Manganese(III) complexes of bis(hydroxyphenyl)dipyrromethenes are potent orally active peroxynitrite scavengers

Article abstract of DOI:10.1021/ja110427e

We report a new series of biscyclohexano-fused Mn(III) complexes of bis(hydroxyphenyl)dipyrromethenes, 4a-c, as potent and orally active peroxynitrite scavengers. Complexes 4a-c are shown to reduce peroxynitrite through a two-electron mechanism, thereby forming the corresponding Mn(V)O species, which were characterized by UV, NMR, and LC-MS methods. Mn(III) complex 4b and its strained BODIPY analogue 9b were analyzed by X-ray crystallography. Finally, complex 4a is shown to be an orally active and potent analgesic in a model carrageenan-induced hyperalgesia known to be driven by the overproduction of peroxynitrite.

Full text of DOI:10.1021/ja110427e

COMMUNICATION
pubs.acs.org/JACS
Manganese(III) Complexes of Bis(hydroxyphenyl)dipyrromethenes
Are Potent Orally Active Peroxynitrite Scavengers
Smita Rausaria,† Andrew Kamadulski,† Nigam P. Rath,§ Leesa Bryant,‡ Zhoumou Chen,‡
Daniela Salvemini,‡ and William L. Neumann*,†
†Department of Pharmaceutical Sciences, School of Pharmacy, Southern Illinois University Edwardsville, Edwardsville,
Illinois 62026, United States
‡Department of Pharmacology and Physiology, St. Louis University, St. Louis, Missouri 63104, United States
§Department of Chemistry and Biochemistry and Center for Nanoscience, University of Missouri - St. Louis, St. Louis,
Missouri 63121, United States
S Supporting Information
b
ABSTRACT: We report a new series of biscyclohexano-
fused Mn(III) complexes of bis(hydroxyphenyl)dipyrro-
methenes, 4a-c, as potent and orally active peroxynitrite
scavengers. Complexes 4a-c are shown to reduce perox-
ynitrite through a two-electron mechanism, thereby forming
the corresponding Mn(V)O species, which were character-
ized by UV, NMR, and LC-MS methods. Mn(III) complex
4b and its strained BODIPY analogue 9b were analyzed by
X-ray crystallography. Finally, complex 4a is shown to be an
orally active and potent analgesic in a model carrageenan-
induced hyperalgesia known to be driven by the over-
production of peroxynitrite.
he overproduction of reactive oxygen species (ROS) in vivo
is now widely recognized as a key contributor to numerous
T
pathologies.1 One particularly damaging situation results from
the diffusion-controlled radical coupling of the central ROS,
superoxide, with nitric oxide to form peroxynitrite.2 The highly
reactive peroxynitrite is a powerful biological oxidant that leaves a
trail of dysfunctional oxidized and nitrated proteins, lipids, and
nucleotides in its wake.3 From a pharmacological perspective,
peroxynitrite is considered a potent proinflammatory and proa-
poptotic species that plays a critical role in pain of several
etiologies, as demonstrated initially by our team and then by
others.4-6 Accordingly, the discovery of pharmaceutically rele-
vant agents that can effectively decompose peroxynitrite should
have significant therapeutic value.2,3
Figure 1. (A) Peroxynitrite decomposition catalysts 1 and 2. (B)
Bis(hydroxyphenyl)dipyrromethene analogues 3 and 4.
are the most well studied, both as peroxynitrite reductase
catalysts and superoxide dismutase (SOD) mimics.9,10
Recently, Gross has reported that Mn(III) and Fe(III) corroles
are also excellent peroxynitrite decomposition catalysts.11 Remark-
ably, the Mn(III) corroles operate through a two-electron cycle,
reducing peroxynitrite to nitrite instead of nitrogen dioxide through
a novel disproportionation mechanism. The most important finding
from that work was that Mn(III) corroles can decompose perox-
ynitrite in a catalytic fashion [in contrast to Mn(III) porphyrins] and
therefore do not require the assistance of endogenous coreductants.
Although Mn(III) porphyrins such as 1 and Mn(III) corrole
systems such as 2 have proven to be powerful pharmacological
tools in animal studies, where they have demonstrated the benefits
of destroying peroxynitrite in vivo,11-14 they are not optimal as
therapeutic candidates. While these types of polycationic com-
plexes have excellent catalytic activities and their high water
solubility is useful for laboratory measurements, their corre-
sponding high polarity renders them poorly membrane-soluble.
As a result of the early discoveries of Groves7 and Stern,8
Mn(III) and Fe(III) porphyrins have emerged as important
classes of peroxynitrite reductase and isomerase catalysts, re-
spectively (Figure 1A). Elegant mechanistic studies have revealed
that the more pharmacologically suitable Mn(III) porphyrins
decompose peroxynitrite primarily in a one-electron fashion and
require a biological coreductant such as ascorbate to complete
the reductase catalytic cycle.9 One-electron reduction of perox-
ynitrite produces the potentially damaging nitrogen dioxide
radical, which is also thought to undergo rapid reduction by
endogenous antioxidants.5 Thus, if endogenous antioxidants are
plentiful, Mn(III) pophryins can fully detoxify peroxynitrite
in vivo. From this class, the isomeric Mn(III) tetrakis(meso-N-
alkylpyridinium)porphyrins [e.g., Mn(III)-4-TMPyP5þ (1)]
Received: November 19, 2010
Published: March 03, 2011
r
2011 American Chemical Society
4200
dx.doi.org/10.1021/ja110427e J. Am. Chem. Soc. 2011, 133, 4200–4203
|

Journal of the American Chemical Society
COMMUNICATION
More amphiphilic metallocorroles have indeed shown great
promise as orally active peroxynitrite decomposition cat-
alysts.12c Unfortunately, synthetic methods for accessing polyfunc-
tional corrole systems remain quite challenging, and thus, these
systems are not particularly suited for iterative structure-activity
relationship (SAR) studies. In view of this, we have been keenly
interested in the design, synthesis, and evaluation of new catalyst
systems with enhanced druglike properties.
Scheme 1. Synthesis of Mn(III) Complexes of Bishydroxy-
phenyl-DIPYsa
In our search for alternative trianionic ligand systems capable
of supporting a Mn(V)O intermediate arising from the two-
electron reduction of peroxynitrite [similar to Mn(III) corroles],
we were intrigued by the B,O-chelated boron dipyrromethene
(BODIPY) dye 3 first reported by Burgess (Figure 1B).15 This
and related dyes are constrained because of the chelate effect and
thus have improved fluorescence properties relative to their
nonchelated and well-known BODIPY congeners. Not only is
the overall “ligand set” in these systems perfect for mimicking the
trianionic corrole, but these compounds are also amenable to
modular synthesis in good to excellent yields.16 Here we report
the synthesis and evaluation of new biscyclohexano-fused
Mn(III) complexes of bis(hydroxyphenyl)dipyrromethenes as
potent peroxynitrite scavengers with druglike properties.
The syntheses commenced with readily available tetrahydroisoin-
dole 5,17 which was converted to Boc-protected 3-bromotetrahydroi-
soindole 6 in 95% yield over two steps (Scheme 1). Next, compound
6 underwent smooth Suzuki couplings with a representative set of
protected hydroxyphenylboronic acids (7a-c) in70-89% yield, and
this was followed by a one-pot decarboxylation/deprotection proce-
dure that furnished the corresponding 2-benzyloxy- or 2-methoxy-
phenyltetrahydroisoindole derivatives 8a-c. Compounds 8a-cwere
then treated with benzaldehyde and trifluoroacetic acid (TFA) under
Lindsey conditions18 to form the corresponding dipyrromethane
derivatives; this was followed by oxidation to the dipyrromethenes
(DIPYs) with p-chloranil19 and subsequent phenol ether deprotec-
tion with boron tribromide.19,20 This sequence was carried out
without intervening purifications. The boron tribromide deprotection
step afforded the crude BODIPY systems 9a-c, which were then
directly converted to the Mn(III) complexes 4a-c by reaction with
manganese(II) chloride under basic aerobic conditions. The dark-
emerald-green complexes 4a-c behave as simple lipophilic “organic”
molecules and were thus amenable to purification by flash chroma-
tography on silica gel, which provided analytical materials for
characterization and activity studies. The overall yield of this sequence
ranged from 31 to 56% (unoptimized) and is therefore quite
competitive with porphyrin and corrole syntheses, which require
the preparation of functionalized pyrrole and/or dipyrromethane
units followed by an often low-yield cyclization/oxidation step.18
Magnetic susceptibility measurements confirmed representative
complex 4b to be a high-spin d4 Mn(III) complex. 1H NMR spectra
of 4a in CD2Cl2 revealed broad, shifted peaks indicative of the
paramagnetic Mn(III) complex. Oxidation of 4a with m-CPBA in
CD2Cl2 afforded normal sharpened peaks characteristic of the
corresponding diamagnetic low-spin d2 Mn(V)O complex
(Figures S21 and S22 in the Supporting Information).21
a Conditions: (a) NBS, THF, 100%; (b) Boc2O, 4-DMAP, CH3CN,
95%; (c) 5% Pd(PPh3)4, Na2CO3/H2O, CH3OH, toluene, 70-89%;
(d) (CH2OH)2, KOH, 195 °C, 70-85%; (e) PhCHO, cat. TFA, 97-
99%; (f) p-chloranil, CH2Cl2; (g) BBr3, CH2Cl2; (h) MnCl2, CHCl3,
CH3OH, 2,6-lutidine, air, 68-79% for three steps (from f).
in Goldberg's elegant studies of highly functionalized Mn(V)O
corrolazines.21 For all three complexes 4a-c, the oxo species
were clearly observable as [M þ H]þ species using positive-ion-
mode LC-MS, confirming the formal oxidation state of
[Mn(V)O]3þ (accounting for the trianionic ligand set). Treat-
ment of the methanolic solutions of Mn(V)O species with 5
equiv of ascorbate in phosphate buffer (pH 7.2) resulted in the
apparent instantaneous conversion back to the green Mn(III)
form, effecting a possible reductase mode of action.
The results shown in Figure 2 are of interest only for identifying
the putative two-electron oxidation of 4b with subsequent study of
the Mn(V)O species 10b by spectroscopic methods. However, to
confirm that the complexes can indeed decompose peroxynitrite
more rapidly than its spontaneous decomposition at physiological
pH, a relevant rapid-throughput in vitro assay was sought.
Complexes 4a-c were therefore assayed for their peroxynitrite
decomposition activity by determining their ability to inhibit aryl
boronate oxidation.24 Oxidation of 4-acetylphenylboronic acid to
4-acetylphenol by peroxynitrite is a clean conversion devoid of any
observable intermediates, and the second-order rate constant for
this reaction has been accurately measured to be k = 1.6 ꢀ 106 M-1
s-1 using stopped-flow spectrophotometric methods.24 Inhibition
results for complexes 4a-cand 1arepresentedinTable1. Fromthe
percent inhibition values, we calculated the corresponding apparent
second-order rate constants for oxidation of the Mn(III) form to the
Mn(V)O form for 4a-c and the Mn(IV)O form for 1 at 25 °C in
phosphate buffer (pH 7.2). The numbers match up well with the
rate constants reported in the literature for the well-known complex
1.25 Thus, this assay provides a reliable method for the rapid in vitro
measurement of activity toward decomposition of peroxynitrite
prior to in vivo studies. Our data reveal that analogues 4a-c have
estimated apparent rate constants in the range of those observed for
manganese corrole systems.11 If the resulting Mn(V)O forms are
more rapidly reduced back to the Mn(III) resting state by endo-
genous reductants, the k values in Table 1 would be the catalytic rate
Treatment of green-colored methanolic solutions of the Mn-
(III) complexes 4a-c with excess peroxynitrite (in 0.1N NaOH)22
afforded the corresponding red-colored Mn(V)O intermediates
(Figure 2). In methanol solution, the Mn(V)O species persisted for
20-30 min and were stable enough to be confirmed by LC-MS
(Figure 2 inset) and UV-vis spectroscopy. The UV-vis spectral
changes are analogous to those observed during the oxidative
generation and subsequent reduction of Mn(V)O corroles23 and
4201
dx.doi.org/10.1021/ja110427e |J. Am. Chem. Soc. 2011, 133, 4200–4203

Journal of the American Chemical Society
COMMUNICATION
Table 1. Inhibition of Arylboronic Acid Oxidation
% inhibition
k (M-1 s-1 b
)
catalyst
LogPa
at 25 °C
at 25 °C
4a
4b
4c
1
þ4.18 ( 0.19
þ4.31 ( 0.02
þ4.05 ( 0.15
-4.54 ( 0.16
32.0 ( 2.40
25.3 ( 2.21
31.5 ( 2.50
62.4 ( 0.68
(7.5 ( 0.6) ꢀ 105
(5.4 ( 0.5) ꢀ 105
(7.3 ( 0.6) ꢀ 105
(2.6 ( 0.2) ꢀ 106, 1.8 ꢀ 106 c
a Octanol-water partition coefficients measured using the slow-stir meth-
od.27 b Apparent second-order rate constant for the oxidation of complex (1
equiv) by peroxynitrite (1 equiv) estimated from the % inhibition in
100 mM phosphate buffer (pH 7.2) with no secondary antioxidants added.
Inhibition data were determined by LC-MS after 1 min of reaction time.
c Second-order rate constant measured using stopped-flow methods.25
Figure 2. UV-vis spectral changes for 4b (33 μM, MeOH, 25 °C) after
treatment with 0-20 equiv of peroxynitrite (spectra were collected
every 2 min; see the Supporting Information). Center inset: samples of
4b and 10b. Right inset: LC-MS data ([M þ H]þ) for 10b.
constants for reductase-type activity.9 In that manganese corroles
have been shown to possess peroxynitrite decomposition activity
without the need for endogenous reductants,11 complexes 4a-c
may also operate via a similar catalytic cycle, as they possess a similar
trianionic ligand environment. Future mechanistic studies will
address this possible mode of catalysis.
Single-crystal X-ray analysis of 4b and its BODIPY analogue, the
synthetic precursor 9b, provided very interesting structural informa-
tion for this new ligand class (Figure 3). The BODIPY system 9b
shows significant distortion of the tetrahedral boron center, similar to
that observed for the related non-cyclohexano system.15 In the case of
9b, the O-B-O angle is 108.4°, but the N1-B-O2 and N2-B-
O1 angles are 114.4 and 113.1°, respectively. The N-B-N angle is
pinchedinwardto106.5°, most likely in response to the phenyl group
that bisects the planar dipyrromethene unit and therefore feels a close
steric interaction with the CH2 groups of the neighboring fused
cyclohexano rings. The Mn(III) complex 4b crystallized with co-
ordinated axial methanol molecules (from the CH2Cl2/MeOH
solution) to afford a Jahn-Teller-distorted octahedral array around
the manganese atom. The phenyldipyrromethene unit in 4b is
displaced upward relative to the equatorial plane, with the coordinat-
ing phenolate groups displaced slightly downward. The release in
strain associated with the distorted tetrahedron of 9b most likely
drives its direct conversion to 4b without the need for isolation of the
free phenol ligand. The two axial Mn-O(MeOH) bonds are
considerably longer than those in similar structures observed for
Mn(III) corroles (2.19 Å)11c and Mn(III) corrolazines (2.107 Å),21b
most likely because of the Jahn-Teller effect.26 The Mn-O3
bond distance is 2.226 Å and the longer Mn-O4 bond distance is
2.342 Å, possibly also influenced by steric interactions with the
saddled hydroxyphenyl groups.
Figure 3. Single-crystal structural analysis of BODIPY analogue 9b and
Mn(III) complex 4b.
(Figure 4). Nearly 100% inhibition for 2 h was seen, with a
substantial inhibitory effect maintained out to 5 h. At the
2 h time point, a similar degree of inhibition was observed with
the nonselective COX-1/COX-2 inhibitor ibuprofen at 300 mg/
kg (99 ( 5% inhibition, n = 6). Under the same conditions and at
300 mg/kg, acetaminophen or aspirin attenuated hyperalgesia by
20 ( 4% and 50 ( 6% respectively (n = 6, P < 0.05 relative to
carrageenan alone at the 2 h time point). Complexes 4b and 4c
were also shown to possess potent oral activity in this model. All
three new complexes have octanol-water partition coefficient
(LogP) values in the range of þ4, indicating high lipid solubility,
while 1 and related catalysts possess highly negative values
(Table 1).30 Both 4b and 4c were designed to divert or block
the potential metabolic hydroxylation para to the chelated phenol
In the well-known Hargreaves model,28 intraplantar injection of
carrageenan leads to the time-dependent development of thermal
hyperalgesia in rats, an inflammatory response known to be driven
by high levels of peroxynitrite flux (Figure 4).29 This hyperalgesic
effect was potently inhibited when 4a was given by oral gavage
4202
dx.doi.org/10.1021/ja110427e |J. Am. Chem. Soc. 2011, 133, 4200–4203

Journal of the American Chemical Society
COMMUNICATION
(5) Salvemini, D.; Neumann, W. L. Trends Pharmacol. Sci. 2009, 30, 194.
(6) Salvemini, D. Arch. Biochem. Biophys. 2009, 484, 238.
(7) Groves, J. T.; Marla, S. S. J. Am. Chem. Soc. 1995, 117, 9578.
(8) Stern, M. K.; Jensen, M. P.; Kramer, K. J. Am. Chem. Soc. 1996,
118, 8735.
(9) Lee, J.; Hunt, J. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 6053.
(10) Ferrer-Sueta, G.; Vitturi, D.; Batinic-Haberle, I.; Fridovich, I.;
Goldstein, S.; Czapski, G.; Radi, R. J. Biol. Chem. 2003, 278, 27432.
(11) (a) Mahammed, A.; Gross, Z. Angew. Chem., Int. Ed. 2006,
45, 6544. (b) Kupershmidt, L.; Okun, Z.; Amit, T.; Mandel, S.; Saltsman,
I.; Mahammed, A.; Bar-Am, O.; Gross, Z.; Youdim, M. B. J. Neurochem.
2010, 113, 363. (c) Gershman, Z.; Goldberg, I.; Gross, Z. Angew. Chem.,
Int. Ed. 2007, 46, 4320.
(12) (a) Okun, Z.; Kupershmidt, L.; Amit, T.; Mandel., S.; Bar-Am,
O.; Youdim, M. B. H.; Gross, Z. ACS Chem. Biol. 2009, 4, 910. (b)
Kanamori, A.; Catrinescu, M.-M.; Mahammed, A.; Gross, Z.; Levin, L. A.
J. Neurochem. 2010, 114, 488. (c) Haber, A.; Mahammaed, A.; Fuhrman,
B.; Volkova, N.; Coleman, R.; Hayek, T.; Aviram, M.; Gross, Z. Angew.
Chem., Int. Ed. 2008, 47, 7896.
(13) Drel, V. R.; Pacher, P.; Vareniuk, I.; Pavlov, I.; Ilnytska, O.;
Lyzogubov, V. V.; Tibrewala, J.; Groves, J. T.; Obrosova, I. G. Eur. J.
Pharmacol. 2007, 569, 48.
(14) Vareniuk, I.; Pavlov, I. A.; Drel, V. R.; Lyzogubov, V. V.;
Ilnytska, O.; Bell, S. R.; Tibrewala, J.; Groves, J. T.; Obrosova, I. G.
Exp. Neurol. 2007, 205, 425.
(15) Kim, H.; Burghart, A.; Welch, M. B.; Reibenspies, J.; Burgess, K.
Chem. Commun. 1999, 1889.
(16) Burghart, A.; Kim, H.; Welch, M. B.; Thoresen, L. H.; Reibenspies,
J.; Burgess, K. J. Org. Chem. 1999, 64, 7813.
(17) May, D. A., Jr.; Lash, T. D. J. Org. Chem. 1992, 57, 4820.
(18) (a) Lindsey, J. S.; Schreiman, I. C.; Hsu, H. C.; Kearney, P. C.;
Marguerettaz, A. M. J. Org. Chem. 1987, 52, 827. (b) Littler, B. J.;
Ciringh, Y.; Lindsey, J. S. J. Org. Chem. 1999, 64, 2864.
Figure 4. Time-dependent development of carrageenan-induced ther-
mal hyperalgesia in rats (n = 3) was blocked by oral administration of 4a
(100 mg/kg, n = 5) (n = number of animals). Results are expressed as
means ( standard errors of the mean and were analyzed by two-way
ANOVA with Bonferroni post hoc tests; ** denotes P < 0.05 and
** denotes P < 0.001 vs vehicle.
through methyl and fluoro substitution, respectively. Full SAR
studies incorporating further electron-donating and -withdrawing
functionalities will be the subject of future reports.
In conclusion, the results presented herein demonstrate the
modular synthesis of a new class of orally active Mn(III) com-
plexes that function as peroxynitrite scavengers. These studies suggest
that this new complex design may afford improved in vivo perfor-
mance through the two-electron reduction of peroxynitrite to nitrite.
(19) Parhi, A. K.; Kung, M. P.; Ploessl, K.; Kung, H. F. Tetrahedron
Lett. 2008, 49, 3395.
’ ASSOCIATED CONTENT
(20) Loudet, A.; Bandichhor, R.; Burgess, K.; Palma, A.; McDonnell,
S. O.; Hall, M. J.; O’Shea, D. F. Org. Lett. 2008, 10, 4771.
(21) Similar behavior was observed for the Mn(V)O corrolaz-
ines. See: (a) Mandimutsira, B. S.; Ramdhanie, B.; Todd, R. C.; Wang, H.;
Zareba, A. A.; Czernuszewicz, R. S.; Goldberg, D. P. J. Am. Chem. Soc. 2002,
124, 15170. (b) Lansky, D. E.; Mandimutsira, B. S.; Ramdhanie, B.; Clausen,
M.; Penner-Hahn, J.; Zvyagin, S. A.; Telser, J.; Kryzstek, J.; Zhan, R.; Ou, Z.;
Kadish, K. M.; Zakharov, L.; Rheingold, A. L.; Goldberg, D. P. Inorg. Chem.
2005, 44, 4485. (c) Goldberg, D. P. Acc. Chem. Res. 2007, 40, 626.
(22) Uppu, R. M.; Pryor, W. A. Anal. Biochem. 1996, 236, 242.
(23) Liu, H. Y.; Yam, F.; Xie, Y. T.; Li, X. Y.; Chang, C. K. J. Am.
Chem. Soc. 2009, 131, 12890.
S
Supporting Information. Experimental procedures; char-
b
acterization data; LC-MS, NMR, and UV-vis data; and crystal-
lographic data (CIF). This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
wneuman@siue.edu
’ ACKNOWLEDGMENT
(24) Sikora, A.; Zielonka, J.; Lopez, M.; Joseph, J.; Kalyanaraman, B.
Free Radical Biol. Med. 2009, 47, 1401.
Support of this research by the NIH (NIAMS, RC1AR058231)
is gratefully acknowledged. The authors also thank Dr. A. Michael
Crider of the SIUE School of Pharmacy and Dr. Michael Shaw of
the SIUE Department of Chemistry for many helpful discussions.
The authors thank the Donald Danforth Plant Science Center
Proteomics and Mass Spectrometry Group and the Washington
University Resource for Biomedical and Biological Mass Spectro-
metry for HRMS analyses. Funding from the National Science
Foundation (MRI, CHE-0420497) for the purchase of the Apex
II diffractometer is acknowledged.
(25) (a) Hunt, J. A.; Lee, J.; Groves, J. T. Chem. Biol. 1997, 4, 845.
(b) Differences in pH (7.2 vs 7.4) for the boronate assay vs the stopped-flow
experiments are in part responsible for the minor differences in the kinetic data.
(26) (a) Lieb, D.; Zahl, A.; Shubina, T. E.; Ivanovic-Burmazovic, I.
J. Am. Chem. Soc. 2010, 132, 7282. (b) Hoogenraad, M.; Ramkisoensing,
K.; Driessan, W. L.; Kooijman, H.; Spek, A. L.; Brouwman, E.; Haasnoot,
J. G.; Reedijk, J. Inorg. Chim. Acta 2001, 320, 117.
(27) Lodge, K. J. Chem. Eng. Data 1999, 4, 1321.
(28) For measurement of paw-withdrawl latency from a thermal
stimulus (paw withdrawl from a warm surface), see: Hargreaves, K.;
Dubner, R.; Brown, F.; Flores, C.; Joris, J. Pain 1988, 32, 77.
(29) Salvemini, D.; Wang, Z. Q.; Bourdon, D. M.; Stern, M. K.;
Currie, M. G.; Manning, P. T. Eur. J. Pharmacol. 1996, 303, 217.
(30) (a) Kos, I.; Benov, L.; Spasojevic, I.; Reboucas, J. S.; Batinic-
Haberle, I. J. Med. Chem. 2009, 52, 7868. (b) Batinic-Haberle, I.;
Ndengele, M. M.; Cuzzocrea, S.; Reboucas, J. S.; Spasojevic, I.; Salve-
mini, D. Free Radical Biol. Med. 2009, 46, 212.
’ REFERENCES
(1) Batinic-Haberle, I.; Reboucas, J. S.; Spasojevic, I. Antioxid. Redox
Signaling 2010, 13, 877.
(2) Ferrer-Sueta, G.; Radi, R. ACS Chem. Biol. 2009, 4, 161.
(3) Szabo, C.; Ischiropoulos, H.; Radi, R. Nat. Rev. Drug Discovery
2007, 6, 662.
(4) Salvemini, D.; Neumann, W. Life Sci. 2010, 86, 604.
4203
dx.doi.org/10.1021/ja110427e |J. Am. Chem. Soc. 2011, 133, 4200–4203