Synthesis and in vitro cytotoxic evaluation of novel 3,4,5-trimethoxyphenyl substituted β-carboline derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.03.030

To elucidate further our SARs' study on the chemistry and cytotoxic activity and probe the structural requirement for the potent antitumor activity of β-carbolines, a series of novel 1,9-disubstituted and 1,3,9-trisubstituted β-carboline derivatives were

Full text of DOI:10.1016/j.ejmech.2008.03.030

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 533e540
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro cytotoxic evaluation of novel 3,4,
5-trimethoxyphenyl substituted b-carboline derivatives
b
b
a
Qifeng Wu b, Rihui Cao a, , Manxiu Feng , Xiangdong Guan , Chunming Ma ,
*
Jinbing Liu a, Huacan Song a, Wenlie Peng
b,**
a School of Chemistry and Chemical Engineering, Sun Yat-sen (Zhongshan) University, 135 Xin Gang Xi Road, Guangzhou 510275, PR China
b School of Life Sciences, Sun Yat-sen (Zhongshan) University, 135 Xin Gang Xi Road, Guangzhou 510275, PR China
Received 11 January 2008; received in revised form 25 March 2008; accepted 27 March 2008
Available online 4 April 2008
Abstract
To elucidate further our SARs’ study on the chemistry and cytotoxic activity and probe the structural requirement for the potent antitumor
activity of b-carbolines, a series of novel 1,9-disubstituted and 1,3,9-trisubstituted b-carboline derivatives were designed and synthesized from
the starting material L-tryptophan and 3,4,5-trimethoxybenezaldehyde. Cytotoxic activities of these compounds in vitro were investigated, and
the SARs associated with position-1, 3 and 9 substituents in b-carbolines have also been discussed. It has been observed that these compounds
only displayed moderate to weak cytotoxic activities. Interestingly, most of the investigated compounds displayed selectively cytotoxic activities
to human BCG-823 cell lines with IC50 value lower than 100 mM. In addition, the short alkyl substituents in position-9 increased the cytotoxic
activities with the tendency of n-butyl > ethyl > methyl. These data confirmed that (1) an alkyl substituent at position-9 of b-carboline nucleus
plays an important role in determining their antitumor activities; (2) different b-carbolines bearing various substituents in b-carboline nucleus
interacted selectively with specific targets leading to the difference of biochemical and pharmacological effects.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: b-Carboline; Synthesis; Antitumor; Cytotoxic activities; SARs
1. Introduction
Previous reports [17e22] and our recent investigations
[23e30] on the synthesis of a variety of b-carboline deriva-
tives and the evaluation of their antitumor activities unraveled
that b-carbolines demonstrated potent antitumor activities and
the activity was correlated to both the planarity of the mole-
cule and the presence of the ring substituents. Subsequently,
a systematic structureeactivity relationships (SARs) analysis
was carried out to determine the influence on antitumor activ-
ity of various substituents in b-carboline ring, and preliminary
SARs were also depicted [31]. Clearly, to acquire more infor-
mation about the structural requirements for the possible
improvement of the cytotoxic potential and to elucidate
SARs between substituent properties in b-carboline and antitu-
mor activities, design and synthesis of more novel b-carboline
derivatives with various substituents at different positions of
the b-carboline nucleus are needed.
The b-carboline alkaloids are a large group of natural and
synthetic indole alkaloids that possess a common tricyclic pyr-
ido[3,4-b]indole ring structure [1,2]. These compounds pos-
sess a wide diversity of important biochemical effects and
pharmacological properties. Numerous previous reports inves-
tigated the effects of b-carboline alkaloids on the central ner-
vous system (CNS), such as their affinity with benzodiazepine
receptors (BZRs) [3e12] and 5-hydroxy serotonin receptors
[13e16]. Recently, interests in these alkaloids were stimulated
by their potent antitumor activities.
* Corresponding author. Tel.: þ86 20 84110918; fax: þ86 20 84112245.
** Corresponding author.
Inspired by these results of previous and our recent investiga-
tion, in the present investigation, we designed and synthesized
E-mail addresses: caorihui@mail.sysu.edu.cn (R. Cao), pengwl@mail.
sysu.edu.cn (W. Peng).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.030

534
Q. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 533e540
several series of novel b-carboline derivatives bearing 3,4,5-
trimethoxyphenyl in position-1 on the basis of harmine chemi-
cal structure, it is possible to discover and develop novel
antitumor agents. The design of substituents in position-9 was
based on the previous experience [24e27], and the choice of
substituents in position-3 was limited to carboxylate, carboxyl,
acyl, hydroxymethyl and aldehyde. The focus of this investiga-
tion was to probe the optimal structural requirement of these
compounds with regard to antitumor activities and further elu-
cidate the SARs. To the best of our knowledge, all b-carboline
derivatives are novel. We report here the preparation of novel
b-carboline derivatives and their cytotoxic activities.
3. Results and discussion
The cytotoxic potential of all newly synthesized b-carbo-
line derivatives was evaluated in vitro against a panel of hu-
man tumor cell lines. Compounds 7aec were converted into
its water-soluble sodium salts, and the other compounds exam-
ined were all prepared in the form of hydrochloride in order to
enhance the solubility in aqueous solution by the usual
methods before use. The results are summarized in Table 1.
As shown in Table 1, compound 4 and its N9-alkyl substituted
derivatives 4aec showed moderate cytotoxic activities against
human tumor cell lines. Interestingly, the compounds bearing
a carboxylate (6aed), carboxyl (7aec), and acylamide (8ae
c) substituents in position-3 of b-carboline ring system
displayed selectively cytotoxic activities against human BGC-
823 cell lines but failed to show cytotoxic effect in other tumor
cell lines at the concentration of 100 mM. In addition, the com-
pounds with a hydroxymethyl (9aed) and carboxyaldehyde
(10aed) substituents in position-3 were almost inactive to all
tumor cell lines investigated at the concentration of 100 mM.
As predicted, introducing a short alkyl substituent into posi-
tion-9 of compounds 4 and 6 led to compounds 4a and 6a
(methyl), 4b and 6b (ethyl), 4c and 6c (butyl), which all showed
more potent cytotoxic activities than their parent compounds 4
and 6 with the tendency of methyl > ethyl > butyl. Similarly,
the cytotoxic potency of compounds 7aec and 8aec followed
the sequence of 7c > 7b > 7a and 8c > 8b > 8a.
As a whole, the present results showed that 9-position short
alkyl substituents might be a favorable group to exploit in
searching for new antitumor leading compounds, and these
data further substantiated our previous observations, indicating
that position-9 substituted with short alkyl led to derivatives
with enhanced cytotoxic acitivities. In addition, substituents
at position-3 of b-carboline were detrimental to cytotoxic
activity of b-carboline derivatives, and these results also con-
firmed our previous reports that substituents in position-3 were
unfavorable for their cytotoxic activities. Although the b-
carboline derivatives presented here showed modest cytotoxic
activities, the investigations of these structural modifications
and preliminary SARs would be helpful to further design
and develop more potent compounds.
2. Chemistry
1-(3,4,5-Trimethoxy)phenyl-1,2,3,4-tetrahydro-b-carboline-
3-carboxylic acid 3 was prepared by the condensation of
L-tryptophan with 3,4,5-trimethoxybenzaldehyde via well-
known PicteteSpengler condensation in acid media, and subse-
quently oxidated and decarboxylated in a single step according
to the method previously described bySnyderet al. [32] to afford
1-(3,4,5-trimethoxy)phenyl-b-carboline 4. Esterification of
compound 3 with ethanol in the presence of SOCl2 by heating
yielded the corresponding ethyl 1-(3,4,5-trimethoxy)phenyl-
1,2,3,4-tetrahydro-b-carboline-3-carboxylate 5, and followed
by dehydrogenation with sulfur in refluxing xylene to afford
fully unsaturated compound 6. This sequence of reactions can
be easily scaled up to give enough material 6 for further
transformation.
Substitution of alkyl or benzyl groups at the N9-position of b-
carboline ring system would be expected to enhance cytotoxic-
ity, consequently a number of derivatives were designed and
synthesized with substituents ranging in size from methyl group
to phenylpropyl in position-9 of b-carboline ring system. The
N9-position of compounds 4 and 6 was alkylated or arylated
by the action of sodium hydride in dry DMF followed by addi-
tion of the relevant appropriate alkylating and arylating agents
to provide various 9-substituted b-carboline 4aec and b-
carboline-3-carboxylate 6aed, respectively. Compounds 6ae
c were further hydrolyzed in alkaline solution to produce the
corresponding b-carboline-3-carboxylic acids 7aec.
In this paper we reported only preliminary results on the
relationship between structure and cytotoxic activities. To
acquire more information about the structural requirements
for improving cytotoxic activities, the synthesis of more new
b-carboline derivatives with different substituents at other
positions is needed. Further investigations and biological eval-
uations on these and other unreported b-carboline derivatives
in animal models are in progress in our laboratories, and the
data will be reported in a separated paper.
The amidation of carboxylate in position-3 of compounds
6aec with ethanolamine could be easily reacted in dry ethanol
followed by addition of sodium hydride to give the corre-
sponding amidated derivatives 8aec in good yield. The ester
group in position-3 of compounds 6aed was reduced to its
corresponding alcohols by lithium borohydride (LiBH4) in
dry THF according to the method reported by Cain et al. [4]
to provide 9aed, and further oxidized by MnO2 in CH3CN
to afford 3-carboxaldehyde derivatives 10aed. Unfortunately,
in our studies, compounds 6aed failed to be reduced to alco-
hols by LiAlH4 as described by Srivastava et al. [33], whereas,
they could be easily reduced to alcohols by LiBH4 in good
yield (see Scheme 1). The chemical structures of all the
synthesized novel compounds were confirmed by FAB-MS,
4. Experimental
4.1. Chemistry
All reagents were purchased from commercial suppliers
and were dried and purified when necessary. Harmine was
1
IR, H NMR and elemental analyses data.

Q. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 533e540
535
COOH
COOH
N
NH
NH2
N
H
CHO
N
H
N
H
OCH3
OCH3
OCH3
OCH3
1
H3CO
H3CO
OCH3
H3CO
OCH3
4
3
2
COOH
N
COOC2H5
N
N
N
NH
R9
OCH3
OCH3
R9
N
H
OCH3
OCH3
H3CO
H3CO
OCH3
OCH3
4a R9=CH3
4b R9=C2H5
4c R9=n-C4H9
H3CO
7a R9=CH3
7b R9=C2H5
7c R9=n-C4H9
5
COOC2H5
CONHCH2CH2OH
COOC2H5
N
N
N
N
R9
H3CO
N
N
H
R9
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H3CO
H3CO
6a R9=CH3
6b R9=C2H5
6c R9=n-C4H9
6d R9=(CH2)3C6H5
8a R9=CH3
8b R9=C2H5
8c R9=n-C4H9
6
CHO
N
CH2OH
N
N
N
R9
R9
OCH3
OCH3
OCH3
OCH3
H3CO
H3CO
10a R9=CH3
9a R9=CH3
10b R9=C2H5
10c R9=n-C4H9
10d R9=(CH2)3C6H5
9b R9=C2H5
9c R9=n-C4H9
9d R9=(CH2)3C6H5
Scheme 1. Synthesis of 1,9 and 1,3,9-substituted b-carboline derivatives.
1
extracted from Peganum multisectum Maxim, a plant indige-
nous to western China, according to the method by Duan
et al. [34]. Melting points were determined in capillary tubes
on an electrothermal PIF YRT-3 apparatus without correction.
FAB-MS spectra were obtained from VG ZAB-HS spectrom-
eter. FT-IR was run on a Bruker Equinox 55 Fourier
Transformation Infrared Spectrometer. H NMR spectra were
recorded on a Varian INOVA 500NB spectrometer. Elemental
analyses were carried out on an ElementarVario EL CHNS
Elemental Analyzer. Silica gel F254 was used in analytical
thin-layer chromatography (TLC) and silica gel was used in
column chromatography.

536
Q. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 533e540
1404, 1346, 1232, 1126, 1000, 827, 751; 1H NMR
(500 MHz, CDCl3): d 8.79 (1H, s, NH ); 8.52e8.53 (1H, d,
J ¼ 5.5 Hz, H-3); 8.14e8.16 (1H, d, J ¼ 8.0 Hz, H-5); 7.91e
7.92 (1H, d, J ¼ 5.5 Hz, H-8); 7.52e7.57 (2H, m, H-6, H-7);
7.29e7.32 (1H, m, H-4); 7.13 (2H, s, PhH ); 3.90 (9H, s,
OCH3); Anal. Calcd for C20H18N2O3: C, 71.84; H, 5.43; N,
8.38. Found: C, 71.70; H, 5.48; N, 8.32.
Table 1
Cytotoxicity of b-carboline derivatives in vitroc
IC50 (mM)a
Compounds
Helab
Bel-7402b
BGC-823b
HepG2b
MCF-7b
Harmine
4
4a
60.0
54.0
43.8
68.0
43.1
46.0
63.2
72.0
31.7
19.6
71.8
50.9
44.2
91.1
43.9
43.2
35.2
86.8
>100
51.9
74.2
72.7
4b
4c
42.5
27.5
96.6
51.8
92.3
4.1.3. General procedure for the preparation of 9-
substituted 1-(3,4,5-trimethoxy)-phenyl-b-car-boline (4aec)
A mixture of 4 (1.67 g, 5 mmol) and anhydrous DMF
(50 ml) was stirred at room temperature until clear, and then
60% NaH (0.3 g, 7.5 mmol) and halogenated alkane (10e
15 mmol) were added. The mixture was stirred at room
temperature for 0.5e2 h. 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 or ethyl ether-petroleum ether.
6
6a
>100
>100
>100
>100
>100
>100
>100
61.4
>100
>100
>100
>100
>100
>100
>100
>100
79.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
56.6
53.2
>100
>100
43.6
6b
6c
27.3
95.4
6d
7a
>100
>100
>100
>100
36.3
91.1
66.5
7b
7c
>100
>100
>100
>100
>100
>100
>100
47.5
51.1
36.2
7d
8a
90.9
74.7
>100
>100
87.4
95.5
53.8
8b
8c
37.2
31.6
9a
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9b
9c
9d
10a
10b
10c
10d
a
>100
>100
>100
>100
>100
4.1.3.1. 1-(3,4,5-Trimethoxy)phenyl-9-methyl-b-carboline (4a).
Afforded white crystals (1.4 g, 80%), mp 147e148 ꢀC. FAB-
MS m/z (M þ 1): 349; IR (KBr): 3005, 2938, 2836, 1618,
1581, 1502, 1460, 1401, 1353, 1229, 1123, 1006, 840, 752;
1H NMR (500 MHz, CDCl3):
d 8.50e8.51 (1H, d,
Cytotoxicity as IC50 for each cell line is the concentration of compound
which reduced by 50% the optical density of treated cells with respect to un-
treated cells using the MTT assay.
J ¼ 5.5 Hz, H-3); 8.18e8.19 (1H, d, J ¼ 7.0 Hz, H-8); 7.98e
7.99 (1H, d, J ¼ 5.0 Hz, H-5); 7.61e7.64 (1H, m, H-7);
7.43e7.45 (1H, d, J ¼ 8.0 Hz, H-6); 7.31e7.34 (1H, m, H-
4); 6.86 (2H, s, PhH ); 3.91e3.93 (9H, m, OCH3); 3.55 (3H,
s, NCH3); Anal. Calcd for C21H20N2O3: C, 72.40; H, 5.79;
N, 8.04. Found: C, 72.53; H, 5.86; N, 7.99.
b
Cell lines include cervical carcinoma (Hela), liver carcinoma (HepG2 and
Bel -7402), gastric carcinoma (BGC-823), and breast carcinoma (MCF-7).
c
Data represent the mean values of three independent determinations.
4.1.1. 1-(3,4,5-Trimethoxy)phenyl-1,2,3,4-tetrahydro-
b-carboline-3-carboxylic acid (3)
4.1.3.2. 1-(3,4,5-Trimethoxy)phenyl-9-ethyl-b-carboline (4b).
Afforded white crystals (1.5 g, 83%), mp 144e145 ꢀC. FAB-
MS m/z (M þ 1): 363; IR (KBr): 2962, 2931, 2826, 1617,
1584, 1504, 1447, 1402, 1352, 1229, 1129, 1001, 832, 748;
A mixture of L-tryptophan (40.8 g, 200 mmol), 3,4,5-trime-
thoxybenzaldehyde (39.2 g, 200 mmol) and acetic acid
(300 ml) was refluxed for 3 h, then cooled and adjusted to
pH 5 with concentrated ammonium hydroxide, the precipitated
product was collected by filtration and washed well with water
and then dried. White solid (74.0 g, 97%) was obtained.
Further purification was not necessary and used directly for
the next steps.
1H NMR (500 MHz, CDCl3):
d 8.49e8.50 (1H, d,
J ¼ 5.0 Hz, H-3); 8.18e8.19 (1H, d, J ¼ 8.0 Hz, H-8); 7.98e
7.99 (1H, d, J ¼ 5.0 Hz, H-5); 7.58e7.61 (1H, m, H-7);
7.45e7.46 (1H, d, J ¼ 8.5 Hz, H-6); 7.30e7.33 (1H, m, H-
4); 6.83 (2H, s, PhH ); 4.08e4.09 (2H, m, NCH2CH3);
3.89e3.93 (9H, m, OCH3); 1.05e1.08 (3H, s, NCH2CH3);
Anal. Calcd for C22H22N2O3: C, 72.91; H, 6.12; N, 7.73.
Found: C, 72.67; H, 6.21; N, 7.64.
4.1.2. 1-(3,4,5-Trimethoxy)phenyl-b-carboline (4)
Compound 3 (38.2 g, 100 mmol) was diluted to 3000 ml
with water, and then heated to boiling. To the hot solution
was added potassium dichromate (150 g) and glacial acetic
acid (200 ml). The brown suspension was heated for 10 min
and then cooled under the tap. After treating the cold solution
with sodium sulfite to remove the excess oxidizing agent, the
mixture was made definitely alkaline with sodium hydroxide.
The solution was extracted exhaustively with ethyl acetate
(10 L), the extracts dried over anhydrous sodium sulfate, and
the solvent removed. The residue was crystallized from anhy-
drous ethanol, and white crystals of 4 were obtained (20 g,
60%), mp 167e169 ꢀC. FAB-MS m/z (M þ 1): 335; IR
(KBr): 3556, 3309, 2937, 2832, 1624, 1583, 1502, 1456,
4.1.3.3. 1-(3,4,5-Trimethoxy)phenyl-9-n-butyl-b-carboline (4c).
Afforded white crystals (1.4 g, 83%), mp 118e119 ꢀC. FAB-
MS m/z (M þ 1): 391; IR (KBr): 3013, 2959, 2931, 2835,
1617, 1582, 1505, 1450, 1410, 1362, 1238, 1123, 997, 831,
751; 1H NMR (500 MHz, CDCl3): d 8.49e8.50 (1H, d,
J ¼ 5.0 Hz, H-3); 8.17e8.19 (1H, d, J ¼ 7.5 Hz, H-8); 7.98e
7.99 (1H, d, J ¼ 5.0 Hz, H-5); 7.57e7.61 (1H, m, H-7);
7.43e7.45 (1H, d, J ¼ 8.0 Hz, H-6); 7.30e7.32 (1H, m, H-
4); 6.83 (2H, s, PhH ); 3.98e4.01 (2H, m, NCH2(CH2)2CH3);
3.89e3.92 (9H, m, OCH3); 1.44e1.47 (2H, m,
NCH2CH2CH2CH3); 0.94e1.01 (2H, m, NCH2CH2CH2CH3);

Q. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 533e540
537
0.70e0.73 (3H, m, NCH2CH2CH2CH3); Anal. Calcd for
C24H26N2O3: C, 73.82; H, 6.71; N, 7.17. Found: C, 73.72;
H, 6.79; N, 7.11.
OCH2CH3); Anal. Calcd for C24H24N2O5: C, 68.56; H, 5.75;
N, 6.66. Found: C, 68.39; H, 5.86; N, 6.61.
4.1.6.2. Ethyl 1-(3,4,5-trimethoxy)phenyl-9-ethyl-b-carboline-
3-carboxylate (6b). Afforded white crystals (1.3 g, 60%), mp
170e171 ꢀC. FAB-MS m/z (M þ 1): 435; IR (KBr): 3059,
2977, 2939, 2826, 1730, 1695, 1619, 1583, 1505, 1465,
4.1.4. Ethyl 1-(3,4,5-trimethoxy)phenyl-1,2,3,
4-tetrahydro-b-carboline-3-carboxylate (5)
A mixture of compound 3 (76.4 g, 200 mmol), anhydrous
ethanol (500 ml) and SOCl2 (20 ml) was heated at reflux for
4 h, and then evaporated in reduced pressure. The resulting mix-
ture was poured into H2O (300 ml) and neutralized with sodium
hydrogen carbonate. The solution was extracted with ethyl
acetate (3 ꢁ 300 ml). The organic phase was washed with water
and brine, then dried over anhydrous sodium sulfate, filtered
and evaporated. The residue was crystallized from ethyl acetate
to afford white solid (73 g, 91%). Further purification was not
necessary and used directly for the next steps.
1
1369, 1246, 1123, 1005, 747; H NMR (500 MHz, CDCl3):
d 8.89 (1H, s, H-4); 8.25e8.27 (1H, d, J ¼ 7.0 Hz, H-8);
7.63e7.66 (1H, m, H-5); 7.49e7.50 (1H, d, J ¼ 8.5 Hz, H-
6); 7.37e7.40 (1H, m, H-7); 6.84 (2H, s, PhH ); 4.51e4.55
(2H, m, OCH2CH3); 4.05e4.07 (2H, m, NCH2CH3); 3.89e
3.92 (9H, m, OCH3); 1.46e1.49 (3H, m, NCH2CH3); 1.07e
1.10 (3H, m, OCH2CH3); Anal. Calcd for C25H26N2O5: C,
69.11; H, 6.03; N, 6.45. Found: C, 69.00; H, 6.13; N, 6.42.
4.1.6.3. Ethyl 1-(3,4,5-trimethoxy)phenyl-9-n-butyl-b-carbo-
line-3-carboxylate (6c). Afforded white crystals (1.6 g,
69%), mp 138e139 ꢀC. FAB-MS m/z (M þ 1): 463; IR
(KBr): 3065, 2933, 1711, 1621, 1583, 1505, 1450, 1413,
1368, 1260, 1237, 1128, 1006, 739; 1H NMR (500 MHz,
CDCl3): d 8.89 (1H, s, H-4); 8.25e8.26 (1H, d, J ¼ 8.5 Hz,
H-8); 7.62e7.65 (1H, m, H-5); 7.47e7.49 (1H, d,
J ¼ 8.0 Hz, H-6); 7.36e7.39 (1H, m, H-7); 6.83 (2H, s,
PhH ); 4.51e4.55 (2H, m, OCH2CH3); 3.95e3.98 (2H, m,
NCH2CH2CH2CH3); 3.89e3.91 (9H, m, OCH3); 1.46e1.49
(5H, m, OCH2CH3, NCH2CH2CH2CH3); 0.95e0.99 (2H, m,
NCH2CH2CH2CH3); 0.70e0.73 (3H, m, NCH2CH2CH2CH3);
Anal. Calcd for C27H30N2O5: C, 70.11; H, 6.54; N, 6.06.
Found: C, 69.83; H, 6.65; N, 5.98.
4.1.5. Ethyl 1-(3,4,5-trimethoxy)phenyl-b-carboline-
3-carboxylate (6)
A suspension of compound 5 (41 g, 100 mmol) and sulfur
(9.6 g, 300 mmol) in xylene (250 ml) was heated at reflux for
8 h. The solution was cooled and stored at 4 ꢀC for 3 h, and
then filtered and washed generously with petroleum ether, the
solid was dried and recrystallized from ethyl acetate to afford
white crystals (27 g, 68%), mp 229e230 ꢀC. FAB-MS m/z
(M þ 1): 407; IR (KBr): 3252, 2991, 2940, 2832, 1711, 1625,
1588, 1501, 1455, 1391, 1248, 1127, 1006, 840, 745; 1H
NMR (500 MHz, CDCl3): d 9.28 (1H, s, NH ); 8.80 (1H, s, H-
4); 8.20e8.22 (1H, d, J ¼ 7.5 Hz, H-8); 7.58e7.63 (2H, m,
H-5, H-6); 7.36e7.39 (1H, m, H-7); 6.82 (2H, s, PhH );
4.51e4.55 (2H, m, OCH2CH3); 3.87e3.89 (9H, m, OCH3);
1.47e1.50 (3H, m, OCH2CH3); Anal. Calcd for C23H22N2O5:
C, 67.97; H, 5.46; N, 6.89. Found: C, 67.79; H, 5.52; N, 6.83.
4.1.6.4. Ethyl 1-(3,4,5-trimethoxy)phenyl-9-phenylpropyl-b-
carboline-3-carboxylate (6d). Afforded white crystals (1.8 g,
68%), mp 158e159 ꢀC. FAB-MS m/z (M þ 1): 525; IR
(KBr): 3061, 3025, 2938, 2840, 1729, 1704, 1621, 1583,
1504, 1452, 1412, 1367, 1242, 1124, 1008,745; 1H NMR
(500 MHz, CDCl3): d 8.94 (1H, s, H-4); 8.47e8.49 (1H, d,
J ¼ 10.0 Hz, H-6); 7.72e7.74 (1H, d, J ¼ 10.5 Hz, H-8);
7.64e7.68 (1H, m, H-5); 7.36e7.39 (1H, m, H-7); 7.01e
7.29 (7H, m, PhH ); 4.34e4.41 (2H, m, NCH2CH2CH2Ph);
4.02e4.06 (2H, m, OCH2CH3); 3.79 (9H, m, OCH3); 2.16e
2.20 (2H, m, NCH2CH2CH2Ph); 1.74e1.76 (2H, m,
NCH2CH2CH2Ph); 1.34e1.38 (3H, m, OCH2CH3); Anal.
Calcd for C32H32N2O5: C, 73.26; H, 6.15; N, 5.34. Found:
C, 73.43; H, 6.26; N, 5.29.
4.1.6. General procedure for the preparation of ethyl
1-(3,4,5-trimethoxy)-phenyl-9-substituted
b-carboline-3-carboxylate (6aed)
A mixture of compound 6 (2.0 g, 5 mmol) and anhydrous
DMF (50 ml) was stirred at RT until clear, and then 60%
NaH (0.3 g, 7.5 mmol) and halogenated alkane (10e
15 mmol) were added. The mixture was stirred at RT for
0.5e2 h. 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 sul-
fate, filtered and evaporated. The resulting oil was crystallized
from ethyl ether or ethyl ether-petroleum ether.
4.1.7. General procedure for the preparation of 1-(3,4,5-
trimethoxy)phenyl-9-substituted-b-carboline-3-carboxylic
acid (7aec)
A mixture of compound 6aec (5 mmol), NaOH (25 mmol),
ethanol (50 ml) and H2O (50 ml) was refluxed for 1 h, and eth-
anol was removed on the rotary evaporator. The mixture was
neutralized (pH 5) with 5 M HCl and cooled. The precipitate
was collected, washed well with H2O and dried in vacuum.
4.1.6.1. Ethyl 1-(3,4,5-trimethoxy)phenyl-9-methyl-b-carbo-
line-3-carboxylate (6a). Afforded white crystals (1.5 g, 71%),
mp 173e175 ꢀC. FAB-MS m/z (M þ 1): 421; IR (KBr): 2939,
2834, 1702, 1622, 1583, 1504, 1461, 1369, 1319, 1259, 1128,
1
1005, 736; H NMR (500 MHz, CDCl3): d 8.87 (1H, s, H-4);
8.24e8.26 (1H, d, J ¼ 7.5 Hz, H-8); 7.64e7.67 (1H, m, H-5);
7.47e7.48 (1H, d, J ¼ 8.5 Hz, H-6); 7.37e7.40 (1H, m, H-7);
6.86 (2H, s, PhH ); 4.50e4.54 (2H, m, OCH2CH3); 3.90e3.92
(9H, m, OCH3); 3.55 (3H, s, NCH3); 1.46e1.49 (3H, m,
4.1.7.1. 1-(3,4,5-Trimethoxy)phenyl-9-methyl-b-carboline-3-
carboxylic acid (7a). Yellow solid was obtained (1.9 g,

538
Q. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 533e540
97%), mp 246e248 ꢀC. FAB-MS m/z (M þ 1): 393; IR (KBr):
1
1239, 1126, 1004, 751; H NMR (500 MHz, DMSO): d 8.93
(1H, s, H-4); 8.46e8.47 (1H, d, J ¼ 7.5 Hz, H-8); 7.66e7.71
(2H, m, H-5, H-6); 7.37e7.40 (1H, m, H-7); 6.94 (2H, s,
PhH ); 3.78e3.84 (9H, m, OCH3); 3.56 (3H, s, NCH3);
Anal. Calcd for C22H20N2O5: C, 67.34; H, 5.14; N, 7.14.
Found: C, 67.26; H, 5.16; N, 7.10.
s, PhH ); 3.92e3.97 (9H, m, OCH3); 3.88e3.90 (2H, m,
NHCH2CH2OH); 3.68e3.70 (2H, m, NHCH2CH2OH); 3.55
(3H, s, NCH3); Anal. Calcd for C24H25N3O5: C, 66.19; H,
5.79; N, 9.65. Found: C, 66.01; H, 5.86; N, 9.68.
3192, 2944, 2826, 1747, 1619, 1585, 1501, 1466, 1421, 1362,
4.1.8.2.
N-(2-Hydroxyethyl)-1-(3,4,5-trimethoxy)phenyl-9-
ethyl-b-carboline-3-formamide (8b). White crystals were ob-
tained (1.8 g, 80%), mp 168e169 ꢀC. FAB-MS m/z (M þ 1):
450; IR (KBr): 3400, 2966, 2933, 1666, 1583, 1533, 1503,
1464, 1368, 1234, 1125, 1005, 750, 728; 1H NMR
(500 MHz, CDCl3): d 8.97 (1H, s, H-4); 8.25e8.27 (1H, d,
J ¼ 6.5 Hz, H-8); 7.62e7.66 (1H, m, H-5); 7.47e7.49 (1H,
d, J ¼ 8.0 Hz, H-6); 7.36e7.39 (1H, m, H-7); 6.81 (2H, s,
PhH ); 4.03e4.08 (2H, m, NCH2CH3); 3.91e3.98 (9H, m,
OCH3); 3.85e3.87 (2H, m, NHCH2CH2OH); 3.68e3.69
(2H, m, NHCH2CH2OH); 1.08e1.11 (3H, s, NCH2CH3);
Anal. Calcd for C25H27N3O5: C, 66.80; H, 6.05; N, 9.35.
Found: C, 66.89; H, 6.14; N, 9.32.
4.1.7.2. 1-(3,4,5-Trimethoxy)phenyl-9-ethyl-b-carboline-3-car-
boxylic acid (7b). Yellow solid was obtained (1.9 g, 94%),
mp 215e216 ꢀC. FAB-MS m/z (M þ 1): 407; IR (KBr):
3525, 3368, 2942, 2356, 1707, 1620, 1584, 1502, 1455,
1413, 1362, 1234, 1126, 993, 786; 1H NMR (500 MHz,
DMSO): d 9.05 (1H, s, H-4); 8.53e8.54 (1H, d, J ¼ 7.5 Hz,
H-8); 7.70e7.78 (2H, m, H-5, H-6); 7.40e7.43 (1H, m, H-
7); 6.99 (2H, s, PhH ); 4.07e4.11 (2H, m, NCH2CH3);
3.79e3.82 (9H, m, OCH3); 1.03e1.06 (3H, s, NCH2CH3);
Anal. Calcd for C23H22N2O5: C, 67.97; H, 5.46; N, 6.89.
Found: C, 67.87; H, 5.50; N, 6.83.
4.1.8.3. N-(2-Hydroxyethyl)-1-(3,4,5-trimethoxy)phenyl-9-n-
butyl-b-carboline-3-formamide (8c). White crystals were ob-
tained (1.8 g, 76%), mp 197e198 ꢀC. FAB-MS m/z (M þ 1):
478; IR (KBr): 3354, 2955, 2932, 2871, 1644, 1620, 1584,
4.1.7.3. 1-(3,4,5-Trimethoxy)phenyl-9-n-butyl-b-carboline-
3-carboxylic acid (7c). Yellow solid was obtained (2.0, 97%),
mp 203e204 ꢀC. FAB-MS m/z (M þ 1): 435; IR (KBr): 3300,
2962, 2873, 1736, 1620, 1584, 1506, 1456, 1410, 1362, 1304,
1238, 1125, 1002, 957, 824, 737; 1H NMR (500 MHz,
DMSO): d 8.93 (1H, s, H-4); 8.45e8.47 (1H, d, J ¼ 7.5 Hz, H-
8); 7.64e7.73 (2H, m, H-5, H-6); 7.35e7.38 (1H, m, H-7);
6.91 (2H, s, PhH ); 3.99e4.02 (2H, m, NCH2CH2CH2CH3);
3.78e3.82 (9H, m, OCH3); 1.39e1.45 (2H, m,
NCH2CH2CH2CH3); 0.91e0.95 (2H, m, NCH2CH2CH2CH3);
0.65e0.68 (3H, m, NCH2CH2CH2CH3); Anal. Calcd for
C25H26N2O5: C, 69.11; H, 6.03; N, 6.45. Found: C, 68.98; H,
6.13; N, 6.42.
1
1533, 1503, 1446, 1368, 1231, 1129, 1055, 1004, 735; H
NMR (500 MHz, CDCl3): d 8.96 (1H, s, H-4); 8.25e8.26
(1H, d, J ¼ 6.5 Hz, H-8); 7.61e7.65 (1H, m, H-5); 7.46e7.47
(1H, d, J ¼ 8.5 Hz, H-6); 7.35e7.38 (1H, m, H-7); 6.80 (2H,
s, PhH ); 3.91e3.98 (11H, m, OCH3, NCH2CH2CH2CH3);
3.85e3.87 (2H, m, NHCH2CH2OH); 3.66e3.69 (2H, m,
NHCH2CH2OH); 1.44e1.51 (2H, m, NCH2CH2CH2CH3);
0.94e1.02 (2H, m, NCH2CH2CH2CH3); 0.71e0.74 (3H, s,
NCH2CH2CH2CH3); Anal. Calcd for C27H31N3O5: C, 67.91;
H, 6.54; N, 8.80. Found: C, 67.74; H, 6.51; N, 8.71.
4.1.9. General procedure for the preparation of 1-(3,4,
5-trimethoxy)phenyl-3-hydroxymethyl-9-substituted-b-
carboline (9aed)
4.1.8. General procedure for the preparation of
N-(2-hydroxyethyl)-1-(3,4,5-trimethoxy)-phenyl-
9-substituted-b-carboline-3-formamide (8aec)
A fine suspension of compounds 6aed (10 mmol) in dry
THF (100 ml) was treated with LiBH4 (30 mmol), and the mix-
ture 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 solu-
tion 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 puri-
fied by silica column chromatography with ethyl acetate as the
eluent. Upon recrystallization, white crystals were obtained.
A solution of compounds 6aec (5 mmol) in ethanol (50 ml)
containing ethanolamine (40 mmol) and 60% NaH (0.4 g,
10 mmol) was refluxed for 15e30 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 re-
sulting oil obtained was purified by silica column chromatogra-
phy with ethyl acetate as the eluent, and crystallized from ethyl
acetate or ethyl acetate-petroleum ether to give white crystals.
4.1.9.1. 1-(3,4,5-Trimethoxy)phenyl-3-hydroxymethyl-9-methyl-
b-carboline (9a). White crystals were obtained (3.0 g, 79%),
mp 151e152 ꢀC. FAB-MS m/z (M þ 1): 379; IR (KBr):
3275, 3060, 2998, 2930, 2833, 1622, 1581, 1504, 1462,
1413, 1370, 1234, 1126, 1063, 1003, 836, 738; 1H NMR
(500 MHz, CDCl3): d 8.30e8.32 (1H, d, J ¼ 10.0 Hz, H-4);
8.19 (1H, s, H-6); 7.60e7.61 (2H, m, H-5, H-8); 7.27e7.31
(1H, m, H-7); 6.86 (2H, s, PhH ); 4.73e4.75 (2H, d,
J ¼ 7.0 Hz, CH2OH); 3.82 (6H, s, OCH3); 3.76 (3H, s,
4.1.8.1. N-(2-Hydroxyethyl)-1-(3,4,5-trimethoxy)phenyl-9-
methyl-b-carboline-3-formamide (8a). White crystals were ob-
tained (1.6 g, 74%), mp 206e207 ꢀC. FAB-MS m/z (M þ 1):
436; IR (KBr): 3387, 2935, 2835, 1658, 1583, 1538, 1500,
1457, 1363, 1320, 1232, 1125, 1053, 1004, 842, 742; 1H
NMR (500 MHz, CDCl3): d 8.98 (1H, s, H-4); 8.26e8.27
(1H, d, J ¼ 6.5 Hz, H-8); 7.65e7.68 (1H, m, H-5); 7.46e7.48
(1H, d, J ¼ 8.5 Hz, H-6); 7.37e7.40 (1H, m, H-7); 6.83 (2H,

Q. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 533e540
539
OCH3); 3.50 (3H, s, NCH3); Anal. Calcd for C22H22N2O4: C,
69.83; H, 5.86; N, 7.40. Found: C, 69.70; H, 5.92; N, 7.35.
74%), mp 200e201 ꢀC. FAB-MS m/z (M þ 1): 377; IR
1
1463, 1367, 1238, 1123, 997, 837, 740; H NMR (500 MHz,
(KBr): 3057, 2986, 2939, 2815, 1684, 1620, 1585, 1501,
4.1.9.2. 1-(3,4,5-Trimethoxy)phenyl-3-hydroxymethyl-9-ethyl-
b-carboline (9b). White crystals were obtained (2.9 g, 74%),
mp 154e155 ꢀC. FAB-MS m/z (M þ 1): 393; IR (KBr):
3328, 3068, 2968, 2936, 2835, 1621, 1582, 1504, 1450,
1410, 1370, 1231, 1125, 1052, 1013, 832, 742; 1H NMR
(500 MHz, CDCl3): d 8.30e8.32 (1H, d, J ¼ 10.5 Hz, H-4);
8.20 (1H, s, H-6); 7.57e7.65 (2H, m, H-5, H-8); 7.26e7.30
(1H, m, H-7); 6.86 (2H, s, PhH ); 4.73e4.74 (2H, d,
J ¼ 7.0 Hz, CH2OH); 4.02e4.07 (2H, s, NCH2CH3); 3.81
(6H, s, OCH3); 3.77 (3H, s, OCH3); 0.93e0.97 (3H, m,
NCH2CH3); Anal. Calcd for C23H24N2O4: C, 70.39; H, 6.16;
N, 7.14. Found: C, 70.28; H, 6.19; N, 7.09.
CDCl3): d 10.27 (1H, s, CHO); 8.76 (1H, s, H-4); 8.25e8.27
(1H, d, J ¼ 9.5 Hz, H-6); 7.66e7.70 (1H, m, H-5); 7.49e
7.51 (1H, d, J ¼ 9.5 Hz, H-8); 7.40e7.44 (1H, m, H-7); 6.84
(2H, s, PhH ); 3.94 (3H, s, OCH3); 3.92 (6H, s, OCH3); 3.59
(3H, s, NCH3); Anal. Calcd for C22H20N2O4: C, 70.20; H,
5.36; N, 7.44. Found: C, 69.96; H, 5.46; N, 7.35.
4.1.10.2.
1-(3,4,5-Trimethoxy)phenyl-9-ethyl-b-carboline-3-
carboxaldehyde (10b). White crystals were obtained (1.6 g,
82%), mp 209e210 ꢀC. FAB-MS m/z (M þ 1): 391; IR
(KBr): 3065, 3001, 2935, 2829, 1687, 1621, 1580, 1503,
1457, 1404, 1325, 1233, 1127, 1008, 865, 743; 1H NMR
(500 MHz, CDCl3): d 10.27 (1H, s, CHO); 8.77 (1H, s, H-
4); 8.26e8.28 (1H, d, J ¼ 9.5 Hz, H-6); 7.67e7.69 (1H, m,
H-5); 7.50e7.53 (1H, d, J ¼ 10.5 Hz, H-8); 7.39e7.43 (1H,
m, H-7); 6.84 (2H, s, PhH ); 4.07e4.12 (2H, m, NCH2CH3);
3.94 (3H, s, OCH3); 3.91 (6H, s, OCH3); 3.59 (3H, s,
NCH3); 1.12e1.15 (3H, m, NCH2CH3); Anal. Calcd for
C23H22N2O4: C, 70.75; H, 5.68; N, 7.17. Found: C, 70.51;
H, 5.79; N, 7.10.
4.1.9.3.
1-(3,4,5-Trimethoxy)phenyl-3-hydroxymethyl-9-n-
butyl-b-carboline (9c). White crystals were obtained (3.2 g,
76%), mp 127e128 ꢀC. FAB-MS m/z (M þ 1): 421; IR
(KBr): 3338, 3181, 3061, 2986, 2832, 1621, 1583, 1505,
1465, 1409, 1366, 1297, 1235, 1179, 1126, 1051, 998, 833,
739; 1H NMR (500 MHz, CDCl3): d 8.30e8.31 (1H, d,
J ¼ 10.5 Hz, H-4); 8.20 (1H, s, H-6); 7.57e7.65 (2H, m, H-
5, H-8); 7.25e7.29 (1H, m, H-7); 6.84 (2H, s, PhH ); 4.72e
4.74 (2H, d, J ¼ 7.0 Hz, CH2OH); 3.93e3.97 (2H, m,
NCH2CH2CH2CH3); 3.80 (6H, s, OCH3); 3.75 (3H, s,
OCH3); 1.31e1.39 (2H, m, NCH2CH2CH2CH3); 0.86e0.92
(2H, m, NCH2CH2CH2CH3); 0.62e0.66 (3H, m,
NCH2CH2CH2CH3); Anal. Calcd for C25H28N2O4: C, 71.41;
H, 6.71; N, 6.66. Found: C, 71.19; H, 6.81; N, 6.58.
4.1.10.3. 1-(3,4,5-Trimethoxy)phenyl-9-n-butyl-b-carboline-3-
carboxaldehyde (10c). White crystals were obtained (1.5 g,
72%), mp 150e151 ꢀC. FAB-MS m/z (M þ 1): 419; IR
(KBr): 3059, 3002, 2958, 2809, 1694, 1618, 1582, 1505,
1464, 1410, 1363, 1244, 1203, 1124, 1001, 969, 851, 754;
1H NMR (500 MHz, CDCl3): d 10.26 (1H, s, CHO); 8.76
(1H, s, H-4); 8.25e8.27 (1H, d, J ¼ 10.0 Hz, H-6); 7.64e
7.68 (1H, m, H-5); 7.49e7.51 (1H, d, J ¼ 10.5 Hz, H-8);
7.38e7.42 (1H, m, H-7); 6.84 (2H, s, PhH ); 3.98e4.02 (2H,
m, NCH2CH2CH2CH3); 3.93 (3H, s, OCH3); 3.91 (6H, s,
OCH3); 1.48e1.56 (2H, m, NCH2CH2CH2CH3); 0.96e1.06
(2H, m, NCH2CH2CH2CH3); 0.73e0.76 (3H, m,
NCH2CH2CH2CH3); Anal. Calcd for C25H26N2O4: C, 71.75;
H, 6.26; N, 6.69. Found: C, 71.62; H, 6.29; N, 6.63.
4.1.9.4. 1-(3,4,5-Trimethoxy)phenyl-3-hydroxymethyl-9-phe-
nylpropyl-b-carboline (9d). White crystals were obtained
(3.5 g, 72%), mp 124e125 ꢀC. FAB-MS m/z (M þ 1): 483; IR
(KBr): 3324, 3062, 2938, 2836, 1621, 1582, 1503, 1459, 1410,
1
1364, 1235, 1177, 1126, 1052, 838, 746; H NMR (500 MHz,
CDCl3): d 8.30e8.32 (1H, d, J ¼ 10.5 Hz, H-4); 8.21 (1H, s,
H-6); 7.56e7.63 (2H, m, H-5, H-8); 6.98e7.30 (6H, m, H-7,
PhH ); 6.87 (2H, s, PhH ); 4.73e4.74 (2H, d, J ¼ 7.0 Hz,
CH2OH); 4.03e4.05 (2H, m, NCH2CH2CH2Ph); 3.79 (9H, s,
OCH3); 2.12e2.16 (2H, m, NCH2CH2CH2Ph); 1.62e1.70
(2H, m, NCH2CH2CH2Ph); Anal. Calcd for C30H30N2O4: C,
74.67; H, 6.27; N, 5.81. Found: C, 74.76; H, 6.35; N, 5.76.
4.1.10.4. 1-(3,4,5-Trimethoxy)phenyl-9-phenylpropyl-b-carbo-
line-3-carboxaldehyde (10d). White crystals were obtained
(1.8 g, 75%), mp 174e175 ꢀC. FAB-MS m/z (M þ 1): 481; IR
(KBr): 3065, 2998, 2957, 2934, 2828, 1689, 1621, 1581,
1
1504, 1456, 1405, 1366, 1236, 1127, 1006, 746; H NMR
4.1.10. General procedure for the preparation of 1-(3,4,5-
trimethoxy)phenyl-9-substituted-b-carboline-3-
carboxaldehyde (10aed)
To a solution of 9aed (5 mmol) in CH3CN (300 ml) was
added activated MnO2 (20 mmol). The suspension was re-
fluxed 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 or ace-
tone-petroleum ether to afford white crystals.
(500 MHz, CDCl3): d 10.30 (1H, s, CHO); 8.76 (1H, s, H-4);
8.24e8.26 (1H, d, J ¼ 10.0 Hz, H-6); 7.62e7.66 (1H, m, H-
5); 7.38e7.42 (2H, m, H-7, H-8); 6.97e7.26 (5H, m, PhH );
6.85 (2H, s, PhH ); 4.05e4.09 (2H, m, NCH2CH2CH2Ph);
3.97 (3H, s, OCH3); 3.89 (6H, s, OCH3); 2.25e2.29 (2H, m,
NCH2CH2CH2Ph); 1.80e1.88 (2H, m, NCH2CH2CH2Ph);
Anal. Calcd for C30H28N2O4: C, 74.98; H, 5.87; N, 5.83. Found:
C, 74.82; H, 5.90; N, 5.79.
4.2. Cytotoxicity assays in vitro
4.1.10.1. 1-(3,4,5-Trimethoxy)phenyl-9-methyl-b-carboline-3-
carboxaldehyde (10a). White crystals were obtained (1.4 g,
Cytotoxicity assays in vitro were carried out using 96-
microtitre plate cultures and MTT staining according to the

540
Q. Wu et al. / European Journal of Medicinal Chemistry 44 (2009) 533e540
[12] E.D. Cox, H. Diaz-Arauzo, Q. Huang, M.S. Reddy, C. Ma, B. Harris,
R. McKernan, P. Skolnick, J.M. Cook, J. Med. Chem. 41 (1998)
2537e2552.
procedures described by Al-Allaf and Rashan [35] with a slight
modification. Cells were grown in RPMI-1640 medium con-
taining 10% (v/v) fetal calf serum and 50 mg/ml penicillin
and 50 mg/ml streptomycin. Cultures were propagated at
37 ꢀC in a humified atmosphere containing 5% CO2. Cell lines
were obtained from Shanghai Cell Institute, Chinese Academy
of Science. Drug stock solutions were prepared in DMSO. The
final concentration of DMSO in the growth medium was 2%
(v/v) or lower, concentration without effects on cell replica-
tion. The human tumor cell line panel consisted of cervical
carcinoma (Hela), liver carcinoma (HepG2 and Bel-7402),
gastric carcinoma (BGC-823) and breast carcinoma (MCF-
7). In all of these experiments, three replicate wells were
used to determine each point.
[13] B. Grella, M. Teitler, C. Smith, K. Herrick-Davis, R.A. Glennon, Bioorg.
Med. Chem. Lett. 13 (2003) 4421e4425.
[14] J.E. Audia, D.A. Evrard, G.R. Murdoch, J.J. Droste, J.S. Nissen,
K.W. Schenck, P. Fludzinski, V.L. Lucaites, D.L. Nelson, M.L. Cohen,
J. Med. Chem. 39 (1996) 2773e2780.
[15] B. Grella, M. Dukat, R. Young, M. Teitler, K. Herrick-Davis,
C.B. Gauthierr, R.A. Glennon, Drug Alcohol Depend. 50 (1998) 99e107.
[16] R.A. Glennon, M. Dukat, B. Grella, S.-S. Hong, L. Costantino,
M. Teitler, C. Smith, C. Egan, K. Davis, M.V. Mattson, Drug Alcohol
Depend. 60 (2000) 121e132.
[17] J. Ishida, H.-K. Wang, K.F. Bastow, C.-Q. Hu, K.-H. Lee, Bioorg. Med.
Chem. Lett. 9 (1999) 3319e3324.
[18] W. Jiang, C. Charlet-Fagnere, J. Sapi, J.-Y. Laronze, P. Renard,
B. Pfeiffer, S. Leonce, J. Enzyme Inhib. Med. Chem. 17 (2002) 369e374.
[19] Y.-C. Shen, C.-Y. Chen, P.-W. Hsieh, C.-Y. Duh, Y.-M. Lin, C.-L. Ko,
Chem. Pharm. Bull. 253 (2005) 32e36.
[20] S. Xiao, W. Lin, C. Wang, M. Yang, Bioorg. Med. Chem. Lett. 11 (2001)
437e441.
Acknowledgements
This work was supported by grants from the National New
Drug Funds for Doctors (96-901-06-36) of the Ministry of Sci-
ence and Technology, the Guangzhou Bureau of Science and
Technology (97-Z-12-01), and Xinjiang Huashidan Pharma-
ceutical Co. Ltd.
[21] Y. Boursereau, I. Coldham, Bioorg. Med. Chem. 14 (2004) 5841e5844.
[22] M. Zhao, L. Bi, W. Wang, C. Wang, M. Baudy-Floc’h, J. Ju, S. Peng,
Bioorg. Med. Chem. 14 (2006) 6998e7010.
[23] Q. Chen, R. Chao, H. Chen, X. Hou, H. Yan, S. Zhou, W. Peng, A. Xu,
Int. J. Cancer 114 (2004) 675e682.
[24] R. Cao, Q. Chen, X. Hou, H. Chen, H. Guan, Y. Ma, W. Peng, A. Xu,
Bioorg. Med. Chem. 12 (2004) 4613e4623.
References
[25] R. Cao, W. Peng, H. Chen, X. Hou, H. Guan, Q. Chen, Y. Ma, A. Xu, Eur.
J. Med. Chem. 40 (2005) 249e257.
[1] R.A. Abramovitch, I.D. Spenser, Adv. Heterocycl. Chem. 3 (1964) 79e207.
[2] J.R.F. Allen, B.R. Holmstedt, Phytochemistry 19 (1980) 1573e1582.
[3] H.A. Robertson, G.B. Baker, R.T. Coutts, A. Benderly, R.A. Locock,
I.L. Martin, Eur. J. Pharmacol. 76 (1981) 281e284.
[26] R. Cao, H. Chen, W. Peng, Y. Ma, X. Hou, H. Guan, X. Liu, A. Xu, Eur.
J. Med. Chem. 40 (2005) 991e1001.
[27]
R. Cao, W. Peng, H. Chen, Y. Ma, X. Liu, X. Hou, H. Guan, A. Xu,
Biochem. Biophys. Res. Commun. 338 (2005) 1557e1563.
[28] X. Hou, Q. Chen, R. Cao, W. Peng, A. Xu, Acta Pharmacol. Sin. 25
(2004) 959e965.
[4] M. Cain, R.W. Weber, F. Guzman, J.M. Cook, S.A. Barker,
K.C. Rice, J.N. Crawley, S.M. Paul, P. Skolnick, J. Med. Chem.
25 (1982) 1081e1091.
[5] A.M. Morin, Brain Res. 321 (1984) 151e154.
[6] K.P. Lippke, W.G. Schunack, W. Wenning, W.E. Muller, J. Med. Chem.
26 (1983) 499e503.
[29] H. Guan, X. Liu, W. Peng, R. Cao, Y. Ma, H. Chen, A. Xu, Biochem.
Biophys. Res. Commun. 342 (2005) 894e901.
[30] H. Guan, H. Chen, W. Peng, Y. Ma, R. Cao, X. Liu, A. Xu, Eur. J. Med.
Chem. 41 (2006) 1167e1179.
[31] R. Cao, W. Peng, Z. Wang, A. Xu, Curr. Med. Chem. 14 (2007)
479e500.
[32] H.R. Snyder, S.M. Parmerter, L. Katz, J. Am. Chem. Soc. 70 (1948)
222e225.
[7] T.J. Hagen, P. Skolnick, J.M. Cook, J. Med. Chem. 30 (1987) 750e753.
[8] R.H. Dodd, C. Ouannes, L.P. de Carvalho, A. Valin, P. Venault, J. Med.
Chem. 28 (1985) 824e828.
[9] M.S. Allen, T.J. Hagen, M.L. Trudell, P.W. Codding, P. Skolnick,
J.M. Cook, J. Med. Chem. 31 (1988) 1854e1861.
[10] S.P. Hollinshead, M.L. Trudell, P. Skolnick, J.M. Cook, J. Med. Chem. 33
(1990) 1062e1069.
[33] S.K. Srivastava, A. Agarwal, P.M.S. Chauhan, S.K. Agarwal,
A.P. Bhaduri, S.N. Singh, N. Fatima, R.K. Chatterjee, J. Med. Chem. 7
(1999) 1223e1236.
[34] J. Duan, R. Zhou, S. Zhao, M. Wang, C. Che, J. China Pharm. Univ. 29
(1998) 21e23.
[35] T.A.K. Al-Allaf, L.J. Rashan, Eur. J. Med. Chem. 33 (1998) 817e820.
[11] M.S. Allen, A.J. LaLoggia, L.J. Dorn, M.J. Martin, G. Costantino,
T.J. Hagen, K.F. Koehler, P. Skolnick, J.M. Cook, J. Med. Chem. 35
(1992) 4001e4010.