Dual mechanisms of HNO generation by a nitroxyl prodrug of the diazeniumdiolate (NONOate) class

Article abstract of DOI:10.1021/ja106552p

Here we describe a novel caged form of the highly reactive bioeffector molecule, nitroxyl (HNO). Reacting the labile nitric oxide (NO)- and HNO-generating salt of structure iPrHN-N(O)=NO^-Na⁺ (1, IPA/NO) with BrCH₂OAc produced a stable derivative of structure iPrHN-N(O)=NO-CH₂OAc (2, AcOM-IPA/NO), which hydrolyzed an order of magnitude more slowly than 1 at pH 7.4 and 37 °C. Hydrolysis of 2 to generate HNO proceeded by at least two mechanisms. In the presence of esterase, straightforward dissociation to acetate, formaldehyde, and 1 was the dominant path. In the absence of enzyme, free 1 was not observed as an intermediate and the ratio of NO to HNO among the products approached zero. To account for this surprising result, we propose a mechanism in which base-induced removal of the N-H proton of 2 leads to acetyl group migration from oxygen to the neighboring nitrogen, followed by cleavage of the resulting rearrangement product to isopropanediazoate ion and the known HNO precursor, CH₃-C(O)-NO. The trappable yield of HNO from 2 was significantly enhanced over 1 at physiological pH, in part because the slower rate of hydrolysis for 2 generated a correspondingly lower steady-state concentration of HNO, thus, minimizing self-consumption and enhancing trapping by biological targets such as metmyoglobin and glutathione. Consistent with the chemical trapping efficiency data, micromolar concentrations of prodrug 2 displayed significantly more potent sarcomere shortening effects relative to 1 on ventricular myocytes isolated from wild-type mouse hearts, suggesting that 2 may be a promising lead compound for the development of heart failure therapies.

Full text of DOI:10.1021/ja106552p

Published on Web 10/29/2010
Dual Mechanisms of HNO Generation by a Nitroxyl Prodrug of
the Diazeniumdiolate (NONOate) Class
†
‡
|,⊥,§
|
Daniela Andrei, Debra J. Salmon, Sonia Donzelli,
Azadeh Wahab,
@
¶
¶
#
John R. Klose, Michael L. Citro, Joseph E. Saavedra, David A. Wink,
,
‡
,†
Katrina M. Miranda,* and Larry K. Keefer*
Chemistry Section, Laboratory of ComparatiVe Carcinogenesis, National Cancer Institute at
Frederick, Frederick, Maryland 21702, United States, Department of Chemistry and
Biochemistry, UniVersity of Arizona, Tucson, Arizona 85721, United States, Department of
Experimental and Clinical Pharmacology and Toxicology, UniVersity Medical Center
Hamburg-Eppendorf, Hamburg, Germany, Department of Neurology, UniVersity Medical Center
Hamburg-Eppendorf, Hamburg, Germany, CardioVascular Research Center, UniVersity Medical
Center Hamburg-Eppendorf, Hamburg, Germany, Laboratory of Proteomics and Analytical
Technologies, SAIC Frederick, Frederick, Maryland 21702, United States, Basic Research
Program, SAIC Frederick, Frederick, Maryland 21702, United States, and Radiation Biology
Branch, National Cancer Institute, Bethesda, Maryland 20892, United States
Received July 23, 2010; E-mail: kmiranda@email.arizona.edu; keeferl@mail.nih.gov
Abstract: Here we describe a novel caged form of the highly reactive bioeffector molecule, nitroxyl (HNO).
-
+
Reacting the labile nitric oxide (NO)- and HNO-generating salt of structure iPrHN-N(O)dNO Na (1, IPA/
NO) with BrCH OAc produced a stable derivative of structure iPrHN-N(O)dNO-CH OAc (2, AcOM-IPA/
2
2
NO), which hydrolyzed an order of magnitude more slowly than 1 at pH 7.4 and 37 °C. Hydrolysis of 2 to
generate HNO proceeded by at least two mechanisms. In the presence of esterase, straightforward
dissociation to acetate, formaldehyde, and 1 was the dominant path. In the absence of enzyme, free 1 was
not observed as an intermediate and the ratio of NO to HNO among the products approached zero. To
account for this surprising result, we propose a mechanism in which base-induced removal of the N-H
proton of 2 leads to acetyl group migration from oxygen to the neighboring nitrogen, followed by cleavage
of the resulting rearrangement product to isopropanediazoate ion and the known HNO precursor,
CH -C(O)-NO. The trappable yield of HNO from 2 was significantly enhanced over 1 at physiological pH,
3
in part because the slower rate of hydrolysis for 2 generated a correspondingly lower steady-state
concentration of HNO, thus, minimizing self-consumption and enhancing trapping by biological targets such
as metmyoglobin and glutathione. Consistent with the chemical trapping efficiency data, micromolar
concentrations of prodrug 2 displayed significantly more potent sarcomere shortening effects relative to 1
on ventricular myocytes isolated from wild-type mouse hearts, suggesting that 2 may be a promising lead
compound for the development of heart failure therapies.
Introduction
Nitroxyl (HNO) has been shown to possess intriguing
in the mechanism of the inhibitory effect of cyanamide on
aldehyde dehydrogenase in treating alcohol abuse and has been
4
demonstrated to enhance contractility in experimental models
1-21
biological properties.
For instance, HNO has been implicated
9
of heart failure. That HNO rapidly and irreversibly dimerizes
2
2,23
and dehydrates to produce nitrous oxide (eq 1),
making it
†
Laboratory of Comparative Carcinogenesis, National Cancer Institute
impossible to isolate in pure form, has been a major barrier to
achieving a quantitative understanding of its chemical and
at Frederick.
‡
University of Arizona.
Department of Experimental and Clinical Pharmacology and Toxicol-
2
4
|
bioeffector behavior.
ogy, University Medical Center Hamburg-Eppendorf.
⊥
Department of Neurology, University Medical Center Hamburg-
2
HNO f [HONdNOH] f N O + H O
2
2
6
Eppendorf.
§
-1 -1
Cardiovascular Research Center, University Medical Center Hamburg-
(
k ) 8 × 10 M s ) ref 24 (1)
Eppendorf.
@
Laboratory of Proteomics and Analytical Technologies, SAIC Frederick.
¶
Basic Research Program, SAIC Frederick.
As an approach to studying the chemistry and biology of
#
Radiation Biology Branch, National Cancer Institute.
HNO, considerable effort has been devoted to designing
(
(
(
1) Lee, M. J. C.; Nagasawa, H. T.; Elberling, J. A.; DeMaster, E. G.
J. Med. Chem. 1992, 35, 3648–3652.
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1
6526 9 J. AM. CHEM. SOC. 2010, 132, 16526–16532
10.1021/ja106552p  2010 American Chemical Society

Dual Mechanisms of HNO Generation
A R T I C L E S
Scheme 1. Structures of the Two Related, Spontaneously
HNO-Generating Salts, Angeli’s Salt and IPA/NO (1)
the related compound IPA/NO (1, the sodium salt of diazeni-
umdiolated isopropylamine; Scheme 1) has been shown to
34
mimic the chemical and biological properties of Angeli’s salt.
Unfortunately, neither of these salts is itself ideal from the drug
development perspective, as they are prone to deterioration on
storage and they are difficult to purify. Additionally, their similar
half-lives of ∼2 min for spontaneous hydrolysis at physiological
pH and temperature do not allow for prolonged exposure of
target tissues.
A possible approach to circumventing the limitations but
harnessing the advantages of such ions is to alkylate the salts
to produce stable neutral species that can be rigorously purified
and repurified as necessary, then induced to release HNO by
way of enzymatic, hydrolytic, or other tunable activation
pathways. To our knowledge, alkylating or otherwise suitably
derivatizing Angeli’s salt has been unsuccessful to date. In
compounds capable of serving as caged HNO donors, or
1
-5,20,21,25-33
prodrugs.
We are among those working to
develop a platform for systematically generating reliable,
controlled fluxes of HNO at programmable rates in physi-
ological media.
Historically, Angeli’s salt (Scheme 1) has been the reagent
3
1
of choice for generating HNO in aqueous solution. Recently,
35
contrast, 1 has proven amenable to alkylation. Here, we report
that acetoxymethylation of 1 as shown in eq 2 leads to prodrug
(
6) Wink, D. A.; Miranda, K. M.; Katori, T.; Mancardi, D.; Thomas, D. D.;
Ridnour, L.; Espey, M. G.; Feelisch, M.; Colton, C. A.; Fukuto, J. M.;
Pagliaro, P.; Kass, D. A.; Paolocci, N. Am. J. Physiol.: Heart Circ.
Physiol. 2003, 285, H2264–H2276.
2
(AcOM-IPA/NO), which is easily purifiable by column
chromatography and functions as an advantageous HNO donor
with an unexpected mechanism of activation.
(
(
7) Feelisch, M. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4978–4980.
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9) Paolocci, N.; Katori, T.; Champion, H. C.; St. John, M. E.; Miranda,
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Ferlito, M.; Paolocci, N.; Fukuto, J. M. Prog. Inorg. Chem. 2005, 54,
Results and Discussion
Compound 2 Hydrolyzes to HNO More Slowly and Ef-
ficiently than 1. A key goal of this research is to introduce an
array of agents that display a broad range of half-lives for HNO
release while minimizing the simultaneous generation of NO that
accompanies the spontaneous dissociation of salt 1 at physiological
pH. Ideally, this would come in the form of a series of water-
soluble compounds that produce reliable fluxes of pure HNO at
rates that are tunable for any given experimental application.
Compound 2 appears to represent a step in that direction.
3
49–384.
(
(
12) Miranda, K. M. Coord. Chem. ReV. 2005, 249, 433–455.
13) Paolocci, N.; Jackson, M. I.; Lopez, B. E.; Miranda, K.; Tocchetti,
C. G.; Wink, D. A.; Hobbs, A. J.; Fukuto, J. M. Pharmacol. Ther.
2
007, 113, 442–458.
(
(
(
(
(
(
(
(
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96.
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Hypertension 2007, 49, 885–892.
Under physiological conditions, Angeli’s salt is primarily an
HNO donor (<0.1% NO in the presence of a metal chelator
18) Fukuto, J. M.; Jackson, M. I.; Kaludercic, N.; Paolocci, N. Methods
Enzymol. 2008, 440, 411–431.
36
such as 50 µM diethylenetriaminepentaacetic acid (DTPA)).
3
4
In contrast, 1 generates mixtures of NO and HNO. Although
19) Lancel, S.; Zhang, J.; Evangelista, A.; Trucillo, M. P.; Tong, X.; Siwik,
D. A.; Cohen, R. A.; Colucci, W. S. Circ. Res. 2009, 104, 720–723.
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1 decomposes with nearly the same half-life as Angeli’s salt
37
(
1
2.3 min in pH 7.4 phosphate buffer at 37 °C), conversion of
5
212.
to 2 increased the half-life to 41 min (pH 7.4, 37 °C), which
21) Kovacic, P.; Edwards, C. L. J. Recept. Signal Transduct. [Early
Online]. DOI: 10.3109/10799893.2010.497152. Published online: Jun
offers a useful addition to the otherwise short half-lives available
for spontaneous generation of HNO in aqueous media.
The methods available to detect NO are quite varied and
adaptable, but quantitative analysis of HNO is limited by its
3
0, 2010.
(
(
22) Smith, P. A. S.; Hein, G. E. J. Am. Chem. Soc. 1960, 82, 5731–5740.
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5
796.
(
24) Shafirovich, V.; Lymar, S. V. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
2
rapid and irreversible conversion to N O (eq 1). The most
7
340–7345.
common method for identifying HNO and establishing a lower
limit for the amount formed from a given donor is to trap HNO
(
(
25) Ware, R. W., Jr.; King, S. B. J. Org. Chem. 2000, 65, 8725–8729.
26) Zeng, B. B.; Huang, J.; Wright, M. W.; King, S. B. Bioorg. Med.
Chem. Lett. 2004, 14, 5565–5568.
38,39
with metmyoglobin (metMb)
according to eq 3. This method
(
(
27) King, S. B. Free Radical Biol. Med. 2004, 37, 735–736.
was used earlier to suggest that 1 is a donor of both NO and
28) Huang, J.; Kim-Shapiro, D. B.; King, S. B. J. Med. Chem. 2004, 47,
3
495–3501.
(
(
29) King, S. B. Curr. Top. Med. Chem. 2005, 5, 665–673.
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2
005, 15, 2331–2334.
(
(
(
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Chem. Soc. 2006, 128, 9687–9692.
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Biomol. Chem. 2009, 7, 1954–1962.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 46, 2010 16527

A R T I C L E S
Andrei et al.
For longer-lived donors of HNO such as 2, the rate of
autoxidation of MbNO back to metMb (eq 4) requires use of
anaerobic conditions to detect HNO.
+
metMb + HNO f MbNO + H
5
-1 -1
(
k ) 8 × 10 M s ) ref 8 (3)
-
3
MbNO + O f metMb + NO
2
-
4 -1
(
k ) 2 × 10 s ) ref 40 (4)
GSH + HNO f GSNHOH
6
-1 -1
(
k ) 2 × 10 M s ) ref 8 (5)
metMb + NO h metMbNO
4
-1 -1
-1
(
kon ) 4.8 × 10 M s , k ) 43 s ) ref 41 (6)
off
metMbNO + GSH f MbNO + products -
k ) 47 M s ) ref 42 (7)
1 -1
(
Both 1 and 2 reductively nitrosylated metMb (Figure 1), but
2
did so more efficiently. We conclude that the slower hydrolysis
rate of 2 decreases the steady-state concentration of HNO to
impede self-consumption such that MbNO is formed in higher
yield. The rates of MbNO formation were comparable to the
decomposition rates of 1 and 2, respectively.
Another biological target known to react rapidly with HNO
is glutathione (GSH). To confirm the production of HNO in
the spontaneous hydrolysis of 2, the metMb assay was repeated
in the presence of excess GSH, which reacts with HNO more
rapidly than metMb (eqs 3 vs 5) but does not interact appreciably
with low concentrations of NO. GSH inhibited reductive
nitrosylation by 2 under deaerated conditions, as expected based
on the respective rate constants and concentrations employed
(Figure 2A, compare Figure 1B). Interestingly, under deaerated
Figure 1. Reductive nitrosylation of metMb (50 µM) by (A) 1 (100 µM),
or (B) 2 (100 µM). The assay was performed in phosphate-buffered saline
(
PBS) (pH 7.4) containing 50 µM DTPA at 37 °C under deaerated conditions
until there were no further spectral changes at 543 and 575 nm (1, 2, 4, 6,
, 14, 23, and 46 min for 1; 3-107 min in 4- to 10-min intervals shown
for 2). Concentrations of MbNO produced under these conditions by 1 and
were ∼18 and 45 µM, respectively.
9
2
HNO while Angeli’s salt is a donor of only HNO under
3
4
physiological conditions. Here, we employed this established
method to determine whether the products of hydrolyzing 2
include HNO, as would be expected for a prodrug form of 1.
Figure 2. Effect of GSH on the reductive nitrosylation of metMb (50 µM) by (A) 2 (100 µM), (B) 1 (100 µM), (C) DEA/NO (50 µM), or (D) Angeli’s salt
100 µM). The assay was performed in PBS (pH 7.4) containing 50 µM DTPA and 250 µM GSH at 37 °C under deaerated conditions. Scans are plotted at
.5 and 60 min in panel A, 2, 4, 6, 9, 14, 23, and 56 min in panel B, and at 1-min intervals to 8 min and then at 11, 14, 18, 23, 28, and 40 min in panel
(
0
C. Panel D includes spectra at 500 s (green trace), where the decomposition of Angeli’s salt was complete, and at 60 min (blue trace). The effect of GSH
appears to be dependent on the batch of myoglobin, indicating the possible interaction with a contaminating protein such as Cu, Zn superoxide dismutase
(
data not shown; product formation is variable).
1
6528 J. AM. CHEM. SOC. 9 VOL. 132, NO. 46, 2010

Dual Mechanisms of HNO Generation
A R T I C L E S
Figure 3. The pH-dependence of the first-order rate constant of decomposi-
2
tion measured at 250 nm (mean ( SEM, n g 3, all R values >0.995 for 1
and >0.98 for 2) of 1 (A, 100 µM) and 2 (B, 100 µM) at 37 °C in PBS
containing 50 µM DTPA and adjusted to the indicated pHs as necessary
by adding NaOH or HCl; measured pH was the same at the end of each
kinetic run as it was at time zero.
conditions, GSH also enhanced MbNO formation during the
hydrolysis of 1 (Figure 2B), which hydrolyzes to produce both
3
4
HNO and NO. In deaerated solution, GSH has been shown
to enhance the interaction of NO with metMb (eqs 6 and 7).
42,43
Figure 4. Spontaneous (A and B), and esterase-catalyzed (C) hydrolysis,
of 2. In panel A, spectra were collected every 60 s (10-min intervals shown)
after dissolving 2 in PBS (pH 7.4) at 37 °C, and only loss of the parent
compound was observed. In panel B, the pH was elevated to 12, and scans
were collected every 0.5 s (through 2.5 s and 3.5, 5.5, 9.5, and 54.5 s shown).
In panel C, porcine liver esterase (1.4 U/mL) was added (pH 7.4), and
spectra were collected every 10 s. Hydrolysis of 2 (λmax 236 nm) to 1 (λmax
Note that the spectral changes for 1 were comparable to those
3
7
observed when the NO donor DEA/NO was hydrolyzed in
the presence of both metMb and GSH (Figure 2C, compare
Figure 2B). Quenching by GSH of the reaction of 2 with metMb
(
Figure 2A) was similar to that of the HNO donor Angeli’s salt
-
1
2
50 nm) was complete within 40 s (0.067 s ; red spectra) under these
conditions. The rate of decay of 1 in panel C (blue spectra at 40, 100, 160,
40, 400, 600, and 2460 s) was comparable to that shown in Figure 3A
(
Figure 2D), confirming that HNO generation by 2 under these
conditions is comparable to that of Angeli’s salt but enhanced
relative to that of 1.
2
-
1
-1
(0.0033 s vs 0.0012 s , respectively). Elevation of the intensity near
200 nm indicates formation of autoxidation products such as nitrite.
Deaeration inhibited this peak (data not shown).
Compound 2 Generates Negligible NO on Spontaneous
Hydrolysis, in Contrast to 1. As mentioned above, 1 produces
3
4
both NO and HNO on spontaneous hydrolysis at neutral pH.
Surprisingly, the total amount of NO detected on hydrolyzing
under the same conditions while purging gases formed in the
reaction into an NO-specific chemiluminescence detector was
1% of theoretical (data not shown), suggesting a different
hydrolysis mechanism.
To gain insight into the mechanistic origins of this unexpected
finding, we explored the pH/rate profiles of the two diazeni-
umdiolates. Hydrolysis of 1 slowed as pH was increased from
3
to 10 (Figure 3A). This is quite the opposite of the results for
2
2, whose hydrolysis rate was essentially first order in hydroxide
ion concentration between pH 6 and 12 (Figure 3B). Below pH
<
6
, the long half-life of 2 (g2 days) precluded quantitation.
Interestingly, at and above pH 8, the rate of decomposition
of 2 exceeded that of 1 (Figure 3), further indicating a mecha-
nism more complicated than simple hydrolysis of 2 to 1.
Correspondingly, absorbance at 250 nm due to 1 was not
observed in the spontaneous hydrolysis of 2 at pH 7.4 (Figure
4A). Of further importance, as the pH was raised to 12, the
higher energy shoulder resolved into a peak at 217 nm of
significant intensity, and there was a shift in the 236-nm peak
to 278 nm, consistent with ionization of the N-H bond (Figure
(
(
(
(
(
(
38) Bazylinski, D. A.; Hollocher, T. C. J. Am. Chem. Soc. 1985, 107,
7
982–7986.
39) Doyle, M. P.; Mahapatro, S. N.; Broene, R. D.; Guy, J. K. J. Am.
Chem. Soc. 1988, 110, 593–599.
40) Andersen, H. J.; Skibsted, L. H. J. Agric. Food. Chem. 1992, 40, 1741–
1
750.
41) Laverman, L. E.; Wanat, A.; Oszajca, J.; Stochel, G.; Ford, P. C.; van
Eldik, R. J. Am. Chem. Soc. 2001, 123, 285–293.
3
5
4B).
42) Reichenbach, G.; Sabatini, S.; Palombari, R.; Palmerini, C. A. Nitric
Oxide 2001, 5, 395–401.
In contrast to the results in simple buffer solutions, 1 was an
observable intermediate in the hydrolysis of 2 when active
esterase was present. As shown in Figure 4C, the 250-nm peak
43) Ford, P. C.; Fernandez, B. O.; Lim, M. D. Chem. ReV. 2005, 105,
2
439–2456.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 46, 2010 16529

A R T I C L E S
Andrei et al.
Scheme 2. Two Limiting Hydrolysis Mechanisms for Generation of
NO versus HNO from a Diazeniumdiolated Primary Amine Anion
Figure 5. NMR spectra of products generated on exhaustive hydrolysis of
1
7
(A) and 2 (B) in 0.1 M phosphate buffer containing 50 µM DTPA at pH
.4 and 37 °C. Note the near-absence of isopropylamine, a byproduct of
the NO-forming pathway, in the hydrolysis of 2.
With the assumption that paths a and b are the only routes
by which 1 can dissociate, the yield of amine observed in such
a dissociation can be taken as a quantitative measure of the
preponderance of path a, while the combined yields of olefin
and RX species quantitatively reflect the extent of competing
route b. With this in mind, we followed the course of hydrolysis
for both 1 and 2 by NMR and quantified the organic products.
Consistent with the HNO/NO yield data reported above,
complete dissociation of 1 at pH 7.4 generated similar amounts
of isopropylamine and isopropanol, with essentially saturating
concentrations of propene also being observed (Figure 5A). In
contrast, methyl peaks attributable to the amine bordered on
undetectable when 2 was hydrolyzed under the same conditions,
pointing to a hydrolysis mechanism for 2 that does not involve
for 1 dominated the spectrum within a short time after adding
the enzyme to 2 in PBS at pH 7.4. Additional studies confirmed
that the esterase does not significantly affect the stability of 1
or the ability of metMb and GSH to trap the hydrolytically
produced HNO (Figure S2 in Supporting Information).
1
as an intermediate (Figure 5B).
Quantitative Insight into the NO/HNO Partition by Anal-
ysis of the Organic Products. Two limiting mechanisms of
hydrolysis for an ionic primary amine diazeniumdiolate are
Differences in time course as well as stoichiometry in the
alkaline hydrolysis of 1 and 2, illustrated in Figure 6, also
matched expectation based on the HNO/NO yield and rate data.
Ionic 1 hydrolyzed slowly at pH 11. Compound 2, on the other
hand, completely dissociated within 5 min at 37 °C in pH 11
carbonate buffer. Peaks for formaldehyde and acetate were seen
in addition to those for the carbocation-derived alcohol and
alkene, but the amine was undetectable.
4
4
depicted in Scheme 2. Path a is simply the reverse of the
synthesis reaction, with the ion dissociating on N-protonation
to yield the parent amine and 2 equiv of NO. Path b also begins
with a protonation step, but at a different nitrogen, yielding an
intermediate that dissociates to form 1 equiv of HNO and a
primary nitrosamine; the latter species then rearranges to a
diazoate that, in a protonating medium, loses a molecule of water
to form a highly reactive alkyldiazonium ion. This intermediate
can then react rapidly with available nucleophiles (represented
in Scheme 2 as water) to displace a dinitrogen molecule and
produce a nucleophile/carbocation adduct (shown as the alcohol
in Scheme 2); if the R+ group bears a hydrogen atom in the
Mechanism of Base-Induced Hydrolysis. Although 1 hydro-
lyzed 2 orders of magnitude more slowly than 2 at pH 11 (Figure
3
), the characteristic 250-nm wavelength maximum of 1 was
detected only at low intensity during the rapid hydrolysis of 2
at this pH (Figure 4B), and the corresponding NMR signals for
1
could not be separated from the noise (Figure 6). One way to
rationalize the near absence of free 1 as a product of hydrolyzing
is to postulate that base-induced removal of the N-H proton
2
ꢀ
-position, loss of that proton would produce an olefin.
of 2 generates 3, as in Scheme 3. This intermediate might be
expected to undergo 1-4 acyl migration via cyclic intermediate
4. Expulsion of formaldehyde from 4 could give 5, which could
(
44) Dutton, A. S.; Suhrada, C. P.; Miranda, K. M.; Wink, D. A.; Fukuto,
J. M.; Houk, K. N. Inorg. Chem. 2006, 45, 2448–2456.
1
6530 J. AM. CHEM. SOC. 9 VOL. 132, NO. 46, 2010

Dual Mechanisms of HNO Generation
A R T I C L E S
Figure 6. Distribution of products in the hydrolysis at 37 °C of 1 (left side panels) and 2 (right side panels) in pH 11 aqueous 0.05 M sodium carbonate
containing 10% deuterium oxide. Note that hydrolysis of 1 to isopropanol and propene proceeded to the extent of only ∼15% during 1 h while dissociation
of 2 was complete within 5 min. Since 1 is more stable than 2 at this pH, the absence of a peak for it in the spectrum of fully dissociated 2 indicates that
free 1 is not significantly produced in the hydrolysis of 2 under these conditions. An expanded view of the spectrum at the lower right is included as Figure
S1 in Supporting Information. It provides integrals for all the organic products including formaldehyde, and shows that the combined yield of propene plus
isopropanol essentially matches that of acetate, as expected for a hydrolysis mechanism for 2 that produces an NO/HNO ratio approaching zero.
Scheme 3. Proposed Mechanism of Hydrolytic HNO Generation
from 2 in the Absence of Esterase
7 are known to hydrolyze to HNO with the derived carboxylate
21,46
as byproduct.
2
Precedent for ionization of O -substituted IPA/NO derivatives
such as 2 with an accompanying shift in absorbance maximum
from ∼236 to ∼278 nm (see Figure 4B) has been reported for
47
a
methylated IPA/NO, with a measured pK of 12.3. For 2, with
increasing alkalinity this shift became apparent at pH 11,
indicating a comparable pK . The importance of anionic
a
resonance form 3 as a nucleophilic site is supported by the
finding that glucosylated IPA/NO can be methylated at that site
48
in basic media. Examples of 1-4 acyl migrations reminiscent
of that shown in Scheme 3 for conversion of 3 to 5 have been
49
reported, and cleavage of 5 to the two well established reactive
intermediates 6 and 7 would not be surprising. Consistent with
the view that the base-catalyzed dissociation of 2 according to
Scheme 3 is the only pathway by which it decomposes in
enzyme-free media, product ratios were the same in the slow
pH 7.4 reaction (Figure 5) and its faster pH 11 counterpart
(
Figure 6), except that propene appeared to be present in
somewhat higher concentration at the higher pH. The observed
integral for acetate corresponded to a yield (taken as 100%)
essentially matching that expected for the sum of propene plus
isopropanol (26% + 79%, respectively; see Figure S1 in the
Supporting Information), suggesting that analysis of the organic
products in these reactions can provide reliable quantitative
insight into the partition between the NO- and HNO-forming
pathways.
Effects of 1 and 2 on Isolated Cardiac Myocytes. To deter-
mine whether the slower and more efficient generation of HNO
by 2 relative to 1 translates to enhanced bioactivity, we turned
then fragment with N-N bond cleavage to produce diazoate
ion 6 and acylnitroso derivative 7. Aliphatic diazoates are well-
known progenitors of unstable carbenium ions that can indis-
criminately alkylate ambient nucleophiles (such as water to
produce the corresponding alcohol) with cogeneration of an
(
(
46) Cohen, A. D.; Zeng, B.-B.; King, S. B.; Toscano, J. P. J. Am. Chem.
Soc. 2003, 125, 1444–1445.
47) Wang, Y.-N.; Bohle, D. S.; Bonifant, C. L.; Chmurny, G. N.; Collins,
J. R.; Davies, K. M.; Deschamps, J.; Flippen-Anderson, J. L.; Keefer,
L. K.; Klose, J. R.; Saavedra, J. E.; Waterhouse, D. J.; Ivanic, J. J. Am.
Chem. Soc. 2005, 127, 5388–5395.
4
5
equivalent of dinitrogen, and acylnitroso compounds such as
(
48) Bohle, D. S.; Keefer, L. K.; Saavedra, J. E. Tetrahedron Lett. 2009,
5
0, 5917–5919.
(
45) Smith, M. B.; March, J. March’s AdVanced Organic Chemistry.
Reactions, Mechanisms, and Structure. Fifth ed.; John Wiley & Sons,
Inc.: New York, 2001; pp 447-448.
(49) Banthorpe, D. V. Rearrangements involving amino groups. In The
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A R T I C L E S
Andrei et al.
suitable derivatization of 1 can dramatically slow the rate of
spontaneous HNO generation, with concomitant improvement
in the efficiency of its capture by biological targets. Furthermore,
conversion of relatively unstable 1 to 2 provides a rigorously
purifiable derivative with alternate mechanisms of hydrolysis
to HNO and improved ability to strengthen contraction of
beating cardiac myocytes at low micromolar concentrations.
Work currently in progress is focused on further exploring the
structure-activity and -reactivity relationships in the primary
amine diazeniumdiolate series, with the aim of developing a
variety of improved tools for probing the chemistry and
pharmacology of the intriguing bioeffector molecule HNO.
Figure 7. Relative increases in contractility in ventricular myocytes isolated
from wild-type mouse hearts. Dose-response effects of 1 and 2 on cell
shortening. ***P < 0.001 vs control. Number of cells ) 6 at 1 µM, 5 at 2
µM, and 15 for 3 and 50 µM.
Acknowledgment. This work was supported by: the Marie
Curie Intra European Fellowship within the 7th European Com-
munity Framework Programme (PIEF-GA-2008-221666 to S.D.);
the Forschungsf o¨ rderungsfonds (NWF-08/04 to S.D.); the Intra-
mural Research Program of the NIH, National Cancer Institute,
Center for Cancer Research; NCI contract HHSN261200800001E;
National Institutes of Health grants R01-GM076247 to K.M.M. and
1F31AA018069-01A1 (National Institute on Alcohol Abuse and
Alcoholism) to D.J.S.
to a model for one of the most exciting applications of HNO
donors, the treatment of heart failure. Donors of HNO enhance
contractility in failing hearts. As might be expected for an agent
9
that generates substantially more HNO than 1, prodrug 2 proved
more potent at micromolar concentrations in ventricular myo-
cytes isolated from wild-type mice. As shown in Figure 7, 2
elicited a roughly 4-fold higher change than 1 in sarcomere
shortening relative to baseline at 3 µM. While this finding
suggests potential pharmacological utility on the part of 2, it is
important to note its apparently negative inotropic effect at 50
µM, a possible reflection of toxicity not seen with 1 (Figure 7).
We are currently exploring the origin of this undesirable effect
in an attempt to infer a means of circumventing it.
Supporting Information Available: Complete ref 34; experi-
mental procedures including synthesis and characterization of
2
; details of the analytical and biological methodologies
employed; and additional spectral data from studies of the
esterase-mediated hydrolyses of 1 and 2. This material is
available free of charge via the Internet at http://pubs.acs.org.
Summary and Significance. Our results suggest that diazeni-
umdiolate chemistry may offer a versatile platform for develop-
ment of advantageous HNO sources for fundamental research
and biomedical applications. The present comparison shows that
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6532 J. AM. CHEM. SOC. 9 VOL. 132, NO. 46, 2010