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Synthesis, acute toxicities, and antitumor

effects of novel 9-substituted β-carboline
derivatives

Article in Bioorganic & Medicinal Chemistry · October 2004

DOI: 10.1016/j.bmc.2004.06.038 · Source: PubMed

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 Bioorganic & Medicinal Chemistry 12 (2004) 4613–4623

Synthesis, acute toxicities, and antitumor effects of novel

9-substituted b-carboline derivatives
Rihui Cao, Qi Chen, Xuerui Hou, Hongsheng Chen, Huaji Guan, Yan Ma,
Wenlie Peng and 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, PR China
Received 3 May 2004; revised 27 June 2004; accepted 28 June 2004

Abstract—A series of novel 9-substituted b-carboline derivatives was synthesized from harmine and L -tryptophan, respectively. Cy-
totoxic activities of these compounds in vitro were investigated. The results showed that most compounds of 9-substituted b-carb-
oline derivatives had more remarkable cytotoxic activities in vitro than their corresponding parent compounds. Acute toxicities and
antitumor effects of the selected b-carboline derivatives in mice were also examined. The results demonstrated that a short alkyl or
benzyl substituent at position-9 increased the antitumor activities significantly and a ethoxycarbonyl or carboxyl substituent at posi-
tion-3 reduced the acute toxicity and neurotoxicity of these b-carboline derivatives dramatically. Moreover the compounds both
with an alkoxycarbonyl or carboxyl substituent at position-3 and a short alkyl or benzyl substituent at positon-9 exhibited more
significant antitumor activities and lower acute toxicities and neurotoxicities than the other compounds. The compound 8c, having
an n-butyl and a carboxyl substituent at position-9 and 3, respectively, was found to have the highest antitumor effect and the lowest
acute toxicity and neurotoxicity. These data suggested that (1) appropriate substituents at both position-9 and 3 of b-carboline
derivatives might play a crucial role in determining their enhanced antitumor activities and decreased acute toxicities and neurotoxic
effects; (2) the b-carboline derivatives have the potential to be used as antitumor drug leads.

2004 Published by Elsevier Ltd.

1. Introduction our preliminary investigation results demonstrated that

the compounds with b-carboline nucleus have potential
b-Carbolines are a large group of naturally occurring antitumor activities. Yet we also found that this class of
and synthetic indole alkaloids with different degrees of compounds caused remarkable acute neurotoxicity
aromaticity, some of which are widely distributed in nat- characterized by tremble, twitch, and jumping in exper-
ure, including various plants,1–3 marine creatures,4 in- imental mice models.
sects,5 mammalians as well as human tissues and body
fluids.6–9 These compounds are of great interest due to Many previous reports focused on the effects of these
their various biological activities such as intercalating compounds on the central nervous system (CNS), such
into DNA,10,11 inhibiting CDK,12 and Topisom- as their affinity with benzodiazepine receptors,17–19 5-
erase,13,14 inhibiting monoamine oxidase15,16 as well as HT2A and 5-HT2C receptors20 and imidazoline recep-
interacting with benzodiazepine receptors17–19 and 5- tor32, and so on. However so far there have been few
hydroxy serotonin receptors,20 and their broad spectrum such literatures about their cytotoxic activities of these
pharmacological properties including anxiolytic, hyp- compounds in vitro, even no reports are available deal-
notic, anticonvulsant,21–23 parasiticidal,24 antiviral25 as ing with systematic and detailed studies of structure–
well as antimicrobial activities.30 Recent work26–28 and activity relationships on both antitumor activities and
neurotoxic activities in vivo. A probable reason for this
is that many of the b-carboline derivatives of interest are
Keywords: b-Carboline; Synthesis; Acute toxicity; Antitumor; Neuro- not readily available. One goal of the present investiga-
toxicity. tions was to synthesize a series of novel b-carboline
* Corresponding author. Tel.: +86-20-84113655; fax: +86-20- derivatives and elucidate their preliminary relationships
84038377; e-mail: [email protected] between structures and antitumor activities as well as

0968-0896/$ - see front matter
2004 Published by Elsevier Ltd.

doi:10.1016/j.bmc.2004.06.038
4614 R. Cao et al. / Bioorg. Med. Chem. 12 (2004) 4613–4623

neurotoxicities, and the other aim of this study was to COOR3 COOR3
increase the antitumor activities and decrease the neuro- DMF/ THF
toxic activities of these compounds. The work would N N
help search for new antitumor leading compounds with NaH, RI or RBr
N N
significant antitumor activities and lower neurotoxicities H R9
by simple chemical structural modifications on the basis 5a R3=CH3 R9=CH3
5 R3=CH3
of b-carboline ring. To the best of our knowledge, all 9- 5b R3=CH3 R9=CH2CH3
6 R3=CH2CH3
substituted b-carboline derivatives except 2a–c are novel 3 5c R3=CH3 R9=n-C4H9
7 R =n-C4H9
compounds, and this is the first time to report the anti- 5d R3=CH3 R9=CH2C6H5
tumor activities and acute toxicities as well as neurotoxi- 6a R3=CH2CH3 R9=CH3
cities of this class of compounds in mice. 6b R3=CH2CH3 R9=CH2CH3
6c R3=CH2CH3 R9=n-C4H9
6d R3=CH2CH3 R9=CH2C6H5
2. Results and discussion 6e R3=CH2CH3 R9=CH2C6F5
6f R3=CH2CH3 R9=(CH2)3C6H5
2.1. Synthetic approach 7a R3=n-C4H9 R9=CH3
7b R3=n-C4H9 R9=CH2CH3
N-alkylation and N-benzylation derivatives of harmine 7c R3=n-C4H9 R9=n-C4H9
1 could be easily obtained in a good yield through react- 7d R3=n-C4H9 R9=CH2C6H5
ing with alkyl or benzyl bromide in the presence of so-
Scheme 3. Synthesis of 9-substituted b-carboline-3-carboxylate deriv-
dium hydride in DMF and THF (see Scheme 1). The
atives.
starting material L -tryptophan reacted with formalde-
hyde via well-known Pictet–Spengler condensation and
then the products were esterified with relevant alcohol
to produce 1,2,3,4-tetrahydro-b-carboline-3-carboxy- COOC2 H5 COOH
lates, respectively. The 1,2,3,4-tetrahydro-b-carboline- NaOH
N N
3-carboxylates reacted with SeO2 in acetic acid solution
by oxidative decarboxylation to produce b-carboline 3 N N
and reacted with sulfur in anhydrous xylene by dehydro- R9 R9
genation to afford b-carboline-3-carboxylate 5, 6, 7, 6, 6a-6f
9
8 R =H
respectively. The N-9 position of compound 3, 5, 6, 7 8a R9=CH3
was further alkylated or benzylated by the action of so- 8b R9=CH2CH3
dium hydride in anhydrous DMF followed by addition 8c R9=n-C4H9
of the relevant appropriate alkylating and benzylating 8d R9=CH2C6H5
agents (see Schemes 2 and 3) to produce various 9-sub- 8e R9=CH2C6F5
8f R9=(CH2)3C6H5

Scheme 4. Synthesis of 9-substituted b-carboline-3-carboxylic acid

derivatives.
DMF/ THF
N N
H3 CO H3 CO
N NaH, RI or RBr N
H CH3
R9
CH3 stituted b-carboline-3-carboxylate (5a–d, 6a–e, 7a–d).
The compounds 6 and 6a–f were hydrolyzed in sodium
1 2a R9=CH3 hydroxide solution to afford the compounds 8 and 8a–
2b R9=CH2CH3 f (see Scheme 4). In our studies, we found that N-alkyl-
2c R9=n-C4H9 ation and N-benzylation of b-carboline-3-carboxylate
2d R9=CH2CH2OH would be accompanied by ester exchange and ester
2e R9=CH2C6H5 hydrolysis unexpectedly. As a result, separating and
2f R9=CH2C6F5 purifying the products would become difficult. Conse-
2g R9=(CH2)3C6H5 quently, the key step was to use anhydrous DMF so
Scheme 1. Synthesis of 9-substituted harmine derivatives.
as to guarantee the dryness of reaction system of alkyla-
tion and benzylation of b-carboline-3-carboxylate 5, 6,
7. The chemical structures of all the synthesized novel
DMF/ THF compounds were confirmed by FAB-MS, UV, IR, 1H
N
N NMR, and elemental analyses data.
NaH, RI or RBr N
N
H R9 2.2. Cytotoxicity assays
9
4a R =CH3
3
4b R9=CH2CH3
Thirty-seven b-carboline derivatives were examined for
4c R9=n-C4H9 cytotoxic activities against a panel of human tumor cell
4d R9=CH2C6H5 lines. In order to enhance the solubility in aqueous solu-
tion, compounds 8 and 8a–f were converted into their
Scheme 2. Synthesis of 9-substituted b-carbolines. water-soluble sodium salts and the other compounds
 R. Cao et al. / Bioorg. Med. Chem. 12 (2004) 4613–4623 4615

Table 1. Cytotoxicity of b-carboline derivatives in vitroc (IC50,a lM)

Compounds PLA-801b HepG2b Bel-7402b BGC-823b Helab Lovob
Harmine 45 46 54 68 60 66
2a 29 16 63 52 8 43
2b 22 14 28 45 17 170
2c 111 62 58 69 28 87
2d 78 313 244 204 25 27
2e 65 85 57 95 270 52
2f 42 46 43 22 28 31
2g 50 36 44 46 24 27
3 17 228 379 274 353 35
4a 219 102 302 204 327 220
4b 130 167 311 171 234 159
4c 133 99 164 101 88 134
4d 124 107 77 73 48 123
5 295 241 293 278 100 160
5a 125 119 195 181 181 175
5b 25 117 124 79 17 107
5c 136 464 433 310 289 166
5d >1000 >1000 >1000 >1000 >1000 312
6 275 >1000 >1000 >1000 567 >1000
6a 170 108 260 141 136 69
6b 87 109 107 108 137 77
6c 71 476 784 760 487 173
6d 78 38 45 38 13 122
6e 47 116 9 52 78 37
6f >1000 >1000 387 >1000 >1000 >1000
7 >1000 >1000 >1000 >1000 >1000 >1000
7a 675 266 313 394 295 104
7b 109 70 74 82 67 83
7c >1000 >1000 >1000 72 954 45
7d 994 >1000 >1000 >1000 >1000 >1000
8 327 354 369 330 262 198
8a 267 147 226 126 89 88
8b 212 179 130 163 183 62
8c 92 73 92 116 60 11
8d 100 102 112 52 86 41
8e 86 31 61 35 44 13
8f 57 31 53 28 100 5
a
Cytotoxicity as IC50 for each cell line, is the concentration of compound, which reduced by 50% the optical density of treated cells with respect to
untreated cells using the MTT assay.
b
Cell lines include nonsmall cell lung carcinoma (PLA-801), liver carcinoma (HepG2 and Bel-7402), gastric carcinoma (BGC-823), cervical carci-
noma (Hela), colon carcinoma (Lovo).
c
Data represent the mean values of three independent determinations.

investigated were all prepared in the form of hydrochlo- pound 7 were found to be less active with IC50 values of
ride by the usual methods before use. The results were more than 100 lM against tumor cell lines, suggesting
summarized in Table 1. that the substitution of 7-methoxy and 1-methyl groups
might contribute to their increased cytotoxic activities
As shown in Table 1, most studied compounds showed while a long chain alkoxy at 3-position might not be
significant cytotoxic activities against several human tu- preferable. Compared with the IC50 values of other com-
mor cell lines. 9-Substituted harmines, b-carbolines, b- pounds, the IC50 values of the compounds 2a, 2b, 2f,
carboline-3-carboxylates, and b-carboline-3-carboxylic and 2g was lower than 50 lM, which implicated that
acids (except 5d, 6f, and 7d) displayed more remarkable the alkyl and benzyl substituents had almost equal po-
cytotoxic activities than their parent compounds, respec- tency to enhance their cytotoxic activities in vitro.
tively, indicating that the introduction of an appropriate
alkyl or benzyl group into 9-position of b-carboline ring As for the harmine series, the compound 2b with an
system facilitates the increase of their cytotoxic activi- ethyl group at 9-position exhibited the highest cytotoxic
ties. activities (except for Lovo cell line). The compound 2a
having a methyl at position-9 showed selective cytotoxic
Among all the compounds investigated, 9-substituted activities against the screened tumor cell lines with the
harmine series demonstrated the highest cytotoxicities lowest IC50 value (8 lM) against Hela. The compounds
while 9-substituted butyl b-carboline-3-carboxylates 7a, 2f and 2g with a pentafluorobenzyl and a phenylpropyl
7c, 7d (except 7b), and their corresponding parent com- at position-9, respectively, demonstrated the prominent
4616 R. Cao et al. / Bioorg. Med. Chem. 12 (2004) 4613–4623

and broader spectrum of cytotoxic activities against all Table 2. Acute toxic effects of b-carboline derivatives in mice
six human tumor cell lines with IC50 values of all lower Compound Acute toxicity
than 50 lM. LD50 (mg/kg, 95% CL) Neurotoxic effect

In contrast to the harmine series, 9-substituted b-carbo- 1 59.00 (43.50–110.00) ++a

line series failed to exhibit obviously increased cytotoxi- 2b 24.25 (22.24–26.44) ++
2c 26.45 (23.91–29.26) ++
cities. The compound 3 having no substituent on ring
2e 147.82 (132.55–164.85) ++
system showed a good cytotoxic activity against PLA- 5a 70.61 (64.17–77.70) +
801 and Lovo cell lines. Compounds 4a–d displayed al- 6d 240.38 (211.38–273.50)

most equal cytotoxic activities while the compound 4d 8 135.22 (121.62–150.34)

showed higher and selective cytotoxic activity against 8c >500

Lovo cells with IC50 values of 48 lM. 8d 163.48 (141.56–188.76)

8e 219.19 (193.25–248.61)

Of all 9-substituted b-carboline-3-carboxylate deriva- a
Acute neurotoxic manifestation were denoted by Ô+Õ and Ô
Õ. A Ô+Õ
tives, the compound 6d having a benzyl at position-9 represents toxic reactions including tremble, twitch, jumping, teta-
displayed strong cytotoxic activities against HepG2, nus, and supination, Ô++Õ means the same reactions with more
Bel-7402, BGC-823, and Hela with IC50 values of 38, severity, while Ô
Õ means no such reaction.
45, 38, and 13 lM, respectively. However compounds
5d and 7d, having a benzyl at 9-position, had little or
even no cytotoxic effects on the tested cell lines, indicat-
ing that ethoxycarbonyl substituent was preferable for receiving the b-carboline derivatives via intraperitoneal
improving cytotoxic activities of this type of com- (i.p.) route. All the tested compounds resulted in acute
pounds. The compounds 5b, 6b, and 7b with an ethyl toxic manifestation. Of all the compounds investigated,
at position-9, respectively, displayed higher cytotoxic 1, 2b, 2c, 2e, and 5a caused remarkable acute toxic
activities against human tumor cell lines tested. Mean- effects including tremor, twitch, jumping, tetanus, and
while the compound 5b showed highly selective cytotox- supination. Death occurred mostly in high dosage group
ic effects against PLA-801 and Hela with IC50 values 25 within 1 min after administration and then reached a
and 17 lM, respectively. All these results, together with peak 1 h later. For survived animals, the tremor and
the cytotoxic activity of the compound 2b, confirmed jumping lasted for about 20 min and then relieved grad-
that the ethyl group at position-9 might play a crucial ually and returned to normal in the next day. Animals
role in eliciting distinct cytotoxic activities of these com- were drowsy and exhibited a decrease in locomotor
pounds. activity after the administration of the compound 8,
6d, 8d, and 8f. Death only occurred in high dosage
For the b-carboline-3-carboxylic acid series (8 and 8a– group 10–20 min after administration and reached a
e), the cytotoxic activities were also enhanced by intro- peak 1 h later. However, the compounds 8c caused no
duction of short alkyl or benzyl substituent into posi- obvious neurotoxic reaction and no death at tested dos-
tion-9 of the b-carboline ring. The compounds 8c and age. Autopsy of the animals that died in the course of
8e, having an n-butyl and a benzyl at positon-9, respec- experiment and the necropsy findings in surviving ani-
tively, showed remarkable cytotoxic activities against mals at the end of experimental period (14 days) revealed
Lovo cell lines with IC50 values of 11 and 13 lM, respec- no apparent changes in any organs. The results of toxi-
tively, furthermore the compound 8f exhibited the most cities suggested that an alkoxycarbonyl or carboxyl sub-
significant cytotoxic activity against Lovo cell lines with stituent at position-3 of the b-carboline ring might play
IC50 values 5 lM. a vital role in determining their decreased acute toxici-
ties and neurotoxicities, and perhaps the carboxyl sub-
A total analysis of the cytotoxic activities of b-carboline stituent is more favorable.
derivatives in vitro clearly suggested that (1) the b-carb-
oline nucleus might be an important basis for the design 2.4. Evaluation of antitumor activity
and synthesis of new antitumor drugs; (2) the cytotoxic
potencies of b-carboline derivatives depended upon the The tumor inhibition rates of the selected b-carboline
presence and location and nature of the subtituents, derivatives in mice bearing Lewis lung cancer and Sar-
which were introduced into the b-carboline ring. (3) coma180 (S180) were summarized in Table 3. Harmine
The cytotoxic activities of b-carboline derivatives were (compound 1) only showed a moderate antitumor effect
enhanced by the introduction of an appropriate substit- (34.1% and 15.3% against Lewis lung cancer and S180,
uent into position-9 of b-carboline ring; (4) the Lovo cell respectively), however the selected 9-substituted b-carb-
lines were more sensitive to the tested compounds than oline derivatives all exhibited more potent antitumor
other tumor cells. activities. Compounds 2c, 2d, 2e, 6d, and 8c displayed
remarkable antitumor activities with the tumor inhibi-
2.3. Assessment of acute toxicity tion rate of more than 40% against mice bearing Lewis
lung cancer and S180. Moreover the compounds 2e
The LD50 values and scores for neurotoxicity of the se- and 8c demonstrated the highest antitumor effect with
lected b-carboline derivatives in mice after administra- the tumor inhibition rate of 46.9% against mice bearing
tion were summarized in Table 2. Neurotoxicities were Lewis lung cancer among all the tested compounds. The
the principal acute toxic effects observed in mice after results indicated that short alkyl or benzyl substituent at
 R. Cao et al. / Bioorg. Med. Chem. 12 (2004) 4613–4623 4617

Table 3. Antitumor effects of b-carboline derivatives against mice thesis of more new b-carboline derivatives with
bearing Sarcoma 180 and Lewis lung carcinoma different substituents at other positions is needed.
Compound Tumor inhibition rate (%)
Lewis lung carcinoma Sarcoma180
4. Experimental
1 34.1 15.3
2b 42.0 37.6
2c 44.0 40.9
4.1. Materials
2e 46.9 45.2
5a 35.0 31.1 All reagents were purchased from commercial suppliers
6d 43.3 42.1 and were dried and purified when necessary. Compound
8 33.4 32.2 1 (Harmine) was extracted from Peganum multisectum
8c 46.9 43.1 Maxim, a plant indigenous to western China, according
8d 43.2 34.4 to the method by Duan et al.2 Compounds 3 and 5–8
8e 39.6 32.2 were synthesized from the starting material L -trypto-
CTX 88.6 87.5 phan according to the procedures described by Hagen
et al.19 and Lippke et al.18 with a slight modification,
respectively.

position-9 of the b-carboline ring facilitated the increase Melting points were determined in capillary tubes on an
of their antitumor activities. electrothermal PIF YRT-3 apparatus and without cor-
rection. UV spectra were measured on Shimadzu UV
2501PC Spectrometer. FAB-MS spectra were obtained
3. Conclusions from VG ZAB-HS spectrometer. FTIR were run on a
Bruker Equinox 55 Fourier Transformation Infarred
The present investigation reported for the first time that Spectrometer. 1H NMR spectra were recorded on a Var-
b-carboline derivatives had significant antitumor activi- ian INOVA 500NB spectrometer. Elemental analyses
ties in mice bearing Lewis lung cancer and Sarcoma 180 were carried out on an Elementar Vario EL CHNS Ele-
but also exhibited remarkable acute neurotoxicity in mental Analyzer. Silica gel F254 were used in analytical
experimental mice models. Alkyl or benzyl substituent thin-layer chromatography (TLC) and silica gel were
at position-9 of b-carboline ring might facilitate their used in column chromatography, respectively.
antitumor activities and minimize their acute toxicities
but not neurotoxicities. However alkoxycarbonyl or 4.1.1. 7-Methoxy-1,9-dimethyl-b-carboline (2a). A mix-
carboxyl substituent at position-3 played a crucial role ture of Harmine 1 (2.12 g, 10 mmol), anhydrous DMF
in determining their decreased acute toxicities and neu- (50 mL), and anhydrous THF (50 mL) was stirred at rt
rotoxicities. Furthermore, the compounds with appro- until clear, and then 60% NaH (0.6 g, 15 mmol) and
priate substituents both at positon-3 and 9 displayed iodomethane (2 mL, 30 mmol) were added and stirred
enhanced antitumor activities and decreased neurotoxi- at rt for 30 min. Later the mixture was evaporated in re-
cities simultaneously. The compound 8c, having an n- duced pressure. The resulting solution was poured into
butyl and a carboxyl at position-9 and 3, respectively, H2O (100 mL), and extracted with ethyl acetate
exhibited the highest antitumor effect and the lowest (3 · 150 mL). The organic phase was washed with water
acute neurotoxicity and was found to be the most prom- and brine, then dried over anhydrous sodium sulfate, fil-
ising leading compound for further investigation. These tered, and evaporated. The oil obtained was purified by
data suggested that (1) appropriate substituents at both silica column chromatography with ethyl acetate as the
position-9 and 3 of b-carboline derivatives might play a eluent. Upon recrystallization, white crystals of 2a were
crucial role in determining their enhanced antitumor obtained (1.8 g, 80%), mp 121–123 C (from ether);
activities and decreased acute toxicities and neurotoxic FAB-MS m/e 227 (M+1); UV kmax 345, 332, 302, 264,
effects; (2) the b-carboline derivatives have the potential 244, 214 nm; IR (KBr): 3380, 2744, 1628, 1570, 1469,
to be used as antitumor drug leads. 1348, 1249, 1152, 1045, 810 cm
1; 1H NMR (500 MHz,
CDCl3): 8.24–8.25 (1H, m, H-5), 7.93–7.96 (1H, m, H-
Although the antitumor effects of the studied b-carbo- 3), 7.69–7.71 (1H, m, H-4), 6.86–6.89 (1H, m, H-8),
line derivatives presented here remained relatively mod- 6.81–6.83 (1H, m, H-6), 4.04–4.07 (3H, m, NCH3),
est, it should be noted that the acute neurotoxicities of 3.94–3.95 (3H, m, OCH3), 3.04–3.05 (3H, m, CH3).
some compounds decreased dramatically by introduc- Anal. Calcd for C14H14N2O: C, 74.33; H, 6.19; N,
tion of an alkoxycarbonyl or carboxyl substituent into 12.39. Found: C, 74.21; H, 6.37; N, 12.31.
position-3 of the b-carboline ring. This investigation
and finding would be helpful to further design and de- 4.1.2. 7-Methoxy-9-ethyl-1-methyl-b-carboline (2b). A
velop more potent antitumor agents together with low mixture of Harmine 1 (2.12 g, 10 mmol), anhydrous
neurotoxicities. Further investigations are in progress DMF (50 mL), and anhydrous THF (50 mL) was stirred
in our laboratory to elucidate the molecular mechanisms at rt until clear, and then 60% NaH (0.6 g, 15 mmol) and
involved in antitumor activities and neurotoxicities of b- iodoethane (2.5 mL, 30 mmol) were added. Later the
carboline derivatives. To acquire more information mixture was treated in a manner similar to that de-
about the structural requirements for enhancing antitu- scribed for 2a to afford 2b (2.0, 83%), mp 99–101 C
mor activities and minimizing neurotoxicities, the syn- (from ether); FAB-MS m/e 241 (M+1); UV kmax 345,
4618 R. Cao et al. / Bioorg. Med. Chem. 12 (2004) 4613–4623

332, 303, 265, 244, 213 nm; IR (KBr): 3362, 3128, 1622, H-8, H-6, Ar–H), 6.98–7.00 (1H, d, J = 7.0 Hz, Ar–H),
1565, 1451, 1346, 1217, 1136, 812 cm
1; 1H NMR 6.90–6.92 (1H, m, Ar–H), 6.76 (1H, s, Ar–H), 5.75
(500 MHz, CDCl3): 8.26–8.27 (1H, d, J = 5 Hz, H-5), (2H, s, NCH2Ar), 3.85 (3H, s, OCH3), 2.88 (3H, s,
7.96–7.98 (1H, d, J = 5 Hz, H-3), 7.74–7.75 (1H, d, CH3). Anal. Calcd for C20H18N2O: C, 79.47; H, 5.96;
J = 4.5 Hz, H-4), 6.86–6.90 (2H, m, H-8, H-6), 4.52– N, 9.27. Found: C, 79.29; H, 6.19; N, 9.21.
4.57 (2H, m, CH2CH3), 3.95 (3H, s, OCH3), 3.05 (3H,
s, CH3), 1.43–1.46 (3H, m, CH2CH3). Anal. Calcd for 4.1.6. 9-(2 0 ,3 0 ,4 0 ,5 0 ,6-Pentafluoro)benzyl-7-methoxy-1-
C15H16N2O: C, 75.00; H, 6.67; N, 11.67. Found: C, methyl-b-carboline (2f). A mixture of Harmine 1
74.89; H, 6.87; N, 11.59. (0.53 g, 2.5 mmol), anhydrous DMF (15 mL), and anhy-
drous THF (15 mL) was stirred at rt until clear, and then
4.1.3. 7-Methoxy-9-butyl-1-methyl-b-carboline (2c). A 60% NaH (0.15 g, 3.8 mmol) and a-bromo-2,3,4,5,6-pen-
mixture of Harmine 1 (2.12 g, 10 mmol), anhydrous tafluorotoluene (0.7 mL, 4.5 mmol) were added. Later
DMF (50 mL), and anhydrous THF (50 mL) was stirred the mixture was treated in a manner similar to that
at rt until clear, and then 60% NaH (0.6 g, 15 mmol) and described for 2a to afford 2f (0.64 g, 65%), mp 173–
n-butyl iodide (6 mL, 50 mmol) were added and refluxed 174 C (from ether); FAB-MS m/e 393 (M+1); UV
for 1 h. Later the resulting mixture was treated in a man- kmax 338, 325, 300, 241, 209 nm; IR (KBr): 2961, 1622,
ner similar to that described for 2a to afford 2c (2.1, 1502, 1446, 1256, 1026, 816 cm
1; 1H NMR (500 MHz,
78%), mp 104–105 C (from ether); FAB-MS m/e 269 CDCl3): 8.32–8.33 (1H, d, J = 5 Hz, H-5), 7.94–7.95
(M+1); UV kmax 346, 334, 303, 265, 244, 213 nm; IR (1H, d, J = 7.5 Hz, H-3), 7.72–7.73 (1H, d, J = 5.5 Hz,
(KBr): 3428, 2959, 2927, 1621, 1563, 1497, 1448, 1356, H-4), 7.26 (1H, s, H-8), 6.87–6.89 (1H, m, H-6), 5.85
1243, 1197, 1137, 812 cm
1; 1H NMR (500 MHz, (2H, s, CH2Ar), 3.88 (3H, s, OCH3), 3.05 (3H, s,
CDCl3): 8.26–8.28 (1H, d, J = 5.5 Hz, H-5), 7.95–7.97 CH3). Anal. Calcd for C20H13F5N2O: C, 61.22; H,
(1H, d, J = 9 Hz, H-3), 7.71–7.72 (1H, d, J = 5 Hz, H-4), 3.32; N, 7.14. Found: C, 61.09; H, 3.46; N, 7.06.
6.85–6.88 (2H, m, H-8, H-6), 4.43–4.46 (2H, m,
J = 8 Hz, CH2CH2CH2CH3), 3.94 (3H, s, OCH3), 3.01 4.1.7. 9-Phenylpropyl-7-methoxy-1-methyl-b-carboline
(3H, s, CH3), 1.78–1.84 (2H, m, CH2CH2CH2CH3), (2g). A mixture of Harmine 1 (1.05 g, 5 mmol), anhy-
1.41–1.48 (2H, m, CH2CH2CH2CH3), 0.97–1.00 (3H, drous DMF (25 mL), and anhydrous THF (25 mL) were
m, CH2CH2CH2CH3). Anal. Calcd for C17H20N2O: C, stirred at rt until clear, and then 60% NaH (0.3 g,
76.12; H, 7.46; N, 10.45. Found: C, 75.96; H, 7.69; N, 7.5 mmol) and 1-bromo-3-phenylpropane (3 mL,
10.53. 20 mmol) were added and refluxed for 3 h. Later the mix-
ture was treated in a manner similar to that described
4.1.4. 7-Methoxy-9-hydroxyethyl-1-methyl-b-carboline for 2a to afford 2g (0.84 g, 51%), mp 117–118 C (from
(2d). A mixture of Harmine 1 (1.05 g, 5.0 mmol) and ether); FAB-MS m/e 331 (M+1); UV kmax 346, 332,
anhydrous DMF (25 mL), and anhydrous THF 302, 265, 244, 211 nm; IR (KBr): 2995, 2931, 1623,
(25 mL) was stirred at rt until clear, and then 60% 1563, 1449, 1238, 1156, 1043, 801 cm
1; 1H NMR
NaH (0.3 g, 7.5 mmol) and 2-iodoethanol (3 mL, (500 MHz, CDCl3): 8.25–8.26 (1H, d, J = 5 Hz, H-5),
40 mmol) were added and refluxed for 5 h. Then the 7.92–7.94 (1H, d, J = 8.5 Hz, H-3), 7.69–7.70 (1H, d,
resulting mixture was treated in a manner similar to that J = 5 Hz, H-4), 7.29–7.32 (2H, m, H-8, H-6), 7.20–7.25
described for 2a to afford 2d (0.7 g, 54%), mp 204–206 C (3H, m, Ar–H), 6.84–6.86 (1H, m, Ar–H), 6.63–6.64
(from ether); FAB-MS m/e 257 (M+1); UV kmax 328, (1H, m, Ar–H), 4.42–4.45 (2H, m, NCH2CH2CH2Ar),
304, 249, 211 nm; IR (KBr) 3295, 2696, 1629, 1569, 3.84–3.85 (3H, s, OCH3), 2.88 (3H, s, CH3), 2.74–2.77
1467, 1352, 1155, 1051, 810 cm
1; 1H NMR (500 MHz, (2H, m, NCH2CH2CH2Ar), 2.12–2.18 (2H, m,
CDCl3): 8.15–8.16 (1H, d, J = 4 Hz, H-5), 7.93–7.94 NCH2CH2CH2Ar). Anal. Calcd for C22H22N2O: C,
(1H, d, J = 3.5 Hz, H-3), 7.63–7.64 (1H, d, J = 5.0 Hz, 80.00; H, 6.67; N, 8.48. Found: C, 79.87; H, 6.82; N,
H-4), 6.88–6.95 (2H, m, H-8, H-6), 4.66–4.71 (2H, t, 8.39.
J = 5.5 Hz, NCH2CH2OH), 4.06–4.08 (2H, m,
NCH2CH2OH), 3.94 (3H, s, OCH3), 2.99 (3H, s, 4.1.8. 9-Methyl-b-carboline (4a). A mixture of norhar-
CH3). Anal. Calcd for C15H16N2O2: C, 70.31; H, 6.25; man 3 (1.68 g, 10 mmol) and anhydrous DMF (50 mL)
N, 10.94. Found: C, 70.13; H, 6.46; N, 10.87. was stirred at rt till clear. Then 60% NaH (0.6 g,
15 mmol) and iodomethane (2 mL, 30 mmol) were
4.1.5. 9-Benzyl-7-methoxy-1-methyl-b-carboline (2e). A added, and the mixture was stirred at rt for 30 min. La-
mixture of Harmine 1 (2.12 g, 10 mmol), anhydrous ter the resulting mixture was poured into H2O (150 mL),
DMF (50 mL), and anhydrous THF (50 mL) was stirred and extracted with ethyl acetate (3 · 150 mL). The organ-
at rt until clear, and then 60% NaH (0.6 g, 15 mmol) and ic phase was washed with water and brine, then dried
benzyl bromide (5 mL, 40 mmol) were added. Later the over anhydrous sodium sulfate, filtered, and evaporated.
mixture was treated in a manner similar to that de- The oil obtained was purified by silica column chroma-
scribed for 2a to afford 2e (2.2, 67%), mp 131–133 C tography with petroleum ether–acetone (2:1) as the elu-
(from ether); FAB-MS m/e 303 (M+1); UV kmax 343, ent. Upon recrystallization, white crystals of 4a were
330, 301, 244, 210 nm; IR (KBr): 3421, 2958, 1620, obtained (1.4 g, 77%), mp 108–109 C (from petroleum
1565, 1498, 1447, 1361, 1256, 1197, 1172, 1044, ether–acetone); FAB-MS m/e 183 (M+1); UV kmax
825 cm
1; 1H NMR (500 MHz, CDCl3): 8.29–8.30 (1H, 360, 346, 289, 236, 216 nm; IR (KBr): 2509, 1638,
d, J = 5.5 Hz, H-5), 8.01–8.02 (1H, d, J = 8.5 Hz, H-3), 1504, 1335, 1259, 835 cm
1; 1H NMR (500 MHz,
7.80–7.81 (1H, d, J = 5.5 Hz, H-4), 7.23–7.30 (3H, m, CDCl3): 8.88 (1H, s, H-4), 8.46–8.47 (1H, d, J = 5 Hz,
 R. Cao et al. / Bioorg. Med. Chem. 12 (2004) 4613–4623 4619

H-1), 8.13–8.14 (1H, d, J = 6 Hz, H-8), 7.94–7.95 (1H, d, added. Later the mixture was stirred at rt for 30min.
J = 5 Hz, H-3), 7.59–7.62 (1H, m, H-5), 7.45–7.46 (1H, d, The resulting mixture was poured into iced-water
J = 8.5 Hz, H-6), 7.25–7.30 (1H, m, H-7), 3.93 (3H, s, (150 mL) and extracted with ethyl acetate (3· 100mL).
CH3). Anal. Calcd for C12H10N2: C, 79.12; H, 5.49; N, The combined organic phase was washed with water and
15.38. Found: C, 78.93; H, 5.71; N, 15.45. brine. Then dried over anhydrous sodium sulfate, filtered,
and evaporated. The yellow oil obtained was purified by
4.1.9. 9-Ethyl-b-carboline (4b). A mixture of norharman silica column chromatography with ethyl acetate as the
3 (1.68 g, 10 mmol) and anhydrous DMF (50 mL) was eluent. After that the solid was collected, it was recrystal-
stirred at rt till clear, and then 60% NaH (0.6 g, 15 mmol) lized from ethyl ether to give a white crystals (1.8 g, 75%),
and iodoethane (2.5 mL, 30 mmol) were added. Later the mp 215–216C (from ether); FAB-MS m/e 241 (M+1); UV
mixture was treated following the method described for kmax 358, 343, 307, 272, 236, 219 nm; IR (KBr): 3387, 2548,
4a to afford yellow oil 4b (1.5 g, 76%), FAB-MS m/e 197 2056, 1730, 1631, 1334, 1282, 1207 cm
1; 1H NMR
(M+1); UV kmax 365, 342, 291, 239, 218 nm; IR(KBr): (500 MHz, CDCl3): 8.94 (1H, s, H-4), 8.88 (1H, s, H-1),
2485, 2022, 1633, 1500, 1462, 1355, 1239, 831 cm
1; 1H 8.20–8.21 (1H, d, J = 7.5Hz, H-8), 7.65–7.68 (1H, m, Ar–
NMR (500 MHz, CDCl3): 8.84 (1H, s, H-4), 8.42–8.43 H), 7.50–7.52 (1H, d, J= 8Hz, Ar–H), 7.36–7.39 (1H, m,
(1H, d, J = 5 Hz, H-1), 8.04–8.05 (1H, d, J = 8 Hz, H-8), J = 8 Hz, Ar–H), 4.06 (3H, s, OCH3), 4.00 (3H, s,
7.85–7.86 (1H, d, J = 5 Hz, H-3), 7.50–7.53 (1H, m, H- NCH3). Anal. Calcd for C14H12N2O2: C, 70.00; H, 5.00;
5), 7.34–7.36 (1H, d, J = 8 Hz, H-6), 7.20–7.23 (1H, m, N, 11.67. Found: C, 69.83; H, 5.23; N, 11.59.
H-7), 4.26–4.30 (2H, m, CH2CH3), 1.35–1.38 (3H, m,
CH2CH3). Anal. Calcd for C13H12N2: C, 79.59; H, 4.1.13. Methyl 9-ethyl-b-carboline-3-carboxylate (5b). A
6.12; N, 14.29. Found: C, 79.32; H, 6.43; N, 14.13. mixture of 5 (2.26 g, 10 mmol) and anhydrous DMF
(60 mL) was stirred at rt for 10 min, then 60% NaH
4.1.10. 9-Butyl-b-carboline (4c). A mixture of norharman (0.6 g, 2 mmol) and iodoethane (2.5 mL, 30 mmol) were
3 (1.68 g, 10 mmol) and anhydrous DMF (50 mL) was added. Later the mixture was treated in a manner similar
stirred at rt till clear, and then 60% NaH (0.6 g, 15 mmol) to that described for 5a to afford white crystals 5b (2.0 g,
and n-butyl iodide (6 mL, 50 mmol) were added and ref- 79%), mp 155–156 C (from ether); FAB-MS m/e 255
luxed for 1 h. Later the mixture was treated following (M+1); UV kmax 358, 345, 306, 273, 236, 205 nm; IR
the method described for 4a to afford white crystals 4c (KBr): 3248, 1694, 1629, 1337, 1250 cm
1; 1H NMR
(1.6 g, 71%), mp 108–109 C (from light petroleum (500 MHz, CDCl3): 8.90–9.00 (2H, m, H-4, H-1), 8.21–
ether–acetone); FAB-MS m/e 225 (M+1); UV kmax 8.23 (1H, d, J = 8 Hz, H-8), 7.65–7.68 (1H, m, H-5),
362, 348, 290, 237, 217 nm; IR (KBr): 2525, 2030, 7.52–7.54 (1H, d, J = 8 Hz, H-6), 7.36–7.39 (1H, m, H-
1632, 1500, 1458, 1355, 1235, 823 cm
1; 1H NMR 7), 4.51 (2H, s, NCH2CH3), 4.07 (3H, s, OCH3), 1.52
(500 MHz, CDCl3): 8.86 (1H, s, H-4), 8.43–8.44 (1H, (3H, s, NCH2CH3). Anal. Calcd for C15H14N2O2: C,
d, J = 5.5 Hz, H-1), 8.06–8.08 (1H, d, J = 8 Hz, H-8), 70.87; H, 5.51; N, 11.02. Found: C, 70.65; H, 5.68; N,
7.88–7.89 (1H, d, J = 4.5 Hz, H-3), 7.52–7.55 (1H, m, 10.93.
H-5), 7.38–7.40 (1H, d, J = 8.5 Hz, H-6), 7.21–7.25 (1H,
m, H-7), 4.25–4.28 (2H, m, CH2CH2CH2CH3), 4.1.14. Methyl 9-butyl-b-carboline-3-carboxylate (5c). A
1.79–1.85 (2H, m, CH2CH2CH2CH3), 1.30–1.38 (2H, mixture of 5 (2.26 g, 10 mmol) and anhydrous DMF
m, CH2CH2CH2CH3), 0.86–0.91 (3H, m, CH2CH2- (60 mL) was stirred at rt for 10 min, then 60% NaH
CH2CH3). Anal. Calcd for C15H16N2: C, 80.36; H, (0.6 g, 2 mmol) and n-butyl iodide (6 mL, 50 mmol) were
7.14; N, 12.50. Found: C, 80.15; H, 7.35; N, 12.39. added and refluxed for 1 h. Later the mixture was treated
in a manner similar to that described for 5a to afford
4.1.11. 9-Benzyl-b-carboline (4d). A mixture of norhar- white crystals 5c (2.3 g, 82%), mp 181–183 C (from
man 3 (1.68 g, 10 mmol) and anhydrous DMF (50 mL) petroleum ether–ethyl ether 1:2); FAB-MS m/e 283
was stirred at rt till clear, and then 60% NaH (0.6 g, (M+1); UV kmax 358, 344, 306, 273, 236, 222 nm; IR
15 mmol) and benzyl bromide (5 mL, 40 mmol) were (KBr): 2959, 1735, 1625, 1361, 1246 cm
1; 1H NMR
added and refluxed for 3 h. Later the mixture was trea- (500 MHz, CDCl3): 8.94 (1H, s, H-4), 8.89 (1H, s, H-1),
ted following the method described for 4a to afford 8.19–8.21 (1H, d, J = 7 Hz, H-8), 7.62–7.65 (1H, m, H-
white crystals 4d (1.8 g, 69%), mp 118–120 C (from 5), 7.50-7.52 (1H, d, J = 7.5 Hz, H-6), 7.34–7.37 (1H, m,
ether); FAB-MS m/e 259 (M+1); UV kmax 358, 344, H-7), 4.41–4.44 (2H, m, CH2CH2CH2CH3), 4.06 (3H,
289, 237, 213 nm; IR (KBr): 3023, 1619, 1448, 1332, s, OCH3), 1.87–1.93 (2H, m, CH2CH2CH2CH3), 1.35–
1258, 821 cm
1; 1H NMR (500 MHz, CDCl3): 8.84 1.42 (2H, m, CH2CH2CH2CH3), 0.93–0.96 (3H, m,
(1H, s, H-4), 8.48 (1H, s, H-1), 8.15–8.16 (1H, d, CH2CH2CH2CH3). Anal. Calcd for C17H18N2O2: C,
J = 8 Hz, H-8), 7.98 (1H, s, H-3), 7.53–7.56 (1H, m, H- 72.34; H, 6.38; N, 9.93. Found: C, 72.21; H, 6.57; N,
5), 7.41–7.43 (1H, d, J = 8 Hz, H-6), 7.13–7.31 (6H, m, 9.86.
J = 8 Hz, H-7, Ar–H), 5.55 (2H, s, CH2Ar). Anal. Calcd
for C18H14N2: C, 83.72; H, 5.43; N, 10.85. Found: C, 4.1.15. Methyl 9-benzyl-b-carboline-3-carboxylate (5d). A
83.57; H, 5.69; N, 10.76. mixture of 5 (2.26 g, 10 mmol) and anhydrous DMF
(60 mL) was stirred at rt for 10 min, then 60% NaH
4.1.12. Methyl 9-methyl-b-carboline-3-carboxylate (5a). A (0.6 g, 2 mmol) and benzyl bromide (5 mL, 40 mmol) were
mixture of 5 (2.26 g, 10 mmol) and anhydrous DMF added and refluxed for 3 h. Later the mixture was treated
(60 mL) was stirred at rt for 10 min, then 60% NaH in a manner similar to that described for 5a to afford white
(0.6 g, 2 mmol) and iodomethane (2 mL, 30mmol) were crystals 5d (2.3 g, 73%), mp 187–188C (from petroleum
4620 R. Cao et al. / Bioorg. Med. Chem. 12 (2004) 4613–4623

ethyl ether); FAB-MS m/e 317 (M+1); UV kmax 356, 341, 4.1.19. Ethyl 9-benzyl-b-carboline-3-carboxylate (6d). A
304, 272, 235, 205 nm; IR (KBr): 3026, 2945, 1731, 1622, mixture of 5 (2.4 g, 10 mmol) and anhydrous DMF
1335, 1242 cm
1; 1H NMR (500MHz, CDCl3): 8.89– (60 mL) was stirred rt for 10 min, then 60% NaH (0.6 g,
8.90 (2H, d, J = 4 Hz, H-4, H-1), 8.21–8.22 (1H, d, 2 mmol) and benzyl bromide (5 mL, 40 mmol) were
J = 8 Hz, H-8), 7.58–7.61 (1H, m, H-5), 7.47–7.48 (1H, d, added and refluxed for 3 h. Later the mixture was treated
J = 8.5 Hz, H-6), 7.35–7.38 (1H, m, H-7), 7.24–7.28 (3H, in a manner similar to that described for 5a to afford
m, Ar–H), 7.13–7.15 (2H, m, Ar–H), 5.60 (2H, s, CH2Ar), white crystals 6d (2.4 g, 70%), mp 126–127 C (from ethyl
4.05 (3H, s, OCH3). Anal. Calcd for C20H16N2O2: C, ether); FAB-MS m/e 331 (M+1); UV kmax 356, 342, 305,
75.95; H, 5.06; N, 8.86. Found: C, 75.75; H, 5.29; N, 8.78. 273, 234 nm; IR (KBr): 3425, 3060, 3027, 2974, 1723,
1622, 1335, 1214 cm
1; 1H NMR (500 MHz, CDCl3):
4.1.16. Ethyl 9-methyl-b-carboline-3-carboxylate (6a). A 8.91 (2H, m, H-4, H-1), 8.23–8.25 (1H, d, J = 8 Hz, H-
mixture of 6 (2.4 g, 10 mmol) and anhydrous DMF 8), 7.59–7.62 (1H, m, H-5), 7.49–7.50 (1H, d, J = 8 Hz,
(60 mL) was stirred at rt for 10 min, then 60% NaH H-6), 7.36–7.39 (1H, m, H-7), 7.25–7.27 (3H, s, Ar–H),
(0.6 g, 15mmol) and iodomethane (2 mL, 30 mmol) were 7.14–7.16 (2H, m, Ar–H), 5.62 (2H, s, CH2Ar), 4.51–
added. Later the mixture was treated in a manner similar 4.55 (2H, m, OCH2CH3), 1.47–1.50 (3H, m, OCH2CH3).
to that described for 5a to afford white crystals 6a (1.9 g, Anal. Calcd for C21H18N2O2: C, 76.36; H, 5.45; N, 8.48.
75%), mp 139–140 C (from ethyl ether); FAB-MS m/e Found: C, 76.19; H, 5.67; N, 8.38.
255 (M+1); UV kmax 358, 342, 306, 272, 236, 219 nm;
IR (KBr): 3446, 3398, 2603, 1721, 1628, 1328, 4.1.20. Ethyl 9-(2 0 ,3 0 ,4 0 ,5 0 ,6 0 -pentafluoro)benzyl-b-carbo-
1280cm
1; 1H NMR (500 MHz, CDCl3): 8.94 (1H, s, line-3-carboxylate (6e). A mixture of 5 (2.4 g,10 mmol)
H-4), 8.86 (1H, s, H-1), 8.19–8.20 (1H, d, J = 7.5 Hz, H- and anhydrous DMF(60 mL) was stirred at rt for
8), 7.66–7.67 (1H, m, H-5), 7.49–7.51 (1H, d, J = 8.5 Hz, 10 min, then 60% NaH (0.6 g, 15 mmol) and a-bromo-
H-6), 7.35–7.37 (1H, m, H-7), 4.52–4.56 (2H, m, 2,3,4,5,6-pentafluorotoluene (3 mL, 20 mmol) were
OCH2CH3), 3.98 (3H, s, NCH3), 1.48–1.51 (3H, s, added. Later the mixture was treated in a manner simi-
OCH2CH3). Anal. Calcd for C15H14N2O2: C, 70.87; H, lar to that described for 5a to afford white crystals 6d
5.51; N, 11.02. Found: C, 70.73; H, 5.73; N, 11.09. (2.6 g, 62%), mp 153–154 C (from ethyl ether); FAB-
MS m/e 421 (M+1); UV kmax 350, 335, 299, 271, 236,
4.1.17. Ethyl 9-ethyl-b-carboline-3-carboxylate (6b). A 217 nm; IR (KBr): 3399, 3065, 2987, 1709, 1627, 1337,
mixture of 6 (2.4 g, 10 mmol) and anhydrous DMF 1245 cm
1; 1H NMR (500 MHz, CDCl3): 9.07 (1H, s,
(60 mL) was stirred rt for 10 min, then 60% NaH H-4), 8.86 (1H, s, H-1), 8.19–8.21 (1H, d, J = 8 Hz, H-
(0.6 g, 2 mmol) and iodoethane (2.5 mL, 30 mmol) were 8), 7.65–7.68 (1H, m, H-5), 7.59–7.61 (1H, d,
added. Later the mixture was treated in a manner simi- J = 8.5 Hz, H-6), 7.38–7.41 (1H, m, H-7), 5.67 (2H, s,
lar to that described for 5a to afford white crystals 6b CH2Ar), 4.52–4.56 (2H, m, OCH2CH3), 1.49–1.51 (3H,
(1.8 g, 67%), mp 117–118 C (from ethyl ether); FAB- m, OCH2CH3). Anal. Calcd for C21F5H13N2O2: C,
MS m/e 269 (M+1); UV kmax 359, 344, 306, 273, 237, 60.00; H, 3.10; N, 6.67. Found: C, 59.87; H, 3.29; N,
221 nm; IR (KBr): 3413, 2984, 1717, 1633, 1334, 6.58.
1257 cm
1; 1H NMR (500 MHz, CDCl3): 8.93 (1H, s,
H-4), 8.86 (1H, s, H-1), 8.18–8.21 (1H, d, J = 8 Hz, H- 4.1.21. Ethyl 9-phenylpropyl-b-carboline-3-carboxylate
8), 7.62–7.64 (1H, m, H-5), 7.48–7.50 (1H, d, J = 8 Hz, (6f). A mixture of 5 (2.4 g, 10 mmol) and anhydrous
H-6), 7.33–7.36 (1H, m, H-7), 4.42–4.56 (4H, m, DMF (60 mL) was stirred at rt for 10 min, then 60%
OCH2CH3, NCH2CH3), 1.46–1.52 (6H, m, OCH2CH3, NaH (0.6 g, 2 mmol) and 1-bromo-3-phenylpropane
NCH2CH3). Anal. Calcd for C16H16N2O2: C, 71.64; (6 mL, 40 mmol) were added and refluxed for 3 h. Later
H, 5.97; N, 10.45. Found: C, 71.41; H, 6.21; N, 10.38. the mixture was treated in a manner similar to that de-
scribed for 5a to afford white crystals 6e (2.0 g, 56%),
4.1.18. Ethyl 9-butyl-b-carboline-3-carboxylate (6c). A mp 140–142 C (from ethyl ether); FAB-MS m/e 359
mixture of 6 (2.4 g, 10 mmol) and anhydrous DMF (M+1); UV kmax 358, 343, 306, 273, 236, 217 nm; IR
(60 mL) was stirred rt for 10 min, then 60% NaH (KBr): 3026, 2983, 2933, 1724, 1622, 1333, 1246 cm
1;
1
(0.6 g, 15 mmol) and n-butyl iodide (6 mL, 50 mmol) were H NMR (500 MHz, CDCl3): 8.89 (2H, m, H-4, H-1),
added and refluxed for 1 h. Later the mixture was trea- 8.20–8.22 (1H, d, J = 7.5 Hz, H-8), 7.61–7.64 (1H, m,
ted in a manner similar to that described for 5a to afford H-5), 7.41–7.42 (1H, m, H-6), 7.34–7.37 (1H, m, H-7),
white crystals 6c (2.3 g, 78%), mp 76–77 C (from petro- 7.26–7.30 (2H, m, Ar–H), 7.19–7.22 (1H, m, Ar–H),
leum ether–ethyl ether 1:2); FAB-MS m/e 297 (M+1); 7.13–7.15 (2H, m, Ar–H), 4.52–4.57 (2H, m,
UV kmax 359, 343, 307, 273, 235, 221 nm; IR (KBr): NCH2CH2CH2Ar), 4.41–4.44 (2H, m, NCH2CH2-
3438, 2956, 1731, 1623, 1333, 1242 cm
1; 1H NMR CH2Ar), 2.70–2.73 (2H, m, OCH2CH3), 2.24–2.30 (2H,
(500 MHz, CDCl3): 8.98 (1H, s, H-4), 8.89 (1H, s, H- m, NCH2CH2CH2Ar), 1.49–1.52 (3H, m, OCH2CH3).
1), 8.21–8.23 (1H, d, J = 8 Hz, H-8), 7.63–7.66 (1H, m, Anal. Calcd for C23H22N2O2: C, 77.09; H, 6.15; N,
H-5), 7.51–7.53 (1H, d, J = 8.5 Hz, H-6), 7.36–7.38 (1H, 7.82. Found: C, 76.88; H, 6.38; N, 7.75.
m, H-7), 4.52–4.56 (2H, m, OCH2CH3), 4.42–4.44 (2H,
m, CH2CH2CH2CH3), 1.88–1.94 (2H, m, CH2CH2- 4.1.22. Butyl 9-methyl-b-carboline-3-carboxylate (7a). A
CH2CH3), 1.49–1.52 (3H, m, OCH2CH3), 1.35–1.42 mixture of 7 (2.68 g, 10 mmol) and anhydrous DMF
(2H, m, CH2CH2CH2CH3), 0.93–0.96 (3H, m, CH2CH2- (80 mL) was stirred at rt for 10 min, then 60% NaH
CH2CH3). Anal. Calcd for C18H20N2O2: C, 72.97; H, (0.6 g, 15 mmol) and iodomethane (2 mL, 30 mmol) were
6.76; N, 9.46. Found: C, 72.78; H, 6.98; N, 9.37. added and stirred at rt for 30 min. Later the mixture was
 R. Cao et al. / Bioorg. Med. Chem. 12 (2004) 4613–4623 4621

treated in a manner similar to that described for 5a to af- (from ethyl ether); FAB-MS m/e 359 (M+1); UV kmax
ford white crystals 7a (2.0 g, 70%), mp 235–238 C (from 356, 342, 305, 273, 235, 205 nm; IR (KBr): 3051, 2959,
ethyl ether); FAB-MS m/e 283 (M+1); UV kmax 358, 343, 2930, 2869, 1700, 1620, 1362, 1246 cm
1; 1H NMR
305, 272, 236, 219 nm; IR (KBr): 3402, 2956, 2869, 1726, (500 MHz, CDCl3): 8.89 (1H, s, H-4), 8.85 (1H, s, H-
1625, 1333, 1238 cm
1; 1H NMR (500 MHz, CDCl3): 1), 8.19–8.20 (1H, d, J = 8 Hz, H-8), 7.56–7.59 (1H, m,
8.94 (1H, s, H-4), 8.83 (1H, s, H-1), 8.18–8.20 (1H, d, H-5), 7.45–7.46 (1H, d, J = 8.5 Hz, H-6), 7.33–7.36 (1H,
J = 7.5 Hz, H-8), 7.63–7.66 (1H, m, H-5), 7.48–7.50 m, H-7), 7.22–7.26 (3H, m, ArH), 7.11–7.13 (2H, m,
(1H, d, J = 8.5 Hz, H-6), 7.34–7.37 (1H, m, H-7), 4.46– ArH), 5.56 (2H, s, CH2Ar), 4.45–4.48 (2H, m,
4.49 (2H, m, OCH2CH2CH2CH3), 3.97 (3H, s, NCH3), OCH2CH2CH2CH3), 1.82–1.88 (2H, m, OCH2-
1.84–1.90 (2H, m, J = 7.0 Hz, OCH2CH2CH2CH3), CH2CH2CH3), 1.47–1.54 (2H, m, OCH2CH2CH2CH3),
1.48–1.56 (2H, m, OCH2CH2CH2CH3), 0.99–1.02 (3H, 0.98–1.01 (3H, m, OCH2CH2CH2CH3). Anal. Calcd
m, OCH2CH2CH2CH3). Anal. Calcd for C17H18N2O2: for C23H22N2O2: C, 77.09; H, 6.15; N, 7.82. Found: C,
C, 72.34; H, 6.38; N, 9.93. Found: C, 72.25; H, 6.58; 76.89; H, 6.35; N, 7.75.
N, 9.85.
4.1.26. General procedure for the preparation of b-carb-
4.1.23. Butyl 9-ethyl-b-carboline-3-carboxylate (7b). A oline-3-carboxylic acids 8a–e. A mixture of the corre-
mixture of 6 (2.4 g, 10 mmol) and anhydrous DMF sponding b-carboline-3-carboxylate (6a–f, 10 mmol),
(60 mL) was stirred at rt for 10 min, then 60% NaH NaOH (50 mmol), ethanol (50 mL), and H2O (100 mL)
(0.6 g, 15 mmol) and iodoethane (2.5 mL, 30 mmol) were was refluxed for 1 h, and the ethanol was removed on
added. Later the mixture was treated in a manner simi- the rotary evaporator. The mixture was neutralized
lar to that described for 5a to afford white crystals 6b (pH 5) with 5 M HCl and cooled. The precipitate was
(2.0 g, 65%), mp 76–77 C (from ethyl ether); FAB-MS collected, washed well with H2O, and dried in vacuo.
m/e 297 (M+1); UV kmax 360, 345, 306, 273, 240,
220 nm; IR (KBr): 3053, 2963, 2401, 1999, 1722, 1627, 4.1.27. 9-Methyl-b-carboline-3-carboxylic acid (8a). Yel-
1337, 1269 cm
1; 1H NMR (500 MHz, CDCl3): 9.02 low solid was obtained (2.32 g, 99%). Mp 267–269 C;
(1H, s, H-4), 8.86 (1H, s, H-1), 8.20–8.22 (1H, d, FAB-MS m/e 227 (M+1); UV kmax 384, 360, 273, 241,
J = 8 Hz, H-8), 7.63–7.67 (1H, m, H-5), 7.51–7.53 (1H, 217 nm; IR (KBr): 3250–2100, 1716, 1630, 1405,
d, J = 7.5 Hz, H-6), 7.35–7.38 (1H, m, H-7), 4.47–4.51 1200 cm
1; 1H NMR (500 MHz, DMSO-d6) d 12.15
(4H, m, OCH2CH2CH2CH3, NCH2CH3), 1.84–1.90 (1H, s, COOH), 9.19 (1H, s, H-4), 9.12 (1H, s, H-1),
(2H, m, OCH2CH2CH2CH3), 1.49–1.56 (2H, m, OCH2- 8.42–8.44 (1H, d, J = 8.0 Hz, H-8), 7.81–7.88 (2H, m, H-
CH2CH2CH3), 0.99–1.02 (3H, m, OCH2CH2CH2CH3). 5, H-6), 7.50–7.53 (1H, m, H-7), 4.16 (1H, s, NCH3).
Anal. Calcd for C18H20N2O2: C, 72.97; H, 6.76; N, Anal. Calcd for C13H10N2O2: C, 69.03; H, 4.42; N,
9.46. Found: C, 72.83; H, 6.97; N, 9.39. 12.39. Found: C, 68.96; H, 4.57; N, 12.31.

4.1.24. Butyl 9-butyl-b-carboline-3-carboxylate (7c). A 4.1.28. 9-Ethyl-b-carboline-3-carboxylic acid (8b). Yel-

mixture of 6 (2.4 g, 10 mmol) and anhydrous DMF low solid was obtained (2.41 g, 98%). Mp 201–202 C;
(60 mL) was stirred at rt for 10 min, then 60% NaH FAB-MS m/e 241 (M+1); UV kmax 387, 359, 273, 239,
(0.6 g, 15 mmol) and n-butyl iodide (6 mL, 50 mmol) were 220 nm; IR (KBr): 3250–2250, 1713, 1630, 1407, 1336,
added and refluxed for 1 h. Later the mixture was trea- 1198 cm
1; 1H NMR (500 MHz, DMSO-d6) d 12.10
ted in a manner similar to that described for 5a to afford (1H, s, COOH), 9.16 (1H, s, H-4), 8.97 (1H, s, H-1),
white crystals 6c (2.2 g, 74%), mp 94–95 C (from petro- 8.31–8.33 (1H, d, J = 8.0 Hz, H-8), 7.75–7.81(2H, m, H-
leum ether–ethyl ether 1:2); FAB-MS m/e 325 (M+1); 5, H-6), 7.43–7.46 (1H, m, H-7), 4.63–4.67 (2H, s,
UV kmax 359, 344, 307, 274, 238, 221 nm; IR (KBr): NCH2CH3), 1.46–1.52 (3H, s, NCH2CH3). Anal. Calcd
3061, 2956, 2866, 1728, 1624, 1358, 1247 cm
1; 1H for C14H12N2O2: C, 70.00; H, 5.00; N, 11.67. Found: C,
NMR (500 MHz, CDCl3): 8.95 (1H, s, H-4), 8.85 (1H, 69.89; H, 5.19; N, 11.59.
s, H-1), 8.19–8.20 (1H, d, J = 7 Hz, H-8), 7.60–7.64
(1H, m, H-5), 7.49–7.50 (1H, d, J = 8.5 Hz, H-6), 7.32– 4.1.29. 9-n-Butyl-b-carboline-3-carboxylic acid (8c). Yel-
7.36 (1H, m, H-7), 4.47–4.49 (2H, m, OCH2CH2CH2- low solid was obtained (2.71 g, 99%). Mp 182–184 C;
CH3), 4.37–4.42 (2H, m, NCH2CH2CH2CH3), 1.84– FAB-MS m/e 269 (M+1); UV kmax 387, 359, 272, 238,
1.92 (4H, m, OCH2CH2CH2CH3, NCH2CH2CH2CH3), 221 nm; IR (KBr): 3250–2250, 1710, 1630, 1334,
1.49–1.56 (2H, m, OCH2CH2CH2CH3), 1.34–1.40 (2H, 1213 cm
1; 1H NMR (500 MHz, DMSO-d6) d 12.11
m, NCH2CH2CH2CH3), 0.99–1.02 (3H, m, OCH2CH2- (1H, s, COOH), 9.14 (1H, s, H-4), 8.94 (1H, s, H-1),
CH2CH3), 0.92–0.95 (3H, m, NCH2CH2CH2CH3). 8.42–8.44 (1H, d, J = 8.0 Hz, H-8), 7.77–7.79 (1H, d,
Anal. Calcd for C20H24N2O2: C, 74.07; H, 7.41; N, J = 8.5 Hz, H-5), 7.65–7.68 (1H, m, H-6), 7.34–7.37 (1H,
8.64. Found: C, 73.88; H, 7.65; N, 8.57. m, H-7), 4.56–4.59 (2H, m, NCH2CH2CH2CH3), 1.79–
1.85 (2H, m, NCH2CH2CH2CH3), 1.28–1.32 (2H, m,
4.1.25. Butyl 9-benzyl-b-carboline-3-carboxylate (7d). A NCH2CH2CH2CH3), 0.87–0.90 (3H, m,
mixture of 5 (2.4 g, 10 mmol) and anhydrous DMF NCH2CH2CH2CH3). Anal. Calcd for C16H16N2O2: C,
(60 mL) was stirred at rt for 10 min, then 60% NaH 71.64; H, 5.97; N, 10.45. Found: C, 71.45; H, 6.19; N,
(0.6 g, 15 mmol) and benzyl bromide (5 mL, 40 mmol) 10.36.
were added and refluxed for 3 h. Later the mixture was
treated in a manner similar to that described for 5a to 4.1.30. 9-Benzyl-b-carboline-3-carboxylic acid (8d). Yel-
afford white crystals 7d (2.4 g, 67%), mp 105–106 C low solid was obtained (2.96 g, 98%). Mp 261–262 C;
4622 R. Cao et al. / Bioorg. Med. Chem. 12 (2004) 4613–4623

FAB-MS m/e 303 (M+1); UV kmax 355, 342, 267, approximately 24 ± 2 C, with a relative humidity 60–
239 nm; IR (KBr): 3457, 3409, 3250–2250, 1683, 1624, 70%, and in 12 h light-dark cycle. The sterile food and
1334, 1225 cm
1; 1H NMR (500 MHz, DMSO-d6) d water were provided according to institutional guide-
12.18 (1H, s, COOH), 9.15 (1H, s, H-4), 8.96 (1H, s, lines. All animals were provided by Shanghai Labora-
H-1), 8.44–8.45 (1H, d, J = 8.0 Hz, H-8), 7.79–7.80 (1H, tory Animal Center of Chinese Academy of Science.
d, J = 8.0 Hz, H-5), 7.63–7.66 (1H, m, H-6), 7.35–7.38 All animal procedures were approved by the Animal
(1H, m, H-7), 7.22–7.31 (5H, m, Ar–H), 5.85 (2H, s, Ethical Committee of the Sun Yat-sen University. Prior
NCH2Ar). Anal. Calcd for C19H14N2O2: C, 75.50; H, to each experiment, mice were fastened overnight and al-
4.64; N, 9.27. Found: C, 75.38; H, 4.85; N, 9.18. lowed free access to water. Various doses of the b-carb-
oline derivatives ranging from 10 to 300 mg/kg dissolved
4.1.31. 9-(2 0 ,3 0 ,4 0 ,5 0 ,6 0 -Pentafluoro)benzyl-b-carboline-3- in 0.5% carboxymethyl cellulose sodium (CMC-Na) salt
carboxylic acid (8e). White solid was obtained (3.84 g, solution were given via intraperitoneal (i.p.) to different
98%). Mp >270 C; FAB-MS m/e 393 (M+1); UV kmax groups of healthy C57BL/6 mice, and each group con-
351, 338, 261, 239, 218 nm; IR (KBr): 3500–2250, tained 10 mice (five males and five females). After the
1714, 1628, 1335 cm
1; 1H NMR (500 MHz, DMSO- administration of the compounds, mice were observed
d6) d 12.14 (1H, s, COOH), 9.10 (1H, s, H-4), 8.83 continuously for the first 2 h for any gross behavioral
(1H, s, H-1), 8.45–8.47 (1H, d, J = 8 Hz, H-8), 7.65– changes and deaths, then intermittently for the next
7.68 (1H, m, H-5), 7.59–7.61 (1H, d, J = 8.5 Hz, H-6), 24 h and occasionally thereafter for 14 days, and for
7.38–7.41 (1H, m, H-7), 5.67 (2H, s, CH2Ar). Anal. Cal- the onset of any delayed effects. All animals were sacri-
cd for C19F5H9N2O2: C, 58.16; H, 2.30; N, 7.14. Found: ficed at the 14th day after drug administration and
C, 58.03; H, 2.39; N, 7.18. checked macroscopically for possible damage to the
heart, liver, and kidneys. Mice of immediate death fol-
lowing drug administration were also examined for
4.1.32. 9-Phenylpropyl-b-carboline-3-carboxylic acid (8f). any possible organ damage. LD50 values were calculated
Yellow solid was obtained (3.2 g, 97%). Mp 213–215 C; graphically as described.31
FAB-MS m/e 331 (M+1); UV kmax 358, 346, 268, 239,
218 nm; IR mmax 3500–2250, 1692, 1629, 1335,
1215 cm
1; 1H NMR (500 MHz, DMSO-d6) d 12.09 4.4. Assay of antitumor activity
(1H, s, COOH), 9.13 (1H, s, H-4), 9.00 (1H, s, H-1),
8.46–8.48 (1H, d, J = 7.5 Hz, H-8), 7.76–7.78 (1H, d, Lewis lung cancer and S180 sarcoma cell lines were pro-
J = 8.0 Hz, H-5), 7.69–7.72 (1H, m, H-6), 7.38–7.41 vided by Shanghai Institute of Pharmaceutical Industry.
(1H, m, H-7), 7.21–7.26 (2H, m, ArH), 7.13–7.17 (3H, Tumor cells of Lewis lung cancer and S180 sarcoma
m, Ar–H), 4.63–4.66 (2H, m, NCH2CH2CH2Ar), 2.49– were inoculated to mice. After 7 days, tumors were taken
2.51 (2H, m, NCH2CH2CH2Ar), 2.14–2.20 (2H, m, out and cells harvested. Viable tumor cells (2 · 106 cells/
NCH2CH2CH2Ar). Anal. Calcd for C21H18N2O2: C, mouse) were inoculated to the armpit of mice by subcu-
76.36; H, 5.45; N, 8.48. Found: C, 76.22; H, 5.69; N, taneous injection. Each compound was injected by intra-
8.38. peritoneal (i.p.) to different group mice (each group
containing 10 female mice) 24 h after the inoculation at
4.2. In vitro cytotoxicity assays a dosage of 7.5 mg/kg once a day for consecutive 7 days.
This dose was the maximum tolerated dose for most
Cytotoxicity assays in vitro were carried out using 96 compounds based on our preliminary studies. Cyto-
microtiter plate cultures and MTT staining according phosphane (CTX) at 30 mg/kg was used as a positive
to the procedures described by Al-Allaf and Rashan26 control and vehicle as negative control. The weights of
with a slight modification. Cells were grown in RPMI- animals were recorded every 3 days. All animals were
1640 medium containing 10% (v/v) fetal calf serum sacrificed at the 21st day after tumor inoculation and
and 100 lg/mL penicillin and 100 lg/mL streptomycin. the tumors were excised and weighed. The inhibition
Cultures were propagated at 37 C in a humified atmos- rate was calculated as follows:
phere containing 5% CO2. Cell lines were obtained from
Shanghai Cell Institute, Chinese Academy of Science. ðC
T Þ=C  100
Drug stock solutions were prepared in DMSO. The final
concentration of DMSO in the growth medium was 2% T: average tumor weight of treated group; C: average
(v/v) or lower, concentration without effects on cell rep- tumor weight of negative control group.
lication. The human tumor cell line panel consisted of
nonsmall cell lung carcinoma (PLA-801), liver carci-
noma (HepG2 and Bel-7402), gastric carcinoma
(BGC-823), cervical carcinoma (Hela), colon carcinoma Acknowledgements
(Lovo). In all of these experiments, three replicate wells
were used to determine each point. We are thankful for the financial support of this work
by Xinjiang Huashidan Pharmaceutical Co. Ltd and
4.3. Assay of acute toxicities Guangzhou Commission of Science and Technology
(97-Z-12-01). We are also grateful to Prof. Huifang
Healthy C57BL/6 mice (9–12 weeks) weighing 18–22 g Yan and co-workers for performance of the antitumor
were housed in rooms where the temperature was and acute toxic assays in mice.
 R. Cao et al. / Bioorg. Med. Chem. 12 (2004) 4613–4623 4623

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