of 3.63 min), and the internal
standard 4-hydroxybenzophenone (retention time of 10.89 min) were
confirmed by MS (LC−MS chromatograms are provided as
Supporting Information). The amount of compound 19 produced
upon incubation of the test compound was determined from a
standard curve (that was linear over a concentration range of 0.2−10
μg/mL, R2 > 0.997) prepared using an authentic sample of compound
19. The LC−MS HNO release data presented in Table 1 are the mean
of triplicate experiments. A control experiment involving incubation of
compound 17 and 4-hydroxybenzophenone under identical con-
ditions, but not containing a test compoud, and subsequent LC−MS
analysis showed the absence of 19.
O2-(2-Methylsulfonylethyl) 1-(N-Boc-isopropylamino)-
diazen-1-ium-1,2-diolate (13). O2-(2-Methylthioethyl) 1-(N-Boc
isopropylamino)diazen-1-ium-1,2-diolate (12, 100 mg, 0.341 mmol)
was dissolved in THF−MeOH (1:1, v/v; 3 mL). A solution of
potassium peroxymonosulfate (420 mg, 0.682 mmol) in water (3 mL)
was added dropwise at 0 °C, and the reaction was allowed to proceed
for 2 h at 25 °C with stirring. Water (10 mL) was added to the
reaction mixture. This solution was extracted with EtOAc (3 × 10
mL). The combined organic fractions were washed with brine (10
mL), and the organic fraction was dried (MgSO4). Removal of the
solvent in vacuo afforded a residue which was purified by flash column
chromatography using EtOAc−hexane (1:1, v/v) as eluent to furnish
the title compound 13 (62 mg, 56%) as a yellowish oil, which
solidified upon storage in a refrigerator to give 13 as a yellow powder;
mp 61−62 °C. IR (film): 2983, 2933, 1747, 1267, 1136 cm−1. ESI-MS:
348 [M + Na]+, 364 [M + K]+. 1H NMR (CD3OD): δ 1.25 (d, J = 7.2
Hz, 6H, (CH3)2CH), 1.48 (s, 9H, C(CH3)3), 2.99 (s, 3H, SO2CH3),
3.35 (t, J = 5.4 Hz, 2H, OCH2CH2S), 4.25−4.29 (m, 1H, (CH3)2CH),
4.68 (t, J = 5.4 Hz, 2H, OCH2CH2S). 13C NMR (DMSO-d6): δ 19.6,
28.0, 43.0, 51.9, 53.9, 67.7, 84.2, 151. Anal. Calcd for C11H23N3O4S: C,
40.60; H, 7.12; N, 12.91. Found: C, 40.67; H, 7.06; N, 12.66.
O2-(Methylsulfonylmethyl) 1-(Isopropylamino)diazen-1-
ium-1,2-diolate (15). O2-(Methylthiomethyl) 1-(isopropylamino)-
diazen-1-ium-1,2-diolate (14, 190 mg, 1.06 mmol) was dissolved in
THF−MeOH (1:1, v/v; 5 mL). A solution of potassium
peroxymonosulfate (1305 mg, 2.12 mmol) in water (5 mL) was
added dropwise at 0 °C, and the reaction was allowed to proceed for 2
h at 25 °C with stirring. Water (15 mL) was added to the reaction
mixture. This solution was extracted with EtOAc (3 × 15 mL). The
combined organic fractions were washed with brine (15 mL), and the
organic fraction was dried (MgSO4). Removal of the solvent in vacuo
afforded a residue which was purified by flash column chromatography
using EtOAc−hexane (1:1, v/v) as eluent to furnish the title
compound 15 (141 mg, 63%) as a colorless oil, which solidified
upon storage in a refrigerator to give 15 as a white powder; mp 64−65
°C. IR (film): 3263, 2990, 2938, 1305, 1143, 1084 cm−1. ESI-MS: 212
[M + H]+, 234 [M + Na]+, 250 [M + K]+. 1H NMR (CD3OD): δ 1.14
(d, J = 6.6 Hz, 6H, (CH3)2CH), 2.93 (s, 3H, SO2CH3), 3.93−3.97 (m,
Determination of Cardiac Hemodynamic Properties for
Compound 5. C57BL/6 mouse hearts were perfused in the
Langendorff mode as described.31−33 Briefly, hearts were perfused in
a retrograde fashion at constant pressure (90 cmH2O) with
continuously aerated (95%O2/5%CO2) Krebs−Henseleit buffer at
37 °C. Hearts were first stabilized for 20 min with buffer and then
perfused with compound 5 (100 μM) or isoproterenol (100 μM,
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Journal of Medicinal Chemistry
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positive ionotropic control) for 20 min. Heart rate (HR), diastolic and
systolic rates, and left ventricular developed pressure (LVDP) were
obtained.
(6) Pagliaro, P.; Mancardi, D.; Rastaldo, R.; Penna, C.; Gattullo, D.;
Miranda, K. M.; Feelisch, M.; Wink, D. A.; Kass, D. A.; Paolocci, N.
Nitroxyl affords thiol-sensitive myocardial protective effects akin to
early preconditioning. Free Radical Biol. Med. 2003, 34, 33−43.
(7) DuMond, J. F.; King, S. B. The chemistry of nitroxyl-releasing
compounds. Antioxid. Redox Signaling 2011, 14, 1637−1648.
(8) Huang, Z.; Velazquez, C.; Abdellatif, K. R. A.; Chowdhury, M.;
Jain, S.; Reisz, J. A.; DuMond, J. F.; King, S. B.; Knaus, E. E. Acyclic
triaryl olefins possessing a sulfohydroxamic acid pharmacophore:
synthesis, nitric oxide/nitroxyl release, cyclooxygenase inhibition, and
anti-inflammatory studies. Org. Biomol. Chem. 2010, 8, 4124−4130.
(9) Huang, Z.; Velazquez, C.; Abdellatif, K. R. A.; Chowdhury, M.;
Reisz, J. A.; DuMond, J. F.; King, S. B.; Knaus, E. E.
Ethanesulfohydroxamic acid ester prodrugs of nonsteroidal antiin-
flammatory drugs (NSAIDs): synthesis, nitric oxide and nitroxyl
release, cyclooxygenase inhibition, anti-inflammatory, and ulcer-
ogenicity index studies. J. Med. Chem. 2011, 54, 1356−1364.
(10) Dutton, A. S.; Suhrada, C. P.; Miranda, K. M.; Wink, D. A.;
Fukuto, J. M.; Houk, K. N. Mechanism of pH-dependent
decomposition of monoalkylamine diazeniumdiolates to form HNO
and NO, deduced from the model compound methylamine
diazeniumdiolate, density functional theory, and CBS-QB3 calcula-
tions. Inorg. Chem. 2006, 45, 2448−2456.
In Vivo Blood Pressure Measurement. Noninvasive blood
pressure and heart rate measurements were performed according to a
protocol approved by the Health Sciences Animal Welfare Committee
at the University of Alberta (Edmonton, Canada). Following oral
administration of test compound 5 (60.345 μmol/kg or 22.5 mg/kg po
dose) dissolved in water containing 1% methylcellulose, changes in
BPsys, BPdia, BPmean, and HR in C57 black mice were measured in a
conscious state at 1, 3 and 6 h time intervals using our previously
reported method.34
ASSOCIATED CONTENT
■
S
* Supporting Information
Preparation and characterization data of compounds 6−12, 14,
and 17−19; half-lives determination studies for 2−5;
mechanistic decomposition studies using 1H NMR; and
numerical blood pressure and heart rate data for control and
treated mice. This material is available free of charge via the
Internet at http://pubs.acs.org.
(11) 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. HNO and NO release from a primary
amine-based diazeniumdiolate as a function of pH. Inorg. Chem. 2011,
50, 3262−3270.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (780) 492-5993. Fax: (780) 492-1217. E-mail:
eknaus@pharmacy.ualberta.ca.
(12) 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.
Dual mechanisms of HNO generation by a nitroxyl prodrug of the
diazeniumdiolate (NONOate) class. J. Am. Chem. Soc. 2010, 132,
16526−16532.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(13) Huang, Z.; Knaus, E. E. O2-(N-Hydroxy(methoxy)-2-ethane-
sulfonamido) protected diazen-1-ium-1,2-diolates: nitric oxide release
via a base-induced β-elimination cleavage. Org. Lett. 2011, 13, 1178−
1181.
We thank Dr. Vishwanatha Somayaji for spectrometry services.
We are also grateful to the Canadian Institutes of Health
Research (Grant MOP-14712 to E.E.K and Grant MOP-
115037 to J.M.S.) for financial support of this research.
(14) Chakrapani, H.; Showalter, B. M.; Kong, L.; Keefer, L. K.;
Saavedra, J. E. V-PROLI/NO, a prodrug of the nitric oxide donor,
PROLI/NO. Org. Lett. 2007, 9, 3409−3412.
ABBREVIATIONS USED
■
−
(15) Sunko, D.; Jursic, B.; Ladika, M. Leaving group rate ratios in
solvolytic displacement reactions. Effect of neighboring sulfur. J. Org.
Chem. 1987, 52, 2299−2301.
NO, nitric oxide; HNO, nitroxyl; NO2 , nitrite; N2O, nitrous
oxide; GSH, glutathione; KHSO5, potassium peroxymonosul-
fate; PBS, phosphate buffered solution; TBS, Tris buffer
solution; AS, Angeli’s salt; IPA/NO, isopropylamine diaze-
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