Discovery of 6-[4-(6-nitroxyhexanoyl)piperazin-1-yl)]-9H-purine, as pharmacological post-conditioning agent

Article abstract of DOI:10.1016/j.bmc.2012.07.037

Novel purine analogues bearing nitrate esters were designed and synthesized in an effort to develop compounds triggering endogenous cardioprotective mechanisms such as ischemic preconditioning (IPC) or postconditioning (PostC). The majority of the compounds reduced infarct size compared to the control group in anesthetized rabbits, whereas administration of the most active analogue 16 at a dose of 3.8 μmol/kg resulted on a significant reduction of infarct size, compared to PostC group (13.4 ± 1.9% vs 26.4 ± 2.3%). These findings introduce a novel class of promising pharmacological compounds that could be used as mimics or enhancers of PostC.

Full text of DOI:10.1016/j.bmc.2012.07.037

Bioorganic & Medicinal Chemistry 20 (2012) 5948–5956
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of 6-[4-(6-nitroxyhexanoyl)piperazin-1-yl)]-9H-purine,
as pharmacological post-conditioning agent
Maria Koufaki a, , Theano Fotopoulou , Efstathios K. Iliodromitis , Sophia-Iris Bibli ,
⇑
,
a
c
b
c
c
b,
Anastasia Zoga , Dimitrios Th. Kremastinos , Ioanna Andreadou
National Hellenic Research Foundation, Institute of Biology, Medicinal Chemistry and Biotechnology, 48, Vas. Constantinou Ave. 11635 Athens, Greece
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Athens, Panepistimioupolis-Zografou, 15771 Athens, Greece
Second University Department of Cardiology, Medical School, Attikon General Hospital, University of Athens, 12462 Athens, Greece
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 April 2012
Revised 5 July 2012
Accepted 23 July 2012
Available online 31 July 2012
Novel purine analogues bearing nitrate esters were designed and synthesized in an effort to develop com-
pounds triggering endogenous cardioprotective mechanisms such as ischemic preconditioning (IPC) or
postconditioning (PostC). The majority of the compounds reduced infarct size compared to the control
group in anesthetized rabbits, whereas administration of the most active analogue 16 at a dose of
3.8
lmol/kg resulted on a significant reduction of infarct size, compared to PostC group (13.4 ± 1.9% vs
2
6.4 ± 2.3%). These findings introduce a novel class of promising pharmacological compounds that could
Keywords:
Purine analogues
Nitrate ester
be used as mimics or enhancers of PostC.
Ó 2012 Elsevier Ltd. All rights reserved.
Ischemia/reperfusion
Pre- and postconditioning
1
. Introduction
artery applied at the onset of reperfusion, reduce the infarct size
and coronary artery endothelial dysfunction.
Acute myocardial infarction (AMI) is a serious and often fatal
Major signalling pathways and mediators involved in IPC and
PostC are: ATP-sensitive potassium mitochondrial channels (mito-
consequence of coronary artery disease (CAD). Although restora-
tion of blood flow to the jeopardized myocardium is a prerequisite
for myocardial salvage, reperfusion itself may lead to additional
KATP), nitric oxide (NO), cyclic GMP (cGMP), activation of specific
RISK kinases (Reperfusion Injury Salvage Kinases) such as PI3K,
Akt, and ERKs, inhibition of GSK-3b, which preserve the mitochon-
drial function by preventing the mitochondrial permeability tran-
sition pore (mPTP) opening which is considered the end point of
1
tissue injury, known as reperfusion injury.
To solve this problem and to improve clinical outcome and pre-
vention of the lethal reperfusion injury, an intervention is needed
that would make the heart muscle resistant to necrosis. During
the last two decades, scientific efforts focus on improving means
for limiting infarct size. However, the morbidity and mortality of
CAD remain significant worldwide and lay the ground for the
development of novel cardioprotective therapies.
5
cardioprotection.
Since KATP channels, nitric oxide (NO) and adenosine receptor
activation are involved in IPC, pharmacological interventions,
which have only recently been identified, involve nicorandil and
adenosine derivatives. A variety of known compounds such as
adenosine A2B-selective agonists, cyclosporin, volatile anesthetics,
natriuretic peptides, applied in the early minutes of reperfusion,
Protection of the ischemic myocardium is known to occur as a
result of ischemic preconditioning (IPC), in which repetitive brief
periods of ischemia protect the heart from a subsequent prolong
3
b,5h,6
seem to trigger PostC.
2
ischemic insult. Although IPC is a powerful form of protection, it
However, no accepted adjunctive drug or therapy to acutely fur-
ther limit myocardial infarct size above and beyond reperfusion
exists. Thus, there is an unmet need for a safe and effective adjunc-
tive therapy, which can be administered together with reperfusion,
to further reduce the size of a myocardial infarction and improve
clinical outcomes such as reducing mortality and decreasing heart
failure.
is of limited clinical application for obvious ethical and practical
reasons.3 Another endogenous form of cardioprotection, similar
to IPC but applicable at the time of reperfusion, termed postcondi-
4
tioning (PostC) has been recently described. Short series of repet-
itive cycles of brief reperfusion and re-occlusion of the coronary
Our group has been involved in the design and synthesis of no-
vel molecules, which may confer their protection by using intracel-
lular pathways involved in IPC. Our preliminary in vivo data in an
⇑
Corresponding author. Tel.: +30 2107273818; fax: +30 2107273831.
E-mail address: mkoufa@eie.gr (M. Koufaki).
These authors contributed equally to this work.
0
968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.037

M. Koufaki et al. / Bioorg. Med. Chem. 20 (2012) 5948–5956
5949
the NMR spectrum of this compound H2 and H8 appear at 8.13
and 7.70 ppm, respectively, while for 3-isomer at 7.97 and
N
N
7
.93 ppm. Treatment of 1 with ethanolamine, followed by nitration
N
N
using acetyl nitrate gave analogue 3 (Scheme 1)
N
The acetyl protected nucleoside 4 was obtained by condensa-
ONO2
tion of 1,2,3,5-tetra-O-acetyl-b-D-ribofuranose with the silylated
4
6
-piperidinyl-purine, under Vorbrüggen conditions, as shown in
Figure 1. Compound A.
Scheme 2. The reaction was performed at room temperature in
dichloroethane (DCE) in the presence of trimethylsilyl triflate
(
TMSOTf) affording 4 in 85% yield. NOESY spectrum of 4 showed
0
0
experimental model of ischemia/reperfusion in rabbits showed
that the presence of a purine moiety and of a nitrate ester (NO do-
nor) is able to confer cardioprotection by means of infarct size
reduction, when the compounds were administered before ische-
a correlation between the anomeric H-1 (6.15 ppm) and H-4
(4.38 ppm) protons indicating the formation of b-isomer.8 More-
over, HMBC spectrum of 4 confirmed the N-9 substitution of the
0
9
purine ring (correlation between H-1 and C-8 and C-4). N-7 ana-
logue was obtained in 15% yield. After removal of acetyl groups and
protection of 2 and 3 hydroxyls of compound 5, the resulting iso-
propylidene derivative 6 was nitrated and subsequently deprotec-
ted to afford 7.
mia at very low doses (3.8 lmol/kg). Among the tested com-
0
0
pounds, we identified a 6-piperidinyl purine derivative bearing a
nitroxyhexyl chain at position 9, compound A (Fig. 1) as a pharma-
cological tool able to induce IPC in vivo.7
The aim of the present study was (i) the design and synthesis of
analogues of the lead compound A, (ii) evaluation of the new
derivatives as pharmacological IPC agents and (iii) evaluation of
the cardioprotective activity of the lead compound and the new ana-
loguesbymeansofinfarctsizereduction, whenthecompoundswere
Compound 9, was synthesized starting from the THP protected
6-piperidinyl-purine II (Scheme 3) which was lithiated and subse-
quently reacted with methylchloroformate to furnish ester 8. Con-
densation of 8 with ethanolamine followed by nitration, using
1
0
100% HNO and H SO
4
3
2
afforded 9.
administered before reperfusion at a dose of 3.8
l
mol/kg. Thus we
The synthesis of N-(nitroxyethylamino)carbonylmethyl ana-
logue 12 is depicted in Scheme 4. Reaction of THP-protected
purine III with glycine methyl ester, gave 10 which converted
to 12 upon condensation with ethanolamine, nitration and
deprotection.
Purine analogue III was also used as starting material for the
synthesis of 6-piperazinyl-purine analogues (Scheme 5). Amina-
tion of III with excess of piperazine, followed by acylation with
bromoacetyl bromide or activated bromohexanoic acid gave 13
synthesized the 6-piperidinyl purine analogues in which the nitro-
xyhexyl chain has been replaced by the nitroxyethyl carboxamide
group or a 5-nitroxy-ribosyl moiety, as well as 6-piperidinyl purine
bearing a nitrate ester at position 8. In addition 6-piperazinyl-purine
analogues containing nitrate esters have been synthesized.
2
. Chemistry
and 14, respectively, which upon nitration using AgNO
tection produced 15 and 16.
3
and depro-
N-acetylation of 6-piperidinyl-purine with ethyl bromoacetate
afforded the 9-substituted purine analogue 1 as major isomer. In
N
N
N
N
N
N
N
N
a
N
N
b
N
N
c
N
N
N
N
N
N
N
H
O
O
N
O
OH
ONO2
OEt
N3-alkylated analogue
N
N
I
1
2
3
H
H
Scheme 1. Synthesis of compound 3. Reagents and conditions: (a) BrCH
8%.
2
CO
2 2
CH CH
3
, Cs
2
CO
3 2 2 2 3 2
, anh DMF, 87%; (b) H N(CH ) OH, 100 °C, 98%; (c) 65% HNO , Ac O, AcOH, AcOEt,
8
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
a
c
N
N
N
O
d, e
O
O
O
N
H
RO
HO
2
O NO
I
OR
OH
RO
O
HO
4
5
R= Ac
R= H
6
7
b
Scheme 2. Synthesis of compound 7. Reagents and conditions: (a) (i) HMDS, (NH
)
4 2
SO
4
, 140 °C, (ii) 1,2,3,5-tetra-O-acetyl-b-
D-ribofuranose, TMSOTf, DCE, 85%; (b) NaOMe,
MeOH, 95%; (c) p-TsOH, 2,2-dimethoxypropane, acetone, 91%; (d) 65% HNO , Ac O, CH
3
2
2
Cl , 0 °C, 93%; (e) 80% TFA/H O, 81%.
2
2

5
950
M. Koufaki et al. / Bioorg. Med. Chem. 20 (2012) 5948–5956
N
N
N
N
N
N
O
O
N
N
b,c
N
N
a
N
N
N
N
OCH3
ONO2
N
H
N
H
THP
THP
II
8
9
Scheme 3. Synthesis of compound 9. Reagents and conditions: (a) ClCO
4%.
2
CH
3
, LDA, THF, ꢁ78 °C, 70%; (b) H
2
2 2
N(CH ) OH, 100 °C, 100%; (c) 100% HNO
3
, H
2
SO
4 2
, CH Cl
2
, ꢁ5 °C,
8
H
N
H
N
H CO
3
O NO
NH
N
Cl
N
NH
N
HO
b
NH
N
2
O
O
O
N
N
N
a
N
N
N
N
c,d
N
N
N
N
N
H
THP
THP
THP
III
10
11
12
Scheme 4. Synthesis of compound 12. Reagents and conditions: (a) H
2
NCH
2
CO
2
CH
3
3 2 2 2 3 2 2 2
ꢂHCl, Et N, MeOH, 70 °C, 97%; (b) H N(CH ) OH, 100 °C, 95%; (c) 65% HNO , Ac O, CH Cl ,
0
2 2
°C, 67%; (d) 50% TFA/CH Cl , 55%.
O
O
Given prior to sustained ischemia, compound 3, bearing a N-
nitroxyethyl) aminocarbonylmethyl group at position 9 of purine
H
N
(
N
N
Br
N
N
ONO2
n
n
moiety, reduced infarct size (28.4 ± 1.2%) compared with the con-
trol group, but it was less potent than the lead compound A. This
suggests that not only the presence of a nitrate ester but also the
nature of the alkyl chain influences the cardioprotective activity.
N
a
b, c
N
N
N
N
N
N
N
N
N
0
N
H
Replacement of the nitroxyalkyl chain by 5 -nitro-ribosyl group
N
N
THP
THP
n= 0
n= 4
(
compound 7) results in significant improvement of the protection
1
3
4
15 n= 0
16 n= 4
IV
in terms of infarct size (17.9 ± 2.6%). This adenosine analogue is not
expected to interact with adenosine receptors. According to litera-
ture data, the presence of a secondary amine, such as piperidine, at
position 6 of the purine abolishes the activity at the human
adenosine receptors (ARs), indicating that, at least one hydrogen
atom at the N6-position is essential for hydrogen bonding in the
1
Scheme 5. Synthesis of compounds 15 and 16. Reagents and conditions: (a)
BrCH COBr, Et N, CH Cl , 0 °C (13), Br(CH COOH, CDI, Et N, THF (14), 71% and
8%, respectively; (b) AgNO , CH CN, 80 °C, 71% and 85%, respectively; (c) 50% TFA/
CH Cl , 57% and 81%, respectively.
2
3
2
2
)
2 5
3
6
3
3
2
2
1
1
binding site of the AR. The analogue 9 which has the same struc-
tural characteristics that nicorandil and its indole and quinoline
derivatives (heteroaromatic compound substituted by a N-(nitr-
oxyethyl) aminocarbonyl group) was less active (34.4 ± 4.3%) than
its indole and quinoline counterparts previously tested.7
The analogues 12 and 15 bearing the nitrate ester at position 6
of purine skeleton, administered before ischemia did not reduce in-
farct size. Compound 16 bearing the nitroxyhexyl chain, was
slightly less potent than A.
An additional goal of this study was to compare the activity of
the new analogues with the effect of PostC. Thus, the compounds
were administered bolus at the 20th min of ischemia and the
1st min of reperfusion. Administration of the lead compound A
did not result on significant reduction of infarct size (36.8 ± 2.0%)
compared to the control group.
3
. Discussion
All the compounds were administered in anesthetized rabbits
before ischemia or before reperfusion, at the same dose as the lead
compound A in our previous study (3.8 lmol/kg). The effect of the
7
tested compounds on infarct size reduction is compared with the
control group as well as to the IPC or PostC groups. The schematic
presentation of the experimental protocol is shown in Figure 2.
Specifically, the rabbits were randomized into groups (n = 6–8
per group), anesthetized and subjected to 30 min of myocardial
ischemia and 3 h of reperfusion with the following additional
interventions: Control group, no intervention, groups in which
the compounds were administered bolus at the 40th min and at
the 1st min before sustained ischemia, in
.8 mol/kg, groups in which the compounds were administered
bolus at the 20th min of ischemia and the 1st min of reperfusion
in a total dose of 3.8 mol/kg, a IPC group, consisted of two cycles
of preconditioning, each composed of 5 min of regional ischemia-
0 min reperfusion and a PostC group, consisted of eight cycles
a total dose of
3
l
Administration of compounds 3, 9 and 12, bearing the N-(nitr-
oxyethyl) aminocarbonyl group, resulted in the death of animals
at the 10th and 15th min of reperfusion. The death was due to
development of intractable ventricular arrhythmias at the time of
reperfusion and thus animals were excluded from the study and
these analogues were not further tested.
l
1
of post conditioning (8 ꢀ 30 s) immediately after completion of in-
dex ischemia.
The effect of compound 7 on infarct size reduction (22.7 ± 3.6%)
was comparable to that of PostC (26.4 ± 2.3%) indicating that the
replacement of the nitroxyhexyl chain of A or the N-(nitroxyeth-
After the end of the experiments the infarct size (I) and the area
at risk (R) were estimated in % I/R. The hemodynamic variables of
the different study groups are presented in Table 1. No significant
differences were observed among the groups. The effects of the
compounds on infarct size are summarized in Table 2.
0
yl)aminocarbonylmethyl group of 3 by a 5 nitro-ribosyl moiety re-
sults in significant improvement of the cardioprotective activity. As
mentioned above, compound 7 is not expected to act at the

M. Koufaki et al. / Bioorg. Med. Chem. 20 (2012) 5948–5956
5951
3
0 min
180 min
Reperfusion
Control
3
0 min
180 min
Reperfusion
Administration of the new
Administration of
the new
analogues 1.9µmol/Kg
analogues 1.9µmol/Kg
3
0 min
180 min
Reperfusion
Administration of
analogues 1.9µmol/Kg
the new
Administration of
the new
analogues 1.9µmol/Kg
3
0 min
180 min
PC
Reperfusion
3
0 min
180 min
Reperfusion
PostC
-
40΄
-20΄ -18΄
-1΄ 0΄
20΄
0΄
1΄
210΄
3
Figure 2. Schematic presentation of the study protocol.
Table 1
Hemodynamic variables of the different study groups.
Study group
Baseline
20-min Ischemia
180-min Reperfusion
HR
BP
HR
BP
HR
BP
Control
A
IPC
PostC
3
7
9
286 ± 12
272 ± 10
283 ± 13
276 ± 11
283 ± 14
275 ± 13
277 ± 14
279 ± 12
280 ± 10
274 ± 12
276 ± 14
281 ± 13
114 ± 3
122 ± 5
126 ± 4
119 ± 3
127 ± 2
125 ± 4
117 ± 5
119 ± 6
123 ± 3
128 ± 5
113 ± 4
110 ± 6
284 ± 13
276 ± 12
284 ± 10
279 ± 12
280 ± 14
279 ± 12
274 ± 15
282 ± 11
278 ± 13
278 ± 15
279 ± 10
284 ± 12
104 ± 6
112 ± 4
121 ± 6
114 ± 3
115 ± 5
113 ± 3
112 ± 6
110 ± 5
114 ± 4
117 ± 7
106 ± 5
102 ± 3
280 ± 14
270 ± 13
280 ± 15
275 ± 11
276 ± 12
271 ± 15
269 ± 10
273 ± 12
274 ± 13
269 ± 11
271 ± 14
275 ± 12
100 ± 4
104 ± 3
116 ± 5
107 ± 4
102 ± 5
110 ± 4
103 ± 7
104 ± 6
109 ± 5
112 ± 3
101 ± 6
98 ± 5
12
15
16
17
18
HR: mean heart rate in beats/min; BP: mean blood pressure in mm Hg.
2
0-min ischemia is the period of the initial ischemic insult, 180-min reperfusion is the period of the reperfusion.
0
adenosine receptors. The known adenosine receptor agonist 5 -ni-
benefit of compound 16, this analogue was administered before
reperfusion to the PostC protocol and the infarct size was deter-
mined. The I/R was found to be 12.3 ± 0.8% (P <0.05 vs PostC
group), indicating that this compound produced a stronger stimu-
lus that PostC in reducing the infarct size and that PostC can
achieve maximal protection when a pharmacological agent is co-
administered. Cyclosporine, a mPTP inhibitor has been shown to
reduce the I/R with an average of 24 ± 4% when administered
(10 mg/kg) 10 min before coronary occlusion and of 25 ± 3% when
administered 1 min before the reperfusion period in anesthetized
rabbits (the same surgical procedure than ours) suggesting that
specific inhibition of mPTP opening at reperfusion following
acute myocardial infarction provides a powerful antinecrotic
1
2
tro-adenosine (analogue 18, Table 2) did not reduce infarct size
(
40.2 ± 2.9%).
The 6-piperazinyl-purine derivatives were active when admin-
istered before reperfusion. The effect of the analogue 15
26.2 ± 4.6%) was similar to PostC (26.4 ± 2.3%) while the most ac-
tive compound 16 reduced infarct size statistically significant com-
pared to PostC (13.4 ± 1.9% vs 26.4 ± 2.3%, P <0.05). According to
our previous findings, the proper choice of timing and number of
cycles of PostC is important for success. Thus, a PostC protocol con-
sisted of eight cycles of ischemia-reperfusion (8 ꢀ 30 s) immedi-
ately after completion of index ischemia, offered the maximum
protection to the rabbits.13 In order to investigate an additional
(

5
952
M. Koufaki et al. / Bioorg. Med. Chem. 20 (2012) 5948–5956
Table 2
Effect of the compounds on infarct size reduction
Compound
Given before ischemia (%)
Given before reperfusion (%)
36.8 ± 2.0
*
A
3
7
9
19.8 ± 3.1
*
a
28.4 ± 1.2
Not tested
*
*
17.9 ± 2.6
22.7 ± 3.6
a
34.4 ± 4.3
34.8 ± 4.0
37.3 ± 4.2
Not tested
a
1
1
1
2
5
6
Not tested
*
26.2 ± 4.6
*
**
24.9 ± 4.1
13.4 ± 1.9
O
N
N
N
N
S
*
1
7
S
28.6 ± 3.2
N
HN
5
-Nitro-adenosine 18
40.2 ± 2.9
26.4 ± 2.3
control
IPC
PostC
48.1 ± 2.0
15.7 ± 2.3
*
*
*
*
P <0.05 versus Control.
*
P <0.05 versus postC.
a
These analogues were not further tested due to the death of animals at the 10th and 15th min of reperfusion.
protection.14 Many studies have failed to show an enhanced car-
It has been shown that the protective effect of IPC can be mim-
icked pharmacologically with NO donors. However, the effect of
15
5a
dioprotection when IPC and PostC are combined, probably be-
cause both IPC and PostC protect against myocardial reperfusion
injury by the same mechanisms, including the PI3K–Akt signal
transduction pathway, NO-PKG pathway and mitochondrial ATP
sensitive channels, as well as adenosine, oxygen free radicals and
NO donors in PostC, has not been determined in vivo. Nitroglycerin
(NTG), given at reperfusion in isolated hearts, failed to reduce the
infarct size; the authors suggested that this may occur because of
the surge of superoxide radical that is generated at the onset of
reperfusion and forms the peroxynitrite anion via NO from
1
6
the mPTP. Compound 16 exhibited more significant cardiopro-
tection than PostC and cyclosporine in terms of infarct size limiting
effect and this may be due to alternative intracellular mechanisms
that need to be explored. Further work to uncover the specific
mode of action of this compound is currently under investigation.
Based on our experience on lipoic acid analogues17 and the lit-
erature data on the ability of lipoic acid and the piperazinyl-purine
derivatives to modulate various kinases,18 we synthesized and
tested the analogue 17 bearing 1,2-dithiolane group instead of ni-
trate ester. This compound was equipotent (28.6 ± 3.2%) with 15
and less active than 16 indicating that both the length of the alkyl
chain and the presence-of the nitrate ester (NO donor) influences
the ability of these compounds to reduce infarct size.
1
9
NTG. NO is an important mediator in PostC-mediated cardiopro-
2
0
tection, while the reduction of peroxynitrite formation plays a
2
1
significant role in the PostC protective effect. However, NO may
react with superoxide anions, whose production is increased dur-
ing post-ischemic reperfusion to form the toxic nitrating and oxi-
ꢁ
ꢁ
dant agent peroxynitrite (ONOO ). ONOO is one of the major
22
triggers of cardiomyocyte apoptosis. Peroxynitrite is formed after
NO reaction with superoxide anion at a very fast rate; this causes
an irreversible inhibition of the mitochondrial respiratory chain.
It also disturbs the cellular function, modifies iron/sulfur centers
as well as protein thiol and tyrosine residues. The latter leads to
the formation of nitrotyrosine (NT), which serves as a surrogate
Figure 3. Nitrotyrosine (NT) formation at baseline, and reperfusion ( P <0.05 vs baseline, #P <0.05 vs control, ⁄⁄P <0.05 vs all the other groups).
⁄

M. Koufaki et al. / Bioorg. Med. Chem. 20 (2012) 5948–5956
5953
⁄
Figure 4. Western Blot analyses: total endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS (A, p <0.05 vs Control and compound A post); total Akt and
⁄
phosphorylated Akt (B, p <0.05 vs Control and compound A post).
marker of the reactive nitrogen species.23 We have previously
shown a significant reduction in myocardial and circulating NT in
PostC compared to control group.1 In order to investigate the ef-
fect of the novel compounds on nitrosative stress, we determined
the NT concentration in circulation at the 10th min of reperfusion,
when the compounds administered prior to reperfusion and we
compared the levels of NT with those of control and PostC groups
ventions previously described (Fig. 2). The tissue from ischemic
area of myocardium was quickly excised and stored in ꢁ80 °C for
Akt and eNOS assessment with western blot analysis as previously
3a
1
3
described. In both endogenous cardioprotective mechanisms IPC
and PostC, activation of proapoptotic kinase Akt by phosphoryla-
2
4
tion exerts infract limiting results. Additionally, subsequent acti-
vation of eNOs in ser 1177, activates further signal transduction
1
5c
(Fig. 3). All the tested compounds significantly reduced the NT lev-
and plays a crucial role in cell survival.
Induction of PostC and
els independently of the infarct limiting results. However, a further
significant decrease was noted in the group treated with com-
pound 16 compared to the other groups. Thus, the effect of com-
pound 16 on nitrosative stress may be involved in the
mechanism by which the drug reduces the infarct size in a larger
extent that PostC.
Moreover, we performed additional experiments in order to
investigate the difference observed between pre-ischemic and
reperfusion treatment with compound A and to study the intracel-
lular alterations. In a second series of experiments 20 additional
animals (4 for each group) from groups control, PostC, IPC, com-
pound A pre (administration of compound A in two doses,
IPC as well as administration of compound A before and in the
beginning of ischemia raised the phosphorylation level of both
eNOs (Fig. 4A) and Akt (Fig. 4B) in contrast to control group and
the administration of compound A during ischemia and in the
beginning of reperfusion. These data, which are in agreement with
I/R results, suggest that the observed difference in infract size lim-
itation, in terms of time administration of the new compounds,
might be due to molecular mechanism differentiation and to the
pharmacokinetic properties as the activation time of half-life.
However specific pharmacokinetic studies are required.
4. Conclusion
4
0 min before ischemia and 1st min of ischemia) and compound
A post (administration of compound A in two doses, 20 min of
ischemia and 1st min of reperfusion) were subjected in 30 min
ischemia followed by 10 min of reperfusion with the same inter-
Our data suggest that administration of the new analogues in-
duces cardioprotection activating different intracellular signaling
pathways depending on the timing of the administration (before

5
954
M. Koufaki et al. / Bioorg. Med. Chem. 20 (2012) 5948–5956
ischemia or before reperfusion). Thus 6-piperidinyl-purine ana-
logues A, 3 and 7 are effective when given before ischemia, while
solid (mp 166–168 °C). HPLC: gradient elution 90/10 H
CH CN_0.1%TFA to 20/80 O_0.1%TFA/CH CN_0.1%TFA over
30 min, flow rate: 1.5 mL/min, t : 7.32 min, detection at 216 nm,
2
O_0.1%TFA/
3
H
2
3
6
-piperazinyl-purine derivatives 15 and 16 are active when admin-
R
1
istered before reperfusion. The administration of compound 16 at
reperfusion reduced significantly the infarct size in anesthetized
rabbits compared to PostC. Moreover, a significant decrease of
nitrotyrosine (NT) levels, at the 10th min of reperfusion, was ob-
served in the group treated with compound 16 compared to the
other groups. These findings introduce a novel class of promising
pharmacological compounds that could be used as mimics or
enhancers of PostC. In order to further investigate the molecular
pathways by which compound 16 offers cardioprotection, pharma-
cological studies are currently in progress.
purity 100%. H NMR (600 MHz, CDCl
3
) d: 8.15 (s, 1H, H-2), 7.86
0
(s, 1H, H-8), 5.88 (d, J = 3.2 Hz, 1H, 1 -H), 4.81 (dd, J = 11.9,
0
0
2.6 Hz, 1H, 5 -H), 4.67 (dd, J = 11.9, 4.4 Hz, 1H, 5 -H), 4.37 (d,
0
0
0
J = 3.6 Hz, 1H, 4 -H), 4.29–4.22 (m, 2H, 2 -H and 3 -H), 4.13 (br s,
0
0
4H, –NCH
2
), 3.40–3.27 (m, 2H, 2 -OH and 3 -OH), 1.64 (br s, 6H, –
). C NMR (75 MHz, CDCl ) d: 153.7, 152.0, 149.4, 135.9,
120.2, 89.8, 80.3, 74.1, 71.3, 69.9, 26.0, 24.6. MS m/z: 381.06
1
3
CH
2
3
+
+
+
(M+H , 100%), 403.04 (M+Na , 15%), 782.77 (2 M+Na , 40%). HRMS:
+
21 6 6
calcd for C15H O N (M+H ) 381.1517; found: 381.1507
5
.1.3. N-(2-Nitroxyethyl)-6-(piperidine-1-yl)-purine-8-
carboxamide (9)
4
Concentrated H SO (2 equiv) was slowly and dropwise added
5
5
. Experimental section
2
.1. Chemistry
at ꢁ5 °C to 100% HNO (2 equiv) and the mixture was stirred for
3
additional 10 min. N-(2-Hydroxyethyl)-6-(piperidin-1-yl)-9-(tet-
All starting materials and common laboratory chemicals were
rahydropyran-2-yl)-purine-8-carboxamide (0.04 g, 0.09 mmol) in
CH Cl was then dropped slowly to the reaction solution and the
purchased from commercial sources and used without further
purification. All reactions involving air- or moisture-sensitive re-
agents were performed under an argon atmosphere. Melting points
2
2
reaction mixture was stirred at 0 °C for 30 min. When the reaction
was complete, 10% aqueous solution of sodium bicarbonate was
added, dried over Na SO and concentrated in vacuo. The resulting
1
were determined on a Buchi 510 apparatus and are uncorrected. H
2
4
NMR spectra were recorded on Varian spectrometers operating at
3
using CDCl as solvent. Silica gel plates Macherey-Nagel Sil G-25
3
UV254 were used for thin layer chromatography. Chromatographic
purification was performed with silica gel (200–400 mesh). The
purity of the tested compounds was determined by HPLC (Thermo
Scientific HPLC Spectra System) column Nucleosil 100-5
2 2 3
residue was purified by column chromatography (CH Cl /CH OH,
00 MHz or 600 MHz and 13C spectra were recorded at 75 MHz
95:5) to provide 9 (0.03 g, 84%) as a white solid (mp 180–
182 °C). HPLC: gradient elution 90/10 H O_0.1%TFA/
CH CN_0.1%TFA to 20/80 H O_0.1%TFA/CH CN_0.1%TFA over
2
3
2
3
30 min, flow rate: 1.5 mL/min, t : 7.93 min, detection at 216 nm,
R
1
purity 100%. H NMR (600 MHz, CDCl ) d: 8.49 (s, 1H, H-2), 7.61
3
(t, J = 6.0 Hz, 1H, –CONHCH –), 4.70 (t, J = 5.2 Hz, 2H, –CH ONO ),
2
2
2
1
50 ꢀ 4.6 mm C18 5
l
, Macherey-Nagel. Mass spectra were ob-
4.42–4.10 (br s, 4H, –NCH2 piperidine), 3.87 (t, J = 5.2 Hz, 2H, –
n
13
tained on HPLC–MS Fleet-Thermo, in the ESI mode. HRMS spectra
were recorded, in the ESI mode, on UPLC-MS Orbitrap Velos-
Thermo.
NHCH CH –), 1.80–1.69 (m, 6H, –CH2 piperidine).
C NMR
2
2
n
(75 MHz, CDCl ) d: 158.7, 154.0, 153.1, 151.2, 140.0, 119.7, 71.2,
3
+
36.5, 25.9, 24.4. HRMS: calcd for C13H18O N (M+H ) 336.1415;
4
7
found: 336.1407.
5
.1.1. N-(2-Nitroxyethyl)-6-(piperidin-1-yl)-9H-purine
acetamide (3)
5
.1.4. N-(2-Nitroxyethyl)-2-(9H-purin-6-ylamin)-acetamide (12)
To a solution of 11 (0.10 g, 0.27 mmol) in CH Cl (1.1 mL) was
Nitric acid (0.1 mL, 65%), acetic acid (0.5 mL) and acetic anhy-
dride (0.5 mL) were stirred for 10 min at 0 °C and this mixture
was added dropwise to a solution of 2 (0.1 g, 0.32 mmol) in CH Cl .
2 2
The reaction mixture was stirred at 0 °C for an additional 90 min.
After completion of the reaction, 10% aqueous solution of sodium
bicarbonate was added and then extracted with ethyl acetate. The
2
2
added trifluoroacetic acid (TFA) (1.1 mL, 13.7 mmol) and then
was stirred for 30 min. When the reaction was complete, the mix-
ture was neutralized by addition 10% aqueous NaHCO
Na SO and concentrated in vacuo. The resulting residue was puri-
fied by column chromatography (CH Cl /CH OH, 95:5 to 90:10) to
provide 12 (0.04 g, 55%) as a white solid (mp 158–160 °C). HPLC:
gradient elution 95/5 O_0.1%TFA/CH CN_0.1%TFA to 50/50
O_0.1%TFA/CH CN_0.1%TFA over 30 min, flow rate: 0.8 mL/min,
2.56 min, detection at 216 nm, purity 100%.
300 MHz CD OD) d: 8.82 (s, 1H, H-2), 8.40 (s, 1H, H-8), 4.55 (t,
3
, dried over
2
4
2
2
3
organic layer was washed with brine, dried over Na
trated in vacuo. The crude residue was purified by column chroma-
tography (CH Cl /CH OH, 97:3) to provide 3 (0.1 g, 88%) as a white
solid (mp 172–174 °C). HPLC: gradient elution 90/10 H O_0.1%TFA/
CH CN_0.1%TFA to 20/80 O_0.1%TFA/CH CN_0.1%TFA over
0 min, flow rate: 1.5 mL/min, t : 6.53 min, detection at 216 nm,
purity 100%. H NMR (300 MHz, CDCl ) d: 8.33 (s, 1H, H-2), 7.81
s, 1H, H-8), 7.58 (br s, 1H, –CONH–), 4.87 (s, 2H, –NCH CONH–),
.52 (t, J = 5.2 Hz, 2H, –CH ONO ), 4.37–4.13 (br s, 4H, CH ), 3.60
t, J = 5.2 Hz, 2H, –NHCH CH –), 1.82–1.58 (br s, 6H, CH
) d: 166.9, 153.9, 152.4, 150.1, 138.1, 119.8,
2 4
SO , concen-
H
2
3
2
2
3
H
2
3
2
1
t
R
:
H NMR
3
H
2
3
(
3
3
R
J = 5.1 Hz, 2H, –CH
J = 5.1 Hz, 2H, –CH CH
1
2
ONO
2
), 4.24 (s, 2H, –NHCH
2
–), 3.55 (t,
1
3
1
3
2
2
ONO
2
). C NMR (75 MHz, CDCl ) d: 170.3,
3
(
4
(
2
55.8, 154.1, 152.6, 138.2, 134.5, 81.9, 71.1, 68.8. MS m/z: 282.08
2
2
2
+
+
(
(
M+H , 100%), 304.08 (M+Na , 95%). HRMS: calcd for C
M+H ) 282.0945; found: 282.0942.
H
9 12
O
4
N
7
1
3
2
2
2
).
C
+
NMR (75 MHz, CDCl
3
+
7
1.0, 47.6, 37.1, 26.1, 24.7. HRMS: calcd for C14
20 4 7
H O N
(M+H )
3
50.1571; found: 350.1564.
5.1.5. 6-(4-Nitroxyacetylpiperazin-1-yl)-9H-purine (15)
White solid, yield 57%, mp 166–168 °C. HPLC: gradient elution
95/5 O_0.1%TFA/CH CN_0.1%TFA to 50/50 O_0.1%TFA/
CH CN_0.1%TFA over 30 min, flow rate: 0.8 mL/min, t : 2.49 min,
detection at 216 nm, purity 100%. H NMR (300 MHz CDCl ) d:
8.29 (s, 1H, H-2), 7.88 (s, 1H, H-8), 5.08 (s, 2H, –COCH ONO ),
4.37 (d, J = 17.2 Hz, 4H, –CH ), 3.76 (s, 2H, –CH ), 3.56 (s, 2H, –
). C NMR (75 MHz, CDCl ) d: 163.4, 153.6, 151.8, 150.8,
5
.1.2. 9-(5-O-Nitro-1b-
D-ribofuranosyl)-6-(piperidin-1-yl)-9H-
H
2
3
H
2
purine (7)
A suspension of 9-(2,3-O-isopropylidene-5-O-nitro-1b-
3
R
1
D
-ribo-
3
furanosyl)-6-(piperidin-1-yl)-9H-purine (0.12 g, 0.29 mmol) in a
mixture of TFA (1.6 mL) and water (4:1) was stirred at room tem-
perature for 1 h. The resulting reaction mixture was concentrated
in vacuo and the residue was coevaporated several times with
MeOH. The crude residue was purified by column chromatography
2
2
2
2
1
3
CH
2
3
+
137.1, 119.1, 67.6, 44.6, 41.6. MS m/z: 308.14 (M+H , 100%),
+
+
330.19 (M+Na , 25%). HRMS: calcd for
11 14 4 7
C H O N (M+H )
(
2
CH Cl
2
/CH
3
OH, 95:5 to 90:10) affording 7 (0.09 g, 81%) as a white
308.1102; found: 308.1100.

M. Koufaki et al. / Bioorg. Med. Chem. 20 (2012) 5948–5956
5955
5
.1.6. 6-[4-(6-Nitroxyhexanoyl)piperazin-1-yl]-9H-purine (16)
White solid, yield 81%, mp 161–163 °C. HPLC: gradient elution
for blood pressure monitoring via a transducer attached to a mul-
tichannel recorder. The chest was opened via a left thoracotomy
and after pericardiotomy the beating heart was exposed. A 3-0 silk
thread was passed through the myocardium around a prominent
branch of the left coronary artery. Ischemia was induced by pulling
the ends of the suture through a small segment of a soft tube,
which was firmly attached against the artery with a clamp. The
successful induction of ischemia was verified by ST segment eleva-
tion on the electrocardiogram and by visual inspection (cyanosis)
of the heart. Reperfusion was achieved by releasing the clamp
and was verified by the refilling of the artery.
9
CH
0/10
2 3
H O_0.1%TFA/CH CN_0.1%TFA to 20/80
H
2
O_0.1%TFA/
: 7.65 min,
detection at 216 nm, purity 100%. H NMR (300 MHz CDCl ) d:
.41 (br s, 1H, H-2), 7.98 (s, 1H, H-8), 4.44 (t, J = 6.5 Hz, 2H, CH O-
), 4.39–4.21 (br s, 4H), 3.89–3.72 (br s, 2H), 3.71–3.54 (br s,
), 1.81–1.66 (m, 4H), 1.56–
.41 (m, 2H). C NMR (75 MHz, CDCl ) d: 171.3, 155.2, 153.8,
51.3, 137.3, 119.9, 73.1, 45.5, 41.6, 32.9, 26.7, 25.5, 24.6. MS m/
3
CN_0.1%TFA over 30 min, flow rate: 1.5 mL/min, t
R
1
3
8
NO
2
2
2
1
1
H), 2.40 (t, J = 7.2 Hz, 2H, NCOCH
2
1
3
3
+
+
z: 364.17 (M+H , 30%), 386.08 (M+Na , 50%). HRMS: calcd for
C
+
15
H
22
O
4
N
7
(M+H ) 364.1728; found: 364.1725.
5
.2.2. Experimental protocol
5
.1.7. 6-[4-(1,2-Dithiolan-3-pentanoyl)piperazin-1-yl]-9H-
The rabbits were randomized into groups consisted of male rab-
purine (17)
-(Piperazin-1-yl)-9-(tetrahydropyran-2-yl)-9H-purine
bits (n = 6–8 per group), which were anesthetized and subjected to
30 min of myocardial ischemia and 3 h of reperfusion with the fol-
lowing additional interventions:
6
IV
(
(
0.08 g, 0.27 mmol) and the activated with CDI lipoic acid (LA)
0.11 g, 0.54 mmol) were treated as described for 14. After removal
of the THP group 17 was obtained as a white solid (0.03 g, 60%; mp
52–154 °C). HPLC: gradient elution 90/10 O_0.1%TFA/
CH CN_0.1%TFA to 20/80 O_0.1%TFA/CH CN_0.1%TFA over
0 min, flow rate: 1.5 mL/min, t : 9.0 min, detection at 216 nm,
purity 100%. H NMR (600 MHz CDCl ) d: 8.40 (s, 1H, H-2), 7.97
s, 1H, H-8), 4.36 (br s, 4H, –CH piperazine), 3.78 (s, 2H, –CH
piperazine), 3.63 (s, 2H, –CH piperazine), 3.57 (dd, J = 8.4, 6.2 Hz,
H, –CHSS–), 3.13 (ddd, J = 17.9, 8.9, 4.8 Hz, 2H, –CH SS–), 2.48–
.43 (m, 1H, –HCHCH SS), 2.40 (t, 2H, J = 7.5 Hz, –NHCOCH -),
.92–1.87 (m, 1H, –HCHCH SS–), 1.76–1.66 (m, 4H, –CH –), 1.54–
–). C NMR (75 MHz, CDCl ) d: 171.7, 154.0,
51.2, 150.9, 137.4, 119.7, 56.6, 45.7, 40.4, 38.6, 34.9, 33.2, 29.2,
(1) Control group, no intervention.
(2) Groups in which compounds were administered bolus at the
40th min and 1st min before sustained ischemia, in a total
1
H
2
3
H
2
3
3
R
dose of 3.8 lmol/kg. The dose was defined according to
1
7
3
our previous experiments.
(
2
2
(3) Groups in which compounds were administered bolus at the
20th min of ischemia and the 1st min of reperfusion in a
2
1
2
1
1
1
2
2
total dose of 3.8 lmol/kg. Administration of compounds 3
2
2
and 9, and 12, resulted in the death of animals at reperfu-
sion. Since these analogues seem to be toxic using this
experimental protocol, they were not further tested.
2
2
13
.44 (m, 2H, –CH
2
3
(4) PC group, consisted of two cycles of preconditioning, each
composed of 5 min of regional ischemia—10 min reperfusion.
(5) postC group consisted of eight cycles of post conditioning
+
+
5.1. MS m/z: 393.22 (M+H , 40%), 806.86 (2M+Na , 100%). HRMS:
+
6 2
calcd for C17H25ON S (M+H ) 393.1526; found: 393.1514.
(
8 ꢀ 30 s) immediately after completion of index ischemia.
0
5
.1.8. 5 -O-Nitro adenosine (18)
Nitro adenosine 18 was obtained as described in WO 2005/
17910 A2 with minor modifications. White solid, yield 63%, mp
Heart rate and blood pressure were measured immediately be-
fore sustained ischemia (baseline), at the 20th minute of sustained
ischemia and at the end of long reperfusion. Blood samples from all
groups were collected at baseline, and at the 10th min of reperfu-
sion for analysis of NT.
1
7
CH
2–74 °C.
CN_0.1%TFA to 50/50
0 min, flow rate: 0.8 mL/min, t
HPLC:
gradient
elution
O_0.1%TFA/CH
: 2.5 min, detection at 216 nm,
95/5
2
H O_0.1%TFA/
3
H
2
3
CN_0.1%TFA over
3
R
1
purity 100%. H NMR (300 MHz CDCl
3
0
) d: 8.20 (s, 1H, H-2), 7.98
After the end of the experiments the infarct size (I) and the area
at risk (R) were estimated in % I/R.
(
s, 1H, H-8), 5.93 (d, J = 3.4 Hz, 1H, 1 -H), 4.86 (d, J = 2.8 Hz, 1H,
0
0
0
5
2
1
3
C
-H), 4.75–4.68 (m, 1H, 5 -H), 4.57–4.45 (m, 1H, 4 -H), 4.34 (m,
0
0
13
H, 2 -H and 3 -H). C NMR (75 MHz, (CD
3
)
2
CO) d: 156.2, 152.7,
5.2.3. Risk area and infarct size measurement
49.6, 139.9, 119.9, 89.4, 80.4, 73.5, 73.00, 71.0, 54.1. MS m/z:
After the end of reperfusion, the hearts were harvested,
mounted on an apparatus, and perfused retrogradely via the aorta
with normal saline for 2 min. When all residual blood had been
removed from the coronary arteries, the coronary ligature was
retightened at the same site and 10 ml of green fluorescent micro-
+
+
13.11 (M+H , 100%), 335.00 (M+Na , 16%). HRMS: calcd for
+
10
13 6 6
H O N (M+H ) 313.0891; found: 313.0885.
5
5
3
.2. In vivo experiments
spheres (1–10 lm diameter, Duke Scientific Corp., Palo Alto, CA,
.2.1. Surgical preparation
New Zealand White male rabbits with a weight between 2.3 and
.1 kg received proper care in compliance with the Principles of
Laboratory Animal Care formulated by the National Society for
Medical Research and the Guide for the Care and Use of Laboratory
Animals prepared by the National Academy of Sciences and pub-
lished by the National Institute of Health (NIH Publication No.
5-23, revised 1996).
Approval from the Ethical Committee and the veterinary
authorities of East Attica prefecture was obtained before the study
was started.
All animals were anesthetized by injecting 30 mg/kg sodium
thiopentone (Pentothal, Abbott) into an ear vein, intubated and
mechanically ventilated with a respirator for small animals (MD
Industries, Mobile, AL, USA) at a rate adjusted to keep blood gases
within the normal range. Two catheters were inserted; one in the
jugular vein for fluids and anesthetic and one in the carotid artery
USA, suspended in saline) was infused for the delineation of the
normally perfused tissue from the risk zone. Hearts were then fro-
zen for 24 h at ꢁ10 °C and later sliced into 3-mm-thick sections
from the apex to the base. The slices were then incubated in 1% tri-
phenyl tetrazolium chloride (TTC) in isotonic phosphate buffer, pH
7.4 for 20 min at 37 °C. The heart slices were immersed in 10%
formaldehyde solution to delineate the infarcted areas more
clearly. To identify the borders between the risk zone and the nor-
mal area, slices were examined under UV light (wavelength
366 nm). The infarcted, the risk, and the normal areas were traced
onto an acetate sheet, which had been placed over the top glass
plate. The tracings were subsequently scanned with the Abode
Photoshop 6.0 and measured with the Scion Image program. The
areas of myocardial tissue at risk and that infarcted were automat-
ically transformed into volumes. Infarct- and risk area volumes
8
3
were expressed in cm and the percent of infarct to risk area ratio
1
3a,25
(%I/R) was calculated.

5
956
M. Koufaki et al. / Bioorg. Med. Chem. 20 (2012) 5948–5956
5
.2.4. Measurement of circulating nitrotyrosine levels
Blood samples at the 10th min of reperfusion after administra-
gramme (FP7-REGPOT-2009-1) under grant agreement No.
245866.
tion of compounds A, 7, 15, 16, 17 and 18 during and after sus-
tained ischaemia as well as from PostC and control groups were
centrifuged in 4000 rpm in 4 °C for 10 min. Nitrotyrosine concen-
tration was determined using a commercially available enzyme-
linked immunosorbent assay kit according to the manufacture’s
specifications (Hycult Biotechnology b.v., The Netherlands). For
this measurement the antigen present in samples is captured by
a solid phase monoclonal antibody and then detected with a bio-
tin-labeled goat polyclonal anti-nitrotyrosine. A streptavidin per-
oxidase conjugate then binds to the biotinylated antibody. A
tetramethylbenzidine (TBM) substrate is added and the yellow
product is measured at 450 nm. This kit has a minimum detection
level of 2 nM and measurable concentration range of 2–1500 nM.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.037.
References and notes
1
.
(a) Yellon, D. M.; Hausenloy, D. J. N. Engl. J. Med. 2007, 357, 1121; (b) Dirksen, M.
T.; Laarman, G. J.; Simoons, M. L.; Duncker, D. J. Cardiovasc. Res. 2007, 74, 343.
Murry, C. E.; Jennings, R. B.; Reimer, K. A. Circulation 1986, 74, 1124.
2
.
3. (a) Andreadou, I.; Koufaki, M.; Iliodromitis, E.; Kremastinos, T. D. Mini-Rev. Med.
Chem. 2008, 8, 952; (b) Andreadou, I.; Iliodromitis, E. K.; Farmakis, D.; Koufaki,
M.; Tsotinis, A.; Kremastinos, D. Th. Curr. Med. Chem. 2008, 15, 3204.
4
.
Zhao, Z. Q.; Corvera, J. S.; Halkos, M. E.; Kerendi, F.; Wang, N. P.; Guyton, R. A.;
Vinten-Johansen, J. Am. J. Physiol. Heart Circ. Physiol. 2003, 285, H579.
(a) Iliodromitis, E. K.; Georgiadis, M.; Cohen, M. V.; Downey, J. M.; Bofilis, E.;
Kremastinos, D. Th. Basic Res. Cardiol. 2006, 101, 502; (b) Skyschally, A.; Caster,
P.; Iliodromitis, E. K.; Schulz, R.; Kremastinos, D. Th.; Heusch, G. Basic Res.
Cardiol. 2009, 104, 469; (c) Iliodromitis, E. K.; Downey, J. M.; Heusch, G.;
Kremastinos, D. Th. J. Cardiovasc. Pharmacol. Therap. 2009, 14, 269; (d)
Kharbanda, R. K. Heart 2010, 96, 1179; (e) Ovize, M.; Baxter, G. F.; Di Lisa, F.;
Ferdinandy, P.; Garcia-Dorado, D.; Hausenloy, D. J. Cardiovasc. Res. 2010, 87,
406; (f) Hansen, P. R.; Thibaul, H.; Abdulla, J. Int. J. Cardiol. 2010, 144, 22; (g)
Vinten-Johansen, J.; Shi, W. J. Cardiovasc. Pharmacol. Therap. 2011, 16, 260; (h)
Sanada, S.; Komuro, I.; Kitakaze, M. Am. J. Physiol. Heart Circ. Physiol. 2011, 301,
H1723.
5
.2.5. Western blot analysis
In a second series of experiments twenty additional animals (4
5
.
for each group) from groups control, PostC, IPC, Compound A pre
administration of compound A in two doses, 40 min before ische-
(
mia and 1st min of ischemia) and Compound A post (administra-
tion of compound A in two doses, 20 min of ischemia and 1st
min of reperfusion) were subjected in 30 min ischemia followed
by 10 min of reperfusion with the same interventions previously
described where tissue from ischemic area of myocardium was
quickly excised and stored in ꢁ80 °C for Akt and eNOS assessment
with Western blot analysis as previously described.1 In brief tis-
sue-samples were lysated (1% Triton X100, 20 mM Tris pH 7.4–7.6,
6
.
.
Kloner, R. A.; Schwartz Longacre, L. J. Cardiovasc. Pharmacol. Ther. 2011, 16,
223.
Fotopoulou, T.; Iliodromitis, E. K.; Koufaki, M.; Tsotinis, A.; Zoga, A.; Gizas, V.;
Pyriochou, A.; Papapetropoulos, A.; Andreadou, I.; Kremastinos, D. Th. Bioorg.
Med. Chem. 2008, 16, 4523.
3b
7
1
50 mM NaCl, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM Glycer-
olphosphatase, 1% SDS, 100 mM PMSF, 0,1% Protease phosphatase
inhibitor cocktail) and homogenized. After centrifugation at
8. Besada, P.; Costas, T.; Teijeira, M.; Terán, C. Eur. J. Med. Chem. 2010, 45,
114.
Huang, Q.; Herdewijn, P. Eur. J. Org. Chem. 2011, 3450.
6
9.
1
1,000g for 15 min at 4 °C, supernatants were collected, mixed
10. Fang, L.; Appenroth, D.; Decker, M.; Kiehntopf, M.; Roegler, C.; Deufel, T.; Fleck,
C.; Peng, S.; Zhang, Y.; Lehmann, J. J. Med. Chem. 2008, 51, 713.
with Dave’s buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol,
1
1. Jeong, L. S.; Lee, H. W.; Kim, H. O.; Jung, J. Y.; Gao, Z. G.; Duong, H. T.; Rao, S.;
Jacobson, K. A.; Shin, D. H.; Lee, J. A.; Gunaga, P.; Lee, S. K.; Jin, D. Z.; Chun, M.
W.; Moon, H. R. Bioorg. Med. Chem. 2006, 14, 4718.
0
.004% bromophenyl blue, 0.125 M Tris/HCl) and heated in
1
00 °C for 10 min and stored at ꢁ80 °C. The protein concentration
was determined based on the Bradford dye-binding procedure
12. WO2005117910 Inotek Pharmaceuticals.
1
3. (a) Iliodromitis, E. K.; Andreadou, I.; Prokovas, E.; Zoga, A.; Farmakis, D.;
Fotopoulou, T.; Ioannidis, K.; Paraskevaidis, I. A.; Kremastinos, D. Th. Basic Res.
Cardiol. 2010, 105, 193; (b) Andreadou, I.; Farmakis, D.; Prokovas, E.; Sigala, F.;
Zoga, A.; Spyridaki, K.; Papalois, A.; Papapetropoulos, A.; Anastasiou-Nana, M.;
Kremastinos, D. Th.; Iliodromitis, E. K. Cardiovasc. Res. 2012, 94, 501.
4. Argaud, L.; Gateau-Roesch, O.; Muntean, D.; Chalabreysse, L.; Loufouat, J.;
Robert, D.; Ovize, M. J. Mol. Cell. Cardiol. 2005, 38, 367.
5. (a) Manintveld, O. C.; Hekkert, M. L.; van der Ploeg, N. T.; Verdouw, P. D.;
Duncker, D. J. Exp. Biol. Med. (Maywood) 2009, 234, 1345; (b) Halkos, M. E.;
Kerendi, F.; Corvera, J. S.; Wang, N. P.; Kin, H.; Payne, C. S., et al Ann. Thorac.
Surg. 2004, 78, 961; (c) Tsang, A.; Hausenloy, D. J.; Mocanu, M. M.; Yellon, D. M.
Circ. Res. 2004, 95, 230.
(
Biorad, Protein Assay).
An equal amount of protein was loaded in each well and then
was separated by sodium dodecylsulfate–polyacrylamide gel elec-
trophoresis 7.5–11% and transferred onto a polyvinylidene difluo-
ride membrane. After blocking with 5% non-fat dry milk,
membranes were incubated overnight at 4 °C with the following
primary antibodies: phospho-eNOS (Ser1177), eNOS, phospho-
Akt (Ser473) and Akt (Cell Signaling Technology, Beverly, MA,
USA). Membranes were then incubated with secondary antibodies
for 1–2 h at room temperature (Biorad goat anti-mouse and goat
anti-rabbit HRP) and developed using the GE Healthcare ECL Wes-
tern Blotting Detection Reagents (Thermo Scientific Technologies).
Relative densitometry was determined using a computerized soft-
ware package (NIH Image) and the values for phosphorylated eNOS
and Akt were normalized to the values for total eNOS and Akt.
1
1
16. (a) Granfeldt, A.; Lefer, D. J.; Vinten-Johansen, J. Cardiovasc. Res. 2009, 83, 234;
b) Hu, X.; Jiang, H.; Ma, F.; Xu, C.; Bo, C.; Wen, H., et al Int. J. Cardiol. 2009, 144,
35.
(
1
1
7. Koufaki, M.; Detsi, A.; Theodorou, E.; Kiziridi, C.; Calogeropoulou, T.;
Vassilopoulos, A.; Kourounakis, A. P.; Rekka, E.; Kourounakis, P. N.; Gaitanaki,
C.; Papazafiri, P. Bioorg. Med. Chem. 2004, 12, 4835.
18. Petersen Shay, K.; Hagen, T. M. Biogerontology 2009, 10, 443; (a) Yamada, T.;
Hashida, K.; Takarada-Iemata, M.; Matsugo, S.; Hori, O. Neurochem. Int. 2011,
5
9, 1003; (b) WO2005117909. Exelixis. INC.
1
2
9. Salloum, F. N.; Takenoshita, Y.; Ockaili, R. A.; Daoud, V. P.; Chou, E.; Yoshida, K.;
Kukreja, R. C. J. Mol. Cell. Cardiol. 2007, 42, 453.
0. Heusch, G.; Boengler, K.; Schulz, R. Circulation 2008, 118, 1915.
5
.2.6. Data analysis and statistics
All results are presented as means ± standard error (SEM). Data
of myocardial infarct size (%I/R), were compared by one-way anal-
ysis of variance (ANOVA) with Bonferroni correction and Tukey
post-hoc analysis. A calculated P value of less than 0.05 was con-
sidered to be statistically significant.
21. Fan, Q.; Yang, X. C.; Liu, Y.; Wang, L. F.; Liu, S. H.; Ge, Y. G.; Chen, M. L.; Wang,
W.; Zhang, L. K.; Irwin, M. G.; Xia, Z. Clin. Sci. 2011, 120, 251.
2
2
2. Levrand, S.; Vannay-Bouchiche, C.; Pesse, B.; Pacher, P.; Feihl, F.; Waeber, B.;
Liaudet, L. Free Radic. Biol. Med. 2006, 41, 886.
3. Andreadou, I.; Sigala, F.; Iliodromitis, E. K.; Papaefthimiou, M.; Sigalas, C.;
Aligiannis, N.; Savvari, P.; Gorgoulis, V.; Papalabros, E.; Kremastinos, D. Th. J.
Mol. Cell. Cardiol. 2007, 42, 549.
Acknowledgments
24. (a) Downey, J. M.; Davis, A. M.; Cohen, M. V. Heart Fail. Rev. 2007, 12, 181; (b)
Gross, E. R.; Gross, G. J. Cardiovasc. Res. 2006, 70, 212.
This work was supported by Novartis Hellas, the Hellenic Cardi-
ological Society and the European Union’s Seventh Framework Pro-
25. Kremastinos, D. Th.; Bofilis, E.; Karavolias, G.; Papalois, A.; Kaklamanis, L.;
Iliodromitis, E. K. Atherosclerosis 2000, 150, 81.