Synthesis and in vitro cytotoxic evaluation of 1,3-bisubstituted and 1,3,9-trisubstituted β-carboline derivatives

Article abstract of DOI:10.1016/j.ejmech.2004.11.005

A series of novel 1,3-bisubstituted and 1,3,9-trisubstituted β-carboline derivatives was synthesized from the starting material l-tryptophan. Cytotoxic activities of these compounds were investigated in vitro. The results showed that 1,3,9-trisubstituted β-carboline derivatives had higher cytotoxic activities in vitro than the corresponding 1,3-bisubstituted compounds. Among all the synthesized 1,3,9-trisubstituted β-carboline derivatives, the compounds with a methyl substituent at position-1 displayed more potent cytotoxic activities, furthermore compound 5e having an ethoxycarbonyl substituent at position-3 and a pentafluorobenzyl at position-9, respectively, was found to be the most potent compounds of this series with IC₅₀ value of 4:uM against BGC-823 cell lines. These data suggested that (1) the cytotoxic potencies of β-carboline derivatives were enhanced by the introduction of appropriate substituents into position-1 and position-9 in β-carboline; (2) the β-carboline structure might be an important basis for the design and synthesis of new antitumor drugs; (3) the methyl substituent at position-1, the pentafluorobenzyl group at position-9 and the ethoxycarbonyl substituent at position-3 were the optimal combination for the improvement of cytotoxic activity of the β-carboline derivatives.

Full text of DOI:10.1016/j.ejmech.2004.11.005

European Journal of Medicinal Chemistry 40 (2005) 249–257
www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro cytotoxic evaluation of 1,3-bisubstituted
and 1,3,9-trisubstituted b-carboline derivatives
Rihui Cao, Wenlie Peng, Hongsheng Chen, Xuerui Hou, Huaji Guan, Qi Chen,
Yan Ma, Anlong Xu *
Department of Biochemistry and Center for Biopharmaceutical Research, College of Life Sciences, Sun Yat-sen (Zhongshan) University,
135 Xin Gang Xi Road, Guangzhou 510275, China
Received 28 June 2004; received in revised form 8 November 2004; accepted 9 November 2004
Available online 13 January 2005
Abstract
A series of novel 1,3-bisubstituted and 1,3,9-trisubstituted b-carboline derivatives was synthesized from the starting material L-tryptophan.
Cytotoxic activities of these compounds were investigated in vitro. The results showed that 1,3,9-trisubstituted b-carboline derivatives had
higher cytotoxic activities in vitro than the corresponding 1,3-bisubstituted compounds. Among all the synthesized 1,3,9-trisubstituted
b-carboline derivatives, the compounds with a methyl substituent at position-1 displayed more potent cytotoxic activities, furthermore com-
pound 5e having an ethoxycarbonyl substituent at position-3 and a pentafluorobenzyl at position-9, respectively, was found to be the most
potent compounds of this series with IC50 value of 4 uM against BGC-823 cell lines. These data suggested that (1) the cytotoxic potencies of
b-carboline derivatives were enhanced by the introduction of appropriate substituents into position-1 and position-9 in b-carboline; (2) the
b-carboline structure might be an important basis for the design and synthesis of new antitumor drugs; (3) the methyl substituent at position-1,
the pentafluorobenzyl group at position-9 and the ethoxycarbonyl substituent at position-3 were the optimal combination for the improvement
of cytotoxic activity of the b-carboline derivatives.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Synthesis; Cytotoxic; b-Carboline; Structure–activity relationships
1. Introduction
toxic activities in vitro against human tumor cell lines. How-
ever, many previous reports mainly concentrated on neuro-
chemical and neuropharmacological roles of this class of
compounds, so far few investigations have been reported on
the antitumor activity of this class of compounds against
human tumor cell lines in vitro, especially there was no infor-
mation of systematic and detailed studies of structure–
activity relationships on their antitumor activities and neuro-
toxic activities in vivo. In the present work, we reported such
an investigation of synthesis and cytotoxicity evaluation of
novel b-carboline derivatives on the basis of previous inves-
tigations and reports. This paper aims at elucidating the pre-
liminary structure–activity relationships by simple chemical
structural modification and probing structural requirement for
the marked antitumor activity of these compounds. Another
objective of the study was to search for and develop novel
and more potent antitumor drugs with lower toxicity, based
on this investigation.
b-Carboline derivatives, containing planar polycyclic sys-
tems, represent a large number of naturally and synthetic
indole alkaloids [1–8]. In the past several decades, these com-
pounds attracted considerable attention owing to their bio-
logical and pharmaceutical importance [9–16]. The reported
effects of this class of compounds comprise anticonvulsive,
anxiolytic, sedative [9–11], antimicrobial [12], antithrom-
botic [13], anti-HIV [4], parasiticidal [14], intercalation DNA
[15,16], CDK inhibition [17] as well as inhibition Topi-
somerase [18,19]. Recent work [20–23] had reported that this
class compounds with b-carboline nucleus had potent cyto-
* Corresponding author. Tel.: +86 20 8411 3655;
fax: +86 20 8403 8377.
E-mail address: ls36@zsu.edu.cn (A. Xu).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.11.005

250
R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 249–257
2. Chemistry
Pictet-Spengler cyclization [5,24,33] of L-tryptophan (1)
in the presence of the appropriate aldehyde (R1CHO) afforded
the corresponding diastereoisomeric mixture 1,2,3,4-
tetrahydro-bcarboline-3-carboxylic acid (2a–f). Esterifica-
tion [5] of 2a–f with ethanol or methanol in the presence of
SOCl2 yielded the respective 1,2,3,4-tetrahydro-b-carboline-
3-carboxylate (3a–f), followed by dehydrogenation [25,29–
31] with sulfur in xylene gave 1-substituted b-carboline-3-
carboxylate (4a–f), respectively, as described in Scheme 1.
All of the above tetrahydro-b-carbolines was devoid of cyto-
toxic activity against human tumor cell lines, therefore, in
the present work the separation and purification of these com-
pounds were not necessary.
Substitution of alkyl or benzyl groups at the N9-position
of b-carboline ring system would be expected to enhance cyto-
toxic activities, consequently a number of derivatives were
designed and synthesized with substituents ranging in size
from methyl group to phenylpropyl. The N9-position of com-
pounds 4a, 4c and 4d were alkylated [26,27] or benzylated
[27] by the action of sodium hydride in anhydrous DMF fol-
lowed by addition of the relevant appropriate alkylating and
benzylating agent to afford compounds 5a–e, 6a–d, 7a–b,
followed by hydrolyzation [5] in alkaline solution to provide
the corresponding b-carboline-3-carboxylic acids (8a–f, 9a–d,
10a–b) as outlined in Scheme 2. The chemical structures of
all the synthesized novel compounds were confirmed by FAB-
MS, UV, IR, 1H-NMR and elemental analyses data.
Scheme 2. Synthesis of 1,3,9-trisubstituted b-carboline derivatives.
3. Biological results
cell lines. Compounds were converted into their water-
soluble hydrochlorides (4a–f, 5a–e, 6a–d, 7a–b) or sodium
salts (8a–e, 9a–d, 10a–b) by the usual methods before use.
The results were summarized in Table 1.
All the new synthesized compounds were tested for their
cytotoxic activities in vitro against a panel of human tumor
As shown in Table 1, some compounds (e.g. 4a, 5a, 5b,
5d, 5e) showed significant cytotoxic activities while others
(e.g. 6a, 6b, 9c, 9d, 10a, 10b) displayed medium cytotoxic
activities against human tumor cell lines, and others (e.g. 4b,
4c, 4f) only had marginal or no cytotoxic effect in any cell
lines. Among all the synthesized b-carboline derivatives,
1,3,9-trisubstituted-b-carboline-3-carboxylate demonstrated
higher cytotoxic activities in vitro than the corresponding 1,3-
bisubstituted compounds. Furthermore, the Lovo cell lines
were more sensitive to these compounds than other cell lines.
These data indicated that the cytotoxic potencies of
b-carboline were enhanced by the introduction of appropri-
ate substituent into N9-position of b-carboline ring.
Of all 1,3-bisubstituted b-carboline derivatives, com-
pound 4a, having a methyl group at positon-1, displayed
potent cytotoxic activity, however, the others were inactive
against tumor cell lines tested. These results implied that the
methyl group represented the most optimal structure at posi-
tion-1 in b-carboline for these compounds class to exhibit
remarkable cytotoxicity.
Scheme 1. Synthesis of 1,3-substituted b-carboline derivatives.

R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 249–257
251
Table 1
Cytotoxicity of b-carboline derivatives in vitroc (µM)
IC50 (µM)a
Bel-7402b
Compound
Harmine
4a
PLA-801b
45
HepG2b
46
BGC-823b
68
Helab
60
Lovob
66
54
262
86
227
111
97
83
4b
4c
4d
4e
4f
5a
>1000
668
>1000
>1000
>1000
>1000
>1000
102
>1000
>1000
>1000
>1000
>1000
485
>1000
>1000
>1000
965
>1000
>1000
318
>1000
>1000
>1000
619
>1000
52
>1000
>1000
>1000
138
154
>1000
131
>1000
112
5b
158
83
84
73
63
50
5c
5d
386
276
209
784
255
174
25
111
40
78
43
42
5e
47
35
38
4
19
39
6a
6b
6c
6d
7a
7b
8a
8b
142
103
462
109
>1000
95
61
706
107
326
232
104
>1000
59
157
79
>1000
911
>1000
>1000
>1000
>1000
641
807
259
120
62
>1000
>1000
>1000
>1000
405
>1000
>1000
>1000
>1000
280
624
>1000
>1000
>1000
967
>1000
>70
435
594
204
53
380
>1000
>1000
692
>1000
278
8c
8d
373
208
144
560
270
222
8e
93
118
120
156
114
84
9a
9b
9c
9d
>1000
714
>1000
536
>1000
569
545
955
210
256
137
83
395
421
270
168
184
267
158
226
127
204
238
136
10a
10b
652
163
282
210
173
110
101
113
165
142
254
136
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.
b Cell lines include non-small cell lung carcinoma (PLA-801), liver carcinoma (HepG2 and Bel-7402), gastric carcinoma (BGC-823), cervical carcinoma
(Hula), colon carcinoma (Lovo).
c Data represent the mean values of three independent determinations.
Among all 1,3,9-trisubstituted b-carboline derivatives,
compounds 5a–e (except 5c), which all had a methyl at
position-1 demonstrated more cytotoxic activities than the
other two series which had an n-propyl (6a–d) and a phenyl
(7a–b) at position-1, respectively. Furthermore, compound
5e, which has a methyl group at position-1, an ethoxycarbo-
nyl substituent at position-3 and a pentafluorobenzyl group
at position-9, respectively, was the most active compound with
IC50 values of lower than 50 uM against all cell lines screened,
and particularly exhibited the highest cytotoxic activity against
BGC-823 cell lines with IC50 value of 4 uM. These data sug-
gested that the cytotoxicity was maximal with a methyl group
at position-1 and a pentafluorobenzyl substituent at position-
9 of the b-carboline.
Unlike compounds 5a–e, having an ethoxycarbonyl sub-
stituent at positon-3, their hydrolyzed congener 8a–e (except
8c), which had a free carboxylic acid group at position-
3 exhibited comparatively weaker cytotoxic activities against
all tested human tumor cell lines. Similarly, compared to com-
pounds 6a–b, compounds 9a–b had no significant cytotoxic-
ity in any cell lines. Conversely, 9c–d and 10a–b displayed
significant cytotoxic activities, but their congener 6c–d and
7a–b exhibited weaker or no cytotoxic effect on tumor cell
lines. In contrast to compounds with an ethoxycarbonyl at
position-3, their hydrolyzed products having improved solu-
bility in aqueous solution, presented a marked difference in
the cytotoxic activity. These results implied that the cyto-
toxic potencies of these b-carboline derivatives depend upon
the presence and location and nature of the subtituents, which
were introduced into the b-carboline nucleus.
An overview of the cytotoxic activities data of all new syn-
thesized b-carboline derivatives clearly indicated that sub-
stituents at position-1 and position-9 in b-carboline played a
very vital role for exerting cytotoxic effect on human tumor
cell lines. Meanwhile 3-ethoxycarbonyl substituent might be
superior to 3-carboxylic acid for the series of compounds with
a methyl group at position-1. The optimal combination of sub-
stituents in b-carboline nucleus was a methyl group at
position-1, an ethoxycarbonyl at position-3 and a pentafluo-
robenzyl at position-9, respectively, which led to the most
active compound 5e with the lowest IC50 values of 4 uM
against BGC-823.
A total analysis to the cytotoxic activities of b-carboline
derivatives which had been reported above and of the previ-

252
R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 249–257
ous investigation [26] clearly suggested that (1) the cytotoxic
potencies of b-carboline derivatives were enhanced by the
introduction of appropriate substituents into position-1 and
position-9 in b-carboline nucleus; (2) the b-carboline struc-
ture might be an important basis for the design and synthesis
of new antitumor drugs; (3) the methyl substituent at
position-1, the pentafluorobenzyl group at position-9 and the
ethoxycarbonyl substituent at position-3 were the optimal
combination for enhancing cytotoxic activity of the
b-carboline derivatives.
We have explored the influence of substituents at
position-1, 3 and 9 in b-carboline nucleus on the ability of
these compounds against human tumor cell lines. To acquire
more information about the structural requirements for the
possible improvement of the cytotoxic properties, synthesis
of additional new b-carboline derivatives with various sub-
tistuents at other positions of the b-carboline nucleus is desir-
able. Further biological evaluation, which are required to con-
firm these results in animal models on these b-carboline
derivatives are in progress in our laboratories. Moreover, the
molecular mechanisms of these compounds are ongoing to
further design and develop more potent compounds.
4.1.1.2. 1-Ethyl-1,2,3,4-tetrahydro-b-carboline-3-carboxy-
lic acid (2b). Compound 2b was obtained as white solid in
82% yield.
4.1.1.3. 1-n-Propyl-1,2,3,4-tetrahydro-b-carboline-3-
carboxylic acid (2c). Compound 2c was obtained as white
solid in 75% yield.
4.1.2. General procedure for the preparation of 1,2,3,4-
tetrahydro-b-carboline-3-carboxylic acids (2d–f)
A mixture of L-tryptophan (10.2 g, 50 mmol), acetic acid
(200 ml) and aldehyde (55 mmol, benzaldehyde for 2d,
homoanisaldehyde for 2e, p-hydroxybenzaldehyde for 2f) was
refluxed for 2 h, then cooled and adjusted pH to 5 with con-
centrated ammonium hydroxide, the prepricitated product was
collected by filtration and washed well with water and then
dried. Further purification was not necessary and used directly
for the next steps.
4.1.2.1. 1-Phenyl-1,2,3,4-tetrahydro-b-carboline-3-carboxy-
lic acid (2d). Compound 2d was obtained as white solid in
87% yield.
4.1.2.2. 1-(4-Methoxy) phenyl-1,2,3,4-tetrahydro-b-carboline-
3-carboxylic acid (2e). Compound 2e was obtained as white
solid in 79% yield.
4. Experimental
4.1. Chemistry
4.1.2.3. 1-(4-Hydroxy) phenyl-1,2,3,4-tetrahydro-b-carboline-
3-carboxylic acid (2f). Compound 2f was obtained as white
solid in 75% yield.
All reagents were purchased from commercial suppliers
and were dried and purified when necessary. Harmine (purity
99.85%) was extracted from Peganum multisectum Maxim,
a plant indigenous to western China, according to the method
by Duan et al. [28]. Melting points (m.p.) were determined in
capillary tubes on an electrothermal PIFYRT-3 apparatus and
without correction. UV spectra were measured on Shimadzu
UV 2501PC Spectrometer. FAB-MS spectra were obtained
fromVG ZAB-HS spectrometer. FT-IR spectra were run on a
Bruker Equinox 55 Fourier Transformation Infared Spectrom-
4.1.3. General procedure for the preparation of 1,2,3,4-
tetrahydro-b-carboline-3-carboxylates (3a–f)
A solution of the corresponding 1,2,3,4-tetrahydro com-
pounds 2a–f (20 mmol) and alcohol (500 ml, ethanol for 2a–c
and methanol for 2d–f) and SOCl2 (20 ml) was heated at reflux
for 2 h, then evaporated in reduced pressure. The resulting
mixture was poured into H2O (200 ml), and extracted with
ethyl acetate (3 × 200 ml). The organic phase was washed
with water and brine, then dried over anhydrous sodium sul-
fate, filtered and evaporated.
1
eter. H-NMR spectra were recorded on a Varian INOVA
500NB spectrometer. Elemental analyses were carried out on
an ElementarVario EL CHNS ElementalAnalyzer. Silica gel
F254 was used in analytical thin-layer chromatography (TLC)
and silica gel was used in column chromatography.
4.1.3.1. Ethyl 1-methyl-1,2,3,4-tetrahydro-b-carboline-3-
carboxylate (3a). Compound 3a was obtained as yellow oil.
4.1.1. General procedure for the preparation
of 1-substituted 1,2,3,4-tetrahydro-b-carboline-3-
carboxylic acids (2a–c)
4.1.3.2. Ethyl 1-ethyl-1,2,3,4-tetrahydro-b-carboline-3-
carboxylate (3b). Compound 3b was obtained as yellow oil.
A mixture of L-tryptophan (20.4 g, 100 mmol), aldehydes
(300 mmol, acetaldehyde for 2a, propionaldehyde for 2b,
n-butyraldehyde for 2c), 0.5 M H2SO4 (2.5 ml) and H2O
(200 ml) was stirred at room temperature for about 10 h and
detected by TLC. The precipitate was filtered and washed
well with water, dried in vacuum. The material was used with-
out further purification for the following steps.
4.1.3.3. Ethyl 1-n-propyl-1,2,3,4-tetrahydro-b-carboline-3-
carboxylate (3c). Compound 3c was obtained as yellow oil.
4.1.3.4. Ethyl 1-phenyl-1,2,3,4-tetrahydro-b-carboline-3-
carboxylate (3d). Compound 3d was obtained as yellow oil.
4.1.3.5. Ethyl 1-(4-methoxy) phenyl-1,2,3,4-tetrahydro-b-
carboline-3-carboxylate (3e). Compound 3e was obtained
as yellow oil.
4.1.1.1. 1-Methyl-1,2,3,4-tetrahydro-b-arboline-3-carboxy-
lic acid (2a). Compound 2a was obtained as white solid in
80% yield.

R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 249–257
253
4.1.3.6. Ethyl 1-(4-hydroxy) phenyl-1,2,3,4-tetrahydro-b-
carboline-3-carboxylate (3f). Compound 3f was obtained as
yellow oil.
CDCl3) d 8.91 (1H, s, NH), 8.86 (1H, s, H-4), 8.20–8.21 (1H,
d, J = 8 Hz, H-8), 7.90–7.91 (2H, m, H-5, H-6), 7.58–7.60
(2H, m, H-7, Ar–H), 7.54–7.57 (2H, m, Ar–H), 7.41–7.44
(1H, m,Ar–H), 7.35–7.37 (1H, m,Ar–H), 4.04 (3H, s, OCH3).
Anal. Calc. for C19H14N2O2: C, 75.50; H, 4.64; N, 9.27.
Found: C, 75.36; H, 4.86; N, 9.18.
4.1.4. General procedure for the preparation
of b-carboline-3-carboxylates (4a–f)
A suspension of the corresponding 1,2,3,4-tetrahydro-b-
carboline-3-carboxylate (3a–f) (10 mmol) and sulfur
(25 mmol) in xylene (200 ml) was heated at reflux for 6 h,
then cooled and stored at 4 °C for 3 h, and then filtered and
washed generously with petroleum, the solid was dried and
crystallized from ethyl acetate.
4.1.4.5. Methyl 1-(4-methoxy) phenyl-b-carboline-3-
carboxylate (4e). Afforded white solid (15 g, 75%). M.p.
229–230 °C (lit [30] m.p. 229–231 °C); FAB-MS m/z (M +
1) 333; UV kmax 357, 347, 284, 268, 230, 215 nm; IR (KBr)
3639, 3320, 1714, 1611, 1512, 1351, 1255, 1103, 1033 cm–1;
1H-NMR (500 MHz, CDCl3) d 9.40 (1H, s, NH), 8.76 (1H, s,
H-4), 8.15–8.16 (1H, d, J = 8 Hz, H-8), 7.69–7.70 (2H, d,
J = 8.5 Hz, H-5, H-6), 7.54–7.56 (2H, m, H-7, Ar–H), 7.31–
7.34 (1H, m, Ar–H), 6.75–6.7 (2H, d, J = 8.5 Hz, Ar–H), 4.00
(3H, s, OCH3), 3.69 (3H, s, Ar–OCH3). Anal. Calc. for
C20H16N2O3: C, 72.29; H, 4.82; N, 8.43. Found: C, 72.08; H,
5.05; N, 8.29.
4.1.4.1. Ethyl 1-methyl-b-carboline-3-carboxylate (4a). Af-
forded white solid (15 g, 75%). M.p. 217–218 °C (lit [29]
m.p. 248–249 °C); FAB-MS m/z (M + 1) 255; UV kmax 345,
330, 303, 270, 236, 219 nm; IR (KBr) 3316, 3041, 2978, 1709,
1
1567, 1499, 1367, 1344, 1254, 1145, 1031 cm–1; H-NMR
(500 MHz, CDCl3) d 10.50 (1H, s, NH), 8.78 (1H, s, H-4),
8.15–8.17 (1H, d, J = 8 Hz, H-8), 7.58–7.60 (1H, d, J = 8 Hz,
H-5), 7.52–7.55 (1H, m, H-6), 7.30–7.33 (1H, m, H-7), 4.44–
4.48 (2H, m, OCH2CH3), 2.68 (3H, s, CH3), 1.32–1.35 (3H,
m, OCH2CH3). Anal. Calc. for C15H14N2O2: C, 70.87; H,
5.51; N, 11.02. Found: C, 70.65; H, 5.69; N, 11.12.
4.1.4.6. Methyl 1-(4-hydroxy) phenyl-b-carboline-3-carbo-
xylate (4f). Afforded white solid (15 g, 75%). M.p. 267–
269 °C (from ethyl acetate); FAB-MS m/z (M + 1) 319; UV
kmax 387, 340, 327, 285, 215 nm; IR (KBr) 3459, 3159, 1715,
1691, 1610, 1513, 1432, 1352, 1260 cm–1; 1H-NMR
(500 MHz, CDCl3) d 9.51 (1H, s, NH), 8.83 (1H, d, J = 8 Hz,
H-4), 8.20–8.21 (1H, d, J = 8 Hz, H-8), 7.83–7.85 (2H, d,
J = 8.5 Hz, H-5, H-6), 7.60–7.61 (2H, m, H-7, Ar–H), 7.38–
7.39 (1H, m, Ar–H), 7.03–7.04 (2H, d, J = 8 Hz, Ar–H), 4.06
(3H, s, OCH3). Anal. Calc. for C19H14N2O3: C, 71.70; H,
4.40; N, 8.81. Found: C, 71.56; H, 4.63; N, 8.72.
4.1.4.2. Ethyl 1-ethyl-b-carboline-3-carboxylate (4b). Af-
forded white solid (15 g, 75%). M.p. 209–210 °C (from ethyl
acetate); FAB-MS m/z (M + 1) 269; UV kmax 345, 331, 303,
270, 236 nm; IR (KBr) 3327, 2974, 2930, 1705, 1566, 1498,
1
1451, 1346, 1257, 1143, 1043 cm–1; H-NMR (500 MHz,
CDCl3) d 9.63 (1H, s, NH), 8.76 (1H, s, H-4), 8.16–8.17 (1H,
d, J = 8 Hz, H-8), 7.60–7.62 (1H, d, J = 8.5 Hz, H-5), 7.54–
7.57 (1H, m, H-6), 7.31–7.34 (1H, m, H-7), 4.47–4.51 (2H,
m, OCH2CH3), 3.11–3.15 (2H, m, CH2CH3), 1.40–1.43 (3H,
m, OCH2CH3), 1.29–1.32 (3H, m, CH2CH3). Anal. Calc. for
C16H16N2O2: C, 71.64; H, 5.97; N, 10.45. Found: C, 71.52;
H, 6.21; N, 10.36.
4.1.4.7. Ethyl 1,9-dimethyl-b-carboline-3-carboxylate (5a). A
mixture of 4a (2.54 g, 10 mmol) and anhydrous DMF (60 ml)
was stirred at RT until clear, and then 60% NaH (0.6 g,
15 mmol) and Iodomethane (2 ml, 30 mmol) were added.
The mixture was stirred at RT for 1 h. The resulting mixture
was poured into H2O (100 ml), and extracted with ethyl
acetate (3 × 150 ml). The organic phase was washed with
water and brine, then dried over anhydrous sodium sulfate,
filtered and evaporated. The oil obtained was purified by silica
column chromatography with ethyl acetate as the eluent. Upon
recrystallization, white crystals of 2a were obtained (2.2 g,
82%), m.p. 141–142 °C (lit [32] m.p. 145 °C); FAB-MS m/z
(M + 1) 269; UV kmax 354, 338, 307, 272, 239 nm; IR (KBr)
3044, 2978, 2903, 1713, 1620, 1557, 1456, 1369, 1249, 1215,
1138 cm–1; 1H-NMR (500 MHz, CDCl3) d 8.71 (1H, s, H-4),
8.14–8.16 (1H, d, J = 8 Hz, H-8), 7.60–7.63 (1H, m, H-5),
7.45–7.47 (1H, d, J = 8.5 Hz, H-6), 7.31–7.34 (1H, m, H-7),
4.50–4.54 (2H, m, OCH2CH3), 4.16 (3H, s, NCH3), 3.15 (3H,
s, CH3), 1.48–1.50 (3H, m, OCH2CH3). Anal. Calc. for
C16H16N2O2: C, 71.64; H, 5.97; N, 10.45. Found: C, 71.39;
H, 6.23; N, 10.35.
4.1.4.3. Ethyl 1-n-propyl-b-carboline-3-carboxylate (4c).
Afforded white solid (15 g, 75%). M.p. 194–195 °C (from
ethyl acetate); FAB-MS m/z (M + 1) 283; UV kmax 346, 331,
302, 271, 237 nm; IR (KBr) 3329, 2963, 1706, 1567, 1498,
1367, 1344, 1252 cm–1; 1H-NMR (500 MHz, CDCl3) d 11.14
(1H, s, NH), 8.79 (1H, s, H-4), 8.15–8.17 (1H, d, J = 7.5 Hz,
H-8), 7.64–7.66 (1H, d, J = 8 Hz, H-5), 7.51–7.54 (1H, m,
H-6), 7.25–7.31 (1H, m, H-7), 4.42–4.46 (2H, m, OCH2CH3),
2.76–2.79 (2H, m, CH2CH2CH3), 1.45–1.51 (2H, m,
CH2CH2CH3), 1.25–1.32 (3H, m, OCH2CH3), 0.37–0.43 (3H,
m, CH2CH2CH3). Anal. Calc. for C17H18N2O2: C, 72.34; H,
6.38; N, 9.93. Found: C, 72.21; H, 6.58; N, 9.85.
4.1.4.4. Methyl 1-phenyl-b-carboline-3-carboxylate (4d).
Afforded white solid (15 g, 75%). M.p. 257–258 °C (lit [30]
m.p. 257–260 °C, [31] m.p. 253 °C); FAB-MS m/z (M + 1)
303; UV kmax 355, 344, 279, 231 nm; IR (KBr) 3315, 1720,
4.1.4.8. Ethyl 9-ethyl-1-methyl-b-carboline-3-carboxylate
(5b). A mixture of 4a (2.54 g, 10 mmol) and anhydrous DMF
(60 ml) was stirred at RT until clear, and then 60% NaH (0.6 g,
1
1623, 1350, 1251, 1215, 1098 cm–1; H-NMR (500 MHz,

254
R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 249–257
15 mmol) and Iodoethane (2.5 ml, 30 mmol) were added.
The mixture was stirred at RT for 2 h. The resulting mixture
was poured into H2O (100 ml), and extracted with ethyl
acetate (3 × 150 ml). The organic phase was washed with
water and brine, then dried over anhydrous sodium sulfate,
filtered and evaporated. The oil obtained was purified by silica
column chromatography with ethyl acetate as the eluent. Upon
recrystallization, white crystals of 5b were obtained (2.3 g,
81%), m.p. 96–98 °C (from ether); FAB-MS m/z (M + 1)
283; UV kmax 354, 338, 307, 273, 240 nm; IR (KBr) 3359,
3057, 2974, 2931, 2902, 1687, 1620, 1556, 1449, 1368, 1342,
1275, 1243, 1133 cm–1; 1H-NMR (500 MHz, CDCl3) d 8.74
(1H, s, H-4), 8.16–8.18 (1H, d, J = 7.5 Hz, H-8), 7.60–7.63
(1H, m, H-5), 7.48–7.50 (1H, d, J = 8.5 Hz, H-6), 7.31–7.35
(1H, m, H-7), 4.62–4.66 (2H, m, NCH2CH3), 4.50–4.55 (2H,
m, OCH2CH3), 3.13 (3H, s, CH3), 1.46–1.50 (6H, m,
NCH2CH3, OCH2CH3). Anal. Calc. for C17H18N2O2: C,
72.34; H, 6.38; N, 9.93. Found: C, 72.21; H, 6.64; N, 9.84.
clear, and then 60% NaH (0.6 g, 15 mmol) and a-bromo-
2,3,4,5,6-pentafluorotoluene (3 ml, 20 mmol) were added,
then reacted and treated in a manner similar to that described
for 5a to afford white crystals 5e (3.0 g, 68%), m.p. 145–
146 °C (from ether); FAB-MS m/z (M + 1) 435; UV kmax
346, 332, 301, 271, 265, 238 nm; IR (KBr) 3398, 2974, 2931,
1
1708, 1629, 1503, 1454, 1341, 1268, 1122 cm–1; H-NMR
(500 MHz, CDCl3) d 8.75 (1H, s, H-4), 8.17–8.19 (1H, d,
J = 8.5 Hz, H-8), 7.56–7.59 (1H, m, H-5), 7.34–7.38 (2H, m,
H-6, H-7), 5.99 (2H, s, NCH2Ar), 4.51–4.56 (2H, m,
OCH2CH3), 3.16 (3H, s, CH3), 1.48–1.51 (3H, m, OCH2CH3).
Anal. Calc. for C22F5H15N2O2: C, 60.83; H, 3.46; N, 6.45.
Found: C, 60.73; H, 3.59; N, 6.38.
4.1.4.12. Ethyl 9-methyl-1-n-propyl-b-carboline-3-carboxy-
late (6a). A mixture of 4c (2.82 g, 10 mmol) and anhydrous
DMF (60 ml) was stirred at RT until clear, and then 60%
NaH (0.6 g, 15 mmol) and Iodomethane (2 ml, 30 mmol)
were added, then reacted and was treated in a manner similar
to that described for 5a to afford white crystals 6a (2.2 g,
74%), m.p. 108–109 °C (from ether); FAB-MS m/z (M + 1)
297; UV kmax 354, 339, 307, 272, 240 nm; IR (KBr) 2957,
4.1.4.9. Ethyl 9-benzyl-1-methyl-b-carboline-3-carboxylate
(5c). A mixture of 4a (2.54 g, 10 mmol) and anhydrous DMF
(60 ml) was stirred at RT until clear, and then 60% NaH (0.6 g,
15 mmol) and benzyl bromide (5 ml, 40 mmol) were added
and heated at reflux for 3 h, and then treated in a manner
similar to that described for 5a to afford white crystals 5c
(2.5 g, 73%), m.p. 155–156 °C (lit [32] m.p. 149 °C); FAB-MS
m/z (M + 1) 345; UV kmax 351, 337, 305, 272, 239 nm; IR
(KBr) 3441, 3059, 2967, 2929, 1694, 1622, 1561, 1455, 1342,
1272, 1238, 1136, 1029 cm–1; 1H-NMR (500 MHz, CDCl3)
d 8.80 (1H, s, H-4), 8.22–8.23 (1H, d, J = 7.5 Hz, H-8), 7.55–
7.58 (1H, m, H-5), 7.40–7.41 (1H, d, J = 8.5 Hz, H-6), 7.34–
7.37 (1H, m, H-7), 7.24–7.28 (3H, m, Ar–H), 6.94–6.96 (2H,
m, Ar–H), 5.85 (2H, s, NCH2Ar), 4.50–4.54 (2H, m,
OCH2CH3), 2.96 (3H, s, CH3), 1.47–1.50 (3H, m, OCH2CH3).
Anal. Calc. for C22H20N2O2: C, 76.74; H, 5.81; N, 8.14.
Found: C, 76.58; H, 6.05; N, 8.05.
1
2868, 1703, 1555, 1466, 1367, 1263, 1135 cm–1; H-NMR
(500 MHz, CDCl3) d 8.69 (1H, s, H-4), 8.14–8.16 (1H, d,
J = 8 Hz, H-8), 7.59–7.62 (1H, m, H-5), 7.46–7.47 (1H, d,
J = 8.5 Hz, H-6), 7.30–7.33 (1H, m, H-7), 4.49–4.53 (2H, m,
OCH2CH3), 4.11 (3H, s, NCH3), 3.36–3.39 (2H, m,
CH2CH2CH3), 1.87–1.92 (2H, m, CH2CH2CH3), 1.46–1.49
(3H, m, OCH2CH3), 1.09–1.12 (3H, m, CH2CH2CH3). Anal.
Calc. for C18H20N2O2: C, 72.97; H, 6.76; N, 9.46. Found: C,
72.83; H, 6.97; N, 9.34.
4.1.4.13. Ethyl 9-ethyl-1-n-propyl-b-carboline-3-carboxy-
late (6b). A mixture of 4c (2.82 g, 10 mmol) and anhydrous
DMF (60 ml) was stirred at RT until clear, and then 60%
NaH (0.6 g, 15 mmol) and Iodoethane (2.5 ml, 30 mmol)
were added, then reacted and treated in a manner similar to
that described for 5a to afford white crystals 6b (2.0 g, 65%),
m.p. 86–87 °C (from ether); FAB-MS m/z (M + 1) 311; UV
kmax 355, 340, 307, 273, 240 nm; IR (KBr) 3052, 2960, 2868,
1694, 1555, 1446, 1344, 1243, 1132 cm–1; 1H-NMR
(500 MHz, CDCl3) d 8.73 (1H, s, H-4), 8.18–8.19 (1H, d,
J = 8 Hz, H-8), 7.60–7.63 (1H, m, H-5), 7.50–7.52 (1H, d,
J = 8.5 Hz, H-6), 7.32–7.35 (1H, m, H-7), 4.49–4.62 (4H, m,
NCH2CH3, OCH2CH3), 3.30–3.34 (2H, m, CH2CH2CH3),
1.87–1.95 (2H, m, CH2CH2CH3), 1.45–1.49 (6H, m,
NCH2CH3, OCH2CH3), 1.10–1.13 (3H, m, CH2CH2CH3).
Anal. Calc. for C19H22N2O2: C, 73.55; H, 7.10; N, 9.03.
Found: C, 73.41; H, 7.38; N, 8.96.
4.1.4.10. Ethyl 9-phenylpropyl-1-methyl-b-carboline-3-
carboxylate (5d). A mixture of 4a (2.54 g, 10 mmol) and
anhydrous DMF (60 ml) was stirred at RT until clear, and
then 60% NaH (0.6 g, 15 mmol) and 1-bromo-3-
phenylpropane (6 ml, 40 mmol) were added and refluxed for
4 h, and then treated in a manner similar to that described for
5a to afford white crystals 5d (2.8 g, 75%), m.p. 101–102 °C
(from ether); FAB-MS m/z (M + 1) 373; UV kmax 355, 339,
308, 273, 240, 220 nm; IR (KBr) 3059, 2977, 2930, 2852,
1694, 1620, 1557, 1454, 1366, 1341, 1257, 1135 cm–1;
1H-NMR (500 MHz, CDCl3) d 8.73 (1H, s, H-4), 8.15–8.17
(1H, d, J = 7.5 Hz, H-8), 7.56–7.59 (1H, m, H-5), 7.18–7.35
(7H, m, H-6, H-7, Ar–H), 4.49–4.58 (4H, m, OCH2CH3,
NCH2CH2CH2Ar), 2.97 (3H, s, CH3), 2.74–2.77 (2H, m,
NCH2CH2CH2Ar), 2.14–2.20 (2H, m, NCH2CH2CH2Ar),
1.47–1.50 (3H, m, OCH2CH3). Anal. Calc. for C24H24N2O2:
C, 77.42; H, 6.45; N, 7.53. Found: C, 77.36; H, 6.63; N, 7.42.
4.1.4.14. Ethyl 9-benzyl-1-n-propyl-b-carboline-3-carboxy-
late (b). A mixture of 4c (2.82 g, 10 mmol) and anhydrous
DMF (60 ml) was stirred at RT until clear, and then 60%
NaH (0.6 g, 15 mmol) and benzyl bromide (5 ml, 40 mmol)
were added and heated at reflux for 3 h, and then treated in a
manner similar to that described for 5a to afford white crys-
4.1.4.11. Ethyl 9-(2′,3′,4′,5′,6′-pentafluoro)benzyl-1-methyl-
b-carboline-3-carboxylate (5e). A mixture of 4a (2.54 g,
10 mmol) and anhydrous DMF (60 ml) was stirred at RT until

R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 249–257
255
tals 6c (1.8 g, 64%), m.p. 158–159 °C (from ether); FAB-MS
m/z (M + 1) 373; UV kmax 352, 337, 304, 272, 240 nm; IR
(KBr) 3430, 2960, 2934, 2868, 1727, 1619, 1556, 1462, 1339,
1259, 1223, 1147 cm–1; 1H-NMR (500 MHz, CDCl3) d 8.78
(1H, s, H-4), 8.22–8.24 (1H, d, J = 8 Hz, H-8), 7.55–7.58
(1H, m, H-5), 7.41–7.42 (1H, d, J = 8.5 Hz, H-6), 7.34–7.37
(1H, m, H-7), 7.23–7.30 (3H, m, Ar–H), 6.94–6.95 (2H, m,
Ar–H), 5.79 (2H, s, NCH2Ar), 4.49–4.54 (2H, m, OCH2CH3),
3.12–3.16 (2H, m, CH2CH2CH3), 1.78–1.84 (2H, m,
CH2CH2CH3), 1.46–1.52 (3H, m, OCH2CH3), 0.96–1.01 (3H,
m, CH2CH2CH3). Anal. Calc. for C24H24N2O2: C, 77.42; H,
6.45; N, 7.53. Found: C, 77.29; H, 6.65; N, 7.40.
170 °C (from ether); FAB-MS m/z (M + 1) 331; UV kmax
360, 345, 309, 275, 234 nm; IR (KBr) 3429, 3054, 2980, 1724,
1
1621, 1556, 1429, 1351, 1247, 1133, 1051 cm–1; H-NMR
(500 MHz, CDCl3) d 8.92 (1H, s, H-4), 8.23–8.26 (1H, d,
J = 8 Hz, H-8), 7.60–7.64 (3H, m, H-5, H-6, H-7), 7.45–7.53
(4H, m, Ar–H), 7.35–7.39 (1H, m, Ar–H), 3.96–4.03 (5H, s,
NCH2CH3, OCH3), 0.98–1.01 (3H, m, NCH2CH3). Anal.
Calc. for C21H18N2O2: C, 76.36; H, 5.45; N, 8.48. Found: C,
76.27; H, 5.68; N, 8.39.
4.1.5. General procedure for the preparation
of b-carboline-3-carboxylic acids 8a–e, 9a–d and 10a–b
A mixture of the corresponding b-carboline-3-carboxylate
(10 mmol), NaOH (40 mmol), ethanol (50 ml) and H2O
(100 ml) was refluxed for 1 h, and the ethanol 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.
4.1.4.15. Ethyl 9-phenylpropyl-1-n-propy-b-carboline-3-
carboxylate (6d). A mixture of 4c (2.82 g, 10 mmol) and
anhydrous DMF (60 ml) was stirred at RT until clear, and
then 60% NaH (0.6 g, 15 mmol) and 1-bromo-3-
phenylpropane (6 ml, 40 mmol) were added and refluxed for
4 h, and then treated in a manner similar to that described for
5a to afford white crystals 6d (1.7 g, 57%), m.p. 92–93 °C
(from ether); FAB-MS m/z (M + 1) 401; UV kmax 355, 339,
307, 273, 241, 201 nm; IR (KBr) 3068, 2994, 2968, 2929,
2870, 1701, 1620, 1556, 1450, 1365, 1258, 1132 cm–1;
1H-NMR (500 MHz, CDCl3) d 8.71 (1H, s, H-4), 8.15–8.17
(1H, d, J = 8 Hz, H-8), 7.55–7.59 (1H, m, H-5), 7.34–7.36
(1H, d, J = 8.5 Hz, H-6), 7.29–7.32 (3H, m, H-7,Ar–H), 7.18–
7.25 (3H, m, Ar–H), 4.46–4.53 (4H, m, NCH2CH2CH2Ar,
OCH2CH3), 3.15–3.18 (2H, m, CH2CH2CH3), 2.74–2.77 (2H,
m, NCH2CH2CH2Ar), 2.11–2.11 (2H, m, NCH2CH2CH2Ar),
1.77–1.84 (2H, m, CH2CH2CH3), 1.46–1.49 (3H, m,
OCH2CH3), 0.97–1.00 (3H, m, CH2CH2CH3). Anal. Calc.
for C26H28N2O2: C, 78.00; H, 7.00; N, 7.00. Found: C, 77.89;
H, 7.26; N, 6.92.
4.1.5.1. 1,9-Dimethyl-b-carboline-3-carboxylic acid (8a). Yel-
low solid was obtained (2.32 g, 97%). M.p. 262–264 °C;
FAB-MS m/z (M + 1) 241; UV kmax 355, 340, 269, 239 nm;
IR (KBr) 3558, 3332, 2250–3250, 1936, 1712, 1621, 1344,
1215 cm–1; 1H-NMR (500 MHz, DMSO-d6) d 12.25 (1H, s,
COOH), 8.89 (1H, s, H-4), 8.44–8.45 (1H, d, J = 8 Hz, H-8),
7.82–7.83 (1H, d, J = 8.5 Hz, H-5), 7.70–7.73 (1H, m, H-6),
7.37–7.40 (1H, m, H-7), 4.50–4.54 (3H, s, NCH3), 3.18 (3H,
s, CH3). Anal. Calc. for C14H12N2O2: C, 70.00; H, 5.00; N,
11.67. Found: C, 69.83; H, 5.21; N, 11.54.
4.1.5.2. 9-Ethyl-1-methyl-b-carboline-3-carboxylic acid
(8b). Yellow solid was obtained (2.48 g, 97%). M.p. 243–
245 °C; FAB-MS m/z (M + 1) 255; UV kmax 355, 270, 240,
223 nm; IR (KBr) 3393, 2250–3250, 1714, 1621, 1589, 1366,
1
1233, 1130 cm–1; H-NMR (500 MHz, DMSO-d6) d 12.26
4.1.4.16. Methyl 1-phenyl-9-methyl-b-carboline-3-carboxy-
late (7a). A mixture of 4d (3.02 g, 10 mmol) and anhydrous
DMF (60 ml) was stirred at RT until clear, and then 60%
NaH (0.6 g, 15 mmol) and Iodomethane (2 ml, 30 mmol)
were added, then reacted and treated in a manner similar to
that described for 5a to afford white crystals 7a (2.4 g, 76%),
m.p. 205–206 °C (from ether); FAB-MS m/z (M + 1) 317;
UV kmax 360, 346, 309, 275, 233 nm; IR (KBr) 3426, 3051,
2944, 1723, 1622, 1557, 1493, 1435, 1356, 1262, 1223,
1129 cm–1; 1H-NMR (500 MHz, CDCl3) d 8.91 (1H, s, H-4),
8.24–8.25 (1H, d, J = 7.5 Hz, H-8), 7.63–7.66 (3H, m, H-5,
H-6, H-7), 7.45–7.53 (4H, m, Ar–H), 7.37–7.40 (1H, m,
Ar–H), 4.03 (3H, s, NCH3), 3.47 (3H, s, OCH3). Anal. Calc.
for C20H16N2O2: C, 75.95; H, 5.06; N, 8.86. Found: C, 75.74;
H, 5.29; N, 8.78.
(1H, s, COOH), 8.89 (1H, s, H-4), 8.45–8.46 (1H, d,
J = 7.5 Hz, H-8), 7.83–7.85 (1H, d, J = 8.5 Hz, H-5), 7.70–
7.73 (1H, m, H-6), 7.37–7.41 (1H, m, H-7), 4.72–4.76 (2H,
m, NCH2CH3), 3.14 (3H, s, CH3), 1.40–1.42 (3H, m,
NCH2CH3). Anal. Calc. for C15H14N2O2: C, 70.87; H, 5.51;
N, 11.02. Found: C, 70.73; H, 5.69; N, 10.94.
4.1.5.3. 9-Benzyl-1-methyl-b-carboline-3-carboxylic acid
(8c). Yellow solid was obtained (3.12 g, 98%). M.p. 246–
248 °C; FAB-MS m/z (M + 1) 317; UV kmax 352, 338, 268,
239 nm; IR (KBr) 2250–3750, 1720, 1615, 1340, 1206 cm–1;
1H-NMR (500 MHz, DMSO-d6) d 12.21 (1H, s, COOH), 8.80
(1H, s, H-4), 8.31–8.35 (1H, d, J = 7.5 Hz, H-8), 7.65–7.68
(1H, d, J = 8.0 Hz, H-5), 7.58–7.60 (1H, m, H-6), 7.15–7.30
(6H, m, H-7, Ar–H), 5.78 (2H, m, NCH2Ar), 2.95 (3H, s,
CH3).Anal. Calc. for C20H16N2O2: C, 75.95; H, 5.06; N, 8.86.
Found: C, 75.82; H, 5.24; N, 8.74.
4.1.4.17. Methyl 1-phenyl-9-ethyl-b-carboline-3-carboxylate
(7b). A mixture of 4d (3.02 g, 10 mmol) and anhydrous DMF
(60 ml) was stirred at RT until clear, and then 60% NaH (0.6 g,
15 mmol) and Iodoethane (2.5 ml, 30 mmol) were added,
then reacted and treated in a manner similar to that described
for 5a to afford white crystals 7b (2.3 g, 69%), m.p. 169–
4.1.5.4. 9-Phenylpropyl-1-methyl-b-carboline-3-carboxylic
acid (8d). Yellow solid was obtained (3.4 g, 98%). M.p. 186–
188 °C; FAB-MS m/z (M + 1) 345; UV kmax 356, 269, 240 nm;

256
R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 249–257
IR (KBr) 3417, 2500–3250, 1740, 1624, 1591, 1355,
1061 cm–1; 1H-NMR (500 MHz, DMSO-d6) d 12.16 (1H, s,
COOH), 8.75 (1H, s, H-4), 8.36–8.37 (1H, d, J = 7.5 Hz,
H-8), 7.68–7.69 (1H, d, J = 8.0 Hz, H-5), 7.61–7.64 (1H, m,
H-6), 7.18–7.34 (6H, m, H-7, Ar–H), 4.64–4.67 (2H, m,
NCH2CH2CH2Ar), 2.90 (3H, s, CH3), 2.73–2.76 (2H, m,
CH2CH2CH2Ar), 2.06–2.12 (2H, m, NCH2CH2CH2Ar).Anal.
Calc. for C22H20N2O2: C, 76.74; H, 5.81; N, 8.14. Found: C,
76.64; H, 5.98; N, 8.06.
CH2CH2CH3), 0.86–0.89 (3H, m, CH2CH2CH3). Anal. Calc.
for C22H20N2O2: C, 76.74; H, 5.81; N, 8.14. Found: C, 76.58;
H, 5.98; N, 8.19.
4.1.5.9. 9-Phenylpropyl-1-n-propyl-b-carboline-3-carboxy-
lic acid (9d). Yellow solid was obtained (3.6 g, 97%). M.p.
176–178 °C; FAB-MS m/z (M + 1) 373; UV kmax 356, 343,
269, 240 nm; IR (KBr) 3163, 3068, 2963, 2933, 1745, 1620,
1583, 1460, 1362, 1245 cm–1; 1H-NMR (500 MHz, DMSO-
d6) d 12.15 (1H, s, COOH), 8.75 (1H, s, H-4), 8.36–8.38 (1H,
d, J = 7.5 Hz, H-8), 7.71–7.72 (1H, d, J = 8.0 Hz, H-5), 7.62–
7.65 (1H, m, H-6), 7.19–7.35 (6H, m, H-7, Ar–H), 4.57–4.60
(2H, s, NCH2CH2CH2Ar), 3.06–3.09 (2H, m, CH2CH2CH3),
2.74–2.77 (2H, m, NCH2CH2CH2Ar), 2.05–2.11 (2H, m,
CH2CH2CH3), 1.73–1.79 (2H, m, NCH2CH2CH2Ar), 0.92–
0.96 (3H, m, CH2CH2CH3). Anal. Calc. for C24H24N2O2: C,
77.42; H, 6.45; N, 7.53. Found: C, 77.31; H, 6.56; N, 7.48.
4.1.5.5. 9-(2′,3′,4′,5′,6′-Pentafluoro)benzyl-1-methyl-b-
carboline-3-carboxylic acid (8e). White solid was obtained
(4.0 g, 98%). M.p. 191–193 °C; FAB-MS m/z (M + 1) 407;
UV kmax 349, 336, 268, 238 nm; IR (KBr) 3422, 2250–3250,
1
1754, 1652, 1625, 1592, 1491, 1360, 1131 cm–1; H-NMR
(500 MHz, DMSO-d6) d 12.18 (1H, s, COOH), 8.80 (1H, s,
H-4), 8.40–8.42 (1H, d, J = 8.0 Hz, H-8), 7.62–7.64 (2H, m,
H-5, H-6), 7.34–7.37 (1H, m, H-7), 6.10 (2H, s, NCH2Ar),
3.01–3.02 (3H, s, CH3). Anal. Calc. for C20F5H11N2O2: C,
59.11; H, 2.71; N, 6.90. Found: C, 58.92; H, 2.83; N, 6.83.
4.1.5.10. 1-Phenyl-9-methyl-b-carboline-3-carboxylic acid
(10a). Yellow solid was obtained (3.0 g, 98%). M.p. 223–
224 °C; FAB-MS m/z (M + 1) 303; UV kmax 360, 273, 237 nm;
IR (KBr) 2000–3250, 1754, 1681, 1622, 1557, 1392, 1262,
1051 cm–1; 1H-NMR (500 MHz, DMSO-d6) d 12.18 (1H, s,
COOH), 8.93 (1H, s, H-4), 8.45–8.46 (1H, d, J = 7.5 Hz,
H-8), 7.65–7.70 (4H, m, H-5, H-6, H-7, Ar–H), 7.56–7.60
(3H, m, Ar–H), 7.36–7.39 (1H, m, Ar–H), 3.44–3.47 (3H, s,
NCH3). Anal. Calc. for C19H14N2O2: C, 75.50; H, 4.64; N,
9.27. Found: C, 75.39; H, 4.77; N, 9.16.
4.1.5.6. 1-n-Propyl-9-methyl-b-carboline-3-carboxylic acid
(9a). Yellow solid was obtained (2.6 g, 97%). M.p. 181–
183 °C; FAB-MS m/z (M + 1) 269; UV kmax 356, 342, 269,
240 nm; IR (KBr) 3142, 3059, 2960, 2869, 1739, 1620, 1582,
1470, 1359, 1248, 1128 cm–1; 1H-NMR (500 MHz, DMSO-
d6) d 8.76 (1H, s, H-4), 8.36–8.37 (1H, d, J = 7.5 Hz, H-8),
7.75–7.77 (1H, d, J = 8.0 Hz, H-5), 7.64–7.67 (1H, m, H-6),
7.32–7.35 (1H, m, H-7), 4.17 (3H, s, NCH3), 3.35–3.38 (2H,
m, CH2CH2CH3), 1.85–1.89 (2H, m, CH2CH2CH3), 1.04–
1.07 (3H, m, CH2CH2CH3). Anal. Calc. for C16H16N2O2: C,
71.64; H, 5.97; N, 10.45. Found: C, 71.44; H, 6.19; N, 10.38.
4.1.5.11. 1-Phenyl-9-ethyl-b-carboline-3-carboxylic acid
(10b). Yellow solid was obtained (3.1 g, 97%). M.p. 194–
195 °C; FAB-MS m/z (M + 1) 317; UV kmax 360, 273, 239 nm;
IR (KBr) 3283, 2974, 1730, 1620, 1559, 1451, 1355,
1299 cm–1; 1H-NMR (500 MHz, DMSO-d6) d 12.24 (1H, s,
COOH), 8.98 (1H, s, H-4), 8.46–8.48 (1H, d, J = 8 Hz, H-8),
7.71–7.73 (1H, d, J = 8 Hz, H-5), 7.56–7.68 (6H, m, H-6,
H-7, Ar–H), 7.36–7.39 (1H, m, Ar–H), 4.02–4.06 (2H, m,
NCH2CH3), 0.86–0.89 (3H, m, NCH2CH3). Anal. Calc. for
C20H16N2O2: C, 75.95; H, 5.06; N, 8.86. Found: C, 75.73; H,
5.23; N, 8.78.
4.1.5.7. 1-n-Propyl-9-ethyl-b-carboline-3-carboxylic acid
(9b). Yellow solid was obtained (2.7 g, 97%). M.p. 172–
174 °C; FAB-MS m/z (M + 1) 283; UV kmax 356, 342, 268,
240 nm; IR (KBr) 3191, 2965, 2871, 1745, 1619, 1558, 1456,
1
1354, 1228, 1120 cm–1; H-NMR (500 MHz, DMSO-d6) d
8.77 (1H, s, H-4), 8.37–8.39 (1H, d, J = 7.5 Hz, H-8), 7.77–
7.79 (1H, d, J = 8.5 Hz, H-5), 7.64–7.67 (1H, m, H-6), 7.32–
7.36 (1H, m, H-7), 4.63–4.68 (2H, m, NCH2CH3), 3.26–3.30
(2H, m, CH2CH2CH3), 1.86–1.93 (2H, m, CH2CH2CH3),
1.37–1.40 (3H, m, NCH2CH3), 1.05–1.08 (3H, m,
CH2CH2CH3).Anal. Calc. for C17H18N2O2: C, 72.34; H, 6.38;
N, 9.93. Found: C, 72.11; H, 6.56; N, 9.82.
4.2. Cytotoxicity assays in vitro
Cytotoxicity assays in vitro were carried out using 96 mi-
crotitre plate cultures and MTT staining according to the pro-
cedures described byAl-Allaf and Rashan [20] with a slightly
modification. Cells were grown in RPMI-1640 medium con-
taining 10% (v/v) fetal calf serum and 100 ug ml–1 penicillin
and 100 ug ml–1 streptomycin. Cultures were propagated at
37 °C in a humified atmosphere containing 5% CO2. Cell
lines were obtained from Shanghai Institute of Biochemistry
and Cell Biology, 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, con-
centration without effect on cell replication. The tumor cell
line panel consisted of non-small cell lung carcinoma (PLA-
4.1.5.8. 9-Benzyl-1-n-propyl-b-carboline-3-carboxylic acid
(9c). Yellow solid was obtained (3.4 g, 96%). M.p. 193–
194 °C; FAB-MS m/z (M + 1) 345; UV kmax 353, 339, 268,
240 nm; IR (KBr) 3064, 2957, 2923, 2866, 1752, 1643, 1589,
1456, 1353, 1209, 1130 cm–1; 1H-NMR (500 MHz, DMSO-
d6) d 12.15 (1H, s, COOH), 8.83 (1H, s, H-4), 8.43–8.45 (1H,
d, J = 8 Hz, H-8), 7.70–7.71 (1H, d, J = 8.0 Hz, H-5), 7.60–
7.63 (1H, m, H-6), 7.35–7.38 (1H, m, H-7), 7.22–7.29 (3H,
m, Ar–H), 6.93–6.95 (2H, m, Ar–H), 5.92 (2H, s, NCH2Ar),
3.07–3.10 (2H, m, CH2CH2CH3), 1.67–1.75 (2H, m,

R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 249–257
257
[13] N. Lin, M. Zhao, C. Wang, S. Peng, Bioorg. Med. Chem. Lett. 12
(2002) 585–587.
[14] P. Rivas, B.K. Cassels, A. Morello, Y. Repetto, Comp. Biochem.
Physiol. 122 (1999) 27–31.
[15] K. Hayashi, M. Nagao, T. Sugimura, Nucleic Acids Res. 4 (1977)
3679–3685.
801), liver carcinoma (HepG2 and Bel-7402), gastric carci-
noma (BGC-823), cervical carcinoma (Hela), colon carci-
noma (Lovo). In all of these experiments, three replicate wells
were used to determine each point.
[16] D. Csanyi, G. Hajos, Z. Riedl, Bioorg. Med. Chem. Lett. 10 (2000)
1767–1769.
[17] Y. Song, J. Wang, S.F. Teng, D. Kesuma,Y. Deng, J. Duan, J.H. Wang,
R. Zhong-Qi, M.M. Sim, Bioorg. Med. Chem. Lett. 12 (2002) 1129–
1132.
[18] H. Nii, Mutation Res. 541 (2003) 123–136.
[19] A.M. Deveau, M.A. Labroli, C.M. Dieckhaus, Biorg. Med. Chem.
Lett. 11 (2001) 1251–1255.
[20] T.A.K. Al-Allaf, L.J. Rashan, Eur. J. Med. Chem. 33 (1998) 817–820.
[21] J. Ishida, H.-K. Wang, K.F. Bastow, C.-Q. Hu, K.-H. Lee, Bioorg.
Med. Chem. 9 (1999) 3319–3324.
Acknowledgements
This work was supported by grants from Xinjiang Huash-
idan Pharmaceutical Co. Ltd. and Guangzhou Commission
of Science and Technology (97-Z-12-01). We also thank Cen-
ter forAnalysis and Test of SunYat-sen (Zhongshan) Univer-
sity for UV, FAB-MS, IR and 1H-NMR spectra.
[22] P. Molina, P.M. Fresneda, S. Garcia-Zafra, P. Almendros, Tetrahedron
Lett. 36 (1995) 3581–3582.
References
[23] S.G. Abdel-Moty, S. Sakai, N. Aimi, H. Takayama, M. Kitajima,
A. E1-Shorbagi, A.N. Ahmed, N.M. Omar, Eur. J. Med. Chem. 32
(1997) 1009–1017.
[24] A. Brossi, A. Focella, S. Teitel, J. Med. Chem. 16 (1973) 418–420.
[25] S.K. Srivastava, A. Agarwal, P.M.S. Chauhan, S.K. Agarwal,
A.P. Bhaduri, S.N. Singh, et al., Bioorg. Med. Chem. 7 (1999) 1223–
1236.
[26] J. Ishida, H.-K. Wang, K.F. Bastow, C.-Q. Hu, K.-H. Lee, Bioorg.
Med. Chem. 9 (1999) 3319–3324.
[27] M. Bertrand, G. Poissonnet, M.-H. Theret-Bettiol, C. Gaspard,
G.H. Werner, B. Pfeiffer, P. Renard, S. Leonce, R.H. Dodd, Bioorg.
Med. Chem. 9 (2001) 2155–2164.
[28] J. Duan, R. Zhou, S. Zhao, M. Wang, C. Che, J. China Pharm. Univ. 29
(1998) 21–23.
[29] G. Cauzzo, G. Jordi, J. Org. Chem. 37 (1972) 1429–1433.
[30] F. Gatta, D. Mitisi, J. Heterocyclic Chem. 24 (1987) 1183–1187.
[31] M. Cain, R.W. Weber, F. Guzman, J.M. Cook, S.A. Barker, K.C. Rice,
et al., J. Med. Chem. 25 (1982) 1081–1091.
[1] M.M. Airaksinen, I. Kari, Med. Biol. 59 (1981) 21–34.
[2] T.S. Zhou, W.C. Ye, Z.T. Wang, Phytochemistry 49 (1998) 1807–
1809.
[3] G.M. Carbrera, A.M. Seldes, J. Nat. Prod. 62 (1999) 759–760.
[4] R.S. Kusurkar, S.K. Goswami, S.M. Vyas, Tetrahedron Lett. 44
(2003) 4761–4763.
[5] K.P. Lippke, W.G. Schunack, W. Wenning, W.E. Muller, J. Med.
Chem. 26 (1983) 499–503.
[6] M. Cain, R.W. Weber, F. Guzman, J.M. Cook, S.A. Barker, K.C. Rice,
et al., Med. Chem. 25 (1982) 1081–1091.
[7] T.J. Hagen, P. Skolnick, J.M. Cook, J. Med. Chem. 30 (1987) 750–
753.
[8] R.H. Dodd, C. Ouannes, L.P. Carvalho, A. Valin, P. Venault,
G. Chapouthier, et al., Med. Chem. 28 (1985) 824–828.
[9] C. Braestrup, M. Nielsen, C.E. Olsen, Proc. Natl. Acad. Sci. USA 77
(1980) 2288–2292.
[10] W. Schlecker, A. Huth, E. Ottow, J. Mulzer, Synthesis (Mass.) 131
(1995) 1225–1227.
[32] P. Van Broeck, P. Van Doren, G. Hoornaert, Synthesis (1992) 473–
476.
[33] J.M. Bobbitt, J.P. Willis, J. Org. Chem. 45 (1980) 1978–1984.
[11] A. Batch, R.H. Dodd, J. Org. Chem. 63 (1998) 872–877.
[12] P. Molina, P.M. Fresneda, S. Garcia-Zafra, P. Almendros, Tetrahedron
Lett. 35 (1994) 8851–8853.