A new class of β-carboline alkaloid-peptide conjugates with therapeutic efficacy in acute limb ischemia/reperfusion injury

Article abstract of DOI:10.1016/j.ejmech.2011.01.021

We describe a novel class of β-carboline alkaloid-peptide conjugates that possess both free radical scavenging and thrombolytic activity. These conjugates demonstrate therapeutic efficacy in a rat arterial thrombosis assay, as well as free radical scavenging capacity as evaluated in a PC12 cell survival assay. Our results indicate that β-carboline alkaloid-peptide conjugate 26a exerts a significant protective effect against local and remote organ injury induced by limb I/R injury in the rat.

Full text of DOI:10.1016/j.ejmech.2011.01.021

European Journal of Medicinal Chemistry 46 (2011) 1453e1462
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A new class of
b
-carboline alkaloid-peptide conjugates with therapeutic efficacy
in acute limb ischemia/reperfusion injury
,
d
c
c
c
c
c
Wei Bi c , Yue Bi , Ping Xue , Yanrong Zhang , Xiang Gao , Zhibo Wang , Meng Li ,
**
Michèle Baudy-Floc’h e, Nathaniel Ngerebara a, K. Michael Gibson b, Lanrong Bi a
,
*
a Department of Chemistry, Michigan Technological University, Houghton, MI 49931, USA
b Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, USA
c Department of Surgery, Second Hospital of HeBei Medical University, Shijiazhuang 050000, PR China
d School of Basic Medical Sciences, HeBei Medical University, Shijiazhuang 050000, PR China
e UMR 6226 CNRS, ICMV, Sciences Chimiques de Rennes, Université de Rennes I, F-35042 Rennes Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe a novel class of
b-carboline alkaloid-peptide conjugates that possess both free radical
Received 14 September 2010
Received in revised form
31 December 2010
Accepted 12 January 2011
Available online 21 January 2011
scavenging and thrombolytic activity. These conjugates demonstrate therapeutic efficacy in a rat arterial
thrombosis assay, as well as free radical scavenging capacity as evaluated in a PC12 cell survival assay.
Our results indicate that
against local and remote organ injury induced by limb I/R injury in the rat.
Published by Elsevier Masson SAS.
b-carboline alkaloid-peptide conjugate 26a exerts a significant protective effect
Keywords:
Ischemia-reperfusion injury
b
-Carboline alkaloid-peptide conjugates
Antioxidant
Thrombolytic activity
1. Introduction
(streptokinase, urokinase, recombinant tissue plasminogen acti-
vator, and reteplase) [4e7]. Although reperfusion is unavoidable,
there is extensive evidence that reperfusion itself induces addi-
tional cellular injury. The literature indicates that free radicals (such
as superoxide and hydroxyl radicals) are responsible for reperfu-
sion injury. More than 90% of cellular ROS (reactive oxygen species),
even under resting conditions, is produced in the mitochondria.
Antioxidant supplementation, therefore, is the logical counter-
measure to counteract oxidative stress in patients with critical limb
ischemia. Along these lines, several studies have employed anti-
oxidants to attenuate oxidative stress and improve initial claudi-
cation distance in patients with critical limb ischemia [8e10].
Acute limb ischemia associates with significant morbidity and is
generally linked with thrombosis, embolism, or trauma. In the
absence of timely intervention, the outcome encompasses irre-
versible tissue damage and necrosis [1,2]. Acute limb ischemia can
be divided into three progressive stages: stage 1 features continued
growth of the arterial thrombus until all collateral vessels are
occluded; stage 2 includes tissue edema and fluid accumulation
leading to elevated compartmental pressure which induce
compression of further restriction of blood flow; and stage 3
involves narrowing and obstruction of the microvasculature due to
edema [1e3]. Occlusion at the stage 3 level is linked to irreversible
tissue edema. Intervention for acute limb ischemia usually
encompasses the intraarterial application of thrombolytic agents
b-carboline alkaloids derived from tryptophan are naturally
occurring substances generated during food production, processing
and storage. These alkaloids may occur in foods under mild
conditions following a Pictet-Spengler condensation of indole-
amines (e.g.,
aromatic aldehydes may generate novel tetrahydro-
foods during processing or cooking via condensation reaction with
-tryptophan. Some phenolic tetrahydro- -carbolines demonstrate
antioxidant properties that exceed ascorbic acid and the water-
soluble vitamin E analogue, Trolox [11e16].
L
-tryptophan) and reactive aldehydes. As well, some
b
-carbolines in
Abbreviations: I/R, ischemia/reperfusion; ROS, reactive oxygen species; HOMO,
highest occupied molecular orbital; MDA, malondialdehyde; GSH, glutathione.
* Corresponding author. Tel.: þ1 906 487 1868; fax: þ1 906 487 2061.
** Corresponding author. Tel./fax: þ86 311 66002578.
L
b
E-mail addresses: biwei8115@sohu.com (W. Bi), lanrong@mtu.edu (L. Bi).
0223-5234/$ e see front matter Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2011.01.021

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W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 1453e1462
5
4
COOH
NBoc
COOH
4b
4a
9a
3
6
7
O
H
2
H
H
1
COOH
NH
ii
i
R1
R2
8a
N
H
N
H
8
+
NH2
R1
R1
R2
N
H
R4
R3
1a,b,c
R4
R2
R4
R3
3a,b,c
R3
2a,b,c
Scheme 1. Synthesis of
R3 ¼ OCH3, R4 ¼ H; in 1c, 2c and 3c: R1 ¼ H, R2 ¼ OCH3, R3 ¼ OH, R4 ¼ H.
b
-carboline alkaloids 3aec. Regents and condition: (i) H2SO4; (ii) Boc-N3. In 1a, 2a and 3a: R1 ¼ OH, R2 ¼ H, R3 ¼ H, R4 ¼ H; in 1b, 2b and 3b: R1 ¼ H, R2 ¼ H,
HCl Lys(Z)-OBzl
5
Boc-Lys(Z)-OBzl
4
Boc-Ala-Lys(Z)-OBzl
6
HCl Ala-Lys(Z)-OBzl
7
Boc-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl
10
HCl Pro-Ala-Lys(Z)-OBzl
9
Boc-Pro-Ala-Lys(Z)-OBzl
8
HCl Arg(Tos)-Pro-Ala-Lys(Z)-OBzl
11
Boc-AA-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl
12-14
HCl AA-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl
15-17
COAA-Arg-Pro-Ala-Lys-OH
NH
R1
COAA-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl
H
NBoc
N
H
ARPAK
QRPAK
GRPAK
N
H
H
R1
18-20
R4
R2
R4
R2
R3
R3
21a,b,c-23a,b,c
24a,b,c-26a,b,c
Scheme 2. Synthesis of
21ce26c: R1 ¼ H, R2 ¼ OCH3, R3 ¼ OH, R4 ¼ H.
b
-carboline alkaloid-peptide conjugates 24a,b,ce26a,b,c. In 21ae26a: R1 ¼ OH, R2 ¼ H, R3 ¼ H, R4 ¼ H; in 21be26b: R1 ¼ H, R2 ¼ H, R3 ¼ OCH3, R4 ¼ H; in
The pentapeptide Ala-Arg-pro-Ala-Lys (ARPAK) was recently
isolated from products generated by plasmin degradation from the
including Gly-Arg-Pro-Ala-Lys (GRPAK) and Gln-Arg-Pro-Ala-Lys
(QRPAK), have demonstrated thrombolytic activity [19,20]. In light of
the preceding observations, and considering the antioxidant prop-
fibrinogen
b chain, and ARPAK was identified as the active species
responsible for enhanced microvascular permeability in both rat and
human skin [17,18]. Furthermore, ARPAK and other pentapeptides,
erties of
b-carboline alkaloids coupled with the thrombolytic activity
observed with ARPAK, GRPAK and QRPAK pentapeptides, we
Fig. 1. Reduction of thrombus mass (X ꢀ SD mg) by synthesized peptide conjugates. aP<0.001 as compared to NS, bP < 0.01 as compared with NS, cP<0.05 as compared with NS,
dP < 0.05 as compared with UK. NS (normal saline) ¼ 3 mL/kg; UK (urokinase) ¼ 20000 IU/kg; 18e20 ¼ 10.0
m
mol/kg; 24a,b,ce26a,b,c ¼ 10.0 mol/kg; n ¼ 8.
m

W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 1453e1462
1455
160
140
120
100
80
H
H2O2
60
40
20
0
2a
2b
2c
26a
26b
26c
Fig. 2. Free radical scavenging activities in PC 12 cell survival assays.
hypothesized that
ated from
b
-carboline alkaloids peptide conjugates gener-
the N-terminal free intermediates 15e17, which were then coupled
with intermediate 3 to generate protective peptide conjugates
b
-carboline alkaloids and thrombolytic peptides would
exhibit both enhanced antioxidant and thrombolytic activities.
21a,b,ce23a,b,c, respectively. Finally, the target b-carboline alka-
loid-peptide conjugates 24a,b,ce26a,b,c were obtained by removal
of all protecting groups of 21a,b,ce23a,b,c in the presence of
anhydrous HF, respectively.
2. Chemistry
2.1. Synthesis of
b
-carboline alkaloids
3. Biological results and discussion
To prepare
b
-carboline alkaloid-peptide conjugates, L-trypto-
3.1. Evaluation of thrombolytic activity in vivo
phan was initially reacted with various aldehydes (salicylaldehyde,
anisaldehyde and vanillin) in acidic-aqueous solution in order to
The thrombolytic activity of 24a,b,ce26a,b,c was assessed as
previously described and expressed as thrombus mass reduction in
comparison to thatof parent compounds (18e20) [22]. The results are
presented in Fig. 1. We found that, similar to the parent peptides
18e20, theb-carboline alkaloid-peptideconjugates (24a,b,ce26a,b,c)
could significantly reduce thrombus mass.
generate 1,3-disubstituted tetrahydro-b-carbolines. This reaction
proceeds via a PicteteSpengler intramolecular cyclization of an
intermediate Schiff base to produce the 1,3-disubstituted tetrahy-
dro-
b
-carbolines [13]. 1,3-Disubstituted tetrahydro-b-carbolines
derived from
L
-tryptophan and aldehydes (Scheme 1:1) provided
key intermediates (Scheme 1:2), which were isolated via RP-HPLC.
Diastereoisomer assignments were based upon acquired positive
NOE spectra of proton signals at C-1 and C-3 for the cis-isomers. C-1
position proton signals (consistent with the cis-isomers) appeared
at higher field than the same signals generated from the trans-
isomers; Conversely, C-1 and C-3 NMR (consistent with the trans-
isomers) appeared at higher field than those of the corresponding
cis-isomers. This was consistent with the expected pattern for 1,3-
3.2. Free radicals scavenging activities determined by cell survival
assay in PC 12 cells
During ischemia/reperfusion, various reactive oxygen species
(ROS) induce cellular and tissue damage. For example, H2O2 leads to
DNA strand breaks and base damage in conjunction with transition
metal ions. Metal (i.e., copper ion) present in biological systems can
disubstituted tetrahydro-
these intermediates with Boc-N3 yielded the N-Boc protected 1,3-
disubstituted tetrahydro- -carbolines (Scheme 1:3). For the tetra-
hydro- -carboline derivatives, our recent results suggested that
b-carbolines. Subsequent treatment of
b
Table 1
b
MDA and GSH levels in the Sham, I/R and I/R þ 26a groups.
trans-isomers exhibited more pronounced biological effects than
cis-isomers [21]. Accordingly, our current research efforts were
focused on the trans-isomers although the precise biological effects
and action mechanism of the cis-isomers remains to be determined.
Groups
Muscle
MDA (nmol/mg)
GSH (nmol/mg)
Sham
I/R
I/R þ 26a
Sham
I/R
I/R þ 26a
Sham
I/R
I/R þ 26a
0.6 ꢀ 0.1
7.8 ꢀ 0.3a
2.8 ꢀ 0.2b
1.3 ꢀ 0.1
5.1 ꢀ 0.4a
1.5 ꢀ 0.1b
2.5 ꢀ 0.1
5.8 ꢀ 0.3a
2.8 ꢀ 0.2b
1.3 ꢀ 0.2
0.2 ꢀ 0.05a
1.0 ꢀ 0.1b
16.8 ꢀ 2.1
4.9 ꢀ 1.7a
14.7 ꢀ 3.2b
11.2 ꢀ 0.4
4.1 ꢀ 0.3a
9.1 ꢀ 0.2b
Heart
Blood
2.2. Synthesis of
b-carboline alkaloid-peptide conjugates
Starting from the
L-Lys(Z)-OBzl, protected peptide intermediates
12e14 were prepared via the solution-phase peptide synthesis
method (from C- to N-terminus) of the peptides described in
Scheme 2. Removal of the Boc protecting moiety in 12e14 yielded
a
Compared with Sham group, P < 0.05.
Compared with I/R group, P < 0.05; n ¼ 8.
b

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W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 1453e1462
react with H2O2 via Fenton reactions to produce hydroxyl radicals
which also induce DNA strand breakage. Hydroxyl radicals can rapidly
react with almost any cellular bio-molecule. We hypothesized that
markedly decreased after PC 12 cells were exposed to free radicals.
The free radical scavenging activities of 26aec against NO, H2O2, and
$OH were evaluated in PC12 cells and compared with that of the
b-carboline alkaloid-peptide conjugates (26aec) with potent throm-
parent species (2aec) [23]. The results were expressed as EC50 (mM)
bolytic activity would also display free radical scavenging activity
against ROS, especially hydroxyl radicals. To test this hypothesis, we
employed rat pheochromocytoma (PC 12) cells that originate from the
adrenal medulla and synthesize and release catecholamines. This cell
line is very sensitive to oxidative stress [23], and the PC 12 cell model
system has been routinely employed for in vitro ischemia studies. We
found that cell survival, as determined by MTT (3-(4,5-di-methyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, was
values. Reduction of viability induced by exposure to free radicals was
curtailed with incubation of the test compounds. The scavenging
capacity (EC50) among all tested species ranged from 77to 110
mM for
NO, 38e66 M for H2O2, and 72e94 M for $OH. These results sug-
m
m
gested that all test compounds had reasonable scavenging capacity
toward the radical species. The EC50 values of 26aec were comparable
to those of 2aec (Fig. 2), indicating that they were effective radical
scavengers. The scavenging capacity of 26a was more pronounced for
Fig. 3. Hematoxylin-eosinestained frozen cross-section of skeletal muscle. A, B, C: Representative muscle tissue sections taken from the sham cohort; D, E, F: Representative muscle
tissue sections taken from rats exposed to limb I/R; G, H, I: Representative muscle tissue sections taken from I/R þ 26a (20 mg/kg)-treated rats. Magnification: A, D, G: ꢁ 100; B, E,
H: ꢁ 200; C, F, I: ꢁ 400.

W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 1453e1462
1457
$OH and H2O2, suggesting that 26a might act as a scavenger of
3.4. Limb ischemia-reperfusion injury
hydroxyl radicals generated through the Fenton reaction.
Tourniquet application is broadly employed in orthopedic,
vascular and reconstructive surgery of the extremities to provide
a bloodless surgical field. During the ischemic period, however, free
oxygen radicals are produced in the ischemic tissue [24]. Following
tourniquet release, free oxygen and superoxide radicals facilitate
endothelial injury, increased microactivated permeability, and
tissue edema. Additionally, activated adhesion molecules and
cytokines may lead to a systemic inflammatory response. We
employed a rubber band tourniquet model to induce limb I/R injury
in the rat [21], in which collateral blood flow around the femoral
3.3. Stability of 26aec in vitro trypsin intervention
10 mg of 26aec each was dissolved in 1 mL of phosphate buffer
(pH 8) supplemented with 0.5 mg of trypsin. The reaction mixture
was maintained at 37 ꢂC and the concentration of test compound
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 26a, 26b, and 26c observed after the enzyme promoted
hydrolysis was postponed for 4, 3, and 3 h, respectively.
Fig. 4. Histology of myocardial tissues stained with hematoxylin and eosin. A, B, C: Tissue from the sham cohort showed normal myocardium architecture; D, E, F: Representative
myocardium taken from rats exposed to limb I/R; G, H, I: Representative myocardium taken from I/R þ 26a (20 mg/kg)- treated rats. Magnification: A, D, G: ꢁ 100; B, E, H: ꢁ 200; C,
F, I: ꢁ 400.

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W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 1453e1462
and iliac arteries can be fully occluded to ensure complete limb
ischemia. Following 3h of bilateral lower limb ischemia, tourniquet
release was followed by 4h of reperfusion. Based upon preliminary
results, we examined compound 26a to ascertain its capacity for
protection against local and remote organ injury.
observed in the interstitial region and small blood vessels. The
more pronounced effects in heart muscle may reflect this organ’s
higher oxygen requirement as compared to skeletal muscle.
3.5. Simulation of the molecular dynamics of
peptide conjugates
b-carboline alkaloid-
3.4.1. Effect of 26a on lipid peroxidation-malondialdehyde (MDA)
and glutathione (GSH) assays
The antioxidant capacity of many antioxidants may be correlated
with parameters such as the highest occupied molecular orbital
(HOMO) energy, the net charge, and the difference in heat of
formation between antioxidant and its radical, consistent with the
OeH bond dissociation energy. Accordingly, the HOMO ionization
energy may represent a surrogate marker for the capacity to scav-
enge oxidants [27]. Generally, the higher the HOMO energy, the
larger the antioxidant capacity for a given compound. Among the
Malondialdehyde (MDA), a metabolite of free radical-mediated
lipid peroxidation cascade, is a widely employed marker of oxida-
tive stress [25]. Recent studies have shown that tissue I/R associates
with lipid peroxidation, which is an autocatalytic mechanism
resulting in oxidative destruction of the cellular membranes [25].
As a surrogate biomarker of lipid peroxidation, MDA levels are
representative of the level of free radical and inflammatory
response of the tissue. Ischemic skeletal muscle MDA content
increased significantly in muscle I/R groups compared with sham
groups (Table 1; P < 0.01), and treatment with 26a significantly
attenuated this increase (P < 0.01). In addition to local injury, there
is also a systemic component associated with I/R injury in distal
organs, including heart, but also blood, and we also measured both
MDA and GSH levels in these systems (Table 1).
b-carboline alkaloid-peptide conjugates (24a,b,ce26a,b,c) pre-
sented in the current report, the HOMO energy of 26a (EHOMO
¼ ꢃ9.612 ev) and 26b (EHOMO ¼ ꢃ9.729 ev) are the highest (Table 2-
supplemental). Our initial studies indicated that introduction of
pentapeptide moieties (ARPAK, GRPAK, QRPAK) into phenolic
b-carboline alkaloids did not enhance the free radical scavenging
potential of these conjugates. Nonetheless, conjugation of -carbo-
b
Reduced glutathione (GSH), whose levels are maintained by
glutathione reductase, is a key component of the body’s intracel-
lular antioxidant defense. To maintain the cellular redox status,
glutathione disulphide (GSSG) is actively transported out of cells
during oxidative stress, resulting in a transient decrease of the
intracellular GSH/GSSG ratio [26]. GSH levels in tissues and blood
subjected to I/R injury were significantly lower than in sham-
operated groups (Table 1), indicating impaired antioxidant defense
in our model. Decreased GSH, in conjunction with increased lipid
peroxidation, indicates an impaired antioxidant defense system.
Pretreatment with 26a prior to reperfusion reversed the effect on
GSH levels in all systems (P < 0.05; Table 1). We speculate that the
line alkaloids with these peptides resulted in the formation of
a favorable intramolecular hydrogen bond, which induced cyclic
peptide-like structures. For example, cyclization of both 26a/26bwas
facilitated by an enhanced number of hydrogen bonds that were not
present in parent compounds 2a/2b. We speculate that the addition
of active peptide fragments led to the induction of a more “active”
conformation, resulting in an improved conformational rigidity that
increased the stability of our conjugates toward trypsin, while
simultaneously extending their bioavailability.
4. Conclusions
b
-carboline alkaloid fragment of 26a is responsible for this
Acute arterial occlusion induces downstream skeletal muscle
ischemia and restoration of blood flow can result in more necrosis
than the initial ischemic event itself. Reperfusion also associates
with the release of toxic metabolites (lactic acid, potassium,
thromboxane, and myoglobin) into the systemic circulation. These
substances can lead to metabolic acidosis, cardiac arrhythmia, and
renal failure. In an ongoing search for agents with therapeutic
efficacy for I/R damage, we have designed and synthesized a series
compound’s antioxidant capacity.
3.4.2. Histological analysis
Routine histological evaluation (hematoxylin-eosin staining)
frozen cross-sections of skeletal muscle derived from sham rats
revealed myofibers of uniform size and shape, mosaicly arranged
and in almost direct contact separated by a thin level of endomy-
sium. Overall, histological appearance was normal (Fig. 3A,B,C).
Conversely, reperfusion following 3h of ischemia led to extensive
interstitial edema and damage characterized by swelling and
inflammatory cell infiltration (Fig. 3D,E,F). Muscle tissue damage
after reperfusion was concomitant with increased MDA, suggesting
a correlation with enhanced lipid peroxidation associated with
oxygen free radicals. On the other hand, intervention with 26a
prevented interstitial edema, and left the microvessel cross-section
area essentially unchanged (Fig. 3G,H,I). The cumulative data indi-
cated that 26a treatment resulted in only mild edema and slight
inflammatory cell infiltration in the tibialis anterior muscle, and
muscle fibers from this group showed no evidence for discernible
histological changes.
of new b-carboline alkaloid-peptide conjugates, and characterized
both their thrombolytic activity and free radical scavenging
capacities in model rat and cell culture systems. One compound,
26a, was selected and further characterized in a hind limb I/R injury
animal model because of its potent free radical scavenging and
thrombolytic activities. We found that 26a was protective against
local and remote organ injury induced by limb I/R injury in the rat.
Our results suggest that
b-carboline alkaloid-peptide conjugates
may represent a novel therapeutic approach in I/R-related injuries.
5. Experimental section
5.1. Chemistry
Myocardium sections stained with hematoxylin and eosin
revealed normal architecture and tight cardiac muscle alignment
(Fig. 4A,B,C). Reperfusion-induced myocardial damage was evident
in relation to edema in the perivascular spaces and between
myofibers in the I/R cohort. Edema was prevalent within cardiac
muscle fibers and around blood vessels (Fig. 4D,E,F), indicative of
both structural and biochemical changes during I/R injury, and
likely correlated with inflammatory signaling and calcium influx. In
contrast, necrosis was not obvious in the I/R D26a group
(Fig. 4G,H,I), and only a minimal number of inflammatory cells were
General. All chemicals were purchased from Sigma Aldrich.
Unless otherwise stated, all reactions were run under a nitrogen
atmosphere (1 bar). Chromatography was performed on silica gel H
(230e400mesh). The purity (>97%) of the intermediates and the
final products was confirmed using both TLC (Merck silica gel plates
type 60 F254, 0.25 mm layer thickness) and HPLC (Waters, C18
column 4.6 mm ꢁ 150 mm). NMR spectra were obtained on
a Bruker Advance 500 spectrometer. FAB-MS was determined using
a VG-ZAB-MS high resolution GC/MS/DS and HP ES-5989ꢁ.

W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 1453e1462
1459
Reaction of l-tryptophan with aromatic aldehydes to produce tet-
rahydro- -carbolines [13]. As a general procedure, -tryptophan
with petroleum ether to provide the corresponding ester.
0.20 mmol of the C-terminal component and 0.26 mmol of
b
L
(1 mmol) was dissolved in 0.05 N H2SO4 (13 mL) and stirred with
the corresponding aromatic or phenolic aldehyde (1.3 mmol) to
produce a precipitate that was isolated by filtration and washed
with cold water. The filtrate was evaporated to dryness under
vacuum. The reaction was followed by RP-HPLC, and the final
products were analyzed via NMR, MS, and HPLC-MS. Synthesized
N-methylmorpholine were then added to a solution of the
preceding ester in 10 mL of anhydrous THF. The reaction mixture
was stirred at room temperature for 24 h. Following evaporation,
the residue was dissolved in 50 mL of ethyl acetate. The solution
was washed with 5% sodium bicarbonate, followed by 5% citric acid
and saturated sodium chloride, and the organic phase was sepa-
rated and dried over anhydrous sodium sulfate. Following filtration
and evaporation under reduced pressure, the desired product was
obtained by chromatographic purification (CHCl3/CH3OH, 30:1).
General Procedure for Removal of Boc of the C-Terminal Compo-
nent. 0.20 mmol of Boc-protected compound was stirred at room
temperature for 3 h in 2 mL of hydrogen chloride in ethyl acetate
(4 mol/L). Solvent was removed by evaporation. The residue was
dissolved in 10 mL of ethyl acetate, and the solution evaporated to
dryness. The resulting solid was used for the coupling reaction
without additional purification.
1,3-disubstituted tetrahydro-b-carbolines were composed of two
isomers with 1S, 3S-cis and 1R, 3S-trans configurations. The cis/
trans ratios measured via 1H NMR signals correlated well with
those obtained by RP-HPLC that enabled baseline separation of the
diastereoisomers. For RP-HPLC conditions were: Nova-Pak C18
column (Waters, Milford, MA), 50 mM ammonium phosphate
buffer (pH 3) (buffer A), and 20% of A in acetonitrile (buffer B); 0%e
32% B in 8 min, and then 90% B at 18min. The following alkaloids
were obtained.
(1R, 3S)-1-(o-Hydroxyphenyl)-1, 2, 3, 4-tetrahydro-
b-carboline-3-
carboxylic Acid (2a). Following the general procedure,
L
-tryptophan
General Procedure for Removal of Side Chain Protective Groups of
the Peptides. 0.2 mmol of the protected intermediate of the peptide
was added to 1 mL of phenyl methyl ether, 1 mL of dimethyl sulfide,
and 4 mL of HF and stirred at 0 ꢂC for 2 h. The reaction mixture was
taken to dryness by evaporation. The residue was mixed with 1 mL
of phenyl methyl ether, 1 mL of dimethyl sulfide and 8 mL of HF and
stirred at 0 ꢂC for 2 h. The reaction mixture was evaporated under
reduced pressure and the residue was then triturated with petro-
leum ether. To prepare target peptide conjugates, the triturated
residue was purified on Sephadex G-10 followed by HPLC
purification.
Boc-Lys(Z)OBzl (4). 0.20 mmol of Boc-Lys(Z)-OH in 5 mL of
anhydrous ethanol was added to 0.10 mmol of Cs2CO3 in 2 mL of
water, stirred at room temperature for 1 h, and evaporated to
dryness under reduced pressure. The residue was desiccated
overnight. The dried product was dissolved in 2 mL of anhydrous
DMF, to which 34 mg (0.2 mmol) of benzyl bromide was added. The
reaction mixture was stirred at 50 ꢂC for 16 h and then filtered to
remove precipitated CsBr. The filtrate was evaporated to dryness
under reduced pressure. The residue was dissolved in 30 mL of
ethyl acetate and washed successively with 5% sodium bicarbonate,
5% citric acid, and saturated sodium chloride, and the organic phase
was isolated and dried over anhydrous sodium sulfate. Following
filtration and evaporation under reduced pressure, 89 mg (95%) of
the desired compound was obtained.
(1 mmol) and salicylaldehyde (1.3 mmol) (Fluka) were reacted at
40 ꢂC overnight and then at 70 ꢂC for 6 days. The resulting precip-
itate was collected and purified by HPLC to give 2a (31% yield). 1H
NMR (CD3OD þ TFA)
d 3.52, 3.74 (m, 2H, H-4), 4.52 (dd, 1H, H-3,
J ¼ 8.6 Hz, J ¼ 5.6 Hz), 6.57 (s, 1H, H-1), 7.03e7.75 (m, 8H, indol and
Ph); 13C NMR (CD3OD þ TFA)
d 23.43, 52.22, 53.51, 107.73, 112.55,
116.67, 119.11, 120.76, 121.44, 123.89, 127.74, 132.84, 134.84, 138.69,
157.31,171.18; ESI-MS (M þ H)þ 309, (M þ H ꢃ 73) 236. (1S, 3S)-1-(o-
Hydroxyphenyl)-1, 2, 3, 4-tetrahydro-b-carboline-3-carboxylic Acid
(2’a). 2’a: 1H NMR (CD3OD þ TFA)
d
4.80 (dd, 1H, H-3, J ¼ 11.9 Hz,
J ¼ 5.1 Hz), 6.48 (s, 1H, H-1); 13C NMR (CD3OD þ TFA)
d
55.01 (C-1),
57.88 (C-3); other signals similar to 2a.
(1R, 3S)-1-(p-Methoxyphenyl)-1, 2, 3, 4-tetrahydro-
b
-carboline-3-
carboxylic Acid (2b). Following the general procedure,
L-tryptophan
(1.0 mmol) and anisaldehyde (1.30 mmol) were reacted at 40 ꢂC
overnight and at 70 ꢂC for an additional 6 days. A solid precipitate
was collected and purified by HPLC to give 2b (25% yield). 1H NMR
(CD3OD)
J ¼ 7.6 Hz, J ¼ 5.8 Hz), 6.24 (s, 1H, H-1), 7.12e7.81 (m, 8H, indol and
Ph); 13C NMR (CD3OD)
23.30, 53.03, 55.97, 56.58, 107.45, 112.49,
d 3.81 (m, 2H, H-4), 4.00 (s, 3H, OCH3), 4.67 (dd, 1H, H-3,
d
115.69, 119.20, 120.74, 122.87, 123.90, 126.87, 128.39, 132.59, 138.74,
162.74, 170.98; ESI-MS (M þ H)þ 323. (1S, 3S)-1-(p-Methoxyphenyl)-
1, 2, 3, 4-tetrahydro-b
-carboline-3-carboxylic Acid (2’b): 1H NMR
(CD3OD)
d
4.80 (dd,1H, H-3, J ¼ 12.1 Hz, J ¼ 5.3 Hz), 6.08 (s,1H, H-1);
13C NMR (CD3OD)
d
57.50 (C-3), 59.62 (C-1); other signals as in 2b.
HCl$Lys(Z)-OBzl (5). Following the procedure described above
for removal of the Boc of the C-terminal component, 0.2 mmol of
Boc-Lys(Z)-OBzl was converted into HCl$Lys(Z)-OBzl. The resulting
product was directly used for the coupling reaction without further
purification.
Boc-Ala-Lys(Z)-OBzl (6). Following the procedure described above
for coupling of C-terminal and N-terminal components from
0.2 mmol of Boc-Ala-OH and 0.2 mmol of HCl$Lys(Z)-OBzl, 98 mg of
(1R, 3S)-1-(4’-Hydroxy-3’-methoxyphenyl)-1, 2, 3, 4-tetrahydro-
carboline-3-carboxylic Acid (2c). Following the general procedure,
tryptophan (1.0 mmol) and vanillin (1.30 mmol) were reacted at
40 ꢂC overnight and then at 70 ꢂC for an additional 8 days. A solid
precipitate was collected and purified by HPLC to yield 2c (41% yield).
b
-
L-
1H NMR (CD3OD þ TFA)
d 3.49 (m, 2H, H-4), 3.99 (s, 3H, OCH3), 4.30
(dd,1H, H-3, J ¼ 8.1 Hz, J ¼ 5.9 Hz), 6.16 (s,1H, H-1), 6.94e7.74 (m, 7H,
indol and Ph); 13C NMR (CD3OD þ TFA)
d
23.69, 53.30, 56.50, 57.14,
the desired compound was obtained as a colorless powder (90%
20
107.54, 112.62, 114.17, 116.77, 119.20, 120.84, 124.04, 124.50, 126.08,
yield). mp: 88e90 ꢂC. FAB-MS (m/e): 542 [M þ H]þ. [
a]
D
¼ 6.6
127.12, 129.40, 138.75, 149.88, 171.14; ESI-MS (M þ H)þ 339. (1S, 3S)-
(c ¼ 0.2, CHCl3). 1H NMR (CDCl3):
d
8.12 (d, J ¼ 6.69 Hz, 1H), 8.10 (d,
1-(4’-Hydroxy-3’-methoxyphenyl)-1, 2, 3, 4-tetrahydro-
b
-carboline-3-
J ¼ 6.70 Hz,1H), 8.08 (t, J ¼ 4.59 Hz,1H), 7.36 (d, J ¼ 7.86 Hz, 2H), 7.36
(d, J ¼ 7.79 Hz, 2H), 7.31 (t, J ¼ 7.86 Hz, 1H), 7.29 (t, J ¼ 7.79 Hz,1H), 7.21
(t, J ¼ 7.86 Hz, 2H), 7.20 (t, J ¼ 7.79 Hz, 2H), 4.31 (m, 1H), 5.33 (s, 2H),
5.315 (s, 2H), 4.21 (dt, J ¼ 6.70 Hz, J ¼ 4.64 Hz,1H), 2.95 (dt, J ¼ 4.72 Hz,
J ¼ 4.59 Hz, 2H),1.95 (dt, J ¼ 4.77 Hz, J ¼ 4.70 Hz, 2H),1.53 (m, 2H),1.32
(m, 2H), 1.47 (s, 3H), 1.45 (s, 9H).
carboxylic Acid (2’c):1H NMR (CD3OD þ TFA)
d 4.35 (dd, 1H, H-3,
J ¼ 12.0 Hz, J ¼ 5.1 Hz), 5.91 (s, 1H, H-1); 13C NMR (CD3OD þ TFA)
d
57.66 (C-3), 60.29 (C-1); other signals as 2c.
General Procedure for Coupling C-Terminal and N-Terminal
Components. 0.20 mmol of HOBt and 0.25 mmol of dicyclohex-
ylcarbodiimide (DCC) were added to solution containing
a
HCl$AlaeLys(Z)-OBzl (7). Following the procedure described
above for the removal of the Boc of the C-terminal component,
0.2 mmol of Boc-Ala-Lys(Z)-OBzl was converted into HCl$AlaeLys
(Z)-OBzl, which was directly used for the coupling reaction without
further purification.
0.20 mmol of the N-terminal component in 5 mL of anhydrous THF
at 0 ꢂC. The reaction mixture was stirred at 0 ꢂC for 24 h. Precipi-
tated dicyclohexyl urea was isolated by filtration. The filtrate was
evaporated under reduced pressure, and the residue was triturated

1460
W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 1453e1462
Boc-Pro-Ala-Lys(Z)-OBzl (8). Following the procedure described
1.51 (d, J ¼ 6.67 Hz, 3H), 1.49 (d, J ¼ 6.67 Hz, 3H), 1.45 (s, 9H), 1.31
(m, 2H).
above for the coupling of C-terminal and N-terminal components,
0.2 mmol of Boc-Pro-OH and 0.2 mmol of HCl$Ala-Lys(Z)-OBzl
yielded in 113 mg (89% yield) of Boc-Pro-Ala-Lys(Z)-OBzl as
Boc-Gln-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl (13). Following the proce-
dure for coupling C-terminal and N-terminal components from
0.25 mmol Boc-Gln-OH and 0.2 mmol HCl$Arg(Tos)-Pro-Ala-Lys(Z)-
OBzl, 185 mg Boc-Gln-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl was obtained
a colorless powder. mp: 85e87 ꢂC. FAB-MS (m/e): 639 [M þ H]þ.
20
[
a]
¼ ꢃ8.6 (c ¼ 0.2, CHCl3). 1H NMR (CDCl3):
8.12 (d, J ¼ 6.67 Hz,
d
D
1H), 8.11 (d, J ¼ 6.71 Hz, 1H), 8.09 (t, J ¼ 4.62 Hz, 1H), 7.37 (d,
J ¼ 7.88 Hz, 2H), 7.36 (d, J ¼ 7.76 Hz, 2H), 7.31 (t, J ¼ 7.87 Hz,1H), 7.29
(t, J ¼ 7.78 Hz, 1H), 7.21 (t, J ¼ 7.84 Hz, 2H), 7.21 (t, J ¼ 7.76 Hz, 2H),
4.32 (m, 1H), 5.33 (s, 2H), 5.321 (s, 2H), 4.22 (dt, J ¼ 6.72 Hz,
J ¼ 4.66 Hz, 1H), 3.44 (t, J ¼ 4.78 Hz, 2H), 2.95 (dt, J ¼ 4.75 Hz,
J ¼ 4.58 Hz, 2H), 2.23 (t, J ¼ 4.69 Hz, 2H), 1.97 (m, 2H), 1.95 (dt,
J ¼ 4.79 Hz, J ¼ 4.72 Hz, 2H), 1.53 (m, 2H), 1.32 (m, 2H), 1.47 (s, 3H),
1.46 (s, 9H).
as a colorless powder in 86% yield. mp: 90e92 ꢂC. FAB-MS (m/e):
20
1077 [M þ H]þ. [
a
]
¼ ꢃ8.9ꢂ (c ¼ 0.2, CHCl3). 1H NMR (DMSO-d6):
D
d
9.40 (s, 1H), 8.33 (d, J ¼ 6.72 Hz, 1H), 8.31 (d, J ¼ 6.64 Hz, 1H), 8.14
(d, J ¼ 6.66 Hz, 1H), 8.13 (d, J ¼ 6.65 Hz, 1H), 8.02 (t, J ¼ 5.92 Hz, 1H),
7.86 (d, J ¼ 7.73 Hz, 2H), 7.40 (d, J ¼ 7.75 Hz, 2H), 7.34 (t, J ¼ 7.60 Hz,
1H), 7.32 (t, J ¼ 7.60 Hz, 1H), 7.23 (d, J ¼ 7.60 Hz, 2H), 7.22 (d,
J ¼ 7.60 Hz, 2H), 7.215 (t, J ¼ 7.50 Hz, 2H), 7.20 (t, J ¼ 7.62 Hz, 2H),
6.46 (t, J ¼ 6.69 Hz, 2H), 5.35 (s, 2H), 5.338 (s, 2H), 4.74 (dt,
J ¼ 6.71 Hz, J ¼ 4.81 Hz, 1H), 4.69 (dt, J ¼ 6.72 Hz, J ¼ 4.66 Hz, 1H),
4.66 (m, 1H), 4.67 (m, 1H), 4.43 (t, J ¼ 5.66 Hz, 1H), 3.46 (t,
J ¼ 5.60 Hz, 2H), 2.96 (dt, J ¼ 5.98 Hz, J ¼ 4.85 Hz, 2H), 2.66 (dt,
J ¼ 6.74 Hz, J ¼ 4.70 Hz, 2H), 2.39 (s, 3H), 2.26 (t, J ¼ 5.64 Hz, 2H),
2.20 (t, J ¼ 6.76 Hz, 1H), 2.19 (t, J ¼ 6.69 Hz, 1H), 2.15 (t, J ¼ 6.72 Hz,
1H), 2.07 (t, J ¼ 6.67 Hz, 1H), 1.98 (m, 2H), 1.92 (dt, J ¼ 6.67 Hz,
J ¼ 4.85 Hz, 2H), 1.93 (dt, J ¼ 6.65 Hz, J ¼ 4.83 Hz, 2H), 1.79 (m, 2H),
1.62 (m, 2H), 1.55 (m, 2H), 1.51 (d, J ¼ 6.66 Hz, 3H), 1.44 (s, 9H), 1.31
(m, 2H).
HCl$Pro-Ala-Lys(Z)-OBzl (9). Following the procedure described
above for the removal of the Boc of the C-terminal component,
0.2 mmol of Boc-Pro-Ala-Lys(Z)-OBzl was converted into HCl$Pro-
Ala-Lys(Z)-OBzl, which was directly used for the following coupling
reaction without further purification.
Boc-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl (10). Following the procedure
described above for the removal of the Boc of the C-terminal
component, 0.2 mmol Boc-Pro-Ala-Lys(Z)-OBzl was converted into
HCl$Pro-Ala-Lys(Z)-OBzl, which was then coupled with Boc-Arg
(Tos)-OH following the procedure for the coupling of C-terminal
and N-terminal components to provide 154 mg Boc-Arg(Tos)-Pro-
Boc-Gly-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl (14). Following the proce-
dure for coupling C-terminal and N-terminal components, 187 mg
Boc-Gly-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl was prepared from 0.23 mmol
Boc-Gly-OH and 0.20 mmol HCl$Arg(Tos)-Pro-Ala-Lys(Z)-OBzl
Ala-Lys(Z)-OBzl as a colorless powder in 79% yield. mp: 76e78 ꢂC.
FAB-MS (m/e): 977 [M þ H]þ. [
a]
¼ ꢃ9.4ꢂ (c ¼ 0.2, CHCl3). 1H
20
D
NMR (DMSO-d6):
d
9.35 (s, 1H), 8.23 (d, J ¼ 6.72 Hz, 1H), 8.12 (d,
and yielded a colorless powder (93% yield). mp: 93e95 ꢂC. FAB-
20
J ¼ 6.69 Hz, 1H), 8.12 (d, J ¼ 6.68 Hz, 1H), 8.01 (t, J ¼ 5.98 Hz, 1H),
7.85 (d, J ¼ 7.81 Hz, 2H), 7.38 (d, J ¼ 7.81 Hz, 2H), 7.32 (t, J ¼ 7.54 Hz,
1H), 7.32 (t, J ¼ 7.49 Hz, 1H), 7.228 (d, J ¼ 7.62 Hz, 2H), 7.22 (d,
J ¼ 7.63 Hz, 2H), 7.21 (t, J ¼ 7.55 Hz, 2H), 7.20 (t, J ¼ 7.61 Hz, 2H), 5.34
(s, 2H), 5.33 (s, 2H), 4.78 (d, J ¼ 6.76 Hz, 2H), 4.74 (dt, J ¼ 6.72 Hz,
J ¼ 4.84 Hz, 1H), 4.69 (dt, J ¼ 6.72 Hz, J ¼ 4.68 Hz, 1H), 4.67 (m, 1H),
4.42 (t, J ¼ 5.64 Hz, 1H), 3.46 (t, J ¼ 5.561 Hz, 2H), 2.95 (dt,
J ¼ 5.98 Hz, J ¼ 4.84 Hz, 2H), 2.65 (dt, J ¼ 6.78 Hz, J ¼ 4.65 Hz, 2H),
2.38 (s, 3H), 2.25 (t, J ¼ 5.64 Hz, 2H), 2.18 (t, J ¼ 6.78 Hz, 1H), 2.15 (t,
J ¼ 6.76 Hz, 1H), 1.97 (m, 2H), 1.92 (dt, J ¼ 6.69 Hz, J ¼ 4.88 Hz, 2H),
1.79 (m, 2H), 1.61 (m, 2H), 1.55 (m, 2H), 1.51 (d, J ¼ 6.69 Hz, 3H), 1.44
(9H), 1.30 (m, 2H).
MS (m/e): 1006 [M þ H]þ. [
a]
¼ ꢃ10.6ꢂ (c ¼ 0.2, CHCl3). 1H
D
NMR (DMSO-d6):
d
9.35 (s, 1H), 8.32 (d, J ¼ 6.69 Hz, 1H), 8.28
(d,
J
¼
6.70 Hz, 1H), 8.14 (d,
J
¼
6.70 Hz, 1H), 8.13 (d,
J ¼ 6.66 Hz, 1H), 8.01 (t, J ¼ 5.87 Hz, 1H), 7.86 (d, J ¼ 7.74 Hz,
2H), 7.39 (d, J ¼ 7.79 Hz, 2H), 7.34 (t, J ¼ 7.58 Hz, 1H), 7.32 (t,
J ¼ 7.52 Hz, 1H), 7.23 (d, J ¼ 7.65 Hz, 2H), 7.22 (d, J ¼ 7.58 Hz,
2H), 7.214 (t, J ¼ 7.47 Hz, 2H), 7.20 (t, J ¼ 7.66 Hz, 2H), 5.35 (s,
2H), 5.35 (s, 2H), 4.75 (dt, J ¼ 6.72 Hz, J ¼ 4.81 Hz, 1H), 4.69
(dt, J ¼ 6.76 Hz, J ¼ 4.65 Hz, 1H), 4.67 (d, J ¼ 6.69 Hz, 2H), 4.66
(m, 1H), 4.43 (t, J ¼ 5.67 Hz, 1H), 3.46 (t, J ¼ 5.60 Hz, 2H), 2.95
(dt, J ¼ 5.94 Hz, J ¼ 4.85 Hz, 2H), 2.66 (dt, J ¼ 6.74 Hz,
J ¼ 4.65 Hz, 2H), 2.39 (s, 3H), 2.26 (t, J ¼ 5.64 Hz, 2H), 2.20 (t,
J ¼ 6.76 Hz, 1H), 2.15 (t, J ¼ 6.72 Hz, 1H), 1.97 (m, 2H), 1.92 (dt,
J ¼ 6.67 Hz, J ¼ 4.86 Hz, 2H), 1.79 (m, 2H), 1.62 (m, 2H), 1.55
(m, 2H), 1.48 (d, J ¼ 6.64 Hz, 3H), 1.44 (s, 9H), 1.32 (m, 2H).
HCl$AlaeArg(Tos)-Pro-Ala-Lys(Z)-OBzl (15). Following the
procedure for the removal of the Boc of the C-terminal component,
204 mg (0.2 mmol) of Boc-Ala-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl was
converted to HCl$Ala-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl. The resulting
solid was directly used for the coupling reaction without further
purification.
HCl$Arg(Tos)-Pro-Ala-Lys(Z)-OBzl (11). A solution of 195 mg
(0.20 mmol) of Boc-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl in 2 mL of
hydrochloride in ethyl acetate (6 mol/L) was stirred at room
temperature for 3 h. The mixture was evaporated to dryness. The
residue was dissolved in 10 mL of ethyl acetate, and taken to
dryness. The resulting solid was directly used for the following
coupling reaction without further purification.
Boc-Ala-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl (12). Employing the
procedure for the C-terminal/N-terminal component coupling,
185 mg Boc-Ala-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl was obtained as
a colorless powder (91% yield) from 0.2 mmol Boc-Ala-OH and
HCl$Gln-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl (16). Following the proce-
dure for the removal of the Boc of the C-terminal component, 216 mg
(0.2 mmol) of Boc-Gln-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl was converted
to HCl$Gln-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl. The resulting solid was
directly used for the coupling reaction without further purification.
HCl$Gly-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl (17). Following the proce-
dure for the removal of the Boc of the C-terminal component,
202 mg (0.2 mmol) of Boc-Gly-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl was
converted into the desired compound. The product was directly
used for the coupling reaction without further purification.
Ala-Arg-Pro-Ala-Lys-OH (18). Following the procedure for the
removal of side chain protective groups of the peptides, 204 mg
(0.2 mmol) of Boc-Ala-Arg(Tos)-Pro- Ala-Lys(Z)-OBzl yielded 98 mg
of the desired compound was obtained as colorless crystals (91%
0.2 mmol HCl$Arg(Tos)-Pro-Ala-Lys(Z)-OBzl. mp: 98e101 ꢂC. FAB-
20
MS (m/e) 1020 [M þ H]þ. [
a]
¼ ꢃ8.8ꢂ (c ¼ 0.2, CHCl3). 1H NMR
D
(DMSO-d6):
d
9.36 (s, 1H), 8.32 (d, J ¼ 6.70 Hz, 1H), 8.29 (d,
J ¼ 6.69 Hz, 1H), 8.13 (d, J ¼ 6.68 Hz, 1H), 8.12 (d, J ¼ 6.67 Hz, 1H),
8.01 (t, J ¼ 5.89 Hz, 1H), 7.86 (d, J ¼ 7.75 Hz, 2H), 7.39 (d, J ¼ 7.77 Hz,
2H), 7.33 (t, J ¼ 7.56 Hz, 1H), 7.32 (t, J ¼ 7.54 Hz, 1H), 7.23 (d,
J ¼ 7.64 Hz, 2H), 7.22 (d, J ¼ 7.61 Hz, 2H), 7.21 (t, J ¼ 7.49 Hz, 2H),
7.20 (t, J ¼ 7.64 Hz, 2H), 5.34 (s, 2H), 5.33 (s, 2H), 4.75 (dt,
J ¼ 6.70 Hz, J ¼ 4.83 Hz, 1H), 4.69 (dt, J ¼ 6.74 Hz, J ¼ 4.67 Hz, 1H),
4.66 (m, 1H), 4.67 (m, 1H), 4.43 (t, J ¼ 5.65 Hz, 1H), 3.46 (t,
J ¼ 5.57 Hz, 2H), 2.96 (dt, J ¼ 5.96 Hz, J ¼ 4.87 Hz, 2H), 2.66 (dt,
J ¼ 6.76 Hz, J ¼ 4.67 Hz, 2H), 2.390 (s, 3H), 2.26 (t, J ¼ 5.62 Hz, 2H),
2.20 (t, J ¼ 6.78 Hz,1H), 2.16(t, J ¼ 6.74 Hz,1H), 1.97 (m, 2H), 1.92 (dt,
J ¼ 6.66 Hz, J ¼ 4.85 Hz, 2H), 1.79 (m, 2H), 1.61 (m, 2H), 1.55 (m, 2H),
yield). mp: 224e226 ꢂC. FAB-MS (m/e): 542 [M
þ
H]þ.
20
[a
]
¼ ꢃ40.4ꢂ (c ¼ 2, H2O). 1H NMR (DMSO-d6):
d 9.98 (s, 1H), 8.43
D

W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 1453e1462
1461
(s, 2H), 8.28 (d, J ¼ 6.68 Hz, 1H), 8.22 (d, J ¼ 6.72 Hz, 1H), 8.01 (d,
J ¼ 6.70 Hz, 1H), 7.58 (d, J ¼ 6.65 Hz, 1H), 7.16 (d, J ¼ 6.68 Hz, 1H),
4.26 (1H), 4.21 (dt, J ¼ 6.69 Hz, J ¼ 4.84 Hz, 1H), 4.22 (dt, J ¼ 6.72 Hz,
J ¼ 4.65 Hz, 1H), 4.02 (m, 1H), 3.67 (m, 1H), 3.53 (t, J ¼ 6.74 Hz, 2H),
3.35 (t, J ¼ 5.55 Hz, 2H), 3.07 (t, J ¼ 5.93 Hz, 2H), 2.73 (t, J ¼ 5.64 Hz,
2H), 2.24 (t, J ¼ 6.75 Hz, 1H), 2.24 (m, 2H), 2.02 (dt, J ¼ 6.64 Hz,
J ¼ 4.83 Hz, 2H), 2.01(m, 2H),1.86 (m, 2H),1.67 (m, 2H),1.53 (m, 2H),
1.53 (m, 2H), 1.28 (d, J ¼ 6.65 Hz, 3H), 1.27 (d, J ¼ 6.64 Hz, 3H).
Gln-Arg-Pro-Ala-Lys-OH (19). Following the procedure for the
removal of the side chain protective groups of the peptides, 215 mg
(0.2 mmol) of Boc-Gln-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl yielded
(Tos)-Pro-Ala-Lys(Z)OBzl (0.2 mmol), 232 mg (85%) of 23a was
obtained as a colorless powder. FAB-MS (m/e): 1367 [M þ H].
23b: Following the procedure for the coupling of C-terminal and
N-terminal components from 3b (0.2 mmol) and HCl$Gln-Arg(Tos)-
Pro-Ala-Lys(Z)OBzl (0.2 mmol), 237 mg (86%) of 23b was obtained
as a colorless powder. FAB-MS (m/e): 1381 [M þ H].
23c: Following the procedure for the coupling of C-terminal and
N-terminal components from 3c (0.2 mmol) and HCl$Gln-Arg(Tos)-
Pro-Ala-Lys(Z)OBzl (0.2 mmol), 229 mg (82%) of 23c was obtained
as a colorless powder. FAB-MS (m/e): 1397 [M þ H].
24a: Following the procedure for the removal of side chain
protective groups, 0.1 mmol of 21a was converted to 62 mg (75%) of
compound 24a as a colorless powder. FAB-MS (m/e): 832 [M þ H].
24b: Following the procedure for the removal of side chain
protective groups, 0.1 mmol of 21b was converted to 70 mg (83%) of
compound 24b as a colorless powder. FAB-MS (m/e): 846 [M þ H].
24c: Following the procedure for the removal of side chain
protective groups, 0.1 mmol of 21c was converted to 61 mg (71%) of
compound 24c as a colorless powder. FAB-MS (m/e): 862 [M þ H].
25a: Following the procedure for the removal of side chain
protective groups, 0.1 mmol of 22a was converted to 51 mg (62%) of
compound 25a as a colorless powder. FAB-MS (m/e): 818 [M þ H].
25b: Following the procedure for the removal of side chain
protective groups, 0.1 mmol of 22b was converted to 64 mg (77%) of
compound 25b as a colorless powder. FAB-MS (m/e): 832 [M þ H].
25c: Following the procedure for the removal of side chain
protective groups, 0.1 mmol of 22c was converted to 69 mg (81%) of
compound 25c as a colorless powder. FAB-MS (m/e): 848 [M þ H].
26a: Following the procedure for the removal of side chain
protective groups, 0.1 mmol of 23a was converted to 65 mg (73%) of
compound 26a as a colorless powder. FAB-MS (m/e): 889 [M þ H].
26b: Following the procedure for the removal of side chain
protective groups, 0.1 mmol of 23b was converted to 64 mg (71%) of
compound 26b as a colorless powder. FAB-MS (m/e): 903 [M þ H].
26c: Following the procedure for the removal of side chain
protective groups, 0.1 mmol of 23c was converted to 60 mg (65%) of
compound 26c as a colorless powder. FAB-MS (m/e): 919 [M þ H].
110 mg of the desired species as colorless crystals (92% yield). mp:
20
180e182 ꢂC. FAB-MS(m/e): 599 [M þ H]þ. [
a
]
D
¼ ꢃ35.0ꢂ (c ¼ 2.01,
H2O). 1H NMR (DMSO-d6):
d 9.99 (s, 1H), 8.45 (s, 2H), 8.28 (d,
J ¼ 6.74 Hz, 1H), 8.21 (d, J ¼ 6.66 Hz, 1H), 8.15 (d, J ¼ 6.68 Hz, 1H),
7.57 (d, J ¼ 6.66 Hz,1H), 7.16 (t, J ¼ 5.94 Hz,1H), 6.40 (s, 2H), 4.28 (dt,
J ¼ 6.73 Hz, J ¼ 4.84 Hz, 1H), 4.27 (dt, J ¼ 6.70 Hz, J ¼ 4.68 Hz, 1H),
4.22 (m, 1H), 4.11 (m, 1H), 4.24 (t, J ¼ 5.67 Hz, 1H), 3.54 (dt,
J ¼ 5.96 Hz, J ¼ 4.87 Hz, 2H), 3.36 (t, J ¼ 5.62 Hz, 2H), 3.07 (t,
J ¼ 5.64 Hz, 2H), 2.74 (t, J ¼ 5.62 Hz, 2H), 2.39 (t, J ¼ 6.67 Hz, 2H),
2.29 (t, J ¼ 6.65 Hz, 2H), 2.23 (t, J ¼ 6.64 Hz, 2H), 2.19 (t, J ¼ 6.70 Hz,
2H), 2.03 (m, 2H),1.91 (dt, J ¼ 6.65 Hz, J ¼ 4.83 Hz, 2H), 1.76 (dt,
J ¼ 6.67 Hz, J ¼ 4.81 Hz, 2H), 1.67 (m, 2H), 1.53 (m, 2H), 1.53 (m, 2H),
1.26 (d, J ¼ 6.64 Hz, 3H).
Gly-Arg-Pro-Ala-Lys-OH (20). Following the procedure for the
removal of side chain protective groups, 201 mg (0.2 mmol) of Boc-
Gly-Arg(Tos)-Pro-Ala-Lys(Z)-OBzl yielded 95 mg of the desired
species as colorless crystals (91% yield). mp: 168e170 ꢂC. FAB-MS
20
(m/e): 528 [M þ H]þ. [
a]
¼ ꢃ20.0ꢂ (c ¼ 1.98, H2O). 1H NMR
D
(DMSO-d6):
d
9.99 (s, 1H), 8.56 (s, 2H), 8.10 (d, J ¼ 6.67 Hz, 1H), 7.98
(d, J ¼ 6.72 Hz, 1H), 7.97 (d, J ¼ 6.71 Hz, 1H), 7.62 (d, J ¼ 6.67 Hz, 1H),
7.07 (t, J ¼ 5.86 Hz, 1H), 4.62 (dt, J ¼ 6.70 Hz, J ¼ 4.83 Hz, 1H), 4.52
(dt, J ¼ 6.75 Hz, J ¼ 4.64 Hz, 1H), 4.25 (d, J ¼ 6.67 Hz, 2H), 4.23 (m,
1H), 4.11 (t, J ¼ 5.65 Hz, 1H), 3.22 (t, J ¼ 5.62 Hz, 2H), 3.38 (dt,
J ¼ 5.96 Hz, J ¼ 4.82 Hz, 2H), 3.06 (t, J ¼ 5.48 Hz, 2H), 2.73 (dt,
J ¼ 6.71 Hz, J ¼ 4.63 Hz, 2H), 2.53 (t, J ¼ 5.65 Hz, 2H), 2.53 (t,
J ¼ 5.66 Hz, 2H), 2.04 (m, 2H), 1.98 (dt, J ¼ 6.65 Hz, J ¼ 4.84 Hz, 2H),
1.86 (m, 2H), 1.675 (m, 2H), 1.53 (m, 2H), 1.53 (m, 2H), 1.24 (d,
J ¼ 6.61 Hz, 3H).
5.2. Methods for in vitro biological assessments
21a: Following the procedure for the coupling of C-terminal and
N-terminal components from 3a (0.2 mmol) and HCl$Ala-Arg(Tos)-
Pro-Ala-Lys(Z)OBzl (0.2 mmol), 235 mg (90%) of 21a was obtained
as a colorless powder. FAB-MS (m/e): 1310 [M þ H].
5.2.1. PC12 cell survival assay [23]
The free radical scavenging capacity of compounds 26a,b,c and
associated precursors (2aec) were tested against NO, H2O2, and
21b: Following the procedure for the coupling of C-terminal and
N-terminal components from 3b (0.2 mmol) and HCl$Ala-Arg(Tos)-
Pro-Ala-Lys(Z)OBzl (0.2 mmol), 233 mg (88%) of 21b was obtained
as a colorless powder. FAB-MS (m/e): 1324 [M þ H].
$OH in PC12 cells and expressed as EC50 (mM). PC12 cells were
grown in Dulbecco’s modified Eagle’s medium (DMEM) supple-
mented with 10% heat inactivated horse serum (Hyclone), 5% fetal
bovine serum (GIBCO), 1.0 mM sodium pyruvate, 100 U/ml peni-
21c: Following the procedure for the coupling of C-terminal and
N-terminal components from 3c (0.2 mmol) and HCl$Ala-Arg(Tos)-
Pro-Ala-Lys(Z)OBzl (0.2 mmol), 227 mg (85%) of 21c was obtained
as a colorless powder. FAB-MS (m/e): 1340 [M þ H].
cillin and 100
During the exponential phase of growth, PC12 cells were seeded in
96-well plates coated with poly- -lysine at a density of 20,000 cells
per well. After a 24 h attachment period, fresh media containing
12.5 M, 25 M, 50 M, 100 M, or 200 M of the test compounds
m
g/mL streptomycin at 37 ꢂC in a 5% CO2 atmosphere.
L
22a: Following the procedure for the coupling of C-terminal and
N-terminal components from 3a (0.2 mmol) and HCl$Gly-Arg(Tos)-
Pro-Ala-Lys(Z)OBzl (0.2 mmol), 238 mg (92%) of 22a was obtained
as a colorless powder. FAB-MS (m/e): 1296 [M þ H].
m
m
m
m
m
were added to each well and incubated for 1 h. NO damage was
then induced by adding 2 mM of sodium nitroprusside (SNP) fol-
lowed by 1 h of incubation. After replacement with fresh media,
cells were incubated for 14 h after which cell survival was
measured by a colorimetric assay with MTT according to estab-
lished methods. Similarly, H2O2 damage was induced by 1 h of
incubation with 1 mM H2O2, and $OH damage was induced by 1 h of
22b: Following the procedure for the coupling of C-terminal and
N-terminal components from 3b (0.2 mmol) and HCl$Gly-Arg(Tos)-
Pro-Ala-Lys(Z)OBzl (0.2 mmol), 233 mg (89%) of 22b was obtained
as a colorless powder. FAB-MS (m/e): 1310 [M þ H].
22c: Following the procedure for the coupling of C-terminal and
N-terminal components from 3c (0.2 mmol) and HCl$Gly-Arg(Tos)-
Pro-Ala-Lys(Z)OBzl (0.2 mmol), 231 mg (87%) of 22c was obtained
as a colorless powder. FAB-MS (m/e): 1326 [M þ H].
incubation with 1 mM H2O2/30 mM Fe (II).
5.3. In vivo antithrombotic activity determinations in the rat [22]
23a: Following the procedure for the coupling of C-terminal and
N-terminal components from 3a (0.2 mmol) and HCl$Gln-Arg
Assessments were performed based upon a protocol reviewed
and approved by the Ethics Committee of HeBei Medical University.

1462
W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 1453e1462
Animals were maintained in accordance with the requirements of
the animal welfare act and according to the guide for care and use
of laboratory animals. Tested compounds were dissolved in normal
saline (NS) prior to use and kept in an ice bath. Male Wistar rats
(250e300 g) were anaesthetized with sodium pentobarbital
(80.0 mg/kg, i.p.) and the right carotid artery and left jugular vein
were separated. A 6 cm thread with predetermined weight was
placed into the middle of a polyethylene tube, filled with sodium
heparin (50 IU/mL of NS) and one end inserted into the left jugular
vein. From the other end of the polyethylene tube either sodium
heparin, NS or the test compound in NS was injected and the tube
inserted into the right carotid artery. Blood flow was directed from
the right carotid artery to the left jugular vein via the polyethylene
tube for 15 min. The thread was removed and weighed and the
weight of the wet thrombus was recorded. The thread was
desiccated for 2 weeks and the weight of the dry thrombus re-
recorded (Fig. 1).
& Bio 3D 12.0). Parameters employed were: step interval: 2.0 fs;
frame interval: 10 fs; terminate after: 10000 steps; heating/cooling
Rate: 1.000 Kcal/atom/ps; target temperature: 300 K.
Acknowledgments
The authors are grateful to NASA (MSGC R85197), Research
Excellence Fund (R01121), Natural Science Foundation of HeBei,
China (No. 062761266) and the Administration of Traditional
Chinese Medicine of HeBei Province (No. 2007134), for support of
this research. The authors are also grateful to Professor Herve
Galons for many helpful suggestions.
Appendix. Supplementary material
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.01.021.
5.4. Hind limb ischemia-reperfusion injury assessment [21]
Male Wistar rats (250e300 g; food and water ad libitum) were
randomly allocated into 3 groups: (1) Sham group: rats subjected to
the procedures described below, except for ischemia/reperfusion (I/
R) (n ¼ 6); (2) I/R group: rats subjected to the procedures described
below underwent limb ischemia for 3h followed by reperfusion for
4 h (n ¼ 8); (3) I/R injury þ drug treatment group: rats received
compound 26a (20 mg/kg) 15 min before the reperfusion and again
1h following initiation of reperfusion (n ¼ 8). Animals in groups (1)
and (2) were administrated saline vehicle in lieu of drug. All
animals were maintained under anesthesia for the duration of the
experiment.
Anesthesia was achieved via intraperitoneal injection of sodium
pentobarbital (80 mg/kg), and body temperature was maintained at
37 ꢂC with the aid of a heating pad. Bilateral rubber bands were
applied above the greater trochanter to interrupt the arterial blood
supply to the hind limbs. After 3 h of hind limb ischemia, the rubber
bands were removed, thereby inducing hind limb reperfusion (4 h).
Fifteen minutes prior to the reperfusion and again 1h after initia-
tion of reperfusion, compound 26a (20 mg/kg) was administered
intraperitoneally to rats assigned to the I/R þ drug treatment group,
while sham rats received saline. At the conclusion of experiments,
rats were euthanized via sodium pentobarbital overdose, muscle
and heart tissues were immediately isolated, and blood was also
rapidly obtained from the ascending aorta. Tissue samples were
divided into two sections, one for determination of lipid perox-
idation, and the other for tissue sections (Leica CM1850 UV clinical
cryostat) at ꢃ30 ꢂC for histological examination.
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