A class of Trp-Trp-AA-OBzl: Synthesis, in vitro anti-proliferation/in vivo anti-tumor evaluation, intercalation-mechanism investigation and 3D QSAR analysis

Article abstract of DOI:10.1016/j.ejmech.2011.05.004

From the anti-tumor active N-tryptophanyl-β-carboline-3-carboxylic acid benzyl ester and β-carboline-3-carbonyltryptophan benzyl ester, a pharmacophore, Trp-Trp-OBzl, was drawn. Based on the DOCK scores amino acid residue was inserted into the C-terminus

Full text of DOI:10.1016/j.ejmech.2011.05.004

European Journal of Medicinal Chemistry 46 (2011) 3410e3419
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A class of Trp-Trp-AA-OBzl: Synthesis, in vitro anti-proliferation/in vivo anti-tumor
evaluation, intercalation-mechanism investigation and 3D QSAR analysis
**
Xiaoyi Zhang, Yifan Yang, Ming Zhao , Liu Liu, Meiqing Zheng, Yuji Wang, Jianhui Wu, Shiqi Peng
*
College of Pharmaceutical Sciences, Capital Medical University, No. 10, Beijing 100069, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
From the anti-tumor active N-tryptophanyl-
b
-carboline-3-carboxylic acid benzyl ester and
b
-carboline-
Received 26 November 2010
Received in revised form
20 April 2011
Accepted 2 May 2011
Available online 12 May 2011
3-carbonyltryptophan benzyl ester, a pharmacophore, Trp-Trp-OBzl, was drawn. Based on the DOCK
scores amino acid residue was inserted into the C-terminus of Trp-Trp-OBzl and twenty Trp-Trp-AA-
OBzls (AA ¼ amino acid residues) were provided as DNA intercalators. On the in vitro and in vivo
models seventeen Trp-Trp-AA-OBzls were anti-tumor active, and twelve Trp-Trp-AA-OBzls were more
active than cytarabine. In acute toxicity assay Trp-Trp-AA-OBzls did not damage the immunologic
function and had an LD50 of more than 500 mg/kg. The relationships of structure and activity were
analyzed with 3D QSAR. The action mechanism studies revealed that the in vivo anti-tumor action of Trp-
Trp-AA-OBzls was the result of DNA intercalation.
Keywords:
Trp-Trp-AA-OBzl
Anti-tumor
Intercalation
Docking
Ó 2011 Elsevier Masson SAS. All rights reserved.
3D QSAR
1. Introduction
It is well documented that Trp-Trp is biologically important
either as a dipeptide or as a fragment of some peptides [8,10]. For
For some anti-tumor agents such as groove binders, alkylating
compounds and intercalating agents, the DNA recognition is a crit-
ical step in their anti-tumor action. The intercalation is not only one
kind of the interactions in DNA recognition but also a pivotal step of
several clinically used anti-tumor drugs such as anthracyclines,
acridines and anthraquinones [1]. To push clinical cancer therapy,
the discovery of new DNA intercalators has been considered
a practical approach. A number of intercalators such as harmines
instance, via a similar ring-opening modification both the anti-
tumor active -carboline-3-carboxyltryptophan benzyl ester and
N-trytophanyl- -carboline-3-carboxylic acid benzyl ester forms
Trp-Trp-OBzl (Fig. 1). This suggests that Trp-Trp-OBzl is their
pharmacophore. In this context, Trp-Trp-OBzl was used as a lead,
and twenty novel tripeptide benzyl esters, Trp-Trp-AA-OBzl, were
provided as the DNA intercalators.
b
b
have been reported [2e4]. Harmine and its derivatives belong to b-
carboline intercalators and were characterized by their cytotoxicity
[5,6]. Via intercalation they are able to inhibit topoisomerases I/II
2. Results and discussion
2.1. Docking of Trp-Trp-AA-OBzl toward d(CGATCG)2
and finally damage DNA. In our ongoing efforts a series of b-car-
boline-3-carbonylamino acid benzyl esters were presented by
mimicking the natural products with potent anti-tumor activity. It
was demonstrated that their in vitro cytotoxicity depended on the
DNA intercalators have been widely noticed [9e11], and the
LigandFit/LigandScore in DS Modeling2.1 and d(CGATCG)2 (from
Protein Data Bank, 1D12) have been widely used to carry out the
automated docking studies of the intercalators [9e16]. To evaluate
the possibility of Trp-Trp-AA-OBzl (4aet) as DNA intercalators an
automated docking of 4aet toward the cavity of d(CGATCG)2 was
performed by following a general procedure, the DOCK scores were
calculated and are listed in Table 1. It is shown that the DOCK scores
of Trp-Trp-AA-OBzls are 31e62, and ten of them are 51e62. While
our previous work indicated that the DOCK scores of the inter-
calators with high in vivo anti-tumor activity were 50e60 [8,10].
This means that at least ten Trp-Trp-AA-OBzls possibly have high
in vivo anti-tumor activity.
building blocks, i.e. b-carboline-3-carboxylic acid, amino acid and
benzyl moieties [7]. These requirements were generally met in the
design of N-(3-carboxyl-9-benzylcarboline-1-yl) ethylamino acids.
The docking studies demonstrated that their in vitro anti-
proliferation and in vivo anti-tumor activities could be predicted
with the DOCK scores [8].
* Corresponding author. Tel./fax: þ86 10 8391 1528.
** Corresponding author. Tel./fax: þ86 10 8280 2482.
E-mail address: sqpeng@mail.bjmu.edu.cn (S. Peng).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.004

X. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3410e3419
3411
O
H
O
O
N
O
O
H
N
N
H
O
N
N
H
NH
N
H N
2
H
H
O
O
N
O
N
H
NH
2
N
H
Fig. 1. Structural correlation of the derivatives of 2-Trp and 3-Trp modified b-carboline-3-carboxylic acid benzyl ester with Trp-Trp-OBzl.
2.2. Synthesis of Trp-Trp-AA-OBzl
2.3. Anti-proliferation activities of 4aet
Trp-Trp-AA-OBzls (4aet, AA ¼
L
-amino acid residues) were
To evaluate the in vitro anti-proliferation activities of 4aet the
carcinoma cell lines, HepG2 (human hepatocellular liver carcinoma
cell line), S180 (mouse sarcoma cell line), H22 (mouse hepatocel-
lular carcinoma cell line), K562 (human immortalized myelogenous
leukemia line) and B16 (mouse melanoma cell line), were used. In
the assays the cells were exposed to 4aet, cytarabine and doxo-
rubicin (positive control), underwent the standard MTT tests and
gave their proliferation inhibitions for calculate IC50 velues
(Table 2). It can be seen that 4aet possess distinct in vitro anti-
prepared according to the route depicted in Scheme 1. When Boc-
Trp was coupled with Trp-OBzl the protective dipeptide Boc-Trp-
Trp-OBzl (1) was obtained (96% yield). In aqueous NaOH (2 M)
the benzyl group of 1 was removed and Boc-Trp-Trp (2) was
provided in 90% yield. The coupling reaction of Boc-Trp-Trp with
AA-OBzl provided Boc-Trp-Trp-AA-OBzls (3aet, 41%e98% yields).
In the solution of hydrogen chloride in ethyl acetate the Boc groups
of 3aet were removed, Trp-Trp-AA-OBzls (4aet) were obtained
and the yields were 70%e99%. To ensure the purity, 3aet were
purified on a chromatographic column, while 4aet were purified
by repeatedly using ether-promoted solidification and the HPLC
purities were more than 97%. These data demonstrate that the used
procedures and conditions are suitable for preparing of Trp-Trp-
AA-OBzls in high quality and acceptable yield.
proliferation activities, the IC50 values are 8.1 mMe154.0 mM, most
of them are able to inhibit the proliferation of the carcinoma cells,
the IC50 values of 4aec,e,f,h,j,l,n,o,q,r are significantly lower than
that of cytarabine, and the IC50 value of 4s is equal to that of
cytarabine. Beside, the IC50 values of 4aef,h,j,l,n,o,qes against S180
are significantly lower than that of doxorubicin, the IC50 values of
4b,c,e,f,h,j,l,n,q,r against K562 are significantly lower than that of
doxorubicin, the IC50 values of 4c,f,h,j,n,q against B16 are signifi-
cantly lower than that of doxorubicin, the IC50 values of
4aec,e,f,h,j,l,n,o,qes against H22 are significantly lower than that
of doxorubicin, and the IC50 values of 4f,h,j,n,q against HepG2 are
significantly lower than that of doxorubicin. These suggest that the
lead, Trp-Trp-OBzl, works well, and inserting an amino acid residue
into its C-terminus influences on the in vitro efficacy.
Table 1
DOCK scores of Trp-Trp-AA-OBzl (4aet) toward d(CGATCG)2.
2.4. Tumor weights of 4aet treated S180 mice
The in vivo anti-tumor activities of 4aet were represented with
the tumor weights of the treated mice. Via the in vivo assays the
¢¡
¢¢
Boc-Trp
Boc-Trp-Trp-OBzl
Boc-Trp-Trp
+ Trp-OBzl
2
1
AA-OBzl
¢£
Trp-Trp-AA-OBzl/AA
DOCK score
Trp-Trp-AA-OBzl/AA
DOCK score
¢¤
Trp-Trp-AA-OBzl
Boc-Trp-Trp-AA-OBzl
4a/Ala
4b/Gly
4c/Phe
4d/Leu
4e/Ile
4f/Val
4g/Ser
4h/Thr
4i/Tyr
4j/Pro
55.6
55.5
53.0
30.7
51.7
61.1
39.5
58.6
32.1
56.5
4k/Met
4l/Glu
4m/Asp
4n/Cys
4o/Arg
4p/Lys
4q/Gln
4r/Asn
4s/His
4t/Trp
32.1
51.2
37.4
59.6
43.0
40.7
62.1
49.4
38.4
41.0
4a-t
3a-t
Scheme 1. Synthetic route of Trp-Trp-AA-OBzl. i) DCC, HOBt and NMM; ii) Aqueous
solution of NaOH (2 M); iii) DCC, HOBt and NMM; ⅳ) Hydrogen chloride in ethyl
acetate (4 M). In 3a & 4a AA ¼ Ala, 3b & 4b AA ¼ Gly, 3c & 4c AA ¼ Phe, 3d & 4d
AA ¼ Leu, 3e & 4e AA ¼ Ile, 3f & 4f AA ¼ Val, 3g & 4g AA ¼ Ser, 3h & 4h AA ¼ Thr, 3i &
4i AA ¼ Tyr, 3j & 4j AA ¼ Pro, 3k & 4k AA ¼ Met, 3l & 4l AA ¼ Glu(OBzl), 3m &
4m AA ¼ Asp(OBzl), 3n & 4n AA ¼ Cys(4-MeOBzl), 3o & 4o AA ¼ NG-NO2-Arg, 3p & 4p
AA ¼ Nu-CBz-Lys, 3q & 4q AA ¼ Gln, 3r & 4r AA ¼ Asn, 3s & 4s AA ¼ His, 3t & 4t
AA ¼ Trp.

3412
X. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3410e3419
Table 2
IC50 (m
M) of 4aet inhibiting carcinoma cell lines.a
Compd.
S180
25.43 ꢀ 1.64
K562
B16
30.62 ꢀ 3.14
H22
25.98 ꢀ 1.60
HepG2
Cytarabine
Doxorubicin
24.29 ꢀ 1.60
17.23 ꢀ 1.43
18.37 ꢀ 1.69e
16.21 ꢀ 1.43b
14.92 ꢀ 1.22b
116.23 ꢀ 10.33
19.40 ꢀ 1.56c
26.23 ꢀ 1.77
17.54 ꢀ 1.75
20.06 ꢀ 1.97d
17.76 ꢀ 1.43c
16.77 ꢀ 1.40c
122.83 ꢀ 10.99
22.23 ꢀ 1.99d
9.49 ꢀ 0.97b
46.55 ꢀ 3.98
13.69 ꢀ 1.19b
43.16 ꢀ 3.92
14.73 ꢀ 1.39b
52.73 ꢀ 4.94
21.02 ꢀ 1.89d
120.78 ꢀ 11.94
11.39 ꢀ 1.09b
35.90 ꢀ 3.90
30.42 ꢀ 2.68
10.16 ꢀ 0.98b
17.95 ꢀ 1.48c
27.29 ꢀ 1.72e
154.03 ꢀ 10.11
29.2 ꢀ 6.02
18.52 ꢀ 1.39b
19.50 ꢀ 1.42b
14.53 ꢀ 0.99b
118.47 ꢀ 10.30
19.21 ꢀ 1.49b
8.14 ꢀ 0.98b
41.22 ꢀ 3.80
11.79 ꢀ 1.02b
35.95 ꢀ 2.68
12.50 ꢀ 0.90b
49.05 ꢀ 4.10
13.39 ꢀ 0.95b
132.01 ꢀ 9.30
9.77 ꢀ 0.97b
23.69 ꢀ 1.95c
32.65 ꢀ 2.98
8.74 ꢀ 0.89b
15.54 ꢀ 1.05b
26.52 ꢀ 1.64c
114.56 ꢀ 8.89
21.50 ꢀ 1.86
22.98 ꢀ 1.94d
24.30 ꢀ 1.98c
19.28 ꢀ 1.48b
124.06 ꢀ 11.00
25.93 ꢀ 1.99c
11.15 ꢀ 1.01b
54.33 ꢀ 4.11
15.98 ꢀ 1.34b
35.97 ꢀ 3.03
17.23 ꢀ 1.49b
65.41 ꢀ 5.01
20.75 ꢀ 1.84d
152.72 ꢀ 10.31
13.38 ꢀ 1.11b
36.32 ꢀ 2.89
55.70 ꢀ 4.51
11.95 ꢀ 1.02b
21.32 ꢀ 1.89d
31.69 ꢀ 3.10e
156.81 ꢀ 10.00
36.51 ꢀ 3.56
20.84 ꢀ 1.95b
22.44 ꢀ 1.90b
17.44 ꢀ 1.51b
121.26 ꢀ 10.93
21.65 ꢀ 1.38b
9.97 ꢀ 0.98b
46.33 ꢀ 3.90
13.98 ꢀ 1.18b
39.57 ꢀ 3.47
15.45 ꢀ 1.41b
49.08 ꢀ 4.01
13.65 ꢀ 0.96b
99.19 ꢀ 9.17
11.96 ꢀ 1.08b
25.43 ꢀ 2.15c
36.56 ꢀ 3.24
10.68 ꢀ 0.92b
18.67 ꢀ 1.52b
26.97 ꢀ 1.65c
98.14 ꢀ 9.00
4a
4b
4c
4d
4e
4f
4g
4h
4i
b
8.27 ꢀ 0.99
41.56 ꢀ 3.75
12.01 ꢀ 1.13b
45.69 ꢀ 3.37
12.90 ꢀ 0.92b
41.58 ꢀ 3.97
10.95 ꢀ 0.99b
130.15 ꢀ 9.18
9.92 ꢀ 1.04b
20.78 ꢀ 1.77c
32.31 ꢀ 2.99
8.86 ꢀ 0.90b
16.29 ꢀ 1.45b
25.33 ꢀ 1.65d
125.47 ꢀ 9.43
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
a
n ¼ 6.
b
For S180: Compared to cytarabine and doxorubicin p < 0.01; For K562: Compared to cytarabine and doxorubicin p < 0.01; For B16: Compared to cytarabine and
doxorubicin p < 0.01; For H22: Compared to cytarabine and doxorubicin p < 0.01; For HepG2: Compared to cytarabine and doxorubicin p < 0.01.
c
For S180: Compared to doxorubicin p < 0.01; For K562: Compared to cytarabine p < 0.01; For B16: Compared to cytarabine p < 0.01; For H22: Compared to doxorubicin
p < 0.01 and to cytarabine p > 0.05; For HepG2: Compared to cytarabine p < 0.01 and to doxorubicin p > 0.05.
d
For K562: Compared to cytarabine p > 0.05; For B16: Compared to cytarabine p < 0.01 and to doxorubicin p > 0.05; For HepG2: Compared to cytarabine p < 0.01.
For K562: Compared to doxorubicin p > 0.05; For B16: Compared to cytarabine p > 0.05; For HepG2: Compared to cytarabine p > 0.05.
e
tumor weights of 4aet treated S180 mice were obtained and are
shown in Table 3. When the mice were given a daily i.p injection of
8.9 mmol/kg of 4aet for seven consecutive days, seventeen tripep-
tides (4aec,eel,nes) were anti-tumor active (the tumor weights of
the treated mice were 0.170 ge0.688 g were significantly lower
than that, 0.827 g, of NS treated mice), and three tripeptides
(4d,m,t) were anti-tumor inactive. Among the anti-tumor active
tripeptides the efficacies of three tripeptides (4i,p,s, tumor weights
were 0.544 ge0.660 g) were essentially equal to that of cytarabine
(positive control, tumor weight 0.563 g), the efficacies of twelve
weight 0.429 g), the efficacies of eight tripeptides (4b,c,f,h,j,l,n,q,
tumor weights were 0.170e0.359 g) were significantly higher than
that of doxorubicin. These suggest that the lead, Trp-Trp-OBzl,
works well, and inserting an amino acid residue into its C-
terminus influences on the in vivo efficacy.
2.5. Effect of 4aet on the organ and body weights of treated S180
mice
The spleen index and the body weight are the important
parameters to evaluate the immunologic function of the treated
mice. To know the immunologic function of 4aet treated S180 mice
the spleen indexes and the increased body weights were measured
and are listed in Table 4. The data indicate that the liver, brain and
kidney weights as well as the spleen indexes of 4aet treated S180
tripeptides
(4aec,e,f,h,j,l,n,o,q,r,
tumor
weights
were
0.170e0.434 g) were significantly higher than that of cytarabine.
Besides, among the anti-tumor active tripeptides the efficacies of
three tripeptides (4e,o,r, tumor weights were 0.401e0.434 g) were
essentially equal to that of doxorubicin (positive control, tumor
Table 3
Effects of 4aet on tumor weights of S180 mice.a
Compd
Tumor weight
Inhibition%
Compd
Tumor weight
Inhibition%
NS
4a
4b
4c
4d
4e
4f
4g
4h
4i
0.827 ꢀ 0.165
0.413 ꢀ 0.087d
0.359 ꢀ 0.078e
0.358 ꢀ 0.077e
0.800 ꢀ 0.157
0.401 ꢀ 0.080d
0.170 ꢀ 0.035e
0.678 ꢀ 0.129b
0.321 ꢀ 0.066e
0.660 ꢀ 0.073b
0.348 ꢀ 0.070e
0.688 ꢀ 0.127b
0.00
Cytarabine
Doxorubicin
4l
4m
4n
4o
4p
4q
4r
0.563 ꢀ 0.102
0.429 ꢀ 0.011
0.370 ꢀ 0.063f
1.037 ꢀ 0.200
0.300 ꢀ 0.054e
0.434 ꢀ 0.079d
0.544 ꢀ 0.111c
0.231 ꢀ 0.046e
0.409 ꢀ 0.083d
0.573 ꢀ 0.112c
0.985 ꢀ 0.176
31.93 ꢀ 5.59
48.22 ꢀ 1.20
55.32 ꢀ 7.91
ꢁ30.68 ꢀ 4.10
63.71 ꢀ 4.54
47.51 ꢀ 5.01
34.30 ꢀ 4.82
72.03 ꢀ 6.98
50.59 ꢀ 6.35
30.69 ꢀ 4.31
ꢁ19.07 ꢀ 4.01
50.05 ꢀ 7.06
56.56 ꢀ 6.64
56.73 ꢀ 6.40
3.29 ꢀ 0.52
51.47 ꢀ 6.12
79.45 ꢀ 9.07
18.06 ꢀ 2.82
61.25 ꢀ 5.06
20.24 ꢀ 3.23
57.98 ꢀ 5.88
16.86 ꢀ 1.91
4s
4t
4j
4k
a
Dose ¼ 8.9 mol/kg, n ¼ 12, tumor weight is expressed by x ꢀ SD g.
m
b
c
d
e
f
Compared to NS p < 0.05.
Compared to NS p < 0.01.
Compared to NS and cytarabine p < 0.01, to doxorubicin p > 0.05.
Compared to NS, cytarabine and doxorubicin p < 0.01.
Compared to NS and cytarabine p < 0.01, to doxorubicin p < 0.05.

X. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3410e3419
3413
Table 5
mice are substantially equal to those of NS, cytarabine and doxo-
Anti-tumor activity of 4f at different doses against S180 mice.a
rubicin treated S180 mice, but the increased body weights of
4aec,eej,nes treated S180 mice are significantly higher than that
of NS, cytarabine and doxorubicin treated S180 mice. The signifi-
cant differences in the increased body weights demonstrate that
the damage resulted from the therapy of 4aec,eej,nes is lower
than that from the therapy of NS, cytarabine and doxorubicin.
Compd.
Dose (
m
mol/kg)
Tumor weight (g)
%Inhibition
Cytarabine
8.9
0.563 ꢀ 0.102b
0.657 ꢀ 0.113c
0.762 ꢀ 0.129
0.170 ꢀ 0.035d
0.246 ꢀ 0.044e
0.482 ꢀ 0.049f
0.827 ꢀ 0.165
31.93 ꢀ 5.59
20.57 ꢀ 3.02
7.86 ꢀ 1.18
79.45 ꢀ 9.07
70.23 ꢀ 6.07
41.72 ꢀ 4.39
0.89
0.089
8.9
0.89
0.089
0.2 ml
4f
NS
2.6. Dose dependent in vivo anti-tumor activities of 4f
a
n ¼ 12, tumor weight is expressed by x ꢀ SD g.
b
c
d
e
f
The most potent tripeptide (4f) was selected to examine the
dose dependent action of 4aet. At the doses of 8.9, 0.89 and
0.089 mmol/kg, 4f showed tumor inhibitions of 79.5%, 70.2%, and
41.7%, respectively (Table 5). Therefore, 4f possesses dose-
dependent anti-tumor action.
Compared to 0.89
m
mol/kg of cytarabine p < 0.05.
mol/kg of cytarabine p < 0.05.
mol/kg of 4f and 8.9 mol/kg of cytarabine p < 0.01.
mol/kg of 4f and 8.9 mol/kg of cytarabine p < 0.01.
mol/kg of cytarabine p < 0.01.
Compared to 0.089
Compared to 0.89
Compared to 0.089
Compared to NS and 0.89
m
m
m
m
m
m
2.8.1. Viscosity of CT DNA without and with 4f
In the intercalation in order to accommodate and bind small
molecule the base pairs of the DNA can be pushed apart and
consequently could result in the increase of the viscosity. Thus the
increase of the viscosity has been widely used to reflect the inter-
calation [19,20]. Herein the relative viscosities of the sample solu-
tions of [4f]/[CT DNA] in the ratios of 0e0.36 were measured. Fig. 2
explains that the relative viscosity of CT DNA is gradually increased
with the increase of the concentration of 4f added. This is in
consistent with the intercalation of 4f toward CT DNA.
2.7. Acute toxicity of 4aec,eel,nes treated mice
A series of intercalators were known to be strong neurotoxic
[17]. To know the toxicity of Trp-Trp-AA-OBzl the anti-tumoral
active 4aec,eel,nes were examined for the LD50 and neurotox-
icity in mouse model. The examination indicates that even
receiving 500 mg/kg of 4aec,eel,nes the mice neither exhibit
tremor, twitch, jumping, tetanus, and supination, nor occur death.
On the 7th day the necropsy findings in 500 mg/kg of 4aec,eel,nes
receiving mice revealed that the therapy led no apparent changes in
any organs. These suggest that 4aec,eel,nes are comparatively
non-toxic, and their LD50 values should be more than 500 mg/kg.
2.8.2. Melting temperature of CT DNA without and with 4f
The thermal melting of DNA is widely used to measure the
thermostability of the double helix [21,22]. If the buffer solution of
DNA was heated at a properly elevated temperature its double
strands will dissociate, the corresponding single stands will form,
and the absorbance of the solution will increase. The temperature
that induces the absorbance of DNA solution a 50% increase is
termed melting temperature (Tm). Here the temperature of the
2.8. Action mechanism of 4f
Small molecule induced variation of UV, circular dichroic (CD),
fluorescence, viscosity and melting temperature has been widely
used to identify DNA intercalation [18ꢁ21]. In the examination of
the intercalation of 4aet toward DNA calf thymus DNA (CT DNA)
was used as the model DNA and 4f was used as model tripeptide,
and the viscosity and melting temperature of CT DNA alone were
compared to those of CT DNA plus 4f.
solution of CT DNA alone (PBS, pH 7.4, 100
mM) and it plus 4f (PBS,
pH 7.4, 18
m
M) was risen at a rate of 1 ꢂC/min and the absorbance
was monitored at 279 nm. The melting curves of CT DNA alone and
CT DNA plus 4f are shown with Fig. 3. The fact that the Tm of CT
Table 4
Effect of 4aet on the spleen index and organ weights of S180 mice.a
Compd.
Liver
Brain
Kidney
Spleen index
Increased BW
NS
Cytarabine
Doxorubicin
1.85 ꢀ 0.23
1.62 ꢀ 0.11
1.61 ꢀ 0.06
1.77 ꢀ 0.13b
1.80 ꢀ 0.21b
1.89 ꢀ 0.21b
1.70 ꢀ 0.17
1.85 ꢀ 0.19b
1.89 ꢀ 0.20b
1.73 ꢀ 0.20
1.81 ꢀ 0.05b
1.78 ꢀ 0.19b
1.81 ꢀ 0.20b
1.74 ꢀ 0.25
1.86 ꢀ 0.13b
1.77 ꢀ 0.15b
1.75 ꢀ 0.143
1.87 ꢀ 0.27b
1.88 ꢀ 0.27b
1.81 ꢀ 0.18b
1.82 ꢀ 0.16b
1.78 ꢀ 0.22b
1.78 ꢀ 0.21b
0.27 ꢀ 0.03
0.29 ꢀ 0.02
0.25 ꢀ 0.01
0.27 ꢀ 0.03
0.27 ꢀ 0.02
0.28 ꢀ 0.03
0.26 ꢀ 0.02
0.28 ꢀ 0.03
0.27 ꢀ 0.01
0.28 ꢀ 0.05
0.30 ꢀ 0.07
0.27 ꢀ 0.03
0.27 ꢀ 0.01
0.28 ꢀ 0.03
0.27 ꢀ 0.02
0.27 ꢀ 0.03
0.27 ꢀ 0.01
0.27 ꢀ 0.01
0.27 ꢀ 0.02
0.27 ꢀ 0.01
0.27 ꢀ 0.01
0.28 ꢀ 0.02
0.29 ꢀ 0.03
0.16 ꢀ 0.02
0.16 ꢀ 0.03
0.15 ꢀ 0.02
0.16 ꢀ 0.01
0.16 ꢀ 0.02
0.16 ꢀ 0.01
0.16 ꢀ 0.02
0.17 ꢀ 0.02
0.15 ꢀ 0.02
0.16 ꢀ 0.02
0.15 ꢀ 0.02
0.16 ꢀ 0.03
0.15 ꢀ 0.01
0.16 ꢀ 0.03
0.17 ꢀ 0.02
0.15 ꢀ 0.02
0.16 ꢀ 0.03
0.16 ꢀ 0.02
0.16 ꢀ 0.02
0.16 ꢀ 0.02
0.16 ꢀ 0.02
0.16 ꢀ 0.02
0.16 ꢀ 0.02
7.26 ꢀ 1.37
7.50 ꢀ 1.84
7.31 ꢀ 1.61
7.59 ꢀ 1.38
8.11 ꢀ 1.39
7.96 ꢀ 1.36
7.45 ꢀ 1.80
8.12 ꢀ 1.66
7.97 ꢀ 1.63
7.59 ꢀ 1.96
8.21 ꢀ 1.55
7.50 ꢀ 1.84
8.01 ꢀ 1.79
7.69 ꢀ 1.88
7.92 ꢀ 1.60
8.25 ꢀ 1.78
8.21 ꢀ 1.66
7.99 ꢀ 1.82
8.22 ꢀ 1.60
7.96 ꢀ 1.90
7.81 ꢀ 1.32
8.34 ꢀ 1.40
7.75 ꢀ 1.72
8.42 ꢀ 1.52
7.12 ꢀ 1.39
6.12 ꢀ 1.39
9.50 ꢀ 1.05b
9.77 ꢀ 1.25b
9.86 ꢀ 1.22b
8.40 ꢀ 1.45
9.80 ꢀ 1.39b
9.97 ꢀ 1.39b
9.95 ꢀ 1.33b
9.99 ꢀ 1.38b
9.76 ꢀ 1.40b
10.24 ꢀ 1.64b
8.93 ꢀ 1.55c
9.94 ꢀ 1.43b
8.38 ꢀ 1.17
9.69 ꢀ 1.04b
9.93 ꢀ 1.46b
9.76 ꢀ 1.31b
9.89 ꢀ 1.49b
9.96 ꢀ 1.41b
10.04 ꢀ 1.49b
8.71 ꢀ 1.50c
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
a
Dose ¼ 8.9 mol/kg, BW ¼ body weight, body and organ weights are expressed by x ꢀ SD g n ¼ 12.
m
b
For liver weight: Compared to cytarabine and doxorubicin p < 0.05, to NS p > 0.05; For increased body weight: Compared to cytarabine and doxorubicin p < 0.01, to NS
p > 0.05.
c
For increased body weight: Compared to cytarabine p < 0.01, to doxorubicin p < 0.01, to NS p > 0.05.

3414
X. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3410e3419
Fig. 4. Alignment stereoview of 4aet used for molecular field generation.
Fig. 2. Effect of 4f on the relative viscosity of CT DNA (200 mM).
[24]. Both the electrostatic and steric fields of 4aen,p,ret were
created by use of proton, hydroxyl group and methyl group as the
probe. These fields were sampled at each point of a regularly spaced
grid of 1 Å. An energy cutoff of ꢀ30.0 kcal/mol was set for two
fields. The totally generated grid points were 1200. The regression
analysis was carried out by using the genetic partial least square (G/
PLS) method consisting of 10,000 generations with a population
size of 100. The number of the components was set to 5. The cross-
validation was performed with the leave-one-out procedure. PLS
analysis was scaled, with all variables normalized to a variance of
1.0. The MFA model for the tumor weights of 4aen,p,ret treated
mice in terms of the most relevant descriptors including proton,
hydroxyl group and methyl group is expressed by equation (1).
DNA plus 4f is 22.0 ꢀ 0.6 ꢂC higher than that of CT DNA alone
indicates that in the presence of 4f the double strands of CT DNA are
stabilized. This increased stability of the double helix of CT DNA can
be attributed to the intercalation of 4f toward CT DNA.
2.9. 3D QSAR analysis of 4aet
To elucidate the effects of Trp-Trp-AA-OBzl (4aet) on the tumor
weights of the treated S180 mice the 3D QSAR analysis was per-
formed. For 3D QSAR the training set 4aen,p,ret and the test set
4o,q selections were made manually such that they populated over
a wide range of anti-tumor activities in a similar proportion. The 3D
QSAR module of Cerius2 was followed to quantitatively correlate
the tumor weights with the structures of 4aen,p,ret. To obtain
ꢀ
ꢁ
Tumor weight ¼ 0:13 þ 0:0068 Hþ=692
ꢀ
ꢁ
ꢁ 0:0038 Hþ=832 þ 0:0086ðCH3=654Þ
a
consistent alignment, their common moiety, Trp-Trp, was
selected as the template for superposing 4aet. The method used for
performing the alignment was the maximum common subgraph
(MCS) [23]. The stereoview of aligned 4aet is shown in Fig. 4. The
alignment stereoview explores that to superimpose onto Trp-Trp
the C-terminal amino acid residue of each structure of Trp-Trp-
AA-OBzl has to take individual conformation.
þ 0:0062ðCH3=763Þ ꢁ 0:0041ðCH3=833Þ
ꢀ
ꢁ
þ 0:0051 HOꢁ=942
ð1Þ
The data points (n), correlation coefficient (r) and square
correlation coefficient (r2) of equation (1) were 18, 0.974 and 0.948,
respectively. The correlation of the tested tumor weights on S180
Based on the alignment molecular field analysis (MFA) of
4aen,p,ret was performed by using the QSAR module of Cerius2
Fig. 3. Thermal denaturation curves of CT DNA without and with 4f.
Fig. 5. Graph of the tested tumor weights against calculated tumor weights.

X. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3410e3419
3415
13C NMR (125 Hz) spectra were acquired on a Bruker AC 300
spectrometer in CDCl3 or in DMSO-d6 with TMS as internal stan-
dard, and chemical shifts are expressed in ppm. Optical rotations
were determined with a Jasco P-1020 Polarimeter. Statistical anal-
ysis of all the biological data was carried out by use of ANOVA test,
p < 0.05 is considered significant.
Table 6
Predict and test tumor weight of 4o,q treated mice.
Compd.
Tumor weight (g)
Predict value
Test value
Error
4o
4q
0.427
0.249
0.434
0.231
ꢁ0.007
0.018
4.1.1. Boc-Trp-Trp-OBzl (1)
At 0 ꢂC, 1.87 g (6.6 mmol) of 1-hydroxybenzotriazole (HOBt) and
1.48 g (7.2 mmol) of dicyclohexylcarbodiimide (DCC) were added to
a solution of 2.84 g (7.2 mmol) of Boc-Trp and 2.17 g (6.6 mmol) of
HCl$Trp-OBzl in 40 ml of anhydrous THF. At 0 ꢂC the reaction
mixture was adjusted pH 8 with 1.44 ml (6.6 mmol) of NMM and
then stirred for 24 h. The precipitated DCU was removed by
filtration. The filtrate was evaporated under reduced pressure, and
the residue was dissolved in 80 ml of EtOAc. The solution was
washed successively with saturated aqueous NaHCO3, aqueous
KHSO4 (5%) and saturated aqueous NaCl. The organic phase was
dried over anhydrous Na2SO4. After filtration and evaporation
under reduced pressure, the residue was purified by column
chromatography eluting with CHCl3:CH3OH (30:1) to provide
3.68 g (96%) of the title compoundþas colorless powder. Mp
189e191 ꢂC, ESI/MS (m/e) 581 [M þ H] .
mouse model with the calculated tumor weights from equation (1)
is explained with Fig. 5.
In equation (1) one term of Hþ/692 with positive coefficient
from proton descriptor, which means that at this position electron-
releasing group will increase the in vivo anti-tumor activity, one
term of Hþ/832 with negative coefficient from proton descriptor,
which means that at this position electron-releasing group will
decrease the in vivo anti-tumor activity, two terms of CH3/654 and
CH3/763 with positive coefficients from methyl descriptor, which
means that at this positions large group will increase the in vivo
anti-tumor activity, one term of CH3/833 with negative coefficient
from methyl descriptor, which means that at this positions large
group will decrease the in vivo anti-tumor activity, and one term of
HOꢁ/942 with positive coefficient from hydroxyl descriptor, which
means that at this position hydrogen bond forming group will
increase the activity, are involved.
The predict power of equation (1) was demonstrated by the
comparison of the calculated and the tested tumor weights of the
test set (Table 6, 4o,q). The results indicate that equation (1)
rationally gives the tumor weights of 4o and 4q treated mice and
the errors are ꢁ0.007 g and 0.018 g, respectively. The calculated
tumor weight closes the experimental tumor weight means that
equation (1) is practical to accurately predict the anti-tumor
activity of Trp-Trp-AA-OBzl.
4.1.2. Boc-Trp-Trp (2)
At 0 ꢂC to the solution of 1.00 g (1.72 mmol) of Boc-Trp-Trp-OBzl
in 10 ml of methanol 3 ml of aqueous NaOH (2 M) was added to
adjust the solution to pH 12. The reaction mixture was stirred at
0 ꢂC for 30 min, and then was adjusted to pH 5.5 with hydrochloric
acid (2 N). After filtration the filtrate was evaporated under reduced
pressure. The residue was dissolved in 30 ml of methanol and the
solution was filtered. The filtrate was evaporated under reduced
pressure and the residue was solidified in 10 ml of anhydrous ether
to provide 0.76 g (90%) of the title compound as colorless powder.
Mp 200e202 ꢂC, ESI/MS (m/e) 491 [M þ H]þ.
3. Conclusions
A similar ring-opening modification of the anti-tumoral active
N-tryptophanyl-b-carboline-3-carboxylic acid benzyl ester and b-
4.1.3. Boc-Trp-Trp-Ala-OBzl (3a)
carboline-3-carbonyltryptophan benzyl ester a pharmacophore,
Trp-Trp-OBzl, was obtained. With Trp-Trp-OBzl as a lead and
inserting amino acid residue into its C-terminus twenty anti-
tumoral active tripeptides, Trp-Trp-AA-OBzls, were built. The
in vivo anti-tumor activities of twelve of twenty Trp-Trp-AA-OBzls
were higher than that cytarabine. The acute toxicity assay defined
Trp-Trp-AA-OBzls having a LD50 value of more than 500 mg/kg. The
automated docking, UV, CD, fluorescence, viscosity and melting
temperature studies suggested the anti-tumor action mechanism of
Trp-Trp-AA-OBzls to be DNA intercalation. The effect of C-terminal
amino acid residue on the anti-tumor activity can be quantitatively
estimated with 3D QSAR.
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 386 mg (1.1 mmol)
of Tos$Ala-OBzl 641 mg (98%) of the title compound was obtained
as colorless powder. Mp 202e204 ꢂC, ESI/MS (m/e) 652 [M þ H]þ.
4.1.4. Boc-Trp-Trp-Gly-OBzl (3b)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 371 mg (1.1 mmol) of
Tos$Gly-OBzl 414 mg (65%) of the title compound was obtained as
colorless powder. Mp 206e208 ꢂC, ESI/MS (m/e) 638 [M þ H]þ.
4.1.5. Boc-Trp-Trp-Phe-OBzl (3c)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 470 mg (1.1 mmol)
of Tos$Phe-OBzl 682 mg (94%) of the title compound was obtained
as colorless powder. Mp 200e202 ꢂC, ESI/MS (m/e) 638 [M þ H]þ.
4. Experimental
4.1. General
All chemicals were purchased from commercial suppliers and
4.1.6. Boc-Trp-Trp-Leu-OBzl (3d)
were purified when necessary. Protected amino acids with
L
-
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 432 mg (1.1 mmol)
of Tos$Leu-OBzl 675 mg (97%) of the title compound was obtained
as colorless powder. Mp 206e208 ꢂC, ESI/MS (m/e) 694 [M þ H]þ.
configuration were purchased from sigma chemical Co. Chroma-
tography was performed on Qingdao silica gel H. The purities
(>97%) of the intermediates and the products were measured by
TLC analysis (Merck silica gel plates of type 60 F254, 0.25 mm layer
thickness) and HPLC analysis (waters, C18 column, 4.6 ꢃ 150 mm).
Melting points were determined in capillary tubes on an electro-
thermal SM/XMP apparatus and without correction. UV spectra
were measured on Shimadzu UV 2550. ESI-MS was determined by
Micromass Quattro micro TM API, Waters Co. 1H NMR (500 Hz) and
4.1.7. Boc-Trp-Trp-Ile-OBzl (3e)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 432 mg (1.1 mmol)
of Tos$Ile-OBzl 687 mg (99%) of the title compound was obtainþed as
colorless powder. Mp 212e214 ꢂC, ESI/MS (m/e) 694 [M þ H] .

3416
X. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3410e3419
4.1.8. Boc-Trp-Trp-Val-OBzl (3f)
of Tos$Lys(Nu-Z)-OBzl 829 mg (98%) of the title compound was
obtained as colorless powder. Mp 201e203 ꢂC, ESI/MS (m/e) 843
[M þ H]þ.
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 432 mg (1.1 mmol)
of Tos$Val-OBzl 642 mg (95%) of the title compound was obtainþed
as colorless powder. Mp 210e212 ꢂC, ESI/MS (m/e) 680 [M þ H] .
4.1.19. Boc-Trp-Trp-Gln-OBzl (3q)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 300 mg (1.1 mmol)
of HCl$Gln-OBzl 469 mg (66%) of the title compound was obtainþed
as colorless powder. Mp 195e197 ꢂC, ESI/MS (m/e) 709 [M þ H] .
4.1.9. Boc-Trp-Trp-Ser-OBzl (3g)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 404 mg (1.1 mmol)
of Tos$Ser-OBzl 563 mg (84%) of the title compound was obtainþed
as colorless powder. Mp 195e197 ꢂC, ESI/MS (m/e) 668 [M þ H] .
4.1.20. Boc-Trp-Trp-Asn-OBzl (3r)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 286 mg (1.1 mmol)
of HCl$Asn-OBzl 633 mg (91%) of the title compound was obtainþed
as colorless powder. Mp 190e192 ꢂC, ESI/MS (m/e) 695 [M þ H] .
4.1.10. Boc-Trp-Trp-Thr-OBzl (3h)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 419 mg (1.1 mmol) of
Tos$Thr-OBzl 580 mg (85%) of the title compound was obtainþed as
colorless powder. Mp 190e192 ꢂC, ESI/MS (m/e) 682 [M þ H] .
4.1.21. Boc-Trp-Trp-His-OBzl (3s)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 648 mg (1.1 mmol)
of 2Tos$His-OBzl 556 mg (68%) of the title compound was obtained
as colorless powder. Mp 189e191 ꢂC, ESI/MS (m/e) 718 [M þ H]þ.
4.1.11. Boc-Trp-Trp-Tyr-OBzl (3i)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 487 mg (1.1 mmol)
of Tos$Tyr-OBzl 708 mg (95%) of the title compound was obtained
as colorless powder. Mp 220e222 ꢂC, ESI/MS (m/e) 744 [M þ H]þ.
4.1.22. Boc-Trp-Trp-Trp-OBzl (3t)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 363 mg (1.1 mmol)
of HCl$Trp-OBzl 664 mg (87%) of the title compound was obtainþed
as colorless powder. Mp 180e192 ꢂC, ESI/MS (m/e) 767 [M þ H] .
4.1.12. Boc-Trp-Trp-Pro-OBzl (3j)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 266 mg (1.1 mmol)
of HCl$Tyr-OBzl 654 mg (96%) of the title compound was obtainþed
as colorless powder. Mp 182e184 ꢂC, ESI/MS (m/e) 678 [M þ H] .
4.1.23. Trp-Trp-Ala-OBzl (4a)
To 500 mg (0.78 mmol) of Boc-Trp-Trp-Ala-OBzl 7 ml of 4 M
solution of hydrochloride in ethyl acetate was added. The reaction
mixture was stirred at room temperature for 60 min and TLC
(chloroform/methanol, 5:1) indicated the disappearance of Boc-
Trp-Trp-Ala-OBzl. The reaction mixture was evaporated under
reduced pressure, the residue was dissolved in 40 ml of ethyl
acetate, the solution was again evaporated under reduced pressure
and the residue was washed with anhydrous ether to provide
4.1.13. Boc-Trp-Trp-Met-OBzl (3k)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 452 mg (1.1 mmol)
of Tos$Met-OBzl 692 mg (97%) of the title compound was obtainþed
as colorless powder. Mp 218e220 ꢂC, ESI/MS (m/e) 712 [M þ H] .
4.1.14. Boc-Trp-Trp-Glu(OBzl)-OBzl (3l)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 549 mg (1.1 mmol)
of Tos$Glu(OBzl)-OBzl 471 mg (59%) of the title compound was
obtained as colorless powder. Mp 202e204 ꢂC, ESI/MS (m/e) 800
[M þ H]þ.
449 mg (98%) of the title compound as þcolorless powder. Mp.
20
205e207 ꢂC; ESI/MS (m/e) 552 [M þ H] ; [
a]
¼ 11.03 (c 1,
D
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.90 (m, 2H), 8.66
(d, J ¼ 7.3 Hz,1H), 8.59 (d, J ¼ 8.1 Hz,1H), 7.63 (d, J ¼ 7.8 Hz,1H), 7.49
(d, J ¼ 7.8 Hz, 1H), 7.14 (m, 13H), 5.02 (m, 2H), 4.66 (m, 2H), 4.60 (m,
1H), 3.10 (m, 4H), 2.03 (s, 2H), 1.49 (m, 3H); 13C NMR (125 MHz,
4.1.15. Boc-Trp-Trp-Asp(OBzl)-OBzl (3m)
DMSO-d6)
d
/ppm ¼ 172.9, 142.3, 136.7, 130.1, 127.7, 127.5, 127.2,
Using the same procedure as described for Boc-Trp-Trp-OBzl from
490 mg (1.0 mmol) of Boc-Trp-Trp and 533 mg (1.1 mmol) of
Tos$Asp(OBzl)-OBzl473 mg(60%)ofthetitlecompound wasobtaþined
as colorless powder. Mp 208e210 ꢂC, ESI/MS (m/e) 786 [M þ H] .
122.9, 122.2, 120.1, 119.2, 119.0, 111.3, 111.1, 110.9, 68.5, 54.6, 48.7,
31.4, 30.7, 20.5. Anal. Calcd for C32H33N5O4: C, 69.67; H, 6.03; N,
12.70. Found: C, 69.88; H, 6.17; N, 12.91.
4.1.24. Trp-Trp-Gly-OBzl (4b)
4.1.16. Boc-Trp-Trp-Cys(4-MeOBzl)-OBzl (3n)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.79 mmol) of Boc-Trp-Trp-Gly-OBzl 446 mg (99%)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 404 mg (1.1 mmol)
of HCl$Cys(4-OMeBzl)-OBzl 801 mg (99%) of the title compound
was obtained as colorless powder. Mp 221e223 ꢂC, ESI/MS (m/e)
804 [M þ H]þ.
of the title compound was obtained asþcolorless powder. Mp
20
201e203 ꢂC; ESI/MS (m/e) 538 [M þ H] ; [
a
]
D
¼ 8.23 (c 1.2,
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.96 (s, 1H), 10.90
(s, 1H), 8.80 (d, J ¼ 7.3 Hz, 1H), 8.67 (d, J ¼ 8.3 Hz, 1H), 7.60 (d,
J ¼ 7.9 Hz, 1H), 7.52 (d, J ¼ 7.9 Hz, 1H), 7.17 (m, 13H), 5.05 (m, 2H),
4.70 (m, 2H), 3.16 (m, 6H), 2.02 (s, 2H); 13C NMR (125 MHz, DMSO-
4.1.17. Boc-Trp-Trp-Arg(NGNO2)-OBzl (3o)
Using the same procedure as described for Boc-Trp-Trp-OBzl from
490 mg (1.0 mmol) of Boc-Trp-Trp and 404 mg (1.1 mmol) of
HCl$Arg(NO2)-OBzl 317 mg (41%) of the title compound was obtained
as colorless powder. Mp 207e209 ꢂC, ESI/MS (m/e) 782 [M þ H]þ.
d6)
d
/ppm ¼ 172.9, 142.2, 136.7, 130.2, 127.7, 127.5, 127.2, 122.9,
122.2, 120.1, 119.2, 119.0, 111.3, 111.1, 110.9, 68.5, 54.6, 48.8, 43.1, 31.4,
30.6. Anal. Calcd for C31H31N5O4: C, 69.26; H, 5.81; N, 13.30. Found:
C, 69.27; H, 5.95; N, 13.52.
4.1.18. Boc-Trp-Trp-Lys(Nu-Z)-OBzl (3p)
4.1.25. Trp-Trp-Phe-OBzl (4c)
Using the same procedure as described for Boc-Trp-Trp-OBzl
from 490 mg (1.0 mmol) of Boc-Trp-Trp and 596 mg (1.1 mmol)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.69 mmol) of Boc-Trp-Trp-Phe-OBzl 432 mg (97%)

X. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3410e3419
3417
of the title compound was obtained as colorless powder. Mp
54.7, 31.4, 30.6. Anal. Calcd for C32H33N5O5: C, 67.71; H, 5.86; N,
12.34. Found: C, 67.50; H, 5.71; N, 12.11.
20
199e201 ꢂC; ESI/MS (m/e) 628 [M þ H]þ; [
a
]
¼ 11.23 (c 1.1,
D
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.98 (m, 2H), 8.98
(d, J ¼ 8.0 Hz,1H), 8.82 (d, J ¼ 7.2 Hz,1H), 7.64 (d, J ¼ 7.9 Hz,1H), 7.53
4.1.30. Trp-Trp-Thr-OBzl (4h)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.73 mmol) of Boc-Trp-Trp-Thr-OBzl 429 mg (95%)
(d, J ¼ 11.4 Hz,1H), 7.19 (m,18H), 5.03 (m, 2H), 4.68 (m, 2H), 4.41 (m,
1H), 3.10 (m, 6H), 1.98 (s, 2H); 13C NMR (125 MHz, DMSO-d6)
d/
ppm ¼ 172.9, 142.2, 139.6, 136.7, 130.0, 128.6, 127.8, 127.5, 127.2,
126.1, 122.9, 122.2, 120.1, 119.2, 119.0, 111.3, 111.1, 110.9, 68.6, 54.6,
53.7, 40.6, 31.2, 30.6. Anal. Calcd for C38H37N5O4: C, 72.71; H, 5.94;
N, 11.16. Found: C, 72.50; H, 5.80; N, 11.39.
of the title compound was obtained asþcolorless powder. Mp
20
195e197 ꢂC; ESI/MS (m/e) 582 [M þ H] ; [
a]
¼ 6.10 (c 1.2,
D
CH3OH); 1H NMR (500 MHz, DMSO-d6)
(m, 15H), 6.76 (s, 2H), 5.07 (m, 2H), 4.66 (m, 2H), 3.62 (m, 1H), 3.40
d
/ppm ¼ 10.97 (m, 2H), 7.32
(m, 2H), 3.13 (m, 4H), 1.98 (s, 2H), 1.15 (m, 3H); 13C NMR (125 MHz,
4.1.26. Trp-Trp-Leu-OBzl (4d)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.72 mmol) of Boc-Trp-Trp-Leu-OBzl 450 mg (99%)
DMSO-d6)
d
/ppm ¼ 172.9, 142.3, 136.8, 130.1, 127.7, 127.5, 127.2,
122.9, 122.2, 120.1, 119.2, 119.0, 111.4, 111.1, 110.9, 71.0, 68.6, 65.8,
54.6, 31.3, 30.8, 19.2. Anal. Calcd for C33H35N5O5: C, 68.14; H, 6.07;
N, 12.04. Found: C, 68.35; H, 6.22; N, 12.25.
of the title compound was obtained as colorless powder. Mp
20
222e224 ꢂC; ESI/MS (m/e) 594 [M þ H]þ; [
a
]
D
¼ 15.76 (c 1.3,
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.92 (m, 2H), 8.76
4.1.31. Trp-Trp-Tyr-OBzl (4i)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.67 mmol) of Boc-Trp-Trp-Tyr-OBzl 449 mg (98%)
(d, J ¼ 8.1 Hz,1H), 8.71 (d, J ¼ 7.2 Hz,1H), 7.64 (d, J ¼ 7.9 Hz,1H), 7.50
(d, J ¼ 7.9 Hz, 1H), 7.16 (m, 13H), 6.85 (s, 2H), 5.02 (m, 2H), 4.63 (m,
2H), 3.75 (m, 1H), 3.15 (m, 4H), 2.01 (s, 2H), 1.59 (m, 1H), 0.89 (m,
of the title compound was obtained asþcolorless powder. Mp
6H); 13C NMR (125 MHz, DMSO-d6)
d
/ppm ¼ 172.9, 142.2, 136.8,
207e209 ꢂC; ESI/MS (m/e) 644 [M þ H] ; [
a
]
D
¼ 9.77 (c 1.1,
20
130.1, 127.8, 127.5, 127.1, 122.9, 122.2, 120.1, 119.2, 119.0, 111.3, 111.1,
110.9, 68.6, 54.8, 51.3, 43.6, 31.3, 30.6, 22.6, 22.0. Anal. Calcd for
C35H39N5O4: C, 70.80; H, 6.62; N, 11.80. Found: C, 70.98; H, 6.77; N,
12.03.
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.89 (m, 2H), 8.78
(d, J ¼ 7.3 Hz,1H), 8.74 (d, J ¼ 8.2 Hz,1H), 7.66 (d, J ¼ 8.0 Hz,1H), 7.51
(d, J ¼ 7.9 Hz, 1H), 7.07 (m, 17H), 5.90 (s, 1H), 5.02 (m, 2 H), 4.70 (m,
2H), 3.90 (m, 1H), 3.04 (m, 6H), 1.98 (s, 2H); 13C NMR (125 MHz,
DMSO-d6)
d
/ppm ¼ 172.6, 155.9, 142.2, 136.7, 132.2, 130.1, 129.0,
4.1.27. Trp-Trp-Ile-OBzl (4e)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.72 mmol) of Boc-Trp-Trp-Ile-OBzl 429 mg (99%)
127.7, 127.5, 127.2, 122.9, 122.2, 120.1, 119.2, 119.0, 115.2, 111.3, 111.1,
110.9, 68.6, 54.8, 53.1, 40.6, 31.3, 30.8. Anal. Calcd for C38H37N5O5: C,
70.90; H, 5.79; N, 10.88. Found: C, 70.69; H, 5.63; N, 11.12.
of the title compound was obtained as colorless powder. Mp
20
228e230 ꢂC; ESI/MS (m/e) 594 [M þ H]þ; [
a
]
¼ 17.00 (c 1.2,
4.1.32. Trp-Trp-Pro-OBzl (4j)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.74 mmol) of Boc-Trp-Trp-Pro-OBzl 451 mg (99%)
D
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.96 (m, 2 H),
8.70 (d, J ¼ 7.2 Hz, 1H), 8.65 (d, J ¼ 8.0 Hz, 1H), 7.25 (m, 13H), 6.82 (s,
2H), 5.04 (m, 2H), 4.68 (m, 2H), 3.47 (m, 1H), 3.11 (m, 4H), 1.98 (s,
2H), 1.25 (m, 1H), 1.08 (m, 2H), 0.83 (m, 6H); 13C NMR (125 MHz,
of the title compound was obtained as colorless powder. Mp
20
182e184 ꢂC; ESI/MS (m/e) 578 [M þ H]þ; [
a
]
¼ 16.73 (c 1.3,
D
DMSO-d6)
d
/ppm ¼ 172.9, 142.1, 136.6, 130.0, 127.8, 127.5, 127.2,
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.92 (s, 1H), 10.87
122.8, 122.2, 120.1, 119.2, 119.0, 111.3, 111.1, 110.9, 68.6, 57.1, 54.8,
39.5, 31.2, 30.6, 24.9,14.6,10.9. Anal. Calcd for C35H39N5O4: C, 70.80;
H, 6.62; N, 11.80. Found: C, 71.01; H, 6.78; N, 11.58.
(s, 1H), 8.73 (m, 2H), 7.64 (d, J ¼ 7.9 Hz, 1H), 7.50 (d, J ¼ 7.9 Hz, 1H),
7.18 (m, 13H), 5.06 (m, 2H), 4.66 (m, 2H), 4.08 (m, 1H), 3.16 (m, 6H),
2.02 (m, 1H), 1.79 (m, 4H); 13C NMR (125 MHz, DMSO-d6)
d/
ppm ¼ 172.7, 142.2, 136.6, 130.1, 127.8, 127.5, 127.2, 122.9, 122.2,
120.1, 119.2, 119.0, 111.3, 111.1, 110.9, 68.7, 60.5, 54.8, 45.7, 32.3, 31.2,
30.6, 24.9. Anal. Calcd for C34H35N5O4: C, 70.69; H, 6.11; N, 12.12.
Found: C, 70.90; H, 6.27; N, 12.34.
4.1.28. Trp-Trp-Val-OBzl (4f)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.74 mmol) of Boc-Trp-Trp-Val-OBzl 440 mg (97%)
of the title compound was obtained as colorless powder. Mp
20
200e202 ꢂC; ESI/MS (m/e) 580 [M þ H]þ; [
a]
¼ 13.43 (c 1.4,
4.1.33. Trp-Trp-Met-OBzl (4k)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.70 mmol) of Boc-Trp-Trp-Met-OBzl 448 mg (98%)
D
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.90 (m, 2H), 8.72
(d, J ¼ 7.3 Hz,1H), 8.58 (d, J ¼ 7.9 Hz,1H), 8.63 (d, J ¼ 7.9 Hz,1H), 7.49
(d, J ¼ 7.9 Hz, 1H), 7.17 (m, 13H), 5.03 (m, 2H), 4.71 (dd, J ¼ 13.5 Hz,
J ¼ 5.4 Hz,1H), 4.62 (dd, J ¼ 14.3 Hz, J ¼ 7.1 Hz,1H), 3.61 (m,1H), 3.17
(m, 4H), 2.07 (m, 1H), 1.98 (s, 2H), 0.90 (m, 6H); 13C NMR (125 MHz,
of the title compound was obtained asþ colorless powder. Mp
20
215e217 ꢂC; ESI/MS (m/e) 612 [M þ H] ; [
a]
¼ 7.77 (c 1.4,
D
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.96 (m, 2H), 8.79
DMSO-d6)
d
/ppm ¼ 172.9, 142.1, 136.7, 130.0, 127.7, 127.5, 127.2,
(d, J ¼ 7.3 Hz,1H), 8.71 (d, J ¼ 7.9 Hz,1H), 7.72 (d, J ¼ 7.8 Hz,1H), 7.67
(d, J ¼ 7.8 Hz, 1H), 7.19 (m, 13H), 5.03 (m, 2H), 4.71 (dd, J ¼ 14.2 Hz,
J ¼ 7.3 Hz, 1H), 4.63 (dd, J ¼ 14.2 Hz, J ¼ 7.3 Hz, 1H), 3.19 (m, 5H),
2.03 (m, 3 H), 1.98 (s, 2 H), 1.62 (m, 4 H); 13C NMR (125 MHz, DMSO-
122.9, 122.2, 120.1, 119.2, 119.0, 111.3, 111.1, 110.9, 68.5, 59.7, 54.8,
33.6, 31.2, 30.7,17.4. Anal. Calcd for C34H37N5O4: C, 70.45; H, 6.34; N,
12.08. Found: C, 70.26; H, 6.20; N, 11.89.
d6)
d
/ppm ¼ 172.9, 142.0, 136.9, 130.2, 127.7, 127.5, 127.2, 122.9,
4.1.29. Trp-Trp-Ser-OBzl (4g)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.75 mmol) of Boc-Trp-Trp-Ser-OBzl 440 mg (97%)
122.2,120.1,119.2,119.0,111.3,111.1,110.8, 68.6, 54.9, 53.2, 34.5, 31.4,
30.6, 29.5, 17.8. Anal. Calcd for C34H37N5O4S: C, 66.75; H, 6.10; N,
11.45. Found: C, 66.94; H, 6.25; N, 11.23.
of the title compound was obtained as colorless powder. Mp
218e220 ꢂC; ESI/MS (m/e) 568 [M þ H]þ; [
a
]
¼ 25.37 (c 1.3,
4.1.34. Trp-Trp-Glu(OBzl)-OBzl (4l)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.63 mmol) of Boc-Trp-Trp-Glu(OBzl)-OBzl 455 mg
20
D
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.93 (m, 2H), 8.72
(m, 2H), 7.28 (m, 15H), 5.09 (m, 2H), 4.61 (m, 2H), 4.08 (m, 2H), 3.46
(m, 1H), 3.13 (m, 4H), 2.06 (s, 2H), 1.98 (s, 1H); 13C NMR (125 MHz,
(99%) of the title compound was obtainedþas colorless powder. Mp
20
DMSO-d6)
d
/ppm ¼ 172.7, 142.1, 136.8, 130.1, 127.6, 127.5, 127.1,
214e216 ꢂC; ESI/MS (m/e) 700 [M þ H] ; [
a
]
¼ 20.17 (c 1.1,
D
122.9, 122.2, 120.1, 119.2, 119.0, 111.4, 111.1, 110.9, 68.6, 64.1, 56.8,
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.93 (m, 2H), 8.77

3418
X. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3410e3419
(d, J ¼ 7.2 Hz,1H), 8.73 (d, J ¼ 7.7 Hz,1H), 7.64 (d, J ¼ 7.8 Hz,1H), 7.50
(d, J ¼ 7.8 Hz, 1H), 7.16 (m, 18H), 5.03 (m, 4H), 4.68 (m, 2 H), 3.82 (m,
1H), 3.07 (m, 4H), 2.56 (m, 2H), 2.06 (m, 2H), 1.98 (s, 2H); 13C NMR
4.1.39. Trp-Trp-Gln-OBzl (4q)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.71 mmol) of Boc-Trp-Trp-Gln-OBzl 424 mg (93%)
(125 MHz, DMSO-d6)
d
/ppm ¼ 173.3, 172.7, 142.5, 136.8, 130.1, 129.0,
of the title compound was obtained as colorless powder. Mp
20
127.7, 127.5, 127.2, 122.9, 122.2, 120.1, 119.2, 119.0, 111.3, 111.1, 110.9,
68.6, 54.8, 53.6, 31.3, 30.6, 29.2, 27.8. Anal. Calcd for C41H41N5O6: C,
70.37; H, 5.91; N, 10.01. Found: C, 70.16; H, 5.75; N, 10.24.
188e190 ꢂC; ESI/MS (m/e) 609 [M þ H]þ; [
a
]
¼ 13.33 (c 1.4,
D
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.88 (m, 2H), 8.75
(d, J ¼ 7.2 Hz, 1H), 8.70 (d, J ¼ 7.8 Hz,1H), 7.56 (m, 2H), 7.16 (m, 13H),
6.17 (s, 2H), 5.03 (m, 2H), 4.68 (m, 2H), 3.89 (m, 1H), 3.16 (m, 4H),
2.26 (m, 2H), 2.05 (s, 2H), 1.98 (m, 2H); 13C NMR (125 MHz, DMSO-
4.1.35. Trp-Trp-Asp(OBzl)-OBzl (4m)
Using the same procedure as described for Trp-Trp-Ala-OBzl from
500 mg (0.64 mmol) of Boc-Trp-Trp-Asp(OBzl)-OBzl 450 mg (98%) of
d6)
d
/ppm ¼ 174.2,172.9,142.4, 136.8, 130.1, 127.7, 127.5,127.2, 122.9,
122.2, 120.1, 119.2, 119.0, 111.3, 111.1, 110.9, 68.7, 54.6, 53.7, 32.4, 31.3,
30.5, 30.2. Anal. Calcd for C34H36N6O5: C, 67.09; H, 5.96; N, 13.81.
Found: C, 66.88; H, 5.81; N, 13.60.
the title compound was obtained as colorless powder. Mp
20
210e212 ꢂC;ESI/MS(m/e)686[MþH]þ;[
a]
D
¼10.03 (c1.2, CH3OH);
1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.98 (m, 2H), 8.77 (d,
J ¼ 7.9 Hz,1H), 7.72 (d, J ¼ 7.3 Hz,1H), 7.30 (m, 20H), 5.08 (m, 4H), 4.65
4.1.40. Trp-Trp-Asn-OBzl (4r)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.72 mmol) of Boc-Trp-Trp-Asn-OBzl 431 mg (95%)
(m, 2H), 3.36 (m,1H), 3.17 (m, 4H), 2.92 (m, 2H),1.99 (s, 2H); 13C NMR
(125 MHz, DMSO-d6)
d
/ppm ¼ 173.4, 172.9, 142.3, 141.1, 136.8, 130.2,
129.1, 127.8, 127.6, 127.3, 122.9, 122.2, 120.1, 119.2, 119.0, 111.3, 111.1,
110.9, 68.7, 54.5, 49.3, 41.0, 31.3, 30.2. Anal. Calcd for C40H39N5O6: C,
70.06; H, 5.73; N, 10.21. Found: C, 70.25; H, 5.90; N, 10.43.
of the title compound was obtained asþcolorless powder. Mp
20
199e201 ꢂC; ESI/MS (m/e) 595 [M þ H] ; [
a
]
¼ 8.13 (c 1.1,
D
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.96 (m, 2H), 8.74
(m, 2H), 7.59 (m, 2H), 7.21 (m, 13H), 6.18 (s, 2H), 5.05 (m, 2H), 4.62
4.1.36. Trp-Trp-Cys(4-MeOBzl)-OBzl (4n)
(m, 2H), 2.98 (m, 7H), 1.96 (s, 2H); 13C NMR (125 MHz, DMSO-d6)
d/
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.62 mmol) of Boc-Trp-Trp-Cys(4-OMeBzl)-OBzl
441 mg (96%) of the title compound was obtained as colorless
ppm ¼ 174.7, 172.6, 142.3, 136.7, 130.1, 127.7, 127.5, 127.2, 122.9,
122.2,120.1,119.2,119.0,111.3,111.1,110.9, 68.9, 54.5, 49.3, 40.3, 31.0,
30.5. Anal. Calcd for C33H34N6O5: C, 66.65; H, 5.76; N, 14.13. Found:
C, 66.44; H, 5.61; N, 14.35.
20
powder. Mp 219e221 ꢂC; ESI/MS (m/e) 704 [M þ H]þ; [
a
]
D
¼ 14.43
(c 1.5, CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.99 (m,
2H), 8.93 (d, J ¼ 7.9 Hz, 1H), 8.75 (d, J ¼ 7.9 Hz, 1H), 7.63 (d,
J ¼ 7.9 Hz, 1 H), 7.44 (d, J ¼ 7.9 Hz, 1H), 7.10 (m, 17H), 4.98 (m, 2H),
4.72 (dd, J ¼ 10.5 Hz, J ¼ 7.5 Hz, 1H), 4.60 (dd, J ¼ 10.5 Hz, J ¼ 7.5 Hz,
1H), 3.71 (m, 5H), 3.43 (dd, J ¼ 10.5 Hz, J ¼ 7.5 Hz,1 H), 3.00 (m, 6 H),
4.1.41. Trp-Trp-His-OBzl (4s)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.70 mmol) of Boc-Trp-Trp-His-OBzl 320 mg (70%)
of the title compound was obtained aþs colorless powder. Mp
20
1.98 (s, 2H); 13C NMR (125 MHz, DMSO-d6)
d
/ppm ¼ 172.7, 159.2,
211e213 ꢂC; ESI/MS (m/e) 618 [M þ H] ; [
a]
¼ 16.43 (c 1.5,
D
142.2, 136.7, 130.1, 129.9, 129.3, 127.7, 127.5, 127.2, 122.9, 122.2, 120.1,
119.2, 119.0, 114.0, 111.3, 111.1, 110.9, 68.6, 56.2, 54.7, 54.4, 38.4, 36.7,
31.3, 30.7. Anal. Calcd for C40H41N5O5S: C, 68.26; H, 5.87; N, 9.95.
Found: C, 68.04; H, 5.71; N, 10.17.
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 11.03 (s, 1H), 10.98
(s, 1H), 9.02 (s, 1H), 8.90 (m, 2H), 8.43 (s, 3H), 7.66 (d, J ¼ 7.9 Hz, 1H),
7.52 (d, J ¼ 7.9 Hz,1H), 7.29 (m,13H), 5.05 (m, 2H), 4.66 (m, 2H), 3.20
(m, 6H), 2.98 (m, 1H), 2.02 (s, 2 H); 13C NMR (125 MHz, DMSO-d6)
d
/ppm ¼ 172.6, 142.0, 136.5, 135.4, 133.6, 130.1, 127.7, 127.5, 127.2,
4.1.37. Trp-Trp-Arg(NGNO2)-OBzl (4o)
122.9, 122.2, 120.1, 119.8, 119.2, 119.0, 111.3, 111.1, 110.8, 68.6, 54.5,
53.3, 33.0, 31.2, 30.9. Anal. Calcd for C35H35N7O4: C, 68.06; H, 5.71;
N, 15.87. Found: C, 67.85; H, 5.55; N, 15.63.
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.64 mmol) of Boc-Trp-Trp-Arg(NGNO2)-OBzl
445 mg (97%) of the title compound was obtained as colorless
20
powder. Mp 188e190 ꢂC; ESI/MS (m/e) 682 [M þ H]þ; [
a
]
D
¼ 18.00
4.1.42. Trp-Trp-Trp-OBzl (4t)
(c 1.5, CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 11.00 (m,
Using the same procedure as described for Trp-Trp-Ala-OBzl from
500 mg (0.65 mmol) of Boc-Trp-Trp-Trp-OBzl 416 mg (91%) of the title
2H), 8.53 (d, J ¼ 7.5 Hz, 1H), 8.39 (d, J ¼ 7.4 Hz, 1H), 7.34 (m, 15H),
5.10 (m, 2H), 4.19 (m, 1H), 4.04 (m, 2H), 3.14 (m, 4H), 1.73 (m, 2H),
2.02 (s, 2H), 1.95 (m, 3H), 1.58 (m, 4H); 13C NMR (125 MHz, DMSO-
compound wasobtained ascolorlesspowder. Mp212e214 ꢂC;ESI/MS
20
(m/e)667 [Mþ H]þ;[
a]
D
¼ ꢁ6.73 (c1.2, CH3OH);1H NMR(500 MHz,
d6)
d
/ppm ¼ 172.6, 163.1, 142.2, 136.7, 130.1, 127.7, 127.5, 127.2, 122.9,
DMSO-d6)
d
/ppm ¼ 11.10 (m, 3H), 8.03 (m, 2H), 7.39 (m, 20H), 4.57 (m,
122.2, 120.1, 119.2, 119.0, 111.3, 111.1, 110.9, 68.5, 54.7, 53.7, 37.5, 31.8,
31.3, 30.5, 24.7.Anal. Calcd for C35H39N9O6: C, 61.66; H, 5.77; N,
18.49. Found: C, 61.84; H, 5.93; N, 18.26.
4H), 3.59 (s,1H), 3.43(m, 6H), 2.05(s, 2H); 13C NMR (125 MHz, DMSO-
d6)
d
/ppm ¼ 172.8, 142.2, 136.7, 136.3, 130.1, 127.7, 127.5, 127.2, 122.9,
122.2, 120.1, 119.2, 119.0, 111.3, 111.1, 110.9, 68.5, 55.0, 54.7, 34.1, 31.3,
30.7. Anal. Calcd for C40H38N6O4: C, 72.05; H, 5.74; N,12.60. Found: C,
71.86; H, 5.60; N, 12.82.
4.1.38. Trp-Trp-Lys(Nu-Z)-OBzl (4p)
Using the same procedure as described for Trp-Trp-Ala-OBzl
from 500 mg (0.59 mmol) of Boc-Trp-Trp-Lys(Nu-Z)-OBzl 459 mg
4.2. Bioassay
(99%) of the title compound was obtainedþas colorless powder. Mp
20
193e195 ꢂC; ESI/MS (m/e) 743 [M þ H] ; [
a]
¼ 14.77 (c 1.2,
4.2.1. In vitro anti-proliferation assay of 4aet
D
CH3OH); 1H NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 10.99 (m, 2H), 8.97
Following a slightly modified procedure [13] the in vitro anti-
proliferation assays were carried out by use of 96 microtiter plate
cultures and MTT staining. The HepG2, S180, H22, K562 and B16
cells (final concentration in the growth medium was 1 ꢃ 104/ml)
were grown in RPMI-1640 medium or DMEM medium (containing
(d, J ¼ 7.9 Hz,1H), 8.66 (d, J ¼ 7.3 Hz,1H), 8.26 (d, J ¼ 7.1 Hz,1H), 7.78
(d, J ¼ 7.9 Hz, 1H), 7.64 (d, J ¼ 7.9 Hz, 1H), 7.17 (m, 18 H), 5.01 (m,
4 H), 4.72 (m, 1 H), 4.33 (m, 1 H), 3.36 (m, 1H), 3.04 (m, 6H), 1.99 (s,
2H), 1.50 (m, 6H); 13C NMR (125 MHz, DMSO-d6)
d
/ppm ¼ 172.9,
156.1, 142.2, 141.1, 136.7, 130.1, 129.3, 127.7, 127.5, 127.2, 122.9, 122.2,
120.1, 119.2, 119.0, 111.3, 111.1, 110.9, 68.6, 65.5, 54.8, 54.1, 42.2, 34.6,
31.3, 30.5, 29.6. Anal. Calcd for C43H46N6O6: C, 69.52; H, 6.24; N,
11.31. Found: C, 69.30; H, 6.10; N, 11.54.
10%, v/v FCS), penicillin (100
without (for control) or with 4aet (final concentrations ranging
from 1
M). The cultures were propagated at 37 ꢂC in a humidified
atmosphere containing 5% CO2 for 24 h, after the first renew of the
mg/ml) and streptomycin (100 mg/ml)
m

X. Zhang et al. / European Journal of Medicinal Chemistry 46 (2011) 3410e3419
3419
growth medium without (for control) or with 4aet (for test sample)
were propagated for another 48 h, after the second renew of the
growth medium with 25 l of MTT solution (5 mg/ml) were prop-
agated for another 4 h. The growth medium was removed and the
residue was dried in the air. The dried residues were dissolved in
4.2.4. In vivo acute toxicity assay
Health male ICR mice of 24-week old were given a single i.p
injection of 500 mg/kg of the anti-tumoral active 4aec,eel,nes in
0.2 ml of NS. The mice were monitored for 7 days to observe the
neurotoxic behavior, such as tremor, twitch, jumping, tetanus, and
supination, as well as the death. On the 7th day the survival mice
were sacrificed by diethyl ether anesthesia and dissected to
immediately obtain the organs for examination.
m
100 ml of DMSO and the absorption values of light of the formed
purple solutions were recorded on Bio-rad 570 nm microplate (Bio-
rad, USA). The inhibited rates were calculated according to I% ¼ %
(C ꢁ T/C).
Acknowledgments
4.2.2. In vivo anti-tumor assay of 4aet
The assessments described here were performed based on
a protocol reviewed and approved by the ethics committee of
Capital Medical University. The committee assures the welfare of
the male ICR mice was maintained in accordance to the require-
ments of the animal welfare act and according to the guide for care
and use of laboratory animals. The mice, purchased from Peking
University Health Science Center, were 10e12 weeks old at the
beginning of the experiments. S180 cells for the initiation of
subcutaneous tumors were obtained from the ascitic form of the
tumors in mice and serially transplanted once per week. Subcuta-
neous tumors were implanted by injecting 0.2 ml of NS containing
1 ꢃ 107 viable tumor cells under the skin on the right oxter of the
mice. Twenty-four hours after the implantation the tumor-bearing
mice were randomized into 22 experimental groups (12 per group).
All the mice were given a daily i.p. injection of cytarabine and
This work was completed in the Beijing area major laboratory of
peptide and small molecular drugs, supported by PHR (IHLB, KM
200910025009, KM200910025010), the National Natural Scientific
Foundation of China (81072522), and Special Project
(2011ZX09302-007-01) of China.
References
[1] G. Momekov, A. Bakalova, M. Karavanova, Curr. Med. Chem. 12 (2005)
2177e2191.
[2] A. Bolognese, G. Correale, M. Manfra, A. Lavecchia, O. Mazzoni, E. Novellino,
P. La Colla, G. Sanna, R. Loddo, J. Med. Chem. 47 (2004) 849e858.
[3] M.A. Lynch, O. Duval, A. Sukhanova, J. Devy, S.P. Mackay, R.D. Waigh, I. Nabiev,
Bioorg. Med. Chem. Lett. 11 (2001) 2643e2646.
[4] R. Cao, W. Peng, H. Chen, Y. Ma, X. Liu, X. Hou, H. Guan, A. Xu, Biochem.
Biophys. Res. Commun. 338 (2005) 1557e1563.
[5] M. Laronze, M. Boisbrun, S. Leonce, B. Pfeiffer, P. Renard, O. Lozach, L. Meijer,
A. Lansiaux, C. Bailly, J. Sapi, J.Y. Laronze, Bioorg. Med. Chem. 13 (2005)
2263e2283.
[6] A.M. Sobhani, S.A. Ebrahimi, M.J. Mahmoudian, J. Pharm. Pharm. Sci. 5 (2002)
19e23.
[7] M. Zhao, L. Bi, W. Wang, C. Wang, M. Baudy-Floc, J. Ju, S.Q. Peng, Bioorg. Med.
Chem. 14 (2006) 6998e7010.
[8] J.H. Wu, M. Zhao, K.D. Qian, K.H. Lee, S.M. Natschke, S.Q. Peng, Eur. J. Med.
Chem. 44 (2009) 4153e4161.
[9] Z. Cruz-Monserra, H.C. Vervoot, R. Bai, D.J. Newman, S.B. Howell, G. Los,
J.T. Mullaney, M.D. Williams, G.R. Pettit, W. Fenical, E. Hamel, Mol. Pharmacol.
63 (2003) 1273e1280.
doxorubicin (positive control, 2.0
mmol/kg/day in 0.2 ml of NS), or
NS (negative control, 0.2 ml), or 4aet (8.9
mmol/kg/day in 0.2 ml of
NS) for seven consecutive days. Twenty-four hours after the last i.p.
injection, all mice were sacrificed by diethyl ether anesthesia, the
tumors of the treated (Tw) and control (Cw) groups were separated
and weighed to calculate the percentage of the tumor growth
inhibition by using the following equation:
Inhibition ð%Þ ¼ ½1 ꢁ ðTw=CwÞꢄ ꢃ 100
[10] J.H. Wu, C.Y. Li, M. Zhao, W.J. Wang, Y.J. Wang, S.Q. Peng, Bioorg. Med. Chem.
18 (2010) 6220e6229.
[11] A.M. Sobhani, S.R. Amini, J.D.A. Tyndall, E. Azizi, M. Daneshtalab, A. Khalaj,
J. Mol. Graph. Model. 25 (2006) 459e469.
[12] A. Marco, R.C. Loza-Mejía, L.R. Alfonso, J. Mol. Graph. Model. 27 (2009)
900e907.
[13] C.M.A. Alves, S. Naik, J.P.G. Coutinho, Tetrahedron 65 (2009) 10441e10452.
[14] C.M. Venkatachalam, X. Jiang, T. Oldfield, M. Waldman, J. Mol. Graph. Model.
21 (2003) 289e307.
[15] R.P. Queiroz, M.S.E. Castanheir, C.T.T. Lopes, K.Y. Cruz, G. Kirsch, J. Photochem.
Photobiol. A 190 (2007) 45e52.
[16] U. Varadarajan, C. Alfonso, U.N. Balachandran, Polyhedron 26 (2007)
3008e3016.
[17] N. Li, Y. Ma, C. Yang, L.P. Guo, X.R. Yang, Biophys. Chem. 116 (2005) 199e205.
[18] P.U. Maheswari, M. Palaniandavar, J. Inorg. Biochem. 98 (2004) 219e230.
[19] T.S. Pitchumony, H.S. Evans, M. Palaniandavar, J. Inorg. Biochem. 99 (2005)
2110e2118.
[20] F. Arjmand, M. Aziz, Eur. J. Med. Chem. 44 (2009) 834e844.
[21] S. Shi, T. Xie, T.M. Yao, C.R. Wang, X.T. Geng, D.J. Yang, L.J. Han, L.N. Ji, Poly-
hedron 28 (2009) 1355e1372.
[22] L.W. Hsin, H.P. Wang, P.H. Kao, O. Lee, W.R. Chen, H.W. Chen, J.H. Guh,
Y.L. Chan, C.P. His, M.S. Yang, T.K. Li, C.H. Lee, Bioorg. Med. Chem. 16 (2008)
1006e1014.
4.2.3. In vivo dose-dependent assay of 4f
Male ICR mice, purchased from Peking University Health Science
Center, were 10e12 week old at the beginning of the experiments.
The tumor used was S180 that forms solid tumors, when injected
subcutaneously. S180 cells for initiation of the subcutaneous
tumors were obtained from the ascitic form of the tumors in the
mice, which were serially transplanted once per week. Subcuta-
neous tumors were implanted by injecting 1 ꢃ 107 viable tumor
cells in 0.2 ml of NS under the skin of the right oxter of the mice.
Twenty-four hours after the implantation, the mice were
randomized into 6 experimental groups (10 per group), and were
given a daily i.p. injection of cytarabine (positive control, 8.9, 0.89
or 0.089
mmol/kg/day in 0.2 ml of NS), or NS (negative control,
0.2 ml), or 4f (8.9, 0.89 or 0.089
mmol/kg/day in 0.2 ml of NS) for
seven consecutive days. Twenty-four hours after the last i.p.
injection, all mice were sacrificed by diethyl ether anesthesia, the
tumors of the treated (Tw) and control (Cw) groups were separated
and weighed to calculate the percentage of the tumor growth
inhibition accordingly.
[23] A.R. Shaikh, M. Ismael, C.A.D. Carpio, H. Tsuboi, M. Koyama, A. Endou, M. Kubo,
E. Broclawikc, A. Miyamoto, Bioorg. Med. Chem. Lett. 16 (2006) 5917e5925.
[24] M.R. Doddareddy, Y.S. Cho, H.Y. Koh, A.N. Pae, Bioorg. Med. Chem. 12 (2004)
3977e3985.