A class of novel carboline intercalators: Their synthesis, in vitro anti-proliferation, in vivo anti-tumor action, and 3D QSAR analysis

Article abstract of DOI:10.1016/j.bmc.2010.07.043

Based on DOCK scores 18 N-(3-benzyloxycarbonylcarboline-1-yl)ethylamino acid benzylesters (6a-r) were synthesized as anti-tumor agents. Their IC ₅₀ values against five human carcinoma cell lines ranged from 11.1 μM to more than 100 μM. The in vivo assay identified five derivatives of them had no anti-tumor action, the anti-tumor activity of nine derivatives of them equaled that of cytarabine, and the anti-tumor activity of three derivatives of them was higher than that of cytarabine. The UV and fluorescence spectra, as well as the relative viscosity and melting temperature measurements of calf thymus DNA (CT DNA) with and without the representative compound suggested that DNA intercalation could be their action mechanism. The 3D QSAR analysis of N-(3-benzyloxycarbonylcarboline-1-yl)ethylamino acid benzylesters (6a-r) revealed that their in vivo anti-tumor activity significantly depends on the molecular electrostatic and steric fields of the side chain of the amino acid residue.

Full text of DOI:10.1016/j.bmc.2010.07.043

Bioorganic & Medicinal Chemistry 18 (2010) 6220–6229
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A class of novel carboline intercalators: Their synthesis, in vitro
anti-proliferation, in vivo anti-tumor action, and 3D QSAR analysis
*
*
Jianhui Wu, Chunyu Li, Ming Zhao , Wenjing Wang, Yuji Wang, Shiqi Peng
College of Pharmaceutical Sciences, Capital Medical University, 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:
Based on DOCK scores 18 N-(3-benzyloxycarbonylcarboline-1-yl)ethylamino acid benzylesters (6a–r)
Received 15 May 2010
Revised 16 July 2010
Accepted 17 July 2010
Available online 22 July 2010
were synthesized as anti-tumor agents. Their IC50 values against five human carcinoma cell lines ranged
from 11.1 lM to more than 100 lM. The in vivo assay identified five derivatives of them had no anti-
tumor action, the anti-tumor activity of nine derivatives of them equaled that of cytarabine, and the
anti-tumor activity of three derivatives of them was higher than that of cytarabine. The UV and fluores-
cence spectra, as well as the relative viscosity and melting temperature measurements of calf thymus
DNA (CT DNA) with and without the representative compound suggested that DNA intercalation could
be their action mechanism. The 3D QSAR analysis of N-(3-benzyloxycarbonylcarboline-1-yl)ethylamino
acid benzylesters (6a–r) revealed that their in vivo anti-tumor activity significantly depends on the
molecular electrostatic and steric fields of the side chain of the amino acid residue.
Keywords:
Carboline
Anti-tumor activity
Intercalation
3D QSAR
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Docking
1. Introduction
and a series of novel N-(3-benzyloxycarbonylcarboline-1-yl)ethyl-
amino acid benzylesters were investigated.
DNA recognition is known to play a pivotal role in the anti-
tumor efficacy of several anti-tumor agents, including groove
binders, alkylating compounds and intercalating agents. DNA
intercalation is known to play a pivotal role in the anti-tumor effi-
cacy of several clinically used anti-tumor drugs including anthra-
cyclines, acridines, and anthraquinones.1 The discovery of new
DNA intercalators has been a noticeable approach to push clinical
therapy of cancer. Numerous intercalators have been reported
recently,2,3 and some b-carbolines, such as harmine and the deriv-
atives, are involved.4 The intercalation of b-carbolines towards
DNA is correlated with cytotoxicity.5,6 By intercalation b-carbolines
inhibit topoisomerases I/II and finally damage DNA. As part of our
ongoing efforts, we recently explored a series of b-carboline-3-car-
bonylamino acid benzylesters as potent anti-tumor derivatives of
natural products. We demonstrated that the in vitro cytotoxicity
of these benzylesters depended on their building blocks, that is,
b-carboline-3-carboxylic acid, amino acid and benzyl moieties.7
These requirements were successfully used for the design of
N-(3-carboxyl-9-benzylcarboline-1-yl)ethylamino acids, and the
in vitro anti-proliferation and the in vivo anti-tumor activities
were analyzed with DOCK score.8 In the present paper N-(3-car-
boxyl-9-benzylcarboline-1-yl)ethylamino acids were modified
2. Results and discussion
2.1. DOCK scores leading to N-(3-benzyloxycarbonylcarboline-
1-yl)ethylamino acid benzylesters
DNA can be conveniently intercalated by planar heterocy-
cles.9–11 The LigandFit/LigandScore in DS Modeling 2.1 (Accelry
Inc) and d(CGATCG)2 oligonucleotide retrieved from the Protein
Data Bank (1D12) has been widely used for the automated docking
studies.12,9,13 Firstly, the automated docking of N-(3-carboxyl-9-
benzylcarboline-1-yl)ethylamino acids toward the cavity of
d(CGATCG)2 was carried out according to a general procedure,
the DOCK scores were calculated and are listed in Table 1. Sec-
ondly, the possible derivatives of N-(3-carboxyl-9-benzylcarbo-
line-1-yl)ethylamino acids were constructed by changing the
position of the 9-benzyl. Finally, the automated docking of them
toward the cavity of d(CGATCG)2 was similarly carried out to
calculate the DOCK scores. The DOCK scores of N-(3-carboxyl-9-
benzylcarboline-1-yl)ethylamino acids and N-(3-benzyloxycarbon-
ylcarboline-1-yl)ethylaminoacid benzylesters are listed in the left
and right columns of Table 1, respectively. In the DOCK process it
was observed that the binding cavity in the duplex was more suit-
able for benzylester derivatives than for 9-benzyl derivatives. In
the DOCK process it was also observed that a graphic of benzylester
derivative matching the binding cavity of d(CGATCG)2 usually
gained a high DOCK score. This ensured that the graphic could be
* Corresponding authors. Address: College of Pharmaceutical Sciences, Capital
Medical University, Beijing 100069, PR China. Tel./fax: +86 10 8391 1528 (S.P.); tel./
fax: +86 10 8280 2482 (M.Z.).
E-mail addresses: mingzhao@mail.bjmu.edu.cn (M. Zhao), sqpeng@mail.bjmu.
edu.cn (S. Peng).
0968-0896/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.043

J. Wu et al. / Bioorg. Med. Chem. 18 (2010) 6220–6229
6221
Table 1
DOCK scores of carbolines with and without 9-benzyl
CO2H
CO2Bzl
N
N
N
R
H
N
H
R
H
N
N
Bzl
H
H
CO2H
CO2H
DOCK score
Compound/R
DOCK score
55.79
51.12
49.14
46.26
46.71
52.83
39.65
48.05
49.10
59.91
50.81
40.68
33.5
6a/H
6b/CH3
6c/CH(CH3)2
74.21
72.46
65.97
61.84
52.71
65.22
52.36
52.87
62.19
67.37
60.50
54.46
42.8
6d/CH2CH(CH3)2
6e/CH(CH3)CH2CH3
6f/CH2CH2SCH3
6g/NH@NCH2CH2CH2
6h/CH2C6H5
6i/indole-3-ylmethylene
6j/CH2OH
6k/CH(OH)CH3
6l/CH2CONH2
6m/CH2CH2NH2
6n/CH2C6H4-OH-p
6o/CH2CO2Bzl
6p/CH2(CH2)4NH2
6q/imidazole-3-ylmethylene
6r/CH2CH2CO2Bzl
49.03
60.73
30.26
52.77
55.80
68.41
85.38
43.17
60.98
71.72
used as a model for the docking. The DOCK scores of N-(3-benzyl-
oxycarbonylcarboline-1-yl)ethylamino acid benzylesters are sig-
nificantly higher than those of N-(3-carboxyl-9-benzylcarboline-
1-yl)ethylamino acids. This comparison suggests that N-(3-benzyl-
oxycarbonylcarboline-1-yl)ethylamino acid benzylesters may
comfortably adopt the cavity of d(CGATCG)2 and high DOCK score
benefits the in vitro anti-proliferative and in vivo anti-tumor activ-
ities. As seen in Table 2, the IC50 of N-(3-benzyloxycarbonylcarbo-
line-1-yl)ethylamino acid benzylesters against HepG2 and Hela
cells range from 13.5
N-(3-carboxyl-9-benzylcarboline-1-yl)ethylamino acids against
HepG2 and Hela cells range from 26.5 M to more than 400 M.
Besides, on S180 mouse model the effective dose of N-(3-benzyl-
oxycarbonylcarboline-1-yl)ethylamino acid benzylesters is 10-fold
lower than that of N-(3-carboxyl-9-benzylcarboline-1-yl)ethyl-
amino acids.8
l
M to more than 100
l
M, while the IC50 of
l
l
2.2. Synthesis of N-(3-benzyloxycarbonylcarboline-1-yl)-
ethylamino acid benzylesters
N-(3-Benzyloxycarbonylcarboline-1-yl)ethylamino acid benzy-
lesters (6a–r) were synthesized according to the seven-step-route
Table 2
IC50 of 6a–r against HepG2, Hela, Bel7402, K562, and B16 cellsa
depicted in Scheme 1. Using a common procedure,
verted to -Trp-OMe (92% yield). The Pictet-Spengler condensation
of -Trp-OMe and 1,1,3,3-tetramethoxypropane provided methyl
L-Trp was con-
Compd
HepG2
Hela
Bel7402
K562
B16
L
Ara-C
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
0.2 0.1
50.0 8.3
>100
29.2 1.2
25.2 2.1
>100
18.7 1.2
17.1 1.2
>100
60.0 3.2
19.9 1.1
26.0 2.1
>100
35.9 5.2
>100
>100
36.3 4.2
>100
63.1 10.2
—
34.2 2.1
53.2 7.6
100 5.2
20.3 3.1
43.5 1.4
>100
25.8 2.1
52.8 2.2
>100
33.5 1.8
47.1 6.2
48.2 8.4
20.9 1.8
21.5 1.8
>100
13.5 1.0
20.1 1.9
>100
14.9 1.2
19.7 2.1
38.3 3.2
63.6 10.3
>100
—
L
26.8 4.2
76.4 1.2
25.5 2.2
22.5 3.1
>100
28.8 1.5
25.8 1.5
>100
59.5 2.7
20.3 2.0
15.2 1.2
97.2 15.2
71.0 8.2
16.7 2.0
>100
33.9 4.8
63.6 7.2
54.9 8.2
75.4 9.1
20.5 2.3
28.8 1.3
50.2 3.2
19.2 2.1
61.1 2.8
71.8 2.2
28.1 2.1
20.7 1.3
60.3 8.4
38.4 3.3
17.1 4.5
42.5 6.2
24.5 2.8
11.1 4.5
49.4 5.4
60.0 4.6
66.4 5.2
1-(2,2-dimethoxyethyl)-1,2,3,4-tetrahydro-b-carboline-3-carbox-
ylate (1) in 89% yield. In the presence of KMnO4 the piperidine ring
of 1 was aromatized to form b-carboline-3-carboxylic acid methyl-
ester (2) in 80% yield. At 0 °C 2 was treated with 2 N aqueous of
NaOH to saponify and form 3. Esterification of 3 and benzyl bro-
mide provided benzyl 1-(2,2-dimethoxyethyl)-b-carboline-3-car-
boxylate (4) in 74% yield. In the presence of HOAc and HCl the
acetal 4 was hydrolyzed to aldehyde 5 in 67% yield. Reduced ami-
nation of 5 and amino acid banzylesters provided N-(3-benzyloxy-
carbonyl-9-H-carboline-1-yl)ethylamino acid benzylesters (6a–r)
in 35–88% yields.
39.0 2.1
54.6 6.8
>100
>100
81.3 6.7
23.7 2.3
60.3
14.9 3.2
36.9 5.3
>100
18.9 1.9
>100
15.4 2.8
14.4 3.1
18.5 4.5
2.3. IC50 of 6a–r against the proliferation of carcinoma cells
To predict the tumoricidal indication of 6a–r in the in vitro anti-
a
Ara-C = cytarabine, n = 6, IC50 is represented by mean SD lM.
proliferation assays five carcinoma cell lines, HepG2 (human

6222
J. Wu et al. / Bioorg. Med. Chem. 18 (2010) 6220–6229
CO Me
CO Me
CO H
2
2
2
i
ii
iii
iv
L-Trp
H
N
NH
N
N
N
N
H
H
H
CH(OMe)
2
CH(OMe)
CH(OMe)
2
(1S, 3S)-1: 60%
(1R, 3S)-1: 29%
2
3
2
v
CO Bzl
CO Bzl
2
2
CO Bzl
2
vii
vi
N
N
N
N
H
N
N
H
R
H
NH
5
H
6a-q
CHO
4
CH(OMe)
2
CO Bzl
2
vii
CO Bzl
CO Bzl
2
2
O
N
N
N
CH CH CO Bzl
N
H
2
2
2
H
NH
N
H
6r'
CO Bzl
6r
CO Bzl
2
2
Scheme 1. Synthesis of N-(3-benzyloxycarbonyl-9-H-carboline-1-yl)ethylamino acid benzylesters 6a–r. Reagents and conditions: (i) SOCl2, MeOH; (ii) HCl, 1,1,3,3-
tetramethoxypropane, MeOH; (iii) KMnO4, DMF; (iv) NaOH, H2O, MeOH; (v) K2CO3, BrCH2C6H5, DMF/THF; (vi) HCl, HOAc, H2O; (vii) NaOH, NaBH3CN and amino acid
benzylester; 6a R = H, 6b R = CH3, 6c R = CH(CH3)2, 6d R = CH2CH(CH3)2, 6e R = CH(CH3)CH2CH3, 6f R = CH2CH2SCH3, 6g R = NH = NCH2CH2CH2, 6h R = CH2C6H5, 6i R = indole-
3-ylmethylene, 6j R = CH2OH, 6k R = CH(OH)CH3, 6l R = CH2CONH2, 6m R = CH2CH2CONH2, 6n R = CH2C6H4–OH-p, 6o R = CH2CO2Bzl, 6p R = CH2(CH2)4NH2, 6q R = imidazole-
3-ylmethylene, 6r R = CH2CH2CO2Bzl.
hepatocellular liver carcinoma cell line), Hela (human epithelial
cervical cancer), Bel7402 (human hepatoma cell line), K562 (hu-
man immortalized myelogenous leukemia line) and B16 (mouse
melanoma cell line), were used. In the assays the cells were ex-
ing in vitro activity. It was the case that the compounds, such as
6c,d,f,g,j,k, with flexible side chain usually gave low IC50 value.
2.4. Effect of 6a–r on the tumor weight of S180 mice
posed to serial concentrations (0.1–200 lM) of 6a–r or cytarabine
(positive control), and the standard MTT assays were performed.
The IC50 values are summarized in Table 2. The data indicate that
6a–r efficiently inhibit the proliferation of these cells. The IC50 val-
In the previous paper it was observed that due to the in vivo sta-
bility of the b-carboline ring of 9-benzyl derivatives their in vitro
activities did not quite correlate with their in vivo activities.8 To
continue this observation in the present in vivo evaluation all com-
pounds were assayed. In the assays the tumor weight of 6a–r trea-
ted S180 mice was recorded. As shown in Table 3, when the mice
ues range from 11.1 lM to more than 100 lM with 6c as the most
potent compound. It was explored that the cells show different
sensitivity to 6a–r. HepG2 cells were more sensitive to
6c,d,f,g,j,k than to the others, B16 cells was sensitive to 6o only,
Hela cells was sensitive to 6n only, while no cells were practically
sensitive to 6e and 6h. Due 6a–r having common b-carboline ring a
preliminary structure–activity relationship was analyzed by corre-
lating their individual amino acid side chain with the correspond-
were given a daily ip injection of 8.9 lmol/kg of 6a–r in 0.2 ml of
normal saline (NS) for seven consecutive days, the tumor weights
ranged from 0.71 g to 1.70 g (NS receiving mice, 1.62 g), and 13
derivatives of 6a–r significantly decreased the tumor weight of
the treated mice. The efficacy of 6d,j,o (tumor weight ranging from
0.71 g to 1.02 g), the most potent compounds for these five carci-
noma cells, is significantly higher than that of cytarabine (positive
control, tumor weight 1.22 g) and the efficacy of 6b,c,e,k,n,p–r (tu-
mor weight ranging from 1.05 g to 1.26 g) essentially equals that of
cytarabine. The different anti-tumor efficacy and different amino
acid residue of 6a–r suggest that the anti-tumor activity correlates
with the side chain of the amino acid residue. On the other hand,
the comparison of the data of Table 2 and Table 3 suggests that
similarly to 9-benzyl derivatives for the present benzylesters the
in vitro activities also does not quite correlate with the in vivo
activities.
Table 3
Effects of 6a–r on tumor weights of S180 micea
Compd
Tumor weight
%Inhibition
NS
Ara-C
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
1.62 0.28
1.22 0.17b
1.56 0.27
1.26 0.19b
1.07 0.59b
0.80 0.23d
1.09 0.17b
1.37 0.30c
1.50 0.41
1.51 0.31
1.27 0.31c
1.02 0.09d
1.05 0.42b
1.39 0.33
1.70 0.55
1.10 0.24b
0.71 0.18d
1.15 0.32b
1.04 0.32b
1.14 0.25b
24.3 0.5
3.5 0.4
22.0 0.5
34.0 0.8
50.9 0.7
33.9 0.6
15.3 1.1
7.1 1.8
Table 4
Effect of the dose of 6o and 6q on the tumor weight of S180 micea
6.6 0.6
22.2 0.6
36.7 0.6
34.8 0.7
14.0 0.9
ꢀ5.3 0.9
31.7 0.5
56.1 0.7
28.9 1.3
35.4 0.5
29.4 0.4
Compd
Ara-C
Dose (
lmol/kg)
Tumor weight
%Inhibition
89.0
8.9
0.89
89.0
8.9
0.89
89.0
8.9
0.73 0.20b
1.22 0.17c
1.52 0.30
0.43 0.11b
0.71 0.18c
0.96 0.31
0.63 0.27b
1.04 0.32c
1.54 0.39
54.8 14.5
24.3 0.5
6.8 0.7
73.5 4.0
56.1 0.7
40.6 3.6
60.7 14.3
35.4 0.5
6.3 1.2
6o
6q
0.89
a
Ara-C = cytarabine, NS = normal saline, n = 12, dose of Ara-C and 6a–r:
a
b
c
ꢀ
8.9
l
mol/kg, tumor weight is represented by x ꢁ SD g.
ꢀ
Ara-C = cytarabine, n = 12, tumor weight is expressed by x ꢁ SD g.
Compared to 8.9 mol/kg of 6o and 6q p <0.01.
For Ara-C and 6q: compared to 0.89 mol/kg of 6q p <0.01, for 6o: compared to
mol/kg of 6o p <0.05.
b
Compared to NS p <0.01.
Compared to NS p <0.05.
Compared to NS and Ara-C p <0.01.
l
c
l
d
0.89
l

J. Wu et al. / Bioorg. Med. Chem. 18 (2010) 6220–6229
6223
2.5. Dose-dependent in vivo anti-tumor activity of 6o,q
Two representatives (6o and 6q) were selected to examine the
dose-dependent action. The tumor inhibition of 89.0, 8.9, and
0.89 lmol/kg of 6o and 6q is 73.5%, 56.1%, and 40.6% as well as
60.7%, 35.4%, and 6.3%, respectively (Table 4). Therefore, 6o and
6q dose-dependently inhibit the growth of the tumor. The minimal
effective dose of 6o and 6q is 0.89 lmol/kg and 8.9 lmol/kg,
respectively.
2.6. Action mechanism of 6a–r
Small molecule-induced variation of DNA spectra has been
widely used to identify the intercalation.14–17 To examine DNA
intercalation of 6a–r, calf thymus DNA (CT DNA) was selected as
the model DNA, 6d, one of the most potent compounds that their
in vitro activities substantially correlate with their in vivo activi-
ties, was selected as model compound. The UV, fluorescence spec-
tra, viscosity, and melting temperature of CT DNA alone and CT
DNA plus 6d were compared. These comparisons provided spectral
evidence for DNA intercalation of 6d.
Figure 2. Fluorescence spectra of 6d in PBS (concentration 0.5
kex = 254 nm) explain the fluorescence quenching induced by 10
of CT DNA in PBS (8, 16, 24, 32, 40, 48, 56, 64, 72 M).
l
l
M, kem = 382 nm,
l of the solution
l
2.6.3. Viscosity of CT DNA without and with 6d18,19
In DNA intercalation to accommodate and bind small molecule
DNA base pairs are pushed apart and consequently lead to viscosity
increase. Thus viscosity increase is widely used to define the inter-
calation. Here the relative viscosity of the sample solutions of [6d]/
[CT DNA] in the ratios of 0–0.36 were measured. Figure 3 indicates
that 6d concentration-dependently increases the relative viscosity
of CT DNA. This could be considered as the result of DNA interca-
lation of 6d.
2.6.1. UV spectra of CT DNA without and with 6d
In the UV experiments the spectra (Fig. 1) of a solution of CT
DNA in PBS (final concentration 240
solution of CT DNA (final concentration 240
centration 27, 54, 81, 108 M) in PBS were determined on a Shima-
lM) and the spectra of the
l
M) plus 6d (final con-
l
dzu 2550 spectrophotometer over 220–400 nm. Figure 1 explores
that 6d induces a hypochromic effect (53.04%) and hypsochromic
shift (2.5 nm).
2.6.4. Melting temperature of CT DNA without and with 6d20,21
The thermal melting of DNA is widely used to measure the ther-
mostability of DNA double helix. Once the temperature of DNA
solution is high enough the double strands dissociate to single
stand, and the temperature that induces the absorbance to increase
50% is termed as melting temperature (Tm). The temperature of the
2.6.2. Fluorescence spectra of CT DNA without and with 6d
In the fluorescence experiments the spectra (Fig. 2) of CT DNA
and CT DNA plus 6d were determined at 300 K using of fluores-
cence spectroscopy. The solution of 6d in PBS (0.5
with 10 l of the solution of a series concentration (0, 8, 16, 24, 32,
40, 48, 56, 64, and 72 M) of CT DNA in PBS. A series of variations
lM) was titrated
l
l
solution of CT DNA in PBS (100
lM) and the solution of CT DNA
of the fluorescence intensity of 6d were recorded on a Shimadzu
RF-5310PC spectrofluorometer at 382 nm of emission wavelength
(the excitation wavelength is 254 nm). The fluorescence quenching
phenomenon, that is, CT DNA induced concentration-dependent
decrease of fluorescence intensity of 6d. When the concentration
of CT DNA is 72 lM 6d gains the minimal fluorescence intensity
(34.65% of decrease). These suggest 6d having a DNA intercalation
mechanism.
(100 M) plus 6d (18 M) in PBS rose at a rate of 1 °C/min and
l
l
the absorbance was monitored at 260 nm. The melting curves of
CT DNA and CT DNA plus 6d are shown with Figure 4. The biphasic
melting curve of CT DNA plus 6d is similar to that of CT DNA plus
daunomycin.22 The Tm of CT DNA plus 6d in PBS is 13.8 0.5 °C
higher than that of CT DNA alone indicates that in the presence
of 6d the double strands of CT DNA are stabilized. This additional
stability should be attributed to DNA intercalation of 6d.
Figure 1. Hypochromic effect and hypsochromic shift occurred in the UV spectra of
CT DNA (final concentration 240
108 M) in PBS.
lM) plus 6d (final concentration 0, 27, 54, 81,
l
Figure 3. Effect of 6d on the relative viscosity of CT DNA (300 lM).

6224
J. Wu et al. / Bioorg. Med. Chem. 18 (2010) 6220–6229
aligned 6a–r is shown in Figure 5. The alignment stereoview ex-
plores that to superimpose onto carboline-3-carboxylic acid benzyl
ester the 1-ethylamino acid residue of each structure has to take
individual conformation. The individual conformation will influ-
ence the anti-tumor activity.
Molecular field analysis (MFA) was performed for 6a–r by using
the QSAR module of Cerius2.24 A five-step-procedure consisted of
generating conformers, energy minimization, matching atoms
and aligning molecules, setting preferences, and regression analy-
sis was automatically practiced in MFA. Molecular electrostatic and
steric fields were created by use of proton, hydroxyl group and
methyl group as the probe, 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
totally generated grid points were 672. Though the spatial and
structural descriptors such as dipole moment, polarizability, radius
of gyration, number of rotatable bonds, molecular volume, princi-
pal moment of inertia, A log P98 (the calculated logarithm of parti-
tion coefficient from the implementation of the atom-type-based
method using the latest published set of parameters), number of
hydrogen bond donors and acceptors, and molar refractivity were
also considered, only the highest variance holder proton, methyl,
and hydroxyl descriptors were used. Regression analysis was car-
ried out using the genetic partial least squares (G/PLS) method con-
sisting of 50,000 generations with a population size of 100. The
number of components was set to 5. Cross-validation was per-
formed 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 fea-
tures of 6a–r in the training set lead to increase or decrease the
activity were specified. Proton descriptor with positive coefficient
indicates a region favorable for electropositive group, while nega-
tive coefficient indicates electronegative group required at this po-
sition. 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–r in terms of the most relevant descriptors
proton, hydroxyl group and methyl group is expressed by Eq. 1.
Figure 4. Thermal denaturation curves of CT DNA with and without 6d. Tm
measurements were performed in PBS at pH 7.4 with a 6d/DNA ratio of 0.18.
2.6.5. 3D QSAR analysis of 6a–r
To elucidate the effects of the side chain of amino acid on the
anti-tumor activity of 6a–r, the activity is expressed with the tu-
mor inhibition rate. For 3D QSAR the training set (6a–f,h,j,l–r)
and the test set (6g,i,k) selections done manually such that they
populate the wide range of anti-tumor activity in a similar
proportions.
The 3D QSAR module of Cerius2 was followed to quantitatively
correlate the tumor inhibition rate with the structure of 6a–f,h,j,l–
r. To establish valid 3D QSAR models a proper alignment procedure
of 6a–r was performed using the target model align strategy in the
align module within Cerius2. With an assumption that each struc-
ture of 6a–r binds the same site of the receptor and exhibits activ-
ity, they were aligned in a pharmacological active orientation. To
obtain a consistent alignment the common moiety carboline-3-
carboxylic acid benzyl ester was selected as the template for super-
posing 6a–r. The method used for performing the alignment was
the maximum common subgraph (MCS).23 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
carboline-3-carboxylic acid benzyl ester that shared by 6a–f,h,j,l–
r. This subset was used for the alignment. A rigid fit of atom pair-
ings was performed to superimpose each structure onto the target
model carboline-3-carboxylic acid benzyl ester. The stereoview of
%Inhibition ¼ 2:45475 þ 0:498953ðHþ=441Þ
ꢀ 0:729572ðHþ=357Þ þ 1:90153ðCH3=404Þ
ꢀ 0:364508ðHOꢀ=693Þ þ 1:30811ðHOꢀ=694Þ
ð1Þ
Figure 6. Graph of the tested in vivo anti-tumor activities against calculated in vivo
Figure 5. Alignment stereoview of 6a–r used for molecular field generation.
anti-tumor activities.

J. Wu et al. / Bioorg. Med. Chem. 18 (2010) 6220–6229
6225
Figure 7. Electrostatic environment of 6d (a) with high in vivo anti-tumor activity and 6m (b) with low in vivo anti-tumor activities within the grid with 3D points of Eq. 1.
The data points (n), correlation coefficient (r) and square corre-
lation coefficient (r2) of Eq. 1 were 15, 0.981, and 0.963, respec-
tively. The correlation of the tested anti-tumor activities on S180
mouse model with the calculated anti-tumor activities from Eq. 1
is explained with Figure 6.
3. Conclusion
N-(3-Benzyloxycarbonyl-9-H-carboline-1-yl)ethylamino acid
benzylesters 6a–r could be prepared in desirable yields by using
a seven-step route. The in vitro anti-proliferation assays of 6a–r
with five human carcinoma cell lines identified K562 cells to be
more sensitive than the others. The in vivo anti-tumor activity as-
says of 6a–r identified 6d and 6o having the highest efficacy. The
In Eq. 1 one term of H+/441 with positive coefficient from pro-
ton descriptor, which means that at this position electron-releasing
group will decrease the in vivo anti-tumor activity, one term of
H+/357 with negative coefficients from proton descriptor, which
means that at this position electron-releasing group will increase
the in vivo anti-tumor activity, one term of CH3/404 with positive
coefficient from methyl descriptor, which means that at this posi-
tions large group will decrease the in vivo anti-tumor activity, and
one term of HOꢀ/694 with positive coefficient from hydroxyl
descriptor, which means that at this position hydrogen bond form-
ing group will increase the activity, and one term of HOꢀ/693 with
negative coefficients from hydroxyl descriptor, which means that
at this position hydrogen bond forming group will decrease the
activity, are involved.
tumor weight of 8.9
(0.71 0.18 g) treated mice closes that (0.73 0.20 g, p >0.05) of
89 mol/kg of cytarabine treated mice. The UV and fluorescence
lmol/kg of 6d (0.80 0.23 g) or 6o
l
spectra, as well as the relative viscosity and melting temperature
measurements of CT DNA with or without 6d suggest 6a–r been
capable of intercalating toward DNA. The 3D QSAR studies were
carried out by building MFA models for the building of a strategy
and consequently for the improvement of the activity of the com-
pounds. The 3D QSAR analysis of 6a–r reveals that the in vivo anti-
tumor activity depends on the molecular electrostatic and steric
fields of the side chains of the amino acid moieties. In general, elec-
tron-drawing group at 441 position, electron-releasing group at
357 position, small group at 404 position group and hydrogen
bond forming group at 694 position and hydrophobic group at
693 position are necessary for higher anti-tumor. The DOCK study
revealed that these ligands bind in the base pairs of d(CGATCG)2.
The comfort of the b-carboline ring in the binding cavity in the du-
plex is crucial for the in vivo anti-tumor activity. The results of the
in vivo evaluation rationally correlated with the results of the 3D
QSAR and with the results of the DOCK, for instance the experi-
mental tumor inhibition of 6d/6m (50.9%/ꢀ5.3%) rationally corre-
lated with their calculated tumor inhibition (51.4%/ꢀ5.8%) and
with their DOCK score (61.84/42.8).
As the examples Figure 7 gives the electrostatic and environ-
ments of one representative 6d with high in vivo anti-tumor activ-
ity, and 6m with low in vivo anti-tumor activity within the grid
with 3D points of Eq. 1. Besides the same electrostatic and environ-
ments as carboline-3-carboxylic acid benzyl ester moiety 6d has
electron-releasing group near H+/441 thus resulting in increasing
the in vivo anti-tumor activity. Besides the same electrostatic
and environments as carboline-3-carboxylic acid moiety 6m has
electron-withdrawing group near H+/441 thus resulting in decreas-
ing the in vivo anti-tumor activity.
Predict power of Eq. 1 was demonstrated by comparing the cal-
culated and the tested in vivo anti-tumor activities of the test set
(Table 5 6g,i,k). The results indicate that Eq. 1 rationally gives
in vivo anti-tumor activities for the test set and the errors range
from +0.5% to ꢀ1.1%. The calculated activity is approximate to
the experimental activity means that Eq. 1 is practical to accurately
predict the anti-tumor activity of the derivatives of carboline-3-
carboxylic acid benzyl ester.
4. Experimental
4.1. General
All chemicals were purchased from commercial suppliers and
were purified when necessary. Protected amino acids with L-con-
figuration were purchased from Sigma Chemical Co. Chromatogra-
phy 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 ꢂ 150 mm). Melting
points were determined in capillary tubes on an electrothermal
SM/XMP apparatus and without correction. UV spectra were mea-
sured on Shimadzu UV 2550. ESI-MS was determined by Micro-
mass Quattro micro TM API, Waters Co. 1H NMR (500 Hz) and
13C NMR (125 Hz) spectra were acquired on a Bruker AC 300 spec-
Table 5
Predict and test anti-tumor activities of 6g,i,k
Compd
Tumor inhibition (%)
Test value
Predict value
Error
6g
6i
6k
7.6
21.6
33.7
7.1
22.2
34.8
+0.5
ꢀ0.6
ꢀ1.1

6226
J. Wu et al. / Bioorg. Med. Chem. 18 (2010) 6220–6229
trometer in CDCl3 or in DMSO-d6 with TMS as internal standard,
and chemical shifts are expressed in ppm. Optical rotations were
determined with 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.
reaction mixture was stirred at 0 °C for 3.5 h and TLC (CHCl3/
CH3OH, 10:1) indicated the complete disappearance of 2. The reac-
tion mixture was adjusted to pH 7 with aqueous solution of KHSO4.
The solution was evaporated under vacuum to remove methanol,
adjusted pH 2 with aqueous solution of KHSO4 and extracted with
EtOAc (30 ml ꢂ 3). The combined EtOAc was successively washed
with saturated aqueous solution of NaCl (20 ml ꢂ 3) and dried with
anhydrous Na2SO4, filtered and concentrated in vacuum to give the
title compound as pale yellow oil. The oil was used for the next
step without any further purification.
4.2. General procedure for preparing amino acid benzylesters
hydrochloride
At room temperature 0.02 mol amino acid was added to the
solution of 10.0 g polyphosphoric acid and 50 ml of benzyl alcohol.
The reaction mixture was stirred at 90 °C for 8 h. TLC indicated
complete disappearance of amino acid. The reaction mixture was
cooled to room temperature, mixed with the solution of 20 ml of
concentrated sulfuric acid in 200 ml of water, treated with 20 ml
of ether, the aqueous phase was separated and neutralized with
aqueous solution of sodium carbonate adjusted to pH 10. The solu-
tion was extracted with ether (370 ml) and the ether phase was
dried with anhydride magnesium sulfate. After filtration the fil-
trate was bubbled with hydrogen chloride to precipitate the hydro-
chloride of amino acid benzyl ester as colorless powder.
4.6. Benzyl 1-(2,2-dimethoxyethyl)-b-carboline-3-carboxylate
(4)
A mixture of 477 mg (1.59 mmol) of 3, 10 ml of anhydrous DMF
and 329 mg (2.39 mmol) of K2CO3 was stirred at room temperature
for 2 h. To this mixture 297 mg (1.74 mmol) of benzyl bromide was
added and the reaction mixture was stirred for 21 h. To this mix-
ture 200 g of ice was added and the solution was extracted with
EtOAc (20 ml ꢂ 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 res-
idue was purified with silica gel chromatography (petroleum
ether/EtOAc, 2:1) to give 473 mg (74%) of the title compound as
colorless powder. ESI/MS (m/e) 391 [M+H]+; 1H NMR (CDCl3) d:
9.68 (1H, s), 8.81 (1H, s), 8.16 (1H, d, J = 8.0 Hz), 7.55 (4H, m),
7.35 (4H, m), 5.53 (2H, s), 4.82 (1H, s), 3.58 (2H, d, J = 4.0 Hz),
3.41 (6H, s). Anal. Calcd for C23H22N2O4: C, 70.75; H, 5.68; N,
7.17. Found: C, 70.94; H, 5.43; N, 7.38.
4.3. Methyl 1-(2,2-dimethoxyethyl)-1,2,3,4-tetrahydro-b-
carboline-3-carboxylate (1)
A suspension of 5.0 g (24.5 mmol) of L-tryptophan methylester,
50 ml of MeOH and 6.0 ml (23.6 mmol) of 1,1,3,3-tetramethoxy-
propane was adjusted pH 1–2 with HCl (5 N) and stirred at 45 °C
for 48 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 ꢂ 3). The organic phase was sepa-
rated, washed successively with 10% sodium carbonate and satu-
rated 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 yel-
low oil. FAB-MS (m/e) 319 [M+H]+.
4.7. Benzyl 1-carbonylmethyl-b-carboline-3-carboxylate (5)
A mixture of 1.09 g (2.79 mmol) of 4, 10 ml of HOAc, 1.25 ml of
HCl and 1.25 ml of water was stirred at room temperature for 4.5 h.
To the reaction mixture 100 g of ice was added. The formed precip-
itates were collected by filtration to provide 642 mg (66.7%) of the
title compound, which was used directly in the next step without
purification.
4.4. Methyl 1-(2,2-dimethoxyethyl)-b-carboline-3-carboxylate
(2)
4.8. General procedure for preparing N-(3-benzyloxycarbonyl-
carboline-1-yl)ethylamino acid benzylesters (6a–r)
A mixture of 4.3 g (13.5 mmol) of 1 and 100 ml of DMF was stir-
red at 0 °C until a clear solution was formed, to which 3.04 g
(19.3 mmol) of KMnO4 was then added. The reaction mixture
was stirred at 0 °C for 1 h, and then at room temperature for 3 h
until TLC (CHCl3/MeOH, 15:1) indicated the complete disappear-
ance 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 of NaCl. The organic phase
was separated and dried over anhydrous sodium sulfate. After fil-
tration the filtrate was evaporated under vacuum. The residue
was solidified in acetone to give 3.47 g (80.3%) of the title com-
pound as a yellow power. Mp 135–136 °C, ESI/MS (m/e) 315
[M+H]+, 1H NMR (CDCl3) d: 9.63 (1H, s), 8.81 (1H, s), 8.16 (1H, d,
J = 8.0 Hz) 7.54–7.56 (4H, m), 7.30–7.42 (4H, m), 5.53 (2H, s),
4.82 (1H, s), 3.58 (2H, d, J = 4.0 Hz), 3.41 (6H, s); 13C NMR (CDCl3)
d: 166.04, 141.36, 140.74, 137.50, 137.17, 136.31, 128.64, 128.51,
128.45, 128.18, 122.09, 121.89, 120.73, 117.07, 112.12, 105.47,
67.02, 54.79, 40.89. Anal. Calcd for C17H18N2O4: C, 64.96; H, 5.77;
N, 8.91. Found: C, 64.74; H, 5.89; N, 8.70.
A mixture of 0.87 mmol of amino acid benzyl esters hydrochlo-
ride, 0.24 mmol of NaOH, 10 ml of MeOH and 200 mg (0.58 mmol)
of 5 was stirred at room temperature for 10 min, and then 18 mg
(0.29 mmol) of NaBH3CN was added. The reaction mixture was
stirred at room temperature for 20 h, adjusted to pH 2 with HCl
(3 N) and evaporated under vacuum. The residue was diluted with
20 ml of water, and the aqueous solution was washed with ether
(30 ml ꢂ 3). The aqueous phase was adjusted to pH 8 with aqueous
NaOH (1 N), extracted with EtOAc (100 ml ꢂ 3), and the organic
phase was separated and washed with saturated aqueous NaCl,
and dried over anhydrous sodium sulfate. After filtration the sol-
vent was evaporated under vacuum, and the residue was purified
by silica gel chromatography to give 6a–r in 35–88% yields.
4.8.1. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylglycine
benzylester (6a)
Yield: 77%. ESI/MS (m/e) 494 [M+H]+; 1H NMR (CDCl3) d: 10.32
(1H, s), 8.77 (1H, s), 8.15 (1H, d, J = 8.0 Hz), 7.59 (1H, m), 7.43 (1H,
d, J = 8.0 Hz), 7.41 (1H, m), 7.39 (2H, m), 7.37 (2H, m), 7.34 (1H, m),
7.33 (2H, m), 7.30 (2H, m), 7.28 (1H, m), 5.52 (2H, s), 5.25 (2H, s),
3.65 (2H, s), 3.47 (2H, t, J = 5.0 Hz), 3.21 (2H, t, J = 5.0 Hz); 13C NMR
(CDCl3) d: 171.58, 166.15, 144.89, 140.92, 136.93, 136.41, 136.15,
135.28, 128.67, 128.57, 128.53, 128.44, 128.35, 128.13, 126.99,
122.03, 121.67, 120.29, 116.91, 112.61, 67.04, 66.92, 50.52, 47.94,
4.5. 1-(2,2-Dimethoxyethyl)-b-carboline-3-carboxylic acid (3)
At 0 °C to the solution of 499 mg (1.59 mmol) of 2 in 7 ml of
methanol was adjusted to pH 11 with 2 N aqueous of NaOH. The

J. Wu et al. / Bioorg. Med. Chem. 18 (2010) 6220–6229
6227
37.05. Anal. Calcd for C30H27N3O4: C, 73.01; H, 5.51; N, 8.51. Found:
C, 72.78; H, 5.38; N, 8.74.
m), 7.48 (2H, m), 7.43 (1H, m), 7.34 (2H, m), 7.31 (m, 2H), 7.20
(1H, m), 5.41 (2H, s), 5.07 (2H, s), 3.50 (1H, m), 3.37 (2H, m),
3.16 (1H, m), 2.99 (1H, m), 2.47 (2H, m), 2.18 (1H, m), 2.08 (1H,
m), 2.05 (3H, s); 13C NMR (CDCl3) d: 166.30, 165.91, 144.61,
140.85, 136.98, 136.68, 136.07, 135.40, 128.76, 128.53, 128.48,
128.43, 128.32, 128.19, 128.09, 121.70, 121.60, 120.52, 116.62,
113.04, 67.17, 67.00, 61.46, 46.46, 36.34, 31.78, 29.73, 29.29. Anal.
Calcd for C33H33N3O4S: C, 69.82; H, 5.86; N, 7.40. Found: C, 69.61;
H, 5.71; N, 7.62.
4.8.2. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylalanine
benzylesters (6b)
Yield: 68%. ½a 2D0
ꢃ
= 30.49 (c 0.01, CHCl3), ESI/MS (m/e) 508
[M+H]+; 1H NMR (CDCl3) d: 12.32 (1H, s), 8.72 (1H, s), 8.12 (1H,
d, J = 7.8 Hz), 7.59 (1H, m), 7.57 (1H, m), 7.42 (1H, m), 7.39 (2H,
m), 7.36 (2H, m), 7.33 (3H, m), 7.30 (2H, m), 7.28 (1H, m), 5.49
(2H, s), 4.73 (2H, s), 3.65 (1H, m), 3.37(2H, m), 3.20 (2H, m), 1.50
(3H, s,); 13C NMR (CDCl3) d: 173.82, 165.94, 143.75, 140.96,
136.66, 136.31, 135.71, 128.66, 128.59, 128.57, 128.53, 128.47,
128.30, 128.24, 127.02, 122.02, 121.68, 120.41, 116.95, 112.47,
67.24, 65.32, 56.24, 52.48, 45.89, 38.05, 17.78. Anal. Calcd for
4.8.7. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylproline
benzylester (6g)
Yield: 68%. ½a 2D0
= ꢀ1.13 (c 0.01, DMSO), ESI/MS (m/e) 534
ꢃ
[M+H]+; 1H NMR (CDCl3) d: 12.42 (1H, br s), 8.80 (1H, s), 8.17
(1H, d, J = 8.0 Hz), 7.66 (1H, m), 7.64 (1H, m), 7.60 (1H, m), 7.58
(2H, m), 7.56 (2H, m), 7.44 (1H, m), 7.41 (2H, m), 7.38 (2H, m),
7.21 (1H, m), 5.54 (2H, s), 5.11 (2H, s), 3.51 (1H, m), 3.49 (2H,
m), 3.30 (2H, m), 2.99 (1H, m), 2.55 (1H, m), 2.30 (1H, m), 2.09
(1H, m), 1.91 (2H, m); 13C NMR (CDCl3) d: 174.56, 166.26,
145.34, 141.23, 137.04, 136.48, 135.97, 135.16, 128.54, 128.47,
128.36, 128.22, 128.19, 128.14, 127.60, 127.01, 122.04, 121.67,
120.12, 116.93, 112.76, 68.41, 66.98, 66.90, 54.51, 54.12, 36.42,
30.07, 24.00. Anal. Calcd for C33H31N3O4: C, 74.28; H, 5.86; N,
7.87. Found: C, 74.47; H, 5.70; N, 7.66.
C31H29N3O4: C, 73.35; H, 5.76; N, 8.28. Found: C, 73.16; H, 5.62;
N, 8.07.
4.8.3. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylvaline
benzylester (6c)
Yield: 68%. ½a 2D0
ꢃ
= 3.80 (c 0.01, CHCl3), ESI/MS (m/e) 536 [M+H]+;
1H NMR (CDCl3) d: 12.41 (1H, br s), 8.83 (1H, s), 8.16 (1H, d,
J = 8.4 Hz), 7.65 (1H, m), 7.62 (1H, m), 7.54 (1H, m), 7.47 (2H, m),
7.42 (2H, m), 7.40 (1H, m), 7.35 (2H, m), 7.30 (2H, m), 7.27 (1H,
m), 5.48 (2H, s), 5.17 (2H, s), 3.54 (1H, m), 3.38 (2H, m), 3.12
(2H, m), 2.21 (1H, m), 1.15 (3H, m), 1.12 (3H, m); 13C NMR (CDCl3)
d: 173.90, 166.17, 145.23, 140.68, 136.88, 136.40, 136.22, 135.32,
128.64, 128.55, 128.49, 128.47, 128.38, 128.23, 128.15, 122.19,
121.84, 120.33, 116.94, 112.27, 67.61, 66.95, 66.90, 47.02, 37.59,
31.80, 19.58, 18.95. Anal. Calcd for C33H33N3O4: C, 74.00; H, 6.21;
N, 7.84. Found: C, 74.22; H, 6.36; N, 7.62.
4.8.8. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylphenyl-
alanine benzylester (6h)
Yield: 88%. ½a 2D0
ꢃ
= 10.92 (c 0.01, CHCl3); ESI/MS (m/e) 584
[M+H]+; 1H NMR (CDCl3) d: 12.42 (1H, br s), 8.79 (1H, s), 8.17
(1H, d, J = 8.0 Hz, 7.58 (1H, m), 7.55 (1H, m), 7.48 (1H, m), 7.46
(2H, m), 7.44 (2H, m), 7.42 (1H, m), 7.40 (2H, m), 7.38 (2H, m),
7.35 (2H, m), 7.33 (2H, m), 7.20 (1H, m), 7.15 (1H, m), 5.53 (2H,
s), 5.18 (2H, s), 3.80 (1H, m), 3.37 (2H, m), 3.18 (1H, m), 3.13
(1H, m), 3.08 (2H, m); 13C NMR (CDCl3) d: 173.55, 166.18,
144.99, 140.80, 137.07, 136.89, 136.40, 136.23, 135.55, 135.12,
129.34, 128.85, 128.59, 128.53, 128.47, 128.45, 128.34, 128.16,
127.22, 126.84, 122.05, 121.75, 120.32, 116.93, 112.51, 67.05,
66.95, 62.66, 46.54, 39.16, 37.32. Anal. Calcd for C37H33N3O4: C,
76.14; H, 5.70; N, 7.20. Found: C, 75.93; H, 5.55; N, 7.01.
4.8.4. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylleucine
benzylester (6d)
Yield: 66%. ½a 2D0
= ꢀ3.03 (c 0.01, CHCl3), ESI/MS (m/e) 550
ꢃ
[M+H]+; 1H NMR (DMSO): d/ppm = 12.38 (1H, br s), 8.61 (1H, s),
8.06 (1H, d, J = 8.0 Hz), 7.61 (1H, m), 7.59 (1H, m), 7.52 (1H, m),
7.49 (2H, m), 7.44 (2H, m), 7.41 (1H, m), 7.37 (2H, m), 7.31 (2H,
m), 7.16 (1H, m), 5.49 (2H, s), 5.18 (2H, s), 3.68 (1H, m), 3.52
(2H, m), 3.23 (2H, s), 2.01 (1H, m), 1.78 (2H, d, J = 5.0 Hz), 1.00
(3H, d, J = 5.0 Hz), 0.95 (3H, d, J = 5.0 Hz); 13C NMR (DMSO) d:
171.58, 165.78, 144.04, 140.92, 136.40, 136.34, 135.08, 128.63,
128.60, 128.48, 128.42, 128.37, 127.02, 121.82, 120.41, 116.78,
112.44, 67.23, 66.92, 59.97, 46.36, 41.71, 25.12, 22.65, 22.35. Anal.
Calcd for C34H35N3O4: C, 74.29; H, 6.42; N, 7.64. Found: C, 74.08; H,
6.57; N, 7.41.
4.8.9. N-(3-Benzyloxycarbonylcarboline-1-yl)ethyltryptophan
benzylester (6i)
Yield: 54%. ½a 2D0
ꢃ
= 1.13 (c 0.01, CHCl3); ESI/MS (m/e) 623
[M+H]+; 1H NMR (DMSO) d: 12.20 (1H, s), 10.85 (1H, s), 8.83 (1H,
s), 8.39 (1H, d, J = 8.0 Hz), 7.63 (1H, m), 7.59 (1H, m), 7.55 (1H, d,
J = 8.0 Hz), 7.53 (1H, m), 7.51 (1H, m), 7.44 (2H, m), 7.42 (2H, m),
7.40 (1H, m), 7.37 (2H, m), 7.36 (1H, d, J = 8.0 Hz), 7.34 (1H, m),
7.24 (1H, s), 7.20 (1H, m), 7.10 (1H, t, J = 7.0 Hz), 7.06 (1H, t,
J = 7.0 Hz), 5.44 (2H, s), 5.02 (2H, s), 3.80 (1H, m), 3.40 (1H, m),
3.34 (2H, m), 3.01 (1H, m), 2.90 (2H, m); 13C NMR (CDCl3) d:
175.46, 165.96, 145.00, 141.27, 136.63, 136.61, 136.51, 136.48,
136.30, 128.65, 128.57, 128.50, 128.45, 128.39, 128.25, 128.16,
127.86, 124.17, 127.02, 122.62, 121.80, 121.32, 120.60, 118.69,
116.74, 112.82, 111.80, 110.24, 66.37, 65.98, 62.79, 60.50, 46.77,
31.22, 29.34. Anal. Calcd for C39H35N3O6: C, 72.99; H, 5.50; N,
6.55. Found: C, 72.78; H, 5.67; N, 6.78.
4.8.5. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylisoleucine
benzylester (6e)
Yield: 60%. ½a 2D0
ꢃ
= 6.57 (c 0.01, CHCl3), ESI/MS (m/e) 550 [M+H]+;
1H NMR (CDCl3) d: 12.40 (1H, br s), 8.79 (1H, s), 8.17 (1H, d,
J = 8.4 Hz), 7.62 (1H, m), 7.59 (1H, m), 7.51 (1H, m), 7.49 (2H, m),
7.46 (2H, m), 7.42 (1H, m), 7.32 (2H, m), 7.30 (2H, m), 7.28 (1H,
m), 5.52 (2H, s), 5.15 (2H, s), 3.52 (1H, m), 3.47 (2H, m), 3.19
(1H, m), 2.99 (1H, m), 2.07 (1H, m), 1.66 (1H, m), 1.43 (1H, m),
1.08 (3H, d, J = 6.9 Hz), 0.97 (3H, t, J = 7.5 Hz); 13C NMR (CHCl3) d:
173.81, 166.19, 145.24, 140.72, 136.91, 136.41, 136.22, 135.35,
128.62, 128.54, 128.48, 128.46, 128.38, 128.22, 128.15, 122.18,
121.48, 120.32, 116.94, 112.23, 66.95, 66.40, 60.41, 47.08, 38.64,
37.56, 25.92, 15.98, 11.71. Anal. Calcd for C34H35N3O4: C, 74.29;
H, 6.42; N, 7.64. Found: C, 74.48; H, 6.58; N, 7.86.
4.8.10. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylserine
benzylester (6j)
Yield: 68%. ½a 2D0
= ꢀ7.89 (c 0.01, CHCl3); ESI/MS (m/e) 524
ꢃ
[M+H]+; 1H NMR (DMSO) d: 12.29 (1H, s), 8.81 (1H, s), 8.37 (1H,
d, J = 8.30 Hz), 7.67 (1H, m), 7.64 (1H, m), 7.61 (1H, m), 7.59 (2H,
m), 7.56 (2H, m), 7.53 (1H, m), 7.44 (2H, m), 7.31 (2H, m), 7.24
(1H, m), 5.42 (2H, s), 5.11 (2H, s), 3.92 (1H, m), 3.72 (1H, m),
3.57 (1H, m), 3.15 (2H, m), 2.97 (2H, m). 13C NMR (DMSO) d:
173.42, 165.93, 145.04, 141.21, 137.05, 136.59, 136.31, 128.95,
4.8.6. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylmethionine
benzylester (6f)
Yield: 65%. ½a 2D0
= ꢀ26.55 (c 0.01, DMSO), ESI/MS (m/e) 568
ꢃ
[M+H]+; 1H NMR (CDCl3) d: 10.94 (1H, s), 8.51 (1H, s), 8.05 (1H,
d, J = 8.0 Hz), 7.66 (1H, m), 7.64 (1H, m), 7.59 (1H, m), 7.57 (2H,

6228
J. Wu et al. / Bioorg. Med. Chem. 18 (2010) 6220–6229
128.78, 128.51, 128.32, 128.15, 127.60, 122.59, 121.77, 120.58,
116.75, 112.87, 66.35, 65.98, 63.55, 62.79, 46.63, 34.88. Anal. Calcd
for C31H29N3O5: C, 71.11; H, 5.58; N, 8.03. Found: C, 71.30; H, 5.42;
N, 7.81.
(1H, d, J = 8.0 Hz), 7.67 (1H, m), 7.61 (1H, m), 7.59 (1H, m), 7.56
(m, 2H), 7.52 (2H, m), 7.49 (1H, m), 7.41 (5H, m), 7.38 (4H, m),
7.22 (1H, m), 5.40 (2H, s), 5.04 (s, 2H), 4.98 (2H, s), 3.75 (1H, m),
3.28 (1H, m), 3.23 (2H, m), 3.10 (1H, m), 2.74 (2H, m); 13C NMR
(CDCl3) d: 173.24, 170.78, 165.94, 144.87, 141.23, 137.04, 136.57,
136.45, 136.37, 136.32, 128.92, 128.85, 128.81, 128.74, 128.63,
128.45, 128.38, 128.30, 128.24, 127.62, 127.06, 126.87, 122.57,
121.80, 120.59, 116.67, 112.82, 66.34, 66.30, 66.05, 57.80, 46.42,
37.77, 34.47. Anal. Calcd for C39H35N3O6: C, 72.99; H, 5.50; N,
6.55. Found: C, 72.78; H, 5.36; N, 6.33.
4.8.11. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylthreonine
benzylester (6k)
Yield: 62%. ½a 2D0
= ꢀ11.02 (c 0.01, CHCl3); ESI/MS (m/e) 538
ꢃ
[M+H]+; 1H NMR (CDCl3) d: 12.34 (1H, s), 8.73 (1H, s), 8.13 (1H,
d, J = 8.0 Hz), 7.59 (1H, t, J = 8.0 Hz), 7.55 (1H, m), 7.52 (1H, m),
7.41 (2H, m), 7.39 (2H, m), 7.37 (1H, m), 7.34 (2H, m), 7.31 (m,
2H), 7.28 (2H, m), 5.48 (2H, s), 5.17 (2H, s), 4.06 (1H, m), 3.69
(1H, m), 3.32 (2H, m), 3.25 (2H, m), 1.31 (3H, d, J = 4.0 Hz); 13C
NMR (CDCl3) d: 172.68, 166.85, 144.06, 140.86, 136.56, 136.26,
136.16, 135.00, 128.64, 128.64, 128.52, 128.46, 128.29, 128.21,
121.95, 121.69, 120.49, 116.83, 112.47, 68.31, 68.21, 67.26, 52.62,
46.85, 35.95, 21.79. Anal. Calcd for C32H31N3O5: C, 71.49; H, 5.81;
N, 7.82. Found: C, 71.28; H, 5.97; N, 7.60.
a
x
4.8.16. N -(3-Benzyloxycarbonylcarboline-1-yl)-N -(benzyl-
oxycarbonyl)ethyllysine benzylester (6p)
Yield: 54%. ½a 2D0
ꢃ
= 2.53 (c 0.01, CHCl3); ESI/MS (m/e) 699
[M+H]+; 1H NMR (DMSO) d: 12.10 (1H, br s), 8.76 (1H, s), 8.14
(1H, d, J = 8.0 Hz), 7.54 (1H, m), 7.44 (1H, m), 7.40–7.42 (6H, m),
7.35–7.37 (5H, m), 7.32–7.34 (6H, m), 5.24 (2H, s), 5.16 (2H, s),
5.07 (2H, s), 3.74 (1H, m), 3.43 (2H, m), 3.18 (2H, m), 3.08 (2H,
m), 1.77 (2H, m), 1.50 (2H, m), 1.35 (2H, m). 13C NMR (DMSO) d:
175.82, 166.13, 156.39, 145.00, 140.75, 136.86, 136.62, 136.51,
136.39, 135.70, 135.34, 128.63, 128.52, 128.36, 128.12, 122.10,
121.84, 120.38, 116.90, 112.25, 66.95, 66.68, 65.86, 61.40, 46.60,
40.82, 34.36, 29.67, 22.76. Anal. Calcd for C42H42N4O6: C, 72.19;
H, 6.06; N, 8.02. Found: C, 72.00; H, 6.22; N, 8.26.
4.8.12. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylasparagine
benzylester (6l)
Yield: 33%. ½a 2D0
= ꢀ31.14 (c 0.01, CHCl3); ESI/MS (m/e) 551
ꢃ
[M+H]+; 1H NMR (CDCl3) d: 11.19 (1H, s), 8.84 (1H, s), 8.38 (1H,
d, J = 8.0 Hz), 7.68 (1H, d, J = 8.0 Hz), 7.60 (1H, t, J = 8.0 Hz), 7.58
(1H, m), 7.54 (2H, m), 7.52 (2H, m), 7.44 (2H, m), 7.40 (2H, m),
7.30 (2H, m), 7.24 (1H, m), 5.42 (2H, s), 5.07 (2H, s), 3.71 (1H,
m), 3.31 (2H, m), 3.29 (1H, m), 3.09 (1H, m), 2.74 (2H, m); 13C
NMR (CDCl3) d: 177.62, 173.99, 165.94, 144.96, 141.23, 137.05,
136.57, 136.36, 128.97, 128.88, 128.75, 128.55, 12.47, 128.30,
128.18, 127.62, 122.59, 121.78, 120.60, 116.75, 112.92, 66.37,
66.05, 58.07, 46.06, 37.50, 34.69. Anal. Calcd for C32H30N4O5: C,
69.80; H, 5.49; N, 10.18. Found: C, 69.61; H, 5.33; N, 10.39.
4.8.17. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylhistidine
benzylester (6q)
Yield: 42%. ½a 2D0
= ꢀ7.99 (c 0.01, CHCl3); ESI/MS (m/e) 574
ꢃ
[M+H]+; 1H NMR (DMSO) d: 12.82 (1H, br s), 12.45 (1H, br), 8.87
(1H, s), 8.67 (1H, m), 8.40 (1H, d, J = 8.0 Hz), 7.69 (1H, m), 7.61
(1H, m), 7.43 (1H, m), 7.39 (4H, m), 7.35 (4H, m), 7.24 (2H, m),
5.42 (2H, s), 4.49 (2H, s), 3.65 (1H, m), 3.28 (1H, m), 3.23 (2H,
m), 3.10 (1H, m), 2.78 (2H, m).;13C NMR (DMSO) d: 172.51,
166.47, 143.00, 140.96, 136.67, 136.33, 135.70, 133.50, 128.65,
128.58, 128.56, 128.53, 128.47, 128.31, 128.24, 127.04, 122.01,
121.69, 120.41, 119.42, 116.95, 112.47, 63.34, 63.27, 59.90, 45.59,
37.77, 34.87. Anal. Calcd for C34H31N5O4: C, 71.19; H, 5.45; N,
12.21. Found: C, 70.99; H, 5.30; N, 12.00.
4.8.13. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylglutamine
benzylester (6m)
Yield: 68%. ½a 2D0
= ꢀ5.8 (c 0.01, CHCl3); ESI/MS (m/e) 565
ꢃ
[M+H]+; 1H NMR (CDCl3) d: 12.12 (1H, s), 8.86 (1H, s), 8.24 (1H,
d, J = 8.0 Hz), 7.61 (1H, m), 7.59 (1H, m), 7.57 (1H, m), 7.54 (2H,
m), 7.51 (2H, m), 7.43 (1H, m), 7.41 (1H, m), 7.32 (2H, m), 7.26
(1H, m), 5.42 (2H, s), 5.07 (2H, s), 3.65 (1H, m), 3.36 (2H, m),
3.12 (1H, m), 2.97 (1H, m), 2.22 (2H, m), 2.07 (2H, m); 13C NMR
(CDCl3) d: 175.16, 173.69, 166.47, 143.47, 142.44, 137.22, 137.07,
136.82, 128.70, 128.54, 128.53, 128.45, 128.27, 128.21, 121.67,
121.59, 120.58, 116.30, 110.34, 68.20, 67.32, 67.25, 47.40, 37.68,
34.4527.42. Anal. Calcd for C33H32N4O5: C, 70.20; H, 5.71; N,
9.92. Found: C, 70.01; H, 5.56; N, 9.70.
4.8.18. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylglutamic
acid dibenzylester (6r)
Yield: 57%. ½a 2D0
= ꢀ30.05 (c 0.01, DMSO), ESI/MS (m/e) 548
ꢃ
[M+H]+; 1H NMR (CDCl3) d: 11.22 (1H, s), 8.78 (1H, s), 8.17 (1H,
d, J = 8.4 Hz), 7.67 (1H, m), 7.64 (1H, m), 7.60 (1H, m), 7.56 (2H,
m), 7.53 (2H, m), 7.43 (1H, m), 7.40 (2H, m), 7.37 (2H, m), 7.28
(1H, m), 5.54 (2H, s), 5.11 (2H, s), 3.51 (1H, m), 3.30 (2H, m),
2.99 (2H, m), 2.55 (1H, m), 2.30 (1H, m), 2.22 (1H, m), 2.09 (1H,
m). 13C NMR (CDCl3) d: 174.56, 166.26, 145.34, 141.23, 137.04,
136.48, 135.97, 135.16, 128.54, 128.47, 128.36, 128.22, 128.19,
128.14, 127.60, 127.01, 122.04, 121.67, 120.12, 116.93, 112.76,
68.41, 66.98, 66.90, 54.51, 54.12, 36.42, 30.07, 24.00. 177.75,
171.70, 166.04, 142.79, 140.97, 136.63, 135.07, 134.94, 128.77,
128.72, 128.67, 128.62, 128.57, 128.47, 128.40, 128.20, 127.58,
127.00, 121.89, 120.56, 117.30, 112.51, 67.56, 66.96, 61.56, 41.99,
33.67, 29.64, 24.80. Anal. Calcd for C33H29N3O5: C, 72.38; H, 5.34;
N, 7.67. Found: C, 72.16; H, 5.18; N, 7.44.
4.8.14. N-(3-Benzyloxycarbonylcarboline-1-yl)ethyltyrocine
benzylester (6n)
Yield: 69%. ½a 2D0
ꢃ
= 8.83 (c 0.01, CHCl3); ESI/MS (m/e) 600
[M+H]+; 1H NMR (CDCl3) d: 12.43 (1H, s), 8.78 (1H, s), 8.14 (1H,
d, J = 8.0 Hz), 7.56 (1H, m), 7.52 (1H, m), 7.50 (1H, m), 7.40 (2H,
m), 7.38 (2H, m), 7.36 (1H, m), 7.32 (2H, m), 7.28 (2H, m), 7.22
(1H, m), 6.92(2H, m), 6.67(2H, m), 5.47 (2H, s), 5.18 (2H, s), 3.78
(1H, m), 3.32 (1H, m), 3.23 (1H, m), 3.07 (2H, m), 2.98 (2H, m);
13C NMR (CDCl3) d: 173.65, 166.09, 155.66, 144.69, 140.93,
136.85, 136.18, 135.39, 135.09, 130.95, 130.22, 130.03, 128.65,
128.61, 128.55, 128.52, 128.42, 128.22, 127.83, 126.92, 121.90,
121.76, 120.43, 117.02, 115.99, 115.80, 112.58, 67.12, 66.99,
62.79, 46.46, 39.67, 36.30. Anal. Calcd for C37H33N3O5: C, 74.11;
H, 5.55; N, 7.01. Found: C, 74.30; H, 5.40; N, 6.79.
4.9. Bioassay
4.9.1. In vitro anti-proliferation assay of 6a–r
According to the slightly modified procedure of Al-Allaf and Ra-
shan,9 the in vitro anti-proliferation assays were carried out by use
of 96 microtiter plate cultures and MTT staining. The HepG2, Hela,
Bel7402, K562, and B16 cells (final concentration in the growth
medium was 1 ꢂ 104/ml) were grown in RPMI-1640 medium (con-
4.8.15. N-(3-Benzyloxycarbonylcarboline-1-yl)ethylaspartic acid
dibenzylester (6o)
Yield: 50%. ½a 2D0
= ꢀ7.96 (c 0.01, CHCl3); ESI/MS (m/e) 642
ꢃ
[M+H]+; 1H NMR (CDCl3): d/ppm = 12.11 (1H, s), 8.82 (1H, s), 8.37
taining 10%, v/v) FCS, penicillin (100 lg/ml) and streptomycin

J. Wu et al. / Bioorg. Med. Chem. 18 (2010) 6220–6229
6229
(100
l
g/ml) without (for control) or with 6a–r (in a series of final
M to 200 M). The cultures were
tumor growth was calculated according to the formula of Section
4.9.2.
concentrations ranging from 0.1
l
l
propagated at 37 °C in a humidified atmosphere containing 5% CO2
for 24 h, after the first renew of the growth medium without (for
control) or with 6a–r (for test sample) were propagated for another
4.10. Viscometric measurement
48 h, after the second renew of the growth medium with 25
MTT solution (5 mg/ml) were propagated for another 4 h. The
growth medium was removed and the residue was dried in
the air. The dried residues were dissolved in 100 ll of DMSO and
the absorption values of light of the formed purple solutions were
recorded on Bio-rad 570 microplate reader (Biorad, USA). The
inhibited rates were calculated according to I% = % (C ꢀ T/C).
Wherein C presents the absorption value of light of the control
and T presents the absorption value of light of the test sample.
l
l of
Ostwald type viscometer, immersed in a thermostated water
bath maintained at 28 °C, was used. CT DNA or 6d was dissolved
in phosphate buffer to prepare solutions. Sample solutions were
prepared by adding 6d solution separately to DNA solution so as
to give the [6d]/[DNA] ratios in the range of 0–0.36. The flow time
of the sample solutions were measured after a thermal equilibrium
time of 5 min. The flow time of each sample was calculated. The
relative viscosity of DNA in the presence and absence of 6d were
calculated according
served flow time in the absence and presence of 6d. Data has been
presented as ( is the viscosity
1/3 versus binding ratio, where
g
= (t ꢀ t0)/t0, where t0 and t were the ob-
The IC50 values were determined via program GWBASIC.EXE
.
g
/g0)
g
4.9.2. In vivo anti-tumor assay of 6a–r
of DNA in the presence of 6d and g0 is the viscosity of DNA alone.
Male ICR mice, purchased from Peking University Health Sci-
ence Center, were maintained at 21 °C with a natural day/night cy-
cle in a conventional animal colony. The mice were 10–12 weeks
old at the beginning of the experiments. The tumor used was
S180 that forms solid tumors, when injected subcutaneously.
S180 cells for initiation of subcutaneous tumors were obtained
from the ascitic form of the tumors in mice, which were serially
transplanted once per week. Subcutaneous tumors were implanted
by injecting 0.2 ml of NS containing 1 ꢂ 107 viable tumor cells
under the skin on the right oxter. Twenty-four hours after implan-
tation, the tumor-bearing mice were randomized into 20 experi-
mental groups (10 per group). All the mice were given a daily ip
Acknowledgments
This work was completed in the Beijing area major laboratory of
peptide and small molecular drugs, supported by PHR (IHLB, KM
200910025010), the National Natural Scientific Foundation of Chi-
na (30901843, 30801426), and Special Project (2008ZX09401-002)
of China.
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injection of cytarabine (positive control, 89, 8.9 or 0.9
day in 0.2 ml of NS), or a daily oral administration of NS (negative
control, 0.2 ml), or 6j,o (89, 8.9 or 0.9 mol/kg/day in 0.2 ml of NS)
lmol/kg/
l
for seven consecutive days. Twenty-four hours after the last
administration, all mice were sacrificed by diethyl ether anesthesia
and the tumors were dissected and weighed. The inhibitory rate of