Design, synthesis and cardioprotective effect of a new class of dual-acting agents: Phenolic tetrahydro-β-carboline RGD peptidomimetic conjugates

Article abstract of DOI:10.1016/j.bmc.2007.08.022

In this study, a new class of phenolic tetrahydro-β-carboline RGD peptidomimetic conjugates was designed and synthesized. The radical scavenging activities of these newly synthesized compounds 12a-c were evaluated in PC12 cell survival assays. The NO scav

Full text of DOI:10.1016/j.bmc.2007.08.022

Bioorganic & Medicinal Chemistry 15 (2007) 6909–6919
Design, synthesis and cardioprotective effect of a new class
of dual-acting agents: Phenolic tetrahydro-b-carboline
RGD peptidomimetic conjugates
a
a
c
b,
*
Wei Bi,a, Jianhui Cai, Sanguang Liu, Michele Baudy-Floc’h and Lanrong Bi
*
`
aSecond Hospital of HeBei Medical University, Shijiazhuang 050000, PR China
bDepartment of Chemistry, Michigan Technological University, Houghton, MI 49931, USA
cUMR 6226 CNRS, Universite´ de Rennes I, F-35042 Rennes Cedex, France
Received 7 May 2007; revised 8 August 2007; accepted 9 August 2007
Available online 21 August 2007
Abstract—In this study, a new class of phenolic tetrahydro-b-carboline RGD peptidomimetic conjugates was designed and synthe-
sized. The radical scavenging activities of these newly synthesized compounds 12a–c were evaluated in PC12 cell survival assays. The
NO scavenging activities of these compounds were confirmed in the acetylcholine-induced vasorelaxation assay. Compounds 12a–c
were efficacious in a rat arterial thrombosis model, and were active in ADP- or PAF-induced in vitro platelet aggregation assays,
which suggests these compounds also possess anti-thrombotic activity. The beneficial effects of dual-acting agent 12c were demon-
strated on the ischemia-reperfusion induced cardiac infarct size and oxidative change in an in vivo rat model.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
radicals is produced when the myocardium is reperfused
following an episode of ischemia and that free radicals
can injure myocytes and endothelial cells. The release
of free radicals, in combination with ischemia induced
decrease in antioxidant activity renders the myocardium
extremely vulnerable.1–3 Thus, targeting free radicals or
improving overall antioxidant defense can be one of the
important therapeutic strategies, whereby excess free
oxy-radicals produced at the time of reperfusion could
be scavenged. It has been reported that a variety of free
radical scavengers and antioxidants are capable of ame-
liorating ischemia-reperfusion injury.4–10
Cardiovascular disease is a major cause of disability and
mortality in developed countries. Prolonged reduction
in coronary blood flow due to atherosclerotic plaques
or vasospasm can result in severe damage to the myocar-
dium, leading to cellular injury and eventual cellular
death due to apoptosis and/or necrosis. Myocardial
reperfusion by thrombolytic therapy, transluminal angi-
oplasty, and bypass surgery is the first line of treatment
for acute myocardial infarction.1 Although restoration
of blood flow to the jeopardized myocardium is a prere-
quisite for myocardial salvage, reperfusion itself may
lead to additional tissue damage, often referred to as
reperfusion injury.2,3 The mechanisms proposed to
cause reperfusion injury include formation of oxygen
free radicals, calcium overload, neutrophil mediated
myocardial and endothelial injury, progressive decline
in microvascular flow to the reperfused myocardium
and depletion of high-energy phosphate stores.2,3
Numerous studies suggest that a transient burst of free
Tetrahydro-b-carbolines are naturally occurring sub-
stances found in food, alcoholic and non-alcoholic
drinks, and fruit-derived products. It has been reported
that tetrahydro-b-carbolines are potent antioxidants
and may thus be useful for the prevention of diseases
associated with oxidative damage.11–15 Furthermore, it
is well recognized that phenolic compounds in foods
possess several interesting biological and chemical prop-
erties, such as antioxidant activity and the ability to
scavenge reactive oxygen species.16,17 Hence, the pheno-
lic compounds may prevent various diseases associated
with oxidative stress, such as cancers, cardiovascular
diseases, and inflammation. In addition, phenolic tetra-
hydro-b-carbolines have been identified as effective free
radical scavengers and antioxidants.13,24
Keywords: Dual-acting agents; Phenolic tetrahydro-b-carboline RGD
peptidomimetics conjugates; Free radicals scavenger; Antithrombotic
agent.
*
Corresponding authors. Tel.: +1 906 487 1868; fax: +1 906 487 2061
(L.Bi); e-mail: lanrong@mtu.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.022

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W. Bi et al. / Bioorg. Med. Chem. 15 (2007) 6909–6919
Since acute myocardial ischemia is usually due to a
thrombus event in the coronary arteries, causal treat-
ment and secondary prevention always include one or
more forms of anti-thrombotic therapies, such as anti-
platelet agents, heparins, or fibrinolytic drugs.1 RGD
(Arg-Gly-Asp) containing peptides have been investi-
gated to develop therapeutic agents for treating throm-
bosis.18–20 The mechanism of action of RGD peptides
is inhibition cell adhesion to extracellular matrix pro-
teins via binding of RGD peptides to the integrin recep-
tors on the cell surface.21 Several anti-thrombotic agents
have been derived from RGD peptides.18,22 For exam-
ple, Aggrastatꢁ, an RGD peptidomimetic, has been
used clinically for treating patients with thrombosis.23
In our previous studies, RGD-S, RGD-V, and RGD-F
were used as building blocks for modification of the pep-
tide conjugates with anti-thrombotic activity.24
2. Results and discussion
2.1. Synthesis of phenolic tetrahydro-b-carboline
For the synthesis of the phenolic tetrahydro-b-carboline
RGD peptidomimetic conjugates, L-tryptophan was first
reacted with phenolic aldehyde (Scheme 1). Specifically,
syringaldehyde in acidic-aqueous conditions afforded 1
in 46% yield. This reaction occurred through a Pictet-
Spengler intramolecular cyclization of the Schiff base
to afford the 1,3-disubstituted tetrahydro-b-carbolines.
1,3-Disubstituted tetrahydro-b-carbolines that arise
from L-tryptophan and syringaldehyde included two
diastereoisomers (1S,3S-1 and 1R,3S-1), which were
then separated via RP-HPLC. Diastereoisomer assign-
ments were based on acquired positive NOE spectra of
H-1 and H-3 signals in the cis-isomer. H-1 signals corre-
sponding to the cis-isomer appeared at higher field than
in the trans-isomer, whereas C-1 and C-3 NMR signals
corresponding to the trans-isomer appeared at higher
field than those of the cis-isomer. This observation is
in agreement with the expected pattern for 1,3-disubsti-
tuted tetrahydro-b-carbolines. Subsequent treatment of
1a with Boc-N3 gave the N-Boc protected 1b in 50%
yield.24
However, one of the major potential problems limiting
the use of natural peptides as therapeutic agents is their
relatively poor stability. In general, such peptides are
short-lived molecules that are rapidly degraded by pro-
teases present in any biological fluid, particularly in an
inflammatory environment. Therefore, it is important
to develop stable peptide analogues that conserve their
biological activity.
2.2. Synthesis of monomers
Recently, we have developed a new class of peptidomi-
metics containing aza-b3-amino acid residues.28–30 Aza-
b3-amino acid monomers, which can be considered as
Na-substituted hydrazino acetic acid monomers, have
no chiral center. Substitution of a-amino acid residues
by aza-b3-amino acid residues overcomes stereochemical
constraints and may contribute to enhanced proteolytic
resistance. The resulting mixed a- and aza-b3-peptide
analogues have nitrogen-enriched peptide backbones,
with nitrogen atoms carrying side chains that mimic
proteinogenic amino acids.25–30 More recently, we have
investigated the possibility that these new peptidomi-
metics might replace the unstable natural peptides as
therapeutic agents.30
For the synthesis of Fmoc-aza-b3-Arg (Boc)2-OH, it was
critical to protect the trifunctional guanidine group due
to its strong nucleophilic character. The Goodman re-
agent N,N0-di-Boc-N00-trifluoromethane sulfonylguani-
dine was used as a versatile guanidinating reagent.
Briefly, starting from the commercially available diethy-
lacetal of 3-aminopropanal, after the treatment with
Boc2O, 3-t-butoxycarbonyl aminopropanal 2 was read-
ily obtained in good yield (90%), which was then sub-
jected to hydrolysis and condensation with Fmoc
hydrazine to give hydrazone 4. After the reduction with
sodium cynanoborohydride (NaBH3CN), hydrazone 4
was converted to N,N0-disubstituted hydrazine 5 in
82% yield. Subsequently, after the nucleophilic substitu-
tion with benzyl bromoacetate and removal of the NH-
Boc protecting group, Fmoc-Aza-b3-Orn-OBn was
obtained in moderate yields (60%), which was then
converted to 7 by treatment with guanidinating agent.
Finally, deprotection of the benzyl-protecting group
with Pd/C (10%) in ethanol proceeded successfully to
Considering the free radical scavenging properties
possessed by phenolic tetrahydro-b-carboline and the
anti-thrombotic activity demonstrated by RGD pepti-
domimetics, we sought to link a phenolic tetrahydro-
b-carboline moiety to the anti-thrombotic RGD
peptidomimetics in the hope that the resulting phenolic
tetrahydro-b-carboline RGD peptidomimetic conju-
gates would exhibit free radical scavenging activity,
anti-thrombotic activity, and enhanced stability.
give the expected Fmoc-aza-b3-Arg(Boc)2-OH
8
(Scheme 2). In addition, Fmoc-aza-b3-Gly(Boc)-OH 9
and Fmoc-aza-b3-Asp(OtBu)-OH 10 were successfully
COOH
NH
COOH
Boc
H
O
N
COOH
N
H
N
H
ii
i
+
NH2
N
H
OCH3
OH
OCH3
OH
H3CO
OCH3
OH
H3CO
H3CO
1b
1a
Scheme 1. Synthesis of phenolic tetrahydro-b-carboline. Reagents and conditions: (i) H2SO4, 70 ꢂC; (ii) Boc-N3, 0 ꢂC ! rt.

W. Bi et al. / Bioorg. Med. Chem. 15 (2007) 6909–6919
6911
NHBoc
N
OEt
OEt
OEt
ii
i
iii
H
CHO
BocHN
OEt
H2N
BocHN
N
Fmoc
2
3
4
BocHN
HN
NBoc
H2N
BocHN
NHBoc
viii
O
vi
O
vii
iv
v
H
8
O
Fmoc
N
Fmoc
N
N
N
N
H
OBz
N
H
OBz
H
Fmoc
N
N
Fmoc
OBz
H
6a
6b
5
7
Scheme 2. Synthesis of Fmoc-aza-b3-Arg (Boc)2-OH. Reagents and conditions: (i) (Boc)2O/NEt3, 90%; (ii) AcOH, 98%; (iii) FmocNHNH2, 89%; (iv)
NaBH3CN, 82%; (v) BrCH2CO2Bn, K2CO3, toluene, 48 h, 60%; (vi) CF3CO2H, NEt3, 95%; (vii) (BocNH)2NTf, NEt3, 84%; (viii) H2 (1 atm), Pd/C,
96%.
synthesized according to our previously reported meth-
od (Scheme 3).28,29
2.4. Scavenging activities determined by cell survival
assay in PC12 cells
2.3. Solid-phase synthesis of phenolic tetrahydro-b-carb-
oline RGD peptidomimetic conjugates
The free radical scavenging activities of 12a–c against
NO, H2O2, and •OH were evaluated in PC12 cells and
compared with that of 1a using our previously reported
method.24,31–35 The results were expressed as EC50 (lM)
values.
The targeted phenolic tetrahydro-b-carboline RGD pep-
tidomimetic conjugates 12a–c were manually synthe-
sized according to our previously reported method.
The general procedure for the solid-phase synthesis of
hybrid peptides using the Fmoc/tert-butyl strategy was
depicted in Figure 1.28,29
As shown in Table 1, the EC50 values of 12a, 12b, and
12c were found to be similar to that of 1a, which sug-
gested that they were as effective scavengers of NO,
H2O2, and •OH as 1a. The scavenging capacity (EC50)
among all of the tested compounds ranged from ꢀ88
to 96 lM for NO, from ꢀ34 to 75 lM for H2O2, and
ꢀ86 to 95 lM for •OH.
Briefly, the Fmoc-Val-OH Wang resin was first liberated
upon treatment with 20% piperidine in dimethylform-
amide (DMF), for elongating the peptide chain, subse-
quently the resin was treated with a solution of 4
equiv of Fmoc-a-amino acid or Fmoc-aza-b3-amino
acid activated by diisopropyl-carbodiimide (DIC)/1-
hydroxybenzotriazole (HOBt) at room temperature for
30–80 min (depending on the amino acid). Coupling cy-
cles for Fmoc-aza-b3-amino acids and coupling cycles
for a-amino acid with the analogue proceeded from
80 min to 2 h while those for Fmoc-a-amino acids pro-
ceeded for 30–60 min. Deprotection cycles were realized
with 20% piperidine in DMF. The hybrid peptides were
then deprotected and cleaved from the resin with TFA/
phenol/TIS/H2O (88:5:2:5), and after preparative
HPLC, the hybrid peptides (25–45% yield) were isolated
in >98% purity. All the peptidomimetics were character-
ized by MALDI-TOF MS.
2.5. NO scavenging activity determined using rat aortic
strip
The NO scavenging activities of 12a–c were further eval-
uated in the acetylcholine (ACh)-evoked, endothelium-
mediated relaxation assay according to our previously
reported method.24,33–35 The endothelium controls the
tone of the underlying vascular smooth muscle through
the production of vasodilator mediators.36–41 In this
experimental model, ACh acts on the endothelium to re-
lease nitric oxide (NO), a potent vasodilating mediator.
The decreased relaxation response in a rat aortic strip
could be attributed to a reduction in NO synthesis by
the endothelium. Therefore, the present study was
undertaken to assess the NO scavenging capability of
these compounds. The results were expressed as the per-
centage inhibition of acetylcholine (ACh)-induced vaso-
relaxation by test compounds and are summarized in
Table 2. It was observed that ACh-induced relaxation
was reversed significantly by compounds 1a and 12a–c.
At a concentration of 10ꢁ6 mol/L, compound 1a showed
good inhibition ability with 81.03 4.12%, whereas
compounds 12a–c displayed higher inhibition activities
at 90.12 1.63, 95.77 2.93, and 97.15 1.15%, respec-
tively. We hypothesized that the NO scavenging activity
of compounds 12a–c led to attenuation of the NO con-
centration in vitro, which then led to the inhibition of
ACh-induced vasorelaxation.42–44
O
Boc
N
O
H
N
i
ii,iii
Boc
BocNHNH2
iv
N
H
OBz
H2N
OBz
9a
9b
Boc
N
O
Boc
N
O
v
Fmoc
Fmoc
N
H
OH
N
H
OBz
9c
9
Scheme 3. Reagents and conditions: (1) BrCH2CO2Bz, DIEA, toluene,
48 h, 61%; (ii) HClg, NEt3, 85%; (iii) (Boc)2O/NEt3, CH2Cl2, 60%; (iv)
FmocCl, 71%; (v) H2 (1 atm), 10% Pd/C, 96%.

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W. Bi et al. / Bioorg. Med. Chem. 15 (2007) 6909–6919
BocHN
NBoc
HN
OtBu
O
Boc
N
O
O
O
N
N
Fmoc
Fmoc
N
Fmoc
N
OH
OH
N
OH
H
H
H
8
9
10
R
H2N
HN
NH
Linker
Fmoc
N
H
OH
O
O
O
O
O
20% piperidine in DMF
R
H
N
N
OH
N
N
H
N
H
H
O
O
NH
Linker
N
H
H2N
O
OCH3
OH
H2N
HN
12a
H3CO
Fmoc-aa-OH or Fmoc-aza-beta(3)-aa-OH
DMF, HOBT, DIC
coupling
NH
O
R
O
O
H
N
H
N
FmocHN
Linker
OH
aa
N
N
H
N
H
N
n
H
O
H
O
O
O
O
or aza-beta(3)-aa
NH
N
H
OH
20% piperidine in DMF
OCH3
OH
H3CO
12b
deprotection
O
R
H2N
Linker
aa
N
H2N
NH
n
H
O
or aza-beta(3)-aa
HN
TFA/phenol/TIS/water
1b
O
O
O
coupling
11a,b,c
H
N
N
OH
N
N
H
N
H
H
O
O
O
NH
N
H
Cleavage
TFA/phenol/TIS/water
OH
OCH3
OH
H3CO
12c
12a,b,c
H2N
HN
NH
H2N
NH
H2N
HN
NH
HN
OH
N
O
O
O
O
O
O
H
N
H
H
H
N
OH
N
N
OH
N
OH
H2N
N
H
N
H
N
N
H
H2N
N
N
H2N
H
H
H
O
O
O
O
O
O
O
O
O
OH
OH
11b
11c
11a
Figure 1. Solid phase synthesis of compounds 11a–c and 12a–c.
2.6. Antiplatelet aggregation in vitro
Table 1. Free radicals scavenging activities in PC12 cell survival assays
Compound
EC50 (lM) values ðx ꢂ SDÞ
In vitro anti-platelet aggregation activities of com-
pounds 1a, 11a–c, and 12a–c were again evaluated
according to our previously reported method.24,33–35
Platelet-rich plasma was prepared by centrifugation of
normal rabbit blood anti-coagulated with sodium citrate
at a final concentration of 3.8%. The platelet counts
EC50/NO
EC50/H2O2
EC50/ÅOH
1a
88.40 1.39
90.15 2.01
91.20 1.90
96.46 3.56
34.52 2.19
45.68 3.10
41.20 2.00
75.35 1.90
85.90 1.59
93.21 1.67
90.30 3.09
95.12 2.03
12a
12b
12c

W. Bi et al. / Bioorg. Med. Chem. 15 (2007) 6909–6919
6913
Table 2. Inhibition of ACh-induced vasorelaxation
gation curve. The obtained data are listed in Tables 3
and 4, respectively.
Compound
Inhibition percentage ðx ꢂ SD%Þ
10ꢁ6 mol/L
10ꢁ7 mol/L
10ꢁ8 mol/L
An anti-platelet aggregation assay was used to evaluate
the effects of compounds 12a–c utilizing in vitro rabbit
platelet aggregation induced by adenosine 5-diphos-
phate (ADP) or platelet activating factor (PAF). The
in vitro assays indicated that this new class of phenolic
tetrahydro-b-carboline RGD peptidomimetic conju-
gates was very likely to have potent platelet aggregation
inhibition based on in vitro induced platelet aggrega-
tion. From Tables 3 and 4, it was noted that compounds
12a–c displayed a remarkable dual anti-platelet aggrega-
tion activity in both ADP- and PAF-induced platelet
aggregation assays. For the ADP-induced platelet assay,
with 12a–c at a concentration of 10ꢁ6 mol/L, the platelet
aggregation rates were collectively decreased to ꢀ28–
30% (NS: 55.80 4.32; 1a: 47.83 3.58, n = 8,
p < 0.001). A similar observation was observed in the
PAF-induced platelet aggregation assay.
NS
1a
1.53 1.31
81.03 4.12a,b
90.12 1.63a,b
95.77 2.93a,b
97.15 1.15a,b
55.32 1.52a,c
63.72 3.54a,c
71.03 0.95a,c
75.31 2.45a,c
10.81 2.40a
12.98 2.16a
14.07 3.92a
28.30 1.91a
12a
12b
12c
a In comparison with normal saline NS, p < 0.001, n = 6.
b In comparison with 10ꢁ7 and 10ꢁ8 mol/L of the same compound,
p < 0.001.
c In comparison with 10ꢁ8 mol/L of the same compound, p < 0.001.
Table 3. Effect of1a, RGDV, 11a–c, and 12a–c on ADP-induced
platelet aggregation
Compound
Am% ðx ꢂ SDÞ
10ꢁ7 mol/L
10ꢁ6 mol/L
10ꢁ5 mol/L
NS
1a
55.80 4.32
47.83 3.58a
28.76 2.23
40.08 1.29a
28.20 1.03a
20.20 1.41a
27.46 4.79a,c
28.32 2.16a,c
25.29 1.09a,c
54.67 1.88
51.00 2.01
49.10 2.43
45.00 2.15b
35.89 0.23a
44.86 2.56b
52.16 4.02b
36.95 2.16a,c
25.35 1.90a
11.79 1.34
18.03 1.32a
12.09 1.47a
6.58 1.55a
10.65 2.09a,c
15.32 1.29a,c
10.89 4.01a,c
RGDV
11a
11b
11c
12a
12b
12c
2.7. Evaluation of anti-thrombotic effect in vivo
The anti-thrombotic effects of compounds 11a–c and
12a–c were evaluated using an in vivo rat model accord-
ing to our previously reported methods,24,33–35 and were
expressed as the reduction of thrombus mass. The ob-
tained results are summarized in Table 5. As shown in
Table 5, the thrombus weight after treatment with
12a–c was 4.85 0.93, 3.79 0.29, and 2.23 0.16 mg,
respectively (NS: 9.28 1.32, p < 0.01). Thus, the anti-
thrombotic activities of 12a–c were higher than those
of 11a–c at a dosage of 5 lmol/kg.
N = 8; NS = vehicle.
a Compared to NS, p < 0.001.
b Compared to NS, p < 0.01.
c Compared to 1a, p < 0.001.
Table 4. Effect of 1a, RGDV, 11a–c, and 12a–c on PAF-induced
platelet aggregation
2.8. Stability of 12a–c to trypsin in vitro
Compound
Am% ðx ꢂ SDÞ
Ten milligrams of each of tested compounds 12a–c were
dissolved in 1 mL of phosphate buffer (pH 8), and
0.5 mg of trypsin was added. The reaction mixture was
kept at 37 ꢂC and the concentration of the tested com-
pound was monitored every 1 h by HPLC; the mobile
phase was 25% CH3OH in water containing 0.1%
CF3COOH. The results indicated that the depletion of
12a, 12b, and 12c observed after the enzyme promoted
hydrolysis was postponed for 4, 3, and 4 h, respectively.
10ꢁ7 mol/L
10ꢁ6 mol/L
10ꢁ5 mol/L
NS
1a
57.93 3.57
53.40 2.13
34.00 3.23a
37.32 1.69a
36.43 2.94a
22.31 3.25a
33.29 4.10a
32.46 2.15a
24.31 3.39a,c
55.03 1.29
54.23 2.10
56.15 2.63
54.93 1.93
41.03 2.18a
53.01 2.11b
50.26 1.79b
35.10 1.23a,c
37.95 3.56a
20.03 2.89a
22.36 2.96a
20.54 2.46a
8.39 1.67a
21.61 2.14a,c
26.30 1.49a,c
9.18 1.64a,c
RGDV
11a
11b
11c
12a
12b
12c
N = 8; NS = vehicle.
Table 5. Effect of the compounds 1a, 11a–c and 12a–c on thrombus
weight ðx ꢂ SDÞ
a Compare to NS, p < 0.001.
b Compared to NS, p < 0.01.
c Compared to 1a, p < 0.001.
Compound
Dosage
Wet thrombus
(mg)
Dry thrombus
(mg)
NS
1a
3 mL/kg
46.03 3.12
44.01 2.06
35.75 2.19
41.80 3.23
34.98 2.47b
27.04 1.50a
25.46 3.19a
24.54 4.46a
21.05 2.14a
9.28 1.32
8.03 0.57
6.78 2.43
7.83 1.32b
7.05 1.25b
4.39 1.65b
4.85 0.93b
3.79 0.29b
2.23 0.16a
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
5 lmol/kg
were adjusted to 2–105 per lL through addition of autol-
ogous plasma. Platelet aggregation studies were con-
ducted in an aggregometer using the standard
turbidimetric technique. The agonists used were platelet
activating factor (PAF, final concentration 10ꢁ7 mol/L)
and adenosine diphosphate (ADP, final concentration
10ꢁ5 mol/L). The effects of compounds 1a, 11a–c, 12a–
c on PAF- or ADP-induced platelet aggregation were
studied. The maximal rate of platelet aggregation
(Am%) was represented by the peak height of the aggre-
RGDV
11a
11b
11c
12a
12b
12c
N = 10.
a In comparison with normal saline (NS), p < 0.001.
b In comparison with NS, p < 0.01.

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W. Bi et al. / Bioorg. Med. Chem. 15 (2007) 6909–6919
Table 6. Effects of 12c on myocardial MDA content after reperfusion
resulting from I/R was also significantly reduced by the
administration of 12c. These results suggest that this
dual-acting agent may reduce ischemia-reperfusion in-
jury induced damage.
Treatment
MDA (nmol/mg prot.)
Sham I/R + vehicle
Sham I/R + 12c
I/R + vehicle
2.69 0.53
2.52 0.39
4.90 0.87
2.91 0.63a
I/R + 12c
3. Conclusion
N = 8.
a Compared with I/R + vehicle (p < 0.05).
In conclusion, we have demonstrated that phenolic tet-
rahydro-b-carboline RGD peptidomimetic conjugates
12a–c have as good free radicals scavenging activity as
parental compound 1a as shown by the EC50 values in
the PC12 cell survival assay. We have further confirmed
the potent NO scavenging activity of compounds 12a–c
in the ACh-induced vasorelaxation assay. In addition,
we have shown that 12a–c are efficacious in a rat arterial
thrombosis model and active in ADP- or PAF-induced
in vitro platelet aggregation assay, which suggest that
compounds 12a–c also possess anti-thrombotic activity.
To explore the possible cardioprotective effect of one of
these dual-acting pharmacological agents against oxida-
tive stress during I/R injury of the heart, we have further
explored the beneficial effects of 12c on the I/R-induced
cardiac infarct size and oxidative change in an in vivo
rat model.
2.9. Effects of dual-acting agent 12c on MDA formation
Malondialdehyde (MDA), a stable metabolite of the free
radical-mediated lipid peroxidation cascade, is widely
used as marker of oxidative stress. Recent studies have
demonstrated that ischemia-reperfusion of the tissue is
associated with lipid peroxidation, which is an autocat-
alytic mechanism leading to oxidative destruction of the
cellular membranes. The destruction can lead to the pro-
duction of toxic, reactive metabolites and cell death. Li-
pid peroxidation, as the free radicals generating system,
is related to I/R-induced tissue damage, and MDA is a
typical indicator of the rate of lipid peroxidation.45,46
We therefore investigated the MDA levels so as to eval-
uate the effect of this dual-acting agent (i.e., 12c) on
myocardial I/R damage. As shown in Table 6, after
120 min of reperfusion, MDA content was low in
sham-I/R hearts with 12c treatment. In the I/R groups,
without 12c pretreatment, the MDA content was
4.90 0.87 nmol/mg protein. 12c reduced the MDA
content to 2.91 0.63 nmol/mg protein. Clearly, the lev-
els of MDA significantly increased due to I/R. After
administration of the dual-acting agent 12c, the levels
of MDA significantly decreased suggesting that 12c
might cause reduction in lipid peroxidation and cellular
injury. We speculated that the protective effect of this
newly synthesized dual-acting agent might occur, in
part, due to the scavenging of any highly reactive •OH
and OONOꢁ.
Taken together, the overall beneficial effect of the dual-
acting agent probably can be attributed to its activity as
a potent free radical scavenger and anti-thrombotic
agent. In particular, the protective action of the dual-
acting agent most likely can be attributed to the reduc-
tion of free radical-mediated lipid peroxidation, thereby
enhancing anti-oxidative defense system. In addition,
the function of the dual-acting agent as antithrombotic
agent may also play a major synergistic role in aiding
the recovery of I/R injured tissue.
Since this novel class of potential dual-action agents has
anti-thrombotic, free radical scavenging capabilities,
and enhanced stability, we expect they may provide ben-
eficial effects in ischemia-reperfusion related injuries.
2.10. Reduction of infarct size with the dual-acting agent
12c
4. Experimental
4.1. Chemistry
In this study, hearts were arrested by global ischemia for
30 min. The whole ventricle was therefore considered as
the area of risk. Percentage of infarct size was signifi-
cantly reduced for the 12c treated group compared with
the I/R+ vehicle group (Table 7).
4.1.1. Synthesis of the monomers
4.1.1.1. (1S,3S)- and (1R,3S)-1-(40-hydroxy-30,50-
dimethoxyphenyl)-1,2,3,4-tetrahydro-b-carboline-3-car-
boxylic acid (cis-1 and trans–1). L-Tryptophan (306 mg,
1.5 mmol) dissolved in 0.05 N H2SO4 (13 mL) was stir-
red with the syringaldehyde (1.65 mmol) (Aldrich).
The reaction mixture was allowed to react at 70 ꢂC for
6 days, and a precipitate of 1 was obtained (1.1:1)
(254 mg, 46%). cis-1: 1H NMR (CD3OD + TFA) d
3.77 (m, 2 H, H-4), 3.97–4.02 [2s, 6H, (OCH3)2], 4.80
(dd, 1H, H-3, J3,4a = 12.1 Hz, J3,4b = 5.3 Hz), 6.01 (s,
1H, H-1), 6.85, 6.98 (2s, 2H, Ph), 7.2–7.65 (m, 4H, in-
dol); 13C NMR (CD3OD + TFA) d 23.73 (C-4), 56.91
(OCH3), 57.69 (C-3), 60.58 (C-1), 108.30 (C-4a),
108.47 (Ph), 112.56 (C-8), 119.25 (C-5), 120.84 (C-6),
124.04 (C-7), 124.45 (Ph), 127.16 (C-4b), 129.43
I/R increased MDA levels comparative to control and
dual-acting agent 12c administration significantly re-
duced the increased MDA levels. The cardiac infarct size
Table 7. Summary of infarct size data
Groups
Risk zone
(cm3)
Infarct size
(cm3)
Infarct
size/risk
zone (%)
I/R + vehicle
I/R + 12c
46
48
3
2
22
3
50
4
17 2a
35 2a
N = 8.
a Compared with I/R + vehicle (p < 0.05). Dose = 10 mg/kg.

W. Bi et al. / Bioorg. Med. Chem. 15 (2007) 6909–6919
6915
(C-9a), 138.78 (C-8a), 149.73, 158.84, 160.24 (Ph),
171.06 (COOH); RP-HPLC-ESI-MS (tR = 15.95 min)
(M+H)+ 369, (M+H-73) 296. trans-1: 1H NMR
(CD3OD + TFA) d 4.72 (m, 1H, H-3), 6.22 (s, 1H, H-
1); 13C NMR (CD3OD + TFA) d 53.60 (C-3), 57.37
(C-1); other signals as cis-1: RP-HPLC-ESI-MS
(tR = 16.8 min) (M+H)+ 369, (M+H-73) 296.
(d), 120.5 (d), 77.7 (s), 65.8 (t), 47.1 (d), 38.4 (t),
28.6 (q), 28.2 (t). Anal. Calcd for C23H29N3O4
(411.22): C, 67.12; H, 7.11; N, 10.22. Found: C,
67.22; H, 7.19; N, 10.24.
4.1.1.5. Fmoc-Aza-b3-Orn(NBoc)-OBn (6a). A mix-
ture of Fmoc-protected ornithine hydrazine (5) (3.7 g,
9 mmol), benzyl 2-bromoacetate (2.66 g, 11.6 mmol),
toluene (20 mL), and dry K2CO3 (870 mg, 0.7 equiv)
was refluxed under stirring for 28 h. After the workup
followed by flash column chromatography (EtOAc/hex-
ane, 1:3), 3.02 g (60%) of the title compound was ob-
tained as a colorless oil that slowly crystallized in
ether: mp 107 ꢂC; 1H NMR (CDCl3) d 1.50 (s, 9H,
CH3), 1.62 (m, 2H, CH2), 2.97 (m, 2H, CH2), 3.24 (m,
2H, CH2), 3.75 (m, 2H, CH2), 4.20 (t, 1H, J = 6.9 Hz,
CH), 4.50 (d, 2H, J = 6.9 Hz, CH2), 5.19 (s, 2H, CH2),
6.88 (br s, 1H, NH), 7.25–7.90 (m, 13H, Ar). 13C
NMR (DMSO) d 171.23 (s), 156.55 (s), 155.50 (s),
144.13, 141.77 (s), 135.54 C (s), 129.13, 129.04, 128.84
(d), 128.14, 127.47, 125.44, 120.40 (d), 79.38 (s), 67.09
(t), 64.47 (t), 57.63 (t), 54.56 (t), 47.67 (d), 38.83 (t),
28.85 (q), 27.83 (t). Anal. Calcd for C32H37N3O6
(559.27): C, 68.66; H, 6.67; N, 7.51. Found: C, 68.59;
H, 6.86; N, 7.28.
4.1.1.2. 1-Boc-amino-3,3-diethoxypropane (2). A mix-
ture of Boc2O (9 g, 40 mmol) in dioxane (40 mL) was
added dropwise to a stirred solution of 1-amino-3,3-
diethoxypropane (5.52 g, 37 mmol) and Et3N (4.04 g,
40 mmol) in dioxane (5 mL) at 0 ꢂC. After 2 h, the mix-
ture was allowed to warm to rt. Stirring was continued
overnight, and the solvent was evaporated. The residual
oil was taken up in water (10 mL), and the mixture was
acidified with an aqueous solution of 1 N HCl (pH 3–4)
and extracted with EtOAc (60 mL · 3). The organic
layer was dried (Na2SO4) and evaporated to give a crude
oil of 1-Boc-amino-3,3-diethoxypropane 8.89 g (90%)
suitable for further work without further purification.
1H NMR (CDCl3) d 1.25 (t, 6H, J = 8.8 Hz, CH3),
1.48 (s, 9H, CH3), 1.85 (q, 2H, J = 6.3 Hz, CH2), 3.26
(q, 2H, J = 6.1 Hz, CH2), 3.22–3.77 (m, 6H, CH2),
4.58 (t, 1H, J = 6.3 Hz, CH), 4.95 (br s, 1H, NH).
4.1.1.3. 3-Boc-aminopropanal (3). A solution of 1-
Boc-amino-3,3-diethoxypropane (8.89 g, 36 mmol) in
AcOH (15 mL) and H2O (4 mL) was stirred at rt for
10 h, neutralized with Na2CO3, taken up in ether, and
washed with brine. The organic phase was evaporated
under vacuum to give a yellow oil used as such in the
next step (6.31 g, 98%). 1H NMR (CDCl3) d l.47 (s,
9H, CH3), 2.75 (t, 2H, CH2), 3.46 (m, 2H, CH2), 4.95
(br s, 1H, NH), 9.85 (s, 1H, CHO).
4.1.1.6. Fmoc-Aza-b3-Arg(Boc)2OBn (7). To a solu-
tion of 6a (1 g, 1.79 mmol) in CH2Cl2 (5 mL), 5 mL of
TFA was added. After stirring at rt overnight, 25 mL
of CH2Cl2 and 10 mL of water were added. The mixture
was neutralized with solid Na2CO3 (pH 8–9), and the or-
ganic layer was washed with brine and dried over
Na2SO4. After concentration, without further purifica-
tion, the crude product 6b was directly subjected to
the next step. 6b was diluted with 10–15 mL of CH2Cl2,
and then NEt3 (1.1 equiv, 251 L) and (BocNH)2C = NTf
(0.9 equiv, 0.64 g) were added. After stirring at rt for
15 h, the solution was sequentially washed with a 2 N
aqueous solution of NaHSO4 (10 mL), Na2CO3
(10 mL), and brine (10 mL). The organic layer was dried
over Na2SO4. After concentration, the crude product
was further purified by flash chromatography (hexane/
EtOAc, 7:3) to afford the title compound 7 (1.00 g,
80%). 1H NMR (CDCl3) d 1.39 (s, 18H, CH3), 1.60
(m, 2H, CH2), 2.80 (m, 2H, CH2), 3.32 (m, 2H, CH2),
3.66 (m, 2H, CH2), 4.24 (t, 1H, J = 7.1 Hz, CH), 4.45
(d, 2H, J = 7.1 Hz, CH2), 5.05 (s, 2H, CH2), 6.95 (br s,
1H, NH), 7.27–7.82 (m, 13H, Ar), 8.28 (br s, 1H,
NH), 11.45 (br s, 1H, NH). 13C NMR (DMSO) d
171.45 (s), 170.80 (s), 163.97 (s), 156.57 (s), 153.59 (s),
144.17 (s), 141.73 (s), 135.67 (d), 129.06, 128.91,
128.78 (d), 128.09, 127.45, 125.47, 120.34 (d), 83.35 (s),
79.48 (s), 66.93 (t), 60.74 (t), 58.04 (t), 53.89 (t), 47.64
(d), 38.83 (t), 28.67 (q), 28.44 (q), 27.45 (t). Anal. Calcd
for C38H47N5O8 (701.34): C, 65.02; H, 6.75; N, 9.98.
Found: C, 65.10; H, 6.83; N, 9.98.
4.1.1.4. Fmoc-protected ornithine hydrazine (5). Fmoc
carbazate (9.24 g, 36.4 mmol) was added to a stirred
solution of 3-Boc-aminopropanal (6.31 g, 36.4 mmol)
in CH2Cl2 (150 mL) at rt The reaction mixture was stir-
red for 12 h and concentrated under vacuum to give a
crude solid that was triturated with petroleum ether to
afford the hydrazone (4) as a white solid (13.2 g, 89%).
1H NMR (CDCl3) d 1.38 (s, 9H, CH3), 2.31 (m, 2H,
CH2), 3.11 (m, 2H, CH2), 4.28 (t, 1H, J = 6.8 Hz,
CH), 4.41 (d, 2 H, J = 6.8 Hz, CH2), 6.91 (br, 1H,
NH), 7.28–7.93 (m, 9H, Ar + CH), 10.84 (s, 1H, NH).
13C NMR (DMSO) d 155.5 (s), 153.2 (s), 146.2 (s),
143.7 (s), 140.72 (s), 127.6 (d), 127.0 (d), 125.1 (d),
120.1 (d), 77.5 (s), 65.4 (t), 46.6 (d), 37.3 (t), 28.8 (t),
28.2 (q).
Fmoc-protected hydrazone (4) (3 g, 7.31 mmol) was
then reduced with sodium cyanoborohydride (0.55 g,
1.2 equiv). The crude solid was triturated with petro-
leum ether to afford a white solid of 5 (2.48 g 82%):
mp 101 ꢂC; 1H NMR (CDCl3) d 1.40 (s, 9H, CH3),
1.50 (m, 2H, CH2), 2.68 (m, 2H, CH2), 2.98 (m,
2H, CH2), 4.24 (t, 1H, J = 6.9 Hz, CH), 4.32 (d,
2H, J = 6.9 Hz, CH2), 4.70–4.85 (br s, 1H, NH),
6.78 (br s, 1H, NH), 6.80 (br s, 1H, NH), 7.25–7.90
(m, 8H, Ar). 13C NMR (DMSO) d 157.2 (s), 155.9
(s), 144.2 (s), 142.9 (s), 128.0 (d), 127.6 (d), 125.6
4.1.1.7. Fmoc-Aza-b3-Arg(Boc)2-OH (8). Catalytic
hydrogenolysis with 10% Pd/C (50 mg) of 7 (0.86 g,
0.5 mmol) afforded 0.72 g (96%) of the title compound
1
8 as a colorless oil. H NMR (CDCl3) d 1.38 (s, 9H,
CH3), 1.39 (s, 9H, CH3), 1.62 (m, 2H, CH2), 2.89 (m,
2H, CH2), 3.38 (m, 2H, CH2), 3.60 (m, 2H, CH2), 4.14

6916
W. Bi et al. / Bioorg. Med. Chem. 15 (2007) 6909–6919
(t, 1H, J = 7.1 Hz, CH), 4.40 (d, 2H, J = 7.1 Hz, CH2),
7.27–7.82 (m, 8H, Ar), 7.95 (br s, 1H, NH), 8.50 (br
s,1H, NH), 11.50 (br s, 1H, NH). 13C NMR (CDCl3)
d 171.35 (s), 170.70 (s), 163.90 (s), 157.45 (s), 153.57
(s), 143.96 (s), 141.75 (s), 128.18 (d), 127.52 (d), 125.44
(d), 120.38 (d), 83.35 (s), 79.60 (s), 67.42 (t), 58.04 (t),
53.89 (t), 47.61 (d), 38.83 (t), 28.59 (q), 28.46 (q),
27.47 (t). HRMS m/z calcd for C31H41N5O8 (M+):
611.2955, found: 611.2960.
156.5 (s), 155.4 (s), 144.1 (s), 141.9 (s), 135.8 (s), 129.3
(d), 129.2 (d), 129.0 (d), 128.9 (d), 128.5 (d), 125.6 (d),
120.7 (d), 83.1 (s), 68.6 (t), 67.8 (t), 53.5 (t), 47.7 (d),
28.7 (q). HRMS (ESI) m/z calcd for C29H30N2O6Na
[M+Na]+: 525.2001, found: 525.2016.
4.1.1.11. Fmoc-Aza-b3-Gly(Boc)-OH (9). Catalytic
hydrogenolysis with 10% Pd/C (100 mg) of compound
9c (0.75g, 1.5 mmol) afforded 0.60 g (95%) of the title
1
compound (9) as a colorless oil. H NMR (CDCl3) d
4.1.1.8. Boc-Aza-b3-Gly-OBn (9a). To a solution of
Boc carbazate (2.00 g, 15.13 mmol) in toluene (25 mL)
were added diisopropylethylamine (DIEA) (1.96 g,
1 equiv) and benzyl 2-bromoacetate (3.47 g, 1 equiv).
The mixture was stirred at 75 ꢂC for 4 days. After fil-
tration and concentration, the residue was further
purified by flash chromatography (EtOAc/CH2Cl2,
1:9) to give the title compound as a colorless oil
(2.60 g, 61%) that slowly crystallized; mp 40 ꢂC. 1H
NMR (CDCl3) d 1.47 (s, 9H, CH3), 3.72 (s, 2H,
CH2), 5.19 (s, 2H, CH2), 6.43 (br s, 1H, NH), 7.31–
7.42 (m, 5H, CHar). 13C NMR (CDCl3) d 171.5 (s),
156.7 (s), 135.8 (s), 129.0 (d), 128.8 (d), 128.7 (d),
80.9 (s), 67.0 (t), 53.2 (t), 28.7(q). HRMS (ESI) m/z
calcd for C14H20N2O4Na [M+Na]+: 303.1321, found:
303.1336.
1.47 (m, 9H, CH3), 4.05–4.38 (br m, 3H, CH + CH2),
4.52 (d, 2H, J = 6.8 Hz, CH2), 5.21 (s, 2H, CH2), 6.96
(br s, 1H, NH), 7.30–7.79 (m, 8H, CHar). 13CNMR
(CDCl3) d 169.8 (s), 156.5 (s), 155.2 (s), 144.0 (s),
141.7 (s), 135.6 (s), 129.1 (d), 128.9 (d), 128.8 (d),
128.3 (d), 127.6 (d), 125.5 (d), 120.5 (d), 82.9 (s), 68.4
(t), 67.6 (t), 53.3 (t), 47.4 (d), 28.49 (q). HRMS (ESI)
m/z calcd for C22H24N2O6Na [M+Na]+: 435.1532,
found: 435.1538.
4.1.1.12. Fmoc-Aza-b3-Asp(OtBu)-OH (10). The syn-
thetic procedure was similar to the synthesis of 9: H
1
NMR (CDCl3) d ppm: 1.51 (s, 9H, CH3), 3.63 (s, 2H,
CH2), 3.71 (s, 2H, CH2), 4.23 (t, 1H, CH), 4.56 (d,
2H, CH2), 7.29–7.80 (m, 8H, CHar). 13C NMR (CDCl3)
d ppm: 171.8, 169.5, 157.2, 143.4, 141.3, 127.8, 127.2,
125.1, 120.1, 82.9, 67.6, 59.2, 58.6, 47.0, 28.1. HRMS
(ESI) m/z calcd for C23H25N2O6Na [M+Na]+:
449.1689, found: 449.1702.
4.1.1.9. Aza-b3-Gly(Boc)-OBn (9b). A solution of
Boc-Aza-b3-Gly-OBn (2.23 g, 7.95 mmol) in CH2Cl2
30 mL was saturated by HClg and stirred at rt over-
night. The mixture was concentrated and taken up with
ether, giving after filtration the hydrochloride of 9a as a
white powder (1.47 g), which was added into a stirred
solution of Et3N (0.76 g, 1.1 equiv) in 20 mL of CH2Cl2.
Boc2O (1.47 g, 1 equiv) was added, and the mixture was
stirred overnight, concentrated, and taken up with ether.
After cooling for 4 h, the hydrochloride of Et3N was re-
moved by filtration, and the filtrate was concentrated.
The crude oil was purified by chromatography on silica
gel (EtOAc/hexane, 3:7) to give the title compound
4.1.2. General procedure for the synthesis of hybrid
peptides 11a–c. The hybrid peptide was prepared from
1 g of resin loaded with the Fmoc-Val-OH (0.60 mmol/g)
in a manual synthesizer using nitrogen bubbling. The re-
sin was first swelled with DMF for 3 min at a 1.5 mL/
min flow rate. After cleavage of the Fmoc group using
20% piperidine in DMF (5 min, 5 mL/min), the resin
was washed with DMF for 7 min (5 mL/min). Subse-
quently, 4 equiv of the appropriate amino acid was dis-
solved in HOBt/DMF (1.4 mL, 0.6 M) and DIC/DMF
(1.4 mL, 0.6 M), and the activated amino acid was then
transferred to the reaction vessel. After 30–80 min, the
reaction vessel was washed with DMF for 4 min
(5 mL/min). The peptide backbone was elongated step
by step as described in Figure 1. Finally, the Fmoc pro-
tective group was removed by treatment with 20% piper-
idine in DMF (1 and 5 min, 5 mL/min), and the resin
was washed with DMF (7 min, 5 mL/min), removed
from the reaction vessel, washed with DCM and ether,
and dried in vacuum. The anchored hybrid peptide thus
obtained was cleaved from the solid support by treat-
ment with TFA/phenol/TIS/water (88:5:2:5) for 2 h.
The mixture was then filtered, and the resin was washed
thoroughly with TFA and then with DCM. The total fil-
trate was concentrated in vacuum to a volume of
approximately 1–2 mL, and then cold ether (10 mL)
was added to precipitate the peptide. The precipitated
peptide was collected by filtration through a fritted glass
funnel and dried in vacuum. The crude product was
purified by reversed-phase preparative HPLC (XTerra
RP18 19 · 300 10 lm). The HPLC fraction was freeze-
dried to give the target hybrid peptide as a white fluffy
solid in 35–46% yield.
1
(1.15 g, 52%) as colorless oil. H NMR (CDCl3) d 1.40
(s, 9H, CH3), 4.17 (s, 2H, CH2), 5.17 (s, 2H, CH2),
7.34–7.41 (m, 5H, CHar). 13C NMR (CDCl3) d 170.4
(s), 158.0 (s), 136.1 (s), 129.2 (d), 129.0 (d), 128.9 (d),
81.9 (s), 67.4 (t), 54.1 (t), 28.8 (q). HRMS (ESI) m/z
calcd for C14H20N2O4Na [M+Na]+: 303.1320, found:
303.1335.
4.1.1.10. Fmoc-Aza-b3-Gly(Boc)-OBn (9c). To a stir-
red solution of 9b (1.83 g, 6.52 mmol) in THF/water
(15/15 mL) was first added solid NaHCO3 (1.09 g,
2 equiv) and then a mixture of FmocCl (2.02 g,
1.2 equiv) in THF (15 mL) was added dropwise. The
reaction mixture was stirred overnight at rt, and then
ether (50 mL) was added. The organic layer was washed
with brine, dried over Na2SO4, and concentrated to af-
ford an oil that was further purified by chromatography
on silica gel (hexane/EtOAc, 9:1 and 7:3) to give 2.31 g
(71%) of the title compound as a white powder: mp
1
140 ꢂC; H NMR (CDCl3) d 1.44 (s, 9 H, CH3), 4.26
(br s, 2H, CH2), 4.35 (br s, 1H, CH), 4.47 (br, 2H,
CH2), 5.21 (s, 2H, CH2), 6.96 (br s, 1H, NH), 7.30–
7.79 (m, 13H, CHar). 13C NMR (CDCl3) d 170.0 (s),

W. Bi et al. / Bioorg. Med. Chem. 15 (2007) 6909–6919
6917
4.1.2.1. H2N-Arg-Gly-Aza-b3-Asp-Val-OH (11a).
HRMS (ESI) m/z calcd for C17H32N8O7Na [M+H]+:
484.2292, found: 484.2297.
rat aortic strip was examined according to our previ-
ously published method. In brief, immediately after
decapitation rat aortic strips were obtained and put in
a perfusion bath with 5 mL of warmed (37 ꢂC), oxygen-
ated (95% O2, 5% CO2) Krebs solution (pH 7.4). The
aortic strips were mounted to the tension transducers
and the relaxation–contraction curves were recorded.
Noradrenaline (NE, final concentration 10ꢁ9 mol/L)
solution was added to induce contraction. When the
hypertonic contraction reached the maximum level, the
NE was washed away and the vessel strips were stabi-
lized for 30 min. After renewal of the solution, NE (final
concentration 10ꢁ9 mol/L) was added. When the hyper-
tonic contraction value of aortic strips reached their
peak, 15 lL of NS or solutions of 12a–c in 15 lL of
water (final concentration 10ꢁ6 mol/L) were added.
Upon stabilization, 1.5 lL ACh (final concentration
10ꢁ6 mol/L) was added and the percentage inhibition
of ACh-induced vasorelaxation by the test compounds
was determined.
4.1.2.2. H2-NArg-Aza-b3-Gly-Asp-Val-OH (11b).
HRMS (ESI) m/z calcd for C17H32N8O7Na [M+H]+:
484.2292, found: 484.2301.
4.1.2.3. H2N-Aza-b3-Arg-Gly-Asp-Val-OH (11c).
HRMS (ESI) m/z calcd for C17H32N8O7Na [M+H]+:
484.2292, found: 484.2295.
4.1.3. General procedure for the synthesis of hybrid peptides
12a–c. The hybride peptide was prepared from 1 g of resin
loaded with the Fmoc-Val-OH (0.60 mmol/g) in a manual
synthesizer using nitrogen bubbling. The resin was first
swelled with DMF for 3 min at 1.5 mL/min flow rate. After
cleavage of the Fmoc group using 20% piperidine in DMF
(5 min, 5 mL/min), the resin was washed with DMF for
7 min (5 mL/min). Subsequently, 4 equiv of the appropri-
ate amino acid was dissolved in HOBt/DMF (1.4 mL,
0.6 M) and DIC/DMF (1.4 mL, 0.6 M), and the activated
amino acid was then transferred to the reaction vessel.
After 30–80 min, the reaction vessel was washed with
DMF for 4 min (5 mL/min). The peptide backbone was
elongated step by step as described in Figure 1. The last
Fmoc protective group was removed by treatment with
20% piperidine in DMF (5 min, 5 mL/min), and the resin
was washed with DMF (7 min, 5 mL/min). 5 equiv of N-
Boc protected phenolic tetrahydro-b-carboline was dis-
solved in HOBt/DMF and DIC/DMF (1.4 mL, 0.6 M),
and the activated Boc protected compound 1b was then
transferred to the reaction vessel. After 80 min of coupling,
the targeted hybrid peptide analogues thus obtained were
cleavedfromthesolidsupportbytreatmentwithTFA/phe-
nol/TIS/water (88:5:2:5) for 2 h. The mixture was then fil-
tered, and the resin was washed thoroughly with TFA
and then with DCM. The total filtrate was concentrated
in vacuum to a volume of approximately 1–2 mL, and then
cold ether (10 mL) was added to precipitate the peptide.
Theprecipitatedpeptidewascollectedbyfiltrationthrough
a fritted glass funnel and dried in vacuum. The crude prod-
uct was purified by reversed-phase preparative HPLC
(XTerra RP18 19 · 300 10lm). The HPLC fraction was
freeze-dried to give the target hybrid peptide 12a–c as a
white fluffy solid in 30–38% yield.
4.2.2. Evaluation of 12a–c as scavengers of NO, H2O2,
and •OH in PC12 cells.24,33–35 Free radical scavenging
activities of these newly synthesized compounds 12a–c
were evaluated in PC12 cells using the method of
Dawson with minor modifications. In brief, PC12 cells
were grown in Dulbecco’s modified Eagle’s medium
supplemented with 10% of heat-inactivated horse ser-
um (Hyclone), 5% of fetal bovine serum (GIBCO),
1.0 mM sodium pyruvate, 100 U/mL penicillin, and
100 lg/mL streptomycin at 37 ꢂC, in 5% CO2 atmo-
sphere. PC12 cells were seeded in 96-well plates coated
with poly-L-lysine at a density of 20,000 cells per well
during the exponential phase of growth. After a 24 h
attachment period fresh media containing 12.5, 25,
50, 100, or 200 lM of 12a–c, respectively, were added
to each well and were incubated for 1 h. NO damage
was then induced by adding 2 mM of sodium nitro-
prusside (SNP) followed by 2 h of incubation. The
media were replaced with fresh media and cells were
incubated for 14 h, following which cell survival was
measured by a colorimetric assay with MTT according
to the method of Mosmann. Similarly, H2O2 damage
was induced by 1 mM H2O2 followed by 1 h of incu-
bation, while •OH damage was induced by 1 mM
H2O2/30 lM Fe(II) followed by 1 h of incubation.
The statistical analysis of the data was carried out
by use of the ANOVA test, p < 0.05 being considered
significant.
4.1.3.1. Phenolic tetrahydro-b-carboline-Arg-Gly-Aza-
b3-Asp-Val-OH (12a). HRMS (ESI) m/z calcd for
C37H50N10O11Na [M+H]+: 834.3558, found: 834.3560.
4.2.3. In vitro anti-platelet aggregation activity
assay.24,33–35 Platelet-rich plasma was prepared by cen-
trifugation of normal rabbit blood anticoagulation with
sodium citrate at a final concentration of 3.8%. The
platelet counts were adjusted to 2 · 105/lL by addition
of autologous plasma. Platelet aggregation tests were
conducted in an aggregometer using the standard turbi-
dimetric technique. The effects of 1a, 11a–c and 12a–c
on PAF (final concentration 10ꢁ5– 10ꢁ7 mol/L) or
ADP (final concentration 10ꢁ5–10ꢁ7 mol/L) induced
platelet aggregation were observed. The maximal rate
of platelet aggregation (Am%) was represented by the
peak height of the aggregation curve.
4.1.3.2. Phenolic tetrahydro-b-carboline-Arg-Aza-b3-
Gly-Asp-Val-OH (12b). HRMS (ESI) m/z calcd for
C37H50N10O11Na [M+H]+: 834.3558, found: 834.3572.
4.1.3.3. Phenolic tetrahydro-b-carboline-Aza-b3-Arg-
Gly-Asp-Val-OH (12c). HRMS (ESI) m/z calcd for
C37H50N10O11Na [M+H]+: 834.3558, found: 834.3564.
4.2. Biology
4.2.1. NO scavenging activity determined using rat aortic
strip.24,33–35 The NO scavenging activity of 12a–c in the

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W. Bi et al. / Bioorg. Med. Chem. 15 (2007) 6909–6919
4.2.4. In vivo antithrombotic activity in rat model.24,33–35
The assessments described herein were performed based
on a protocol reviewed and approved by the Ethics
Committee of HeBei Medical University. The committee
assured that the welfare of the animals was maintained
in accordance with the requirements of the animal wel-
fare act and according to the guide for care and use of
laboratory animals. The tested compound was dissolved
in NS just before use and kept in an ice bath. Male Wis-
tar rats weighing 250–300 g were anaesthetized with so-
dium pentobarbital (80.0 mg/kg, ip) and the right
carotid artery and left jugular vein were separated. A
6 cm thread with exact weight was put into the middle
of the polyethylene tube. The polyethylene tube was
filled with sodium heparin (50 IU/mL of NS) and one
end was inserted into the left jugular vein. From the
other end of the polyethylene tube sodium heparin was
injected as anticoagulant, NS or the solution of the
tested compound in NS was injected and the polyethyl-
ene tube was inserted into the right carotid artery. The
blood was flowed from the right carotid artery to the left
jugular vein via the polyethylene tube for 15 min. The
thread was taken out and weighed and the weight of
the wet thrombus was recorded. The thread was kept
in desiccator for 2 weeks and the weight of the dry
thrombus was recorded. The data are listed in Table 5.
iment. The coronary artery was again briefly occluded
through tightening the ligature that remained at the site
of the previous occlusion. Immediately after the ligation,
1% Evans blue solution was infused through the catheter
into the beating left ventricular cavity to delineate the
ischemic area at risk (underperfused and then reperfused
area) of the left ventricle. Hearts were quickly taken out
and put at ꢁ20 ꢂC for subsequent processing. Frozen
hearts (including only ventricular tissue) were sliced
transversely in a plane perpendicular to the apical–basal
axis into 1 mm-thick sections, the sections were then
placed in 1% triphenyltetrazolium (TTC) and incubated
for 15 min at 37 ꢂC in a dark room. The heart was divided
into ischemic but viable (TTC-stained) and infarcted
(TTC-unstained) zones within the underperfused and
then reperfused area (Evans blue-unstained) and the
non-ischemic area (Evans blue-stained).
4.2.7. Estimation of malondialdehyde (MDA) forma-
tion.8,9,46 Malondialdehyde (MDA) was assayed as de-
scribed previously to monitor the development of
oxidative stress.8,9,46 Hearts to be used for MDA con-
tent detection were taken at the end of the experiment
and quickly put into ice saline to flush residual blood.
Ischemic myocardial tissues were sheared and a 10%
homogenate was produced in the ice bath. An improved
Grass technique and TBA technique were partly
adopted to detect myocardial MDA content.
4.2.5. Ischemia–reperfusion model preparation.8,9,46
Male Wistar rats weighing 250–300 g underwent myo-
cardial ischemia by a temporary occlusion of the left cor-
onary artery as previously described. Briefly, rats were
anaesthetized with sodium pentobarbital (80.0 mg/kg,
ip) and placed on an operating table. Polyethylene cath-
eters were inserted into the common femoral artery for
the measurement of blood pressure and heart rate, and
from the internal carotid artery into the left ventricle
for measurement of left ventricular maximum systolic
pressures (LVSP) and the maximal first derivative of
developed pressure ðdP=dtmaxÞ. After tracheotomy, the
animals were ventilated with room air using small rodent
respirator. The chest was opened and the ribs were gently
spread. The heart was quickly expressed out of the tho-
racic cavity, inverted and a 4–0 silk ligature was placed
under the left coronary artery. The heart was reposi-
tioned in the chest and the animal was allowed to recover
for 15 min. A small plastic snare was threaded through
the ligature and placed in contact with the heart. Tight-
ening the ligature could occlude the artery and reperfu-
sion was achieved by releasing the tension applied to
the ligature (I/R groups). Sham I/R animals underwent
all the above described surgical procedures, apart from
the fact that the 4–0 silk, passing around the left coro-
nary artery, was not tied (Sham I/R group). Animals
were infused with 12c (10 mg/kg) by vehicle (dimethyl
sulfoxide ꢁ0.9% NaCl, 1:103; v/v) via the transjugular
route 15 min before coronary occlusion. The coronary
artery was occluded for 30 min followed by 120 min
reperfusion and the animals were randomized in the fol-
lowing groups: (1) Sham I/R + vehicle; (2) Sham I/
R + 12c; (3) I/R + vehicle; (4) I/R + 12c.
4.3. Statistical analysis
´
A two-way ANOVA followed by Scheffe’s test was first
carried out using the Origin Program totest for differences
between groups. If differences were established, the values
were compared using Student’s t-test for paired data. The
values were expressed as means SE. The results were
considered significant if p was <0.05.
Acknowledgements
The authors are grateful to Natural Science Foundation
of HeBei Province (No. 062761266), Administration of
Traditional Chinese Medicine of HeBei Province (No.
2007134), and Second Hospital of HeBei Medical Uni-
versity for support of this research. The authors are also
grateful to Drs. James Russo, Roger E. Koeppe II and
Leon L.G. Lu for many helpful discussions.
References and notes
1. Cannon, R. O. Nat. Clin. Pract. Cardiovasc. Med. 2005, 2,
88–94.
2. Neuzil, J.; Rayner, B. S.; Lowe, H. C.; Witting, P. K.
Redox Rep. 2005, 10, 187–197.
3. Saeed, S. A.; Waqar, M. A.; Zubairi, A. J.; Bhurgri, H.;
Khan, A.; Gowani, S. A.; Waqar, S. N.; Choudary, M. I.;
Jalil, S.; Zaidi, A. H.; Ara, I. J. Coll. Physician. Surg. Pak.
2005, 15, 507–514.
4. Zweier, J. L.; Talukder, M. A. Cardiovasc. Res. 2006, 70,
181–190.
4.2.6. Evaluation of infarct size.8,9 Hearts to be used for
infarct size calculations were taken at the end of the exper-
5. Kloner, R. A.; Jennings, R. B. Circulation 2001, 104,
2981–2989.

W. Bi et al. / Bioorg. Med. Chem. 15 (2007) 6909–6919
6919
6. Kerendi, F.; Kin, H.; Halkos, M. E.; Jiang, R.; Zatta, A.
J.; Zhao, Z. Q.; Guyton, R. A.; Vinten-Johansen, J. Basic
Res. Cardiol. 2005, 100, 404–412.
7. Jin, Z. Q.; Zhou, H. Z.; Cecchini, G.; Gray, M. O.;
Karliner, J. S. Am. J. Physiol. Heart Circ. Physiol. 2005,
288, 2986–2994.
26. Cheguillaume, A.; Doubli-Bounoua, I.; Baudy-Floc’h, M.;
Le Grel, P. Synlett 2000, 331–334.
27. Cheguillaume, A.; Lehardy, F.; Bouget, K.; Baudy-Floc’h,
M.; Le Grel, P. J. Org. Chem. 1999, 64, 2924–2927.
28. Busnel, O.; Bi, L.; Baudy-Floc’h, M. Tetrahedron Lett.
2005, 46, 7073–7075.
8. Sahna, E.; Acet, A.; Ozer, M. K.; Olmez, E. J. Pineal. Res.
2002, 33, 234–238.
9. Galaris, D.; Barbouti, A.; Korantzopoulos, P. Curr. Phar.
Des. 2006, 12, 2875–2890.
10. Zhu, B. Q.; Simonis, U.; Cecchini, G.; Zhou, H. Z.; Li, L.;
Teerlink, J. R.; Karliner, J. S. J. Cardiovasc. Pharmacol.
Ther. 2006, 11, 119–128.
11. Herraiz, T.; Galisteo, J. J. Agric. Food Chem. 2003, 51,
7156–7161.
29. Busnel, O.; Bi, L.; Dali, H.; Cheguillaume, A.; Chevance,
S.; Bondon, A.; Muller, S.; Baudy-Floc’h, M. J. Org.
Chem. 2005, 70, 10701–10708.
30. Dali, H.; Busnel, O.; Hoebeke, J.; Bi, L.; Decker, P.;
Briand, J. P.; Baudy-Floch, M.; Muller, S. Mol. Immunol.
2007, 44, 3024–3036.
31. 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, 1–4.
12. Ichikawa, M.; Ryu, K.; Yoshida, J.; Ide, N.; Yoshida, S.;
Sasaoka, T.; Sumi, S. Biofactors 2002, 16, 57–72.
13. Herraiz, T.; Galisteo, J.; Chamorro, C. J. Agric. Food
Chem. 2003, 51, 2168–2173.
14. Herraiz, T.; Galisteo, J. Free Radic. Res. 2002, 36, 923–928.
15. Pless, G.; Frederiksen, T. J.; Garcia, J. J.; Reiter, R. J. J.
Pineal Res. 1999, 26, 236–246.
32. Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.
33. Wu, Y.; Bi, L.; Bi, W.; Zhao, M.; Li, Z.; Ju, J.; Peng, S.
Bioorg. Med. Chem. 2006, 14, 5711–5720.
34. Zhao, M.; Bi, L.; Bi, W.; Wang, C.; Yang, Z.; Ju, J.; Peng,
S. Bioorg. Med. Chem. 2006, 14, 4761–4774.
35. Liu, J.; Zhao, M.; Wang, C.; Peng, S. Bioorg. Med. Chem.
Lett. 2003, 13, 4065–4069.
16. Hsu, L. Y.; Lin, C. F.; Hsu, W. C.; Hsu, W. L.; Chang, T.
C. Biol. Pharm. Bull. 2005, 28, 1211–1215.
36. Mombouli, J. V.; Vanhoutte, P. M. J. Mol. Cell. Cardiol.
1999, 31, 61–74.
17. Yilmaz, Y.; Toledo, R. T. Trends Food Sci. Technol. 2004,
15, 422–433.
18. Nicholson, N. S.; Abood, N. A.; Panzer-Knodle, S. G.;
Frederick, L. G.; Page, J. D.; Salyers, A. K.; Suleymanov,
O. D.; Szalony, J. A.; Taite, B. B.; Anders, R. J. Med. Res.
Rev. 2001, 21, 211–226.
37. De Vriese, A. S.; Verbeuren, T. J.; Van de Voorde, J.;
Lameire, N. H.; Vanhoutte, P. M. Br. J. Pharmacol. 2000,
130, 963–974.
38. 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.
19. Scarborough, R. M.; Gretler, D. D. J. Med. Chem. 2000,
43, 3453–3473.
39. Komori, K.; Vanhoutte, P. M. Blood Vessels 1990, 27,
238–245.
20. Zablocki, J. A.; Rico, J. G.; Garland, R. B.; Rogers, T. E.;
Williams, K.; Schretzman, L. A.; Rao, S. A.; Bovy, P. R.;
Tjoeng, F. S.; Lindmark, R. J., et al. J. Med. Chem. 1995,
38, 2378–2394.
40. Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Pharmacol.
Rev. 1991, 43, 109–142.
41. Calles-Escandon, J.; Cippola, M. Endocr. Rev. 2001, 22,
36–52.
21. Ruoslahti, E. Integrins as Receptors for Extracellular
Matrix. In Cell Biology of Extracellular Matrix; Hay, E.
D., Ed.; Plenum Press: New York, NY, 1991.
22. Cheng, S.; Craig, W. S.; Mullen, D.; Tschopp, J. F.; Dixon,
D.; Pierschbacher, M. D. J. Med. Chem. 1994, 37, 1–8.
23. Batchelor, W. B.; Tolleson, T. R.; Huang, Y.; Larsen, R.
L.; Mantell, R. M.; Dillard, P.; Davidian, M.; Zhang, D.;
Cantor, W. J.; Sketch, M. H., Jr.; Ohman, E. M.; Zidar, J.
P.; Gretler, D.; DiBattiste, P. M.; Tcheng, J. E.; Califf, R.
M.; Harrington, R. A. Circulation 2002, 106, 1470–1476.
24. Bi, W.; Bi, L.; Cai, J.; Liu, S.; Peng, S.; Fischer, N. O.;
Tok, J.; Wang, G. Bioorg. Med. Chem. Lett. 2006, 16,
4523–4527.
42. Chalupsky, K.; Lobysheva, I.; Nepveu, F.; Gadea, I.;
Beranova, P.; Entlicher, G.; Stoclet, J.; Muller, B.
Biochem. Pharmacol. 2004, 67, 1203.
43. Beranova, P.; Chalupsky, K.; Kleschyov, A.; Schott, C.;
Boucher, J.; Mansuy, D.; Munzel, T.; Muller, B.; Stoclet,
J. Eur. J. Pharmacol. 2005, 516, 260.
44. Nakae, I.; Matsumoto, T.; Omura, T.; Takashima, H.;
Ohira, N.; Tarutani, Y.; Yasuda, Y.; Matsuo, S.; Koh, T.;
Nakamura, Y.; Kinoshitaa, M.; Horie, M. Life Sci. 2003,
73, 3083.
45. McCormick, J.; Barry, S. P.; Sivarajah, A.; Stefanutti, G.;
Townsend, P. A.; Lawrence, K. M.; Eaton, S.; Knight, R.
A.; Thiemermann, C.; Latchman, D. S.; Stephanou, A.
FASEB J. 2006, 20, 2115–2117.
25. Cheguillaume, A.; Salaun, A.; Sinbandhit, S.; Potel, M.;
Gall, P.; Baudy-Floc’h, M.; Le Grel, P. J. Org. Chem.
2001, 66, 4923–4929.
46. Gutteridge, J. M. C.; Halliw, B. Trends Biochem. Sci.
1990, 15, 129–135.