C-nitroso donors of nitric oxide

Article abstract of DOI:10.1021/jo802517t

A complete understanding of the biological activity of nitric oxide (NO) is complicated by the different reactivity profiles of its various species and by the often complex decomposition behavior of the NO progenitors in common use. Here, we report that a

Full text of DOI:10.1021/jo802517t

C-Nitroso Donors of Nitric Oxide
Harinath Chakrapani, Michael D. Bartberger,† and Eric J. Toone*
Department of Chemistry, Duke UniVersity, B 120 LeVine Science Research Center,
Durham, North Carolina 27708
eric.toone@ duke.edu
ReceiVed NoVember 11, 2008
A complete understanding of the biological activity of nitric oxide (NO) is complicated by the different
reactivity profiles of its various species and by the often complex decomposition behavior of the NO
progenitors in common use. Here, we report that appropriately substituted C-nitroso compounds act solely
as donors of neutral nitric oxide through a first-order homolytic C-N bond scission to release up to 88%
nitric oxide in DMSO at 25 °C. The reaction produces a carbon radical, and the yield of nitric oxide is
dependent on the availability of radical traps. C-Nitroso compounds are sources of biologically active
neutral NO and display potent NO bioactivity in a rabbit aortic ring assay.
SCHEME 1. Rational Design of C-Nitroso Donors of NO in
Various Redox States
Although the involvement of nitric oxide (NO) in biological
processes is by now clear, many aspects of its activity remain
enigmatic. The varying and often contradictory activities of
1
nitric oxide have in some instances been attributed to differences
in the biological activities of its redox-related congeners: neutral
NO radical, the +1 cation (nitrosonium), and the one-electron
reduced form, HNO (nitroxyl).2 A clearer and more complete
understanding of the physiological activity of NO requires
precursors that selectively produce NO at predictable, controlled,
and well-defined rates and in a single oxidation state; ideal
progenitors for single redox forms of NO are largely lacking.
Several classes of NO/NO+/HNO generators and/or surrogates
are in common use, including nitrate esters, inorganic complexes
of nitric oxide, S-nitrosothiols, various nitrogen heterocycles,
and diazenium diolates.3 In many cases, however, the release
of nitric oxide is both mechanistically complex and dependent
on the availability of exogenous agents as simple as protons or
as complex as enzymes. Decomposition often releases NO in
more than one redox state in ratios dependent on environmental
conditions. Single-redox sources of NO that release at a
predictable rate via well-understood mechanisms are thus vital
to a complete understanding of the biological activity of NO.
C-Nitroso compounds offer the potential for highly selective
delivery of a single redox form of NO, facilitating exquisite
control of the electronic and steric characteristics of the carbon
atom bearing the nitroso group (Scheme 1).4 Stabilization of
an incipient carbon radical should facilitate homolytic scission
of the C-N bond and release of neutral nitric oxide (Scheme
1a), while stabilization of the corresponding carbanion would
promote the activity of an NO+ equivalent, (Scheme 1b)
† Current address: Amgen, Inc., One Amgen Center Drive, M/S 29-M-B,
Thousand Oaks, CA 91320. E-mail: mbartber@amgen.com.
(1) (a) Ignarro, L. J. Angew. Chem., Int. Ed. 1999, 38, 1882. (b) Furchgott,
R. F. Angew. Chem., Int. Ed. 1999, 38, 1870. (c) Moncada, S.; Palmer, R. M. J.;
Higgs, E. A. Pharmacol. ReV. 1991, 43, 109. (d) Thomas, D. D.; Ridnour, L. A.;
Isenberg, J. S.; Flores-Santana, W.; Switzer, C. H.; Donzelli, S.; Hussain, P.;
Vecoli, C.; Paolocci, N.; Ambs, S.; Colton, C. A.; Harris, C. C.; Roberts, D. D.;
Wink, D. A. Free Radical Biol. Med. 2008, 45, 18.
(2) (a) Stamler, J. S.; Singel, D. J.; Loscalzo, J. Science 1992, 258, 1898.
(b) Hughes, M. N. Biochim. Biophys. Acta 1999, 263, 1411. (c) Fukuto, J. M;
Bartberger, M. D.; Dutton, A. S.; Paolocci, N.; Wink, D. A.; Houk, K. N. Chem.
Res. Toxicol. 2005, 18, 790.
(3) (a) Granik, V. G.; Grigor‘ev, N. B. Russ. Chem. ReV. (Engl. Transl.).
2002, 51, 1375. (b) Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wen, Z.; Cai, T.;
Janczuk, A. Chem. ReV. 2002, 102, 1091. (c) Napoli, C.; Ignarro, L. J. Annu.
ReV. Pharmacol. Toxicol. 2003, 43, 97–123. (d) King, S. B. Free Radical Biol.
Med. 2004, 37, 735. (e) Thatcher, G. R. J. Curr. Top. Med. Chem. 2005, 5, 597.
(f) Al-Sa’doni, H. H; Ferro, A. Curr. Top. Med Chem 2004, 11, 2679. (g) Bollal,
M.; Almirante, N.; Benedini, F. Curr. Top. Med. Chem. 2005, 5, 707. (h)
Schonafinger, K. Farmaco 1999, 316. (i) Marks, G. S.; McLaughin, B. E.; Jimmo,
S. L.; Pokleskwa-Koziel, M.; Bien, J. F.; Nakatsu, K. Drug Metab. Dispos. 1995,
23, 1248. (j) Keefer, L. K. Curr. Top. Med. Chem. 2005, 5, 625.
(4) Gooden, D. M.; Chakrapani, H.; Toone, E. J. Curr. Top. Med. Chem.
2005, 5, 687.
1450 J. Org. Chem. 2009, 74, 1450–1453
10.1021/jo802517t CCC: $40.75 2009 American Chemical Society
Published on Web 01/15/2009

C-Nitroso Donors of Nitric Oxide
TABLE 1. Calculated Dimerization Energies for C-Nitroso
Compounds
TABLE 2. Bond Dissociation Energies for C-Nitroso Compounds
species
R
E
C-N BDE (calc.)a C-N BDEb
1
2
3
4
5
6
9
10
11
12
13
H
CH3
H
H
36.0
40.8
26.2
26.8
26.0
28.1
28.0
25.7
37-40
34-40
CH3
CN
CN
a
b
species
R2
E
∆Edimerization
∆Gdimerization
1
2
3
4
5
6
7
8
H
CH3
H
H
-18.9 (-20.3)c -8.8 (-9.9)d
CH3 -10.2 +2.1
CN -12.7 (-15.4)c -1.6 (-4.0)d
CH3
22.7
-(CH2)4- (cyclopentyl) CN
-(CH2)5- (cyclohexyl) CN
H
CHO
CHO
Br
NO2
OAc
CH3
CN
-9.2
-9.3
+3.3
+2.8
+1.9
CH3
CH3
CH3
CH3
-(CH2)4- (cyclopentyl) CN
-(CH2)5- (cyclohexyl) CN -10.0
43.0
29.2
36.1
nitrosoethylene
nitrosobenzene
-14.2 (-15.9)c -3.7 (-5.1)d
-3.9
+7.1
a CBS-QB3 values (0 K); this study. b Reference 13.
a B3LYP/6-31G* + ZPE (0 K) values. b B3LYP/6-31G* values at
298.15 K, 1 M. c CBS-QB3 values (0 K) in bold in parentheses.
d CBS-QB3 values (298.15 K, 1 M) in bold in parentheses. All values in
kcal·mol-1. The 1 M standard state values are as defined in ref 10.
of delocalizing substituents at the nitroso carbon. This observa-
tion is in accord with the known resistance of conjugated
C-nitroso species, such as PhNO (8), to undergo dimerization.11,12
From the CBS-QB3 results on model systems 1, 3, and 7 and
the known experimental ∆G of dimerization of +0.61
kcal·mol-1 for 2 (CCl4 solution, 26 °C),11 the predicted
(B3LYP/6-31G*) free energy of dimerization appears to be too
large (positive) by ca. 1.1 to 2.4 kcal ·mol-1. An approximate
range of ca. -0.5 to +2.2 kcal·mol-1 is therefore predicted for
the ∆G of dimerization of R-cyano species 4-6 at 1 M and 25
°C, with dimerzation increasingly favored at higher concentrations.
C-Nitroso bond dissociation and activation energies have been
reported for a small number of aryl and tertiary C-nitroso
species.13 These studies yield C-N homolytic bond dissociation
energies near ca. 40 kcal·mol-1, depending on substitution.4
To supplement these literature values and obtain accurate
energetic parameters for homolysis of the species of interest in
this study, C-N BDE values were computed using the CBS-
QB3 method.
Electron-withdrawing substituents and substituents capable
of radical stabilization through delocalization (CN, CHO, NO2)
lower C-N homolytic BDEs to 26-29 kcal·mol-1. By com-
parison, alkyl S-nitrosothiols, known to be thermally stable to
S-N homolysis at or near room temperature, possess predicted
S-N BDEs of 31-32 kcal ·mol-1 at the same level of theory,
with measured activation energies for homolysis ca. 1-2
kcal·mol-1 lower than the predicted BDE.9 Species 4-6 are
predicted to show significantly more facile NO dissociation (3-5
kcal·mol-1) than S-nitrosothiols and might thus afford pure
neutral NO at physiologically relevant temperatures.
effecting nitrosation of suitable nucleophiles. Finally, activation
of hydrogen atoms vicinal to the NO function via an electron-
withdrawing group (Scheme 1c) promote extrusion of HNO via
ꢀ-elimination. Here, we provide experimental evidence for the
suitability of R-cyano substituted C-nitroso compounds as
donors of neutral NO. Such compounds decompose by ho-
molytic scission to produce nitric oxide.
We envisioned that selective production of neutral nitric oxide
could occur via homolytic scission of a properly activated C-N
bond of an alkylnitroso compound which was suitably substi-
tuted in order to avoid nitrosonium or HNO formation. Tertiary
nitroso species are required, as C-nitroso compounds bearing
R-hydrogen atoms exist as the thermodynamically favored
oxime.5 The biological activities of a few C-nitroso compounds,
including species bearing R-cyano substituents, have previously
been reported.6 Although these compounds showed measurable
(µM) activity, only small amounts of nitric oxide were released,
(<19%) and no kinetic characterization of the release was
reported.6
C-Nitroso compounds undergo dimerization to the corre-
sponding azodioxy species,7 and the rate of NO release is
dependent not only on the rate of C-N bond homolysis but
also on the rate of monomer-dimer interconversion. To probe
the effect of cyano substitution on dimerization and homolysis,
the thermodynamics of both processes were calculated (Tables
1 and 2, respectively) using density functional theory (B3LYP/
6-31G*) and the CBS-QB3 model of Petersson;8 the latter
approach has been shown to reproduce experimental reaction
energetics to within ( 2 kcal/mol, including processes involving
NO dissociation.9
In principle, electron-withdrawing substituents should also
increase the nitrosonium (NO+) donating potential. However,
pKa’s of simple cyanoalkanes are near 35,14 and CN substitution
is not predicted to lower carbon acidity such that nitrosonium
donation is competitive. We thus set out to prepare tertiary
R-cyano C-nitroso compounds and to evaluate their behavior
as NO donors.
Population of the dimer is significantly disfavored by increas-
ing steric bulk, inductive electron withdrawal, and introduction
(5) Gowenlock, B. G.; Batt, L. THEOCHEM 1998, 454, 103.
(6) (a) Rehse, K.; Herpel, M. Arch. Pharm. 1998, 331, 104. (b) Di Stillo,
A.; Medana, C.; Ferrarotti, B.; Gasco, L.; Ghigo, D.; Bosia, A.; Martorana, P. A.;
Gasco, A. Pharm. Res. 2000, 41, 469.
(7) Hoffmann, R.; Gleiter, R.; Mallory, F. B. J. Am. Chem. Soc. 1970, 92,
1460.
(8) Montgomery, J. A.; Frisch, M. J.; Ochterski, J. W.; Petersson, G. A.
J. Chem. Phys. 1999, 110, 2822.
(9) Bartberger, M. D.; Mannion, J. D.; Powell, S. C.; Stamler, J. S.; Houk,
K. N.; Toone, E. J. J. Am. Chem. Soc. 2001, 123, 8868.
(10) Freccero, M.; Gandolfi, R.; Sarzi-Amade`, M.; Rastelli, A. J. Org. Chem.
1999, 64, 3853. (Supporting Information).
(13) (a) Batt, L.; Milne, R. T. Int. J. Chem. Kinet 1973, 5, 1067. (b) Pepekin,
V. I.; Lebedev, V. P.; Balepin, A. A.; Lebdev, Y. A. Dokl. Akad. Nauk. SSSR
1975, 221, 1118. (c) Choo, K. Y.; Mendenhall, G. D.; Golden, D. M.; Benson,
S. W. Int. J. Chem. Kinet. 1974, 6, 813. (d) Boyd, A. A.; Noziere, B.; Lesclaux,
R. J. Phys. Chem. 1995, 99, 10815. (e) Carmichael, P. J.; Gowenlock, B. G.;
Johnson, C. A. F. Int. J. Chem. Kinet. 1972, 4, 339–343. (f) Day, J. S.;
Gowenlock, B. G.; Johnson, C. A. F.; McInally, I. D.; Pfab, J. J. Chem. Soc.
Perkin Trans. 2 1978, 1110. (g) Fu, Y.; Mou, Y.; Lin, B.; Liu, L.; Guo, Q. X.
J. Phys. Chem. A 2002, 106, 12386.
(11) Stowell, J. C. J. Org. Chem. 1971, 36, 3055.
(12) Keussler, V. V.; Luttke, Z. Elektrochem. 1959, 63, 614.
(14) Bordwell, F. G. Acc. Chem. Res. 1988, 21, 456.
J. Org. Chem. Vol. 74, No. 4, 2009 1451

Chakrapani et al.
TABLE 3. NO Release from r-Cyano C-Nitroso Compounds at
25 °C
SCHEME 2. Proposed Mechanism of Nitric Oxide Release
from r-Cyano C-Nitroso Compounds15,16
compd
initial concn (mM)
conditions
dI watera
% NO formed
4
4
5
6
6
6
6
6
6
10
1.25
1.0
1.0
1.25
1.0
1.0
1.0
0.01
<2b
71
31
28
88
23
35
30
89
DMSO
dI watera
dI watera
DMSO
PBSc
PBSc
d
d
dI water + O2
dI water + O2
23 kcal ·mol-1.9 The observed invariance in rate as a function
of both solvent and structure lends strong support to a homolytic
mechanism of NO release.
a Deionized water. b Head space. c Phosphate-buffered saline, pH 7.4.
d Oxygen (1 mL) was injected into the solution at the start of
experiment.
The yields of nitric oxide reported in Table 3 differ
significantly from those reported by Rehse and Herpel, who in
all cases observed yields of e13% for 6.6a Gasco and co-workers
report a 19% yield of NO from 6,6b suggesting that alternative,
presumably bimolecular, pathways of C-nitroso decomposition
might be operative. C-Nitroso compounds react rapidly with
radicals to form stable adducts. Both Gregor and Gowenlock
have reported isolation of the spin-trapped adducts 4a and 6a
in 20-29% yields during decomposition of 4 and 6,15,16
suggesting that spin trapping may be responsible for collateral
consumption of R-cyano C-nitroso compounds.17 To test this
hypothesis, a 10 µM solution of 6 was prepared in deionized
(dI) water pretreated with O2 (g). The solution decomposed to
yield 89% nitric oxide, suggesting that oxygen effectively
competes with spin-trapping at low concentrations (Table 3).
A mechanism for nitric oxide release is shown in Scheme 2.
Rapid dissociation of the dimer in solution generates 2 equiv
of monomer, which undergoes bond homolysis to produce
neutral nitric oxide and the corresponding cyanoalkyl radical
(R•). Reaction of R• with additional C-nitroso compound forms
the spin-trapped adduct, limiting the yield of neutral nitric oxide
in the absence of efficient radical scavengers.17
Our goal at the outset was to prepare useful donors of neutral
nitric oxide. To ensure that R-cyano C-nitroso compounds do
not act as donors of nitrosonium, DMSO and acetonitrile
solutions of 6 were treated with excess propane thiol and sodium
propanethiolate.18 In no case was S-nitrosothiol, the product of
nitrosonium ion transfer, observed. Furthermore, negligible N2O
(<1%) was detected following decomposition of 6,6 suggesting
that HNO is not a decomposition product of R-cyano C-nitroso
compounds.
TABLE 4. Measured Rate Constants for C-N Homolysis in
Various Solvents at 25 °C
compd
solvent
k × 105 (s-1
)
∆Gq (kcal·mol-1
)
4
5
6
6
6
6
DMF
DMF
DMF
DMSO
CH3CN
CHCl3
6.1 ( 1.9
5.5 ( 0.6
5.5 ( 0.9
6.0 ( 2.1
8.5 ( 3.5
8.0 ( 2.3
+23.2 ( 7.2
+23.3 ( 2.5
+23.3 ( 3.8
+23.2 ( 8.1
+23.0 ( 9.4
+23.0 ( 6.6
Compounds 4-6 were prepared according to literature
methods and isolated as blue or bluish white solids.15,16 The
solid-state infrared spectrum of 6 indicated the presence of both
the trans dimer (1292 cm-1) and monomer (1565 cm-1).6 During
storage at -20 °C, the bluish-white solids 4 and 6 become
colorless, presumably the result of dimer formation.6
When compound 4 was dissolved in DMSO, it quickly
produced a deep blue color (λmax ) 650 nm, ε ) 2 × 10-2 M-1
cm-1) characteristic of monomeric C-nitroso compounds. The
kinetics of dimer dissociation were monitored at 25 °C, yielding
a first-order rate constant of 0.1 ( 0.03 s-1 and a ∆Gq of 16.9
kcal·mol-1. The rate of dissociation is solvent independent, and
essentially equivalent rates were observed in acetonitrile,
methanol, and DMF.
In contrast to the solid-state IR spectra, no IR bands
attributable to dimer were observed for dissolved samples. In
conjunction with the observation of a single set of 13C
resonances, these data suggest that the dimer is populated only
in neat samples and at low temperatures.
The blue color of the R-cyano C-nitroso solutions gradually
disappeared during several hours, and the yield of neutral nitric
oxide was determined by chemiluminescence (Table 3). All three
compounds produce significant quantities of nitric oxide,
although the yields are dependent on both solvent and initial
concentration of the C-nitroso species.
Having prepared compounds that act exclusively as donors
of neutral nitric oxide, we examined biological activity of the
expressed nitric oxide using the rabbit aortic ring assay. All
three compounds relax precontracted aortic rings with nanomolar
potencies. The most active of the three, 1-cyano-1-nitrosocy-
clohexane (6), shows an IC50 of roughly 20 nM, while the least
active of the three, 2-cyano-2-nitrosopropane (4), shows an IC50
value roughly 10-fold higher than that of 6 (Figure 1). The origin
of the difference in activity is unclear, since rates of homolytic
Having identified neutral nitric oxide as the major inorganic
product of decomposition, we fit loss of C-nitroso compound
to first-order decays, obtaining first-order rate constants for C-N
bond homolysis (Table 4). The measured rate constants for
decay of compounds 4-6 lie in a fairly narrow range of
(5.5-8.5) × 10-5 s-1, with corresponding ∆Gq values of ca.
(17) The second-order rate constant for the reaction of 2-methyl-2-nitroso-
propane with alkyl radicals is 106-107 M-1 s-1 in water. Madden, K. P.;
Taniguchi, H J. Am. Chem. Soc. 1991, 113, 5541.
(18) Butler, A. R.; Flitney, F. W.; Williams, D. L. H. Trends Pharmacol.
Sc. 1995, 16, 18.
(15) (a) Gregor, V. Collect. Czech. Chem. Commun. 1958, 23, 1782. (b)
Pritzkow, W.; Rosler, W. Leibigs Ann. Chem. 1967, 703, 66.
(16) Gowenlock, B. G.; Pfab, J.; Kresze, G. Leibigs Ann. Chem. 1975, 1903.
1452 J. Org. Chem. Vol. 74, No. 4, 2009

C-Nitroso Donors of Nitric Oxide
Kinetics of Dissociation of Dimeric 4. Compound 4 (dimer, 2
mg, 10 mmol) was dissolved in 3 mL of DMSO, and the absorbance
at 650 nm was measured. A first-order rate constant of 0.1 s-1 was
extracted by curve fitting. This procedure was used in the
measurement of half-lives in acetonitrile (12 s), acetone (11 s), and
methanol (5 s).
Nitric Oxide Analysis. Nitric oxide was quantified by preparing
DMSO stock solutions of 4-6. This stock solution was then diluted
with DMSO, dI water, or PBS to the desired final concentration.
Calibration of the instrument was carried out using 1% w/v
potassium iodide in glacial acetic acid into which known concentra-
tions of nitrite was injected, and a calibration curve was generated
before each experiment. Injections of suitable volumes of samples
into the NOA chamber were carried out to estimate levels of nitric
oxide in headspace or solution. The value of % NO formation was
calculated assuming 2 mol of NO formed per mole of dimer or 1
mol of NO formed from the monomer, as applicable.
Computational Methodology. All calculations were performed
with the Gaussian 98 program system.19 Zero-point energies are
uncorrected. Where relevant, the conformation of a given compound
with the lowest B3LYP/6-31G* energy (UB3LYP for open-shell
species) was used in the CBS-QB3 calculations.
FIGURE 1. Relaxation activity of C-nitroso compounds 4-6 on
precontracted rabbit aortic rings.
scission are predicted to be equivalent in all three compounds.
The rate of monomer-dimer interconversion is sufficient such
that a Curtin-Hammett condition applies, and population of
the dimer cannot account for differences in activity. Differences
in solubility or cellular penetration may play a role in the
observed differences in biological activity.
In summary, we have demonstrated the utility of R-cyano
C-nitroso compounds as donors of neutral nitric oxide. Although
the compounds decompose via a unimolecular process, they exist
as stable dimers in pure form and can be stored for significant,
perhaps indefinite, periods of time as pure solids. The flexibility
afforded by the available range of carbon substitution may be
useful in controlling the rate of NO release. Finally, the exclusive
production of neutral nitric oxide should permit a better
understanding of the biological roles of the various redox states
of nitric oxide. We continue our studies on the effect of carbon
substitution of the chemical and biological properties of
C-nitroso compounds and will report our results in due course.
Bioassay.20 The thoracic aorta of freshly sacrificed (CO2) New
Zealand White rabbits (2.5-3 kg) were removed, cleaned of fat
and connecting tissue, and cut into 3-mm rings. The rings were
mounted under 2 g of resting tension in tissue baths (25 mL) filled
with Krebs solution (37 °C) containing 118 mM NaCl, 4.8 mM
KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 25 mM
NaHCO3, and 11 mM glucose, pH 7.4. The solution was bubbled
with 20% O2, 5% CO2, and balance N2. Changes in isometric
tension were recorded with Statham (Hato Rey, PR) transducers
and a Grass Instruments (Quincy, MA) polygraph, and contractions
were initiated with phenylephrine. The adducts 4-6 were first
dissolved in DMSO (∼10 mM) and then diluted in PBS (pH 7.4 to
give final concentration of approximately 100 µM). From these
stock solutions of 4-6 and subsequent serial dilutions were
generated the concentration-relaxation curves.
Experimental Section
Acknowledgment. We gratefully acknowledge Prof. J. S.
Stamler, Department of Medicine, Duke University, for as-
sistance with the aortic ring assay.
General Methods. NMR spectra were recorded on a spectrom-
eter operating at 300 MHz for H and 75 MHz for 13C. Organic
1
solvents were freshly distilled and degassed before use or used
directly as received. Deionized water from the MilliQ filtration
apparatus was distilled and purged with nitrogen before use. pH
7.4 phosphate-buffered saline (GIBCO) and DMSO (Hybri-Max,
Sigma) in sealed ampules were used as received. All kinetic plots
were prepared using Origin, and its program for curve fitting was
used for rate analysis. Nitric oxide was determined using a
chemiluminescence-based detector (Sievers 280 Nitric Oxide
Analyzer, NOA). Compounds 4-6 were prepared by literature
methods.6,15,16
Synthesis of C-Nitroso Compounds 4-6. Compounds 4-6
were prepared according to literature protocols and showed physical
and spectroscopic properties identical to those of the previously
reported species.
Supporting Information Available: Cartesian coordinates
of all species and intermediates described in the manuscript and
their absolute energies. This information is available free of
charge via the Internet at http://pubs.acs.org.
JO802517T
(19) Frisch, M. J. et al. Gaussian 98, ReVision A.9; Gaussian, Inc.: Pittsburgh,
PA, 1998.
(20) (a) Osborne, J. A.; Lento, P. H.; Siegrfield, M. R.; Stahl, H. G.; Fusman,
B.; Lefer, A. M. J. Clin. InVest. 1989, 83, 465. (b) Stamler, J. S.; Simon, D. I.;
Osborne, J. A.; Mullins, M.; Jaraki, O.; Singel, D. J.; Loscalzo, J. Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 444.
J. Org. Chem. Vol. 74, No. 4, 2009 1453