Nitroxyl (HNO) release from new functionalized N-hydroxyurea-derived acyl nitroso-9,10-dimethylanthracene cycloadducts

Article abstract of DOI:10.1016/j.bmcl.2004.08.062

A thermal retro-Diels-Alder decomposition of N-hydroxyurea-derived acyl nitroso compounds and 9,10-dimethylanthracene cycloadducts followed by acyl nitroso compound hydrolysis produces nitrous oxide, evidence for the formation of nitroxyl, the one-electron reduced form of nitric oxide that has drawn considerable attention for its potential roles in biological systems. EPR and NMR spectroscopy provide further evidence for nitroxyl formation and kinetic information, respectively. Such compounds may prove to be useful alternative nitroxyl donors.

Full text of DOI:10.1016/j.bmcl.2004.08.062

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5565–5568
Nitroxyl (HNO) release from new
functionalized N-hydroxyurea-derived acyl
nitroso-9,10-dimethylanthracene cycloadducts
Bu-Bing Zeng, Jinming Huang, Marcus W. Wright and S. Bruce King*
Department of Chemistry, Wake Forest University, Salem Hall, Winston-Salem, NC 27109, USA
Received 15 July 2004; revised 20 August 2004; accepted 27 August 2004
Available online 18 September 2004
Abstract—A thermal retro-Diels–Alder decomposition of N-hydroxyurea-derived acyl nitroso compounds and 9,10-dimethylanthra-
cene cycloadducts followed by acyl nitroso compound hydrolysis produces nitrous oxide, evidence for the formation of nitroxyl, the
one-electron reduced form of nitric oxide that has drawn considerable attention for its potential roles in biological systems. EPR and
NMR spectroscopy provide further evidence for nitroxyl formation and kinetic information, respectively. Such compounds may
prove to be useful alternative nitroxyl donors.
ꢀ 2004 Elsevier Ltd. All rights reserved.
An increasing amount of evidence highlights the biolog-
ical importance of nitroxyl (HNO), the one-electron
O
retro
Diels
Alder
reduced form of nitric oxide (NO).1 While nitroxyl-
releasing compounds mimic the action of NO donors
under some conditions, recent studies reveal distinct bio-
logical activities for HNO and NO.2 For example, HNO
and NO show differences in their ability to promote oxi-
dative DNA damage.3 Another group of studies high-
light the discrete effects mediated by nitroxyl and nitric
oxide in both normal and failing heart models.4,5
Specifically, nitroxyl stimulates calcitonin gene-related
peptide release while nitric oxide increases cyclic guany-
late monophosphate (cGMP) and these results suggest
the potential of nitroxyl donors for the treatment of
heart failure.4–6 These studies have prompted both the
theoretical and experimental examination of the thermo-
dynamic properties and chemical reactivity of HNO.7–14
N
NHR
O
O
H2O
HNO
RHN
N
O
nitroxyl
1
2
acyl nitroso
compound
O
O
OEt
OH
N
N
H
N
N
O
H
O
O
4
3
Scheme 1.
AngeliÕs salt (sodium trioxodinitrate, Na2N2O3) is cur-
rently the most widely used HNO donor. However, with
the described emerging picture, new nitroxyl donors will
be of increasing importance as both biochemical and
pharmacological tools and potential therapeutic agents.
Hydrolysis of acyl nitroso species (1, Scheme 1) pro-
duces HNO and represents a reliable chemical approach
for HNO formation.15 Earlier work shows that cycload-
ducts (2, Scheme 1) of the acyl nitroso species derived
from N-hydroxyureas and 9,10-dimethylanthracene
(9,10-DMA) undergo a retro-Diels–Alder reaction to
form acyl nitroso species that hydrolyze to HNO, car-
bon dioxide, and the corresponding amine.16 While the
cycloadducts of N-hydroxyureas derived from simple
alkyl and aryl amines release HNO under mild condi-
tions at useful rates, these compounds demonstrate poor
water solubility.16 We wish to report the synthesis and
Keywords: Nitric oxide; Nitroxyl; Retro-Diels–Alder; Acyl nitroso
compounds.
*
Corresponding author. Tel.: +1 336 758 5774; fax: +1 336 758
4656; e-mail: kingsb@wfu.edu
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.062

5566
B.-B. Zeng et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5565–5568
both proton and carbon NMR spectroscopy, mass
O
H
N
spectrometry, and elemental analysis.18 While 3 does
not show appreciable water solubility, the ester group
provides a point for further derivation for improving
the solubility (conversion to the salt or the attachment
of hydrophilic groups). Cycloadduct 4 readily dissolves
in water to yield a 1mM solution.
NH2OH
HOHN
N
CO2Et
OEt
O
C
O
O
5
1) NaH, BnCl
HO
BnO
NHOBn
NH2
NH
NH
OBn
2)
O
6
Thermal decomposition of cycloadduct 3 in a 1:1 mix-
ture of water–acetonitrile at 40ꢁC under argon yields a
mixture of nitrous oxide (42%) and carbon dioxide after
3h as determined by gas chromatography of the reaction
headspace (Scheme 3).16 Nitrous oxide and carbon diox-
ide likely form through the initial retro-Diels–Alder dis-
sociation of 3 to 9,10-DMA and the acyl nitroso species
(8, Scheme 3). Hydrolysis of 8 produces HNO and a carb-
amic acid that further decomposes to carbon dioxide
and presumably ethyl glycine. The dimerization and
dehydration of HNO produces nitrous oxide and
provides strong evidence for the intermediacy of HNO
in this process (Scheme 3).19 Monitoring the disappear-
ance of the unique methyl singlets of 3 over time at 37ꢁC
by nuclear magnetic resonance (NMR) spectroscopy in
the presence of 1,3-cyclohexadiene, to trap the reactive
acyl nitroso species as previously demonstrated,16 shows
that 3 decomposes in a first order manner (k = 1.33 ·
10À2 minÀ1, t1/2 = 53min). Compound 3 decomposes at
a rate similar to other cycloadducts of 9,10-DMA and
the acyl nitroso species derived from alkyl substituted
N-hydroxyureas.16
O
O2N
HO
O
H2, Pd/C
NH
NH
OH
7
O
N
NHR
1) Bu4N+IO4
2) 9, 10-DMA
-
O
5 or 7
3, R = -CH2CO2Et
4, R = -CH2CH2OH
Scheme 2.
evaluation of 9,10-DMA cycloadducts of the acyl
nitroso species derived from the N-hydroxyureas of
ethyl glycine (3) and ethanolamine (4) that demonstrate
improved water solubility and HNO release properties.
Thermal decomposition of cycloadduct 4 in distilled
water at 40ꢁC under argon similarly produces nitrous
oxide (86%) but no carbon dioxide after 3h (Scheme
4). Examination of the reaction by NMR spectroscopy
also shows the formation of 2-oxazolidone (9, Scheme
4). The intramolecular cyclization of the acyl nitroso
species (10, Scheme 4), formed by the retro-Diels–Alder
decomposition of 4, explains the formation of both ni-
trous oxide (through nitroxyl as above) and 2-oxazoli-
done and the absence of carbon dioxide (Scheme 4).
Monitoring the disappearance of the methyl singlets of
4 over time at 37ꢁC in water by NMR spectroscopy re-
veals that 4 also decomposes in a first order manner
(k = 2.84 · 10À2 minÀ1, t1/2 = 24min). As the cycload-
ducts exist in equilibrium with the acyl nitroso com-
pounds and 9,10-DMA, the immediate trapping of the
acyl nitroso compound (10) by an internal nucleophilic
The synthesis of cycloadducts 3 and 4 requires the prop-
erly substituted N-hydroxyurea derivatives. Addition of
hydroxylamine to ethyl isocyanatoacetate yields the gly-
cine derived N-hydroxyurea in 91% yield (5, mp 96–
98ꢁC, Scheme 2). Protection of the hydroxyl group of
ethanolamine as the benzyl ether17 followed by conden-
sation with 4-nitrophenyl-N-hydroxy-O-benzylcarba-
mate gives the fully protected ethanolamine derived
N-hydroxyurea (6) and hydrogenation gives the ethanol-
amine derived N-hydroxyurea (7, 66% yield for three
steps, Scheme 2). Tetra-n-butyl ammonium periodate
oxidation of the N-hydroxyureas (5 and 7) in the pres-
ence of 9,10-DMA yields the cycloadducts 3 and 4
(14% and 44% yield after chromatography, respectively,
Scheme 2). These compounds were characterized by
O
OEt
N
N
H
retro
Diels
Alder
O
O
O
H2O
N2O
EtO
N
H
N
O
O
3
8
-H2O
HNO
EtO
+ CO2 + HNO
H2N2O2
NH2
O
Scheme 3.

B.-B. Zeng et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5565–5568
5567
O
OH
N
N
H
retro
Diels
Alder
O
internal
cyclization
O
HO
N
H
N
O
3
10
O
-H2O
HNO
NH
O
+ HNO
H2N2O2
N2O
9
Scheme 4.
hydroxyl group may accelerate the observed rate of
decomposition of 4. Solvent effects could also play a role
in the different observed decomposition rates of com-
pounds 3 and 4. Compound 4 decomposes at a rate sim-
ilar to other cycloadducts of 9,10-DMA and the acyl
nitroso species derived from aryl substituted N-
hydroxyureas.16
likely limit the use of these compounds to in vitro studies
of HNO chemistry and biology.21 However, this work
clearly indicates that a retro-Diels–Alder reaction of
properly constructed cycloadducts represent a reliable
method of HNO generation.
In summary, cycloadducts 3 and 4 generate HNO
through a retro-Diels–Alder reaction followed by
hydrolysis of the resulting acyl nitroso species. Function-
alization of the hydroxyurea portion of these com-
pounds improves their water solubility and provides a
point of further derivatization while not affecting the
rate of nitroxyl release at 37ꢁC as compared to similar
compounds. Such compounds provide alternative
HNO donors to AngeliÕs salt that may be useful in dis-
tinguishing the biological effects of HNO from those
of NO in some systems.
Electron paramagnetic resonance (EPR) spectroscopic
experiments provide further evidence for HNO release
from cycloadducts 4. Anaerobic incubation of 4
(1mM) with methemoglobin (1mM) in phosphate buffer
produces iron nitrosyl hemoglobin over 2h (Fig. 1) as
indicated by the increasing characteristic absorbance
of ferrous nitrosyl complexes near g = 2. These results
provide a second independent line of evidence for
HNO production from 4 as HNO reductively nitrosyl-
ates a number of ferric heme proteins, including methemo-
globin, to yield the ferrous–NO complexes.20 The
resonance near g = 6 indicates the presence of un-re-
acted methemoglobin suggesting incomplete HNO trap-
ping by methemoglobin under these conditions. While
these studies clearly indicate HNO formation from the
decomposition of 3 and 4, the carcinogenic and muta-
genic properties of 9,10-DMA, the reaction by-product,
Acknowledgements
This work was supported by the National Institutes of
Health (HL62198, SBK) and the American Heart Asso-
ciation (Established Investigator Award, 0140020N,
SBK). The NMR spectrometers used in this work were
purchased with partial support from NSF (CHE-
9708077) and the North Carolina Biotechnology Center
(9703-IDG-1007). The EPR spectrometer was pur-
chased with support from the North Carolina Bio-
technology Center (2003-IDG-1013).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2004.08.062.
10 min
60 min
120 min
References and notes
1. Wilson, E. K. Chem. Eng. News 2004, 82(10), 39–44.
2. (a) Fukuto, J. M.; Gulati, P.; Nagasawa, H. T. Biochem.
Pharmacol. 1994, 47, 922–924; (b) Fukuto, J. M.; Hszieh,
R.; Gulati, P.; Chiang, K. T.; Nagasawa, H. T. Biochem.
Biophys. Res. Commun. 1992, 137, 1367–1373; (c) Fukuto,
J. M.; Chiang, K.; Hszieh, R.; Wong, P.; Chaudhuri, G. J.
Pharmacol. Exp. Ther. 1992, 263, 546–551.
0
1000
2000
3000
4000
5000
Magnetic Field (Gauss)
Figure 1. EPR spectra at 130K of the anaerobic incubation of 4 with
methemoglobin over time. Solid line (10min), dashed line (60min), and
dotted line (120min).

5568
B.-B. Zeng et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5565–5568
3. Ohshima, H.; Tatemichi, M.; Sawa, T. Arch. Biochem.
Biophys. 2003, 417, 3–11.
15. King, S. B.; Nagasawa, H. T. In Methods in Enzymology;
Packer, L., Ed.; Academic: San Diego, 1998; Vol. 301,
Chapter 22.
4. Paolocci, N.; Katori, T.; Champion, H.; St. John, M. E.;
Miranda, K. M.; Fukuto, J. M.; Wink, D. A.; Kass, D. A.
Proc. Natl. Acad. Sci. 2003, 100, 5537–5542.
5. Miranda, K. M.; Paolocci, N.; Katori, T.; Thomas, D. D.;
Ford, E.; Bartberger, M. D.; Espey, M. G.; Kass, D. A.;
Feelisch, M.; Fukuto, J. M.; Wink, D. A. Proc. Natl.
Acad. Sci. 2003, 100, 9196–9201.
6. Feelisch, M. Proc. Natl. Acad. Sci. 2003, 100, 4978–4979.
7. Bartberger, M. D.; Fukuto, J. M.; Houk, K. N. Proc.
Natl. Acad. Sci. 2001, 98, 2194–2198.
8. Bartberger, M. D.; Liu, W.; Ford, E.; Miranda, K. M.;
Switzer, C.; Fukuto, J. M.; Farmer, P. J.; Wink, D. A.;
Houk, K. N. Proc. Natl. Acad. Sci. 2002, 99, 10958–10963.
9. Shafirovich, V.; Lymar, S. V. Proc. Natl. Acad. Sci. 2002,
99, 7340–7345.
10. Miranda, K. M.; Nims, R. W.; Thomas, D. D.; Espey, M.
G.; Citrin, D.; Bartberger, M. D.; Paolocci, N.; Fukuto, J.
M.; Feelisch, M.; Wink, D. A. J. Inorg. Biochem. 2003, 93,
52–60.
16. Xu, Y.; Alavanja, M. M.; Johnson, V. L.; Yasaki, G.;
King, S. B. Tetrahedron Lett. 2000, 41, 4265–4269.
17. Hu, X. E.; Cassady, J. M. Synth. Commun. 1995, 25, 907–
913.
1
18. For 3: mp 92–94ꢁC; H NMR (300MHz, CDCl3) d 7.48
(m, 4H), 7.29 (m, 4H), 4.89 (s, 1H), 4.07 (q, J = 7.1Hz,
2H), 3.33 (s, 2H), 2.59 (s, 3H), 2.29 (s, 3H), 1.18 (t,
J = 7.1Hz, 3H); 13C NMR (75MHz, CDCl3) 170.0, 163.3,
141.6, 140.5, 127.1, 127.0, 121.3, 120.4, 79.4, 64.4, 60.7,
41.4, 16.3, 14.1, 13.1; ESI-MS: m/z = 389 (M++Na)+;
Anal. Calcd for C22H22N2O4: C, 68.84; H, 6.05; N, 7.65.
Found: C, 68.74; H, 6.07; N, 7.65. For 4: mp 171–174ꢁC;
1H NMR (300MHz, D2O) d 7.42(m, 4H), 7.25 (m, 4H),
3.00 (t, J = 5.9Hz, 2H), 2.82 (t, J = 5.9Hz, 2H), 2.41 (s,
3H), 2.18 (s, 3H); 13C NMR (75MHz, CD3OD) 163.2,
141.1, 139.9, 128.7, 128.6, 123.0, 122.0, 78.9, 64.2, 60.0,
41.2, 16.0, 13.8; ESI-MS: m/z = 347 (M++Na)+; Anal.
Calcd for C19H20N2O3: C, 70.35; H, 6.21; N, 8.64. Found:
C, 70.42; H, 6.19; N, 8.64.
11. Bari, S. E.; Marti, M. A.; Amorebieta, V. T.; Estrin, D. A.;
Doctorovich, F. J. Am. Chem. Soc. 2003, 125, 15272–
15273.
19. Bonner, F. T.; Hughes, M. N. Comments Inorg. Chem.
1988, 7, 215–234.
12. Dutton, A. S.; Fukuto, J. M.; Houk, K. N. J. Am. Chem.
Soc. 2004, 126, 3795–3800.
13. Poskrebyshev, G. A.; Shafirovich, V.; Lymar, S. V. J. Am.
Chem. Soc. 2004, 126, 891–899.
14. Sulc, F.; Immoos, C. E.; Pervitsky, D.; Farmer, P. J.
J. Am. Chem. Soc. 2004, 126, 1096–1101.
20. Bazylinski, D. A.; Goretski, J.; Hollocher, T. C. J. Am.
Chem. Soc. 1985, 107, 7986–7989.
21. Fluckiger-Isler, S.; Baumeister, M.; Braun, K.; Gervais,
¨
V.; Hasler-Nyguyen, N.; Reimann, R.; Van Gompel, J.;
Wunderlich, H. G.; Engelhardt, G. Mutat. Res. 2004, 558,
181–197.