Protective effect of nitronyl nitroxide-amino acid conjugates on liver ischemia-reperfusion induced injury in rats

Article abstract of DOI:10.1016/j.bmcl.2008.02.030

Stable nitroxides are potential antioxidant drugs. In this study, we have linked nitroxide to natural amino acids with the aim to improve therapeutic activity. The radical scavenging activities of two nitronyl nitroxide-amino acid conjugates (NNR and NNK) were evaluated in PC 12 cell survival assays. The NO scavenging activities of these compounds were confirmed in the acetylcholine-induced vasorelaxation assay. In addition, the protective effect of NNR was demonstrated in an in vivo rat model of hepatic ischemia-reperfusion (I/R) induced injury and oxidative change. Because NNR reduced hepatic I/R injury by minimizing oxidative stress, it might be possible to develop it into a possible therapeutic agent for hepatic I/R injury.

Full text of DOI:10.1016/j.bmcl.2008.02.030

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1788–1794
Protective effect of nitronyl nitroxide–amino acid conjugates
on liver ischemia–reperfusion induced injury in rats
Wei Bi,a,* Jianhui Cai,a Ping Xue,a Yanrong Zhang,a Sanguang Liu,a Xiang Gao,a
a
a
c
b
b,
*
`
Meng Li, Zhibo Wang, Michele Baudy-Floc’h, Sarah A. Green and Lanrong Bi
aDepartment of Surgery, Second Hospital of HeBei Medical University, Shijiazhuang 050000, PR China
bDepartment of Chemistry, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA
cUMR 6226 CNRS, ICMV, Sciences Chimiques de Rennes, Universite´ de Rennes I, F-35042 Rennes Cedex, France
Received 18 October 2007; revised 8 February 2008; accepted 13 February 2008
Available online 16 February 2008
Abstract—Stable nitroxides are potential antioxidant drugs. In this study, we have linked nitroxide to natural amino acids with the
aim to improve therapeutic activity. The radical scavenging activities of two nitronyl nitroxide–amino acid conjugates (NNR and
NNK) were evaluated in PC 12 cell survival assays. The NO scavenging activities of these compounds were confirmed in the ace-
tylcholine-induced vasorelaxation assay. In addition, the protective effect of NNR was demonstrated in an in vivo rat model of hepa-
tic ischemia–reperfusion (I/R) induced injury and oxidative change. Because NNR reduced hepatic I/R injury by minimizing
oxidative stress, it might be possible to develop it into a possible therapeutic agent for hepatic I/R injury.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Hepatic ischemia/reperfusion (I/R) is a problem com-
monly encountered in many clinical conditions including
liver transplantation, hepatic failure after shock, and liver
surgery for trauma and cancer. To reduce hemorrhagic
complications, temporary clamping of the hepatic blood
supply is required. When the blood supply is restored,
the organ is usually subjected to a further insult, aggravat-
ing the injury created within the ischemic period. I/R in-
jury is a complex pathophysiological process involving
the reaction of oxygen-derived free radical, cytokines,
and neutrophils. This can eventually lead to cell damage,
cell death, increased vascular permeability, tissue necro-
sis, and multi-organ dysfunction.1–5 Reactive oxygen spe-
cies (ROS) are one of the most important components of
tissue injury after reperfusion of ischemic organs. The ma-
jor ROS include the superoxide and hydroxyl radicals,
and hydrogen peroxide. ROS induced injury targets en-
zymes and other proteins, lipids, nucleic acids, the cyto-
skeleton, and cell membranes, resulting in decreased
mitochondrial function, and lipid peroxidation.6,7
limit the effects of ROS but these systems can become
overwhelmed by large quantities of ROS. Recently, sev-
eral successful therapeutic strategies have been developed
to prevent liver tissue damage after I/R. These include
alternative clamping techniques as well as pharmacologi-
cal intervention with antioxidants.8 Enhancing the liver’s
antioxidant capacity might be a promising therapeutic
strategy to prevent I/R injury.9–16 More recently, various
antioxidants have been developed to counteract I/R in-
jury. For example, a-tocopherol, ascorbic acid, allopuri-
nol, coenzyme Q 10, and superoxide dismutase have
been reported to prevent hepatic I/R injury. It has been
suggested that the antioxidant therapy is a promising ap-
proach to ameliorate liver injury during I/R.17–20
Stable nitroxides have found a wide range of applications
in biology and medicine.21 As a unique class of antioxi-
dants, nitroxides have recently been explored as a comple-
mentary strategy for modulating oxidative injury. It has
been suggested that nitroxides possess potential therapeu-
tic benefits in a variety of diseases including I/R injury.22–28
They have been shown to attenuate oxidative damage in
various experimental models.26–28 The protective effects
of nitroxides can be attributed to their antioxidant capac-
ities. In addition to directly scavenging free radical, nitr-
oxides have also been shown to attenuate the formation
of other reactive oxygen and nitrogen species.29–36
Endogenous antioxidant compounds, such as superoxide
dismutase, catalase, glutathione, and b-carotene, may
Keywords: Free radical scavenger; Ischemia–reperfusion injury.
*
Corresponding authors. Tel.: +1 906 487 1868; fax: +1 906 487 2061
(L.B.); e-mail: lanrong@mtu.edu
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.030

W. Bi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1788–1794
1789
The cellular and in vivo pharmacology of stable nitrox-
ides has been investigated in previous studies.31,32 It has
been reported that nitroxides can be reduced in vivo to
hydroxylamines or oxidized to oxo-ammonium cations
via electron transfer reactions. Thus, all these three
forms (nitroxide radical, oxo-ammonium cation,
hydroxylamine) can be present in the tissue.31,32 In addi-
tion, it was also suggested that the hydroxylamine and
oxo-ammonium cation might com-proportionate, yield-
ing two nitroxide molecules. In other words, the non-
radical species might also com-proportionate to yield
the more stable radical form and thus the nitroxides
can replenish itself.31–35 As a result, unlike antioxidants
that act in a sacrificial mode, nitroxides can provide pro-
tection in a catalytic way. Through the continuous ex-
change between the two forms they can act as self-
replenishing antioxidants thus bestow catalytic protec-
tive activity (Fig. 1). This key feature indicates the po-
tential of this unique class of antioxidants against I/R
injury.
displayed significantly protective effects against liver I/R
injury. The detailed synthesis and structure–activity
relationship studies of those newly synthesized nitronyl
nitroxides–amino acid conjugates will be reported else-
where. In the present study, we report the synthesis
and biological studies of two nitronyl nitroxide–amino
acid (Arg,Lys) conjugates (NNR and NNK) (Fig. 2).
Moreover, the protective effect of the newly synthesized
nitronyl nitroxide–amino acid conjugate (i.e., NNR) on
hepatic I/R induced tissue damage was evaluated by
the measurement of biomedical parameters: serum
aspartate aminotransferase (AST) (a nonspecific marker
for hepatic injury), alanine aminotransferase (ALT) (a
specific marker for hepatic parenchyma injury) and liver
malondialdehyde (MDA) levels (an end product of lipid
peroxidation).
The nitronyl nitroxide derivatives were synthesized
according to Ullman’s procedure with minor modifica-
tion as shown in Scheme 1.53 Synthesis was initiated
with dinitro compound 1, followed by reduction with
zinc in an ammonium chloride buffered solution to yield
the key intermediate bis(hydroxyamine) compound 2
(63% yield), which was subsequently subjected to con-
densation with 4-hydroxyl-benzaldehyde to generate
the corresponding dihydroxy-imidazolidine NN-3 (51%
yield). Oxidation by PbO2 gave the nitronyl nitroxide
derivative NN-4 (52% yield). Subsequent treatment of
NN-4 with BrCH2COOC2H5 followed by hydrolysis in
the presence of NaOH provided NN-6 (90% yield).
Contrary to exogenously added SOD or catalase and
several common antioxidants, nitroxides readily cross
the blood–brain barrier and permeate the cell mem-
brane. Thus, nitroxides would seem to have unique ther-
apeutic potential for diseases and injuries related to
oxidative stress.36–46 In addition, it has been reported
that some amino acids can be useful in preventing oxida-
tive damage during surgery.47–58 For example, pretreat-
ment with L-arginine has a protective effect on multiple
organs I/R injury.51,52 To develop new therapeutic
agents against hepatic ischemia–reperfusion injury, we
sought to link an antioxidant moiety (nitronyl nitroxide)
to a series of amino acids in the hope that the resulting
nitronyl nitroxide–amino acid conjugates would provide
a synergistically protective effect for hepatic ischemic-
reperfusion injury. It turned out that some of these new-
ly synthesized nitronyl nitroxides–amino acid conjugates
Synthesis of nitronyl nitroxide–amino acid conjugates
was straightforward, involving a simple incorporation
of the amino acid residue via the phenyloxyacetyl moi-
ety on the 2-position of the imidazolyl ring. After the
coupling and deprotection, the target compounds,
NNR and NNK, were obtained in moderate to good
yield (73–90%).
ESR spectroscopy is the best tool for the study of free
radical, due to the sensitivity and accuracy of the meth-
od. As shown in Figure 3, different types of radicals
show different patterns in the ESR spectra.
N
R
+
OOH+ H
N
O
O2
N
The NO trapping ability of NNR and NNK were moni-
tored by the direct trapping experiment, as shown in
Figure 3. It was observed that the initial spectrum of
the nitronyl nitroxide–amino acid conjugates (NNR
and NNK) had five lines (with an intensity ratio of 1/2/
3/2/1 and the same aN coupling constants). When NO
gas was bubbled into the solution of NNR and NNK
in deaerated phosphate buffer, the ESR spectrum was
R
N
H+
H+
O2
O2
+
N
O
R
N
+
HO
Figure 1. Schematic representation of the redox transformation of
nitroxide, hydroxylamine, and the oxo-ammonium cation.
HN
NH2
NH
NH2
O
O
N
Me
Me
Me
Me
Me
Me
O
O
N
O
O
Me
Me
N
H
COOH
N
H
COOH
N
O
N
O
NNK
NNR
Figure 2. Structure of nitronyl nitroxide–amino acid conjugates (NNR and NNK).

1790
W. Bi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1788–1794
OH
N
Me
Me
Me
Me
Me
Me
HO
Me
Me
HN NH
NO2
Me
Me
NO2
Me
Me
O2N
i
ii
iii
OH
H3C
CH3
OH
N
OH
NN-3
1
2
O
N
O
N
Me
Me
Me
Me
Me
Me
vi
iv
v
OCH2CO2C2H5
OH
Me
Me
N
O
N
O
NN-4
NN-5
HN
NH2
NH
O
O
Me
Me
Me
Me
vii
N
viii
O
NNR
N
H
COOCH3
O
N
N
O
Me
Me
NN-7
OCH2CO2H
NH Cbz
Me
Me
N
O
O
N
O
Me
Me
Me
Me
NN-6
O
x
ix
NNK
N
COOBz
H
N
O
NN-8
Scheme 1. Synthesis of nitronyl nitroxide–amino acid conjugates (NNR and NNK). Reagents: (i) Br2, NaOH (6 mol/L); (ii) Zn, NH4Cl; (iii) 4-
hydroxyl-benzaldehyde/MeOH; (iv) PbO2; (v) BrCH2COOC2H5, NaOEt, THF; (vi) NaOH (2 mol/L); (vii) HClÆArg-OMe, DCC, HOBt, and NMM,
pH 9; (viii) NaOH (2 mol/L); (ix) HClÆLys(Z)-OBzl, DCC, HOBt, and NMM, pH 9; (x) trifluoroacetic acid/trifluoro-methylsulfonic acid (4:1).
EC50 (lM) values are summarized in Table 1. It was ob-
served that NNR and NNK exhibited comparable scav-
enging capacity with their parent compound NN
(nitronyl nitroxide) toward different active radical spe-
cies. Compared with NN, the differences were not statis-
tically significant.
Figure 3. ESR spectra of NNR at 10À5 mol/L in phosphate buffer (pH
7.4), prior to and after the introduction of NO. (A) ESR spectra of the
nitronyl nitroxide derivative, (B) ESR spectra of the imino-nitroxide.
The NO scavenging activities of NNR and NNK were
further evaluated in the acetylcholine (Ach)-evoked,
endothelium-medicated relaxation assay.53,56 The results
expressed as the percentage inhibition of acetylcholine
(Ach)-induced vasorelaxation by the test compounds
are summarized in Table 2.
O
N
N
R
NO
+
NO2
R
N
N
O
O
The animal experiments were performed in compliance
with the ‘Guide for the Care and Use of Laboratory
Animals’ published by the US National Institutes of
Health. Male Wistar rats were randomly allocated into
three groups: (1) Sham group: rats subjected to the sur-
gical procedures (see Supporting Information), except
for liver I/R (n = 4); (2) I/R group: rats subjected to
Scheme 2. The reaction of nitronyl nitroxides radical with NO.
changed to a seven-line pattern (with an intensity ratio
1/1/2/1/2/1/1 and different aN coupling constants). This
is due to an electron interacting with two inequivalent
nitrogens. Imino nitroxides are produced by the
reaction between nitronyl nitroxide derivatives and
NO (Scheme 2).
Table 1. Free radical scavenging activities of NNR and NNK
Compound
EC50 (lM) values ( SD)*
NO
H2O2
ÅOH
Å
Various free radicals are formed during the ischemia and
reperfusion phases. The free radical scavenging proper-
ties of NNR and NNK against NO, H2O2Æ and ÆOH were
evaluated using PC 12 cell survival assay following the
published method with minor modifications.55 The
NN
NNR
NNK
90.1 2.03
92.8 2.95
85.5 4.64
32.0 2.69
49.0 2.68
26.8 3.66
57.2 2.41
98.9 3.12
92.7 3.06
* SD, standard deviation, n = 6; NN = nitronyl nitroxide.

mg). Treatment with L
-Arg or NNR reversed the eleva-
W. Bi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1788–1794
Table 2. Inhibition of Ach-induced vasorelaxation
1791
Inhibition percentage (XÆSD%)*
tions in the MDA level. The MDA level in the NNR +
I/R group (2.76 0.74 nmol/mg) was lower than that
in the I/R (4.86 2.43 nmol/mg) and I/R + L-Arg treat-
ment groups (3.82 1.79 nmol/mg), but the differences
were not statistically significant.
Compound
100 lmol
10 lmol
1 lmol
NS
NN
1.63 1.42
8.6 3.2a
74.0 3.7a,b
60.7 5.5a,b
31.0 2.6a
98.9 4.9a,b
85.0 5.3a,b
1.8 1.4
28.7 5.4a
24.8 5.6a
NNR
NNK
Serum ALT level was 1530 750 U/L in the I/R group,
which is much higher than that in the sham-operated
control group (52 4 U/L, P < 0.001). The ALT level
in the NNR + I/R group was 173 50 U/L, which was
significantly lower than that of the I/R group
(P < 0.05) but still higher than that in the sham group.
The serum AST level was 1894 875 U/L in the I/R
group, which was much higher than that in the sham
group (182 30 U/L, P < 0.001). Compared with the
I/R group, after NNR treatment, ASL level of
NNR + I/R group was dramatically reduced (510
138 U/L, P < 0.05), but still much higher than that of
the sham-operated control group. Although the ALT
and AST levels in the NNR treatment group were lower
than that in the L-Arg treatment group, the difference
was not statistically significant.
a Compared with NS (normal saline), P < 0.001.
b Compared with NN (nitronyl nitroxide), P < 0.001.
* SD, standard deviation, n = 6.
the surgical procedures underwent liver ischemia for
30 min followed by reperfusion for 2 h (n = 8); (3) I/
R + drug treatment group: rats received NNR (30 mg/
kg) or L-Arg (30 mg/kg) just 10 min before the reperfu-
sion and again 1 h after initiation of reperfusion
(n = 8). Animals in groups (1) and (2) were adminis-
trated saline solution in place of drug. All animals were
maintained under anesthesia (sodium pentobarbital,
80 mg/kg) for the duration of the experiment (i.e.,
30 min + 2 h). At the end of the animal experiments, rats
were sacrificed by a sodium pentobarbital overdose.
Blood and liver samples were collected immediately,
and frozen at À70 ꢁC until analysis.
Tissue sections (4–5 lm) were made using a cryostat
microtome (Leica CM1850 UV clinical cryostat) at
À30 ꢁC, and were stained with hematoxylin–eosin and
examined under a light microscope (Olympus-BX51).
The steady-state level of malondialdehyde (MDA),
which is the end product of lipid peroxidation, in the li-
ver mitochondria was determined by measuring the level
of thiobarbituric acid reactive substances spectrophoto-
metrically according to the Buege and Aust method.60
The serum aspartate aminotransferase (AST) and ala-
nine aminotransferase (ALT) levels were measured to
assess liver function. The results are presented in Tables
3 and 4.
ESR spectrometry revealed the existence of unpaired
electrons, suggesting that NNR and NNK were of the
same free radical characteristics as their parent com-
pound, nitronyl nitroxide (NN). In addition, it was
shown that they are reactive with NO as demonstrated
by the ESR results (Scheme 2 and Fig. 3). Likewise,
NNR and NNK retained the free radical scavenging
activity of NN against NO, H2O2Æ and ÆOH as demon-
strated in the PC 12 cells survival assay (Table 1).
There was a significant increase in MDA level in the I/R
group (4.86 2.43 nmol/mg, P < 0.05) compared with
the sham-operated control group (1.75 0.26 nmol/
Rat pheochromocytoma (PC 12) cells, originated from
the adrenal medulla, synthesize and release catechola-
mines. These cells are very sensitive to oxidative stress.
PC 12 cells model system has been established for
in vitro ischemia studies.55 Cell survival as determined
by MTT reduction was markedly decreased after PC
12 cells were exposed to free radical. In our present
study, pre-incubation of PC 12 cells with NNR/NNK,
or the incubation with the test compounds after expo-
sure to free radical, both methods could prevent the
reduction of viability caused by free radical. However,
when the cells were pre-incubated with the test com-
pounds, free radical induced cell toxicity was signifi-
cantly attenuated. This presumably was due to a
synergetic effect between antioxidant activity and mem-
brane permeability. Free radical accumulation leads to
cellular oxidative stress. Therefore, the elimination of
free radical is critical for reducing oxidative stress.
Decreasing PC 12 cells viability was suppressed when
cells incubated with NNR/NNK. These results indicate
that NNR and NNK have good scavenging capacity to-
ward different active radical species. One possible mech-
anism underlying the effectiveness of NNR/NNK against
cellular death induced by free radical involved their nitr-
Table 3. MDA levels in the hepatic tissues of sham, ischemia/
reperfusion (I/R), I/R + L-Arg and I/R + NNR groups
MDA (nmol/mg protein)
Sham
I/R
1.75 0.26
4.86 2.43a
3.82 1.79a
2.76 0.74a
I/R + L-Arg
I/R + NNR
a P < 0.05 compared with sham group; n = 8.
Table 4. Serum levels of ALT and AST in sham, ischemia/reperfusion
(I/R), I/R + L-Arg and I/R + NNR groups of rats
AST (U/L)
ALT (U/L)
52
Sham
I/R
182 30
4
1894 875a
789 214b,c
510 138b,c
1530 750a
493 180b,c
173 50b,c
I/R + L-Arg
I/R + NNR
a P < 0.001 compared with sham group.
b P < 0.05 compared with sham group.
c P < 0.05 compared with I/R group; n = 8.

1792
W. Bi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1788–1794
oxide structures since it is known that nitroxides are po-
tent antioxidants and free radical scavengers. Alterna-
tively, a possible direct scavenging of free radical by
NNR/NNK during the incubation period cannot be ex-
cluded. On the other hand, we speculated NNR/NNK
might preferentially associate with the plasma mem-
branes at the cellular surface.
cepted as a criterion of tissue injury. Interestingly, NNR
treatment caused a significant inhibition in MDA pro-
duction (Table 3). This decrease indicated that lipid per-
oxidation of liver tissue and cellular injury was reduced.
This protective effect of NNR is most likely due to its
ability to scavenge the very reactive hydroxyl and per-
oxyl radicals thereby limiting hepatic injury.
The endothelium controls the tone of the underlying vas-
cular smooth muscle through the production of vasodila-
tor mediators. In Ach-induced relaxation of the rat
thoracic aorta assay, Ach acts on the endothelium to elicit
the release of nitric oxide (NO), a potent vasodilator.53,56
A decreased relaxation response in the rat aortic strip
could be attributed to a reduction in NO synthesized by
the endothelium. Therefore, this assay was undertaken
to assess the NO scavenging capability of NNR and
NNK. Nitronyl nitroxide (NN) was found to be a weak
inhibitor of Ach-induced vasorelaxation, whereas Ach-
induced relaxation was significantly reversed by NNR
and NNK. At the concentration of 10 lmol, compared
to NN (8.6 3.2%), NNR and NNK showed good inhibi-
tion ability with 74.0 3.7% and 60.7 5.5%, respec-
tively (Table 2). We speculate that the nitric oxide
scavenging activity of NNR and NNK led to attenuation
of the NO concentration in vitro, thereby leading to inhi-
bition of Ach-induced vasorelaxation.
Serum aminotransferase activities have long been re-
garded as indicators of hepatic injury.63 Damage to
the hepatocytes alters their transport function and mem-
brane permeability, leading to leakage of enzymes from
the cells.64 Therefore, the marked release of AST and
ALT into the circulation indicates severe damage to he-
patic tissue membranes during the reperfusion process.
Compared with sham-operated rats, the I/R of the liver
resulted in significant increases in AST and ALT levels
(Table 4), demonstrating development of hepatic cellu-
lar injury. In hepatic I/R injury, it has been reported that
excessive amounts of free radical are generated in the
early phase of reperfusion. To obtain an optimal thera-
peutic effect, NNR was administrated just before the
reperfusion and again 1 h after initiation of reperfusion.
We found that NNR administrated 10 min before and
again 1 h after initiation of reperfusion caused a sub-
stantial reduction in the I/R induced increase in ALT
and AST (Table 4).
Oxygen radical formation is harmful for biomolecules
such as nucleic acids, membrane lipids, enzymes, and
receptors. Oxygen radicals tend to attack the mem-
brane-associated polyunsaturated fatty acids and to
form lipid peroxides.57–59 Peroxidation of membrane lip-
ids can disrupt membrane fluidity and cell compartmen-
tation, which can further lead to cell lysis. In fact, lipid
peroxidation is implicated in the pathogenesis of various
liver injuries and subsequent liver fibrogenesis in experi-
mental animals and humans.57–59 MDA is a major reac-
tive aldehyde that appears during the peroxidation of
biological membrane polyunsaturated fatty acids. There-
fore, the hepatic content of MDA can be used as an indi-
cator of liver tissue damage.60 In this study, hepatic I/R
caused a significant increase in MDA (Table 3). This
observation is in agreement with previous studies.61,62
Tissue damage may result in lipid peroxidation and
necrosis, and the increased tissue MDA levels can be ac-
From the histological results, the sham group revealed
regular morphology of liver parenchyma with intact
hepatocytes and sinusoids (Fig. 4A). In the hepatic I/R
group, there was severe sinusoidal congestion and hemor-
rhage, the enlarged central vein, subendothelial edema,
and degenerated hepatocytes with perinuclear vacuoliza-
tion (Fig. 4B). In the I/R + NNR treatment group, there
was moderate sinusoidal dilatation, and the central vein
and hepatocytes appeared normal in most areas
(Fig. 4C). The histological results indicated that, although
the congestion, necrosis, and hepatocellular changes were
still observed in the hepatic I/R + NNR treatment groups,
the histological improvement was prominent with NNR
treatment in the hepatic I/R + NNR group.
In the present study, it was confirmed that L-Arginine
could reduce lipid peroxidation thereby ameliorating he-
patic ischemia–reperfusion injury, which was in accor-
Figure 4. Histological analysis of livers. Representative photographs (original magnification: 200·) were taken from rat livers 2 h after treatment
with sham-operation (Sham) (A), hepatic I/R (I/R) (B), and hepatic I/R + treatment with NNR (C). N = 8.

W. Bi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1788–1794
1793
dance with the previous reports.65 Our present results
indicate that NNR administered before the onset of
the reperfusion and again 1 h after initiation of reperfu-
sion significantly reduced the liver injury after the I/R.
Liver ischemic-reperfusion injury pathogenesis is multi-
factorial. The effects of other factors maybe involved
in the pathogenesis of liver I/R injury, therefore, the pre-
cise acting mechanism of these newly synthesized nitro-
nyl nitroxide–amino acid conjugates should be further
explored and the interaction between the different path-
ways also should be considered in our further studies.
16. Gross, E. R.; Gross, G. J. Expert Opin. Emerg. Drugs
2007, 12, 367.
17. Glantzounis, G. K.; Yang, W.; Koti, R. S.; Mikhailidis, D.
P.; Seifalian, A. M.; Davidson, B. R. Curr. Pharm. Des.
2006, 12, 2891.
18. Cesaratto, L.; Vascotto, C.; Calligaris, S.; Tell, G. Ann.
Hepatol. 2004, 3, 86.
19. Hines, I. N.; Harada, H.; Wolf, R.; Grisham, M. B. Curr.
Med. Chem. 2003, 10, 2661.
20. Amersi, Farin; Nelson, Sally K.; Shen, Xiu Da; Kato,
Hirohisa; Melinek, Judy; Kupiec-Weglinski, Jerzy W.;
Horwitz, Lawrence D.; Busuttil, Ronald W.; Horwitz,
Marcus A. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 8915.
21. Soule, B. P.; Hyodo, F.; Matsumoto, K.; Simone, N. L.;
Cook, J. A.; Krishna, M. C.; Mitchell, J. B. Free Radic.
Biol. Med. 2007, 42, 1632.
Acknowledgments
22. Fink, M. P.; Macias, C. A.; Xiao, J.; Tyurina, Y. Y.;
Delude, R. L.; Greenberger, J. S.; Kagan, V. E.; Wipf, P.
Crit. Care Med. 2007, 35, S461.
23. Soule, B. P.; Hyodo, F.; Matsumoto, K. I.; Simone, N. L.;
Cook, J. A.; Krishna, M. C.; Mitchell, J. B. Antioxid.
Redox. Signal. 2007 (Epub ahead of print).
24. Zeltcer, G.; Berenshtein, E.; Kitrossky, N.; Chevion, M.;
Samuni, A. Free Radic. Biol. Med. 2002, 32, 912.
25. Steinbauer, M.; Guba, M.; Buchner, M.; Farkas, S.;
Anthuber, M.; Jauch, K. W. Shock 2000, 14, 163.
26. Udassin, R.; Haskel, Y.; Samuni, A. Gut 1998, 42, 623.
27. Martin, J.; Lutter, G.; Sarai, K.; Senn-Grossberger, M.;
Takahashi, N.; Bitu-Moreno, J.; Haberstroh, J.; Beyers-
dorf, F. Eur. J. Cardiothorac. Surg. 2001, 19, 321.
28. Kato, N.; Yanaka, K.; Hyodo, K.; Homma, K.; Nagase,
S.; Nose, T. Brain Res. 2003, 979, 188.
29. Zhang, R.; Pinson, A.; Samuni, A. Free Radic. Biol. Med.
1998, 24, 66.
30. Shankar, R. A.; Hideg, K.; Zweier, J. L.; Kuppusamy, P.
J. Pharmacol. Exp. Ther. 2000, 838.
31. Krishna, M. C.; Grahame, D. A.; Samuni, A.; Mitchell,
J. B.; Russo, A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89,
5537.
This work is dedicated to Professor Nicholas J. Turro
on the occasion of his 70th birthday with all best wishes
for his future endeavors. The authors are grateful to
China Natural Science Foundation of HeBei Province
(No. 062761266), Administration of Traditional Chinese
Medicine of HeBei Province (No. 2007134), and Second
Hospital of HeBei Medical University for support of
this research. The authors are also grateful to Dr. Mi-
chael R. Neuman and Dr. James Russo for many helpful
discussions, and the Department of Chemistry at MTU
for funding.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2008.02.030.
References and notes
32. Krishna, M. C.; Russo, A.; Mitchell, J. B.; Goldstein, S.;
Dafni, H.; Samuni, A. J. Biol. Chem. 1996, 271, 26026.
33. Cighetti, G.; Allevi, P.; Debiasi, S.; Paroni, R. Chem.
Phys. Lipids 1997, 88, 97.
1. Urakami, H.; Abe, Y.; Grisham, M. B. Clin. Exp.
Pharmacol. Physiol. 2007, 34, 912.
2. Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T.;
Mazur, M.; Telser, J. Int. J. Biochem. Cell Biol. 2007, 39,
44.
34. Mitchell, J. B.; DeGraff, W.; Kaufman, D.; Krishna, M.
C.; Samuni, A.; Finkelstein, E. Arch. Biochem. Biophys.
1991, 289, 62.
3. Brady, N. R.; Hamacher-Brady, A.; Westerhoff, H. V.;
Gottlieb, R. A. Antioxid. Redox Signal. 2006, 8, 1651.
4. Galaris, D.; Barbouti, A.; Korantzopoulos, P. Curr.
Pharm. Des. 2006, 12, 2875.
35. Mohsen, M.; Pinson, A.; Zhang, R.; Samuni, A. Mol. Cell.
Biochem. 1995, 145, 103.
36. Monti, E.; Cova, D.; Guido, E.; Morelli, R.; Oliva, C. Free
Radic. Biol. Med. 1996, 21, 463.
5. Zweier, J. L.; Talukder, M. A. Cardiovasc. Res. 2006, 70,
181.
37. Hahn, S. M.; Krishna, C. M.; Mitchell, J. B. Adv. Exp.
Med. Biol. 1994, 366, 241.
6. Wiseman, A. Med. Hypotheses 2006, 66, 329.
7. Jaeschke, H. Chem. Biol. Interact. 1991, 79, 115.
8. Fan, C.; Zwacka, R. M.; Engelhardt, J. F. J. Mol. Med.
1999, 77, 577.
38. Samuni, A. M.; DeGraff, W.; Krishna, M. C.; Mitchell, J.
B. Mol. Cell. Biochem. 2002, 234-235, 327–333.
39. Gelvan, D.; Saltman, P.; Powell, S. R. Proc. Natl. Acad.
Sci. U.S.A. 1991, 88, 4680.
9. Zhou, W.; Zhang, Y.; Hosch, M. S.; Lang, A. R.; Zwacka,
M.; Engelhardt, J. F. Hepatology 2001, 33, 902.
10. Muller, M. J.; Vollmar, B.; Friedl, H. P.; Menger, M. D.
Free Radic. Biol. Med. 1996, 21, 189.
11. Yabe, Y.; Nishikawa, M.; Tamada, A.; Takakura, Y.;
Hashida, M. J. Pharmacol. Exp. Ther. 1999, 289, 1176.
12. Yabe, Y.; Kobayashi, N.; Nishihashi, T., et al. J. Phar-
macol. Exp. Ther. 2001, 298, 894.
40. Oliver, C. N.; Starke-Reed, P. E.; Stadtman, E. R.; Liu, G.
J.; Carney, J. M.; Floyd, R. A. Proc. Natl. Acad. Sci.
U.S.A. 1990, 87, 5144.
41. Aronovitch, Y.; Godinger, D.; Israeli, A.; Krishna, M. C.;
Samuni, A.; Goldstein, S. Free Radic. Biol. Med. 2007, 42,
1317.
42. Dragutan, I.;Mehlhorn, R. J. FreeRadic. Res. 2007, 41, 303.
43. Gariboldi, M. B.; Terni, F.; Ravizza, R.; Meschini, S.;
Marra, M.; Condello, M.; Arancia, G.; Monti, E. Free
Radic. Biol. Med. 2006, 40, 1409.
13. Seal, J. B.; Gewertz, B. L. Ann. Vasc. Surg. 2005, 19,
572.
14. Siemionow, M.; Arslan, E. Microsurgery 2004, 24, 468.
15. Hines, I. N.; Harada, H.; Wolf, R.; Grisham, M. B. Curr.
Med. Chem. 2003, 10, 2661.
44. Schubert, R.; Erker, L.; Barlow, C.; Yakushiji, H.;
Larson, D.; Russo, A.; Mitchell, J. B.; Wynshaw-Boris,
A. Hum. Mol. Genet. 2004, 13, 1793.

1794
W. Bi et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1788–1794
45. Kinugasa, Y.; Ogino, K.; Furuse, Y.; Shiomi, T.; Tsutsui,
H.; Yamamoto, T.; Igawa, O.; Hisatome, I.; Shigemasa,
C. Circ. J. 2003, 67, 781.
46. Lanteri, R.; Acquaviva, R.; Di Giacomo, C.; Caltabiano,
R.; Li Destri, G.; Vanella, L.; Santangelo, M.; Lanzafame,
S.; Di Cataldo, A. Microsurgery 2006, 26, 25.
47. Fotiadis, C.; Adamis, S.; Misiakos, E. P.; Genetzakis, M.;
Antonakis, P. T.; Tsekouras, D. K.; Gorgoulis, V. G.;
Zografos, G. C.; Papalois, A.; Fotinou, M.; Perrea, D.
Acta Chir. Belg. 2007, 107, 192.
48. Spanos, C. P.; Papaconstantinou, P.; Spanos, P.; Kara-
mouzis, M.; Lekkas, G.; Papaconstantinou, C. J. Gastro-
intest. Surg. 2007, 11, 247.
49. Schuster, H.; Blanc, M. C.; Neveux, N.; Bonnefont-
Rousselot, D.; Le Tourneau, A.; De Bandt, J. P.; Cynober,
L. Scand. J. Gastroenterol. 2006, 41, 1342.
50. Chander, V.; Chopra, K. J. Surg. Res. 2005, 128, 132.
51. Burra, P.; Ferraresso, M.; Cadrobbi, R., et al. Transplant.
Proc. 1997, 29, 2992.
73, 2424; (d) Boniece, I. R.; Wagner, J. A. J. Neurosci.
1993, 13, 4220; Boniece, I. R.; Wagner, J. A. J. Neurosci.
Res. 1995, 40, 1; (e) Mosmann, T. J. Immunol. Methods
1983, 65, 55.
56. Cowart, M.; Kowaluk, E. A.; Daanen, J. F.; Kohlhaas, K.
L.; Alexander, K. M.; Wagenaar, F. L.; Kerwin, J. F.
J. Med. Chem. 1998, 41, 2636.
57. Paradis, V.; Kollinger, M.; Fabre, M.; Holstege, A.;
Poynard, T.; Beddosa, P. Hepatology 1997, 26, 135.
58. Liu, J. Y.; Chen, C. C.; Wang, W. H.; Hsu, J. D.; Yang,
M. Y.; Wang, C. J. Food Chem. Toxicol. 2006, 44, 336.
59. Vaca, C. E.; Wilhelm, J.; Harms-Rihsdahl, M. Mutat. Res.
Rev. Genet. Toxicol. 1988, 195, 137; Ohkawa, H.; Ohishi,
N.; Yagi, K. Anal. Biochem. 1979, 95, 351.
60. Buege, J. A.; Aust, S. D. Methods Enzymol. 1978, 52, 302.
61. Omar, R.; Nomikos, I.; Piccorrelli, G.; Savino, J.; Agar-
wal, N. Gut 1989, 30, 510.
62. Sumimoto, K.; Oku, J.; Dohi, K.; Kawasaki, T. Trans-
plant. Proc. 1990, 22, 2023.
52. Nilsson, B.; Yoshida, T.; Delbro, D.; Andius, S.; Friman,
S. Transplant. Proc. 1997, 29, 3111.
63. Molander, D. W.; Wroblewsk, F.; La Due, J. S. Clin. Res.
Proc. 1955, 3, 20.
53. Wu, Y.; Bi, L.; Bi, W.; Li, Z.; Zhao, M.; Wang, C.; Ju, J.;
Peng, S. Bioorg. Med. Chem. 2006, 14, 5711.
54. Shimono, S.; Tamura, R.; Ikuma, N.; Takimoto, T.;
Kawame, N.; Tamada, O.; Sakai, N.; Matsuura, H.;
Yamauchi, J. J. Org. Chem. 2004, 69, 475.
55. (a) Desole, M. S.; Sciola, L.; Sircana, S.; Godani, C.;
Migheli, R.; Delogu, M. R.; Piras, G.; De, N. G.; Miele, E.
Neurosci. Lett. 1998, 247, 1; (b) Hillion, J.; Takahashi, K.;
Maric, D.; Ruetzler, C.; Barker, J.; Hallenbeck, J.
J. Cereb. Blood Flow Metab. 2005, 25, 154; (c) Greene,
L. A.; Tischler, A. S. Proc. Natl. Acad. Sci. U.S.A. 1976,
64. Zimmerman, H. J.; Seeff, L. B. Enzymes in Hepatic
Disease. In Diagnostic Enzymology; Goodley, E. L., Ed.;
Lea and Febiger: Philadelphia, 1970; p 1.
65. Shiraishi, Y.; Lee, J. R.; Laks, H.; Waters, P. F.;
Meneshian, A.; Blitz, A.; Johnson, K.; Lam, L.; Chang,
P. Ann. Thorac. Surg. 1996, 62, 1580; Nakanishi, K.;
Vinten-Johansen, J.; Lefer, D. J.; Zhao, Z.; Fowler, W. C.,
3rd; McGee, D. S.; Johnston, W. E. Am. J. Physiol. Heart
Circ. Physiol. 1992, 263, H1650; Lass, A.; Suessenbacher,
A.; Wolkart, G.; Mayer, B.; Friedrich, B. Mol. Pharmacol.
2002, 61, 1081.