Design, synthesis and in vitro and in vivo antitumor activities of novel β-carboline derivatives

Article abstract of DOI:10.1016/j.ejmech.2005.04.008

To further our SAR study on the chemistry and antitumor activity/neurotoxicity of β-carboline alkaloids, several series of β-carboline derivatives with various substituents were designed and synthesized from the starting material l-tryptophan on the basis

Full text of DOI:10.1016/j.ejmech.2005.04.008

European Journal of Medicinal Chemistry 40 (2005) 991–1001
www.elsevier.com/locate/ejmech
Original article
Design, synthesis and in vitro and in vivo antitumor activities
of novel b-carboline derivatives
R. Cao, H. Chen, W. Peng,Y. Ma, X. Hou, H. Guan, X. Liu, A. 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 4 December 2004; received in revised form 18 April 2005; accepted 22 April 2005
Available online 13 June 2005
Abstract
To further our SAR study on the chemistry and antitumor activity/neurotoxicity of b-carboline alkaloids, several series of b-carboline
derivatives with various substituents were designed and synthesized from the starting material L-tryptophan on the basis of harmine chemical
structure. Cytotoxic activities of these compounds were investigated in vitro. The results showed that some b-carboline derivatives had
significant cytotoxic activities against human tumor cell lines. Among all the synthesized b-carboline derivatives, the compounds 27, 28 and
32, having a benzyl substituent at both position-2 and 9, respectively, were found to be the most potent compounds with IC50 value lower than
50 µM against all human tumor cell lines examined. Acute toxicities and antitumor activities of the selected b-carboline derivatives in mice
were also evaluated. The results demonstrated that a benzyl substituent at position-2 increased the antitumor activity as well as acute toxicity
significantly. However an (ethoxycarbonyl)amino substituent at position-3 reduced the acute toxicity as well as antitumor activity remarkedly.
These data suggested that (1) the antitumor potencies of b-carboline derivatives were enhanced by the introduction of benzyl substituent into
the position-2; (2) the acute toxicity of b-carboline derivatives reduced dramatically by the introduction of an appropriate substituent into the
position-3 and 9; (3) the b-carboline structure might be an important basis for the design and synthesis of new antitumor drugs with significant
antitumor activity and low toxicity.
© 2005 Elsevier SAS. All rights reserved.
Keywords: b-Carboline; Synthesis; Cytotoxic; Acute toxicity; Antitumor; SAR
1. Introduction
activities against human tumor cell lines. Our previous inves-
tigation [22] also showed that harmine and its derivatives had
significant antitumor activities in mice bearing both Lewis
lung cancer and Sarcoma 180, while they exhibited remark-
able neurotoxic effects including tremor, twitch and jumping
in experimental animal models. Furthermore we previously
reported [22] that the introduction of appropriate substitu-
ents into the position-3 and 9 in b-carboline nucleus could
contribute to their enhancing antitumor activities and decreas-
ing acute toxicity besides their loss of neurotoxic effect com-
pletely.
Inspired by these results, in the present investigation, we
designed and synthesized numerous b-carboline derivatives
with various substituents at different position in b-carboline
nucleus on the basis of harmine chemical structure, it is pos-
sible to discover and obtain new compounds with compa-
rable activity and lower toxicity than Harmine. Moreover this
study will further elucidate the structure–activity relation-
ships (SAR) of these compounds with regard to antitumor
activity and neurotoxicity/acute toxicity. We report here the
The malignant tumor has long been one of the serious dis-
eases threatening human health. To discover and develop novel
therapeutic agents for the treatment of malignancy has a vital
importance. One of the successful and effective approaches
in the search for new antitumor agents from natural products
is to synthesize novel compounds by simple chemical modi-
fication on the basis of natural leading compounds.
The b-carboline alkaloids, containing planar tricyclic sys-
tem, are a large group of naturally-occurring and synthetic
alkaloids [1–9]. Recently these compounds attracted a con-
siderable attention due to their biological and pharmaceuti-
cal properties [9–17]. Harmine is the most representative
b-carboline alkaloid, which was originally isolated from seeds
of Pegannum harmala L. [4]. Recent work [18–21] demon-
strated that harmine and its derivatives were highly cytotoxic
* 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 © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.04.008

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R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 991–1001
preparation of novel b-carboline derivatives, their antitumor
activities and toxicological effects.
in anhydrous methanol or ethanol to give the methyl and ethyl
carbamates 8 and 9 [9], respectively (see Scheme 1).
Following the same procedures as given above, the ethyl
9-ethyl-b-carboline-3-carboxylate 10 and ethyl 9-benzyl-b-
carboline-3-carboxylate 11, prepared by N9-ethylation and
N9-benzylation through reacting with ethyl or benzyl bro-
mide in the presence of sodium hydride in DMF and THF as
previously reported [22], were reacted with 85% hydrazine
hydrate in ethanol gave the hydrazide 12 and 13, respec-
tively, which after treatment with sodium nitrite, yielded the
corresponding acyl azide 14 and 15. When compounds 14
and 15 were refluxed in anhydrous methanol or ethanol to
produce the methyl and ethyl carbamates 16–18, respec-
tively (see Scheme 2).
Previous studies [22] with Harmine (7-methoxy-1-methyl-
b-carboline) and Norharman (b-carboline) derivatives have
shown that substitution of alkyl or benzyl groups at the
N9-position of b-carboline ring system frequently promotes
the cytotoxic activity of these compounds. For this reason, a
series of 9-substituted 1-methyl-b-carboline (Harman) deriva-
tives were designed and synthesized with substituents rang-
ing in size from methyl to phenylpropyl group. The 1-methyl-
b-carboline 20, prepared by the condensation of L-tryptophan
with acetaldehyde in acid solution and followed by aromati-
zation, oxidation and decarboxylation in a single step through
the action of potassium dichromate according to the method
previously described by Synder et al. [29], were alkylated or
benzylated by the action of sodium hydride in anhydrous DMF
followed by the addition of the relevant appropriate alkylat-
ing and benzylating agents to afford compounds 21–26.Acci-
dentally, we found that both the N9 and N2-position of the
compound 20 were benzylated simultaneously at 50–60 °C
2. Chemistry
The preparation of the 1,2,3,4-tetrahydro-b-carboline
derivatives employed in this investigation was accomplished
via the well-known Pictet–Spengler reaction [6,23,24] utiliz-
ing the appropriate aldehyde in either alkaline [25,26] or acid
media [23–27]. In general, conversion of the 1,2,3,4-
tetrahydro-b-carboline derivatives to the corresponding
b-carboline derivatives was carried out either by oxidation of
the tetrahydro derivatives with sulfur in refluxing xylene [28]
or with chloranil in refluxing s-tetrachloroethane [24] or with
lead tetraacetate in glacial acetic acid [25].
The 1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid 2 was
prepared by the condensation of L-tryptophan with formalde-
hyde according to the method of Lippke et al. [25] and was
subsequently converted to the ethyl 1,2,3,4-tetrahydro-b-
carboline-3-carboxylate 3 by heating ethanolic hydrogen chlo-
ride solution. The compound 3 was then subjected to the oxi-
dation with sulfur in refluxing xylene to provide ethyl
b-carboline-3-carboxylate 4 (Scheme 1). This sequence of
reactions can be easily scaled up to give enough material 4
for further transformations.
In order to examine the effect of amino substitutents at posi-
tion-3 on the antitumor activities of the compounds, 3-amino-
b-carboline 7 was prepared by a Curtius-type reaction start-
ing with compound 4 [7,9,25]. Refluxing of compound 4 with
85% hydrazine hydrate in ethanol gave the hydrazide 5 [9],
and then was treated with sodium nitrite, yielding the corre-
sponding acyl azide 6 [9]. Compound 6 was further refluxed
in acetic acid, and rearranged to afford the amine 7 [9] in
good yield. Alternatively, the compound 6 could be refluxed
Scheme 1
.
Scheme 2.

R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 991–1001
993
for 2 h led to novel compound 27 in good yield (see Scheme
3). This reaction condition can be employed to prepare other
2,9-bisubstitued b-carboline derivatives 28 and 30–32 (see
Scheme 4).
The b-carboline-3-carboxylic acid 33 [25], prepared by
hydrolyzation the compound 4, was reacted with benzyl bro-
mide in anhydrous DMF solution in the presence of sodium
hydride to yield compound 34 in a good yield. Compound 4
was reacted with ethyl diamine according to the method
described by Xiao et al. [30] to provided the 3-carboxamide
derivative 35 (see Scheme 5). The conversion of ethyl
b-carboline-3-carboxylate 4 and ethyl 9-benzyl-b-carboline-
3-carboxylatate 11 to the 3-hydroxymethyl derivatives 36 and
38, respectively, can be accomplished easily by the treatment
of 4 and 11 with lithium borohydride according to the method
reported by Cain et al. [7], which after refluxing in acetic
acid solution, yielded the corresponding derivatives 37 and
39 (see Schemes 5 and 6).
Scheme 3
.
The 1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic
acid 19 was prepared by condensation of L-tryptophan with
acetaldehyde according to the method of Synder et al. [24].
Esterification of 19 with ethanol in the presence of SOCl2
yielded the corresponding ester 40, followed by dehydroge-
nation with sulfur in xylene to give ethyl 1-methyl-b-
carboline-3-carboxylate 41. The N9-position of 41 was fur-
ther n-butylated by the action of sodium hydride in anhydrous
DMF gave derivatives 42, followed by hydrolyzation in alka-
line solution to provide the corresponding 3-carboxylic acid
derivative 43 (see Scheme 7). The chemical structures of all
the synthesized novel compounds were confirmed by FAB-
MS, UV, IR, 1H-NMR and elemental analyses data.
3. Results and discussion
3.1. Cytotoxicity in vitro
The cytotoxic potential of all synthesized b-carboline
derivatives was evaluated in vitro against a panel of human
tumor cell lines. Compound 43 was converted into its water-
soluble sodium salts, and the other compounds (except 27,
Scheme 4
.
Scheme 5
.

994
R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 991–1001
Scheme 6
.
Scheme 7
.
28 and 30–32) examined were all prepared in the form of
hydrochloride by the usual methods before use. The results
were summarized in Table 1.
As shown in Table 1, the compounds 27, 28 and 32 dem-
onstrated the highest cytotoxic activity with all IC50 values
lower than 50 µM against human tumor cell lines tested. The
compounds 8, 20–23, 35 and 38 displayed moderate cyto-
toxic activities, and other compounds only had marginal or
no cytotoxic effect in any cell lines.
Among all the compounds screened, 3-amino-b-carboline
7 and its derivatives 9 and 16–18 (except 8) only showed mar-
ginal or no cytotoxic activity, suggesting that the amino sub-
stituent at position-3 was unfavorable for their cytotoxic
activities.
Of all 1-methyl-b-carboline derivatives, the compound 20
having no substituent at position-9 in b-carboline showed
moderate cytotoxic activity. Introducing a short chain alkyl
substituent into position-9 in b-carboline led to compound
21 (methyl) and 22 (ethyl) and 23 (butyl), which all exhibited
more potent cytotoxic activities than the parent compound
20. The compound 24 with a benzyl group at position-
9 displayed selective cytotoxic activities against HepG2 and
Hela cell lines. Whereas introducing a pentafluorobenzyl or
phenylpropyl into position-9 led to the compounds 25 and
26, respectively, which all demonstrated decreased cytotoxic
activities remarkedly. Interestingly, further introducing a ben-
zyl into position-2 in b-carboline afforded the compound 27,
which displayed the prominent and broader spectrum of cyto-
toxic activities against all six human tumor cell lines tested
with all IC50 values lower than 50 µM.
position-2 and 9 simultaneously led to the compounds 30,
which failed to displayed significant cytotoxic activities
against PLA-801 and Lovo cell lines. Furthermore, the com-
pound 31 having an ethyl group at position-2 and 9 simulta-
neously was inactive as cytotoxic agent. It is noteworthy that
incorporation of benzyl substituent at position-2 and 9 in
b-carboline simultaneously afforded the compound 32, which
had prominent cytotoxic activities against all the screened
tumor cell lines. In order to further corroborate the influence
of benzyl group at position-2 on cytotoxic activity of
b-carboline derivatives, we designed and synthesized the com-
pound 28. As we predicted, the compound 28 exhibited sig-
nificant cytotoxic activities against all six human tumor cell
lines with all IC50 values lower than 40 µM. These results,
together with the cytotoxic activity of the compound 27, fur-
ther confirmed that the benzyl substituent at position-2 might
play a vital role in determining significant cytotoxic activi-
ties of these compounds.
The cytotoxic activity decreased greatly after converting
the compounds 4 and 11 into its 3-hydroxymethyl derivatives
36–37 and 38–39, respectively, whereas the cytotoxic activi-
ties increased significantly after converting the compound 4
into its 3-carboxamide derivative 35. The benzyl at position-
9 in b-carboline, the compound in which the ethoxycarbonyl
at position-3 was converted into the corresponding benzy-
loxycarbonyl (34), led to complete loss of cytotoxic activity.
These results indicated that the introduction of an appropri-
ate carboxamide substituent into 3-position of b-carboline ring
system facilitates the increase of their cytotoxic activities,
while 3-hydroxymethyl group was unfavorable.
The compound 29 [22] having no substituent in b-carboline
nucleus showed significant cytotoxic activity against PLA-
801 and Lovo cell lines. However, introducing a methyl into
An overview of the cytotoxic activities data of all new syn-
thesized b-carboline derivatives and of the earlier report [22]
clearly indicate two results: (i) the position and nature of sub-

R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 991–1001
995
Table 1
Cytotoxicity of b-carboline derivatives in vitro c (µM a)
Compound
PLA-801b
HepG2 b
Bel-7402 b
54
BGC-823 b
68
Hela b
60
Lovo b
66
Harmined
4d
45
46
295
241
365
337
233
>1000
38
293
278
100
395
312
170
>1000
13
160
5
7
8
9
741
>1000
>1000
134
741
310
572
856
242
199
211
134
>1000
78
>1000
45
>1000
38
>1000
122
11 d
12
13
16
17
18
20
21
22
23
24
25
26
27
28
29 d
30
31
32
34
35
36
37
38
39
41
42
43
512
262
139
>1000
>1000
>1000
115
100
148
122
84
371
113
238
247
>1000
>1000
>1000
186
159
165
102
78
191
516
60
267
250
>1000
>1000
>1000
317
>1000
>1000
>1000
151
>1000
>1000
>1000
215
>1000
>1000
>1000
275
163
96
160
128
271
203
282
213
150
145
364
152
324
126
>1000
251
324
>1000
297
>1000
948
7
370
477
>1000
13
>1000
297
449
987
11
43
15
40
14
20
36
40
34
31
17
228
197
425
35
379
274
353
72
35
275
371
263
>1000
>1000
20
>1000
32
>1000
45
>1000
39
857
32
>1000
138
>1000
80
>1000
164
>1000
84
>1000
86
>1000
102
>1000
775
579
>1000
89
739
783
617
779
105
497
97
485
588
638
248
161
112
111
101
254
298
86
128
412
123
262
227
111
83
199
104
229
288
224
89
316
264
449
487
>1000
634
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
(Hela), colon carcinoma (Lovo).
c Data represent the mean values of three independent determinations.
d See Ref. [22].
stituents on the b-carboline nucleus seem to modulate cyto-
toxic activity and (ii) the nature of substituents specially at
position-2, 3 and 9 significantly contribute to the cytotoxic
efficacy.
Table 2
Acute toxic effects of b-carboline derivatives in mice
Compound
Acute toxicity
LD50 (mg kg–1, 95% CL)
Neurotoxic effect
3.2. Assessment of acute toxicity
Harmine b
59.00 (43.50–110.00)
>500
+
_
_
_
_
_
_
_
_
a
The LD50 values and scores for neurotoxicity of the selected
b-carboline derivatives in mice after administration by i.p. route
were summarized in Table 2.All the tested compounds resulted
in acute toxic manifestation but caused no obvious neuro-
toxic reaction including tremor, twitch, jumping, tetanus and
supination just like Harmine.Animals were drowsy and exhib-
ited a decrease in locomotor activity after the administration
of the compounds. Of all the tested compounds, the com-
pound 27 displayed the highest acute toxicity with LD50 value
of 5.82 mg kg–1. The compounds 20, 24, 28 and 43 also exhib-
ited remarkable acute toxicity with LD50 value of 32.63,
18
20
24
25
27
28
38
43
32.63 (28.79–36.99)
68.63 (60.96–77.27)
348.44 (303.43–400.12)
5.82 (4.97–6.82)
65.73 (58.25–74.11)
247.13 (213.51–286.03)
65.30 (57.65–73.96)
a Acute neurotoxic manifestation were denoted by “+” and “–”. A “+”
represents toxic reactions including tremble, twitch, jumping, tetanus and
supination, while “–” means no such reaction.
b See Ref. [22].

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R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 991–1001
68.63, 65.73 and 65.30 mg kg–1, respectively, while for the
compounds 25 and 38, acute toxicities were much less with
LD50 value of 348.44 and 247.13 mg kg–1, respectively. Espe-
cially, the compound 18 caused no death at tested dosage,
except for partial contortion at injection point at the highest
dosage used. Autopsy of the animals that died in the course
of experiment and the necropsy findings in surviving animals
at the end of experimental period (14 days) revealed no appar-
ent changes in any organs.
A total analysis to the acute toxicity and neurotoxic effect
of b-carboline derivatives examined above and of the previ-
ous investigation [22] clearly suggested that (1) the acute tox-
icity and neurotoxic effects of b-carboline derivatives de-
pended upon the presence, location and nature of the
substituent introduced into the b-carboline nucleus; (2) the
acute toxicity increased greatly by introducing a benzyl into
position-2 while it decreased dramatically by introducing
ethoxycarbonylamino into position-3; (3) the methoxy group
at position-7 in b-carboline nucleus might be play a very cru-
cial role in determining their remarkable neurotoxic effects.
substituent into position-2 of b-carboline nucleus improved
their antitumor activities significantly, while incorporation of
hydroxymethyl or ethoxycarbonylamino group at position-
3 in b-carboline nucleus impaired their antitumor activities.
4. Conclusion
Our current investigation corroborated the previous obser-
vations [22] that the antitumor activity and acute toxicity as
well as neurotoxic effect of b-carboline derivatives are
substituent-dependant. An overview of the present study and
of the previous report [22], we arrived at the following con-
clusions: (1) the methoxyl group at position-7 in b-carboline
nucleus plays a very crucial role in determining their remark-
able neurotoxic effects including tremor, twitch, jumping, teta-
nus and supination; (2) introducing appropriate substituents
into position-9 and 2 of b-carboline derivatives increases
greatly their antitumor activities; (3) the nature of substitu-
ents specially at position-3 and 1 significantly contributes to
the decreased acute toxicity of b-carboline derivatives; (4)
b-carboline framework is an important pharmacophore for
the design and synthesis of novel antitumor agents with sig-
nificant pharmacological activity and low toxicity.
In this paper we described a preliminary SAR between
b-carboline derivatives and antitumor activities, while the anti-
tumor activities of the studied b-carboline derivatives pre-
sented here remained relatively moderate. It is necessary to
further design and synthesize more potent antitumor agents
together with low neurotoxicities by simple chemical struc-
tural modification on the basis of the present as well as the
previous investigation. Further investigation to elucidate the
pharmacological mechanisms leading to the improved anti-
tumor effects are underway in our laboratory. We hope that
these easy-to-synthesize b-carboline derivatives should serve
as the basic system for searching for more efficient antitumor
drugs.
3.3. Evaluation of antitumor activity
Eight b-carboline derivatives were selected for evaluation
in vivo against mice bearing Lewis lung cancer and Sar-
coma180. The tumor inhibition rates of these compounds were
summarized in Table 3. All the tested compounds exhibited
potent antitumor activities. Compounds 24, 27, 28 and 43
demonstrated remarkable antitumor activities with the tumor
inhibition rate of over 40% against mice bearing Lewis lung
cancer at dose 22.0, 2.0, 20.0 and 22.0 mg kg–1, respectively.
Furthermore, compound 24 showed the highest antitumor
effect with the tumor inhibition rate of 47.3% against mice
bearing Lewis lung cancer, and compound 28 exhibited almost
equal antitumor activity against mice both bearing Lewis lung
cancer and Sarcoma 180. The other compounds tested showed
moderate antitumor activity with tumor inhibition rates rang-
ing from 28.1% to 38.6% at dose ranging from 10.0 to
100.0 mg kg–1. It is interesting to note that the Lewis lung
cancer was more susceptible to all tested compounds than the
Sarcoma 180. The results indicated that introducing a benzyl
5. Experimental
5.1. Materials
Table 3
Antitumor effects of b-carboline derivatives against mice bearing Sarcoma
180 and Lewis lung carcinoma
Melting points were determined in capillary tubes on an
electrothermal PIF YRT-3 apparatus and without correction.
UV spectra were measured on Shimadzu UV 2501PC Spec-
trometer. FAB-MS spectra were obtained from VG ZAB-HS
spectrometer. FT-IR spectra were run on a Bruker Equinox
Compound
Dose
Tumor inhibition rate (%)
(mg kg–1
)
Lewis lung carcinoma Sarcoma180
Harminea
18
20
24
25
27
7.5
100.0
10.0
22.0
100.0
2.0
34.1
30.9
36.3
47.3
38.6
43.5
41.6
34.5
41.3
86.4
15.3
29.1
35.2
35.5
31.5
39.0
41.1
28.1
36.0
87.3
1
55 Fourier transformation infrared spectrometer. H-NMR
spectra were recorded on a Varian INOVA 500NB spectrom-
eter. Elemental analyses (C, H, N) were carried out on an
Elementar Vario EL CHNS Elemental Analyzer. Silica gel
28
38
43
CTX
20.0
100.0
22.0
30.0
F254 was used in analytical thin-layer chromatography (TLC)
and silica gel was used in column chromatography.
All chemicals were purchased from commercial suppliers
and were dried and purified when necessary. 1,2,3,4-tetra-
a See Ref. [22].

R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 991–1001
997
hydro-b-carboline-3-carboxylic acid (2) [25], ethyl 1,2,3,4-
tetrahydro-b-carboline-3-carboxylate (3) [25], ethyl b-carbo-
line-3-carboxylate (4) [25], b-carboline-3-carbohydrazide (5)
[9], 3-azidocarbonyl-b-carboline (6) [9], 3-amino-b-carboline
(7) [9], 3-[(methoxycarbonyl)amino]-b-carboline (8) [9],
3-[(ethoxycarbonyl)amino] b-carboline (9) [9], ethyl 9-ehtyl-
b-carboline-3-carboxylate (10) [22], ethyl 9-benzyl-b-
carboline-3-carboxylate(11) [22], 1-methyl-1,2,3,4-tetra-
hydro-b-carboline-3-carboxylic acid (19) [29], 1-methyl-b-
carboline (20) [29], b-carboline (29) [8], b-carboline-3-
carboxylic acid (33) [25], b-carboline-3-carboxamide (35)
[30], 3-hydroxymethyl-b-carboline (36) [7] and ethyl
1-methyl-b-carboline-3-carboxylate 41 [32] are known com-
pounds.
H-8), 7.56–7.59 (1H, m, H-5), 7.40–7.42 (1H, d, J = 8.0 Hz,
H-6), 7.23–7.26 (1H, m, H-7), 4.37–4.41 (2H, m, NCH2CH3),
3.87 (3H, s, OCH3), 1.45–1.48 (3H, m, NCH2CH3). Anal.
Calc. for C15H15N3O2: C, 66.91; H, 5.58; N, 15.61. Found:
C, 66.78; H, 5.79; N, 15.52.
5.1.4. 3-[(Ethoxycarbonyl)amino]-9-ethyl-b-carboline (17)
The same conditions as for the preparation of 16 were used
for the preparation of 17 except that ethanol was the reaction
solvent. White crystals of 17 were obtained (0.8 g, 56%); m.p.
217–218 °C; FAB-MS m/z (M + 1) 284; UV kmax 376, 364,
296, 240, 202 nm IR (KBr) 3420, 3202, 2974, 1721, 1627,
1
1585, 1532, 1471, 1281, 1219 cm–1; H-NMR (500 MHz,
CDCl3) d 8.68 (1H, s, H-4), 8.59 (1H, s, H-1), 8.15–8.17 (1H,
d, J = 8.0 Hz, H-8), 7.55–7.59 (1H, m, H-5), 7.39–7.40 (1H,
d, J = 8.5 Hz, H-6), 7.23–7.26 (1H, m, H-7), 4.32–4.38 (4H,
m, NCH2CH3, OCH2CH3), 1.41–1.47 (6H, m, NCH2CH3,
OCH2CH3). Anal. Calc. for C16H17N3O2: C, 67.84; H, 6.00;
N, 14.84. Found: C, 67.73; H, 6.16; N, 14.75.
5.1.1. 3-Carbohydrazide-9-ethyl-b-carboline (12)
A solution of compound 10 (2.68 g, 10 mmol) in ethanol
(100 ml) containing 85% hydrazine hydrate (15 ml) was
refluxed for 8 h. The resulting mixture was cooled and the
precipitate was collected by filtration, washed with ethanol,
and then air-dried. The solid was recrystallized from ethanol,
giving white crystals 12 (2.0 g, 83%), m.p. 195–196 °C;
FAB-MS m/z (M + 1) 255; UV kmax 359, 343, 303, 272, 238,
221 nm IR (KBr) 3298, 3202, 2955, 1627, 1590, 1531, 1458,
5.1.5. 3-Carbohydrazide-9-benzyl-b-carboline (13)
A solution of compound 11 (3.3 g, 10 mmol) in ethanol
(100 ml) containing 85% hydrazine hydrate (20 ml) was
refluxed for 10 h. Then the resulting mixture was treated in a
manner similar to that described for 12 to afford white crys-
tals 13 (2.8 g, 87%), m.p. 209–211 °C; FAB-MS m/z (M + 1)
317; UV kmax 356, 342, 272, 238,207 nm IR (KBr) 3349,
3300, 3201, 3059, 1619, 1556, 1496, 1461, 1336, 1200 cm–1;
1H-NMR (500 MHz, DMSO-d6) d 9.62 (1H, s, NH), 9.04
(1H, s, H-4), 8.85 (1H, s, H-1), 8.43–8.45 (1H, d, J = 8.0 Hz,
H-8), 7.77–7.79 (1H, d, J = 8.5 Hz, H-5), 7.61–7.65 (1H, m,
H-6), 7.33–7.36 (1H, m, H-7), 7.21–7.30 (5H, m, ArH), 5.83
(2H, s, NCH2Ar), 4.54 (2H, s, NH2), 1.37–1.40 (3H, m,
NCH2CH3). Anal. Calc. for C19H16N4O: C, 72.15; H, 5.06;
N, 17.72. Found: C, 72.06; H, 5.21; N, 17.77.
1
1331, 1261, 1128 cm–1; H-NMR (500 MHz, DMSO-d6) d
9.61 (1H, s, NH), 9.03 (1H, s, H-4), 8.82 (1H, s, H-1), 8.40–
8.42 (1H, d, J = 7.5 Hz, H-8), 7.75–7.76 (1H, d, J = 8.5 Hz,
H-5), 7.64–7.67 (1H, m, H-6), 7.32–7.35 (1H, m, H-7), 4.58–
4.62 (2H, m, NCH2CH3), 4.54 (2H, s, NH2), 1.37–1.40 (3H,
m, NCH2CH3).Anal. Calc. for C14H14N4O: C, 66.14; H, 5.51;
N, 22.05. Found: C, 65.92; H, 5.76; N, 22.14.
5.1.2. 3-Azidocarbonyl-9-ethyl-b-carboline (14)
A suspension of compound 12 (2.54 g, 10 mmol) in water
(200 ml) was dissolved by the addition of concentrated hydro-
chloric acid (20 ml). The yellow solution was cooled to 0 °C
before the addition of a solution of sodium nitrite (2.1 g,
30 mmol) in water (30 ml). After 40 min at 0 °C, the mixture
was made basic with saturated aqueous sodium hydrogen car-
bonate, and then the precipitate was collected by filtration,
washed well with water, and dried in a desiccator under
vacuum, yielding yellowish solid 14 (2.3 g, 87%) with a ten-
dency to decompose. The material was used without further
purification for the following steps.
5.1.6. 3-Azidocarbonyl-9-benzyl-b-carboline (15)
A suspension of compound 13 (3.16 g, 10 mmol) in water
(500 ml) was dissolved by the addition of concentrated hydro-
chloric acid (20 ml). The yellow solution was cooled to
0 before the addition of a solution of sodium nitrite (2.1 g,
30 mmol) in water (30 ml). After 40 min at 0 °C, the resulting
mixture was treated in a manner similar to that described for
14, yielding yellowish solid 15 (2.9 g, 89%) with a tendency
to decompose. The material was used without further purifi-
cation for the following steps.
5.1.3. 3-[(Methoxycarbonyl)amino]-9-ethyl-b-carboline (16
)
A mixture of the azide 14 (1.32 g, 10 mmol) in methanol
(100 ml) was refluxed for 10 h. The reaction mixture was
concentrated in vacuum, whereupon the product 16 precipi-
tated. Then the precipitate was collected by filtration, washed
with cooled ethanol. The solid was recrystallized from etha-
nol, giving white crystals 16 (0.8 g, 59%), m.p. 213–214 °C;
FAB-MS m/z (M + 1) 270; UV kmax 376, 365, 296, 251, 241,
203 nm IR (KBr) 3435, 3207, 2984, 1727, 1630, 1595, 1472,
1282, 1228, 1079 cm–1; 1H-NMR (500 MHz, CDCl3) d 8.67
(1H, s, H-4), 8.55 (1H, s, H-1), 8.16–8.17 (1H, d, J = 8.0 Hz,
5.1.7. 3-[(Ethoxycarbonyl)amino]-9-benzyl-b-carboline (18
)
A mixture of the azide 15 (2.9 g, 8.86 mmol) in ethanol
(150 ml) was refluxed for 10 h. The reaction mixture was
concentrated in vacuum, whereupon the product 18 precipi-
tated. Then the precipitate was collected by filtration, washed
with cooled ethanol. The solid was recrystallized from ethyl
acetate, giving white crystals 18 (1.8 g, 57%), m.p. 218–
219 °C; FAB-MS m/z (M + 1) 346; UV kmax 373, 362, 295,
251 nm IR (KBr) 3248, 3200, 2981, 1722, 1629, 1534, 1467,

998
R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 991–1001
1281, 1217 cm–1; 1H-NMR (500 MHz, CDCl3) d 8.88 (1H, s,
H-4), 8.78 (1H, s, H-1), 8.21–8.22 (1H, d, J = 7.5 Hz, H-8),
8.06–8.08 (2H, d, J = 8.0 Hz, H-5, H-6), 7.67–7.70 (1H, m,
H-7), 7.54–7.60 (3H, m,ArH), 7.35–7.38 (1H, m,ArH), 7.31–
7.32 (1H, d, J = 8.0 Hz, ArH), 5.79 (2H, s, NCH2Ar), 4.51–
4.55 (2H, m, OCH2CH3), 1.47–1.50 (3H, m, OCH2CH3).
Anal. Calc. for C21H19N3O2: C, 73.04; H, 5.51; N, 12.17.
Found: C, 72.94; H, 5.67; N, 12.09.
1H-NMR (500 MHz, CDCl3) d 8.31–8.32 (1H, d, J = 5.5 Hz,
H-4), 8.11–8.13 (1H, d, J = 7.5 Hz, H-8), 7.84–7.85 (1H, d,
J = 5.5 Hz, H-3), 7.56–7.59 (1H, m, H-5), 7.45–7.47 (1H, d,
J = 8.5 Hz, H-6), 7.25–7.28 (1H, m, H-7), 4.53–4.56 (2H, m,
NCH2CH2CH2CH3), 3.06 (3H, s, CH3), 1.80–1.86 (2H, m,
NCH2CH2CH2CH3), 1.42–1.49 (2H, m, NCH2CH2CH2CH3),
0.97–1.00 (3H, m, NCH2CH2CH2CH3). Anal. Calc. for
C16H18N2: C, 80.67; H, 7.56; N, 11.77. Found: C, 80.58; H,
7.77; N, 11.63.
5.1.8. 1,9-Dimethyl-b-carboline (21)
A mixture of 20 (1.82 g, 10 mmol) and anhydrous DMF
(50 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. The organic phase was washed with water and brine,
then dried over anhydrous sodium sulfate, filtered and evapo-
rated. The oil obtained was purified by silica column chro-
matography with ethyl acetate as the eluent. Upon recrystal-
lization, white crystals of 21 were obtained (1.6 g, 82%), m.p.
103–104 °C (from ether); FAB-MS m/z (M + 1) 197; UV
kmax 358, 343, 288, 243, 236 nm IR (KBr) 3050, 2920, 1614,
5.1.11. 9-Benzyl-1-methyl-b-carboline (24)
A mixture of 20 (1.82 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 21 to afford white crystals 24
(2.3 g, 85%), m.p. 111–112 °C (from ether); FAB-MS m/z
(M + 1) 273; UV kmax 355, 341, 287, 242, 237 nm IR (KBr)
3029, 1615, 1561, 1446, 1356, 1238, 1131 cm–1; H-NMR
1
(500 MHz, CDCl3) d 8.33–8.34 (1H, d, J = 5.5 Hz, H-4),
8.12–8.14 (1H, d, J = 8.0 Hz, H-8), 7.84–7.86 (1H, d,
J = 5.5 Hz, H-3), 7.48–7.51 (1H, m, H-5), 7.32–7.33 (1H, d,
J = 8.0 Hz, H-6), 7.21–7.28 (4H, m, H-7, ArH), 6.96–6.97
(2H, m, ArH), 5.74 (2H, s, CH2Ar), 2.85 (3H, s, CH3). Anal.
Calc. for C19H16N2: C, 83.82; H, 5.88; N, 10.29. Found: C,
83.67; H, 6.07; N, 10.22.
1
1560, 1446, 1288, 1231, 1134 cm–1; H-NMR (500 MHz,
CDCl3) d 8.28–8.29 (1H, d, J = 5.0 Hz, H-4), 8.08–8.09 (1H,
d, J = 8.0 Hz, H-8), 7.79–7.80 (1H, d, J = 5.5 Hz, H-3), 7.55–
7.59 (1H, m, H-5), 7.41–7.42 (1H, d, J = 8.5 Hz, H-6), 7.24–
7.27 (1H, m, H-7), 4.11 (3H, s, NCH3), 3.07 (3H, s, CH3).
Anal. Calc. for C13H12N2: C, 79.59; H, 6.12; N, 14.29. Found:
C, 79.39; H, 6.35; N, 14.20.
5.1.12. 9-(2′,3′,4′,5′,6′-Pentafluoro)benzyl-1-methyl-b-car-
boline (25)
A mixture of 20 (1.82 g, 10 mmol) and anhydrous DMF
(60 ml) was stirred at RT until clear, and 60% NaH (0.6 g,
15 mmol) and aa-bromo-2,3,4,5,6-pentafluorotoluene (3 ml,
20 mmol) were added, and then reacted and treated in a man-
ner similar to that described for 21 to afford white crystals 25
(2.8 g, 77%), m.p. 195–196 °C (from ether); FAB-MS m/z
(M + 1) 363; UV kmax 349, 335, 286, 235 nm IR (KBr) 3056,
5.1.9. 9-Ethyl-1-methyl-b-carboline (22)
A mixture of 20 (1.82 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.
The mixture was stirred at RT for 2 h, and then treated in a
manner similar to that described for 21 to afford yellow oil
22 (1.7 g, 81%); FAB-MS m/z (M + 1) 211; UV kmax 358,
344, 289, 243, 237 nm IR (KBr) 3046, 2980, 2512, 1623,
1
1618, 1564, 1505, 1445, 1347, 1207, 1120 cm–1; H-NMR
(500 MHz, CDCl3) d 8.36–8.38 (1H, d, J = 5.5 Hz, H-4),
8.10–8.12 (1H, d, J = 8.0 Hz, H-8), 7.84–7.85 (1H, d,
J = 5.0 Hz, H-3), 7.51–7.55 (1H, m, H-5), 7.28–7.32 (2H, m,
H-6, H-7), 5.93 (2H, s, CH2Ar), 3.07 (3H, s, CH3). Anal.
Calc. for C19F5H11N2: C, 62.98; H, 3.04; N, 7.73. Found: C,
62.75; H, 3.25; N, 7.67.
1
1537, 1462, 1334, 1231, 1139 cm–1; H-NMR (500 MHz,
CDCl3) d 8.21–8.22 (1H, d, J = 5.5 Hz, H-4), 8.01–8.02 (1H,
d, J = 8.0 Hz, H-8), 7.69–7.70 (1H, d, J = 5.0 Hz, H-3), 7.47–
7.49 (1H, m, H-5), 7.34–7.36 (1H, d, J = 8.5 Hz, H-6), 7.15–
7.17 (1H, m, H-7), 4.24–4.27 (2H, m, NCH2CH3), 3.03 (3H,
s, CH3), 1.37–1.40 (3H, m, NCH2CH3). Anal. Calc. for
C14H14N2: C, 80.00; H, 6.67; N, 13.33. Found: C, 79.78; H,
6.96; N, 13.18.
5.1.13. 9-Phenylpropyl-1-methyl-b-carboline (26)
A mixture of 20 (1.82 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 2 h, and then treated in a manner
similar to that described for 21 to afford white crystals 26
(2.4 g, 80%), m.p. 119–120 °C (from ether); FAB-MS m/z
(M + 1) 301; UV kmax 358, 344, 289, 243, 237 nm IR (KBr)
3028, 2931, 1617, 1560, 1447, 1363, 1234, 1118 cm–1;
1H-NMR (500 MHz, CDCl3) d 8.29–8.30 (1H, d, J = 5.0 Hz,
H-4), 8.07–8.09 (1H, d, J = 8.0 Hz, H-8), 7.79–7.80 (1H, d,
J = 5.0 Hz, H-3), 7.51–7.54 (1H, m, H-5), 7.17–7.31 (7H, m,
5.1.10. 9-n-Butyl-1-methyl-b-carboline (23)
A mixture of 20 (1.82 g, 10 mmol) and anhydrous DMF
(60 ml) was stirred at RT until clear, and then 60% NaH (0.6 g,
15 mmol) and n-butyl iodide (6 ml, 50 mmol) were added.
The mixture was stirred at RT for 2 h, and then treated in a
manner similar to that described for 21 to afford white crys-
tals 23 (1.8 g, 76%), m.p. 124–126 °C (from ether); FAB-MS
m/z (M + 1) 239; UV kmax 358, 344, 289, 242, 236 nm IR
(KBr) 2962, 2589, 1618, 1564, 1472, 1357, 1255, 1119 cm–1;

R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 991–1001
999
H-6, H-7, ArH), 4.49–4.52 (2H, m, NCH2CH2CH2Ar), 2.89
(3H, s, CH3), 2.72–2.75 (2H, m, NCH2CH2CH2Ar), 2.12–
2.18 (2H, m, NCH2CH2CH2Ar). Anal. Calc. for C21H20N2:
C, 84.00; H, 6.67; N, 9.33. Found: C, 79.86; H, 6.87; N, 9.25.
C13H13IN2: C, 48.15; H, 4.01; N, 8.64. Found: C, 48.03; H,
4.25; N, 8.52.
5.1.17. 2,9-Diethyl-b-carbolinium iodate (31)
A mixture of 29 (0.84 g, 5 mmol) and anhydrous DMF
(30 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.
The mixture was stirred at 50–60 °C for 2 h, and then follow-
ing the method described for 27 to afford yellow crystals 31
(1.1 g, 63%), m.p. 214–215 °C. FAB-MS m/z 225; UV kmax
389, 310, 261, 257, 220, 210 nm IR (KBr) 3437, 2977, 1815,
1639, 1509, 1458, 1243,1158 cm–1; 1H-NMR (CDCl3) d 9.61
(1H, s, H-4), 8.68–8.69 (1H, d, J = 6.5 Hz, H-1), 8.61–8.63
(1H, d, J = 6.5 Hz, H-8), 8.42–8.44 (1H, d, J = 8.0 Hz, H-3),
7.84–7.89 (2H, m, H-5, H-6), 7.48–7.51 (1H, m, H-7), 4.83–
4.87 (2H, m, N+CH2CH3), 4.67–4.72 (2H, m, NCH2CH3),
1.75–1.78 (3H, m, N+ CH2CH3), 1.51–1.54 (3H, m,
NCH2CH3).Anal. Calc. for C15H17IN2: C, 51.14; H, 4.83; N,
7.95. Found: C, 50.97; H, 5.03; N, 7.86.
5.1.14. 2,9-Dibenzyl-1-methyl-b-carbolinium bromate (27)
A mixture of 20 (1.82 g, 10 mmol) and anhydrous DMF
(50 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.
The mixture was stirred at 50–60 °C for 2 h. The resulting
mixture was poured into H2O, and extracted with ethyl acetate.
The organic phase was washed with water and brine, and then
dried over anhydrous sodium sulfate, filtered and evaporated.
The yellow solid was recrystallized from ethanol, and golden
crystals of 27 were obtained (3.6 g, 72%), m.p. > 270 °C;
FAB-MS m/z 363; UV kmax 383, 312, 260 nm IR (KBr) 3032,
1
3004, 1623, 1573, 1456, 1336, 1247, 1221 cm–1; H-NMR
(500 MHz, DMSO-d6) d 8.95–8.96 (1H, d, J = 6.5 Hz, H-4),
8.89–8.91 (1H, d, J = 6.5 Hz, H-8), 8.61–8.62 (1H, d,
J = 8.0 Hz, H-3), 7.89–7.91 (1H, d, J = 9.0 Hz, H-5), 7.83–
7.87 (1H, m, H-6), 7.53–7.56 (1H, m, H-7), 7.24–7.40 (6H,
m, ArH), 7.15–7.17 (2H, d, J = 8.0 Hz, ArH), 7.01–7.02 (2H,
d, J = 7.0 Hz, ArH), 6.08 (2H, s, N+CH2Ar), 6.05 (2H, s,
NCH2Ar), 2.94 (3H, s, CH3). Anal. Calc. for C26H23BrN2: C,
70.43; H, 5.19; N, 6.32. Found: C, 70.32; H, 5.43; N, 6.42.
5.1.18. 2,9-Dibenzyl-b-carbolinium bromate (32)
A mixture of 29 (1.68 g, 10 mmol) and anhydrous DMF
(60 ml) was stirred at RT until clear, and then 60% NaH (1.2 g,
30 mmol) and benzyl bromide (5 ml, 40 mmol) were added.
The mixture was stirred at 50–60 °C for 2 h, and then follow-
ing the method described for 27 to afford golden crystals 32
(3.6 g, 76%), m.p. > 270 °C. FAB-MS m/z 349; UV kmax 390,
313, 262, 235, 205 nm IR (KBr) 3409, 2982, 1644, 1511,
1453, 1337, 1211, 1134 cm–1; 1H-NMR (CDCl3) d 9.57 (1H,
s, H-4), 8.71 (2H, s, H-1, H-8), 8.42–8.44 (1H, d, J = 8.5 Hz,
H-3), 7.81–7.82 (2H, m, H-5, H-6), 7.40–7.50 (6H, m, H-7,
ArH), 7.19–7.28 (5H, m, ArH), 5.95 (2H, s, N+CH2Ar), 5.86
(2H, s, N CH2Ar). Anal. Calc. for C25H21BrN2: C, 69.93; H,
4.90; N, 6.53. Found: C, 69.84; H, 5.13; N, 6.46.
5.1.15. 2,9-Dibenzyl-3-ethoxycarbonyl-1-methyl-b-carbo-
linium bromate (28)
A mixture of 4 (1.2 g, 5 mmol) and anhydrous DMF (30 ml)
was stirred at RT until clear, and then 60% NaH (1.2 g,
30 mmol) and benzyl bromide (2.5 ml, 20 mmol) were added.
The mixture was stirred at 50–60 °C for 2 h, and following
the method described for 27 to afford golden crystals 28 (2.3 g,
84%), m.p. > 270 °C. FAB-MS m/z 421; UV kmax 397, 317,
284, 243 nm IR (KBr) 3421, 2976, 1726, 1630, 1457, 1367,
1257, 1096 cm–1; 1H-NMR (500 MHz, CDCl3) d 9.88 (1H, s,
H-4), 9.30 (1H, s, H-1), 8.55–8.57 (1H, d, J = 7.5 Hz, H-8),
7.87–7.92 (2H, m, H-5, H-6), 7.55–7.59 (1H, m, H-7), 7.20–
7.36 (10H, m, ArH), 6.39 (2H, m, N+CH2Ar), 5.98 (2H, s,
NCH2Ar), 4.44–4.48 (2H, m, OCH2CH3), 1.34–1.37 (3H, m,
OCH2CH3).Anal. Calc. for C28H25BrN2O2: C, 67.07; H, 4.99;
N, 5.59. Found: C, 66.89; H, 5.23; N, 5.47.
5.1.19. Benzyl 9-benzyl-b-carboline-3-carboxylate (34)
A mixture of compound 33 (2.12 g, 10 mmol) and anhy-
drous DMF (50 ml) was stirred at RT till clear, and then 60%
NaH (1.2 g, 30 mmol) and benzyl bromide (5 ml, 40 mmol)
were added and stirred at RT for 2 h. The resulting solution
was poured into H2O (100 ml), and extracted with ethyl
acetate (3 × 200 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 34 were obtained (2.3 g,
57%), m.p. 169–170 °C. FAB-MS m/z (M + 1) 393; UV kmax
356, 341, 305, 273, 236 nm IR (KBr) 3400, 3064, 2935, 1709,
5.1.16. 2,9-Dimethyl-b-carbolinium iodate (30)
A mixture of 29 (0.84 g, 5 mmol) and anhydrous DMF
(30 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 50–60 °C for 1 h, and then follow-
ing the method described for 27 to afford yellow crystals 30
(1.2 g, 73%), m.p. > 270 °C. FAB-MS m/z 197; UV kmax 390,
309, 261, 257, 220, 210 nm IR (KBr) 3447, 2985, 1642, 1517,
1467, 1376, 1335, 1262, 1153 cm–1; 1H-NMR (CDCl3) d 9.42
(1H, s, H-4), 8.66–8.67 (1H, d, J = 6.5 Hz, H-1), 8.51–8.52
(1H, d, J = 6.0 Hz, H-8), 8.43–8.45 (1H, d, J = 8.0 Hz, H-3),
7.83–7.91 (2H, m, H-5, H-6), 7.50–7.53 (1H, m, H-7), 4.57
(3H, m, N+CH3), 4.13 (3H, m, NCH3). Anal. Calc. for
1
1623,1584, 1497, 1461, 1336, 1244, 1109 cm–1; H-NMR
(CDCl3) d 8.96 (1H, s, H-4), 8.91 (1H, s, H-1), 8.22–8.24
(1H, m, H-8), 7.60–7.64 (1H, m, H-5), 7.49–7.55 (3H, m,
H-6, H-7, ArH), 7.31–7.40 (4H, m, ArH), 7.25–7.29 (3H, m,
ArH), 7.12–7.14 (2H, m, ArH), 5.63 (2H, s, OCH2Ar), 5.52
(2H, s, NCH2Ar). Anal. Calc. for C26H20N2O2: C, 79.59; H,
5.10; N, 7.14. Found: C, 79.48; H, 5.26; N, 7.19.

1000
R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 991–1001
5.1.20. 3-Acetyloxymethyl-b-carboline (37)
H-6), 7.19–7.31 (6H, m, H-7, ArH), 5.76 (2H, s, NCH2Ar),
5.28 (2H, s, CH2), 2.11 (3H, s, CH3). Anal. Calc. for
C21H18N2O2: C, 76.36; H, 5.45; N, 8.48. Found: C, 76.24; H,
5.68; N, 8.39.
A mixture of compound 36 (1.98 g, 10 mmol) and acetic
acid (50 ml) was refluxed for 2 h. The solvent was evaporated
in vacuum, and water (50 ml) was added. The aqueous sus-
pension was extracted with ethyl acetate (3 × 200 ml). The
organic extracts were combined, and the solvent was evapo-
rated in vacuum. The residue was chromatographed on silica
gel with petroleum ether/acetone (2:1) as the eluent. Upon
recrystallization, white crystals of 37 were obtained (2.2 g,
92%), m.p. 131–132 °C (from ether); FAB-MS m/z (M + 1)
241; UV kmax 326, 315, 284, 259, 230, 207 nm IR (KBr)
3371, 2938, 1743, 1692, 1616, 1450, 1372, 1238 cm–1;
1H-NMR (500 MHz, CDCl3) d 9.47 (1H, s, NH), 9.03 (1H, s,
H-4), 8.32–8.34 (1H, d, J = 8.0 Hz, H-1), 8.26–8.28 (1H, d,
J = 8.5 Hz, H-8), 8.19–8.20 (1H, m, H-5), 7.67–7.70 (1H, m,
H-6), 7.47–7.51 (1H, m, H-7), 5.29 (2H, s, CH2), 2.16 (3H, s,
CH3). Anal. Calc. for C14H12N2O2: C, 70.00; H, 5.00; N,
11.67. Found: C, 69.91; H, 5.19; N, 11.58.
5.1.23. Ethyl 9-n-butyl-1-methyl-b-carboline-3-carboxylate
(42)
A mixture of 41 (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 n-butyl iodide (6 ml, 50 mmol) were added.
The mixture was stirred at RT for 1 h. The resulting mixture
was poured into H2O, and extracted with ethyl acetate. 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 42 were obtained (2.3 g, 74%), m.p. 83–84 °C
(from ether); FAB-MS m/z (M + 1) 311; UV kmax 355, 338,
308, 273, 240 nm IR (KBr) 3059, 2964, 2868, 1680, 1620,
1557, 1456, 1342, 1270, 1138 cm–1; 1H-NMR (500 MHz,
CDCl3) d 8.72 (1H, s, H-4), 8.14–8.16 (1H, d, J = 8.0 Hz,
H-8), 7.58–7.61 (1H, m, H-5), 7.46–7.48 (1H, d, J = 8.5 Hz,
H-6), 7.30–7.33 (1H, m, H-7), 4.50–4.55 (4H,
NCH2CH2CH2CH3, OCH2CH3), 3.11 (3H, s, CH3), 1.79–
1.85 (2H, m, NCH2CH2CH2CH3), 1.42–1.50 (5H, m,
NCH2CH2CH2CH3, OCH2CH3), 0.96–0.99 (3H, m,
NCH2CH2CH2CH3). Anal. Calc. for C19H22N2O2: C, 73.55;
H, 7.10; N, 9.03. Found: C, 73.43; H, 7.29; N, 8.96.
5.1.21. 9-Benzyl-3-hydroxymethyl-b-carboline (38)
A suspension of compound 11 (3.3 g, 10 mmol) in THF
(400 ml) was treated with LiAlH4 (1.2 g, 30 mmol), and the
mixture was refluxed for 10 h. The reaction mixture was
cooled and treated with H2O and stirred overnight. The sol-
vent was evaporated in vacuum and H2O was added. The aque-
ous suspension was extracted with ethyl acetate. The com-
bined organic extracts was washed with water and brine, then
dried over anhydrous sodium sulfate, filtered and evaporated.
The oil obtained was purified by silica column chromatogra-
phy with ethyl acetate as the eluent. Upon recrystallization,
white crystals of 2a were obtained (1.8 g, 80%), m.p. 120–
122 °C (from ethyl acetate); FAB-MS m/z (M + 1) 289; UV
kmax 361, 347, 291, 284, 239, 215 nm IR (KBr) 3170, 2938,
5.1.24. 9-n-Butyl-1-methyl-b-carboline-3-carboxylic acid (43
)
A mixture of compound 42 (1.55 g, 5 mmol), NaOH (0.8 g,
20 mmol), ethanol (50 ml) and H2O (100 ml) was refluxed
for 1 h, and the ethanol was removed on the rotary evapora-
tor. The mixture was neutralized (pH 5) with 5 M HCl and
cooled. The precipitate was collected, washed well with H2O
and dried in vacuum, yellow solid was obtained (1.4 g, 99%).
M.p. 187–188 °C; FAB-MS m/z (M + 1) 283; UV kmax 356,
341, 271, 240 nm IR (KBr) 3250–2100, 1745, 1622, 1586,
1365, 1205 cm–1; 1H-NMR (500 MHz, DMSO-d6) d 8.76 (1H,
s, H-4), 8.35–8.37 (1H, d, J = 7.5 Hz, H-8), 7.74–7.76 (1H, d,
J = 8.5 Hz, H-5), 7.63–7.66 (1H, m, H-6), 7.32–7.35 (1H, m,
H-7), 4.60–4.64 (2H, m, NCH2CH2CH2CH3), 3.04 (3H, s,
CH3), 1.73–1.79 (2H, m, NCH2CH2CH2CH3), 1.35–1.43
(2H, m, NCH2CH2CH2CH3), 0.91–0.94 (3H, m,
NCH2CH2CH2CH3). Anal. Calc. for C17H18N2O2: C, 72.34;
H, 6.38; N, 9.93. Found: C, 72.23; H, 6.57; N, 9.97.
1
1627, 1559, 1467, 1363, 1264 cm–1; H-NMR (500 MHz,
CDCl3) d 8.73 (1H, s, H-4), 8.13–8.15 (1H, d, J = 8.0 Hz,
H-1), 7.96 (1H, s, H-8), 7.54–7.58 (1H, m, H-5), 7.41–7.43
(1H, d, J = 8.5 Hz, H-6), 7.28–7.31 (1H, m, H-7), 7.23–7.27
(3H, m, ArH), 7.11–7.12 (2H, m, ArH), 5.51 (2H, s, CH2Ar),
4.94 (2H, s, CH2OH), 4.01 (1H, s, CH2OH). Anal. Calc. for
C19H16N2O: C, 79.17; H, 5.56; N, 9.72. Found: C, 79.10; H,
5.72; N, 9.63.
5.1.22. 9-Benzyl-3-acetyloxymethyl-b-carboline (39)
A mixture of compound 38 (1.44 g, 5 mmol) and acetic acid
(30 ml) was refluxed for 2 h. The solvent was evaporated in
vacuum, and water (30 ml) was added. The aqueous suspen-
sion was extracted with ethyl acetate (3 × 100 ml). The organic
extracts were combined, and the solvent was evaporated in
vacuum. The residue was chromatographed on silica gel with
ethyl acetate as the eluent. Upon recrystallization, white crys-
tals of 39 were obtained (1.5 g, 94%), m.p. 141–142 °C (from
ether); FAB-MS m/z (M + 1) 331; UV kmax 360, 346, 291,
284, 239, 214 nm IR (KBr) 3428, 3027, 2942, 1726, 1622,
1452, 1352, 1245 cm–1; 1H-NMR (500 MHz, CDCl3) d 9.01
(1H, s, H-4), 8.30–8.31 (1H, d, J = 8.0 Hz, H-1), 8.20 (1H, s,
H-8), 7.73–7.75 (1H, d, J = 8.5 Hz, H-5), 7.58–7.61 (1H, m,
5.2. Pharmacology
5.2.1. 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 by Al-Allaf and Rashan [18]. Briefly, cells
were grown in RPMI-1640 medium containing 10% (v/v) fetal
bovine serum and 100 ug ml–1 penicillin and 100 ug ml–1
streptomycin. Cultures were propagated at 37 °C in a humi-
fied atmosphere containing 5% CO2. Drug stock solutions

R. Cao et al. / European Journal of Medicinal Chemistry 40 (2005) 991–1001
1001
were prepared in DMSO. The final concentration of DMSO
Acknowledgements
in the growth medium was 2% (v/v) or lower, the concentra-
tion without effect on cell replication. The tumor cell line
panel consisted of non-small cell lung carcinoma (PLA-
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.
This work was supported by Xinjiang Huashidan Pharmaceu-
tical Co. Ltd. and Guangzhou Commission of Science and Tech-
nology (97-Z-12-01). We also thank Center forAnalysis and Test
of SunYat-sen (Zhongshan) University for UV, FAB-MS, IR and
1H-NMR spectra as well as elemental analyses data. We are also
grateful to Professor Huifang Yan and co-workers for perfor-
mance of the antitumor and acute toxic assays in mice.
5.2.2. Assay of acute toxicity
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tutional guidelines. Prior to each experiment, mice were fas-
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day after drug administration and checked macroscopically
for possible damage to the heart, liver and kidneys. Mice of
immediate death following drug administration were also
examined for any possible organ damage. LD50 values were
calculated graphically as described [31].
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5.2.3. Assay of antitumor activity
Antitumor activity against Lewis lung cancer and Sar-
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of Pharmaceutical Industry. Tumor cells of Lewis lung can-
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mice by subcutaneous injection. Each compound was injected
by intraperitoneal (i.p.) to different group mice (each group
containing 10 female mice) 24 h after the inoculation at dose
about one third of LD50 value once a day for consecutive
7 days. Cytophosphane (CTX) at 30.0 mg kg–1 was used as a
positive control and vehicle as negative control. The weights
of animals were recorded every 3 days. All animals were sac-
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lated as follows: C–T ⁄C×100
͑
͒
T: average tumor weight of treated group; C: average tumor
weight of negative control group.