Optimization of HNO production from N, O - Bis -acylated hydroxylamine derivatives

Article abstract of DOI:10.1021/ol203016c

A wide range of N,O-bis-acylated hydroxylamine derivatives with chloro or arenesulfonyl leaving groups, and a related set of N-hydroxy-N-acylsulfonamides, have been synthesized and evaluated for nitroxyl (HNO) production. Mechanistic studies have revealed that the observed aqueous chemistry is more complicated than originally anticipated, and have been used to develop a new series of efficient HNO precursors (4u-4x, 7c-7d) with tunable half-lives.

Full text of DOI:10.1021/ol203016c

ORGANIC
LETTERS
2012
Vol. 14, No. 2
472–475
Optimization of HNO Production from
N,O-bis-Acylated Hydroxylamine Derivatives
Art D. Sutton,† Morgan Williamson,‡ Hilary Weismiller,‡ and John P. Toscano*,†
Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218,
United States, and Department of Chemistry, Edinboro University, Edinboro,
Pennsylvania 16444, United States
jtoscano@jhu.edu
Received November 8, 2011
ABSTRACT
A wide range of N,O-bis-acylated hydroxylamine derivatives with chloro or arenesulfonyl leaving groups, and a related set of N-hydroxy-N-
acylsulfonamides, have been synthesized and evaluated for nitroxyl (HNO) production. Mechanistic studies have revealed that the observed
aqueous chemistry is more complicated than originally anticipated, and have been used to develop a new series of efficient HNO precursors
(4uꢀ4x, 7cꢀ7d) with tunable half-lives.
Recent research has shown that nitroxyl (HNO), the
one-electron reduced and protonated relative of nitric
oxide (NO), has important and unique biological activity,
especially as a potential alternative tocurrent treatments of
cardiac failure.1ꢀ4 HNO is a reactive molecule (especially
withthiols) thatspontaneously dimerizesand subsequently
dehydrates to form nitrous oxide (N2O). Thus, in order to
study HNO chemistry or biology, donor molecules for the
generation of HNO in situ are required; however, the range
of HNO donor molecules suitable for use under physiolo-
gically relevant conditions is currently quite limited.5,6 These
include Angeli’s salt (Na2N2O3),7ꢀ9 Piloty’s acid derivatives
(ArSO2NHOH),10,11 primary amine-based diazenium-
diolates,12 acyloxy nitroso compounds,13 and precursors
to acyl nitroso compounds.14ꢀ16
Acyl nitroso (AN) compounds are transient electro-
philes that react with nucleophiles, including water, to
produce HNO. Notable among the strategies that have
been employed to generate acyl nitroso compounds is the
work of Nagasawa and co-workers, who examined a series
(9) Miranda, K. M.; Dutton, A. S.; Ridnour, L. A.; Foreman, C. A.;
Ford, E.; Paolocci, N.; Katori, T.; Tocchetti, C. G.; Mancardi, D.;
Thomas, D. D.; Espey, M. G.; Houk, K. N.; Fukuto, J. M.; Wink, D. A.
J. Am. Chem. Soc. 2005, 127, 722–731.
† Johns Hopkins University.
‡ Edinboro University.
(10) Bonner, F. T.; Ko, Y. Inorg. Chem. 1992, 31, 2514–2519.
(11) Toscano, J. P.; Brookfield, F. A.; Cohen, A. D.; Courtney, S. M.;
Frost, L. M.; Kalish, V. J. U.S. Patent 8,030,356, 2011.
(12) 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.
(13) Sha, X.; Isbell, T. S.; Patel, R. P.; Day, C. S.; King, S. B. J. Am.
Chem. Soc. 2006, 128, 9687–9692.
(14) Corrie, J. E. T.; Kirby, G. W.; Mackinnon, J. W. M. J. Chem.
Soc., Perkin Trans. 1 1985, 883–886.
(15) Cohen, A. D.; Zeng, B.-B.; King, S. B.; Toscano, J. P. J. Am.
Chem. Soc. 2003, 125, 1444–1445.
(1) Paolocci, N.; Saavedra, W. F.; Miranda, K. M.; Martignani, C.;
Isoda, T.; Hare, J. M.; Espey, M. G.; Fukuto, J. M.; Feelisch, M.; Wink,
D. A.; Kass, D. A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 10463–10468.
(2) Paolocci, N.; Katori, T.; Champion, H. C.; St. John, M. E.;
Miranda, K. M.; Fukuto, J. M.; Wink, D. A.; Kass, D. A. Proc. Natl.
Acad. Sci. U.S.A. 2003, 100, 5537–5542.
(3) Tocchetti, C. G.; Wang, W.; Froehlich, J. P.; Huke, S.; Aon,
M. A.; Wilson, G. M.; Di Benedetto, G.; O’Rourke, B.; Gao, W. D.;
Wink, D. A.; Toscano, J. P.; Zaccolo, M.; Bers, D. M.; Valdivia, H. H.;
Cheng, H.; Kass, D. A.; Paolocci, N. Circ. Res. 2007, 100, 96–104.
(4) Kemp-Harper, B. K. Antioxid. Redox Signaling 2011, 14, 1609–
1613.
(5) Miranda, K. M.; Nagasawa, H. T.; Toscano, J. P. Curr. Top. Med.
Chem. 2005, 5, 649–664.
(6) DuMond, J. F.; King, S. B. Antioxid. Redox Signaling 2011, 14,
1637–1648.
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1976, 703–707.
(16) Evans, A. S.; Cohen, A. D.; Gurard-Levin, Z. A.; Kebede, N.;
Celius, T. C.; Miceli, A. P.; Toscano, J. P. Can. J. Chem. 2011, 89, 130–
138.
(17) Nagasawa, H. T.; Lee, M. J. C.; Kwon, C. H.; Shirota, F. N.;
DeMaster, E. G. Alcohol 1992, 9, 349–353.
(18) Lee, M. J. C.; Elberling, J. A.; Nagasawa, H. T. J. Med. Chem.
1992, 35, 3641–3647.
r
10.1021/ol203016c
Published on Web 12/23/2011
2011 American Chemical Society

of esterase- and cytochrome P-450-activated precursors
(Scheme 1).17ꢀ24 These precursors, which were presumed
to form N-hydroxy intermediate 2, were used to demonstrate
that HNO is a potent inhibitor of aldehyde dehydrogenase
(AlDH) and has potential for the treatment of alcoholism.
Indeed, Nagasawa also demonstrated that cyanamide
(H2NCN), an alcohol deterrent used clinically in Europe,
Canada, and Japan, generates HNO following metabolic
activation to an unstable N-hydroxycyanamide.25ꢀ27
for HNO generation by gas chromatographic (GC) head-
space analysis to quantify the amount of its dimerization
product, N2O, produced following decomposition in phos-
phate-buffered saline (PBS). (See Supporting Information
(SI) for experimental details.)
Although low HNO yields are observed for all N-chloro
compounds in PBS, yields are greatly enhanced when decom-
positions are carried out in deionized (DI) water (Table 1).
The major organic byproduct observed in PBS is the corre-
sponding bis-acylated hydroxylamine, formed by dechlorina-
tion of the precursor. We find that dechlorination is the
dominant process in 3-(N-morpholino)-propanesulfonic acid
(MOPS) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES) buffers as well. N-Chloro compounds 3 have
structures similar to those of the electrophilic chlorinating
agents, N-chlorosuccinimide and trichloroisocyanuric acid,
presumably making them susceptible to dechlorination.
Scheme 1. Enzymatically Activated Acyl Nitroso Precursors
Table 1. HNO Production from N-Chloro-N-acyloxyamides 3
In addition, Fukuto, Nagasawa, and co-workers also
reported the nonenzymatic production of HNO from
several N,O-bis-acylated hydroxylamine derivatives 1.
Low HNO yields (<15%, as determined by N2O analysis)
at neutral or basic pH were observed for N-cyano-N-
benzoyloxybenzamide (1, X = CN, R,R0 = Ph) and also
for N-4-chlorobenzenesulfonyl derivatives (1, X = 4-Cl-
PhSO2 with R,R0 = Me or with R = t-BuO and R0 = Me
or Ph).17,19,20 We wished to build upon these initial studies
to optimize the nonenzymatic production of HNO at
neutral pH from this class of precursors. Herein, we are
pleased to report studies of a wide range of bis-acylated
hydroxylamines 1 and also several N-hydroxy intermedi-
ates 2, the latter of which have been synthesized and
evaluated for HNO production for the first time. Mechan-
istic studies have revealed that the observed aqueous
chemistry is more complicated than originally anticipated,
and have been used to develop a new series of efficient
HNO precursors with tunable half-lives.
%HNO
(PBS)a
%HNO
(DI)a
compd
R
R0
3a
3b
3c
3d
3e
3f
Ph
Ph
<1
<1
3
22
24
42
44
36
28
23
60
36
74
4-Cl-Ph
4-NO2-Ph
Ph
4-Cl-Ph
4-NO2-Ph
4-NO2-Ph
2,6-F-Ph
t-Bu
4
b
Ph
t-Bu
<1
9
3g
3h
3i
EtO
EtO
Me
Me
11
4
t-Bu
Me
3j
t-BuO
Me
29
a Donor compounds were incubated at 37 °C in PBS (pH 7.4) or DI
water. HNO yields were determined from N2O headspace analysis using
a calibration standard following complete decomposition (SEM ( 5%;
n = 3). b Not determined.
We first examined compounds 1 with chloride as an
alternative leaving group X. A series of N-chloro-N-acy-
loxyamides 3 were synthesized by chlorination of the
corresponding N,O-bis-acylatated hydroxylamine with tri-
chloroisocyanuric acid. These compounds were examined
Given our observations that the use of N-chloro com-
pounds 3 as HNO donors would be extremely limited, we
focused on arenesulfonyl analogues 4 (Scheme 2), which
were synthesized following modified literature procedures.19
(See SI for experimental details concerning synthesis of all
precursors.) Initially, we examined HNO generation in PBS
from a series of N-arenesulfonyl-N-acetoxyacetamides
4aꢀ4j. An expected byproduct of HNO release from these
compounds is sulfinic acid 5 (Scheme 2, Path 1a). HPLC
analysis of the aqueous (PBS, pH 7.4) decomposition of
derivative 4f confirms sulfinic acid production (26%) that
corresponds nicely to the HNO yield (26%) (Table 3). This
analysis also indicates that the main non-HNO producing
pathway is amide hydrolysis, producing an O-acylated
Piloty’s acid derivative 6 (Scheme 2, Path 2).
(19) Lee, M. J. C.; Nagasawa, H. T.; Elberling, J. A.; DeMaster, E. G.
J. Med. Chem. 1992, 35, 3648–3652.
(20) Fukuto, J. M.; Hszieh, R.; Gulati, P.; Chiang, K. T.; Nagasawa,
H. T. Biochem. Biophys. Res. Commun. 1992, 187, 1367–1373.
(21) Nagasawa, H. T.; Kawle, S. P.; Elberling, J. A.; DeMaster, E. G.;
Fukuto, J. M. J. Med. Chem. 1995, 38, 1865–1871.
(22) Nagasawa, H. T.; DeMaster, E. G.; Goon, D. J.; Kawle, S. P.;
Shirota, F. N. J. Med. Chem. 1995, 38, 1872–1876.
(23) Conway, T. T.; DeMaster, E. G.; Lee, M. J.; Nagasawa, H. T.
J. Med. Chem. 1998, 41, 2903–2909.
(24) Conway, T. T.; DeMaster, E. G.; Goon, D. J.; Shirota, F. N.;
Nagasawa, H. T. J. Med. Chem. 1999, 42, 4016–4020.
(25) Nagasawa, H. T.; DeMaster, E. G.; Redfern, B.; Shirota, F. N.;
Goon, D. J. W. J. Med. Chem. 1990, 33, 3120–3122.
(26) Shirota, F. N.; Goon, D. J. W.; DeMaster, E. G.; Nagasawa,
H. T. Biochem. Pharmacol. 1996, 52, 141–147.
(27) DeMaster, E. G.; Redfern, B.; Nagasawa, H. T. Biochem.
Pharmacol. 1998, 55, 2007–2015.
We were initially surprised to observe such facile hydro-
lysis of the amide functionality but realized that compounds
4 bear some similarity to N-alkyl-N-acylsulfonamides,
Org. Lett., Vol. 14, No. 2, 2012
473

Table 3. Decomposition Product Yields and Half-Lives of
N-Arenesulfonyl-N-acyloxamides 4
Scheme 2. Reactivity of Potential HNO Precursors 4
t1/2
compd
%5a
%6a
%8a
(min)
b
4f
26
74
58
0
15
31
52
65
<1
280
4m
4u
4v
4w
4x
23
19
0
100c
100c
100c
100c
0
0
0
0
0
0
a Determined by HPLC and reported as relative yields (SEM ( 5%;
n = 3). b In this case, byproducts 6 and 8 are identical. c Sulfinic acid 5
was the only HPLC detected product.
Table 2. HNO Production from N-Arenesulfonyl-N-acyloxa-
mides 4
compda
R
R0
Ar
%HNOb
detected in 58% yield along with a new product (8, R=i-Pr),
detected in 19% yield (Table 3). This newly observed
O-acylated Piloty’s acid derivative is presumably derived via
acyl migration from N-hydroxy intermediate 7 (Scheme 2,
Path 1b) in another non-HNO producing pathway.
4a
4b
4c
Me
Me
Ph
2
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
CH2Cl
i-Pr
2,4,6-i-Pr-Ph
4-Cl-Ph
2-NO2-Ph
2-Cl-Ph
2-Br-Ph
2-Br-4,6-F-Ph
2,6-F-Ph
2,6-Cl-Ph
2,6-Br-Ph
2,6-Cl-Ph
2,6-Cl-Ph
2-Br-Ph
2-Br-Ph
2,6-Cl-Ph
2,6-Cl-Ph
Ph
4
Me
3
4d
4e
Me
10
17
26
29
17
42
53
2
Me
To confirm this proposed N to O acyl migration, we
wished to examine the chemistry of N-hydroxy-N-acylsul-
fonamides 7 themselves. Although these species have been
proposed as intermediates in the chemistry of N,O-bis-
acylated hydroxylamine derivatives,17ꢀ24 they have, to our
knowledge, not been previously synthesized or evaluated
for HNO production. We accomplished the synthesis of
a series of N-hydroxy-N-acylsulfonamides 7 by either
N-acylation and subsequent deprotection of an N-(tetra-
hydropyranyloxy)-arenesulfonamide to give 7a (R = Me)
or 7b (R = t-Bu) (Scheme 3a) or, since the sulfonamide
nitrogen could not be acylated directly using N,N-dimethyl-
carbamoyl chloride, a related sequence (Scheme 3b) to give
7c or 7d. These compounds can be handled and stored for
short periods (days to weeks at ꢀ20 °C) but, as expected,
readily decompose in aqueous solutions.
Decomposition analysis of 7a (R = Me) revealed not
only substantial HNO production (60%) along with a
corresponding amount of sulfinic acid but also a significant
amount (40%) of O-acylated Piloty’s acid derivative
8 (R = Me), confirming potentially significant N to O acyl
migration in these compounds (Table 4). Indeed, HPLC
analysis of 7b (R = t-Bu) indicates over 90% acyl migration
demonstrating that even if amide hydrolysis were diminished
in 4n (R = t-Bu, R0 = Me), production of 7b would result in
very little HNO formation, as is observed (Table 2).
•4f
4g
4h
4i
Me
Me
Me
Me
4j
Me
4k
4l
CHCl2
Ph
13
24
<1
43
16
2
•4m
4n
4o
4p
4q
4r
i-Pr
t-Bu
EtO
BnO
t-BuO
t-BuO
t-BuO
t-BuO
Me2N
Me2N
Me2N
Me2N
2-Br-Ph
2,6-Cl-Ph
2,6-Br-Ph
2-Br-Ph
Ph
5
4s
3
4t
2
•4u
•4v
•4w
•4x
AS
94
99
99
97
Ph
Ph
75
a Compoundsindicated with a bullet (•) are analyzedin more detail in
Table 3. b Donor compounds were incubated at 37 °C in PBS (pH 7.4).
HNO yields were determined from N2O headspace analysis using a
calibration standard following complete decomposition (SEM ( 5%;
n = 3). The N2O yield observed from Angeli’s salt (AS) is provided for
comparison. HNO was confirmed as the source of N2O by complete
quenching with added glutathione.
which are known to undergo facile hydrolysis.28 In an
attempt to inhibit this amide hydrolysis, we examined
derivatives 4kꢀ4x, which contained either bulky substitu-
ents R (4lꢀ4n), carbamate linkages (4oꢀ4t), or carbamide
linkages (4uꢀ4x). Enhanced HNO production was ob-
served only for urea derivatives 4uꢀ4x (Table 2), which will
be discussed in more detail below.
Scheme 3. Synthesis of N-Hydroxy-N-acylsulfonamides 7
HPLC decomposition analysis of derivative 4m (R = i-Pr,
R0 = Me) revealed two O-acylated Piloty’s acid derivatives.
The expected product of amide hydrolysis (6, R0 = Me) is
(28) Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. J. Chem.
Soc. 1971, 636–637.
474
Org. Lett., Vol. 14, No. 2, 2012

Given the high yields of HNO observed following
decomposition of precursors 4uꢀ4x, we synthesized the
related urea derivatives 7c, 7d and analyzed these com-
pounds for HNO production. Acyl migration was not
observed by HPLC in either case. 7c cleanly decomposes
to HNO and sulfinic acid 5 with a very short half-life
(t1/2 < 1 min) (Table 4). Precursor 7d, which replaces the
2-Br-phenyl group in 7c with an unsubstituted phenyl
group, expectedly decomposes with a longer half-life
(t1/2 = 13 min). Again, essentially quantitative production
of HNO and sulfinic acid is observed, but in this case a small
amount of an additional intermediate, which decomposes
over a slightly longer time frame, is detected by HPLC. This
intermediate has been identified as acyl sulfone 9
(Ar = Ph) (Scheme 4) by coinjection with an authentic
sample; we estimate that it is formed in <5% yield.
precursor 4u in 18O-labeled water. Although somewhat
complicated by the CO2 formed from dissociation of the
carbamic acid byproduct (Me2NCO2H), mass spectrometric
analysis of the HNO-derived N2O revealed very small
amounts (<2%) of labeled N2O (see SI), indicating an
insignificant contribution from Scheme 4, Path 2.
Scheme 4. Potential HNO-Forming Reaction Pathways
Table 4. Decomposition Product Yields and Half-Lives of
N-Hydroxy-N-acylsulfonamides 7
t1/2
compd
R
Ar
%HNOa
%5b
%8b
(min)
Our mechanistic analysis of N,O-bis-acylated hydroxy-
lamine derivatives 4aꢀ4x and related N-hydroxy com-
pounds 7aꢀ7d has identified urea-based precursors 4uꢀ4x
and 7cꢀ7d that avoid both amide hydrolysis (from 4) and
acyl migration (from 7) and efficiently produce HNO.
These experiments also indicate that Scheme 4, Path 1a is
the major reaction pathway. The half-life of HNO gen-
eration at pH 7.4, 37 °C from these precursors can be
tuned from seconds to hours by varying the ester R0
group and/or the sulfonyl leaving group (Tables 3, 4),
providing flexibility in biological applications and the
ability to probe the effects of either rapid or slow HNO
production.
7a
7b
7c
7d
Me
2-Br-Ph
2-Br-Ph
2-Br-Ph
Ph
60
5
60
40
91
0
2
t-Bu
9
<1
<1
13
Me2N
Me2N
96
99
100c
100c
0
a Donor compounds were incubated at 37 °C in PBS (pH 7.4). HNO
yields were determined from N2O headspace analysis using a calibration
standard following complete decomposition (SEM ( 5%; n = 3). HNO
was confirmed as the source of N2O by complete quenching with added
glutathione. b Determined by HPLC and reported as relative yields (SEM (
5%; n = 3). c Sulfinic acid 5 was the only HPLC detected product.
Based on Glover’s extensive studies of bis-heteroatom-
substituted amides,29 we propose that 9 forms along with
HNO from 7d via a HERON (heteroatom rearrangement
on nitrogen) reaction as shown in Scheme 4, Path 1b (and
2b). Examination of 9(Ar=Ph) itselfdemonstratesthatin
buffer(pH7.4, 37°C) ithydrolyzescleanlytosulfinicacid5
(Ar = Ph) with a t1/2 of 22 min. A small amount of
HERON product 9 is also observed by HPLC from
decomposition of 4w, which decomposes cleanly (t1/2 <
1 min) to N-hydroxy compound 7d, and ultimately HNO
and sulfinic acid (Table 3).
Glover’s work on bis-heteroatom-substituted amides also
suggests that HNO production could be initiated by water
attack at the amide nitrogen (Scheme 4, Path 2) in addition to
the expected attack at the ester carbonyl (Scheme 4, Path 1).
To probe this possibility, we examined the decomposition of
Acknowledgment. J.P.T. gratefully acknowledges the
National Science Foundation (CHE-0911305) and Cardi-
oxyl Pharmaceuticals for generous support of this re-
search. We thank Dr. I. Phil Mortimer (JHU) for his
excellent technicalsupport inmassspectrometry, Meredith
Cline (JHU) for her assistance with 18O-labeling studies,
Huong Nyguen (JHU) for synthetic assistance, and Pro-
fessor Naod Kebede (Edinboro University) for his insight
during mechanistic studies.
Supporting Information Available. Experimental pro-
cedures, compound characterization data, and details
concerning N2O, HPLC, and UVꢀvis analysis. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(29) For a recent review, see: Glover, S. A. In The Chemistry of
Hydroxylamines, Oximes, and Hydroxamic Acids, Part 2; Rappoport, Z.,
Liebman, J. F., Eds.; John Wiley & Sons: Chichester, West Sussex, 2009;
pp 839ꢀ923.
Org. Lett., Vol. 14, No. 2, 2012
475