Novel β-carboline-tripeptide conjugates attenuate mesenteric ischemia/reperfusion injury in the rat

Article abstract of DOI:10.1016/j.ejmech.2011.03.029

We have synthesized a series of new β-carboline-tripeptide conjugates, and examined their anti-inflammatory properties in a mouse model of xylene-induced ear edema. The analgesic capacity of these compounds was further evaluated in a rodent tail flick ass

Full text of DOI:10.1016/j.ejmech.2011.03.029

European Journal of Medicinal Chemistry 46 (2011) 2441e2452
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel
b-carboline-tripeptide conjugates attenuate mesenteric
ischemia/reperfusion injury in the rat
,
d
c
c
c
c
c
Wei Bi c , Yue Bi , Ping Xue , Yanrong Zhang , Xiang Gao , Zhibo Wang , Meng Li ,
*
Michele Baudy-Floc’h e, Nathaniel Ngerebara a, Xiaoxu Li f, 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
f Columbia University College of Physicians and Surgeons, Columbia University, New York, NY 10027, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized a series of new
b-carboline-tripeptide conjugates, and examined their anti-
Received 6 January 2011
Received in revised form
28 February 2011
Accepted 14 March 2011
Available online 21 March 2011
inflammatory properties in a mouse model of xylene-induced ear edema. The analgesic capacity of these
compounds was further evaluated in a rodent tail flick assay. Our results indicate that -carboline
conjugate 4a manifests potent anti-inflammatory and analgesic activity while exerting a protective effect
against mesenteric ischemia/reperfusion (I/R) injury in the rat.
Published by Elsevier Masson SAS.
b
Keywords:
Ischemia/reperfusion injury
Anti-inflammatory activity
Analgesic activity
b
-carboline-tripeptide conjugate
1. Introduction
neutrophils in this tissue enhance ROS and cytotoxic protein
production, leading to an induction of the inflammatory cascade
Mesenteric ischemia is frequently encountered in multiple
disease states and surgical procedures, including cardiovascular
interventions, small bowel transplantation mesenteric thrombosis,
shock syndromes, and severe burns [1,2]. The ensuing reperfusion
is unavoidable and may induce downstream tissue injury. Intestinal
ischemia and reperfusion injury can accelerate interactions
between endothelium and different cell types resulting in micro-
vascular injury, cellular necrosis, and apoptosis [3e7]. During
intestinal ischemia/reperfusion, reactive oxygen species (ROS)
attack constituents of the cell membrane constituents, resulting in
lipid peroxidation, membrane disintegration, and increased
microvascular permeability. The latter induces activation and
adhesion of polymorphonuclear neutrophils (PMNs), the release of
proinflammatory substances, and increased formation of ROS. The
intestine is highly sensitive to ischemic injury. Activated
[3,4], resulting in cell death and organ failure. To counter these
processes, various therapeutic strategies have been attempted to
attenuate intestinal ischemia/reperfusion injury, including anti-
leukocyte and anti-inflammatory therapies, as well as glutamine,
glycine and antioxidant supplementation [5,8].
b-Carbolines possess potential antioxidant properties. For
example, harmane, harmaline and harmalol inhibit lipid perox-
idation in microsomal hepatic preparations [9] and through
attenuation of oxidative damage of hyaluronic acid, cartilage
collagen and immunoglobulin G [10]. Other findings suggest that
b
-carboline alkaloids exert a protective effect via ROS scavenging
[11e13].
It has been reported that some short peptides with certain
sequences may be used as analgesic agents [14]. In addition, our
recent research findings indicated that some tripeptides with AA-
Phe-Trp sequence present good analgesic activities. Since reactive
oxygen species (ROS) and inflammatory leukocytes play an
important role in the pathogenesis of I/R injury, antioxidant
therapy and inhibition of post-ischemic neutrophil infiltration may
have therapeutic efficacy during I/R injury. Ideally, I/R injury can be
* Corresponding author. Tel./fax: þ86 311 66002578.
** Corresponding author. Tel.: þ1 906 487 1868; fax: þ1 906 487 2061.
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.2011.03.029

2442
W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 2441e2452
O
O
O
OH
OH
OH
Boc
i
ii
NH2
NH
N
N
H
N
H
N
H
2
1
O
O
iii
iv
AA-Phe-Trp-OCH3
Boc
AA-Phe-Trp
N
NH
N
N
H
H
3a-r
4a-r
Scheme 1. Synthetic scheme for b-carboline-tripeptide conjugates preparation. Reagents and conditions: (i) formaldehyde and sulfuric acid; (ii) BoceN3 and triethylamine; (iii)
HCl$L-AA-Phe-TrpeOCH3 and DCC; (iv) HF.
treated by a combination of the therapeutic agents. Generally,
when two synergistic agents are administered individually but
simultaneously, they will be transported to the site of action with
different efficiencies. However, there are potential advantages in
giving the co-administered agents as a single chemical entity. It is
desirable to have the two synergistic agents reach a site simulta-
alkaloids coupled with the analgesic activity observed with
AA-Phe-Trp tripeptides, we hypothesized that -carboline and tri-
peptide (AA-Phe-Trp) conjugates might exhibit both enhanced
antioxidant and analgesic activities. To test this hypothesis, we have
synthesized a series of novel b-carboline-tripeptide (AA-Phe-Trp)
conjugates, and evaluated their biological capacity to protect
against mesenteric I/R injury.
b
neously. Considering the antioxidant properties of
b-carboline
Table 1
In vivo orally analgesic activities of 4aer tested using tail flick method in mice.
Compd.
Pain threshold variation (X ꢀ SD%)
30 min 60 min
5.03 ꢀ 10.26 7.45 ꢀ 8.03
90 min
7.53 ꢀ 11.54
120 min
150 min
180 min
NS
8.09 ꢀ 13.28
98.78 ꢀ 38.43
33.24 ꢀ 18.32a
34.55 ꢀ 38.43a
49.09 ꢀ 29.03a
18.79 ꢀ 15.74
35.79 ꢀ 9.32b
42.03 ꢀ 6.41a
45.46 ꢀ 23.54b
27.32 ꢀ 14.88b
28.55 ꢀ 12.36
42.43 ꢀ 27.22b
39.38 ꢀ 13.48b
41.28 ꢀ 13.33a
32.33 ꢀ 22.89
39.25 ꢀ 17.48b
38.45 ꢀ 19.66b
30.25 ꢀ 31.49b
37.12 ꢀ 15.25b
40.67 ꢀ 33.57a
39.23 ꢀ 27.12b
9.02 ꢀ 12.19
64.55 ꢀ 25.61
30.41 ꢀ 8.98a
25.46 ꢀ 12.12b
22.37 ꢀ 34.30b
25.05 ꢀ 18.13b
25.88 ꢀ 11.77b
28.79 ꢀ 10.13b
21.12 ꢀ 14.45b
15.25 ꢀ 22.46
16.69 ꢀ 10.33
23.19 ꢀ 15.36b
24.33 ꢀ 6.49b
29.04 ꢀ 22.58b
18.32 ꢀ 37.12
22.12 ꢀ 26.22b
26.47 ꢀ 27.55b
28.87 ꢀ 21.93b
29.38 ꢀ 16.45b
13.54 ꢀ 18.46
12.25 ꢀ 26.18
8.75 ꢀ 9.73
Morphine
Aspirin
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
142.20 ꢀ 35.43
32.85 ꢀ 10.46a
48.25 ꢀ 45.02a
10.36 ꢀ 22.24
23.17 ꢀ 25.32
39.47 ꢀ 25.44a
37.58 ꢀ 20.23a
31.34 ꢀ 31.32a
33.01 ꢀ 38.16a
35.05 ꢀ 29.27a
30.75 ꢀ 24.24a
37.25 ꢀ 21.12a
44.27 ꢀ 42.46a
41.83 ꢀ 39.35a
21.24 ꢀ 15.74b
30.56 ꢀ 21.46a
35.75 ꢀ 30.35a
38.16 ꢀ 19.26a
22.17 ꢀ 28.57b
18.24 ꢀ 11.56
175.78 ꢀ 63.21
38.45 ꢀ 12.13a
59.37 ꢀ 37.02a
40.78 ꢀ 32.03b
49.74 ꢀ 35.11a
45.77 ꢀ 26.32a
48.23 ꢀ 14.23a
37.20 ꢀ 15.13b
38.01 ꢀ 29.23a
36.45 ꢀ 27.36a
38.20 ꢀ 32.09a
32.19 ꢀ 14.78b
52.22 ꢀ 33.12a
47.33 ꢀ 19.47b
45.23 ꢀ 11.14a
42.32 ꢀ 16.41b
41.18 ꢀ 17.45a
35.13 ꢀ 22.25a
28.12 ꢀ 19.81b
17.23 ꢀ 13.55
139.03 ꢀ 49.35
41.23 ꢀ 15.76a
44.67 ꢀ 41.08a
46.45 ꢀ 5.46b
21.35 ꢀ 27.20
47.36 ꢀ 28.21a
46.48 ꢀ 9.15a
40.56 ꢀ 8.32b
41.23 ꢀ 44.46a
35.19 ꢀ 21.33a
40.36 ꢀ 23.49a
22.23 ꢀ 14.66
54.39 ꢀ 23.67a
51.25 ꢀ 11.84a
34.56 ꢀ 35.77b
44.13 ꢀ 28.22a
43.23 ꢀ 32.49a
46.08 ꢀ 19.94a
34.55 ꢀ 13.29b
29.25 ꢀ 17.12b
57.43 ꢀ 31.08
21.72 ꢀ 9.74b
18.86 ꢀ 19.46
14.36 ꢀ 28.12
15.68 ꢀ 11.32
16.03 ꢀ 8.96
19.22 ꢀ 17.46
15.32 ꢀ 11.18
16.13 ꢀ 13.39
14.23 ꢀ 12.18
19.25 ꢀ 14.39
13.29 ꢀ 12.47
25.69 ꢀ 11.36b
22.45 ꢀ 43.22b
19.45 ꢀ 21.79
25.07 ꢀ 11.21b
20.94 ꢀ 14.46b
21.55 ꢀ 17.29b
18.77 ꢀ 13.45
12.32 ꢀ 14.94
4q
4r
NS ¼ vehicle; n ¼ 10; oral dose of 4aer ¼ 0.15 mmol/kg; intraperitoneal dose of morphine ¼ 0.15 mmol/kg. Oral dose of aspirin ¼ 0.15 mmol/kg.
a
Compared with vehicle P < 0.01.
Compared with vehicle P < 0.05.
b
Table 2
Analgesic activities of 4a,k at different doses using tail flick method in mice.
Compd.
Dose
Pain threshold variation (X ꢀ SD%)
30 min
60 min
90 min
120 min
150 min
180 min
NS
5.03 ꢀ 10.26
7.45 ꢀ 8.03
7.53 ꢀ 11.54
8.09 ꢀ 13.28
9.02 ꢀ 12.19
8.75 ꢀ 9.73
4a
0.15 mmol/kg
0.10 mmol/kg
0.05 mmol/kg
48.25 ꢀ 45.02a
35.41 ꢀ 13.56a
29.22 ꢀ 10.26b
59.37 ꢀ 37.02a
37.65 ꢀ 28.53a
23.45 ꢀ 15.31b
44.67 ꢀ 41.08a
32.73 ꢀ 18.69a
20.65 ꢀ 18.94
34.55 ꢀ 38.43a
25.45 ꢀ 15.37b
17.97 ꢀ 14.64
25.46 ꢀ 12.12b
22.13 ꢀ 14.89b
8.23 ꢀ 12.04
18.86 ꢀ 19.46
13.42 ꢀ 8.68
7.46 ꢀ 8.95
4k
0.15 mmol/kg
0.10 mmol/kg
0.05 mmol/kg
44.27 ꢀ 42.46a
32.32 ꢀ 19.55b
11.55 ꢀ 9.28
52.22 ꢀ 33.12a
34.48 ꢀ 21.03a
26.54 ꢀ 10.33b
54.39 ꢀ 23.67a
38.33 ꢀ 11.25a
23.58 ꢀ 11.46b
41.28 ꢀ 13.33a
23.08 ꢀ 13.89b
18.99 ꢀ 8.96
29.04 ꢀ 22.58b
21.39 ꢀ 15.60b
15.43 ꢀ 10.94
25.69 ꢀ 11.36b
14.38 ꢀ 20.13
9.06 ꢀ 15.43
LFW
KFW
0.15 mmol/kg
0.15 mmol/kg
25.38 ꢀ 11.85b
20.16 ꢀ 9.88b
28.79 ꢀ 12.32b
25.43 ꢀ 8.79b
20.13 ꢀ 8.73
23.47 ꢀ 15.20b
18.92 ꢀ 9.20
9.77 ꢀ 2.41
7.98 ꢀ 2.94
8.41 ꢀ 5.24
20.14 ꢀ 11.57
11.23 ꢀ 7.89
NS ¼ vehicle; n ¼ 10.
a
Compared with NS, P < 0.01.
Compared with NS, P < 0.05.
b

W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 2441e2452
2443
Table 3
Anti-inflammatory activities of 4aer against xylene-induced ear edema in mice.
Compd. Edema weight Inhibition Compd. Edema weight Inhibition
(X ꢀ SD mg)
(%)
(X ꢀ SD mg)
(%)
NS
Aspirin
4a
4b
4c
4d
4e
4f
4g
3.28 ꢀ 0.49
1.80 ꢀ 0.68a
1.29 ꢀ 0.35a
2.05 ꢀ 0.76b
1.73 ꢀ 0.31a
1.85 ꢀ 0.54a
1.55 ꢀ 0.43a
1.70 ꢀ 0.41a
1.43 ꢀ 0.38a
1.59 ꢀ 0.44a
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
1.62 ꢀ 0.43a
1.39 ꢀ 0.28a
1.47 ꢀ 0.27a
1.89 ꢀ 0.60a
1.45 ꢀ 0.42a
1.78 ꢀ 0.35a
1.61 ꢀ 0.32a
1.41 ꢀ 0.49a
1.97 ꢀ 0.53b
2.35 ꢀ 0.55
50.6
57.6
55.2
42.4
55.8
45.7
50.9
57.0
40.0
28.4
45.1
60.7
37.5
47.3
43.6
52.7
48.2
56.4
51.5
4h
NS ¼ vehicle; n ¼ 10; oral dose of 4aer ¼ 0.15 mmol/kg.
a
Fig. 2. GSH levels (
m
mol/g tissue) in the intestinal tissue of sham, ischemia/reperfusion
Compared to NS, P < 0.01.
b
(I/R), I/R þ 4a and I/R þ melatonin groups. *: Compared with the sham group, P < 0.01;
Compared to NS, P < 0.05.
#: Compared with the I/R group, P < 0.01; n ¼ 8.
Table 4
Anti-inflammatory activities of 4a,c,k,o,p at different doses.
t-butyloxycarbonyl azide leads to selective acylation of the amino
group. Subsequently, compound 2 coupled with tripeptides to
generate a series of the key intermediates 3aer. After deprotection,
the desired b-carboline-tripeptide conjugates 4aer can be readily
prepared (Scheme 1).
Compd.
NS
Dose (mmol/kg)
Edema weight (X ꢀ SD mg)
3.28 ꢀ 0.49
1.29 ꢀ 0.35a
1.65 ꢀ 0.51a
2.01 ꢀ 0.48b
1.55 ꢀ 0.43a
1.81 ꢀ 0.38a
2.21 ꢀ 0.47
1.47 ꢀ 0.27a
1.79 ꢀ 0.54a
2.35 ꢀ 0.43
1.61 ꢀ 0.32a
2.20 ꢀ 0.43
2.75 ꢀ 0.66
1.41 ꢀ 0.49a
2.41 ꢀ 0.58
2.91 ꢀ 0.67
Inhibition (%)
4a
4e
4k
4o
4p
0.15
0.10
0.05
60.7
49.6
38.7
0.15
0.10
0.05
52.7
44.8
32.6
3. Biological results and discussion
3.1. In vivo oral analgesic activities of 4aer
0.15
0.10
0.05
55.2
45.4
28.3
Analgesic activity was evaluated by the tail flick procedure
[15,16]. The analgesic capacity was expressed by the pain threshold
variation, defined as the difference of pain threshold after drug
administration minus basic pain threshold/basic pain threshold.
All compounds were prescreened for analgesic activity at a dose
of 0.15 mmol/kg. For each animal, pain thresholds were estimated
at 30, 60, 120, 150 and 180 min following vehicle or drug admin-
0.15
0.10
0.05
50.9
32.9
16.2
0.15
0.10
0.05
57.0
26.5
11.3
istration. b-Carboline-tripeptide conjugates exhibited moderate to
NS ¼ vehicle; n ¼ 10.
a
good analgesic activity, with compounds 4a,e,k,l,o,p demonstrating
especially good analgesic properties (Table 1). All pain thresholds
were increased after 30 min, yet maximal pain thresholds occurred
at different time points for various synthetic derivatives. For
example, the maximal pain thresholds for 4a,c,e,h,m were
observed after 60 min, whereas for 4d,g,k,l,n,o,p this value was
90 min, and for 4b,f,i,q,r, 120 min post drug administration. The
duration of the analgesic action of 4a,b,c,d,e,f,i,j was 150 min, while
the same value for 4k,l,m,n,o,p was 180 min. We speculate that the
physiological difference in analgesic activity is closely related to the
Compared to NS, P < 0.01.
Compared to NS, P < 0.05.
b
2. Synthesis of
b
-carboline-tripeptide conjugates
-tryptophan, compound 1 was
Starting from optically active
L
synthesized via the PictecteSpengler reaction, and then subjected
to protection. The t-butyloxycarbonyl (Boc) group has been widely
used as a protecting group of various amino groups because of
the mild deprotection condition. Treatment of compound 1 with
tripeptide sequences of the b-carbolines.
Fig. 3. MPO activities (U/g) in the intestinal tissue of sham, ischemia/reperfusion (I/R),
Fig. 1. MDA activities (nmol/g tissue) in the intestinal tissue of sham, ischemia/
reperfusion (I/R), I/R þ 4a and I/R þ melatonin groups. *: Compared with the sham
group, P < 0.01; #: compared with the I/R group, P < 0.01; n ¼ 8.
#
I/R þ 4a and I/R þ melatonin groups. *: Compared with the sham group, P < 0.05;
:
Compared with the I/R group, P < 0.05; n ¼ 8.

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W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 2441e2452
Fig. 4. Representative photomicrographs of histological sections of small intestine. (A)e(C): Representative tissue sections were taken from the rats exposed to ischemia/reper-
fusion; (D)e(F): tissues sections were taken from ischemia/reperfusion þ (20 mg/kg) treated rats. Tissue sections were stained with H&E (hematoxylin and eosin). Magnification:
(A, D) ꢁ100; (B, E) ꢁ200; (C, F) ꢁ400.

W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 2441e2452
2445
Table 5
Table 5 (continued)
Molecular dynamic simulation results.
Predicted lowest
energy conformations
EHOMO (eV)
Distance between
O and H (Å)
Predicted lowest
energy conformations
EHOMO (eV)
Distance between
O and H (Å)
O(32)eH(57): 2.315A
O(31)eH(61): 2.419
O(31)eH(65): 2.129A
O(15)eH(65): 2.060
O(21)eH(76): 3.144
ꢂ8.029
ꢂ9.977
H(71)eO(15): 2.160
O(32)eH(57): 2.252
H(62)eO(45): 2.119
ꢂ7.454
ꢂ9.098
ꢂ9.073
ꢂ8.204
O(15)eH(80): 2.109
O(31)eH(87): 2.201
O(46)eH(71): 2.133
ꢂ9.911
ꢂ10.049
ꢂ8.954
O(15)eH(64): 2.060
O(41)eH(64): 2.008
O(41)eH(78): 1.848
O(46)eH(64): 2.153
O(31)eH(60): 2.346
O(15)eH(56): 2.491
O(15)eH(67): 2.221
O(15)eH(63): 1.846
O(15)eH(71): 2.035
O(15)eH(65): 2.104
O(26)eH(58): 2.036
O(15)eH(62): 2.021
O(46)eH(66): 2.005
ꢂ9.164
ꢂ10.307
ꢂ14.2209
ꢂ9.856
None
O(50)eH(70):2.118
O(46)eH(89): 2.076
O(26)eH(66): 2.291
O(26)eH(88): 2.276
O(46)eH(68): 2.024
O(31)eH(68): 2.262
O(15)eH(69): 2.246
O(26)eH(65): 2.235
O(26)eH(81): 2.708
ꢂ10.091
(continued on next page)

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W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 2441e2452
Table 5 (continued)
doses to establish the detailed pharmacological activity profile.
From Table 4, we noticed a dose-dependent anti-inflammatory
response for 4a,e,k,o,p.
Predicted lowest
energy conformations
EHOMO (eV)
Distance between
O and H (Å)
3.4. Intestinal I/R injury
Acute mesenteric ischemia leading to ischemic damage repre-
sents a serious clinical complication. During I/R, there are complex
interactions between endothelial activation, inflammatory cell
recruitment and production of reactive oxygen species. In addition,
I/R injury induces the transport of proinflammatory molecules into
the systemic circulation, promoting additional systemic inflam-
mation. Based upon the analgesic and anti-inflammatory activities
of 4a, we characterized the effects of this compound on intestinal
I/R injury in this model.
O(15)eH(67): 2.143
O(46)eH(83): 1.858
O(26)eH(59): 2.224
ꢂ8.989
ꢂ8.630
None
3.4.1. Effect of 4a on lipid peroxidation monitored via
malondialdehyde (MDA) and glutathione (GSH) analyses
Lipid peroxidation-induced oxidative stress is one cause of
intestinal I/R, linked to membrane lipid composition. Accordingly,
to assess intestinal lipid peroxidation and antioxidant defenses,
malondialdehyde (MDA) and glutathione (GSH) levels were quan-
tified in intestinal samples. MDA, an end-product of membrane
lipid peroxidation, is a generally accepted surrogate biomarker of
lipid peroxidation. MDA levels were determined following Beuge’s
method [18], while glutathione (GSH; a specific marker for protein
oxidative damage) was determined using Beutler’s method [19]
(Fig. 1).
O(46)eH(68): 2.077
O(26)eH(68): 2.180
ꢂ10.052
I/R significantly elevated MDA levels as compared to the sham
group, suggesting that ROS-induced lipid peroxidation was
underway in rat ileum. Conversely, 4a intervention significantly
attenuated this effect, likely due to the free radical scavenging
capacity of this compound.
O(26)eH(65): 2.288
O(49)eH(81): 2.140
O(49)eH(61): 2.094
ꢂ10.192
Reduced glutathione (GSH) is
a tripeptide of glutamine,
L-cysteine, and glycine and an endogenous antioxidant that reacts
with various free radicals. Oxidation of GSH leads to a disulfide
bridge (glutathione disulfide; GSSG) that can be re-reduced to
generate free GSH. Intracellularly, a dynamic balance is maintained
between GSH synthesis, consumption and oxidation. Typically, the
GSH/GSSG ratio is 9:1 in a healthy cell, and measurement of this
ratio is a useful estimator of intracellular oxidative stress [20]. We
observed GSH during reperfusion (Fig. 2), and this effect was
reversed via intervention with 4a.
THCe: N-(3S-1,2,3,4-Tetrahydro-
b
-carboline-3-carboxyl)e.
3.2. Dose-dependency of analgesic activity for 4a and 4k
To investigate dose effects, the most active species (4a,k) were
examined for dose-dependency of analgesia. Mice were adminis-
trated 0.15, 0.10 and 0.05 mmol/kg of 4a,k orally (Table 2). We
observed a clear correlation between the amount of drug, and even
at 0.05 mmol/kg, the analgesic activities of compounds 4a,k was
still measurable. Of interest,
peptides of 4a,k (Leu-Phe-Trp and Lys-Phe-Trp), all possess anal-
gesic activities, further suggesting that the analgesic activities of
3.4.2. Effect of 4a on MPO (myeloperoxidase) activity
I/R induces an acute inflammatory response in which elevated
vascular permeability and increased adherence and emigration of
leukocytes occurs. Neutrophils initially attach to the damaged
vascular endothelium and accrue in the same vascular bed, and
they are subsequently activated by proinflammatory cytokines and
tissue infiltration. Accordingly, activated neutrophils represent an
important factor in reperfused intestinal tissue injury [22,23].
Myeloperoxidase (MPO), the most abundant protein in neutrophils,
is the focus of inflammatory pathologies. MPO assay is routinely
measured as an index of neutrophil infiltration and a marker for
acute inflammation when polymorphonuclear cell infiltration
occurred [21]. We observed that MPO activity significantly
increased after 3 h of reperfusion (Fig. 3), reflecting neutrophil
infiltration, and this effect was again significantly attenuated in the
presence of 4a.
L-tryptophan, and the precursor
b-carboline-tripeptide conjugates 4a,k might be due in part to the
amino acid moieties or their downstream metabolites.
3.3. Anti-inflammatory activities of 4aer
All compounds were evaluated for anti-inflammatory capacity
in a xylene-induced ear edema model [16]. Briefly, this in vivo assay
involves oral administration of test compounds in 0.5% carbox-
ymethyl cellulose (CMC) suspension. Each compound was initially
tested at 0.15 mmol/kg, and all revealed significant protection
against xylene-induced inflammation (Table 3), suggesting potent
anti-inflammatory capacity. Since compounds (4a,e,k,o,p) exhibit
good analgesic activities in the tail flick assay and superior anti-
inflammatory activities in the xylene-induced ear edema model,
these compounds were then examined in the same assay at lower
3.4.3. Histological analysis
We observed severe mucosal damage consistent with ischemic
injury, ranging from vacuolation of villus tips to denuded villi and
mucosal ulceration in the intestinal I/R group (Fig. 4AeC).

W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 2441e2452
2447
Characteristic features included marked congestion, hemorrage,
desquamation of villum epithelium and inflammatory cell infil-
tration, as well as the presence of crypts and shortening of the villi.
We found mucosal damage on some of the surface epithelium, in
addition to extension of the sub-epithelial space with moderate to
massive epithelial lifting. Intervention with 4a significantly
reversed these findings, with only mild mucosal damage and
minimal focal denudations of the villi epithelium (Fig. 4DeF).
ammonia. The mixture was maintained at 0 ꢃC for 12 h and the
precipitate was collected by filtration. After re-crystallization,
3.97 g (75%) of product was obtained as a colorless powder. Mp:
280e282 ꢃC. EI/MS: 217 [M þ H]þ; IR (KBr): 3450, 3200, 3000,
2950, 2850, 1700, 1601, 1452, 1070, 900 cmꢂ1; 1H NMR (BHSC-500,
DMSO-d6):
d
/ppm ¼ 10.99 (s,1H), 9.89 (s,1H), 7.30 (t, J ¼ 7.5 Hz,1H),
7.22 (t, J ¼ 8.0 Hz, 1H), 7.01 (d, J ¼ 8.0 Hz,1H), 6.81 (d, J ¼ 7.5 Hz, 1H),
4.01 (t, J ¼ 4.8 Hz, 1H), 3.75 (dd, J ¼ 10.5 Hz, J ¼ 5.0 Hz, 1H), 3.64 (dd,
J ¼ 10.5 Hz, J ¼ 2.4 Hz, 1H), 2.91 (d, J ¼ 10.5 Hz, 2H), 2.86 (s, 1H).
Anal. Calcd for C12H12N2O2: C, 66.65; H, 5.59; N, 12.96. Found: C,
66.45; H, 5.72; N, 12.79.
3.5. Molecular dynamic simulation
The electron ionization energy is a quantitative descriptor of the
capacity of a compound to scavenge oxidants. The ionization
energy from the highest occupied molecular orbital (HOMO) can be
estimated by Koopmans theorem, with reasonable accuracy [24].
The HOMO energy is often employed as a primary descriptor of
radical scavenging/antioxidant activity [25]. The high HOMO
4.2.2. N-Boc-3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxylic acid (2)
A suspension of 1.1 g (5.0 mmol) of 1 in 15 mL DMF and 1.4 mL of
triethylamine was vigorously stirred at room temperature, and
supplemented with 1.1 g (7.7 mmol) of BoceN3 over a 30 min
period. The reaction mixture was stirred at room temperature for
24 h and then at 40 ꢃC for 80 h. Citrate solution (5 mL; 20%) was
then added and the solution extracted with ethyl acetate
(3 ꢁ 30 mL). Combined ethyl acetate fractions were dried over
anhydrous MgSO4. The filtrate was dried by evaporation after
removal of MgSO4. The residue was crystallized in CHCl3 to yield
1.20 g (76%) of product. Mp: 165e170 ꢃC. TOF/MS: 317 [M þ H]þ,
339 [M þ Na]þ, 355 [M þ K]; IR (KBr): 3452, 3205, 3001, 2952, 2848,
energy of our indole b-carboline derivatives highlights their elec-
tron-donating potential and coordination capacities, suggesting
they may serve as metal ion coordinators and subsequently as
inhibitors of ion-catalyzed oxidation processes (Table 5).
Generally, the higher the HOMO energy, the more potent the
antioxidant activity. The HOMO energy of the endogenous antiox-
idant melatonin is ꢂ10.425 eV. In comparison, the HOMO energy of
compounds 4a,b,e,k,o,p were ꢂ8.029 eV, ꢂ7.454 eV, ꢂ8.204 eV,
ꢂ8.954 eV, ꢂ8.989 eV and ꢂ8.630 eV, respectively. Accordingly,
4a,b,e,k,o,p should possess higher radical trapping potentials than
melatonin. Moreover, the high positive charge of the hydrogen
1705, 1645, 1600, 1450, 1072, 901 cmꢂ1
DMSO-d6):
7.21 (t, J ¼ 7.9 Hz, 1H), 7.00 (d, J ¼ 7.9 Hz, 1H), 6.84 (t, J ¼ 7.6 Hz, 1H),
4.84 (t, J ¼ 5.0 Hz, 1H), 4.20 (dd, J ¼ 10.2 Hz, J ¼ 4.8 Hz, 1H), 3.98 (dd,
J ¼ 10.2 Hz, J ¼ 3.2 Hz, 1H), 2.93 (d, J ¼ 10.2 Hz, 2H), 1.46 (s, 9H).
Anal. Calcd for C17H20N2O4: C, 64.54; H, 6.37; N, 8.86. Found: C,
64.41; H, 6.25; N, 8.74.
;
1H NMR (BHSC-500,
/ppm ¼ 10.87 (s,1H), 9.86 (s,1H), 7.32 (t, J ¼ 7.6 Hz,1H),
d
atoms connected to the nitrogen atoms in the indole b-carboline
cation radical reflect significant NeH acidity, and these readily
released protons may help to facilitate the scavenging process.
3.6. Conclusion
4.2.3. BocePhe-TrpeOCH3
HOBt (250 mg; 1.75 mmol) and dicyclohexylcarbodiimide
In the present study, we have synthesized a series of new
(400 mg; 1.95 mmol) were added to a solution of 465 mg
b
-carboline-tripeptide conjugates, and examined their anti-
(1.75 mmol) BocePheeOH in THF (15 mL). After stirring for 1 h at
0 ꢃC, 490 mg (1.95 mmol) HCl$TrpeOCH3 was added and the pH
adjusted to 8e9 with NMM. The reaction mixture was stirred
overnight at room temperature. Precipitated dicyclohexylurea was
removed by filtration. The filtrate was evaporated under reduced
pressure, and the residue dissolved in 50 mL of ethyl acetate. The
solution was washed with saturated NaHCO3 (10 mL ꢁ 3), 5% KHSO4
(10 mL ꢁ 3) and saturated sodium chloride, and the organic phase
separated and dried over anhydrous sodium sulfate. Following
filtration and evaporation under reduced pressure, purification by
chromatography provided the final product. Mp: 165e166 ꢃC. ESI-
inflammatory and analgesic activities. In light of potent analgesic
and anti-inflammatory activity, compound 4a was further evalu-
ated in an intestinal ischemia/reperfusion injury animal model and
found to attenuate the structural and functional damage observed
in intestinal I/R. We speculate that these protective effects might
relate to reduced inflammation and an inhibition of lipid
peroxidation.
4. Experimental
4.1. General
MS (m/e) 466 [M þ H]þ; 1H NMR (500 Hz, CDCl3)
/ppm ¼ 10.78 (s,
d
1H), 8.15 (s, 1H), 7.40 (d, J ¼ 7.0 Hz, 1H), 7.35 (d, J ¼ 8.0 Hz, 1H),
7.30e7.23 (m, 5H), 7.20 (t, J ¼ 7.5 Hz, 1H, 1H), 7.09 (t, J ¼ 7.5 Hz, 1H),
6.90 (s, 1H), 6.38 (d, J ¼ 5.5 Hz, 1H), 4.90 (m, 1H), 4.36 (m, 1H), 3.65
(s, 3H), 3.28 (m, 2H), 3.06e3.04 (m, 2H), 1.39 (s, 9H). 13C NMR
All chemicals were purchased from Sigma Aldrich. Unless
otherwise stated, all reactions were run under a nitrogen atmo-
sphere (1 bar). Chromatography was performed on Qingdao silica
gel H (230e400mesh). The purity (>97%) of the intermediates and
the final products was confirmed via 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 recorded on
a Bruker Advance 500 spectrometer. FAB-MS was determined by
VG-ZAB-MS high resolution GC/MS/DS and HP ES-5989x.
(125 Hz, CDCl3)
d
/ppm ¼ 171.75, 170.77, 155.25, 136.59, 136.08,
129.43, 128.62, 127.53, 126.93, 122.26, 119.69, 118.49, 111.30, 109.79,
80.09, 55.68, 52.99, 52.31, 38.36, 28.21, 27.70.
4.2.4. HCl$Phe-TrpeOCH3
BocePhe-TrpeOCH3 (0.2 mmol) in 2 mL of hydrogen chloride in
ethyl acetate (4 mol/L) was stirred for 3 h at room temperature, and
solvent was removed by evaporation. The residue was dissolved in
10 mL of ethyl acetate, and the solution again evaporated to
dryness. The resulting solid was used for the coupling reaction
without further purification. Mp: 176e178 ꢃC. ESI-MS (m/e) 366
4.2. Synthesis of b-carboline-tripeptide conjugates 4aer
4.2.1. 3S-1,2,3,4-Tetrahydro-
b-carboline-3-carboxylic acid (1)
L-Tryptophan (5.0 g; 24.5 mmol) was supplemented with 25 mL
of H2SO4 (1 mol/L), 80 mL of water, and 8 mL of formaldehyde
(36e38%) were added. The reaction mixture was stirred at room
temperature for 2 h and adjusted to pH 6e7 with concentrated
[M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 11.02 (s, 1H), 9.25
d
(d, J ¼ 7.5 Hz, 1H), 8.33 (s, 2H), 7.50 (d, J ¼ 8.0 Hz, 1H), 7.37 (d,
J ¼ 8.0 Hz, 1H), 7.33e7.25 (m, 6H), 7.07 (t, J ¼ 7.5 Hz, 1H), 7.01

2448
W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 2441e2452
(t, J ¼ 7.5 Hz, 1H), 4.59 (m, 1H), 4.13 (m, 1H), 3.25e3.19 (m, 3H), 3.02
127.52, 126.67, 124.16, 121.43, 118.88, 118.41, 111.89, 109.53, 78.53,
60.37, 53.70, 53.57, 52.26, 38.24, 31.00, 28.63, 27.52, 19.55, 18.52.
(dd, J ¼ 7.5 Hz, J ¼ 14.0 Hz, 1H). 13C NMR (125 Hz, DMSO-d6)
d/
ppm ¼ 171.39, 170.41, 155.41, 136.71, 136.28, 129.32, 128.71, 127.02,
122.79, 122.23, 119.65, 118.66, 111.27, 56.29, 52.08, 38.73, 26.30.
4.2.9. BoceAla-Phe-TrpeOCH3 (e)
HCl$Phe-TrpeOCH3 (803 mg; 2.0 mmol) and BoceAlaeOH
(378 mg; 2.0 mmol) were combined to derive the coupled
compound as a colorless solid (967 mg; 90% yield). Mp: 96e98 ꢃC.
4.2.5. BoceLeu-Phe-TrpeOCH3 (a)
HCl$Phe-TrpeOCH3 (603 mg; 1.5 mmol) and BoceLeueOH
(346 mg; 1.5 mmol) were combined to derived the coupled compound
as a colorless solid (521 mg; 60% yield). Mp: 154e156 ꢃC. ESI-MS (m/e)
ESI-MS (m/e) 537 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
d/
ppm ¼ 11.45 (s, 1H), 8.54 (d, J ¼ 7.0 Hz, 1H), 7.91 (d, J ¼ 8.0 Hz, 1H),
7.50 (d, J ¼ 8.0 Hz, 1H), 7.35 (d, J ¼ 8.5 Hz, 1H), 7.26e7.17 (m, 6H),
7.08 (t, J ¼ 7.5 Hz, 1H), 7.00(t, J ¼ 7.5 Hz, 1H), 6.92 (t, J ¼ 6.5 Hz, 1H),
4.60 (m, 1H), 3.57 (s, 3H), 3.46 (m, 1H), 3.18 (dd, J ¼ 6.0 Hz,
J ¼ 14.5 Hz, 1H), 3.09 (dd, J ¼ 7.5 Hz, J ¼ 14.5 Hz, 1H), 2.99 (dd,
J ¼ 4.5 Hz, J ¼ 4.0 Hz, 1H), 2.77 (dd, J ¼ 9.5 Hz, J ¼ 14.0 Hz, 1H), 1.38
579 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.88 (s, 1H), 8.52
d
(d, J ¼ 7.5 Hz,1H), 7.74 (d, J ¼ 8.5 Hz,1H), 7.49(d, J ¼ 8.0 Hz,1H), 7.35 (d,
J ¼ 8.0 Hz, 1H), 7.25e7.18 (m, 5H), 7.16 (s, 1H), 7.08 (t, J ¼ 7.5 Hz, 1H),
6.90 (d, J ¼ 9.0 Hz, 1H), 4.63 (m, 1H), 4.53 (m, 1H), 3.88 (m, 1H), 3.56 (s,
3H), 3.16 (dd, J ¼ 6.5 Hz, J ¼ 14.5 Hz, 1H), 3.09 (dd, J ¼ 7.5 Hz,
J ¼ 14.5 Hz, 1H), 3.00 (J ¼ 4.5 Hz, J ¼ 13.5 Hz, 1H), 2.79 (dd, J ¼ 4.5 Hz,
J ¼ 13.5 Hz, 1H), 1.46 (m, 1H), 1.37 (s, 9H), 1.30 (m, 2H), 0.825 (d,
(s, 9H), 1.25 (d, J ¼ 10.0 Hz, 3H). 13C NMR (125 Hz, DMSO-d6)
d/
ppm ¼ 172.51, 171.52, 169.47, 156.21, 137.99, 136.54, 129.71, 128.46,
127.51, 126.71, 124.23, 121.45, 118.91, 118.42, 111.91, 109.62, 78.54,
53.81, 53.56, 52.29, 43.52, 38.16, 28.65, 27.50, 18.88.
J ¼ 7.0 Hz, 3H), 0.79 (d, J ¼ 7.0 Hz, 3H). 13C NMR (125 Hz, DMSO-d6)
d/
ppm ¼ 172.54, 172.45, 171.52, 155.63, 137.87, 136.55, 129.79, 128.36,
127.53,126.65,124.16, 118.89, 118.41, 111.90, 109.55, 78.57, 53.62, 53.49,
52.26, 41.33, 38.26, 28.63, 27.54, 24.64, 23.34, 21.98.
4.2.10. BocePhe-Phe-TrpeOCH3 (f)
HCl$Phe-TrpeOCH3 (803 mg; 2.0 mmol) and BoceIleeOH
(530 mg; 2.0 mmol) were coupled to derive a yellowsolid (1042 mg;
85% yield). Mp: 90e91 ꢃC. ESI-MS (m/e) 613 [M þ H]þ; 1H NMR
4.2.6. BoceGly-Phe-TrpeOCH3 (b)
HCl$Phe-TrpeOCH3 (804 mg; 2.0 mmol) and BoceGlyeOH
(350 mg; 1.5 mmol) were combined to derive the coupled product
as a colorless solid (635 mg; 81% yield). Mp: 84e86 ꢃC. ESI-MS (m/
(500 Hz, DMSO-d6)
d
/ppm ¼ 10.90 (s, 1H), 8.57 (d, J ¼ 7.5 Hz, 1H),
8.27 (d, J ¼ 8.5 Hz,1H), 7.55 (d, J ¼ 7.5 Hz,1H), 7.35 (d, J ¼ 8.5 Hz,1H),
7.24e7.12 (m,1H), 7.08 (t, J ¼ 7.5 Hz,1H), 7.0 (t, J ¼ 7.5 Hz,1H), 6.87 (d,
J ¼ 8.0 Hz, 1H), 4.65 (m, 1H), 4.55 (m, 1H), 4.12 (m, 1H), 3.58 (s, 3H),
3.18 (dd, J ¼ 6.0 Hz, J ¼ 15.0 Hz, 1H), 3.10 (dd, J ¼ 7.5 Hz, J ¼ 15.0 Hz,
1H), 3.03 (dd, J ¼ 5.0 Hz, J ¼ 14.0 Hz, 1H), 2.82 (dd, J ¼ 9.5 Hz,
J ¼ 14.0 Hz, 2H), 2.62 (dd, J ¼ 10.5 Hz, J ¼ 14.0 Hz,1H),1.30 (s, 9H).13C
e) 523 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.89 (s, 1H),
d
8.54 (d, J ¼ 7.5 Hz, 1H), 7.90 (d, J ¼ 8.5 Hz, 1H), 7.50 (d, J ¼ 8.0 Hz,
1H), 7.36 (d, J ¼ 8.0 Hz, 1H), 7.29e7.20 (m, 5H), 7.19 (s, 1H), 7.10 (t,
J ¼ 7.0 Hz,1H), 7.07 (t, J ¼ 7.0 Hz,1H), 6.92 (t, J ¼ 6.0 Hz,1H), 4.60 (m,
1H), 4.54 (m, 1H), 3.55 (s, 1H), 3.39 (s, 2H), 3.24 (J ¼ 7.5 Hz,
J ¼ 15.0 Hz, 1H), 3.09 (dd, J ¼ 7.5 Hz, J ¼ 15.0 Hz, 1H), 2.95 (dd,
J ¼ 4.5 Hz, J ¼ 11.0 Hz, 1H), 2.68 (dd, J ¼ 7.5 Hz, J ¼ 11.0 Hz, 1H), 1.38
NMR (125 Hz, DMSO-d6)
d
/ppm ¼ 172.47, 171.79, 171.53, 155.51,
138.56, 137.90, 136.54, 129.72, 129.59, 128.46, 128.41, 127.53, 126.74,
126.56, 124.23, 121.44, 118.90, 118.42, 111.90, 109.56, 78.57, 56.24,
53.79, 53.61, 52.29, 38.29, 37.98, 28.56, 27.54.
(s, 9H). 13C NMR (125 Hz, DMSO-d6)
d
/ppm ¼ 172.50, 171.50, 169.47,
156.21, 137.99, 136.55, 129.70, 128.46, 127.52, 126.71, 124.23, 121.45,
118.91,118.42,111.91,109.63, 78.56, 53.82, 53.56, 52.28, 43.55, 38.16,
28.65, 27.51.
4.2.11. BoceIle-Phe-TrpeOCH3 (g)
HCl$Phe-TrpeOCH3 (1003 mg; 2.5 mmol) and BocePheeOH
(577 mg; 2.5 mmol) were combined to derive the end-product as
a colorless solid (1164 mg; 81% yield). Mp: 189e190 ꢃC. ESI-MS (m/e)
4.2.7. BocePro-Phe-TrpeOCH3 (c)
HCl$Phe-TrpeOCH3 (603 mg; 1.5 mmol) and BoceProeOH
(323 mg; 1.5 mmol) were combined to yield coupled product as
a colorless solid (632 mg; 75% yield). Mp: 102e103 ꢃC. ESI-MS (m/e)
576 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.88(s,1H), 8.48
d
(d, J ¼ 7.5 Hz, 1H), 7.89 (d, J ¼ 8.5 Hz, 1H), 7.48 (d, J ¼ 8.0 Hz, 1H), 7.35
(d, J ¼ 8.0 Hz, 1H), 7.24e7.16 (m, 6H), 7.08 (t, J ¼ 7.5 Hz, 1H), 7.00(t,
J ¼ 7.5 Hz,1H), 6.69 (d, J ¼ 9.0 Hz,1H), 4.66 (m,1H), 4.52 (m,1H), 3.76
(m, 1H), 3.54 (s, 3H), 3.15 (dd, J ¼ 6.5 Hz, J ¼ 14.5 Hz, 1H), 3.09 (dd,
J ¼ 7.5 Hz, J ¼ 14.5 Hz, 1H), 2.99 (dd, J ¼ 4.5 Hz, J ¼ 14.0 Hz, 1H), 2.78
(dd, J ¼ 10.0 Hz, 14.0 Hz, 1H), 1.55 (m, 1H), 1.38 (s, 9H), 1.28e1.21 (m,
2H), 0.73 (t, J ¼ 7.5 Hz, 3H), 0.60 (d, J ¼ 7.0 Hz, 3H). 13C NMR (125 Hz,
562 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.91 (s, 1H),
d
8.53 (d, J ¼ 7.0 Hz,1H), 7.82 (d, J ¼ 8.5 Hz,1H), 7.49 (d, J ¼ 8.0 Hz,1H),
7.35 (d, J ¼ 8.0 Hz, 1H), 7.28e7.23 (m, 5H), 7.19 (s, 1H), 7.08 (t,
J ¼ 7.5 Hz,1H), 7.00 (t, J ¼ 7.5 Hz,1H), 4.64 (m,1H), 4.57 (m,1H), 4.00
(m, 1H), 3.56 (s, 3H), 3.41e3.32 (m, 2H), 3.24e3.16 (m, 2H), 3.10 (dd,
J ¼ 8.0 Hz, J ¼ 15.0 Hz, 1H), 3.00 (dd, J ¼ 8.0 Hz, J ¼ 15.0 Hz, 1H), 2.00
(m,1H),1.67e1.60 (m, 3H),1.13 (s, 9H).13CNMR (125 Hz, DMSO-d6)
d/
DMSO-d6)
d
/ppm ¼ 172.42, 171.62, 171.49, 155.66, 138.00, 136.56,
ppm ¼ 172.54, 172.25, 153.80, 138.19, 136.57, 129.63, 128.40, 127.53,
126.67, 124.10, 121.44, 118.88, 118.41, 111.90, 109.62, 78.82, 60.07,
53.94, 53.57, 52.27, 46.92, 38.06, 29.87, 29.47, 28.61, 23.37.
129.69, 128.39, 127.53, 126.65, 124.15, 121.43, 118.41, 111.89, 78.54,
59.46,53.67, 53.59, 52.24,38.24,37.05,28.64, 27.55, 24.69,15.59,11.31.
4.2.12. BoceTrp-Phe-TrpOCH3 (h)
4.2.8. BoceVal-Phe-TrpeOCH3 (d)
HCl$Phe-TrpeOCH3 (804 mg; 2.0 mmol) and BoceTrpeOH
(608 mg; 2.0 mmol) were combined to derive the targeted product
as a colorless solid (723 mg; 78% yield). Mp: 101e104 ꢃC. ESI-MS
HCl$Phe-TrpeOCH3 (803 mg; 2.0 mmol) and BoceValeOH
(434 mg; 2.0 mmol) were combined to derive the end-product as
a colorless solid (813 mg; 72% yield). Mp: 179e180 ꢃC. ESI-MS (m/e)
(m/e) 465 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.90 (s,
d
565 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
d
/ppm ¼ 10.59 (s, 1H),
1H),10.78 (s,1H), 8.57 (d, J ¼ 7.0 Hz,1H), 7.94 (d, J ¼ 8.5 Hz,1H), 7.54
(d, J ¼ 8.0 Hz, 1H), 7.50 (d, J ¼ 8.0 Hz, 1H), 7.35 (d, J ¼ 8.0 Hz, 1H),
7.32 (d, J ¼ 8.0 Hz, 1H), 7.30e7.18 (m, 6H), 7.01e6.96 (m, 5H), 6.79
(d, J ¼ 8.0 Hz, 1H), 4.67 (m, 1H), 4.57 (m, 1H), 4.16 (m, 1H), 3.57 (s,
1H), 3.17 (dd, J ¼ 8.5 Hz, J ¼ 14.5 Hz, 1H), 3.11 (dd, J ¼ 7.5 Hz,
J ¼ 14.5 Hz, 1H), 3.03 (dd, J ¼ 5.0 Hz, J ¼ 14.0 Hz, 1H), 2.93 (dd,
8.51 (d, J ¼ 7.0 Hz, 1H), 7.90 (d, J ¼ 8.5 Hz, 1H), 7.48 (d, J ¼ 8.0 Hz,
1H), 7.35 (d, J ¼ 8.0 Hz,1H), 7.24e7.16 (m, 6H), 7.07 (t, J ¼ 7.5 Hz,1H),
7.00 (t, J ¼ 5.0 Hz, 1H), 6.65 (d, J ¼ 9.0 Hz, 1H), 4.66 (m, 1H), 4.53 (m,
1H), 3.73 (m, 1H), 3.55 (s, 3H), 3.15 (dd, J ¼ 6.0 Hz, J ¼ 14.5 Hz, 1H),
3.09 (dd, J ¼ 7.5 Hz, J ¼ 14.5 Hz, 1H), 2.99 (dd, J ¼ 4.5 Hz, J ¼ 13.5 Hz,
1H), 2.77 (dd, J ¼ 9.5 Hz, J ¼ 13.5 Hz, 1H), 1.80 (m, 1H), 1.38 (s, 9H),
13
C NMR
J ¼ 4.5 Hz, J ¼ 15.0 Hz, 1H), 2.86e2.78 (m, 2H), 1.29 (s, 9H);
(125 Hz, DMSO-d
0.68 (dd, J ¼ 6.5 Hz, J ¼ 17.5 Hz, 6H). 13C NMR (125 Hz, DMSO-d6)
d/
)
d
/ppm ¼ 172.47, 172.05, 171.55, 155.53, 137.92,
6
ppm ¼ 172.44, 171.66, 171.45, 155.74, 138.01, 136.55, 129.69, 128.42,
136.56, 136.50, 129.85, 128.43, 127.85, 127.56, 126.72, 124.21, 123.92,

W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 2441e2452
2449
121.45, 121.26, 118.91, 118.87, 118.62, 118.44, 111.91, 111.71, 110.76,
109.57, 78.59, 55.86, 53.73, 53.66, 52.28, 38.32, 28.56, 28.24, 27.56.
(125 Hz, DMSO-d6)
d
/ppm ¼ 174.46, 172.45, 171.97, 171.46, 155.66,
137.87, 136.54, 129.93, 128.42, 127.53, 126.69, 124.12, 121.44, 118.90,
118.41, 111.89, 109.63, 78.62, 54.53, 53.75, 53.52, 52.27, 38.23, 32.05,
28.64, 28.40, 27.48.
4.2.13. BoceThr-Phe-TrpeOCH3 (i)
HCl$Phe-TrpeOCH3 (804 mg; 2.0 mmol) and BoceThreOH
(438 mg; 2.0 mmol) were combined to derive the end-product as
a colorless solid (637 mg; 75% yield). Mp: 95e97 ꢃC. ESI-MS (m/e)
4.2.17. BoceAsn-Phe-TrpeOCH3 (m)
HCl$Phe-TrpeOCH3 (600 mg; 1.5 mmol) and BoceAsneOH
(350 mg; 1.5 mmol) were combined to derive the end-product as
a yellow solid (773 mg; 89% yield). Mp: 184e185 ꢃC. ESI-MS (m/e) 580
567 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.87 (s, 1H),
d
8.50 (d, J ¼ 7.0 Hz, 1H), 7.91 (d, J ¼ 8.0 Hz, 1H), 7.48 (d, J ¼ 8.0 Hz,
1H), 7.35 (d, J ¼ 8.5 Hz,1H), 7.24e7.16 (m, 6H), 7.08 (t, J ¼ 7.5 Hz,1H),
7.00 (t, 7.5 Hz,1H), 6.38 (d, J ¼ 8.0 Hz,1H), 4.61 (m,1H), 4.51 (m,1H),
3.86e3.79 (m, 2H), 3.55 (s, 3H), 3.16 (dd, J ¼ 6.5 Hz, J ¼ 14.5 Hz, 1H),
3.07 (dd, J ¼ 8.0 Hz, J ¼ 14.5 Hz, 1H), 3.01 (dd, J ¼ 4.5 Hz, J ¼ 14.0 Hz,
1H), 2.79 (dd, J ¼ 9.0 Hz, J ¼ 13.5 Hz, 1H), 1.37 (s, 9H), 0.91 (d,
[M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.88 (s, 1H), 8.52(d,
d
J ¼ 7.0 Hz, 1H), 7.82 (d, J ¼ 8.0 Hz, 1H), 7.49 (d, J ¼ 8.0 Hz, 1H), 7.35 (d,
J ¼ 8.0 Hz,1H), 7.31 (s,1H), 7.31e7.17 (m, 6H), 7.08 (t, J ¼ 7.5 Hz, 1H), 7.00
(t, J ¼ 7.5 Hz, 1H), 6.92 (d, J ¼ 8.0 Hz, 2H), 4.55e4.50 (m, 2H), 4.23 (m,
1H), 3.56 (s, 3H), 3.17 (dd, J ¼ 6.5 Hz, J ¼ 14.5 Hz, 1H), 3.09 (dd,
J ¼ 7.5 Hz, J ¼ 14.5 Hz, 1H), 2.95 (dd, J ¼ 4.5 Hz, J ¼ 14.0 Hz, 1H), 2.80
(dd, J ¼ 8.5 Hz, J ¼ 14.0 Hz, 1H), 2.41 (dd, J ¼ 5.0 Hz, J ¼ 15.0 Hz, 1H),
2.32 (dd, J ¼ 8.0 Hz, J ¼ 15.0 Hz, 1H), 1.37 (s, 9H). 13C NMR (125 Hz,
J ¼ 6.0 Hz, 3H). 13C NMR (125 Hz, DMSO-d6)
/ppm ¼ 172.45, 171.44,
d
170.47, 155.73, 137.87, 136.55, 129.71, 128.45, 126.72, 124.01, 121.49,
118.94, 118.39, 111.93, 109.55, 78.85, 67.22, 60.50, 53.79, 53.68,
52.27, 37.94, 28.57, 27.50, 19.84.
DMSO-d6)
d
/ppm ¼ 172.46,172.14,171.65,171.30,155.52,137.90,136.54,
130.18, 129.74, 128.45, 127.53, 126.68, 124.21, 121.44, 118.90, 118.42,
111.91,109.65, 78.77, 53.95, 53.65, 52.27, 51.77, 37.97, 37.70, 28.63, 27.50.
4.2.14. BoceHis(Boc)-Phe-TrpeOCH3 (j)
HCl$Phe-TrpeOCH3 (804 mg; 2.0 mmol) and BoceHis(Boc)eOH
(710 mg; 2.0 mmol) were combined to derive the end-product as
a colorless solid (996 mg; 71% yield). Mp: 147e148 ꢃC. ESI-MS (m/e)
4.2.18. BoceSer-Phe-TrpeOCH3 (n)
HCl$Phe-TrpeOCH3 (600 mg; 1.5 mmol) and BoceSereOH
(410 mg; 2.0 mmol) were combined to derive the end-product as
a colorless solid (406 mg; 49% yield). Mp: 177e178 ꢃC. ESI-MS (m/e)
703 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.90 (s, 1H),
d
8.58 (d, J ¼ 7.5 Hz, 1H), 8.10 (s, 1H), 7.91 (d, J ¼ 8.0 Hz, 1H), 7.48 (d,
J ¼ 8.0 Hz, 1H), 7.25e7.16 (m, 6H), 7.07 (t, J ¼ 7.5 Hz, 1H), 6.99 (t,
J ¼ 7.5 Hz, 1H), 6.88 (d, J ¼ 8.5 Hz, 1H), 4.61e4.53 (m, 2H), 4.16 (m,
1H), 3.56 (s, 3H), 3.17 (dd, J ¼ 5.5 Hz, J ¼ 14.5 Hz, 1H), 3.08 (dd,
J ¼ 7.5 Hz, J ¼ 14.5 Hz, 1H), 3.00 (dd, J ¼ 4.5 Hz, J ¼ 14.0 Hz, 1H), 2.80
(dd, J ¼ 9.5 Hz, J ¼ 14.0 Hz, 1H), 2.74 (dd, J ¼ 4.0 Hz, J ¼ 15.0 Hz, 1H),
2.63 (dd, J ¼ 9.5 Hz, J ¼ 15.0 Hz, 1H), 1.54 (s, 9H), 1.33 (s, 9H). 13C
553 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.89 (s, 1H),
d
8.51 (d, J ¼ 7.0 Hz, 1H), 7.90 (d, J ¼ 8.5 Hz, 1H), 7.49 (d, J ¼ 8.0 Hz,
1H), 7.35 (d, J ¼ 8.0 Hz, 1H), 7.34e7.17 (m, 6H), 7.10 (t, J ¼ 7.5 Hz, 1H),
7.02 (t, J ¼ 7.5 Hz, 1H), 6.69 (d, J ¼ 8.0 Hz, 1H), 4.93 (t, J ¼ 5.5 Hz, 1H),
4.59 (m, 1H), 4.52 (m, 1H), 3.97 (m, 1H), 3.56 (s, 3H), 3.46 (t,
J ¼ 6.0 Hz, 2H), 3.16 (dd, J ¼ 6.0 Hz, J ¼ 14.5 Hz, 1H), 3.07 (dd,
J ¼ 8.0 Hz, J ¼ 15.0 Hz, 1H), 3.02 (dd, J ¼ 9.5 Hz, J ¼ 14.0 Hz, 1H), 2.79
(dd, J ¼ 9.0 Hz, J ¼ 13.5 Hz, 1H), 1.38 (s, 9H). 13C NMR (125 Hz,
NMR (125 Hz, DMSO-d6)
d
/ppm ¼ 172.48, 171.45, 155.54, 147.15,
137.94, 137.07, 136.54, 129.77, 128.41, 127.54, 126.68, 124.16, 121.43,
118.89, 118.42, 114.61, 111.88, 109.56, 85.53, 78.71, 54.26, 53.85,
53.57, 52.28, 38.01, 31.10, 28.54, 27.82, 27.51.
DMSO-d6)
d
/ppm ¼ 172.43, 171.37, 170.52, 155.60, 137.92, 136.56,
129.76, 128.41, 127.51, 126.68, 124.19, 121.45, 118.91, 118.41, 111.20,
109.59, 78.72, 62.37, 57.30, 53.87, 53.68, 52.26, 37.97, 28.63, 27.54.
4.2.15. BoceLys(Boc)-Phe-TrpeOCH3 (k)
HCl$Phe-TrpeOCH3 (1100 mg; 2.5 mmol) and BoceLys(Boc)eOH
(865 mg; 2.5 mmol) were combined to derive the end-product as
a yellow solid (1559 mg; 90% yield). Mp: 132e134 ꢃC. ESI-MS (m/e)
4.2.19. BoceTyr-Phe-TrpeOCH3 (o)
HCl$Phe-TrpeOCH3 (804 mg; 2.0 mmol) and BoceTyreOH
(560 mg; 2.0 mmol) were combined to derive the end-product as
a colorless solid (1004 mg; 80% yield). Mp: 105e107 ꢃC. ESI-MS (m/
694 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.88 (s, 1H),
d
8.50 (d, J ¼ 7.5 Hz,1H), 7.77 (d, J ¼ 8.0 Hz,1H), 7.48 (d, J ¼ 8.0 Hz,1H),
7.34 (d, J ¼ 8.0 Hz,1H), 7.25e7.16 (m, 6H), 7.08 (t, J ¼ 7.5 Hz,1H), 7.00
(t, J ¼ 7.5 Hz, 1H), 6.83 (d, J ¼ 8.0 Hz, 1H), 6.75 (t, J ¼ 5.0 Hz, 1H), 4.62
(m, 1H), 4.54 (m, 1H), 3.80 (m, 1H), 3.56 (s, 3H), 3.16 (dd, J ¼ 6.5 Hz,
J ¼ 15.0 Hz, 1H), 3.09 (dd, J ¼ 7.5 Hz, J ¼ 14.5 Hz, 1H), 2.99
(dd, J ¼ 4.5 Hz, J ¼ 13.5 Hz, 1H), 2.89e2.82 (m, 2H), 2.78 (dd,
J ¼ 4.0 Hz, J ¼ 13.5 Hz,1H),1.38 (s, 9H),1.37 (s, 9H),1.31e1.25 (m, 2H),
e) 629 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.89 (s, 1H),
d
9.15 (s, 1H), 8.55 (d, J ¼ 7.5 Hz, 1H), 7.91 (d, J ¼ 8.4 Hz, 1H), 7.49 (d,
J ¼ 7.8 Hz, 1H), 7.34 (d, J ¼ 8.1 Hz, 1H), 7.26e7.16 (m, 6H), 7.07
(t, J ¼ 7.5 Hz, 1H), 7.05e6.93 (m, 3H), 6.80 (d, J ¼ 8.7 Hz, 1H), 6.61 (d,
J ¼ 8.4 Hz, 2H), 4.63 (m, 1H), 4.54 (m, 1H), 4.02 (m, 1H), 3.55 (s, 3H),
3.21e2.98 (m, 4H), 2.85e2.68 (m, 2H), 1.29 (s, 9H). 13C NMR
(125 Hz, DMSO-d6)
d
/ppm ¼ 172.44, 171.96, 171.52, 156.13, 155.52,
1.21e1.18 (m, 4H). 13C NMR (125 Hz, DMSO-d6)
172.27, 171.53, 156.03, 155.68, 137.89, 136.54, 129.77, 128.38, 127.54,
126.67, 124.16, 121.43, 118.89, 118.41, 111.89, 109.55, 78.58, 77.79,
55.06, 53.58, 52.26, 38.22, 32.16, 29.65, 28.75, 27.53, 23.22.
d
/ppm ¼ 172.45,
137.90, 136.53, 130.47, 129.81, 128.58, 128.43, 127.53, 126.71, 124.21,
121.43, 118.90, 118.42, 115.22, 111.89, 109.54, 103.51, 78.54, 56.63,
53.77, 53.60, 52.27, 38.27, 37.17, 28.58, 27.53.
4.2.20. BoceArg(NO2)-Phe-TrpeOCH3 (p)
4.2.16. BoceGln-Phe-TrpeOCH3 (l)
HCl$Phe-TrpeOCH3 (600 mg; 1.5 mmol) and BoceArg(NO2)e
OH (480 mg; 1.5 mmol) were combined to derive the end-product
as a colorless solid (779 mg; 78% yield). Mp: 150e151 ꢃC. ESI-MS
HCl$Phe-TrpeOCH3 (804 mg; 2.0 mmol) and BoceThreOH
(438 mg; 2.0 mmol) were combined to derive the end-product as
a colorless solid (771 mg; 65% yield). Mp: 193e194 ꢃC. ESI-MS (m/e)
(m/e) 667 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.88 (s,
d
594 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
d
/ppm ¼ 10.89 (s, 1H),
1H), 8.54 (d, J ¼ 5.0 Hz, 1H), 8.47 (s, 1H), 7.80(d, J ¼ 10.0 Hz, 1H), 7.49
(d, J ¼ 5.0 Hz, 1H), 7.35 (d, J ¼ 5.0 Hz, 1H), 7.25e7.15 (m, 6H), 7.08 (t,
J ¼ 7.5 Hz, 1H), 7.00 (t, J ¼ 7.5 Hz, 1H), 7.00 (t, J ¼ 7.5 Hz, 1H), 6.91(d,
J ¼ 10.0 Hz,1H), 4.63 (m,1H), 4.54 (m, 1H), 3.86 (m,1H), 3.56 (s, 3H),
3.17 (dd, J ¼ 10.0 Hz, J ¼ 15.0 Hz, 1H), 3.11e3.07 (m, 4H), 3.00 (dd,
J ¼ 5.0 Hz, J ¼ 15.0 Hz, 1H), 2.80 (dd, J ¼ 10.0 Hz, J ¼ 15.0 Hz, 1H),
2.09 (s, 2H), 1.49e1.47 (m, 2H), 1.37 (s, 9H), 1.19 (m, 2H). 13C NMR
8.54 (d, J ¼ 7.0 Hz, 1H), 7.87 (d, J ¼ 8.0 Hz, 1H), 7.48 (d, J ¼ 8.0 Hz,
1H), 7.45 (d, J ¼ 8.0 Hz,1H), 7.34e7.16 (m, 6H), 7.08 (t, J ¼ 7.5 Hz,1H),
7.00 (t, J ¼ 7.5 Hz, 1H), 6.89 (d, J ¼ 8.0 Hz, 1H), 6.81 (s, 2H), 4.60 (m,
1H), 4.54 (m, 1H), 3.87 (m, 1H), 3.56 (s, 3H), 3.17 (dd, J ¼ 6.5 Hz,
J ¼ 15.0 Hz, 1H), 3.08 (dd, J ¼ 7.5 Hz, J ¼ 14.5 Hz, 1H), 2.99 (dd,
J ¼ 5.0 Hz, J ¼ 14.0 Hz, 1H), 2.79 (dd, J ¼ 9.0 Hz, J ¼ 13.5 Hz, 1H),
2.11e2.00 (m, 2H), 1.74 (m, 1H), 1.62 (m, 1H), 1.37 (s, 9H). 13C NMR
(125 Hz, DMSO-d6)
d/ppm ¼ 172.47, 171.98, 171.47, 159.79, 155.69,

2450
W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 2441e2452
137.83, 136.55, 129.78, 128.40, 127.53, 126.69, 124.18, 121.45, 118.91,
118.42, 111.91, 105.57, 78.72, 54.64, 53.62, 52.27, 40.77, 38.26, 31.16,
29.68, 28.63, 27.52.
of phenyl methyl ether, 1 mL of dimethyl sulfide and 8 mL of HF and
stirred at 0 ꢃC for 2 h. The reaction mixture was evaporated under
vacuum and the residue triturated with ether. The residue was then
directly purified on Sephadex G-10 and HPLC to provide the desired
product 4aer.
4.2.21. BoceAsp(OCH3)-Phe-TrpeOCH3 (q)
HCl$Phe-TrpeOCH3 (804 mg; 2.0 mmol) and BoceAsp(OCH3)e
OH (480 mg; 2.0 mmol) were combined to derive the end-product
as a colorless solid (962 mg; 81% yield). Mp: 161e162 ꢃC. ESI-MS
4.2.26. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eLeu-
Phe-Trp (4a)
Following deprotection, 0.2 mmol of 3a yielded 107.2 mg of
compound 4a (81% yield). FAB-MS (m/e): 663 [M þ H].
(m/e) 595 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
d/ppm ¼ 10.89 (s,
1H), 8.51 (d, J ¼ 7.5 Hz, 1H), 7.79 (d, J ¼ 8.5 Hz, 1H), 7.49 (d,
J ¼ 8.0 Hz, 1H), 7.35 (d, J ¼ 8.0 Hz, 1H), 7.27e7.17 (m, 6H), 7.11 (d,
J ¼ 8.0 Hz, 1H), 7.08 (t, J ¼ 7.5 Hz, 1H), 7.00 (t, J ¼ 7.5 Hz, 1H),
4.58e4.52 (m, 2H), 4.29 (m, 1H), 3.57 (s, 3H), 3.56 (s, 3H), 3.17 (dd,
J ¼ 6.5 Hz, J ¼ 14.5 Hz, 1H), 3.09 (dd, J ¼ 7.5 Hz, J ¼ 14.5 Hz, 1H), 2.99
(dd, J ¼ 4.5 Hz, J ¼ 14.0 Hz, 1H), 2.82 (dd, J ¼ 8.5 Hz, J ¼ 13.5 Hz, 1H),
2.61 (dd, J ¼ 5.0 Hz, J ¼ 16.0 Hz, 1H), 2.45 (dd, J ¼ 9.0 Hz, J ¼ 16.0 Hz,
4.2.27. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eGly-
Phe-Trp (4b)
Following deprotection, 0.2 mmol of 3b yielded 94.5 mg of
compound 4b (78% yield). FAB-MS (m/e): 607 [M þ H].
1H), 1.37 (s, 9H). 13C NMR (125 Hz, DMSO-d6)
171.21, 170.86, 155.59, 137.77, 136.55, 129.76, 128.62, 128.43, 127.53,
126.72, 124.17, 121.44, 118.90, 118.42, 111.90, 109.58, 78.93, 53.92,
53.60, 52.27, 51.95, 51.49, 38.09, 36.52, 28.59, 27.55.
d
/ppm ¼ 172.44,
4.2.28. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)ePro-
Phe-Trp (4c)
Following deprotection, 0.2 mmol of 3c yielded 95.6 mg of
compound 4c (74% yield). FAB-MS (m/e): 647 [M þ H].
4.2.22. BoceGlu(OCH3)-Phe-TrpeOCH3 (r)
4.2.29. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eVal-Phe-
HCl$Phe-TrpeOCH3 (804 mg; 2.0 mmol) and BoceGlu(OCH3)e
OH (600 mg; 2.2 mmol) were combined to derive the end-product
as a colorless solid (608 mg; 50% yield). Mp: 156e157 ꢃC. ESI-MS (m/
Trp (4d)
Following deprotection, 0.2 mmol of 3d yielded 90.7 mg of
compound 4d (70% yield). FAB-MS (m/e): 649 [M þ H].
e) 609 [M þ H]þ; 1H NMR (500 Hz, DMSO-d6)
/ppm ¼ 10.88 (s, 1H),
d
8.55 (d, J ¼ 5.0 Hz, 1H), 7.85 (d, J ¼ 5.0 Hz, 1H), 7.49 (d, J ¼ 10.0 Hz,
1H), 7.35 (d, J ¼ 10.0 Hz,1H), 7.23e7.17 (m, 6H), 7.08 (t, J ¼ 7.5 Hz,1H),
7.00 (t, J ¼ 7.5 Hz,1H), 6.92 (d, J ¼ 10.0 Hz,1H), 4.62 (m,1H), 4.54 (m,
1H), 3.89 (m, 1H), 3.57 (s, 3H), 3.56 (s, 3H), 3.17 (dd, J ¼ 5.0 Hz,
J ¼ 15.0 Hz, 1H), 3.10 (dd, J ¼ 10.0 Hz, J ¼ 15.0 Hz, 1H), 3.00 (dd,
J ¼ 5.0 Hz, J ¼ 15.0 Hz,1H), 2.79 (dd, J ¼ 10.0 Hz, J ¼ 15.0 Hz,1H), 2.19
(t, J ¼ 5.0 Hz, 2H), 1.78e1.64 (m, 2H), 1.37 (s, 9H). 13C NMR (125 Hz,
4.2.30. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eAla-
Phe-Trp (4e)
Following deprotection, 0.3 mmol of 3e yielded 163.7 mg of
compound 4e (88% yield). FAB-MS (m/e): 621 [M þ H].
4.2.31. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)ePhe-
Phe-Trp (4f)
Following deprotection, 0.3 mmol of 3f yielded 171.2 mg of
compound 4f (82% yield). FAB-MS (m/e): 697 [M þ H].
DMSO-d6)
d
/ppm ¼ 173.26, 172.45, 171.52, 155.60, 137.88, 136.55,
129.75, 128.39, 127.54, 126.68, 124.16, 121.43, 118.89, 109.57, 78.74,
54.12, 53.64, 52.25, 51.74, 38.20, 30.26, 28.61, 27.78, 27.52.
4.2.32. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eIle-Phe-
4.2.23. Preparation of HCl$
L
-AA-Phe-TrpeOCH3
Trp (4g)
Solutions of aer (2.0 mmol) in10 mL ofhydrogenchloride/acetate
(4 mol/L) were stirred at 0 ꢃC until TLC analysis indicated complete
disappearance of the starting species (aer). Following evaporation,
the residue was dissolved in ethyl acetate (3 ꢁ 10 mL) and evaporated
Following deprotection, 0.2 mmol of 3g yielded 105.9 mg of
compound 4g (80% yield). FAB-MS (m/e): 663 [M þ H].
4.2.33. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eTrp-
Phe-Trp (4h)
under reduced pressure to yield crude HCl$
L-AA-Phe-TrpeOCH3.
Following deprotection, 0.2 mmol of 3h yielded 113.2 mg of
compound 4h (77% yield). FAB-MS (m/e): 736 [M þ H].
4.2.24. Preparation of 3aer
HOBt (6.0 mmol) was added to 4.6 mmol of 2 in 30 mL of
anhydrous THF at 0 ꢃC. After stirring for 10 min, 6.0 mmol of DCC
4.2.34. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eThr-
was added. A suspension of 5.0 mmol HCl$L-AA-Phe-TrpeOCH3 in
Phe-Trp (4i)
3 mL anhydrous THF was adjusted with N-methyl morpholine to
pH 8e9 and then stirred at room temperature for another 20 min.
This mixture was then added to solution of 2, and the reaction
mixture stirred at 0 ꢃC for 2 h and then continued stirring at room
temperature for 16 h. Following evaporation, the residue was dis-
solved in 30 mL of ethyl acetate. The solution was washed exten-
sively with 5% sodium bicarbonate, 5% citric acid, and saturated
sodium chloride, and the organic phase separated and dried over
anhydrous sodium sulfate. Following filtration and evaporation
Following deprotection, 0.2 mmol of 3i yielded 94.9 mg of
compound 4i (73% yield). FAB-MS (m/e): 651 [M þ H].
4.2.35. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eHis-
Phe-Trp (4j)
Following deprotection, 0.2 mmol of 3j yielded 96.0 mg of
compound 4j (70% yield). FAB-MS (m/e): 687 [M þ H].
4.2.36. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eLys-Phe-
under reduced pressure, N-(N-Boc-3S-1,2,3,4-tetrahydro-
b
-carbo-
Trp (4k)
line-3-carboxyl)- -AA-Phe-Trp methyl esters (3aer) were obtained,
which were deprotected without further purification.
L
Following deprotection, 0.3 mmol of 3k yielded 180.8 mg of
compound 4k (89% yield). FAB-MS (m/e): 678 [M þ H].
4.2.25. Preparation of 4aer
4.2.37. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eGln-
A mixture of 0.2 mmol of 3aer, 1 mL of phenyl methyl ether,
1 mL of dimethyl sulfide, and 4 mL of HF was stirred at 0 ꢃC for 2 h,
followed by evaporation. The residue was supplemented with 1 mL
Phe-Trp (4l)
Following deprotection, 0.2 mmol of 3l yielded 86.6 mg of
compound 4l (64% yield). FAB-MS (m/e): 678 [M þ H].

W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 2441e2452
2451
4.2.38. N-(3S-1,2,3,4-Tetrahydro-
Phe-Trp (4m)
Following deprotection, 0.3 mmol of 3m yielded 165.1 mg of
compound 4m (83% yield). FAB-MS (m/e): 664 [M þ H].
b
-carboline-3-carboxyl)eAsn-
were evaluated by the tail flick test. Subjects received a single
gavage of each synthetic compound (0.15 mmol/kg in 0.2 mL of
saline mixed with 0.3% CMC-Na). The control cohort received
vehicle only. Pain thresholds were measured 30 min after oral
administration at 30 min intervals for a total of 3 h. Evaluations
were terminated if the animal did not respond within 10 s in order
to avoid tissue damage. The potency of analgesia was estimated by
the pain threshold variation (PTV). The values were calculated as
PTV ¼ AAPT O BPT where BPT ¼ basic pain threshold and
AAPT ¼ pain threshold after administration ꢂ basic pain threshold.
All values over 3 h were averaged and constituted one determina-
tion (repeated in triplicate). Statistical analysis employed one-way
ANOVA with P < 0.05 as the measure of significance.
4.2.39. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eSer-Phe-
Trp (4n)
Following deprotection, 0.3 mmol of 3n yielded 110.7 mg of
compound 4n (58% yield). FAB-MS (m/e): 637 [M þ H].
4.2.40. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eTyr-
Phe-Trp (4o)
Following deprotection, 0.3 mmol of 3o yielded 164.5 mg of
compound 4o (77% yield). FAB-MS (m/e): 713 [M þ H].
4.3.2. Evaluation of in vivo anti-inflammatory activity
4.2.41. N-(3S-1,2,3,4-Tetrahydro-
Phe-Trp (4p)
Following deprotection, 0.3 mmol of 3p yielded 158.6 mg of
compound 4p (75% yield). FAB-MS (m/e): 706 [M þ H].
b
-carboline-3-carboxyl)eArg-
Anti-inflammatory activities of new compounds were evaluated
in a xylene-induced ear edema assay. Briefly, male ICR mice
(20 ꢀ 2 g) were randomly divided into three groups, including the
test group, vehicle control (1% Carboxymethyl cellulose solution,
CMC) and positive control group (aspirin). The positive control
cohort received an oral suspension of aspirin in CMC (10 mg/kg).
The test group received oral suspensions of test species in CMC
(0.15 mmol/kg) initially. Thirty minutes later, 0.03 mL of xylene was
applied to the anterior and posterior surfaces of the right ear, the
left ear serving as control. Two hours following xylene application,
the mice were sacrificed and both ears were removed. Multiple
circular sections were obtained using a cork borer (diameter 7 mm)
and weighed. The increase in weight reflected by xylene irritation
was determined by subtracting the weight of untreated tissue
sections from treated sections.
4.2.42. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eAsp-
Phe-Trp (4q)
Following deprotection, 0.3 mmol of 3q yielded 157.4 mg of
compound 4q (79% yield). FAB-MS (m/e): 665 [M þ H].
4.2.43. N-(3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxyl)eGlu-
Phe-Trp (4r)
Following deprotection, 0.3 mmol of 3r yielded 111.8 mg of
compound 4r (55% yield). FAB-MS (m/e): 679 [M þ H].
4.2.44. HeLeu-Phe-TrpeOH
Mp: 189e192 ꢃC. ESI-MS (m/e) 465 [M þ H]þ; 1H NMR (500 Hz,
4.3.3. Intestinal ischemia/reperfusion model
DMSO-d6)
d/ppm ¼ 12.36 (s,1H),10.98 (s,1H), 8.82 (d, J ¼ 8.0 Hz,1H),
Male Wistar rats (250e300 g) were randomly allocated into 3
groups: (1) sham group: rats subjected to the procedures described
below, with the exception of ischemia/reperfusion (I/R) (n ¼ 6); (2) I/
R group: rats subjected to the procedures described below under-
going intestinal ischemia for 45 min followed by reperfusion for 3 h
(n ¼ 8); (3) I/R þ drug treatment group: rats received melatonin or
4a (20 mg/kg) 15 min prior to reperfusion and again 1 h following
initiation of reperfusion (n ¼ 8). Animals in groups (1) and (2) were
administrated saline vehicle in lieu of drug. Male Wistar rats were
anesthetized with an intraperitoneal injection of sodium pento-
barbital (80 mg/kg). Throughout the experiment body temperature
was maintained at 37 ꢃC with the aid of a heating pad. Laparotomy
was performed through a midline incision into the peritoneal cavity.
The small bowel was gently exposed on moist gauze, followed by
occlusion of the superior mesenteric artery for 45 min using
a microvascular clamp, with 3 h reperfusion afterward. Surgical
incisions were closed during the reperfusion period. Sham-operated
rats underwent an identical surgical protocol but did not undergo
superior mesenteric artery clamping and were anesthetized for the
duration of the experiment. Fifteen minutes prior to reperfusion,
and 1 h after initiation of reperfusion, melatonin or 4a (20 mg/kg)
was administered intraperitoneally to rats assigned to I/R þ drug
treatment group, while sham subjects received only saline. Subjects
were sacrificed by sodium pentobarbital overdose at the conclusion
ofall studiesand small intestine tissueswereimmediatelyharvested
in addition to blood sampling from the ascending aorta. Tissue
samples were divided into two parts, One for determination of lipid
peroxidation, and other for microtome sectioning (Leica CM1850 UV
clinical cryostat) at ꢂ30 ꢃC.
8.69 (d, J ¼ 7.5 Hz, 1H), 8.22 (s, 2H), 7.50 (d, J ¼ 7.5 Hz, 1H), 7.35 (d,
J ¼ 8.0 Hz,1H), 7.29e7.23 (m, 5H), 7.21 (s, 1H), 7.08 (t, J ¼ 7.5 Hz,1H),
7.03 (t, J ¼ 7.5 Hz, 1H), 4.64 (m, 1H), 4.49 (m, 1H), 3.73 (m, 1H), 3.18
(dd, J ¼ 8.5 Hz, J ¼ 15.0 Hz, 1H), 3.09 (dd, J ¼ 7.5 Hz, 1H), 3.04 (dd,
J ¼ 4.5 Hz, J ¼ 14.0 Hz, 1H), 2.86 (dd, J ¼ 9.0 Hz, J ¼ 14.0 Hz, 1H), 1.64
(m, 1H), 1.55e1.52 (m, 2H), 0.86 (t, J ¼ 5.0 Hz, 6H). 13C NMR (125 Hz,
DMSO-d6)
d
/ppm ¼ 171.98, 171.21, 169.40, 137.98, 136.56, 129.68,
128.56, 127.54, 126.8, 124.31, 121.39, 118.83, 118.41, 111.89, 109.67.
4.2.45. HeLys-Phe-TrpeOH
Mp: 268e269 ꢃC. ESI-MS (m/e) 480 [M þ H]þ; 1H NMR (500 Hz,
DMSO-d6)
d
/ppm ¼ 12.63 (s,1H),11.03 (s,1H), 8.90 (d, J ¼ 8.1 Hz,1H),
8.55(d, J ¼ 7.5 Hz,1H), 8.30(s, 2H), 8.13 (s, 2H), 7.55 (d, J ¼ 7.5 Hz,1H),
7.36 (d, J ¼ 8.1 Hz,1H), 7.29e7.15 (m, 6H), 7.07 (t, J ¼ 7.5 Hz,1H), 6.99
(t, J ¼ 7.5 Hz, 1H), 4.58 (m, 1H), 4.48 (m, 1H), 3.73 (m, 1H), 3.20 (dd,
J ¼ 5.1 Hz, J ¼ 15.0 Hz, 1H), 3.09 (dd, J ¼ 8.4 Hz, J ¼ 15.0 Hz, 1H), 3.01
(m, 1H), 2.85 (dd, J ¼ 9.9 Hz, J ¼ 13.5 Hz, 1H), 2.72 (m, 2H), 1.76 (m,
2H), 1.55 (m, 2H), 1.36 (m, 2H). 13C NMR (125 Hz, DMSO-d6)
d/
ppm ¼ 173.59, 171.31, 168.97, 138.06, 136.53, 129.73, 128.54, 127.63,
126.79, 124.36, 121.32, 118.93, 118.57, 111.88, 110.03, 54.99, 53.53,
52.25, 38.58, 37.75, 30.67, 27.48, 26.56, 21.30.
4.3. Biological assays
All animal experiments were performed in compliance with the
“Guide for the Care and Use of Laboratory Animals” published by
the US National Institutes of Health.
4.3.1. Analysis of in vivo pain threshold
Male ICR mice (20 ꢀ 2 g) were housed in a 12/12 light/dark cycle
at 21 ꢀ2 ꢃC for 24 h before use. Pain threshold for each mouse was
measured in triplicate. Analgesic effects of synthetic compounds
4.3.4. Malondialdehyde (MDA) and glutathione (GSH) assays
MDA represents a stable product of oxidative degradation of
polyunsaturated fatty acids, and indicates the level of cellular

2452
W. Bi et al. / European Journal of Medicinal Chemistry 46 (2011) 2441e2452
damage secondary to lipid peroxidation. Accordingly, MDA levels
are routinely employed as a measure of free radical formation
under oxidative stress conditions. MDA levels were determined
following the method of Beuge and Aust [17], and expressed as
nmol/g tissue. GSH was determined using Beutler’s method and
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.
expressed as mmol/g tissue [18].
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differences between groups. If differences were observed, the
values were compared using Student’s t-test for paired data. The
values were expressed as means ꢀ SD, and considered significant
if P < 0.05.
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Acknowledgments
The authors are grateful to NASA (MSGC R85197), Research
Excellence Fund (R01121), Natural Science Foundation of HeBei,