Design, synthesis and 3D-QSAR of β-carboline derivatives as potent antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2010.02.036

In a continuing effort to develop novel β-carbolines endowed with better pharmacological profiles, a series of β-carboline derivatives were designed and synthesized based on the previously developed SARs. Cytotoxicities in vitro of these compounds against

Full text of DOI:10.1016/j.ejmech.2010.02.036

European Journal of Medicinal Chemistry 45 (2010) 2503e2515
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and 3D-QSAR of
antitumor agents
b-carboline derivatives as potent
,
b
a
a
b
b
a
Rihui Cao a , Xiangdong Guan , Buxi Shi , Zhiyong Chen , Zhenhua Ren , Wenlie Peng , Huacan Song
*
a School of Chemistry and Chemical Engineering, Sun Yat-sen University, 135 Xin Gang West Road, Guangzhou 510275, PR China
b School of Life Sciences, Sun Yat-sen University, 135 Xin Gang West Road, Guangzhou 510275, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In a continuing effort to develop novel
b
-carbolines endowed with better pharmacological profiles,
Received 25 July 2009
Received in revised form
13 February 2010
Accepted 16 February 2010
Available online 19 February 2010
a series of
SARs. Cytotoxicities in vitro of these compounds against a panel of human tumor cell lines were also
investigated. The results demonstrated that the N2-benzylated
-carbolinium bromides 56e60 repre-
b-carboline derivatives were designed and synthesized based on the previously developed
b
sented the most potent compounds with IC50 values lower than 10 mM. The application of 3D-QSAR to
these compounds explored the structural basis for their biological activities. CoMFA (q2 ¼ 0.513, r2
¼
0.862) and CoMSIA (q2 ¼ 0.503, r2 ¼ 0.831) models were developed for a set of 47
b-carbolines. The
Keywords:
Synthesis
results indicated that the antitumor pharmacophore of these molecules were marked at position-1, -2,
-3, -7 and -9 of b-carboline ring.
b
-Carboline
Ó 2010 Elsevier Masson SAS. All rights reserved.
Antitumor
3D-QSAR
1. Introduction
(SARs) analysis suggested that the introduction of appropriate
substituents into position-2, -3 and -9 played a vital role in deter-
mining their antitumor effects [37,38,41].
The
b-carboline nucleus is common to many natural and
synthetic products associated with a broad spectrum of biochem-
ical effects and pharmaceutical properties. These compounds have
been shown to intercalate into DNA [1e3], to inhibit CDK [4,5],
topisomerase [1,3,6] and monoamine oxidase [7e9], and to interact
with benzodiazepine receptors (BZ) [10e12], 5-hydroxy serotonin
receptors (5-HT) [13,14], dopamine (DA) [15] and imidazoline
Despitetheserecentundoubtedadvances, b-carbolinederivatives
still present some limitations arising from the relative weak anti-
tumor activities in animal models and the poorly understood action
of mechanism [38]. Obviously, to acquire more information about the
structural requirements for the possible improvement of the cyto-
toxic potential and to elucidate SARs between substituents proper-
receptors [16,17]. In addition, individual
might selectively interact with specific targets so as to lead to
a variety of pharmacological actions in vitro and in vivo. So far,
b
-carboline derivative
ties in
more novel
different position of the
b
-carboline and antitumor activities, design and synthesis of
-carboline derivatives with various substituents at
-carboline nucleus are needed.
b
b
b
-carboline derivatives has been found to have various pharma-
In this context we began a systematic, long term study aiming at
the synthesis of novel antitumor agents endowed with better
pharmacological profiles. In the present report we described the
design and synthesis, and the biological and 3D-QSAR studies of
novel potent antitumor agents bearing various substituents in
ceutical functions including sedative, anxiolytic, hypnotic, anti-
convulsant [18e20], antimicrobial [21,22], antiviral [23,24],
parasiticidal [25,26] as well as antithrombotic activities [27,28].
Previous investigations focused on the effects of b-carbolines on
the central nervous system (CNS). However, interests in these
alkaloids were stimulated by their promising antitumor activities in
the last decades. Recent reports [2, 29e34] our group investigations
position-1, -2, -3, -7 and -9 of
this study was to elucidate the antitumor structureeactivity rela-
tionships (SARs) of -carbolines in finer detail, with the ultimate
b-carboline ring. The aim purpose of
b
[35e40] on the synthesis of a variety of
the evaluation of their antitumor activities unraveled that
b
-carboline derivatives and
-car-
aim of developing a reliable 3D-QSAR model to probe the structural
requirement at the 3D levels for highly potent antitumor activity.
b
bolines had potent antitumor activities and the activities was
correlated to both the planarity of the molecule and the presence of
the ring substituents. Preliminary structureeactivity relationships
2. Chemistry
1-(3,4,5-Trimethoxy)phenyl substituted
synthesized according to already published methods [39]. The
b-carbolines 1e4 were
* Corresponding author. Tel.: +86 20 84110918; fax: +86 20 84112245.
E-mail address: caorihui@mail.sysu.edu.cn (R. Cao).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.036

2504
R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
preparation of compounds 5e8, 10, 13, 16, 25e28, 36e37 and 39
have been already described as antitumor agents in our previous
reports [35,36,40]. Compound 9 was easily prepared in good
yield according to the published method described by our group
[35].
3. Results and discussion
3.1. Cytotoxicities in vitro
The cytotoxic potential of all synthesized b-carboline derivatives
Previously, Xiao et al. [2] and our group [40] reported the
was evaluated in vitro against a panel of human tumor cell lines.
Compound 3 was converted into its water-soluble sodium salts, and
the other compounds (except 29e35 and 56e60) examined were
prepared in the form of hydrochloride by the usual methods before
use. The results were summarized in Table 1.
synthesis of
b-carbolines bearing an alkylamine side chain in
position-3 in 18e27% yield. To improve yield and reaction condi-
tions, in the present investigation, we have developed a straight-
forward cost-effective method involving microwave irradiation for
the preparation of
position-3 in 82e90% yield. The crude mixture of ethyl 9-n-butyl-
b
-carbolines bearing an alkylamine side chain in
The first analysis of the structureeactivity relationship con-
cerned a general comparison of the cytotoxic activities among the
four class of compounds described in this report. Compounds
b
-
carboline-3-carboxylate 5 and ethylenediamine was subjected to
microwave irradiation (150 ꢀC, 15 min) to give the amidated
derivatives 11 in 88% yield. The same synthetic protocol was used
for the preparation of derivatives 12, 14e15 and 17e18 from the
starting materials compounds 6, 7 and 8, respectively (Scheme 1).
Compared to the reaction time of 48 h under the conventional
heating and refluxing, this synthetic protocol only took 15 min to
finish the reaction under microwave irradiation.
bearing a 3,4,5-trimethoxyphenyl substituent at position-1 of b-
carboline ring gave the poorest cytotoxic activities whereas
compounds bearing substituents at position-1, -2, -7 and 9 repre-
sented the most interesting class of antitumor agents with IC50
values lower than 10
investigated.
mM against most of human tumor cell lines
Compounds 1e4 bearing a 3,4,5-trimethoxyphenyl substituent
at position-1 exhibited weak to inactive cytotoxic activities, sug-
gesting that the introduction of 3,4,5-trimethoxyphenyl substit-
uent at position-1 had no positive effects on cytotoxic activities.
Of all 3, 9-disubstituted and 1,3,9-trisubstituted compounds
9e24, compounds 11e12, 14e15 and 17e18, bearing a flexible
aminoalkyl side chain at postion-3, displayed moderate to signifi-
cant cytotoxic activities. Whereas compounds 13, 16 and 19e20,
incorporating a hydroxyethyl substituent in position-3, only had
marginal or no cytotoxic effect in any cell lines. These results
indicated that hydroxyethyl side chain was unsuitable for the
development of potent antitumor agents. Moreover, the substitu-
The ester group in position-3 of compounds 6 and 9 was
reduced to its corresponding alcohols by lithium borohydride
(LiBH4) in dry THF to provide compounds 21 and 22 [39], and
further oxidized by MnO2 in CH3CN to afford 3-carboxaldehyde
derivatives 23 and 24 [39] (Scheme 2).
The N2-alkylated or benzylated
b-carbolinium bromide deriva-
tives 29e35 (Scheme 3) were prepared from the known
compounds 25e28 by the addition of the corresponding alkylating
or benzylating agents in refluxing ethyl acetate [38]. The same
synthetic protocol was used for the preparation of the N2-benzy-
lated b-carbolinium bromide derivatives 56e60 (Scheme 4).
The N9-alkylated harmine derivatives 36e37 and 39 were
prepared according to the synthetic protocol described by our
group [35]. The same synthetic procedure was used for the prep-
aration of compound 38 from harmine and the commercially
available isobutyl bromide. The preparation of compounds 40e43
followed a common synthetic scheme, characterized by demethy-
lation of compound 36e39 using acetic acid and hydrobomic acid
as reaction solvent (Scheme 4).
tion of the b-carboline ring with hydroxymethyl and carbox-
aldehyde determined a negative effect on the cytotoxic activities of
derivatives 21e22 and 23e24, suggesting that this class of
substituents was unfavorable.
The N2-alkylated and arylated derivatives 29e35, as already
reported in the literature [37,38], showed potent cytotoxic activities
and the cytotoxic potency of derivatives followed the tendency of
33 (N2-phenylpropyl) > 34 (N2-benzyl) > 35 (N2-n-butyl). A similar
tendency also applied to derivatives 29 and 30 bearing a benzyl and
phenylpropyl at position-2, respectively.
Compounds 44e55, bearing alkoxy in postion-7 of b-carboline
ring, were synthesized from compounds 40e43 by the action of
sodium hydride in dry DMF followed by addition of the appropriate
alkylating and arylating agents in 71e86% yield.
The cytotoxic potency of most 1,7,9-trisubstituted b-carboline
derivatives 44e55 showed no distinct difference and the IC50
R3
N
COOC2H5
N
N
R9
R1
N
R9
R1
11 R1=H R9=n-C4H9
R3=CONH(CH2)2NH2
R3=CONH(CH2)6NH2
R3=CONH(CH2)2OH
R3=CONH(CH2)2NH2
R3=CONH(CH2)NH2
R3=CONH(CH2)2OH
R3=CONH(CH2)2NH2
R3=CONH(CH2)6NH2
5 R1=H
6 R1=CH3 R9 =n-C4H9
7 R1=H R9=CH2C6H5
R9=n-C4H9
12 R1=H R9=n-C4H9
13 R1=CH3 R9=n-C4H9
14 R1=CH3 R9=n-C4H9
15 R1=CH3 R9=CH2C6H5
16 R1=H R9=CH2C6H5
17 R1=H R9=CH2C6H5
18 R1=H R9=CH2C6H5
8 R1=CH3 R9=CH2C6H5
9 R1=H
R9=CH2C6H4(p-F)
R9=(CH2)3C6H5
10 R1=H
19 R1=H R9=CH2C6H4(p-F) R3=CONH(CH2)2OH
20 R1=H R9=(CH2)3C6H5 R3=CONH(CH2)2OH
Scheme 1. Synthesis of 1,3,9-trisubstituted b-carbolines 11e20.

R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
2505
CH2OH
COOC2H5
CHO
N
N
N
N
R9
N
R9
R1
N
R9
R1
R1
21 R1=CH3 R9=n-C4H9
6 R1=CH3 R9 =n-C4H9
9 R1=H R9=CH2C6H4(p-F)
23 R1=CH3 R9=n-C4H9
22 R1=H
R9=CH2C6H4(p-F)
24 R1=H
R9=CH2C6H4(p-F)
Scheme 2. Synthesis of 1,3,9-trisubstituted
b-carbolines 21e24.
values of this class of compounds were range from 10 to 100
m
M.
reasonable considering the number of compounds used to derive
the models. The standard error of estimate of CoMFA and CoMSIA
models were also reasonably low amounting to 0.223 and 0.247. F-
test results were 42.583 and 33.517 for CoMFA and CoMSIA models,
respectively.
Exceptionally, compound 45 bearing a pentafluorobenzoxyl at
position-7 displayed weak cytotoxic activity against human tumor
cell lines, and the poor water-soluble property might be contrib-
uted to its weak activity.
The N2-alkylated and arylated 1,7,9-trisubstituted
b
-carboline
To further test the validity of the 3D-QSAR models, as well as
their predictive utility, an inactive test set made up of 8 compounds
derivatives 56e60 represented the most interesting cytotoxic
activities. As predicted, the IC50 values of these compounds were
without determinate IC50 values (IC50 > 200 mM) were employed as
lower than 10
estingly, compound 57 was found to be the most active derivative
with IC50 value of 0.93 M against Hela cell line. These results
further confirmed that the position of the N2-arylated substituent
on the -carboline ring played a central role in the modulation of
m
M against most of human tumor cell lines. Inter-
external testing data. The predictive pIC50 values of the test set
were obtained by using the predict property function of the QSAR
module in SYBYL6.9. Considering the pIC50 data of the training set
we selected to construct the 3D-QSAR models were range from 3.63
to 5.80, we considered a predictive pIC50 value lower than 4 here
was reasonably inactive. The predictive results were summarized in
Table 3. Both CoMFA and CoMSIA model shown fairly reliable
predictive abilities as 7 out of 8 compounds exhibited predictive
pIC50 values lower than 4 for CoMFA, while for CoMSIA all predic-
tive pIC50 values were lower than 4.
Figs. 2 and 3 depicted the correlation between the actual pIC50
and the predictive pIC50 of CoMFA and CoMSIA model, respectively,
demonstrating again the fair reliability of CoMFA and CoMSIA
models. PLS Statistics of both CoMFA and CoMSIA models indicated
that CoMFA model was somewhat better than CoMSIA model.
3D-QSAR contour maps of both CoMFA and CoMSIA models
were depicted in Fig. 4, compound 49 with the highest pIC50 value
was chosen to superimpose into the contour maps to better illus-
trate the SAR information. The steric contour maps are represented
in green and yellow while the electrostatic contour maps are rep-
resented in red and blue. The green contours are indicative of
favorable regions for sterically bulkier groups and the yellow
contours are indicative of regions that are sterically less favorable.
In a similar way the red contours represent regions that lead to the
enhancement of activity with electron rich groups, and contrary to
that the blue regions represent electron deficient regions and can
lead to an increase in the activity of compounds.
m
b
the cytotoxic activities.
3.2. 3D-QSAR study
All synthesized
common three-ring structure of
b
-carboline derivatives were aligned to the
-carboline, and compound 57
b
with the highest pIC50 value was chosen as the template molecular.
The aligned compounds were depicted in Fig. 1.
To further investigate the SARs of b-carbolines in more details,
the cytotoxic data of HepG2 cell line were used to build the 3D-
QSAR models. 40 compounds with determinate IC50 values were
employed and their pIC50 values were calculated based on the
original IC50 data.
The PLS statistic parameters of CoMFA and CoMSIA models were
summarized in Table 2. The cross-validation correlation coefficient
q2 which marked the predictive capacity and the conventional
correlation coefficient r2 which marked self-consistence were two
key parameters to evaluate the qualities of PLS analysis. A q2 value
over 0.3 is considered significant for the chance of significant
correlation being <95%. For CoMFA model, the q2 and r2 was 0.513
and 0.862, while for CoMSIA model was 0.503 and 0.831. The
statistic results indicated the good predictive ability of CoMFA and
CoMSIA models. The optimal number of components using to
generate both CoMFA and CoMSIA model is 5, which were
For CoMFA model, the steric term contributed 65.3% to the
interacting energy, indicated the SAR of
b-carboline were more
R3
R3
R2Br
EA
N
N+ R2
Br
R7
R7
-
R1
R1
N
N
H
H
29 R1=CH3
30 R1=CH3
31 R1=CH3
32 R1=CH3
R2=CH2C6H5
R3=R7=H
25 R1=CH3 R3=R7=H
R2=(CH2)3C6H5 R3=R7=H
26 R1=CH3 R3=CO2C2H5 R7=H
27 R1=CH3 R3=H R7=OCH3
28 R1=R3=R7=H
R2=CH2C6H5
R2=CH2C6H5
R3=CO2C2H5 R7=H
R3=H R7=OCH3
33 R1=R3=R7=H R2=n-C4H9
34 R1=R3=R7=H R2=CH2C6H5
35 R1=R3=R7=H R2=(CH2)3C6H5
Scheme 3. Synthesis of N2-alkylated quaternary
b-carbolines 29e35.

2506
R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
N
DMF/NaH
N
H3CO
N
R9
H3CO
R9Br
CH3
N
H
CH3
36 R9=C2H5
37 R9=n-C4H9
38 R9=i-C4H9
27
39 R9=(CH2)3C6H5
DMF/NaH
R7Br
N
HAc/HBr
HO
N
R7O
N
CH3
N
R9
R9
CH3
40 R9=C2H5
41 R9=n-C4H9
42 R9=i-C4H9
44 R9=C2H5 R7=C2H5
45 R9=C2H5 R7=CH2C6F5
46 R9=n-C4H9 R7=C2H5
43 R9=(CH2)3C6H5
47 R9=n-C4H9 R7=CH(CH3)2
48 R9=n-C4H9 R7=n-C4H9
49 R9=n-C4H9 R7=n-C10H21
50 R9=i-C4H9 R7=n-C4H9
51 R9=i-C4H9 R7=CH2C6H5
52 R9=(CH2)3C6H5 R7=CH(CH3)2
53 R9=(CH2)3C6H5 R7=n-C8H17
54 R9=(CH2)3C6H5 R7=CH2C6H5
55 R9=(CH2)3C6H5 R7=CH2C6F5
-
Br
N+ CH2
R2Br
EA
R7O
N
R9
CH3
56 R9=C2H5
57 R9=C2H5
R7=C2H5
R7=CH2C6F5
58 R9=i-C4H9 R7=n-C4H9
59 R9=i-C4H9 R7=CH2C6H5
60 R9=(CH2)3C6H5 R7=n-C8H17
Scheme 4. Synthesis of 1,7,9-trisubstituted and 1, 2, 7, 9-tetrasubsituted b-carbolines 40e60.
influenced by steric effect than by electrostatic effect. The analysis
of the contour signals represented in CoMFA map (Fig. 4A) leads to
a conclusion that a high cytotoxic potency is associated to
compounds placing hydrophobic bulky groups in two green regions
in position-7 and -9 (i.e. compounds 48 and 52); and electroposi-
tive atoms (nitrogen atoms) in the blue region located near
position-2 (i.e. compounds 39 and 45) and position-3 (i.e.
compounds 11 and 21); and electron rich groups such as benzyl or
pentafluorobenzyl in position-2, -7 and -9 due to the electrostatic
attractive effect with the red regions there (i.e. compounds 49 and
51). Noticeably the blue region in position-3 was in good agreement
with the electropositive DNA targeting substituent eCONH
(CH2)nNH2 that favored the antitumor activities.
Low activities compounds are experiencing steric repulsive
contact with the yellow region in the far area of position-7 (i.e.
compounds 33 and 47) due to the long alkyl groups; and electro-
static repulsive interaction with the blue region in position-3 due to
the electronegative groups (i.e. compounds 6 and 34). Another
electrostatic signal was visible in the far area of position-3, where
a red region indicate the electropositive atoms here are unfavorable
for the antitumor cytotoxicity (compounds 23 and 24), this signal
also indicated that the DNA targeting sequence eCONH(CH2)nNH2
with a long carbon side chain (n ¼ 6) were showing lower anti-
tumor activities, this decreasing of cytotoxicity was considered to
be constrained by the steric hindrance between two DNA strands as
we described in our previous investigation.
For CoMSIA model, the SAR signals representing in the resulting
contour map (Fig. 4B) are similar and simpler than the CoMFA map,
confirming the pharmacophore observations made in the CoMFA
map analysis, particularly the contour signals placing in position-7
and -9 that hydrophobic bulky substituents, represented by the
steric green regions and the electronegative red regions, favored
the cytotoxicity; and at position-2 that presence of benzyl
substituted, represented by the blue region in the electropositive
nitrogen atom and red region in the electron rich benzene group,
are associated with high cytotoxicity. Differ from CoMFA map,
a yellow region can be seen near position-1 of CoMSIA map,
explaining the decreasing cytotoxic activities of compounds with
the bulkier substituent 3,4,5-trimethoxyphenyl in position-1 (i.e.
compounds 1 and 2), and also explaining the fairly higher activities
of compounds with relative smaller methyl substituted in position-
1 (i.e. compounds 20 and 41).
In conclusion, the pharmacophore information of b-carbolines
detected by the CoMFA and CoMSIA maps were in good agreement
with most structural features of the SARs analysis elucidated
before.
4. Conclusion
In summary, a number of novel
in this paper proved to be potent antitumor agents. The N2-benzy-
lated -carbolinium bromides 56e60 were found to be the most
b-carboline derivatives described
b

R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
2507
Table 1
Chemical structures and cytotoxic activities of
b
-carboline derivatives in vitro.c
R3
N+ R2
R7
Br
-
R1
N
R9
Compd
Substituents
R1
IC50(m
M)a
d
R2
R3
R7
R9
H
n-C4H9
n-C4H9
n-C4H9
C6H4(p-F)
n-C4H9
n-C4H9
HepG2b
Helab
Bel-7402b
BGC-823b
MCF-7b
pIC50
1
2
3
4
3,4,5-trimethoxyphenyl
3,4,5-trimethoxyphenyl
3,4,5-trimethoxyphenyl
3,4,5-trimethoxyphenyl
H
H
H
CH3
CH3
CH3
H
H
H
H
H
CH3
H
CH3
H
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
CO2C2H5
CO2C2H5
CO2H
CONH(CH2)2OH
COOC2H5
CONH(CH2)2NH2
CONH(CH2)6NH2
CONH(CH2)2OH
CONH(CH2)2NH2
CONH(CH2)2NH2
CONH(CH2)2OH
CONH(CH2)2NH2
CONH(CH2)6NH2
CONH(CH2)2OH
CONH(CH2)2OH
CH2OH
CH2OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
OH
OH
OH
167
>200
208
87.4
>200
82.9
95.8
140
21.9
34.8
>200
104
58.7
>200
>200
128
>200
>200
166
>200
>200
>200
>200
187
>200
>200
115.2
58.5
90.6
56
53.6
>200
51.7
56.8
>200
>200
122
>200
27.3
36.2
37.2
>200
53.2
110.3
45.0
17.3
20.3
>200
11.9
34.8
>200
>200
127
>200
>200
>200
77.4
3.5
55.3
67.4
326
91.8
7.1
160
90.2
140
29.9
57.1
238
37.2
19.1
15.8
28.4
12.1
36.0
16.3
17.4
19.5
7.88
9.2
123
>200
115
3.78
e
3.68
4.06
e
105
9
>200
18.5
42.8
97.3
7.5
11
12
13
14
15
16
17
18
19
20
21
22
23
24
29
30
31
32
33
34
35
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
26.9
4.08
4.02
3.85
4.66
4.46
e
53.0
80.4
13.3
11.0
>200
42.8
24.2
>200
>200
100
>200
169
>200
69.2
49.8
101
77.6
130
85.4
44.3
70.3
89.8
68.3
45.3
30.7
n-C4H9
n-C4H9
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
C6H4(p-F)
(CH2)3C6H5
n-C4H9
C6H4(p-F)
n-C4H9
C6H4(p-F)
H
H
H
H
H
H
H
C2H5
4.7
>200
51.6
54.1
>200
>200
107
3.98
4.23
e
e
3.89
e
>200
143
>200
123
>200
154
CHO
CHO
H
H
3.84
e
>200
69.5
33.0
53.0
54.6
94.0
77.3
44.6
134
81.5
116
28.3
68.9
236
43.3
30.2
15.5
127
12.1
22.2
14.6
105
15.7
147
14.3
1.6
>200
74.0
20.5
56.2
64.2
302
65.1
24.6
165
69.5
114
35.1
27.4
241
36.1
26.8
69.9
18.8
42.5
26.8
36.2
21.8
29.4
18.6
12.6
4.0
>200
89.2
23.8
44.9
76.2
338
91.3
5.7
106
46.9
96.9
34.5
14.4
103
12.0
19.5
17.3
62.7
7.2
54.1
45.3
45.9
63.3
38.7
8.1
CH3
CH3
CH3
CH3
H
CH2C6H5
(CH2)3C6H5
CH2C6H5
CH2C6H5
n-C4H9
CH2C6H5
(CH2)3C6H5
e
4.16
4.48
4.28
4.26
4.03
4.11
4.35
3.87
4.09
3.94
4.55
4.16
3.63
4.36
4.52
4.81
3.90
4.92
4.65
4.84
3.98
4.80
3.83
4.84
5.80
5.74
5.72
5.41
CO2C2H5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
e
n-C4H9
i-C4H9
(CH2)3C6H5
C2H5
e
e
OH
e
OC2H5
OCH2C6F5
OC2H5
OCH(CH3)2
OC4H9
OC10H21
OC4H9
OCH2C6H5
OCH(CH3)2
OC8H17
OCH2C6H5
OCH2C6F5
OC2H5
O CH2C6F5
OC4H9
e
C2H5
258
e
n-C4H9
n-C4H9
n-C4H9
n-C4H9
i-C4H9
i-C4H9
(CH2)3C6H5
(CH2)3C6H5
(CH2)3C6H5
(CH2)3C6H5
C2H5
32.1
21.2
17.4
e
e
e
106
e
20.8
50.5
48.6
69.6
72.6
73.5
2.2
e
e
e
e
e
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
CH2C6H5
C2H5
0.93
4.6
6.8
5.2
3.9
5.7
5.3
3.9
5.3
i-C4H9
i-C4H9
(CH2)3C6H5
1.8
1.9
3.9
2.6
12.4
2.2
5.3
5.0
2.0
OCH2C6H5
OC8H17
a
Cytotoxicity as IC50 for each cell line, is the concentration of compound that causes a 50% growth inhibition to untreated cells using the MTT assay.
Cell lines include liver carcinoma (HepG2 and Bel-7402), gastric carcinoma (BGC-823), cervical carcinoma (Hela), breast adenocarcinoma(MCF-7).
Data represent the mean values of three independent determinations.
b
c
d
pIC50 values of HepG2 human tumor cell lines.
interesting compounds with IC50 values lower than 10
m
M against
Undoubtedly, to develop more refined and more reliable 3D-
QSAR models for predicting the antitumor potency of new
compounds, the synthesis of more larger and structurally more
diverse chemical libraries is needed. Further biological evaluations
a panel of human tumor cell lines, and such compounds can be
considered promising leads for further structural modifications
guided by the valuable information derivable from our detailed SARs.
In addition, 3D-QSAR models were developed to obtain detailed
on these and other unreported b-carboline derivatives in animal
information on structureeactivity relationships between
lines and antitumor potencies, and the models provided effective
tools to predict the antitumor potency of new compounds in silico.
b
-carbo-
models are in progress in our laboratories. Moreover, the molecular
mechanisms of these compounds are ongoing and the relative
possible results will be reported in due course.

2508
R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
Fig. 1. 3D-QSAR structure alignment and superposition of 40 compounds using compound 57 as the template.
5. Experimental section
chromatography (TLC) and silica gel were used in column chro-
matography respectively.
5.1. Reagents and general methods
5.1.1. Preparation of ethyl 9-(4-fluorobenzyl)-b-carboline-3-
All reagents were purchased from commercial suppliers and
carboxylate (9)
were dried and purified when necessary. Harmine (7-methoxy-1-
A mixture of ethyl b-carboline-3-carboxylate (24.0 g, 100 mmol)
methyl-
b
-carboline 27) was extracted from Peganum multisectum
and anhydrous DMF (200 mL) was stirred at room temperature for
10 min, and then 60% NaH (8.0 g, 200 mmol) and 4-fluorobenzyl
bromide (150 mmol) were added. The mixture was stirred at room
temperature. After completion of the reaction as indicated by TLC,
the solution was poured into H2O (500 mL), and extracted with
ethyl acetate. The organic phase was washed with water and brine,
then dried over anhydrous Na2SO4, filtered and evaporated. The
resulting oil was crystallized from ethyl ether to afford white crystal
(30 g, 84%), mp 132e133 ꢀC. FAB-MS m/z 349 (M þ 1); IR (KBr,
cmꢁ1): 3059, 3027, 2986, 2954, 2904, 1726, 1622, 1505, 1371, 1333,
Maxim, a plant indigenous to western China, according to already
published method [42]. The following compounds: ethyl 9-n-butyl-
b
-carboline-3-carboxylate 5 [35], ethyl 9-n-butyl-1-methyl-
boline-3-carboxylate 6 [36], ethyl 9-benzyl- -carboline-3-carbox-
ylate 7 [35], ethyl 9-benzyl-1-methyl- -carboline-3-carboxylate 8
[36], ethyl 9-(3-phenylpropyl)-1-methyl- -carboline-3-carbox-
b-car-
b
b
b
ylate 10 [36], N-(2-hydroxyethyl)-9-n-butyl-1-methyl-
3-carboxamide 13 [40], N-(2-hydroxylethyl)-9-benzyl-
b
b
-carboline-
-carboline-
3-carboxamide 16 [40], 1-methyl-
methyl- -carboline-3-carboxylate 26 [36],
methoxy-9-ethyl-1-methyl- -carboline 36 [35], 7-methoxy-9-n-
butyl-1-methyl- -carboline 37 [35], and 7-methoxy-9-(3-phenyl-
propyl)-1-methyl- -carboline 39 [35], were prepared according to
b
-carboline 25 [37], ethyl 1-
b
b-carboline 28 [35], 7-
1303, 1245, 1107, 1026, 746. 1H NMR (500 MHz, CDCl3)
d 8.90 (1H, d,
b
J ¼ 4.5 Hz); 8.23 (1H, d, J ¼ 8.0 Hz); 7.60e7.61 (1H, t, J ¼ 4.5 Hz); 7.46
(1H, d, J ¼ 8.0 Hz); 7.39 (1H, t, J ¼ 7.5 Hz); 7.26 (1H, s); 6.95e7.14
(4H, m); 5.59 (2H, s); 4.51e4.55 (2H, m); 1.48 (3H, t, J ¼ 7.5 Hz). 13C
b
b
the described procedures.
NMR (75 MHz, CDCl3) d: 162.5, 160.6, 143.6, 137.4, 133.3, 131.9, 131.5,
Melting points were determined in capillary tubes on an elec-
trothermal PIF YRT-3 apparatus and without correction. FAB-MS
spectra were obtained from VG ZAB-HS spectrometer. FT-IR
spectra were run as KBr pellets on a Bruker Equinox 55 Fourier
Transformation Infrared Spectrometer. 1H NMR spectra were
recorded on a Varian INOVA 500NB spectrometer. Chemical shifts
129.8, 129.7, 124.3, 122.8, 121.1, 119.5, 116.4, 116.1, 112.4, 63.0, 46.9,
15.0. Anal. Calcd for C21H17FN2O2: C, 72.40; H, 4.92; N, 8.04. Found:
C, 72.29; H, 4.96; N, 7.98.
5.2. General procedure for the preparation of 3-alkylamino
substituted
b
-carbolines (11e12, 14e15 and 17e18)
are reported in
d (ppm) downfield from an internal solvent peak
and coupling constants, J in hertz. Elemental analyses (C, H and N)
were carried out on an ElementarVario EL CHNS Elemental
Analyzer. Silica gel F254 were used in analytical thin-layer
The mixture of the corresponding
b
-carboline-3-carboxy-lates
(5 mmol) and alkylamines (15 mmol) was stirred under microwave
irradiation at 150 ꢀC for 15 min. The resulting solution was parti-
tioned between water and methylene chloride. The aqueous phase
Table 2
Table 3
PLS Statistics of CoMFA and CoMSIA Models.
CoMFA and CoMSIA predictive pIC50 values of the inactive test set.
q2a
Nb
r2c
SEd
Fe
Fractionf
Steric
Compound
Predictive pIC50
CoMFA
Electrostatic
CoMSIA
CoMFA
CoMSIA
0.513
0.503
5
5
0.862
0.831
0.223
0.247
42.583
33.517
0.653
0.518
0.347
0.482
7
8
9
13
14
18
36
53
3.50
3.58
3.95
3.64
3.45
3.93
3.56
4.10
3.44
3.35
3.75
3.31
3.26
3.66
3.40
3.90
a
Cross-validated correlation coefficient.
Optimum number of components obtained from cross-validated PLS analysis
and same used in final non-cross-validated analysis.
b
c
Non-cross-validated correlation coefficient.
Standard error of estimate.
F-test value.
Field contributions.
d
e
f

R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
2509
1334, 1258, 751. 1H NMR (500 MHz, CDCl3)
d
8.90 (1H, s); 8.74 (1H,
6
5.5
5
CoMFA
s); 8.21 (1H, t, J ¼ 8.0 Hz); 8.15 (1H, d, J ¼ 6.0 Hz); 7.61 (1H, t, J ¼ 7.5
Hz); 7.47 (1H, d, J ¼ 8.5 Hz); 7.32 (1H, t, J ¼ 7.5 Hz); 4.39 (2H, t, J ¼ 7.0
Hz); 3.50e3.54 (2H, m); 2.74e2.77 (2H, m); 1.86e1.91 (2H, m);
1.66e1.69 (2H, m); 1.54e1.55 (2H, m); 1.37e1.46 (6H, m); 0.95 (3H,
t, J ¼ 7.5 Hz). 13C NMR (75 MHz, DMSO-d6)
d: 165.3, 141.8, 140.4,
137.8, 131.6, 129.4, 128.4, 122.9, 121.3, 120.8, 114.3, 111.1, 43.3, 31.7,
31.5, 30.2, 27.1, 26.8, 20.5, 14.4.
5.2.3. N-(2-Aminoethyl)-9-n-butyl-1-methyl-b-carboline-3-
4.5
carboxamide (14)
White solid (1.37 g, 85%) was obtained, mp 157e158 ꢀC (lit. [40]
mp 144e145 ꢀC). FAB-MS m/z (M þ 1) 325. IR (KBr, cmꢁ1): 3377,
3056, 2961, 2865, 1463, 1521, 1457, 1352, 1249, 1110, 751. 1H NMR
4
(500 MHz, CDCl3)
d
8.90(1H, s); 8.76 (1H, s); 8.43 (1H, t, J ¼ 6.0 Hz);
8.17 (1H, d, J ¼ 8.0 Hz); 7.58 (1H, t, J ¼ 7.5 Hz); 7.46 (1H, d, J ¼ 8.0
Hz); 7.29 (1H, t, J ¼ 7.5 Hz); 4.54 (2H, t, J ¼ 7.0 Hz); 3.58e3.62 (2H,
m); 3.04 (3H, s); 2.99 (2H, t, J ¼ 6.0 Hz); 1.80e1.85 (2H, m);
1.41e1.49 (2H, m); 0.98 (3H, t, J ¼ 7.5 Hz).
3.5
3.5
4
4.5
5
5.5
6
Experimental Values
Fig. 2. Predictive vs. experimental pIC50 values derived from the CoMFA model.
5.2.4. N-(2-Aminoethyl)-9-benzyl-1-methyl-b-carboline-3-
carboxamide (15)
was extracted with methylene chloride and the combined organic
layers were washed with brine, dried over Na2SO4, filtered,
evaporated. The residued crude oil was purified by a column
chromatography, using a mixture of chloroform and methanol 20:1
White solid (1.61 g, 90%) was obtained, mp 160e161 ꢀC (lit. [40]
mp 122e124 ꢀC). FAB-MS m/z (M þ 1) 359. IR (KBr, cmꢁ1): 3394,
3054, 2938, 1647, 1620, 1559, 1520, 1452, 1353, 1212, 733. 1H NMR
(500 MHz, CDCl3)
d
8.83 (1H, s); 8.22 (1H, d, J ¼ 7.5 Hz); 7.53e7.56
as an eluent, to successfully afford the desirable
b-carbolines
(1H, m); 7.34e7.39 (2H, m); 7.25e7.27 (4H, m); 6.95 (2H, d, J ¼ 7.5
Hz); 5.82 (2H, s); 3.57e3.61 (2H, m); 2.97e2.99 (2H, t, J ¼ 6.5 Hz);
2.87 (3H, s).
bearing a flexible aminoalkyl side chain in good yield.
5.2.1. N-(2-Aminoethyl)-9-n-butyl-b-carboline-3-carboxamide (11)
Yellow oil was obtained (1.36 g, 88%). FAB-MS m/z (M þ 1) 311. IR
5.2.5. N-(2-Aminoethyl)-9-benzyl-b-carboline-3-carboxamide (17)
(KBr, cmꢁ1): 3366, 2957, 2930, 1655, 1623, 1528, 1496, 1462, 1333,
White solid (1.51 g, 88%) was obtained, mp 193e195 ꢀC. FAB-MS
m/z (M þ 1) 345. IR (KBr, cmꢁ1): 3395, 3052, 2928, 2867, 1660, 1625,
1590, 1554, 1525, 1464, 1334, 1269, 1206, 736. 1H NMR (500 MHz,
1257, 749. 1H NMR (500 MHz, CDCl3)
d 8.79 (1H, s); 8.66 (1H, s);
8.49 (1H, t, J ¼ 6.0 Hz); 8.10 (1H, d, J ¼ 7.5 Hz); 7.54 (1H, t, J ¼ 7.5 Hz);
7.37 (1H, d, J ¼ 8.5 Hz); 7.25 (1H, t, J ¼ 4.5 Hz); 4.26 (2H, t, J ¼ 7.0
Hz); 3.69e3.73 (2H, m); 3.15 (2H, t, J ¼ 6.0 Hz); 1.77e1.83 (2H, m);
1.29e1.36 (2H, m); 0.89 (3H, t, J ¼ 7.5 Hz).
CDCl3)
d
8.94 (1H, s, H-1); 8.70 (1H, s); 8.36 (1H, d, J ¼ 9.0 Hz); 8.25
(1H, d, J ¼ 9.0 Hz); 7.59e7.62 (1H, m); 7.47 (1H, d, J ¼ 10.5 Hz); 7.36
(1H, t, J ¼ 7.5 Hz); 7.25e7.29 (3H, m); 7.14 (2H, d, J ¼ 7.5 Hz); 5.61
(2H, s); 3.58e3.61 (2H, m); 2.97e3.00 (2H, m). 13C NMR (75 MHz,
5.2.2. N-(2-Aminohexyl)-9-n-butyl-b-carboline-3-carboxamide
(12)
DMSO-d6) d: 165.4, 141.9, 140.8, 137.9, 137.7, 131.9, 129.6, 129.3,
128.8, 128.2, 127.5, 123.0, 121.5, 121.1, 114.5, 111.4, 46.9, 42.4, 41.8,
Anal. Calcd for C21H20N4O: C, 73.23; H, 5.85; N, 16.27. Found: C,
72.28; H, 5.89; N, 16.30.
Yellow oil was obtained (1.52 g, 83%). FAB-MS m/z (M þ 1) 367.
IR (KBr, cmꢁ1): 3382, 2931, 2859, 1658, 1624, 1588, 1530, 1497, 1464,
5.2.6. N-(2-Aminohexyl)-9-benzyl-b-carboline-3-carboxamide (18)
White solid (1.64 g, 82%) was obtained, mp 132e134 ꢀC (lit. [40]
mp 162e164 ꢀC). FAB-MS m/z (M þ 1) 401. IR (KBr, cmꢁ1): 3402,
3032, 2925, 2850, 1663, 1526, 1495, 1464, 1333, 1266, 725. 1H NMR
CoMSIA
6
5.5
5
(500 MHz, CDCl3)
d
8.95 (1H, s); 8.68 (1H, s); 8.25 (1H, d, J ¼ 9.0 Hz);
8.10 (1H, d, J ¼ 9.0 Hz); 7.58e7.61 (1H, m); 7.47 (1H, d, J ¼ 10.5 Hz);
7.36 (1H, t, J ¼ 7.5 Hz); 7.26e7.30 (3H, m); 7.14 (2H, d, J ¼ 7.5 Hz);
5.61 (2H, s); 3.50e3.54 (2H, m); 2.68e2.71 (2H, m); 1.38e1.49 (8H,
m). 13C NMR (75 MHz, DMSO-d6)
d: 165.1, 141.9, 140.9, 137.9, 137.7,
131.8, 129.5, 129.3, 128.8, 128.2, 127.4, 123.0, 121.5, 121.1, 114.5, 111.4,
46.9, 41.8, 33.2, 30.3, 27.2, 27.0.
4.5
5.2.7. Preparation of N-(2-hydroxylethyl)-9-(4-fluorobenzyl)-
b-
carboline-3-carboxamide (19)
4
A solution of ethyl 9-(4-fluorobenzyl)-b-carboline-3-carbox-
ylate 9 (3.48 g, 10 mmol) in ethanol (100 mL) containing ethanol-
amine (40 mmol) and 60% NaH (0.8 g, 20 mmol) was refluxed for 30
min. After completion of the reaction, as indicated by TLC, the
solution was cooled and poured into ice-water (200 mL), and
extracted with ethyl acetate. The organic phase was washed with
water and brine, then dried over anhydrous sodium sulfate, filtered
and evaporated. The resulting oil obtained was purified by silica
3.5
3.5
4
4.5
5
5.5
6
Experimental Values
Fig. 3. Predictive vs. experimental pIC50 values derived from the CoMSIA model.

2510
R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
Fig. 4. Contour coefficient map of (A) CoMFA and (B) CoMSIA. Sterically favored regions are shown in green while certain sterically unfavorable regions are shown in yellow; the
region favoring a positive charge are shown in red as do regions favoring a negative charge are shown in blue. The template compound 51 is shown embedded into the final field as
a representative example. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
column chromatography with ethyl acetate as the eluent, and
crystallized from ethyl acetate to give white crystals (2.94 g, 81%),
mp 188e189 ꢀC. FAB-MS m/z (M þ 1) 364. IR (KBr, cmꢁ1): 3378,
3066, 2951, 2917, 2867, 1647, 1626, 1590, 1534, 1497, 1466, 1423,
5.2.9. Preparation of 3-hydroxymethyl-9-n-butyl-1-methyl-b-
carboline (21)
A fine suspension of ethyl 9-n-butyl-1-methyl-b-carboline-3-
carboxylate 6 (3.1 g, 10 mmol) in dry THF (100 mL) was treated with
LiBH4 (30 mmol), and the mixture was stirred at room temperature
for 9 h. The reaction was cooled, treated with 10% aq. HCl (20 mL),
and stirred for 4 h. The reaction mixture was neutralized with 10%
aq NaOH solution and extracted with ethyl acetate. The organic
phase was washed with water and brine, then dried over anhydrous
sodium sulfate, filtered and evaporated. The residue obtained was
purified by silica column chromatography with ethyl acetate as the
eluent. Upon recrystallization, white crystals (2.1 g, 78%) were
1331, 1217, 1075, 825, 746. 1H NMR (500 MHz, CDCl3)
d 8.98 (1H, s);
8.66 (1H, s); 8.23 (1H, d, J ¼ 8.0 Hz); 7.59e7.62 (1H, t, J ¼ 8.5 Hz);
7.44 (1H, d, J ¼ 8.5 Hz); 7.36 (1H, t, J ¼ 8.5 Hz); 7.25 (1H, s);
6.95e7.12 (4H, m); 5.57 (2H, s); 3.88 (2H, t, J ¼ 6.5 Hz); 3.68e3.71
(2H, m). 13C NMR (75 MHz, DMSO-d6)
d: 163.8, 161.7, 160.5, 143.8,
137.0, 134.7, 133.2, 132.0, 129.8, 128.4, 123.7, 122.7, 120.9, 116.3, 116.1,
112.3, 60.1, 46.9, 43.2. Anal. Calcd for C21H18FN3O2: C, 69.41; H, 4.99;
N, 11.56. Found: C, 69.28; H, 5.02; N, 11.59.
obtained. mp 115e116 ꢀC. FAB-MS m/z (M þ 1) 269. IR (KBr, cmꢁ1
)
5.2.8. Preparation of N-(2-hydroxylethyl)-9-phenylpropyl-
b-
3153, 2967, 2918, 2838, 1619, 1557, 1477, 1362, 1278, 1245, 1208,
carboline-3-carboxamide (20)
1075, 988, 871, 741. 1H NMR (500 MHz, CDCl3)
d 8.79e8.81 (1H, m);
Compound 20 was prepared by the same procedure as
compound 19 from ethyl 9-phenylpropyl- -carboline-3-carbox-
ylate 10 (3.58 g, 10 mmol) to give white crystals (2.95 g, 79%), mp
126e127 ꢀC. FAB-MS m/z (M þ 1) 374. IR (KBr, cmꢁ1): 2884, 1659,
1624, 1517, 1474, 1389, 1235, 1131, 1037, 1011, 834, 748. 1H NMR (500
8.52 (1H, s); 8.28e8.32 (1H, m); 8.14 (1H, d, J ¼ 10.5 Hz); 7.96e8.00
(2H, m); 5.61 (2H, s); 5.17 (2H, t, J ¼ 8.0 Hz); 3.77 (3H, s); 2.46e2.54
(2H, m); 2.08e2.18 (2H, m); 1.67 (3H, t, J ¼ 7.5 Hz). 13C NMR (75
b
MHz, DMSO-d6) d: 150.3, 142.1, 140.6, 134.0, 129.7, 128.6, 122.0,
121.3, 120.0, 110.9, 65.0, 44.6, 33.3, 23.7, 20.3, 14.5. Anal. Calcd for
C17H20N2O: C, 76.09; H, 7.51; N, 10.44. Found: C, 75.95; H, 7.54; N,
10.47.
MHz, CDCl3)
d
8.90 (1H, s); 8.63 (1H, s); 8.19 (1H, d, J ¼ 9.5 Hz);
7.58e7.62 (1H, m); 7.14e7.40 (8H, m); 4.40 (2H, t, J ¼ 7.5 Hz);
3.88e3.91 (2H, m); 3.69e3.73 (2H, m); 2.70e2.74 (2H, m);
2.23e2.31 (2H, m). 13C NMR (75 MHz, CDCl3)
d
: 166.8, 141.5, 140.5,
5.2.10. Preparation of 3-hydroxylmethyl-9-(4-fluorobenzyl)-
carboline (22)
b-
139.8, 137.6, 130.0, 129.0, 128.9, 128.8, 128.4, 126.5, 122.4, 121.7,
120.6, 114.6, 109.8, 62.9, 43.1, 33.4, 30.5. Anal. Calcd for C23H23N3O2:
C, 73.97; H, 6.21; N, 11.25. Found: C, 73.90; H, 6.23; N, 11.30.
Compound 22 was prepared by the same procedure as
compound 21 from ethyl 9-(4-fluorobenzyl)- -carboline-3-
b

R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
2511
carboxylate 9 (3.48 g, 10 mmol). White solid (1.8 g, 59%) was
5.3.2. 2-(3-Phenylpropyl)-1-methyl-b-carbolinium bromide (30)
obtained, mp 177e178 ꢀC. FAB-MS m/z (M þ 1) 307. IR (KBr, cmꢁ1):
3307, 3068, 2934, 2867, 2682, 1625, 1602, 1555, 1506, 1465, 1366,
1333, 1261, 1221, 1153, 1097, 1021, 745. 1H NMR (500 MHz, CDCl3)
Yellow crystals (0.58 g, 76%) were obtained, mp >270 ꢀC. FAB-
MS m/z 301. IR (KBr, cmꢁ1): 3250e3600, 2250e3250, 1634, 1574,
1522,1499,1453,1330,1229, 821,754. 1H NMR (500 MHz, DMSO-d6)
d
8.93 (1H, s); 8.19 (1H, d, J ¼ 8.5 Hz); 8.14 (1H, s); 7.65e7.67 (1H,
m); 7.55 (1H, d, J ¼ 9.5 Hz); 7.39e7.41 (1H, m); 6.93e7.27 (4H, m);
5.63 (2H, s); 5.02 (2H, s). 13C NMR (75 MHz, CDCl3)
: 163.6, 160.4,
d
12.87 (1H, s); 8.68 (1H, d, J ¼ 6.5 Hz); 8.64 (1H, d, J ¼ 6.5 Hz); 8.45
(1H, d, J ¼ 8.0 Hz); 7.76e7.81 (2H, m); 7.42e7.46 (2H, m); 7.17e7.31
(5H, m); 4.73 (2H, t, J ¼ 7.5 Hz); 3.10 (3H, s); 2.76 (2H, t, J ¼ 7.5 Hz);
d
151.8, 141.7, 135.8, 134.3, 132.1, 129.5, 129.0, 122.6, 121.4, 120.3, 116.2,
111.8, 110.9, 65.4, 46.0. Anal. Calcd for C19H15FN2O: C, 74.50; H, 4.94;
N, 9.14. Found: C, 74.68; H, 4.96; N, 9.18.
2.22e2.28 (2H, m). 13C NMR (75 MHz, DMSO-d6)
d: 143.7, 141.1,
140.7, 135.3, 134.5, 132.1, 131.0, 129.0, 128.9, 126.7, 124.0, 122.1, 120.1,
116.3, 113.2, 56.9, 32.5, 32.3, 16.1. Anal. Calcd for C21H21BrN2: C,
66.15; H, 5.55; N, 7.35. Found: C, 66.08; H, 5.58; N, 7.37.
5.2.11. Preparation of 9-n-butyl-1-methyl-b-carboline-3-
carboxaldehyde (23)
5.3.3. 3-Ethoxycarbonyl-2-benzyl-1-methyl-b-carbolinium
To a solution of compound 21 (5 mmol) in CH3CN (300 mL) was
added activated MnO2 (20 mmol). The suspension was refluxed for
2 h and then cooled and filtered through Celite. The filtrate was
passed through silica gel and washed with ethyl acetate, and the
solvent was removed under reduced pressure. The residue was
crystallized from acetone-petroleum ether to afford white crystals
(0.95 g, 71%), mp 103e104 ꢀC. FAB-MS m/z (M þ 1) 267. IR (KBr,
cmꢁ1): 3342, 3059, 3019, 2958, 2930, 2869, 1679, 1619, 1570, 1456,
1370, 1337, 1297, 1248, 1196, 1112, 736. 1H NMR (500 MHz, CDCl3)
bromide (31)
Yellow crystals (0.42 g, 49%) were obtained, mp >270 ꢀC. FAB-
MS m/z 345. IR (KBr, cmꢁ1): 3421, 2250e3250, 1721, 1627, 1579,
1514, 1452, 1367, 1334, 1261, 1136, 1105, 1020, 735. 1H NMR (500
MHz, DMSO-d6)
d
13.37 (1H, s); 9.26 (1H, s); 8.61 (1H, d, J ¼ 8.0 Hz);
7.87e7.88 (2H, m); 7.52e7.55 (1H, m); 7.33e7.39 (3H, m); 7.10e7.12
(2H, m); 6.21 (2H, s); 4.33e4.38 (2H, m); 3.14 (3H, s); 1.21 (3H, t, J ¼
7.5 Hz). 13C NMR (75 MHz, DMSO-d6)
d: 162.1, 144.8, 143.2, 142.3,
135.6, 135.2, 132.8, 131.9, 130.1, 129.4, 126.9, 124.3, 122.6, 120.6,
118.1, 113.6, 64.1, 57.6, 17.5, 14.4. Anal. Calcd for C22H21BrN2O2: C,
62.13; H, 4.98; N, 6.59. Found: C, 62.20; H, 5.01; N, 6.62.
d
10.45 (1H, s); 8.67 (1H, s); 8.21 (1H, d, J ¼ 10.0 Hz); 7.40e7.73 (3H,
m); 4.63 (2H, t, J ¼ 8.0 Hz); 3.27 (3H, s); 1.87e1.93 (2H, m);
1.46e1.52 (2H, m); 1.01 (3H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz,
CDCl3)
d: 193.0, 143.0, 142.2, 142.1, 137.1, 129.4, 128.6, 122.4, 121.5,
5.3.4. 7-Methoxy-2-benzyl-1-methyl-b-carbolinium bromide (32)
121.3, 113.8, 111.4, 44.9, 33.3, 23.9, 20.2, 14.4. Anal. Calcd for
C17H18N2O: C, 76.66; H, 6.81; N, 10.52. Found: C, 76.75; H, 6.84; N,
10.56.
Yellow crystals (0.64 g, 84%) were obtained, mp >270 ꢀC. FAB-
MS m/z 303. IR (KBr, cmꢁ1): 1750e3250, 1632, 1505, 1457, 1336,
1286, 1225, 1022, 830, 733; 1H NMR (500 MHz, DMSO-d6)
d 12.79
(1H, s); 8.73 (1H, d, J ¼ 6.5 Hz); 8.57 (1H, d, J ¼ 8.0 Hz); 8.32e8.37
5.2.12. Preparation of 9-(4-fluorobenzyl)-
carboxaldehyde (24)
b-carboline-3-
(1H, m); 7.37e7.44 (3H, m); 7.22e7.24 (2H, m); 7.12e7.13 (1H, m);
7.06e7.09 (1H, m); 5.99 (2H, s). 13C NMR (75 MHz, DMSO-d6)
d:
Compound 24 was prepared by the same procedure as
compound 23 from compound 22 (3.06 g, 10 mmol). White solid
(2.4 g, 79%) was obtained, mp 183e184 ꢀC. FAB-MS m/z (M þ 1) 305.
IR (KBr, cmꢁ1): 3043, 2937, 2843, 2735,1697, 1620, 1579, 1508, 1464,
1358, 1333, 1305, 1263, 1216, 1172, 848, 743. 1H NMR (500 MHz,
163.5,146.4,139.4,135.6,135.1,132.0,129.8,129.1,127.7,125.3,115.2,
114.1, 113.5, 94.9, 59.8, 56.6, 16.3. Anal. Calcd for C20H19BrN2O: C,
62.67; H, 5.00; N, 7.31. Found: C, 62.78; H, 5.02; N, 7.34.
5.3.5. 2-n-Butyl-b-carbolinium bromide (33)
CDCl3)
Hz); 7.76e7.80 (1H, m); 7.60 (1H, d, J ¼ 9.5 Hz); 7.51e7.55 (1H, m);
7.03e7.16 (4H, m); 5.73 (2H, s). 13C NMR (75 MHz, CDCl3)
: 193.0,
d
10.55 (1H, s); 9.11 (1H, s); 8.90 (1H, s); 8.33 (1H, d, J ¼ 9.5
Yellow crystals (0.46 g, 75%) were obtained, mp >270 ꢀC. FAB-
MS m/z 225. IR (KBr, cmꢁ1): 3404, 1750e3250, 1643, 1573, 1518,
1457, 1334, 1255, 1148, 1118, 869. 1H NMR (500 MHz, DMSO-d6)
d
144.6, 141.9, 138.7, 132.3, 131.6, 129.6, 128.4, 122.5, 122.0, 121.6,
116.4, 114.8, 110.4, 110.0, 46.9. Anal. Calcd for C19H13FN2O: C, 74.99;
H, 4.31; N, 9.21. Found: C, 75.10; H, 4.34; N, 9.25.
d
12.86 (1H, s); 9.52 (1H, s); 8.85 (1H, d, J ¼ 6.5 Hz); 8.77 (1H, d, J ¼
6.5 Hz); 8.51 (1H, d, J ¼ 8.0 Hz); 7.79e7.84 (2H, m); 7.45e7.48 (1H,
m); 4.78 (2H, t, J ¼ 8.5 Hz); 1.97e2.03 (2H, m); 1.32e1.39 (2H, m);
0.94 (2H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz, DMSO-d6)
d: 144.2, 135.1,
133.0, 132.6, 132.5, 129.9, 124.1, 122.1, 119.6, 118.5, 113.5, 60.9, 34.0,
19.7, 14.3. Anal. Calcd for C15H17BrN2: C, 59.03; H, 5.61; N, 9.18.
Found: C, 59.18; H, 5.63; N, 9.14.
5.3. General procedure for the preparation of b-carbolinium
derivatives (29e35)
5.3.6. 2-Benzyl-b-carbolinium bromide (34)
A mixture of
b
-carboline derivatives 25e28 (2 mmol) and
Yellow crystals (0.52 g, 77%) were obtained, mp >270 ꢀC. FAB-
halogenated alkane (10 mmol) in ethyl acetate (50 mL) was
refluxed for 5e10 h. After completion of the reaction as indicated by
TLC, the solution was cooled and filtered to afford yellow solid. The
solid was crystallized from ethanol.
MS m/z 259. IR (KBr, cmꢁ1): 3395, 1750e3250, 1642, 1520, 1498,
1337, 1258, 1209, 757, 732. 1H NMR (500 MHz, DMSO-d6)
d 12.86
(1H, s); 9.62 (1H, d, J ¼ 7.0 Hz); 8.83e8.87 (2H, m); 8.50 (1H, d, J ¼
5.5 Hz); 7.79e7.84 (2H, m); 7.43e7.56 (6H, m); 6.00 (2H, s). 13C NMR
(75 MHz, DMSO-d6) d: 144.3, 135.9, 135.2, 133.1, 132.8, 132.6, 129.9,
5.3.1. 2-Benzyl-1-methyl-
b
-carbolinium bromide (29)
129.7, 129.2, 124.1, 122.1, 119.5, 118.8, 113.4, 63.4. Anal. Calcd for
C18H15BrN2: C, 63.73; H, 4.46; N, 8.26. Found: C, 63.68; H, 4.48; N,
8.29.
Yellow crystals (0.46 g, 65%) were obtained, mp >270 ꢀC. FAB-
MS m/z 273. IR (KBr, cmꢁ1): 3420, 1750e3250, 1634, 1575, 1525,
1500, 1452, 1331, 1299, 1258, 1229, 1146, 740. 1H NMR (500 MHz,
DMSO-d6)
Hz); 8.48 (1H, d, J ¼ 7.0 Hz); 7.79e7.80 (2H, m); 7.25e7.47 (6H, m);
6.06 (2H, s); 3.06 (3H, s). 13C NMR (75 MHz, DMSO-d6)
: 144.2,
141.2, 135.8, 135.0, 132.5, 131.8, 129.8, 129.2, 127.7, 124.2, 122.3,
120.3,116.6,113.4, 60.2,16.5. Anal. Calcd for C19H17BrN2: C, 64.60; H,
4.85; N, 7.93. Found: C, 64.49; H, 4.88; N, 7.96.
d
12.94 (1H, s); 8.80 (1H, d, J ¼ 7.0 Hz); 8.73 (1H, d, J ¼ 6.5
5.3.7. 2-(3-Phenylpropyl)-b-carbolinium bromide (35)
Yellow crystals (0.61 g, 83%) were obtained, mp >270 ꢀC. FAB-
MS m/z 287. IR (KBr, cmꢁ1): 3499, 3431, 1750e3250, 1643, 1517,
1496, 1339, 1260, 1137, 826, 754. 1H NMR (500 MHz, DMSO-d6)
d
d
12.85 (1H, s); 9.51 (1H, s); 8.82 (1H, d, J ¼ 6.5 Hz); 8.77 (1H, d, J ¼
6.5 Hz); 8.50 (1H, d, J ¼ 8.0 Hz); 7.77e7.83 (2H, m); 7.43e7.46 (1H,

2512
R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
m); 7.12e7.26 (5H, m); 4.82 (2H, t, J ¼ 8.0 Hz); 2.68 (2H, t, J ¼ 8.0
Hz); 2.31e2.37 (2H, m). 13C NMR (75 MHz, DMSO-d6)
: 144.2, 141.0,
135.0, 133.0, 132.5, 132.4, 129.9, 128.9, 128.8, 126.5, 124.1, 122.0,
119.6, 118.4, 113.4, 60.1, 33.6, 32.4. Anal. Calcd for C20H19BrN2: C,
65.40; H, 5.21; N, 7.63. Found: C, 65.35; H, 5.23; N, 7.66.
8.13 (1H, d, J ¼ 5.0 Hz); 7.95 (1H, d, J ¼ 8.5 Hz); 7.79 (1H, d, J ¼ 5.0
Hz); 6.93 (1H, s); 6.72 (1H, d, J ¼ 8.5 Hz), 4.26 (2H, d, J ¼ 7.5 Hz);
2.90 (3H, s); 2.11e2.17 (1H, m); 0.86 (6H, s). 13C NMR (75 MHz,
DMSO-d6) d: 158.9, 144.2, 140.8, 138.2, 135.4, 129.5, 123.0, 113.8,
112.6, 110.4, 96.6, 51.6, 30.9, 23.9, 20.5. Anal. Calcd for C16H18N2O: C,
75.56; H, 7.13; N, 11.01. Found: C, 75.38; H, 7.15; N, 11.04.
d
5.3.8. Preparation of 7-methoxy-9-isobutyl-1-methyl-b-carboline
(38)
5.4.4. 1-Methyl-9-(3-phenylpropyl)-b-carboline-7-ol (43)
A mixture of 7-methoxy-1-methyl-
b
-carboline 29 (2.12 g, 10
Afforded white crystals (2.5 g, 80%), mp 258e259 ꢀC. FAB-MS m/
z (M þ 1) 317. IR (KBr, cmꢁ1): 3500e1750, 1613, 1567, 1492, 1452,
1412, 1352, 1246, 1194, 1160, 978, 823,746. 1H NMR (500 MHz,
mmol) and anhydrous DMF (50 mL) was stirred at RT until clear,
and then 60% NaH (0.6 g, 15 mmol) and isobutyl bromide (6 mL, 50
mmol) were added. The mixture was continued to stir at RT. After
completion of the reaction as indicated by TLC, the solution was
poured into H2O (150 mL), and extracted with ethyl acetate. The
organic phase was washed with water and brine, then dried over
anhydrous sodium sulfate, filtered and evaporated. The resulting oil
was crystallized from ethyl ether to afford white crystal (2.2 g, 82%),
mp 112e113 ꢀC. FAB-MS m/z (M þ 1) 269. IR (KBr, cmꢁ1): 2961, 1621,
1564, 1496, 1446, 1403, 1338, 1254, 1207, 1139, 1044, 966, 815. 1H
DMSO-d6)
d
9.74 (1H, s); 8.11 (1H, d, J ¼ 5.0 Hz); 7.95 (1H, d, J ¼ 8.5
Hz); 7.77 (1H, d, J ¼ 5.0 Hz); 7.17e7.30 (5H, m); 6.87e6.88 (1H, m);
6.74e6.76 (1H, m); 4.45 (2H, t, J ¼ 7.0 Hz); 2.77 (3H, s); 2.72 (2H, t, J
¼ 7.0 Hz); 1.98e2.05 (2H, m). 13C NMR (75 MHz, DMSO-d6)
d: 175.9,
159.1, 143.7, 141.6, 140.7, 138.0, 135.0, 129.5, 129.0, 126.6, 123.2,
113.9, 112.7, 110.5, 95.8, 44.4, 33.0, 32.5, 23.4. Anal. Calcd for
C21H20N2O: C, 79.72; H, 6.37; N, 8.85. Found: C, 79.68; H, 6.40; N,
8.87.
NMR (500 MHz, CDCl3)
d
8.29 (1H, s); 7.95 (1H, d, J ¼ 8.5 Hz); 7.73
(1H, d, J ¼ 5.0 Hz); 6.85e6.88 (2H, m); 4.26 (2H, d, J ¼ 7.5 Hz); 3.93
5.5. General procedure for the preparation of 7-alkoxyl substituted
(3H, s); 3.01 (3H, s); 2.23e2.28 (1H, m); 0.92 (6H, d, J ¼ 5.5 Hz). 13C
b-carbolines 44e55
NMR (75 MHz, DMSO-d6) d: 160.9, 144.0, 141.1, 138.2, 135.4, 129.2,
122.9, 114.7, 112.8, 109.7, 95.0, 56.3, 51.4, 31.2, 23.9, 20.4. Anal. Calcd
for C17H20N2O: C, 76.09; H, 7.51; N, 10.44. Found: C, 76.27; H, 7.54;
N, 10.39.
A mixture of 7-hydroxyl-1-methyl-b-carboline derivatives (2
mmol) and anhydrous DMF (50 mL) was stirred at room tempera-
ture until clear, and then 60% NaH (3 mmol) and halogenated
alkane (4 mmol) were added. The mixture was stirred at room
temperature for 0.5e2 h. After completion of the reaction as indi-
cated by TLC, the solution was poured into H2O (150 mL), and
extracted with ethyl acetate. The organic phase was made acidic
with concentrated hydrochloric acid. Upon removal of solvent, the
residue was crystallized from acetone to afford yellow solid. The
solid was dissolved in water and made basic with sodium bicar-
bonate, and the aqueous mixture extracted with ethyl acetate. The
organic phase was washed with water and brine, then dried over
anhydrous sodium sulfate, filtered and evaporated. The resulting oil
was crystallized from ethyl ether or ethyl etherepetroleum ether.
5.4. General procedure for the preparation of 7-hydroxyl substitued
b-carbolines 40e43
A mixture of 7-methoxy substituted b-carboline derivatives (10
mmol), acetic acid (100 mL) and 40% hydrobromic acid (50 mL) was
refluxed for 8e12 h. After completion of the reaction as indicated
by TLC, the mixture was cooled and poured onto ice. The aqueous
mixture, made basic with sodium hydroxide, yielded a precipitate
that was filtered and dried. The solid was crystallized from anhy-
drous ethanol.
5.4.1. 9-Ethyl-1-methyl-
b
-carboline7-ol (40)
5.5.1. 7-Ethoxy-9-ethyl-1-methyl-b-carboline (44)
Afforded white crystals (1.8 g, 80%), mp 258e260 ꢀC. FAB-MS m/
z (M þ 1) 227. IR (KBr, cmꢁ1): 3500e1750, 1627, 1568, 1451, 1410,
1347, 1315,1260, 1215, 1092, 983, 821. 1H NMR (500 MHz, DMSO-d6)
White crystals (0.39 g, 76%) were obtained, mp 117e118 ꢀC. FAB-
MS m/z (M þ 1) 255. IR (KBr, cmꢁ1): 2978, 2931, 1620, 1560, 1446,
1407, 1342, 1208, 1139, 1048, 972, 817. 1H NMR (500 MHz, CDCl3)
d
9.74 (1H, s); 8.12 (1H, d, J ¼ 8.0 Hz); 7.96 (1H, d, J ¼ 7.5 Hz); 7.78
d
8.26 (1H, d, J ¼ 5.5 Hz); 7.96 (1H, d, J ¼ 8.5 Hz); 7.74 (1H, d, J ¼ 5.5
Hz); 6.87e6.90 (2H, m); 4.52e4.56 (2H, m); 4.16e4.20 (2H, m); 3.05
(3H, s); 1.43e1.51 (6H, m). 13C NMR (75 MHz, CDCl3)
: 160.3, 142.7,
(1H, d, J ¼ 8.0 Hz); 6.91e6.92 (1H, m); 6.73e6.75 (1H, m);
4.48e4.52 (2H, m); 2.92 (3H, s); 1.32 (3H, t, J ¼ 8.0 Hz). 13C NMR (75
d
MHz, DMSO-d6)
d: 162.0, 146.2, 136.7, 133.6, 133.3, 129.5, 125.1,
140.6, 138.3, 135.2, 129.5, 122.5, 115.3, 112.4, 109.2, 93.9, 64.2, 39.7,
23.7, 15.9, 15.3. Anal. Calcd for C16H18N2O: C, 75.56; H, 7.13; N, 11.01.
Found: C, 75.54; H, 7.15; N, 11.06.
114.3, 113.3, 112.7, 95.5, 18.5, 16.1. Anal. Calcd for C14H14N2O: C,
74.31; H, 6.24; N, 12.38. Found: C, 74.38; H, 6.26; N, 12.43.
5.4.2. 9-n-Butyl-1-methyl-
b
-carboline7-ol (41)
5.5.2. 7-(Perfluorobenzyloxy)-9-ethyl-1-methyl-b-carboline (45)
Afforded white crystals (2.2 g, 87%), mp 205e206 ꢀC. FAB-MS m/
z (M þ 1) 255. IR (KBr, cmꢁ1): 3500e1750, 1618, 1566, 1492, 1451,
1413, 1325, 1238, 1187, 1137, 1112, 980. 1H NMR (500 MHz, DMSO-
White crystals (0.63 g, 78%) were obtained, mp 205e206 ꢀC.
FAB-MS m/z (M þ 1) 407. IR (KBr, cmꢁ1): 3063, 2977, 1620, 1565,
1507, 1442, 1408, 1350, 1208, 1139, 1060, 938, 815. 1H NMR (500
d6)
d
9.72 (1H, s); 8.12 (1H, d, J ¼ 5.0 Hz); 7.95 (1H, d, J ¼ 8.5 Hz);
MHz, CDCl3)
d
8.30 (1H, d, J ¼ 5.0 Hz); 8.01 (1H, d, J ¼ 8.5 Hz); 7.55
7.78 (1H, d, J ¼ 5.0 Hz); 6.90e6.91 (1H, m); 6.72e6.75 (1H, m); 4.43
(2H, t, J ¼ 8.0 Hz); 2.91 (3H, s); 1.67e1.74 (2H, m); 1.36e1.40 (2H,
(1H, d, J ¼ 5.0 Hz); 6.93e6.99 (2H, m); 5.29 (2H, s); 4.55e4.59 (2H,
m); 3.05 (3H, s); 1.46 (3H, t, J ¼ 7.5 Hz). 13C NMR (125 MHz, CDCl3)
d:
m); 0.93 (2H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz, DMSO-d6)
d: 159.3,
153.7, 141.5, 139.5, 137.1, 135.5, 133.1, 131.3, 130.0, 123.8, 117.3, 111.0,
107.0, 104.8, 103.7, 89.5, 52.7, 34.3, 17.9, 10.1. Anal. Calcd for
C21H15F5N2O: C, 62.07; H, 3.72; N, 6.89. Found: C, 61.98; H, 3.73; N,
6.91.
143.9, 140.5, 137.6, 135.0, 129.7, 123.2, 113.9, 112.7, 110.6, 95.9, 44.7,
33.1, 23.3, 20.3, 14.5. Anal. Calcd for C16H18N2O: C, 75.56; H, 7.13; N,
11.01. Found: C, 75.69; H, 7.16; N, 10.97.
5.4.3. 9-Isobutyl-1-methyl-
b
-carboline-7-ol (42)
5.5.3. 7-Ethoxy-9-n-butyl-1-methyl-b-carboline (46)
Afforded white crystals (2.0 g, 79%), mp 247e248 ꢀC. FAB-MS m/
z (M þ 1) 255. IR (KBr, cmꢁ1): 3500e1750, 1626, 1568, 1447, 1392,
White crystals (0.42 g, 75%) were obtained, mp 93e94 ꢀC; FAB-
MS m/z (M þ 1) 283. IR (KBr, cmꢁ1): 3052, 2961, 2928, 2869, 1622,
1562, 1450, 1410,1360, 1255, 1193, 1139, 1048, 948, 809. 1H NMR
1203, 1136, 977, 820. 1H NMR (500 MHz, DMSO-d6)
d 9.70 (1H, s);

R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
2513
(500 MHz, CDCl3)
d
8.26 (1H, d, J ¼ 5.0 Hz); 7.96 (1H, d, J ¼ 8.5 Hz);
5.5.9. 7-Isopropyloxy-1-methyl-9-(3-phenylpropyl)-
(52)
b-carboline
7.75 (1H, d, J ¼ 5.0 Hz); 6.86e6.91 (2H, m); 4.45 (2H, t, J ¼ 8.0 Hz);
4.16e4.20 (2H, m); 3.05 (3H, s); 1.78e1.84 (2H, m); 1.42e1.51 (5H,
White crystals (0.54 g, 76%) were obtained, mp 121e122 ꢀC.
FAB-MS m/z (M þ 1) 359. IR (KBr, cmꢁ1): 2975, 2927, 2858, 1621,
1563, 1494, 1448, 1409, 1374, 1326, 1240, 1158, 1111, 975, 754. 1H
m); 0.98 (3H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz, CDCl3)
d: 160.2, 143.1,
140.6, 138.3, 135.4, 129.4, 122.4, 115.2, 112.4, 109.0, 94.2, 64.1, 44.9,
33.1, 23.8, 20.6, 15.3, 14.3. Anal. Calcd for C18H22N2O: C, 76.56; H,
7.85; N, 9.92. Found: C, 76.70; H, 7.88; N, 9.90.
NMR (500 MHz, CDCl3)
d
8.25 (1H, d, J ¼ 5.5 Hz); 7.92 (1H, d, J ¼
8.0 Hz); 7.71 (1H, d, J ¼ 5.5 Hz); 7.19e7.32 (5H, m); 6.84e6.86
(1H, m); 6.68e6.69 (1H, m); 4.59e4.61 (1H, m); 4.43 (2H, t, J ¼
8.0 Hz); 2.89 (3H, s); 2.74e2.77 (2H, m); 2.12e2.18 (2H, m);
5.5.4. 7-Isopropyloxy-9-n-butyl-1-methyl-b-carboline (47)
Yellow oil (0.41 g, 72%) was obtained, FAB-MS m/z (M þ 1) 297.
1.38e1.39 (6H, m). 13C NMR (75 MHz, CDCl3)
d: 159.1, 143.2,
IR (KBr, cmꢁ1): 3422, 2970, 2930, 1626, 1267, 1447, 1409, 1376, 1243,
140.9, 140.7, 138.4, 135.5, 129.6, 128.8, 128.6, 126.5, 122.5, 115.4,
112.4, 110.5, 95.9, 70.7, 44.4, 33.4, 32.1, 23.6, 22.5. Anal. Calcd
for C24H26N2O: C, 80.41; H, 7.31; N, 7.81. Found: C, 80.37; H,
7.33; N, 7.79.
1191, 1135, 1113, 986, 816. 1H NMR (500 MHz, CDCl3)
d 8.26 (1H, d, J
¼ 5.5 Hz); 7.95 (1H, d, J ¼ 9.0 Hz); 7.73 (1H, d, J ¼ 5.5 Hz); 6.87e6.88
(2H, m); 4.68e4.73 (1H, m); 4.44 (2H, t, J ¼ 8.0 Hz); 3.04 (3H, s);
1.78e1.84 (2H, m); 1.37e1.47 (8H, m); 0.98 (3H, t, J ¼ 7.5 Hz). 13C
NMR (75 MHz, CDCl3)
d
: 159.1, 143.3, 140.6, 138.2, 135.5, 129.5,
5.5.10. 7-Octyloxy-9-(3-phenylpropyl)-1-methyl-b-carboline (53)
122.5, 115.4, 112.4, 110.1, 96.3, 70.8, 45.0, 33.1, 23.7, 22.5, 20.6, 14.3.
White crystals (0.61 g, 71%) were obtained, mp 79e81 ꢀC.
FAB-MS m/z (M þ 1) 429. IR (KBr, cmꢁ1): 2947, 2920, 2852, 1622,
1564, 1496, 1448, 1412, 1244, 1161, 1039, 745. 1H NMR (500 MHz,
5.5.5. 7-n-Butyloxy-9-n-butyl-1-methyl-b-carboline (48)
White crystals (0.52 g, 84%) were obtained, mp 67e68 ꢀC; FAB-
MS m/z (M þ 1) 311. IR (KBr, cmꢁ1): 3054, 2958, 2871, 1622, 1563,
1451, 1410, 1246, 198, 1141, 1044, 1004, 802. 1H NMR (500 MHz,
CDCl3)
d
8.25 (1H, d, J ¼ 5.5 Hz); 7.93 (1H, d, J ¼ 8.5 Hz); 7.74 (1H,
d, J ¼ 5.0 Hz); 7.20e7.33 (5H, m); 6.86e6.89 (1H, m); 6.64e6.65
(1H, m); 4.44 (2H, t, J ¼ 6.0 Hz); 3.97e3.99 (2H, t, J ¼ 7.5 Hz);
2.92 (3H, s); 2.75e2.78 (2H, m); 2.13e2.19 (2H, m); 1.81e1.87
(2H, m); 1.30e1.54 (10H, m); 1.02 (3H, t, J ¼ 7.5 Hz). 13C NMR (75
CDCl3)
d
8.26 (1H, d, J ¼ 5.5 Hz); 7.96 (1H, d, J ¼ 8.5 Hz); 7.76 (1H, d, J
¼ 5.0 Hz); 6.86e6.91 (2H, m); 4.45 (2H, t, J ¼ 7.5 Hz); 4.10 (2H, t, J ¼
8.0 Hz); 3.06 (3H, s, CH3); 1.79e1.88 (4H, m); 1.52e1.60 (2H, m);
MHz, CDCl3) d: 160.5, 143.1, 140.9, 140.7, 138.4, 135.4, 129.6, 128.8,
1.41e1.47 (2H, m); 0.97e1.03 (6H, m). 13C NMR (75 MHz, CDCl3)
d:
128.6, 126.5, 122.4, 115.2, 112.4, 109.6, 93.9, 68.6, 44.3, 33.3, 32.3,
32.1, 29.8, 29.7, 29.6, 26.6, 23.5, 23.1, 14.6. Anal. Calcd for
C29H36N2O: C, 81.27; H, 8.47; N, 6.54. Found: C, 81.30; H, 8.50; N,
6.52.
160.5, 143.2, 140.7, 138.3, 135.5, 129.5, 122.4, 115.3, 112.4, 109.2, 94.4,
68.5, 45.0, 33.1, 31.8, 23.7, 20.6, 19.7,14.3. Anal. Calcd for C20H26N2O:
C, 77.38; H, 8.44; N, 9.02. Found: C, 77.33; H, 8.46; N, 9.03.
5.5.6. 7-Decyloxy-9-n-butyl-1-methyl-
b
-carboline (49)
5.5.11. 7-Benzyloxy-1-methyl-9-(3-phenylpropyl)-b-carboline (54)
White crystals (0.65 g, 81%) were obtained, mp 68e69 ꢀC. FAB-
MS m/z (M þ 1) 395. IR (KBr, cmꢁ1): 3427, 2925, 2852, 1620, 1462,
White crystals (0.68 g, 84%) were obtained, mp 139e140 ꢀC.
FAB-MS m/z (M þ 1) 407. IR (KBr, cmꢁ1): 3060, 3026, 2938, 2863,
1620, 1563, 1495, 1448, 1412, 1385, 1224, 1160, 1001, 742. 1H NMR
1338, 1231, 1194, 1138, 1039, 820. 1H NMR (500 MHz, CDCl3)
d 8.26
(1H, d, J ¼ 5.0 Hz); 7.97 (1H, d, J ¼ 8.5 Hz); 7.78 (1H, d, J ¼ 5.0 Hz);
6.86e6.92 (2H, m); 4.45e4.48 (2H, t, J ¼ 8.0 Hz); 4.08e4.11 (2H, t, J
¼ 8.0 Hz); 3.09 (3H, s); 1.78e1.89 (4H, m); 1.25e1.55 (16H, m); 0.98
(3H, t, J ¼ 7.5 Hz); 0.83 (3H, t, J ¼ 8.0 Hz). 13C NMR (75 MHz, CDCl3)
(500 MHz, CDCl3)
d
8.25 (1H, d, J ¼ 5.5 Hz); 7.94 (1H, d, J ¼ 8.5 Hz);
7.72 (1H, d, J ¼ 5.0 Hz); 7.18e7.48 (10H, m); 6.94e6.96 (1H, m);
6.72e6.73 (1H, m); 5.09 (2H, s); 4.42 (2H, t, J ¼ 6.5 Hz); 2.91(3H, s);
2.71e2.74 (2H, m); 2.09e2.15 (2H, m). 13C NMR (75 MHz, CDCl3)
d:
d
: 160.4, 143.2, 140.6, 138.2, 135.4, 129.5, 122.4, 115.2, 112.4, 109.1,
160.0, 143.0, 140.9, 140.8, 138.4, 137.0, 135.4, 129.5, 128.9, 128.7,
128.3, 127.8, 126.6, 122.6, 115.5, 112.5, 109.8, 94.6, 70.7, 44.4, 33.3,
32.1, 23.6. Anal. Calcd for C28H26N2O: C, 82.73; H, 6.45; N, 6.89.
Found: C, 82.68; H, 6.47; N, 6.91.
94.2, 68.7, 44.9, 33.1, 32.3, 30.0, 29.9, 29.8, 26.5, 23.8, 23.1, 20.6, 14.6,
14.3. Anal. Calcd for C26H38N2O: C, 79.14; H, 9.71; N, 7.10. Found: C,
79.24; H, 9.73; N, 7.08.
5.5.7. 7-n-Butyloxy-9-isobutyl-1-methyl-
b
-carboline (50)
5.5.12. 7-Perfluorobenzyloxy-9-(3-phenylpropyl)-1-methyl-
carboline (55)
b-
Yellow oil (0.48 g, 78%) was obtained. FAB-MS m/z (M þ 1) 311. IR
(KBr, cmꢁ1): 2956, 2868, 2485, 1625, 1574, 1468, 1432, 1336, 1255,
White crystals (0.72 g, 73%) were obtained, mp 149e150 ꢀC. FAB-
MS m/z (M þ 1) 497. IR (KBr, cmꢁ1): 2934, 1623, 1565, 1505, 1448,
1204, 1141, 1043, 804. 1H NMR (500 MHz, CDCl3)
d
8.27 (1H, d, J ¼
5.5 Hz); 7.97 (1H, d, J ¼ 9.5 Hz); 7.79 (1H, d, J ¼ 5.5 Hz); 6.87e6.91
(2H, m); 4.28 (2H, t, J ¼ 8.5 Hz); 4.10 (2H, d, J ¼ 8.0 Hz); 3.07 (3H, s);
2.24e2.27 (1H, m); 1.82e1.88 (2H, m); 1.54e1.58 (2H, m); 1.01 (3H,
1205, 1162, 1135, 1058, 943, 744. 1H NMR (500 MHz, CDCl3)
d 8.27
(1H, d, J ¼ 5.0 Hz); 7.98 (1H, d, J ¼ 9.0 Hz); 7.77 (1H, d, J ¼ 5.0 Hz);
7.19e7.33 (5H, m); 6.91e6.93 (1H, m); 6.74e6.75 (1H, m); 5.14 (2H,
s); 4.45 (2H, t, J ¼ 8.0 Hz); 2.94 (3H, s); 2.76e2.79 (2H, m);
t, J ¼ 7.5 Hz); 0.93e0.95 (6H, m). 13C NMR (75 MHz, CDCl3)
d: 160.3,
143.7, 140.7, 138.3, 135.7, 129.6, 122.3, 115.1, 112.3, 109.2, 95.1, 68.4,
52.1, 31.8, 30.9, 24.0, 20.6, 19.7, 14.3.
2.14e2.20 (2H, m). 13C NMR (75 MHz, CDCl3)
d: 159.1, 147.6, 144.3,
142.8, 140.9, 140.8, 138.5, 135.5, 129.3, 128.8, 126.5, 122.8, 116.2,
112.6, 110.2, 109.5, 94.7, 58.1, 44.4, 33.2, 32.1, 23.5. Anal. Calcd for
C28H21F5N2O: C, 67.74; H, 4.26; N, 5.64. Found: C, 67.82; H, 4.28; N,
5.67.
5.5.8. 7-Benzyloxy-9-isobutyl-1-methyl-b-carboline (51)
White crystals (0.59 g, 86%) were obtained, mp 110e112 ꢀC. FAB-
MS m/z (M þ 1) 345. IR: 3432, 2959, 2928, 2871, 1618, 1568, 1448,
1254, 1196, 1136, 1004, 731. 1H NMR (500 MHz, CDCl3)
d
8.27 (1H, d,
5.6. General procedure for the preparation of b-carbolinium
J ¼ 5.5 Hz); 7.96 (1H, d, J ¼ 8.5 Hz); 7.74 (1H, d, J ¼ 5.5 Hz); 7.30e7.49
(5H, m); 6.95e6.97 (1H, m); 6.92e6.95 (1H, m); 5.20 (2H, s); 4.23
(2H, d, J ¼ 8.0 Hz); 3.01 (3H, s); 2.18e2.21 (1H, m), 0.87 (6H, d, J ¼
derivatives 56e60
A mixture of 7-alkyloxy b-carboline derivatives (2 mmol) and
6.5 Hz). 13C NMR (75 MHz, CDCl3)
d
: 162.3, 147.1, 137.3, 137.0, 133.9,
benzyl bromide (10 mmol) in ethyl acetate (50 mL) was refluxed for
5e10 h. After completion of the reaction as indicated by TLC, the
solution was cooled and filtered to afford yellow solid. The solid
was crystallized from ethanol.
133.6, 129.3, 129.1, 128.6, 124.9, 114.9113.6, 113.4, 96.0, 70.8, 51.6,
31.2, 20.3, 18.4. Anal. Calcd for C23H24N2O: C, 80.20; H, 7.02; N, 8.13.
Found: C, 80.12; H, 7.04; N, 8.16.

2514
R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
5.6.1. 7-Ethoxy-2-benzyl-9-ethyl-1-methyl-
(56)
b
-carbolinium bromide
2.04e2.11 (2H, m); 1.78e1.84 (2H, m); 1.28e1.51 (10H, m); 0.86
(3H, t, J ¼ 7.5 Hz). 13C NMR (125 MHz, DMSO-d6)
d: 163.5, 147.9,
Yellow crystals (0.72 g, 85%) were obtained, mp 260e261 ꢀC.
FAB-MS m/z 345. IR (KBr, cmꢁ1): 3401, 2976, 2874, 1623, 1451, 1376,
1341,1259,1223,1134,1041, 826, 735. 1H NMR (500 MHz, DMSO-d6)
141.2, 139.8, 136.1, 135.3, 135.2, 133.7, 129.7, 128.9, 128.8, 128.7, 127.1,
126.5, 125.1, 114.8, 113.9, 113.0, 94.6, 68.9, 60.2, 45.1, 32.3, 31.8, 31.7,
29.2, 29.1, 29.0, 26.0, 22.5, 16.5, 14.4. Anal. Calcd for C36H43BrN2O: C,
72.11; H, 7.23; N, 4.67. Found: C, 72.18; H, 7.26; N, 4.68.
d
8.78 (1H, d, J ¼ 6.5 Hz, H-3); 8.60 (1H, d, J ¼ 6.5 Hz, H-4); 8.37 (1H,
d, J ¼ 9.0 Hz); 7.36e7.43 (4H, m); 7.19 (2H, d, J ¼ 7.0 Hz); 7.08e7.10
(1H, m); 6.04 (2H, s); 4.69e4.74 (2H, m); 4.26e4.30 (2H, m); 3.10
5.7. Cytotoxicity assays in vitro
(3H, s); 1.37e1.45 (6H, m). 13C NMR (125 MHz, DMSO-d6)
d: 163.1,
147.1, 139.4, 135.6, 134.9, 134.7, 133.2, 129.3, 128.5, 126.8, 124.8,
114.4, 113.5, 112.7, 94.1, 64.4, 59.9, 40.4, 16.2, 15.4, 14.6. Anal. Calcd
for C23H25BrN2O: C, 64.94; H, 5.92; N, 6.59. Found: C, 65.78; H, 5.93;
N, 6.57.
Cytotoxicity assays in vitro were carried out using 96 microtitre
plate cultures and MTT staining according to the procedures
described by our group [35]. Briefly, cells were grown in RPMI-1640
medium containing 10% (v/v) fetal calf serum and 100
m
g mLe1
penicillin and 100
m
g mLe1 streptomycin. Cultures were propagated
at 37 ꢀC in a humidified atmosphere containing 5% CO2. Drug stock
solutions were prepared in DMSO. The final concentration of DMSO
in the growth medium was 2% (v/v) or lower, concentration without
effect on cell replication. The tumor cell line panel consisted of liver
carcinoma (HepG2 and Bel-7402), gastric carcinoma (BGC-823),
cervical carcinoma (Hela), breast adenocarcinoma (MCF-7). In all of
these experiments, three replicate wells were used to determine
each point.
5.6.2. 7-(Perfluorobenzyloxy)-2-benzyl-9-ethyl-1-methyl-
carbolinium bromide (57)
b-
Yellow crystals (0.87 g, 76%) were obtained, mp 196e197 ꢀC.
FAB-MS m/z 497. IR (KBr, cmꢁ1): 3411, 2988, 1624, 1579, 1504, 1454,
1217, 1136, 1057, 973, 942, 828, 732. 1H NMR (500 MHz, DMSO-d6)
d
8.82 (1H, d, J ¼ 6.5 Hz); 8.66 (1H, d, J ¼ 6.5 Hz); 8.43 (1H, d, J ¼ 9.0
Hz); 7.63 (1H, m); 7.36e7.44 (3H, m); 7.14e7.22 (3H, m); 6.07 (2H,
s); 5.48 (2H, s); 4.75e4.76 (2H, m); 3.13 (3H, s); 1.43 (3H, t, J ¼ 7.5
Hz). 13C NMR (125 MHz, DMSO-d6)
d: 161.7,146.5,146.1,144.2,139.6,
138.0, 136.0, 134.7, 134.6, 132.8, 129.1, 128.3, 126.6, 124.8, 114.6,
113.3, 113.0, 109.7, 94.8, 59.8, 58.2, 40.4, 16.1, 15.2. Anal. Calcd for
C28H22BrF5N2O: C, 58.25; H, 3.84; N, 4.85. Found: C, 58.18; H, 3.83;
N, 4.87.
5.8. Data set
In this study, in vitro cytotoxic data against HepG2 cells were
selected to construct the 3D-QSAR models, Table 1 list their struc-
tures and biological data. For CoMFA and CoMSIA analysis, 29
compounds with determinate IC50 values of HepG2 cell line were
selected as the training set, while the rest compounds with inde-
terminate IC50 values (>200 mM) wereassignedto aninactivetestset
to verify if models can correctly identify inactives. IC50 values were
transformed to pIC50 values according to the following formula:
5.6.3. 7-n-Butoxy-2-benzyl-9-isobutyl-1-methyl-b-carbolinium
bromide (58)
Yellow crystals (0.81 g, 84%) were obtained, mp 246e248 ꢀC.
FAB-MS m/z 401. IR (KBr, cmꢁ1): 3410, 2957, 2927, 2867, 1620, 1576,
1457, 1375, 1253, 1209, 1138, 1030, 828. 1H NMR (500 MHz, DMSO-
d6)
d
8.81 (1H, d, J ¼ 6.5 Hz); 8.64 (1H, d, J ¼ 6.5 Hz); 8.37(1H, d, J ¼
1
pIC50 ¼ log
IC50
9.0 Hz); 7.35e7.44 (4H, m); 7.17 (2H, d, J ¼ 7.0 Hz); 7.08e7.10 (1H,
m); 6.04 (2H, s); 4.53 (2H, d, J ¼ 7.5 Hz); 4.21 (2H, t, J ¼ 7.5 Hz); 3.06
(3H, s); 2.04e2.09 (1H, m); 1.77e1.82 (2H, m); 1.47e1.55 (2H, m);
0.97 (3H, t, J ¼ 7.5 Hz); 0.83 (6H, d, J ¼ 6.5 Hz). 13C NMR (125 MHz,
5.9. Structure optimization and alignment
DMSO-d6) d: 163.4, 148.7, 140.0, 136.3, 135.3, 135.2, 133.9, 129.7,
Construction and optimization of the compounds, CoMFA and
CoMSIA modeling were all performed by using SYBYL6.9 molecular
modeling package (TRIPOS Associates Inc.) installed on a Silicon
Graphics Octane2 workstation 2400 with IRIX 6.5 operating
system. Molecules were created by using the sketch module of
SYBYL6.9. A molecular mechanics (MM) optimization was carried
out, MMFF94 force field and MMFF94 charges were employed and
the energy gradient was set to 0.001 kcal/mol. Subsequently,
128.9, 127.1, 125.0, 114.8, 113.9, 112.8, 95.5, 68.7, 60.3, 51.9, 31.1, 30.8,
19.9, 19.2, 16.7, 14.2. Anal. Calcd for C27H33BrN2O: C, 67.35; H, 6.91;
N, 5.82. Found: C, 67.48; H, 6.93; N, 5.85.
5.6.4. 7-Benzyloxy-2-benzyl-9-isobutyl-1-methyl-b-carbolinium
bromide (59)
Yellow crystals (0.86 g, 84%) were obtained, mp 218e220 ꢀC.
FAB-MS m/z 435. IR (KBr, cmꢁ1): 3408, 2959, 2921, 1622, 1581, 1454,
1351, 1253, 1222, 1137, 1029, 817, 743. 1H NMR (500 MHz, DMSO-d6)
compounds were aligned to the common three-ring structure of b-
carboline, and compound 57 with the highest pIC50 value was
chosen as the template molecular.
d
8.80 (1H, d, J ¼ 6.5 Hz); 8.64 (1H, d, J ¼ 6.5 Hz); 8.40 (1H, d, J ¼ 8.5
Hz); 7.34e7.54 (9H, m); 7.17e7.19 (3H, m); 6.04 (2H, s); 5.35 (2H, s);
4.51 (2H, d, J ¼ 7.5 Hz); 3.06 (3H, s); 2.02e2.05 (1H, m); 0.79 (6H, d, J
¼ 6.5 Hz). 13C NMR (75 MHz, DMSO-d6)
d: 162.9, 148.5, 140.2, 137.0,
5.10. CoMFA and CoMSIA studies
136.4, 135.4, 135.3, 134.0, 129.8, 129.2, 128.6, 127.2, 125.2, 115.1,
114.2, 113.2, 96.4, 70.8, 60.6, 52.2, 31.1, 20.2, 17.2. Anal. Calcd for
C30H31BrN2O: C, 69.90; H, 6.06; N, 5.43. Found: C, 69.82; H, 6.08; N,
5.46.
CoMFA computations were setup using default settings. The
steric (LennardeJones) and electrostatic (Coulomb) interaction
energies were generated using a sp3-hybridized carbon steric probe
atom with a positive charge of 1.00 as the electrostatic probe, grid
ꢀ
5.6.5. 7-Octyloxy-9-(3-phenylpropyl)-2-benzyl-1-methyl-
b
-
spacing was set to 2 AA, a cutoff of 30 kcal/mol was adopted.
carbolinium bromide (60)
For CoMSIA computations, the steric and electrostatic fields
were applied, the same grid spacing value and probe atom as
CoMFA was used to evaluate the interaction energies. Different
from the CoMFA, CoMSIA used a Gaussian type distance depen-
dence of physicochemical properties. Thus no singularities
occurred and no arbitrary cutoffs were required. The default value
of 0.3 was used as the attenuation factor (R).
Yellow crystals (0.9 g, 75%) were obtained, mp 185e186 ꢀC. FAB-
MS m/z 519. IR (KBr, cmꢁ1): 3421, 2993, 2926, 2856, 1620, 1579,
1453, 1371, 1248, 1155, 1134, 1031, 827, 727. 1H NMR (500 MHz,
DMSO-d6)
d
8.79 (1H, d, J ¼ 6.5 Hz); 8.60 (1H, d, J ¼ 6.5 Hz); 8.36
(1H, d, J ¼ 9.0 Hz); 7.07e7.43 (12H, m); 6.04 (2H, s); 4.66 (2H, t, J ¼
8.0 Hz); 4.15 (2H, t, J ¼ 8.0 Hz); 2.97 (3H, s); 2.68e2.71 (2H, m);

R. Cao et al. / European Journal of Medicinal Chemistry 45 (2010) 2503e2515
2515
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The partial least squares (PLS) linear regression method was
used to modeling the 3D-QSAR results of CoMFA & CoMSIA, the
CoMFA and CoMSIA descriptors were served as independent vari-
ables, while pIC50 values were served as dependent variables.
SAMPLS leave-one-out (LOO) Cross-validated PLS was performed to
determine the optimal number of components, corresponding to
the highest cross-validated r2 (q2) of the PLS analyses. The q2 was
calculated using the following formula (eq (1)):
ꢀ
ꢁ
[19] W. Schlecker, A. Huth, E. Ottow, J. Mulzer, Synth. (Mass.) 131 (1995)
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2
P
Ypred ꢁ Yactual
Y
q2 ¼ 1:0 ꢁ
(1)
P
ðYactual ꢁ YmeanÞ2
Y
Where Ypred, Yactual and Ymean are predicted, actual, and mean values
of the target property (pIC50), respectively.
Subsequently, the non-cross-validated calculation was
employed using the optimal number of components to generate the
final CoMFA and CoMSIA models. The predictive ability of the
models is expressed by conventional correlation coefficient r2
value, which is analogous to cross-validated r2 (q2). The standard
error of estimate SE and the Fisher test F value were also derived to
evaluate the predictive quality of the models.
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Acknowledgements
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This work was supported by MEGA-Project (2009ZX09103-015)
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