Novel N-(3-carboxyl-9-benzyl-β-carboline-1-yl)ethylamino acids: Synthesis, anti-tumor evaluation, intercalating determination, 3D QSAR analysis and docking investigation

Article abstract of DOI:10.1016/j.ejmech.2009.05.006

Sixteen novel N-(3-carboxyl-9-benzyl-β-carboline-1-yl)ethylamino acids (6a-p) were synthesized as intercalating lead compounds. In the in vitro cytotoxic assay their IC₅₀ values against five human carcinoma cell lines ranged from 10.95 μM to about 400 μM. On S180 mouse model eight of them exhibited anti-tumor action, four of them showed the same anti-tumor potency as that of cytarabine. The preliminary toxicity evaluation revealed that the LD₅₀ values of 6a-p should be more than 500 mg/kg. With CT DNA as model system an intercalating mechanism was explored. Using 3D QSAR analysis the relationship of the in vivo anti-tumor activity and the structure was quantitatively described. By docking 6a-p onto d(CGATCG)₂ oligonucleotides the intercalation was demonstrated.

Full text of DOI:10.1016/j.ejmech.2009.05.006

European Journal of Medicinal Chemistry 44 (2009) 4153–4161
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel N-(3-carboxyl-9-benzyl-b-carboline-1-yl)ethylamino acids: Synthesis,
anti-tumor evaluation, intercalating determination, 3D QSAR analysis
and docking investigation
,
b
b,
***
Jianhui Wu a, Ming Zhao a , Keduo Qian , Kuo-Hsiung Lee
,
**
Susan Morris-Natschke b, Shiqi Peng a
,
*
a College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
b Natural Products Research Laboratories, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7360, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Sixteen novel N-(3-carboxyl-9-benzyl-b-carboline-1-yl)ethylamino acids (6a–p) were synthesized as
Received 4 December 2008
Received in revised form
1 May 2009
Accepted 7 May 2009
Available online 21 May 2009
intercalating lead compounds. In the in vitro cytotoxic assay their IC50 values against five human
carcinoma cell lines ranged from 10.95 M to about 400 M. On S180 mouse model eight of them
m
m
exhibited anti-tumor action, four of them showed the same anti-tumor potency as that of cytarabine. The
preliminary toxicity evaluation revealed that the LD50 values of 6a–p should be more than 500 mg/kg.
With CT DNA as model system an intercalating mechanism was explored. Using 3D QSAR analysis the
relationship of the in vivo anti-tumor activity and the structure was quantitatively described. By docking
6a–p onto d(CGATCG)2 oligonucleotides the intercalation was demonstrated.
Keywords:
N-(3-Carboxyl-9-benzyl-
yl)ethylamino acid
Anti-tumor activity
Intercalation
b-carboline-1-
Ó 2009 Elsevier Masson SAS. All rights reserved.
3D QSAR
Docking
1. Introduction
The synthesis and anticancer activities of a numerous compounds
that intercalate into DNA have been reported recently [7–13]. Some
In medicinal chemistry, discovering new compounds with
potent anticancer activity is one important goal. Among current
anticancer chemotherapeutic agents DNA-recognizing molecules,
including groove binders, alkylating compounds and intercalating
agents, are particularly interesting. DNA intercalating agents are
b
-carboline alkaloids such as harmine and its derivatives, are highly
cytotoxic against human tumor cell lines [14–19]. In studies with
carboline, DNA intercalation and cytotoxicity showed a correlation
[20–23]. Thus, -carbolines act through intercalation to inhibit DNA
topoisomerases I and II and cause DNA damage. As part of our
ongoing efforts, we recently explored a series of -carboline-3-car-
b-
b
important in clinical oncology, and
a
few representative
b
compounds (anthracyclines, acridines, and anthraquinones) are
routinely used [1]. Structurally, DNA intercalating agents have
a planar polycyclic aromatic pharmacophore capable of stacking
between DNA pairs at the intercalation sites [2,3]. Intercalation
results in conformational changes of the double helix, and then
alters the processes of DNA replication, transcription and repair [4].
Thus the discovery of new DNA intercalating agents is considered as
a promising approach toward anticancer drugs [5,6].
bonylamino acid benzyl ester conjugates as potent anticancer
derivatives of natural products. We demonstrated that the in vitro
cytotoxicity of the conjugates depended on all of their building
blocks including
b-carboline-3-carboxylic acid, amino acid and
benzyl moieties [24]. On the other hand, the amino acid moieties of
the conjugates are attractive because they not only possess struc-
turally diverse side chains, but also are capable of improving phar-
macokinetics of the conjugates. With this in mind, sixteen novel
N-(3-carboxyl-9-benzyl-b-carboline-1-yl)ethylamino acids 6a–p
were prepared in the present paper. These compounds were exam-
ined for their ability to inhibit the proliferation of human carcinoma
cell lung carcinoma (H1299), liver carcinoma (HepG2), cervical
carcinoma (Hela), immature granulocyte leukemia (HL-60) and
human sarcoma (MES-SA) cells in vitro. Their in vivo anti-tumor
* Corresponding author. Tel./fax: þ86 10 8391 1528.
** Corresponding author. Tel./fax: þ86 10 8280 2482.
*** Corresponding author. Tel.: þ1 919 962 0066; fax: þ1 919 966 3893.
E-mail addresses: khlee@unc.edu (K.-H. Lee), sqpeng@bjmu.edu.cn (S. Peng).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.05.006

4154
J. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4153–4161
activities were assayed by using an S180 tumor-bearing mouse
model (S180 mice). In order to examine their intercalating mecha-
nism, 6bwas selected as a representative compound and CT DNAwas
selected as a DNA model to construct a simulation system for spectral
analysis of UV, CD and fluorescence. To get insight into the correla-
tion of the in vivo anti-tumor activity with the structure a corre-
sponding 3D QSAR and docking were performed [25–29].
moderate yields of the individual reactions indicate that the
present synthetic route should be suitable for preparing these novel
compounds and their analogs.
2.2. Anti-tumor activities of 6a–p
In the evaluation of the in vitro anti-tumor activity, anti-
proliferation assays with serial concentrations of 6a–p in NS against
H1299, HepG2, Hela, HL-60 and MES-SA cells were performed. In
the evaluation of the in vivo anti-tumor activities of 6a–p, their
effects on the tumor weights of S180 mice were determined. In the
examination of the dose-dependent action of 6a–p the most potent
compound (6b) was selected as the representative compound to
observe the dose-dependent action.
2. Results and discussion
2.1. Synthesis of N-(3-carboxyl-9-benzyl-
ethylamino acids 6a–p
b-carboline-1-yl)
Compounds 6a–p were synthesized according to the seven-step
route depicted in Scheme 1. Using a commonly used procedure,
Trp was converted first to -Trp-OMe (92% yield), and then to tet-
rahydro- -carboline-3-carboxylic acid methyl ester in 89% overall
L-
L
2.2.1. IC50 of 6a–p against the proliferation of five human
carcinoma cells
b
yield as a (1S, 3S) and (1R,3S) diastereoisomeric mixture which
could be resolved for analytical purposes by chromatography over
silica gel. In the presence of KMnO4, the piperidine ring of 1 was
In the anti-proliferation assays, H1299, HepG2, Hela, HL-60 and
MES-SA cells were exposed to serial concentrations (0.1–400 mM)
of 6a–p, and standard MTT assays were performed. The IC50 values
of 6a–p against the five cell lines are summarized in Table 1. The
data indicate that 6a–p selectively inhibit these five carcinoma
types. The highest potencies were found against MES-SA cells, and
aromatized to form
b-carboline-3-carboxylic acid methyl ester (2)
in 80% yield. Benzylation at position-9 of 2 provided 9-benzyl-
b
-
carboline-3-carboxylic acid methyl ester (3, 61% yield). In the
presence of HCl, HOAc and water 3 was underwent a hydrolysis and
the acetal at position-1 was converted to an aldehyde, giving 2-(3-
with IC50 values ranging from 11.0 to 14.5
potent than the remaining compounds. Compounds 6a,b,h,i,k,n
exhibited the lowest IC50 values (ranging from 23.5 to 33.2 M)
against HL-60 cells. Compound 6o was the most potent
mM, 6a,e,l,o were more
methoxycarbonyl-9-benzyl-
b-carboline-1-yl)acetaldehyde (4) in
m
61% yield. Coupling 4 with amino acid methyl esters followed by
reduction of the coupling products provided 9-benzyl-1-carbon-
(IC50 ¼ 20.0
m
M) against H1299 cells, and 6g was the most potent
ylmethyl-
b
-carboline-3-carbonylamino acid methyl esters (5a–o),
(IC50 ¼ 26.5
mM) against Hela cells. However, HepG2 cells were less
in yields ranging from 35% to 88%. However, coupling of 4 with
sensitive than the other four cell lines to these compounds; no IC50
glutamic dimethyl ester and consequent reduction failed to provide
values were less than 70 mM against this cell line.
the desired 9-benzyl-1-carbonylmethyl-
ylglutamic acid trimethyl ester (5p0), Instead, only N-(3-carboxyl-9-
benzyl- -carboline-1-yl)ethyl-5-oxotetrahydropyrole-2-carboxytic
acid, the product of coupling and side chain cyclization (5p), was
obtained in 67% yield. The saponification of 5a–p provided 9-
b-carboline-3-carbon-
2.2.2. In vivo activities of 6a–p on S180 mouse model
In the in vivo anti-tumor assays, the tumor weights of S180 mice
receiving 6a–p were recorded. As shown in Table 2, when the mice
b
were given a daily i.p injection of 89 mmol/kg of 6a–p in 0.2 ml of NS
benzyl-1-carbonylmethyl-
p), in yields ranging from 52% to 72%. The mild conditions and
b
-carboline-3-carbonylamino acids (6a–
for seven consecutive days, their tumor weights ranged from 2.12 g to
0.74 g (NS receiving mice, 1.65 g). Eight compounds significantly
CO2Me
CO2Me
CO2Me
i
ii
iii
L-Trp
iv
H
N
NH
OCH3
OCH3
N
N
N
N
H
H
OCH3
OCH3
OCH3
OCH3
(1S, 3S)-1: 60
(1R, 3S)-1: 29
3
C6H5
2
v
CO2Me
CO2Me
CO2H
N
vi
vii
N
N
N
N
N
N
R
R
NH
NH
6a-o
H
H
C6H5
C6H5
C6H5
5a-o
L-Glu(OCH3)2
CO2Me
4
CO2Me
CO2H
O
NaBH3CN / NaOH
CO2Me
CO2H
O
vii
O
N
N
N
N
N
CH2CH2CO2Me
N
N
NH
C6H5
C6H5
H
C6H5
6p
5p
CO2Me
CO2Me
5p'
CO2Me
Scheme 1. Synthesis of N-(3-carboxyl-9-benzyl-b-carboline-1-yl)ethylamino acids 6a–o. Reaction conditions: (i) SOCl2, MeOH; (ii) HCl, 1,1,3,3-tetramethoxypropane, MeOH; (iii)
KMnO4, DMF; (iv) NaH, BrCH2C6H5, DMF/THF; (v) HCl, HOAc, H2O; (vi) NaOH, NaBH3CN and L-Phe-OMe or L-Ala-OMe or Gly-OMe or L-Arg or L-Asp-OMe or; L-Leu-OMe or L-Met-
OMe or L-Ser-OMe or L-Tyr-OMe or L-Thr-OMe or L-Ile-OMe or L-Trp-OMe or L-Val-OMe or L-Pro-OMe or L-His-OMe; (vii) NaOH/MeOH. 5a and 6a R ¼ CH2C6H5, 5b and 6b R ¼ CH3, 5c
and 6c R ¼ H, 5d and 6d R ¼ CH2(CH2)2NH(NH2)]NH, 5e R ¼ CH2CO2CH3, 6e R ¼ CH2CO2H, 5f and 6f R ¼ CH2CH(CH3)2, 5g and 6g R ¼ CH2CH2SCH3, 5h and 6h R ¼ CH2OH, 5i and 6i
R ¼ CH2C6H4–OH, 5j and 6j R ¼ CH(OH)CH3, 5k and 6k R ¼ CH(CH3)CH2CH3, 5l and 6l R ¼ indole-3-ylmethylene, 5m and 6m R ¼ CH(CH3)2, 5n and 6n R ¼ NH]NCH2CH2CH2, 5o and
6o R ¼ imidazole-3-ylmethylene.

J. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4153–4161
4155
Table 1
IC50 of 6a–p against HL-60, Hela, H1299, HepG2 and MES-SA cell lines.
Table 3
Anti-tumor activity of 6b at different doses against S180 mice.
Compda HL-60
Hela
H1299
HepG2
MES-SA
Compda
Dose (
mmol/kg)
Tumor weight (g)
% Inhibition
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
31.8 ꢀ 10.6
51.7 ꢀ 8.8
59.3 ꢀ 7.5
81.8 ꢀ 8.3
75.6 ꢀ 9.4
86.4 ꢀ 6.2
114.0 ꢀ 7.0
242.0 ꢀ 6.9
>400.0
11.0 ꢀ 3.7
120.0 ꢀ 6.5
178.0 ꢀ 3.0
68.2 ꢀ 8.5
13.0 ꢀ 0.8
71.2 ꢀ 3.8
86.4 ꢀ 15.9
6b
6b
6b
Ara-C
NS
89.00
0.89
0.0089
89.00
0.74 ꢀ 0.09b
1.22 ꢀ 0.27c
1.64 ꢀ 0.38
0.74 ꢀ 0.20
1.65 ꢀ 0.49
55.2 ꢀ 6.7b
25.6 ꢀ 5.8c
1.5 ꢀ 0.3
28.1 ꢀ 7.5
>400.0
122.0 ꢀ 11.0
>400.0
40.9 ꢀ 6.7
43.2 ꢀ 8.4
45.2 ꢀ 3.6
182.0 ꢀ 6.0
23.5 ꢀ 7.6
24.0 ꢀ 1.4
50.4 ꢀ 11.2
33.2 ꢀ 2.6
102.0 ꢀ 4.0
>400.0
259.0 ꢀ 8.4
161.0 ꢀ 3.0
54.8 ꢀ 14.5
99.3 ꢀ 4.0
69.0 ꢀ 10.7 114.0 ꢀ 2.4
189.0 ꢀ 13.5
26.5 ꢀ 1.3
69.9 ꢀ 7.5
176.0 ꢀ 23.1
a
Ara-C ¼ positive control, NS ¼ vehicle, n ¼ 12.
100.0 ꢀ 10.0 108.0 ꢀ 8.0
b
c
Compared to NS and 0.89
m
mol/kg of 6b p < 0.01.
mol/kg of 6b p < 0.05.
210.0 ꢀ 13.0
209.0 ꢀ 17.0
221.0 ꢀ 24.0
200.0 ꢀ 19.0
393.0 ꢀ 20.0
290.0 ꢀ 9.0
161.0 ꢀ 8.0
83.8 ꢀ 8.7
72.5 ꢀ 4.4
86.4 ꢀ 0.6
69.0 ꢀ 9.0
57.3 ꢀ 3.3
199.0 ꢀ 7.0
75.5 ꢀ 1.7
20.0 ꢀ 1.3
174.0 ꢀ 12.0 136.0 ꢀ 3.0
Compared to NS and 0.0089
m
127.0 ꢀ 4.0
>400.0
59.3 ꢀ 4.9
218.0 ꢀ 11.0
254.0 ꢀ 14.0 181.0 ꢀ 15.0
2.3.1. Spleen index and body weight of S180 mice receiving 6a–p
Immunologic function is important to chemotherapy and spleen
index is an important parameter of the immunologic function.
Therefore, the spleen indexes of 6a–p receiving S180 mice were
measured. The data are listed in Table 4. The comparison of the
spleen index of the S180 mice receiving NS and synthetic
compounds 6a–p demonstrates that most of the compounds did
not change the immunologic function of the S180 mice. Exceptions
were 6a,i, which enhanced immunologic function.
During chemotherapy, an increase body weight is an important
parameter of health. Thus, the body weights of the mice receiving
6a–p were measured. The data are listed in Table 4 as increase body
weight from start of treatment. Compounds 6b–e,h–p showed
weight gain similar to that of the vehicle-treat group. Only three
compounds (6a,f,g) showed significantly lower increases in body
weight.
107.0 ꢀ 5.0
>400.0
11.7 ꢀ 4.0
175.0 ꢀ 2.0
55.2 ꢀ 2.3
14.5 ꢀ 1.0
45.9 ꢀ 12.0
28.4 ꢀ 8.2
107.0 ꢀ 1.2
160.0 ꢀ 63.0 136.0 ꢀ 12.0
112.0 ꢀ 19.0 >400.0
70.7 ꢀ 2.0
97.6 ꢀ 12.6 >400.0
a
IC50 value is expressed by x ꢀ SD M and n ¼ 6.
m
decreased the tumor weights of the S180 mice, compounds 6b,i,l,m
(tumor weights ranging from 0.74 g to 0.85 g) were essentially
equipotent to the positive control cytarabine (tumor weight 0.74 g).
Compounds 6d,e,h,p decreased the tumor load (tumor weights
ranging from 1.08 g to 0.94 g), but were less potent than the positive
control.
The anti-tumor action likely correlates with the identity of the
1-substituent (R group). The active compounds contained either
a small aliphatic group (methyl and isopropyl) or an acidic group
(carboxylmethyl, 5-oxotetrahydropyrole-2-carboxylic acid and
phenol) or a basic group (guanidopropyl and indole).
2.3.2. Acute toxicity of mice receiving 6a–e,i–k
Most of the reported 9-substituted-
a class of strong neurotoxic and low survival indole alkaloids, and
the discovery of comparatively non-toxic and highly survival
b-carbolines were known as
2.2.3. Dose dependent in vivo anti-tumor activities of 6b
The most potent compound (6b) was selected as the model
compound to examine the dose-dependent action of 6a–p. At doses
of 0.0089, 0.89 and 89.0 mmol/kg, it showed dose-dependent
inhibition with percentages of 1.5%, 25.6%, and 55.2%, respectively
b
-
carbolines as intercalators has been particularly interesting [29]. To
know the toxic level of 6a–p the representative compounds 6a–e,i–
k were examined for the LD50 and neurotoxicity in mice model. The
results indicate that even the dose of 6a–e,i–k was up to 500 mg/kg
the mice neither exhibited neurotoxic behavior, such as tremor,
twitch, jumping, tetanus, and supination, nor occurred death.
Necropsy findings in 6a–e,i–k receiving mice on the 7th day
revealed that the administration leaded no apparent changes in any
organs. These suggest that 6a–e,i–k are comparatively non-toxic,
and their LD50 values should be more than 500 mg/kg.
(Table 3). Therefore, 6b possesses significant anti-tumor potency at
doses above 89 mmol/kg.
2.3. Toxicity of 6a–e,i–k on mice
To evaluate the preliminary toxicity of 6a–p during the admin-
istration the spleen index of the mice was measured and used to
reflect the immunologic function, the body weights of the mice
were measured and used to reflect the health, and the acute toxicity
of some representatives including the most potent and least potent
compounds was measured and used to estimate LD50 values.
2.4. Intercalation of 6a–d toward CT DNA
Different DNA spectra have been widely used to explore its
intercalation with small molecules. In order to confirm the
Table 2
Table 4
Effects of 6a–p on tumor weights of S180 mice.
Effect of 6a–p on the spleen index and body weight increase of S180 mice.
Compda % Inhibition Tumor weight Compd % Inhibition
Tumor weight
Compda Spleen index Increased BW Compda Spleen index Increased BW
6a
6b
6c
6d
6e
6f
6g
6h
Ara-C
5.2 ꢀ 1.4
55.2 ꢀ 6.7
11.0 ꢀ 2.4
37.7 ꢀ 8.1
34.7 ꢀ 4.8
8.7 ꢀ 1.9
1.56 ꢀ 0.42
0.74 ꢀ 0.09b
1.47 ꢀ 0.32
1.03 ꢀ 0.22c
1.08 ꢀ 0.15c
1.51 ꢀ 0.32
1.63 ꢀ 0.28
1.07 ꢀ 0.21c
0.74 ꢀ 0.20
6i
6j
6k
6l
6m
6n
6o
6p
NS
48.6 ꢀ 12.3 0.85 ꢀ 0.21b
6a
6b
6c
6d
6e
6f
6g
6h
Ara-C
14.41 ꢀ 1.23b
13.32 ꢀ 1.34
13.13 ꢀ 2.18
12.65 ꢀ 1.89
12.99 ꢀ 1.29
10.87 ꢀ 2.10
11.80 ꢀ 3.85
10.49 ꢀ 0.89
10.17 ꢀ 3.32
9.74 ꢀ 2.26b
12.92 ꢀ 1.39
11.34 ꢀ 2.10
11.19 ꢀ 2.78
10.11 ꢀ 3.74
9.04 ꢀ 3.10b
9.12 ꢀ 3.29b
11.46 ꢀ 2.81
9.65 ꢀ 2.50b
6i
6j
6k
6l
6m
6n
6o
6p
NS
13.62 ꢀ 1.51b 13.62 ꢀ 1.51
ꢁ29.1 ꢀ 5.4
1.6 ꢀ 0.2
2.12 ꢀ 0.41
1.62 ꢀ 0.22
11.37 ꢀ 2.12
13.56 ꢀ 1.76
11.67 ꢀ 1.23
10.23 ꢀ 1.52
12.07 ꢀ 2.83
11.87 ꢀ 2.07
12.24 ꢀ 1.10
11.84 ꢀ 2.34
11.37 ꢀ 2.12
13.56 ꢀ 1.76
11.67 ꢀ 1.23
10.23 ꢀ 1.52
12.07 ꢀ 2.83
11.87 ꢀ 2.07
12.24 ꢀ 1.10
12.32 ꢀ 2.94
47.2 ꢀ 13.3 0.85 ꢀ 0.23b
53.8 ꢀ 19.4 0.76 ꢀ 0.28b
7.2 ꢀ 1.9
9.7 ꢀ 2.1
43.0 ꢀ 7.4
1.53 ꢀ 0.43
1.49 ꢀ 0.32
0.94 ꢀ 0.16c
1.65 ꢀ 0.49
1.5 ꢀ 0.3
35.3 ꢀ 6.9
54.8 ꢀ 14.5
a
a
Dose of Ara-C (cytarabine) and 6a–p: 89
m
mol/kg, NS ¼ vehicle, n ¼ 12, tumor
Dose of Ara-C (cytarabine) and 6a–p: 89
m
mol/kg, NS ¼ vehicle, n ¼ 12, spleen
weight is expressed by x ꢀ SD g.
index is expressed by x ꢀ SD mg=kg body weight, increased BW ¼ increased body
weight is expressed by x ꢀ SD g.
b
Compared to NS p < 0.01, to Ara-C p > 0.05.
Compared to NS p < 0.01, to Ara-C p < 0.05.
c
b
Compared to NS p < 0.05.

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J. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4153–4161
intercalation of 6a–p, calf thymus DNA (CT DNA) was selected as the
model DNA, 6b was selected as model compound of 6a–p, and
a combination of CT DNA and 6b was selected as model system for
simulating the interactions of 6a–p toward DNA. UV, CD and fluo-
rescence spectra were measured with this simulation system. The
results provided important spectral evidence for intercalation of 6b
with CT DNA, and also prove that this simulation system should be
1.2
0.8
CT DNA+6b (100μM)
CT DNA
0.4
generally useful to define intercalation of b-carbolines with DNA.
0.0
2.4.1. UV spectra defined intercalation 6b with CT DNA
-0.4
-0.8
-1.2
The UV spectra of a solution of CT DNA alone in PBS buffer (pH
7.4, final concentration, 240
final concentration, 240 M) plus the representative compound
6b (final concentration, 20 M) in PBS buffer were determined on
mM) and a solution of CT DNA (pH 7.4,
m
m
a Shimadzu 2550 spectrophotometer from 220 to 350 nm. Fig. 1
shows the two spectra obtained. Compound 6b induced a hypo-
chromic effect (47.0%) and bathochromic shift (3.2 nm) compared
with the UV spectrum of CT DNA alone. Hypochromic effects and
bathochromic shifts are considered to be evidence of intercalation
of DNA with small molecules [30]. Thus the UV changes seen in UV
experiment are direct evidence for intercalation of 6b with
CT DNA.
240
260
280
300
320
λ / nm
Fig. 2. CD spectra of CT DNA without and with 6b.
2.4.3. Fluorescence spectra defined intercalation of 6b with CT DNA
To further confirm the intercalating mechanism of 6a–p, the
effect of CT DNA on the fluorescence intensity of 6b was determined
at 300 K by the use of fluorescence spectroscopy. In the determi-
2.4.2. CD spectra defined intercalation of 6b with CT DNA
The circular dichroic (CD) spectrum of CT DNA is characterized by
positive and negative bands. The former band is due to base stacking
and the latter band is due to right-handed helicity. The two bands
change in intensity as the result of intercalation interactions of CT
DNA with small molecules [31,32]. In our CD experiments a solution
nations, a solution of 6b in PBS buffer (0.5
titrated with 10 l of solutions containing serial concentrations
(0, 8, 16, 24, 32, 40, 48, 56, 64 and 72
M, pH ¼ 7.4) of CT DNA in PBS
mM, pH ¼ 7.4) was
m
m
buffer. The corresponding changes in the fluorescence intensity of
6b were observed on a Shimadzu RF-5310PC spectrofluorometer at
a fluorescence excitation wavelength of 245 nm. CT DNA caused
a concentration-dependent decrease in the fluorescence intensities
(fluorescence quenching) of 6b. Fig. 3 illustrates the typical course
of the fluorescence quenching, the fluorescence intensity of 6b
gradually decreased as the concentration of added CT DNA gradu-
ally increased. When the concentration of added CT DNA was
of CT DNA alone in PBS buffer (pH 7.4, final concentration, 40
and a solution (pH 7.4) of CT DNA (final concentration, 40 M) plus
M) in
mM)
m
the representative compound 6b (final concentration, 100
m
PBS buffer were incubated at 37 ꢂC for 24 h, and then their CD
spectra were obtained according to the standard procedure. The
parameters of the CD spectra are given in Fig. 2. At 100 mM, 6b
induced an intensity increase in the positive band (molecular
ellipticities [
q
] for CT DNA alone ¼ 18,909 deg cm2 dmolꢁ1; [
q]
increased to 72 mM, the fluorescence intensity of 6b was lowered to
for DNA þ 100
m
M 6b ¼ 26,523 deg cm2 dmolꢁ1) and an intensity
its minimum. At this point, the fluorescence intensity had
decreased by 59.09%. Over the course of the fluorescence quench-
ing, a slight bathochromic shift was also noticed as seen in Fig. 3.
This bathochromic shift is considered to be associated with
a decrease in the energy gap between the highest and lowest
decrease in the negative band ([
q]
for CT DNA
alone ¼ ꢁ21,020 deg cm2 dmolꢁ1
;
[
q]
for CT DNA þ 100
mM
6b ¼ ꢁ9685 deg cm2 dmolꢁ1). These observations reflect intercala-
tion of 6b with CT DNA, characterized by decreasing both the base
stacking and the right-handedness of CT DNA.
700
6b+CT DNA (0 μM)
600
1.6
CT DNA
1.4
1.2
500
400
300
200
CT DNA+ 6b (20 μM)
1.0
0.8
0.6
0.4
0.2
0.0
6b+CT DNA (72 μM)
100
0
350
400
450
500
3.2 nm
260
-0.2
λ / nm
240
280
300
320
λ / nm
Fig. 3. Fluorescence spectra of 6b (concentration 0.5
with serial concentrations (0, 8, 16, 24, 32, 40, 48, 56, 64, 72
buffer.
m
M, pH ¼ 7.4, lmax ¼ 254 nm)
m
M) of CT DNA in PBS
Fig. 1. UV spectra of CT DNA without and with 6b in PBS buffer (pH ¼ 7.4).

J. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4153–4161
4157
9-benzyl-b-carboline-3-carboxylic acid that shared by 6a–p. This
subset was used for the alignment. A rigid fit of atom pairings was
performed to superimpose each structure onto the target model 9-
benzyl-
shown in Fig. 4. The alignment stereoview explores that to super-
impose onto 9-benzyl- -carboline-3-carboxylic acid the 2-group,
b-carboline-3-carboxylic acid. Stereoview of aligned 6a–p is
b
ethylamino acid, of each structure has to take individual confor-
mation. This individual conformation of the 2-group will influence
on the anti-tumor activity.
2.5.2. QSAR module of Cerius2 based MFA of 6a–p
Molecular field analysis (MFA) was performed for 6a–p using the
QSAR module of Cerius2 [35]. A five-step procedure consisted of
generating conformers, energy minimization, matching atoms and
aligning molecules, setting preferences, and regression analysis was
automatically practiced in MFA. Molecular electrostatic and steric
fields were created by use of proton and methyl groups as probes,
respectively. 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
both electrostatic and steric fields. The total grid points generated
were 672. Though the spatial and structural descriptors such as
dipole moment, polarizability, radius of gyration, number of rotat-
able bonds, molecular volume, principal moment of inertia, AlogP98,
number of hydrogen bond donors and acceptors, and molar refrac-
tivity were also considered, only the highest variance holder proton,
methyl and hydroxyl descriptors were used. Regression analysis was
carried out using the genetic partial least squares (G/PLS) method
consisting of 50,000 generations with a population size of 100. The
number of components was set to 5. Cross-validationwas performed
with the leave-one-out procedure. PLS analysis was scaled, with all
variables normalized to a variance of 1.0.
The regions where variations in the steric or electrostatic
features of 6a–p in the training set lead to increase or decrease
activities were specified. Proton descriptor with positive coefficient
indicates a region favorable for electropositive group, while nega-
tive coefficient indicates electronegative group required at this
position. Methyl descriptor with positive coefficient indicates
a region favorable for large group, while negative coefficient indi-
cates small group required at this position. Hydroxyl descriptor
with positive coefficient indicates a region favorable for hydrogen
bond forming group, while negative coefficient indicates hydrogen
bond forming group not required at this position. The MFA model
for the activity of 6a–p in terms of the most relevant descriptors
proton and methyl group is expressed by Equation (1).
Fig. 4. Alignment stereoview of 6a–p used for molecular field generation.
occupied molecular orbitals, and results from intercalation of 6b
with CT DNA [33].
2.5. 3D QSAR analysis of 6a–p
To correlate the structure of 6a-p with the tumor weights of the
mice receiving them a 3D QSAR analysis was performed by use of
Cerius2 module and following standard procedure.
2.5.1. Alignment of 6a–p
To establish valid 3D QSAR models a proper alignment procedure
of 6a–p was performed using the target model align strategy in the
align module within Cerius2. With an assumption that each struc-
ture of 6a–p binds the same site of the receptor and exhibits activity,
they were aligned in a pharmacological active orientation. To obtain
a
consistent alignment, the common 9-benzyl-b-carboline-3-
carboxylic acid moiety was selected as the template for superposing
6a–p. The method used for performing the alignment was the
maximum common subgraph (MCS) [34]. MCS looks at molecules as
points and lines, and uses the techniques out of graph theory to
identify the patterns. Then MCS finds the largest subset of atoms in
Fig. 6. Graph of tested inhibition against predicted inhibition of anti-tumor activities
Fig. 5. Alignment stereoview of 6a–p influencing on in vivo anti-tumor activity.
in vivo.

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J. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4153–4161
Fig. 7. Electrostatic environment of 6b and 6m with high in vivo anti-tumor activities, as well as 6a and 6j with low in vivo anti-tumor activities within the grid with 3D points of
Equation (1).
À
Á
Inhibition ¼ 65:9465 þ 0:263568 Hþ=389
methyl descriptor, which means that at these positions large group
will increase the in vivo anti-tumor activity, four terms of CH3/415,
CH3/394, CH3/537, CH3/278 with negative coefficient from methyl
descriptor, which means that at these positions large group will
decrease the in vivo anti-tumor activity, one term of OHꢁ/333 with
positive coefficients from hydroxyl descriptor, which means that at
this position electron-withdrawing group will increase the in vivo
anti-tumor activity, and two terms of OHꢁ/474, OHꢁ/487 with
negative coefficients from hydroxyl descriptor, which means that at
these positions electron-releasing groups will increase the in vivo
anti-tumor activity, are involved.
À
Á
À
Á
Á
ꢁ 0:296122 Hþ=377 ꢁ 0:616359 Hþ=339
À
Á
À
ꢁ 0:670101 Hþ=402 ꢁ 0:175716 Hþ=290
þ 1:15455ðCH3=331Þ þ 1:20715ðCH3=402Þ
ꢁ 1:51675ðCH3=415Þ ꢁ 0:691845ðCH3=394Þ
ꢁ 0:233533ðCH3=537Þ ꢁ 0:757249ðCH3=278Þ
À
Á
À
Á
ꢁ 1:15548 OHꢁ=474 ꢁ 0:813747 OHꢁ=487
À
Á
þ 0:252125 OHꢁ=333
ð1Þ
As examples Fig. 7 gives the electrostatic environments of
representatives 6b and 6m with high in vivo anti-tumor activities,
as well as 6a and 6j with low in vivo anti-tumor activities within
the grid with 3D points of Equation (1). Besides the same electro-
The correlation of the activities tested on the in vivo anti-tumor
model and the activities calculated using Equation (1) is explained
by Fig. 5 (r2, 0.985). The data points (n), correlation coefficient (r)
and square correlation coefficient (r2) of Equation (1) were 16,
0.992 and 0.985, respectively. The correlation of the anti-tumor
activities tested on S180 mouse model and calculated from Equa-
tion (1) is explained by Fig. 6.
static and environments as 9-benzyl-b-carboline-3-carboxylic acid
moiety 6b has small group near CH3/415 and thus resulting in
increasing the in vivo anti-tumor activity, and 6m has large group
near CH3/402, thus also resulting in increasing the in vivo anti-
tumor activity. Besides the same electrostatic and environments as
In Equation (1) one term of Hþ/389 with positive coefficient
from proton descriptor, which means that at this position electron-
releasing group will increase the in vivo anti-tumor activity, four
terms of Hþ/377, Hþ/339, Hþ/402, Hþ/290 with negative coefficient
from proton descriptor, which means that at these positions elec-
tron-releasing group will decrease the in vivo anti-tumor activity,
two terms of CH3/331, CH3/402 with positive coefficient from
9-benzyl-b-carboline-3-carboxylic acid moiety 6a has large group
near CH3/394, CH3/537 and thus resulting in decreasing the in vivo
anti-tumor activity, and 6j has both electron-releasing and elec-
tron-withdrawing groups near Hþ/339, thus totally resulting in no
change of the in vivo anti-tumor activity.
Fig. 8. (a) Stereoview of 6b,d,e,h,i,l,m,p with in vivo anti-tumor activity intercalating toward d(CGATCG)2, and (b) stereoview of 6a,c,f,g,j,k,n,o without in vivo anti-tumor activity
intercalating toward d(CGATCG)2.

J. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4153–4161
4159
Fig. 9. The docking interactions of the most potent compounds 6b,m and the least potent compounds 6a,j with d(CGATCG)2.
2.6. Docking 6a–p onto d(CGATCG)2 oligonucleotides
anti-proliferation for individual compounds. In vivo assays with
S180 mice revealed eight active anti-tumor compounds, with four
compounds being equipotent with the positive control cytarabine.
Compounds 6a–p neither decreased the immunologic function nor
lowered the body weight increase of the treated S180 mice. By
comparing the UV, CD and fluorescence spectra of CT DNA alone to
those of a combination of CT DNA plus 6b, an intercalating mech-
anism was identified. Docking 6a–p onto d(CGATCG)2 oligonucle-
otides explored that the 9-benzyl of 6a–p to be out of the cavity of
As one of the most important molecular-cellular targets of
several anticancer drugs DNA can be conveniently intercalated by
heterocycle having a planar structure [36]. To further explore the
dependence of the anti-tumor activity on the structure the auto-
mated docking studies of 6a–p were performed using the Ligand-
Fit/LigandScore in Discovery Studio (DS) Modeling 2.1, d(CGATCG)2
oligonucleotide retrieved from the Protein Data Bank (1D12) was
used as the interaction model [37]. Energetically the most favorable
conformation of the docked structure was selected on the basis of
the LigandFit score and visual inspection. Initially hydrogen atoms
were added to the protein, considering all the residues at their
neutral form. The active site of 1D12 was defined from receptor
cavity and was chosen to include not only the active site but also
significant portions of surrounding surface. The grids were 842
points and the stereoview of the compounds with higher and lower
in vivo anti-tumor activities intercalating toward d(CGATCG)2 was
described in Fig. 8a and b, respectively. Fig. 8 indicates that the 9-
benzyl significantly influences the docking of 6a–p into the cavity
of d(CGATCG)2. Only when 9-benzyl is out of the cavity the planar
d(CGATCG)2, the planar
b-carboline ring properly sandwiched
between the base pairs of d(CGATCG)2, and enough hydrogen
bonds occurred between 6a–p and the base pairs of d(CGATCG)2
were essential for in vivo anti-tumor activity.
4. Experimental
4.1. General
All chemicals were purchased from commercial suppliers and
were purified when necessary. Protected amino acids with L-
configuration were purchased from sigma chemical Co. Chroma-
tography was performed on Qingdao silica gel H. The purities 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 electrothermal
SM/XMP apparatus without correction. UV spectra were measured
on Shimadzu UV 2550. FT-IR spectra were run on an infrared
spectrometer. ESI-MS was determined by Micromass Quattro micro
TM API, Waters Co. 1H NMR (500 Hz) and 13C NMR (125 Hz) spectra
were acquired on a Bruker AC 300 spectrometer in CDCl3 or in
DMSO-d6 with TMS as internal standard, and chemical shifts are
b
-carboline ring may be comfortably sandwiched between the base
pairs of d(CGATCG)2 and thus allows to form more hydrogen bonds
between the amino acid residue and the base pairs of d(CGATCG)2.
When 9-benzyl is in the cavity the planar b-carboline ring may be
uncomfortably sandwiched between the base pairs of d(CGATCG)2
and thus allows to form less or no hydrogen bonds between the
amino acid residue and the base pairs of d(CGATCG)2.
To clarify the differences of the docking interactions of
6b,d,e,h,i,l,m,p and 6a,c,f,g,j,k,n,o with d(CGATCG)2 the stereoview
of the most potent compounds 6b,m and the least potent compounds
6a,j docking onto d(CGATCG)2 was analyzed based on Fig. 9. From the
stereoview it was found that a suitable docking consists of
expressed in ppm (d). Optical rotations were determined with
a comfortable insertion of
enough hydrogen bonds between the amino acid residue and the
DNA base pairs. For instance, by comfortably inserting -carboline
ring into the base pairs of d(CGATCG)2 eight hydrogen bonds were
simultaneously formed between the -Ala residue of 6b and the base
b-carboline ring in the DNA base pairs and
a Jasco P-1020 Polarimeter. Statistical analysis of all the biological
data was carried out by use of ANOVA test, p < 0.05 is considered
significant.
b
L
4.2. General procedure for preparing amino acid methyl esters
pairs of d(CGATCG)2. The same docking interactions were also found
At 0 ꢂC, 3.75 ml (50 mmol) of SOCl2 was added dropwise to 50 ml
of MeOH. After stirring at room temperature for 0.5 h, 42 mmol of L-
between 6m and d(CGATCG)2. While docking 6a onto d(CGATCG)2 it
results in an uncomfortable insertion of b-carboline ring in the DNA
base pairs and forming no hydrogen bonds between the amino acid
residue and the DNA base pairs. The same docking interactions were
also found between 6j and d(CGATCG)2.
amino acid was added, and then the reaction mixture was evapo-
rated under vacuum. The residue was solidified and the title
compounds were obtained as colorless powders in yields ranging
from 85% to 99%.
3. Conclusion
4.3. Methyl 1-(2,2-dimethoxyethyl)-1,2,3,4-tetrahydro-b-carboline-3-
In conclusion, novel N-(3-carboxyl-9-benzyl-
b-carboline-1-
carboxylate (1)
yl)ethylamino acids 6a–p were prepared in acceptable yields using
the seven-step route described above. In vitro cytotoxicity assays
against five human carcinoma cell lines explored the cell selective
A suspension of 5.0 g (24.510 mmol) of
50 ml of MeOH and 6.0 ml (23.6 mmol) of 1,1,3,3-tetramethoxypropane
L-tryptophan methyl ester,

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J. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4153–4161
was adjusted topH1–2withHCl(5 N)and stirredat45 ꢂCfor48 h.After
cooling, the solvent was evaporated under vacuum, the residue was
diluted with water and the aqueous solution was extracted with EtOAc
(30 ml ꢃ 3). The organic phase was separated, washed successively
with 10% sodium carbonate and saturated NaCl, dried over anhydrous
sodium sulfate, filtered and the filtrate was evaporated under vacuum.
The residue was purified by silica gel chromatography (CHCl3:MeOH,
30:1) to provide 1.57 g (29%) of (1S,3S)-1 and 3.24 g (60%) of (1R,3S)-1
as pale yellow oil. FAB-MS (m/z) 319 [M þ H]þ.
evaporated under vacuum, and the residue was purified by silica gel
chromatography to give 5a–p in 35.0–88.0% yields.
4.8. General procedure for preparing N-(9-benzyl-3-carboxyl-
carboline-1-yl)-ethylamino acid (6a–p)
b-
At 0 ꢂC, 2 ml methanolic NaOH solution (2 N) was added to
a solution of 5a–p (1.12 mmol) in 8 ml of MeOH and 8 ml of CHCl3.
The reaction mixture was stirred at 0 ꢂC for 10 h. TLC analysis
(CHCl3:MeOH:HOAc, 5:1:0.2) indicated complete disappearance of
5a–p. The resulting mixture was adjusted with HCl (2 N) to pH 2
and the solvent was evaporated under vacuum. The residue was
dissolved in 50 ml of EtOAc and the solution was washed succes-
sively with 5% sodium bicarbonate, 5% citric acid, and saturated
NaCl and then dried over anhydrous sodium sulfate. After filtration
and evaporation under reduced pressure, products 6a–p were
obtained in 52–72% yields.
4.4. Methyl 1-(2,2-dimethoxyethyl)-b-carboline-3-carboxylate (2)
A mixture of 4.3 g (13.522 mmol) of 1 and 100 ml of DMF was
stirred at 0 ꢂC until a clear solution was formed, to which 3.04 g
(19.296 mmol) of KMnO4 was then added. The reaction mixture was
stirred at 0 ꢂC for 1 h, and then at room temperature for 3 h until TLC
(CHCl3:MeOH, 15:1) indicated the complete disappearance of
starting material. The formed precipitate was filtered and the filtrate
was evaporated under vacuum. The residue was diluted with 100 ml
of EtOAc, and the solution was washed successively with water and
saturated aqueous NaCl. The organic phase was separated and dried
over anhydrous sodium sulfate. After filtration, the filtrate was
evaporated under vacuum. The residue was solidified in acetone to
give 3.47 g (80.3%) of the title compound as a yellow power.
4.9. In vitro cytotoxic assay
Cytotoxic assays in vitro were carried out using 96-well plate
cultures and MTT staining according to the procedure described by
Al-Allaf et al. with slight modification. The human-derived cell lines
H1299 (human non-small cell lung carcinoma), HepG2 (human liver
carcinoma), Hela (human cervical carcinoma), HL-60 (human
immature granulocyte leukemia) and MES-SA (human sarcoma cell)
lines were cultured in DMEM or RPIM-1640 (Gibico) medium con-
taining 10% (v/v) fetal bovine serum and 10,000 U/ml penicillin and
4.5. Methyl 9-benzyl-1-(2,2-dimethoxyethyl)-b-carboline-3-
carboxylate (3)
A mixture of 5.50 g (17.525 mmol) of 2, 20 ml of anhydrous DMF
and 20 ml of anhydrous THF was stirred to form a clear solution,
and then 1.05 g (26.32 mmol, 60%) of NaH was added. The mixture
was stirred at room temperature for 30 min, benzyl bromide
(2.98 g, 17.427 mmol) was added and the reaction mixture was
stirred for 4 h. To this mixture, 200 g of ice was added and the
solution was extracted with EtOAc (100 ml ꢃ 3). The organic phase
was separated, washed with saturated aqueous NaCl, and dried
over anhydrous sodium sulfate. After filtration the filtrate was
evaporated under vacuum. The residue was purified with silica gel
chromatography (petroleum ether:acetone, 2:1) to give 4.337 g
(61.2%) of the title compound as colorless powder.
100
m
g/ml streptomycin. Cell suspension (100
m
l of 5 ꢃ104 cells/ml)
was seeded in 96-well plates and incubated at 37 ꢂC in a humidified
atmosphere of 5% CO2 for 4 h. The medium was replaced by medium
containing different concentrations (ranging from 0.1
of 6a–p, which were dissolved in DMSO vehicle. The vehicle control
received only DMSO (1%, v/v). To the wells, 25 l of medium con-
taining test samples of different concentrations of 6a–p was added.
After 48 h, 25 l (final concentration, 0.5 mg/ml) of MTT (Sigma) was
mM to 400 mM)
m
m
added to each well, and the solutions incubated for an additional 4 h.
Then culture medium was removed. The resulting MTT–formazan
product was dissolved in 100 ml DMSO. The amount of formazan was
determined by measuring the optical density at 570 nm. The cyto-
toxic activities were observed to occur in a dose-dependent manner
in the cells. The activity was expressed as an IC50 value, which is the
concentration of test sample to give 50% inhibition of the growth of
tumor cells.
4.6. 2-(3-Methoxycarbonyl-9-benzyl-b-carboline-1-yl)
acetaldehyde (4)
A mixture of 1.0 g (2.475 mmol) of 3, 14 ml of HOAc and 2 ml of
water was stirred at room temperature for 16 h. To the reaction
mixture 200 g of ice was added. The formed precipitates were
collected by filtration to provide 538 mg (60.7%) of the title
compound which was used directly in the next reaction without
purification.
4.10. In vivo anti-tumor assay
Male ICR mice, purchased from Peking University Health Science
Center, were maintained at 21 ꢂC with a natural day/night cycle in
a conventional animal colony. The mice were ten to twelve weeks
old at the beginning of the experiments. S180 ascites tumor cells
were used to form solid tumors after subcutaneous injection. For
initiation of subcutaneous tumors the cells were obtained as an
ascitic form from the tumor-bearing mice, which were serially
transplanted once per week. Subcutaneous tumors were implanted
by injecting 0.2 ml of 0.9% saline containing 1 ꢃ107 viable tumor
cells under the skin on the right armpit. Twenty-four hours after
implantation, the mice (twelve per group) were randomly divided
into experimental groups. The mice of the positive control group
4.7. General procedure for preparing N-(9-benzyl-3-carboxyl-
carboline-1-yl)-ethylamino acid methyl esters (5a–o)
b-
A mixture of 2.0 mmol of amino acid methyl ester, 1.33 mmol of
NaOH, 25 ml of MeOH and 476 mg (1.33 mmol) of 4 was stirred at
room temperature for 10 min, and then 58.2 mg (0.931 mmol) of
NaBH3CN was added. The reaction mixture was stirred at room
temperature for 4 h, adjusted to pH 1–2 with HCl (3 N) and evap-
orated under vacuum. The residue was diluted with 20 ml of water,
and the aqueous solution was washed with ether (30 ml ꢃ 3). The
aqueous phase was adjusted to pH 8 with aqueous NaOH (1 N),
extracted with EtOAc (100 ml ꢃ 3), and the organic phase was
separated and washed with saturated aqueous NaCl, and dried over
anhydrous sodium sulfate. After filtration the solvent was
were given a daily i.p injection of 89 mmol/kg of cytarabine in 0.2 ml
of 0.9% saline for seven consecutive days. The mice of the negative
control group were given a daily i.p injection of 0.2 ml of 0.9% saline
for seven consecutive days. The mice of the treatment groups were
given a daily i.p injection of 89 mmol/kg of 6a–p in 0.2 ml of 0.9%

J. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 4153–4161
4161
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Acknowledgments
This work was finished in Beijing Area Major Laboratory of
Peptide and Small Molecular Drugs, supported by PHR (IHLB,
KZ200810025010), the National Natural Science Foundation of
China (30672513) and Special Project (2008ZX09401-002) of China.
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Appendix. Supplementary data
Thechemicaland physical dataofcompounds2, 3, 5a–p and 6a–p:
These materials are available free of charge via the Internet at http://
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Supplementary data associated with this article can be found in
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