Synthesis and structure-activity relationships of harmine derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2012.11.045

Harmine, a naturally occurring β-carboline alkaloid, showed good antitumor activities together with remarkable neurotoxic effects in animal models. In order to search for novel leading compounds endowed with better antitumor activities and less neurotoxicities, a series of harmine derivatives were designed and synthesized by modification of position-2, 7 and 9 of β-carboline nucleus, and their cytotoxic activities against human tumor cell lines were investigated. Acute toxicities and antitumor activities of the selected compounds in mice were also evaluated. Structure-activity relationships studies confirmed that (1) the 7-methoxy structural moiety was the pharmacophore responsible for the neurotoxic effects of this class of compounds; (2) the substituents in position-2 and 9 played a vital role in modulation of their antitumor activities.

Full text of DOI:10.1016/j.ejmech.2012.11.045

European Journal of Medicinal Chemistry 60 (2013) 135e143
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and structureeactivity relationships of harmine derivatives as potential
antitumor agents
,
c
c
c
a
a
c
Rihui Cao a , Wenxi Fan , Liang Guo , Qin Ma , Guoxian Zhang , Jianru Li , Xuemei Chen ,
*
Zhenghua Ren b , Liqin Qiu
,
a
*
a School of Chemistry and Chemical Engineering, Sun Yat-sen University, 135 Xin Gang Xi Road, Guangzhou 510275, PR China
b School of Life Science, Sun Yat-sen University, 135 Xin Gang Xi Road, Guangzhou 510275, PR China
c Xinjiang Huashidan Pharmaceutical Co. Ltd., 175 He Nan East Road, Urumqi 830011, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Harmine, a naturally occurring
remarkable neurotoxic effects in animal models. In order to search for novel leading compounds
endowed with better antitumor activities and less neurotoxicities, a series of harmine derivatives were
b-carboline alkaloid, showed good antitumor activities together with
Received 22 August 2012
Received in revised form
28 November 2012
Accepted 29 November 2012
Available online 8 December 2012
designed and synthesized by modification of position-2, 7 and 9 of
b-carboline nucleus, and their
cytotoxic activities against human tumor cell lines were investigated. Acute toxicities and antitumor
activities of the selected compounds in mice were also evaluated. Structureeactivity relationships
studies confirmed that (1) the 7-methoxy structural moiety was the pharmacophore responsible for the
neurotoxic effects of this class of compounds; (2) the substituents in position-2 and 9 played a vital role
in modulation of their antitumor activities.
Keywords:
Harmine
b
-Carboline
Ó 2012 Elsevier Masson SAS. All rights reserved.
Cytotoxic
Antitumor
Structureeactivity relationships
1. Introduction
Previous investigations were focused on the neuropharmaco-
logical effects of harmine on the central nervous system (CNS) such
Harmine, the most representative naturally occurring
b
-carbo-
as hallucination, tremor, anxiolytic and sedation. However, recent
interest in harmine has been attracted to its antitumor activity.
Ishida et al. [3] reported the incorporation of various substituents
into position-1, 2, 6, 7 and 9 of harmine and the evaluation of their
biological activities as antitumor agents. Structureeactivity rela-
tionships (SARs) analysis demonstrated that (1) introducing alkoxy
substituents into position-7 of harmine led to enhanced cytotoxic
activities; (2) the length of alkoxy chain affected both cytotoxicity
and cell line specificity; (3) N9-alkylated harmine derivatives
line alkaloid, was originally isolated from Peganum harmala which
is being widely used as a traditional herbal drug as an emmena-
gogue and abortifacient in the Middle East and North Africa [1]. In
Northwest China, the extracts of the seeds of P. harmala have been
traditionally used for hundreds of years to treat the alimentary tract
cancers and malaria [2], and subsequent investigation confirmed
that harmine was the most important active ingredients [3,4]. So
far, numerous previous studies demonstrated that harmine
possessed a wide spectrum of biochemical activities including
intercalation into DNA [5e8], inhibition of topoisomerase I (Topo I)
[8,9] and cyclin-dependent kinases (CDKs) [10,11], inhibition of
monoamine oxidase A (MAO-A) [12,13] and 5-hydroxytryptamine
(5-HT) uptake of human platelet [13]. Moreover, harmine was re-
ported to exhibit a diverse range of pharmacological properties
such as hallucinogenic [14], antitumor [2e4,8], antiviral [15] and
antiparasitic [16] activities.
exhibited strong cytotoxic effects; (4) N2-alkylated
b-carboline
derivatives displayed specific cytotoxic activities. Our previous
investigation [4] also indicated that N9-alkyl and aryl alkyl
substituted harmine derivatives had significant antitumor in mice
bearing both Lewis lung carcinoma and Sarcoma 180, while their
exhibited remarkable neurotoxic effects including tremor, twitch
and jumping in experimental animal models. SARs studies sug-
gested that (1) the introduction of appropriate substituents into
position-9 of harmine remarkably enhanced the antitumor activi-
ties in vitro and in vivo; (2) the methoxy group at position-7 of
harmine might play a very crucial role in determining their
remarkable neurotoxic effects. More recently, our group investi-
gation [17] on the syntheses of harmine derivatives bearing various
* Corresponding authors. Tel.: þ86 20 84110918; fax: þ86 20 84112245.
E-mail addresses: caorihui@mail.sysu.edu.cn (R. Cao), renzhh@mail.sysu.edu.cn
(Z. Ren).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.045

136
R. Cao et al. / European Journal of Medicinal Chemistry 60 (2013) 135e143
substituents at postion-2, 7 and 9 of
b
-carboline nucleus and the
synthesis and structureeactivity relationships of harmine deriva-
evaluation of their antitumor activities in vitro disclosed that the
tives as antitumor agents.
N2-benzyl substituent on the
b-carboline ring played an important
role in the modulation of the cytotoxic activities.
2. Chemistry
In continuing search for novel antitumor agents endowed with
better pharmacological profiles and elucidate the antitumor
structureeactivity relationships (SARs) of harmine derivatives in
finer detail, in the present investigation, we reported the design,
The synthetic routes of harmine derivatives 4aeag and 5aer are
outlined in Scheme 1. The N9-alkylated harmine derivatives 2ae
d were prepared according to the synthetic protocol described by
N
DMF/NaH
N
H3CO
H3CO
N
R9
R9Br
CH3
N
CH3
H
2a R9=C2H5
2b R9=n-C4H9
1
2c R9=CH2CH(CH3)2
2d R9=(CH2)3C6H5
DMF/NaH
R7Br
N
HAc/HBr
HO
N
R7O
N
R9
CH3
N
CH3
R9
3a R9=C2H5
R9=C2H5 R7=C2H5
4a
4b
4c
4d
4e
3b R9=n-C4H9
R9=C2H5 R7=CH(CH3)2
3c R9=CH2CH(CH3)2
3d R9=(CH2)3C6H5
R9=C2H5 R7=n-C4H9
R9=C2H5 R7=CH2CH(CH3)2
R9=C2H5 R7=n-C10H21
CH2Br
Br-
N+ CH2
4f
R9=C2H5 R7=CH2C6H5
R9=C2H5 R7=CH2C6F5
4g
4h
4i
R7O
R9=C2H5 R7=(CH2)3C6H5
R9=n-C4H9 R7=C2H5
EA
N
CH3
R9
R9=n-C4H9 R7=CH(CH3)2
R9=n-C4H9 R7=n-C4H9
4j
R9=C2H5 R7=C2H5
R9=C2H5 R7=n-C4H9
5a
5b
5c
5d
5e
5f
4k
4l
R9=n-C4H9 R7=CH2CH(CH3)2
R9=n-C4H9 R7=CH(CH2CH3)2
R9=n-C4H9 R7=CH2CH(OH)CF3
R9=n-C4H9 R7=n-C8H17
R9=C2H5 R7=CH2CH(CH3)2
R9=C2H5 R7=CH2C6H5
R9=C2H5 R7=CH2C6F5
R9=C2H5 R7=(CH2)3C6H5
R9=n-C4H9 R7=CH2CH(CH3)2
R9=n-C4H9 R7=n-C8H17
R9=n-C4H9 R7=CH2C6H5
R9=CH2CH(CH3)2 R7=n-C4H9
R9=CH2CH(CH3)2 R7=CH2CH(CH3)2
R9=CH2CH(CH3)2 R7=n-C10H21
R9=CH2CH(CH3)2 R7=CH2C6H5
R9=CH2CH(CH3)2 R7=(CH2)3C6H5
R9=(CH2)3C6H5 R7=n-C4H9
4m
4n
4o
4p
4q
4r
R9=n-C4H9 R7=n-C10H21
R9=n-C4H9 R7=CH2C6H5
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
R9=n-C4H9 R7=CH2C6F5
R9=n-C4H9 R7=(CH2)3C6H5
R9=CH2CH(CH3)2 R7=n-C4H9
R9=CH2CH(CH3)2 R7=CH2CH(CH3)2
R9=CH2CH(CH3)2 R7=n-C10H21
R9=CH2CH(CH3)2 R7=CH2C6H5
R9=CH2CH(CH3)2 R7=(CH2)3C6H5
R9=(CH2)3C6H5 R7=CH(CH3)2
R9=(CH2)3C6H5 R7=n-C4H9
R9=(CH2)3C6H5 R7=CH2CH(CH3)2
R9=(CH2)3C6H5 R7=CH(CH2CH3)2
R9=(CH2)3C6H5 R7=CH2CH=C(CH3)2
R9=(CH2)3C6H5 R7=n-C8H17
R9=(CH2)3C6H5 R7=CH2C6H5
R9=(CH2)3C6H5 R7=CH2C6F5
R9=(CH2)3C6H5 R7=(CH2)3C6H5
4s
4t
4u
4v
4w
4x
4y
4z
4aa
4ab
4ac
4ad
4ae
4af
4ag
R9=(CH2)3C6H5 R7=CH(CH2CH3)2
R9=(CH2)3C6H5 R7=n-C8H17
R9=(CH2)3C6H5 R7=(CH2)3C6H5
Scheme 1. Synthesis of harmine derivatives 4aeag and 5aer.

R. Cao et al. / European Journal of Medicinal Chemistry 60 (2013) 135e143
137
our group [4]. The preparation of compounds 3aed followed
a common synthetic scheme, characterized by demethylation of
compounds 2aed using acetic acid and hydrobromic acid as reac-
tion solvent [17]. Compounds 4aeag, bearing alkoxy in postion-7 of
summarized in Table 2. All the tested harmine derivatives resulted
in acute toxic manifestation but caused no obvious neurotoxic
effects including tremor, twitch, jumping and supination just like
harmine 1. Animals were drowsy and exhibited a decrease in
locomotor activity after the administration of harmine derivatives.
Of all investigated compounds, compound 5r exhibited the highest
acute toxicity with LD50 value of 3.75 mg/kg. Compounds 5c, 5e, 5f,
5m and 5p also displayed remarkable acute toxicity with LD50 value
of 12.5, 12.5, 15.0, 5.0 and 6.25 mg/kg, respectively, while for the
compounds 4a, 4h and 4ab, acute toxicities were much less with
LD50 value of 200, 200 and 100 mg/kg, respectively. 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 obvious changes in any organs.
b
-carboline ring, were synthesized from compounds 3aed by the
action of sodium hydride in dry DMF followed by addition of the
appropriate alkylating and arylating agents in 63e87% yield. The
N2-benzylated
b-carbolinium bromates 5aer were prepared from
compounds 4 by the addition of benzyl bromide in refluxing ethyl
acetate [17]. The chemical structures of all the newly synthesized
compounds were characterized by MS, IR, 1H NMR, 13C NMR and
elemental analysis.
3. Results and discussion
A total analysis to the acute toxicity and neurotoxic effect of har-
mine derivatives investigated above and of our previous report [4]
3.1. Cytotoxicity in vitro
confirmed that (1) the methoxy substituent in position-7 of b-carbo-
The cytotoxic potencies of harmine derivatives 4aeag and 5aer
against a panel of human tumor cell lines were investigated and
compared with the reference drugs cisplatin. The human tumor cell
linepanelconsistedofcervicalcarcinoma(Hela), livercarcinoma(Bel-
7402 and HepG2), gastric carcinoma (BGC-823), breast carcinoma
(MCF-7), renal carcinoma (769-P, 786-0 and OS-RC-2), epidermoid
carcinoma of the nasopharynx (KB), non-small cell lung carcinoma
(A549), malignant melanoma (A375), colon carcinoma (HT-29),
bladder squamous carcinoma (SCaBER), malignant bladder carci-
noma (Blu-87), malignant glioma (U251). Compounds 4aeag were
converted into their water-soluble hydrochloride salt by the usual
methods before use. The results were summarized in Table 1.
The cytotoxic potency of most 7,9-disubstituted harmine
derivatives 4aeag showed no distinct difference and the IC50 values
of this class of compounds ranged from 10 to 100 mM. Exceptionally,
compounds 4eeg, 4nep, 4r, 4aa, 4ac, 4v and 4afeag bearing
relatively large and bulky alkoxy substituent in position-7 dis-
played weak or no cytotoxic activities against several human tumor
cell lines, and the poor water-soluble properties might be respon-
sible for their weak activities.
line nucleus played a vital role in determining the remarkable
neurotoxic effects; (2) replacing the methoxy substituent with a bulky
alkoxygroupled to eliminating neurotoxic effectof these compounds;
(3) the acute toxicity increased remarkably by the introduction of
a benzyl substituent into position-2 of b-carboline nucleus.
3.3. Evaluation of antitumor activity
Nine harmine derivatives were selected for evaluation in vivo
against mice bearing Lewis lung cancer and Sarcoma 180 and
compared with the reference drugs harmine 1 and Cyclophospha-
mide (CTX). The tumor inhibition rates of these compounds were
summarized in Table 2. All the tested compounds showed potent
antitumor activities. Harmine 1 exhibited almost equal antitumor
activity against mice both bearing Sarcoma 180 and Lewis lung
cancer with the tumor inhibition rate of 30.8 and 33.7%, respec-
tively. Compounds 5c, 5e, 5f, 5m, 5p and 5r exhibited remarkable
antitumor activities with the tumor inhibition rate of over 40%
against mice bearing Lewis lung cancer and Sarcoma 180 at dose
2.5, 3.0, 1.0, 1.2 and 0.63 mg/kg, respectively. Particularly,
compounds 5f and 5m were found to be the most potent antitumor
agents with the tumor inhibition rate of 53.1 and 52.6% against
mice bearing Sarcoma 180, respectively. The other compounds
tested showed moderate antitumor activities with the tumor
inhibition rate ranging from 20.8 to 36.9% at dose ranging from 2.5
to 40.0 mg/kg. Interestingly, the Sarcoma 180 was more susceptible
to all tested compounds than the Lewis lung cancer, and the results
contradicted with our previous report [4]. The analysis of the
structureeactivity relationships indicated that (1) introducing
Interestingly, compounds 5aer having a benzyl substituent in
position-2 of b-carboline nucleus exhibited the most interesting
cytotoxic potencies with IC50 values of lower than 10
most of human tumor cell lines.
mM against
Of all 2,7,9-trisubstituted harmine derivatives 5aer, compounds
5oer bearing a 3-phenylpropyl substituent in position-9 of -car-
b
boline nucleus showed more potent cytotoxic activities than
compounds 5aef, 5gei and 5jen, which having a ethyl, butyl and
isobutyl group in position-9, respectively. The influence of substit-
uent in position-9 on cytotoxic activities followed the tendency of 3-
phenylpropyl > isobutyl > butyl > ethyl group. Particularly,
compound 5p bearing a benzyl group in position-2, an isobutoxy
group in position-7 and a 3-phenylpropyl group in position-9, was
found to be the most potent cytotoxic agent with IC50 values of lower
a
benzyl substituent into position-2 of
improved significantly their antitumor activities; (2) the arylated
alkoxy group in position-7 of -carboline nucleus was the advisable
b-carboline nucleus
b
pharmacophoric group for their enhanced antitumor activities.
than 5.0
mM against all human tumor cell lines. These results sug-
4. Conclusions
gested that the large and bulky substituent in position-9 might be
advisable pharmacophoric group for enhanced cytotoxic activities.
Anoverviewof thecytotoxicactivitiesdataofallnewlysynthesized
harmine derivatives and of the earlier reports [4,17] clearly confirmed
that (1) the arylated alkyl substituent in position-9 of b-carboline
nucleus was the suitable pharmacophore giving rise to significant
In the present investigation, a series of harmine derivatives
bearing an alkyl substituent in position-9, a benzyl group in
position-2 and an alkoxy chain in position-7 were synthesized and
evaluated as potential antitumor agents. The results corroborated
the previous observations that the antitumor activities and acute
toxicities as well as neurotoxic effects of harmine derivatives were
substituent-dependent. An overview of the present study and of
the previous reports, we arrived at the following conclusions: (1)
cytotoxicagents;(2)theintroductionofbenzylgroupintoposition-2of
b-carboline nucleus facilitated significantly cytotoxic potencies.
3.2. Assessment of acute toxicity
the methoxy substituent in position-7 of b-carboline nucleus
played a vital role in determining the remarkable neurotoxic
effects; (2) replacing the methoxy substituent with a bulky alkoxy
group led to eliminating neurotoxic effects of these compounds; (3)
The LD50 values and scores for neurotoxicity of the selected
harmine derivatives in mice after administration by i.p. route were

138
R. Cao et al. / European Journal of Medicinal Chemistry 60 (2013) 135e143
Ma).
Table 1
Cytotoxic activities of harmine derivatives 4aeag and 5aer in vitroc (IC50
, m
Compd
Helab
Bel-7402
BGC-823
HepG2
MCF
OS-RC-2
A549
A375
786-0
HT-29
SCaBER
Blu-87
769-P
U251
KB
22RV1
1e
4a
4b
4c
4d
4e
60
30.7
27.6
29.2
23.3
>100
>100
>100
54
27.4
43.6
52.9
21.9
>100
>100
>100
13.5
36.1
26.8
69.9
25.6
21.5
62.0
>100
18.8
>100
>100
>100
42.5
42.6
>100
26.8
>100
36.2
34.8
39.3
34.3
24.7
21.8
29.4
18.6
>100
12.6
2.9
68
57.1
59.7
15.6
29.8
15.2
17.2
>100
11.9
37.2
19.1
15.8
23.4
16.7
96.4
15.4
28.4
17.2
18.3
13.8
12.1
26.6
74.1
36.0
18.5
16.3
14.0
23.0
18.3
32.3
17.4
19.5
7.9
19.8
9.2
10.9
10.6
66.1
4.6
7.6
4.6
3.7
21.4
6.8
5.4
2.0
5.2
2.5
4.1
2.5
3.9
2.5
46
68.9
52.6
16.3
20.8
14.1
15.5
>100
11.4
43.3
30.2
15.5
15.3
15.6
>100
13.6
>100
17.8
13.6
15.6
12.1
36.8
>100
22.2
27.7
14.6
12.4
12.9
7.3
21.1
>100
15.7
>100
16.7
14.3
10.0
9.6
49.9
1.6
7.6
4.2
2.7
NDd
14.4
>100
18.5
13.2
45.9
>100
>100
>100
12.0
19.5
17.3
21.6
5.4
33.1
13.6
62.7
16.9
>100
16.0
7.2
29.4
>100
54.1
19.5
45.3
68.9
>100
41.9
>100
45.9
63.3
38.7
>100
8.1
ND
ND
ND
ND
32.0
ND
ND
ND
ND
ND
ND
ND
30.5
26.9
ND
ND
ND
ND
ND
ND
ND
30.5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
7.8
8.8
10.6
15.9
1.8
2.2
3.2
<1.5
3.6
5.8
8.3
4.0
4.6
3.5
6.8
2.5
1.7
3.1
5.6
ND
ND
ND
ND
30.3
ND
ND
ND
22.6
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19.7
ND
ND
ND
18.4
18.8
ND
ND
ND
28.6
ND
21.5
ND
22.2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6.8
6.9
7.8
12.3
3.8
2.4
4.2
<1.5
4.0
2.1
3.9
<1.5
3.3
<1.5
1.5
<1.5
<1.5
<1.5
5.6
ND
ND
ND
ND
24.3
ND
ND
ND
13.2
ND
ND
ND
14.4
13.4
ND
ND
ND
10.1
ND
12.9
ND
18.1
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
9.6
ND
ND
ND
ND
16.9
ND
ND
ND
11.8
ND
ND
ND
20.1
25.1
ND
ND
ND
26.8
ND
51.2
ND
35.3
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
10.2
6.4
4.0
10.9
1.6
4.2
3.1
3.2
3.7
4.0
4.2
1.7
3.4
4.9
3.4
2.0
3.4
2.0
8.7
ND
ND
ND
ND
16.4
ND
ND
ND
6.5
ND
ND
ND
ND
14.2
ND
ND
ND
16.7
ND
ND
ND
17.4
16.0
ND
ND
ND
32.7
ND
26.9
ND
32.3
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
23.4
22.9
25.9
22.1
17.7
17.3
12.3
2.0
ND
ND
ND
ND
27.0
ND
ND
ND
22.6
ND
ND
ND
4.0
22.2
ND
ND
ND
17.1
ND
21.3
ND
30.8
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6.4
ND
ND
ND
ND
44.6
ND
ND
ND
28.0
ND
ND
ND
32.3
25.1
ND
ND
ND
ND
ND
29.9
ND
49.6
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
15.2
14.2
17.3
16.2
6.6
ND
ND
ND
ND
20.3
ND
ND
ND
22.6
ND
ND
ND
19.4
19.8
ND
ND
ND
45.0
ND
41.6
ND
27.5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
40.5
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
<1.4
ND
6.7
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
4x
30.2
32.1
21.2
17.4
40.7
28.2
>100
>100
>100
47.0
>100
24.7
20.8
32.3
2.0
50.5
27.6
48.6
88.7
>100
70.1
>100
ND
ND
ND
15.9
43.8
ND
ND
ND
39.7
ND
20.3
ND
28.8
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
19.8
21.8
34.1
46.3
6.8
ND
30.0
21.0
ND
ND
ND
18.1
ND
28.1
ND
32.4
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
4y
4z
4aa
4ab
4ac
4ad
4ae
4af
4ag
5a
5b
5c
5d
5e
69.6
72.6
73.5
ND
ND
>100
2.2
2.4
4.9
56.3
0.93
11.2
13.0
11.6
19.6
1.8
2.3
3.8
<1.5
4.4
2.2
4.3
<1.5
3.5
<1.5
<1.5
2.0
1.6
3.3
16.0
4.6
5.9
7.8
8.5
15.4
2.9
1.5
4.6
5.2
8.9
13.0
5.0
<1.7
<1.7
8.7
13.8
<1.5
2.0
<1.5
4.0
6.0
<1.5
<1.5
<1.5
<1.5
<1.4
<1.5
<1.5
<1.5
12.1
3.2
3.6
4.0
2.4
2.7
0.86
3.6
5.3
4.3
5.8
5.0
1.3
2.5
0.81
2.0
2.0
11.1
67.6
5.7
2.8
10.4
4.1
15.7
5.3
2.9
1.8
3.9
9.2
3.8
1.8
5.3
6.6
11.3
5f
2.2
3.9
4.0
2.2
2.6
2.1
1.6
12.4
1.4
3.9
1.5
2.2
3.1
7.6
16.2
18.2
4.6
8.5
9.7
12.8
7.8
4.6
3.6
4.7
10.5
12.1
2.0
5.5
1.8
4.5
1.5
<1.5
2.5
3.7
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
2.5
4.6
<1.5
<1.5
2.4
3.4
1.9
<1.5
2.3
2.6
<1.5
<1.5
2.2
<1.5
20.0
2.3
<1.5
<1.5
2.4
3.7
2.9
<1.5
1.8
2.9
2.9
2.1
2.7
1.8
5.7
2.4
1.9
4.0
4.1
3.1
4.6
8.9
5.2
3.2
6.9
3.8
4.4
2.1
<1.6
2.6
<1.5
<1.5
2.8
3.4
4.4
6.1
7.7
12.3
2.1
2.9
0.74
4.6
<1.5
<1.5
2.3
3.9
3.8
6.4
1.9
1.5
6.8
Cisplatin
8.9
10.2
3.9
9.8
6.3
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 cervical carcinoma (Hela), liver carcinoma (Bel-7402 and HepG2), gastric carcinoma (BGC-823), breast carcinoma (MCF-7), renal carcinoma (769-P, 786-
0 and OS-RC-2), epidermoid carcinoma of the nasopharynx (KB), non-small cell lung carcinoma (A549), malignant melanoma (A375), colon carcinoma (HT-29), bladder
squamous carcinoma (SCaBER), malignant bladder carcinoma (Blu-87), malignant glioma (U251).
c
Data represent the mean values of three independent determinations.
d
ND ¼ not tested.
e
See Ref. [4].
the acute toxicity increased remarkably by introducing a benzyl
substituent into position-2 of -carboline nucleus; (4) the anti-
Peganum multisectum Maxim, a plant indigenous to western China
according to the method by Duan et al. [18]. The following interme-
b
tumor activities improved greatly by the introduction of appro-
priate substituents into position-2, 7 and 9.
diates, 7-methoxy-9-ethyl-1-methyl-
9-n-butyl-1-methyl- -carboline 2b [4], 7-methoxy-9-isobutyl-1-
-carboline 2c [17], 7-methoxy-9-(3-phenylpropyl)-1-
-carboline 2d [4], 9-ethyl-1-methyl- -carboline7-ol 3a [17],
-carboline-7-ol 3b[17], 9-isobutyl-1-methyl-
carboline7-ol3c[17]and1-methyl-9-(3-phenyl-propyl)- -carboline-
b-carboline 2a [4], 7-methoxy-
b
methyl-
methyl-
b
b
b
5. Experimental section
9-n-butyl-1-methyl-
b
b-
b
5.1. Reagents and general methods
7-ol 3d [17], were prepared according to the described procedures.
Compounds 4a, 4g, 4iek, 4p, 4t, 4w, 4y, 4ad, 4ae, 4af, 5a, 5e, 5j, 5m
and 5q are known compounds [17].
All reagents were purchased from commercial suppliers and were
dried and purified when necessary. Harmine 1 was extracted from

R. Cao et al. / European Journal of Medicinal Chemistry 60 (2013) 135e143
139
1409, 1343, 1260, 1215, 1140, 1047, 1007, 810 cmꢁ1
(500 MHz, CDCl3)
;
1H NMR
Table 2
Acute toxic effects of harmine derivatives in mice and antitumor activities of these
compounds against mice bearing sarcoma 180 and Lewis lung cancer.
d
8.26 (1H, d, J ¼ 6.0 Hz); 7.95 (1H, d, J ¼ 8.5 Hz);
7.73 (1H, d, J ¼ 6.0 Hz); 6.86e6.90 (2H, m); 4.54 (2H, q, J ¼ 7.0 Hz);
4.10 (2H, t, J ¼ 6.5 Hz); 3.05 (3H, s); 1.84e1.87 (2H, m); 1.54e1.58
(2H, m); 1.45 (3H, t, J ¼ 7.5 Hz); 1.01 (3H, t, J ¼ 7.5 Hz). 13C NMR
Compds
Acute toxicity
Dosage
(mg/kg)
Tumor inhibition rate
(%)
LD50
Neurotoxic
Sarcoma
180
Lewis lung
carcinoma
(75 MHz, CDCl3)
d 160.6, 142.8, 140.6, 138.4, 135.3, 129.6, 122.5,
(mg/kg)
effect
115.5, 112.3, 109.3, 94.1, 68.5, 39.8, 31.8, 23.6, 19.7, 15.9, 14.3. Anal.
Calcd for C18H22N2O: C, 76.56; H, 7.85; N, 9.92. Found: C, 76.48; H,
7.83; N, 9.98.
4d
4h
4ab
5c
5e
5f
5m
5p
5r
1
CTX
200
200
100
12.5
12.5
15.0
5.0
6.25
3.75
59.0
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
40.0
40.0
20.0
2.5
2.5
3.0
1.0
1.2
0.63
7.5
30
27.8
26.0
30.1
44.2
36.9
53.1
52.6
41.5
46.1
30.8
88.7
20.8
21.7
27.1
30.0
23.7
27.2
23.7
28.0
33.7
33.7
85.6
5.2.3. 7-Isobutoxy-9-ethyl-1-methyl-b-carboline (4d)
White crystals (0.97 g, 76%) were obtained, mp 127e128 ꢀC. FAB-
MS m/z (M þ 1) 283; IR (KBr) 3042, 2961, 2925, 2871, 1622, 1566,
1450, 1349, 1263, 1214, 1142, 1101, 1044, 808 cmꢁ1 1H NMR
;
a
(500 MHz, CDCl3)
d
8.26 (1H, d, J ¼ 5.5 Hz); 7.95 (1H, d, J ¼ 8.5 Hz);
þ
7.72 (1H, d, J ¼ 5.5 Hz); 6.85e6.89 (2H, m); 4.54 (2H, q, J ¼ 7.0 Hz);
ꢁ
3.85 (2H, d, J ¼ 6.5 Hz); 3.03 (3H, s); 2.14e2.19 (1H, m); 1.44 (3H, t,
a
Acute neurotoxic manifestation was denoted by “þ” and “ꢁ”. A “þ” represents
toxic responses including tremble, twitch, jumping and supination, while “ꢁ” means
no such reaction.
J ¼ 7.5 Hz), 1.09 (6H, d, J ¼ 6.5 Hz). 13C NMR (75 MHz, CDCl3)
d 160.7,
142.8, 140.4, 138.0, 135.1, 129.6, 122.4, 115.2, 112.3, 109.5, 93.9, 75.1,
39.7, 28.8, 23.4, 19.7, 15.9. Anal. Calcd for C18H22N2O: C, 76.56; H,
7.85; N, 9.92. Found: C, 76.58; H, 7.90; N, 9.97.
Melting points were determined in capillary tubes on an elec-
trothermal PIF YRT-3 apparatus and without correction. FAB-MS
spectra were obtained from VG ZAB-HS spectrometer. FT-IR
spectra were run as KBr pellets on a Bruker Equinox 55 Fourier
Transformation Infrared Spectrometer. 1H NMR spectra were
recorded on a Varian INOVA 500NB spectrometer. Chemical shifts
5.2.4. 7-Decyloxy-9-ethyl-1-methyl-b-carboline (4e)
White crystals (1.52 g, 83%) were obtained, mp 76e77 ꢀC. FAB-
MS m/z (M þ 1) 367; IR (KBr) 3047, 2923, 2850, 1628, 1561, 1443,
1345, 1296,1207, 1147, 1027, 859, 821, 786 cmꢁ1; 1H NMR (500 MHz,
are reported in
d (ppm) downfield from an internal solvent peak
CDCl3)
d
8.29 (1H, d, J ¼ 5.0 Hz); 7.98 (1H, d, J ¼ 9.0 Hz); 7.74 (1H, d,
and coupling constants, J in hertz. Elemental analyses (C, H and N)
were carried out on an Elementar Vario EL CHNS Elemental
Analyzer. Silica gel F254 was used in analytical thin-layer chro-
matography (TLC) and silica gel was used in column chromatog-
raphy respectively.
J ¼ 5.0 Hz); 6.88e6.91 (2H, m); 4.57 (2H, q, J ¼ 7.5 Hz); 4.12 (2H, t,
J ¼ 7.5 Hz); 3.06 (3H, s); 1.85e1.99 (2H, m), 1.51e1.56 (2H, m),
1.47(3H, t, J ¼ 7.5 Hz),1.30e1.44 (12H, m); 0.91 (3H, t, J ¼ 7.5 Hz). 13C
NMR (75 MHz, CDCl3) d 160.6,142.8, 140.6, 138.4,135.2,129.6, 122.4,
115.4, 112.3, 109.3, 94.1, 68.8, 39.7, 32.3, 30.0(2C), 29.8(2C), 29.7,
26.5, 23.6, 23.1, 15.9, 14.5. Anal. Calcd for C24H34N2O: C, 78.64; H,
9.35; N, 7.64. Found: C, 78.48; H, 9.43; N, 7.56.
5.2. General procedure for the preparation of 7-alkoxyl-
b-carboline
derivatives
5.2.5. 7-Benzyloxy-9-ethyl-1-methyl-b-carboline (4f)
A mixture of 7-hydroxyl-
b
-carbolines 3aed (5 mmol) and
White crystals (1.37 g, 87%) were obtained, mp 177e178 ꢀC; FAB-
MS m/z (M þ 1) 317; IR (KBr) 3032, 2967, 2925, 2871, 1621, 1564,
anhydrous DMF (50 ml) was stirred at room temperature until
clear, and then 60% NaH (0.3 g, 7.5 mmol) and alkyl halogenide
(15 mmol) were added. The mixture was stirred at room temper-
ature for 0.5e2 h. After completion of the reaction as indicated by
TLC, the solution was poured into H2O (150 ml), and extracted with
ethyl acetate. The organic phase was made acidic with concentrated
hydrochloric acid. Upon removal of solvent, the residue was crys-
tallized from acetone to afford yellow solid. The solid was dissolved
in water and made basic with sodium bicarbonate, and the aqueous
mixture extracted with ethyl acetate. The organic phase was
washed with water and brine, then dried over anhydrous sodium
sulfate, filtered and evaporated. The resulting oil was crystallized
from ethyl ether or ethyl etherepetroleum ether.
1446, 1261, 1213, 1134, 1001, 837, 810 cmꢁ1 1H NMR (500 MHz,
;
CDCl3)
d
8.27 (1H, d, J ¼ 5.5 Hz); 7.98 (1H, d, J ¼ 8.5 Hz); 7.73 (1H, d,
J ¼ 5.5 Hz); 7.26e7.50 (5H, m); 6.95e6.97 (2H, m); 5.16 (2H, s); 4.53
(2H, q, J ¼ 7.5 Hz); 3.03 (3H, s); 1.40 (3H, t, J ¼ 5.5 Hz). 13C NMR
(75 MHz, CDCl3) d 160.1, 142.8, 140.7, 138.5, 137.1, 135.3, 129.5, 128.8,
128.2, 127.7, 122.6, 115.9, 112.4, 109.5, 94.9, 70.9, 39.8, 23.6, 15.8.
Anal. Calcd for C21H20N2O: C, 79.72; H, 6.37; N, 8.85. Found: C,
79.78; H, 6.35; N, 8.91.
5.2.6. 7-(3-Phenylpropoxy)-9-ethyl-1-methyl-b-carboline (4h)
White crystals (1.31 g, 76%) were obtained, mp 110e111 ꢀC. FAB-
MS m/z (M þ 1) 345; IR (KBr) 3030, 2950, 2924, 2871, 1623, 1561,
1447, 1392, 1210, 1136, 1089, 1040, 966, 805 cmꢁ1 1H NMR
;
5.2.1. 7-Isopropoxy-9-ethyl-1-methyl-
b
-carboline (4b)
(500 MHz, CDCl3)
d
8.27 (1H, d, J ¼ 5.5 Hz); 7.96 (1H, d, J ¼ 8.5 Hz);
White crystals (1.0 g, 79%) were obtained, mp 114e115 ꢀC; FAB-
MS m/z (M þ 1) 269; IR (KBr) 2973, 2926, 1620, 1565, 1446, 1373,
7.72 (1H, d, J ¼ 5.5 Hz); 7.19e7.31 (5H, m); 6.83e6.89 (2H, m); 4.52
(2H, q, J ¼ 7.0 Hz); 4.10 (2H, t, J ¼ 8.0 Hz); 3.02 (3H, s); 2.88 (2H, t,
J ¼ 7.5 Hz); 2.13e2.21 (2H, m); 1.43 (2H, t, J ¼ 7.0 Hz). 13C NMR
1209, 1108, 1038, 978, 813 cmꢁ1; 1H NMR (500 MHz, CDCl3)
d 8.26
(1H, d, J ¼ 5.5 Hz); 7.95 (1H, d, J ¼ 9.0 Hz); 7.70 (1H, d, J ¼ 5.5 Hz);
(75 MHz, CDCl3) d 160.5, 142.8, 141.7, 140.7, 138.4, 135.2, 129.5,
6.85e6.87 (2H, m); 4.68e4.73 (1H, m); 4.53 (2H, q, J ¼ 7.5 Hz); 3.01
128.7(2C), 126.2, 122.5, 115.5, 112.4, 109.4, 94.0, 67.7, 39.7, 32.6, 31.3,
23.7, 15.9. Anal. Calcd for C23H24N2O: C, 80.20; H, 7.02; N, 8.13.
Found: C, 80.03; H, 6.98; N, 8.18.
(3H, s); 1.40e1.45 (9H, m). 13C NMR (75 MHz, CDCl3)
d 159.2, 142.8,
140.6, 138.3, 135.2, 129.5, 122.5, 115.5, 112.3, 110.2, 95.9, 70.8, 39.7,
23.6, 22.5, 15.8. Anal. Calcd for C17H20N2O: C, 76.09; H, 7.51; N,
10.44. Found: C, 75.95; H, 7.48; N, 10.37.
5.2.7. 7-Isobutoxy-9-n-butyl-1-methyl-b-carboline (4l)
White crystals (1.19 g, 77%) were obtained, mp 91e92 ꢀC. FAB-
MS m/z (M þ 1) 311; IR (KBr) 3423, 2962, 2928, 2868, 1622, 1564,
5.2.2. 7-n-Butoxy-9-ethyl-1-methyl-b-carboline (4c)
White crystals (1.2 g, 85%) were obtained, mp 109e110 ꢀC; FAB-
MS m/z (M þ 1) 283; IR (KBr) 2973, 2938, 2875, 1623, 1560, 1453,
1447, 1411, 1366, 1242, 1198, 1139, 1043, 810 cmꢁ1 1H NMR
;
(500 MHz, CDCl3)
d
8.27 (1H, d, J ¼ 5.5 Hz); 7.96 (1H, d, J ¼ 9.0 Hz);

140
R. Cao et al. / European Journal of Medicinal Chemistry 60 (2013) 135e143
7.76 (1H, d, J ¼ 5.5 Hz); 6.85e6.91 (2H, m); 4.47 (2H, t, J ¼ 7.5 Hz);
3.87 (2H, d, J ¼ 6.5 Hz); 3.07 (3H, s); 2.14e2.19 (1H, m); 1.79e1.85
(2H, m); 1.43e1.48 (2H, m); 1.09 (6H, d, J ¼ 4.5 Hz); 1.00 (3H, t,
(1H, d, J ¼ 8.5 Hz); 7.76 (1H, d, J ¼ 5.5 Hz); 6.93e6.96 (2H, m); 5.28
(2H, s); 4.47 (2H, t, J ¼ 7.5 Hz); 3.05 (3H, s); 1.80e1.83(2H, m); 1.44e
1.48 (2H, m); 1.00 (3H, t, J ¼ 7.5 Hz). Anal. Calcd for C23H19F5N2O: C,
63.59; H, 4.41; N, 6.45. Found: C, 63.48; H, 4.36; N, 6.38.
J ¼ 7.5 Hz). 13C NMR (75 MHz, CDCl3)
d 160.6, 143.3, 140.7, 138.4,
135.5, 129.6, 122.4, 115.4, 112.3, 109.3, 94.5, 75.2, 45.0, 33.1, 28.8,
23.8, 20.6, 19.7, 14.2. Anal. Calcd for C20H26N2O: C, 77.38; H, 8.44; N,
9.02. Found: C, 77.29; H, 8.40; N, 8.98.
5.2.13. 7-(3-Phenylpropoxy)-9-n-butyl-1-methyl-b-carboline (4s)
White crystals (1.4 g, 75%) were obtained, mp 89e90 ꢀC. FAB-MS
m/z (M þ 1) 373; IR (KBr) 2954, 2926, 2869, 1622, 1561, 1494, 1445,
5.2.8. 9-n-Butyl-1-methyl-7-(pentan-3-yloxy)-
b
-carboline (4m)
1410, 1366, 1355, 1244, 1190, 1139, 1040, 809, 758 cmꢁ1 1H NMR
;
Yellow oil (1.1 g, 68%) was obtained. FAB-MS m/z (M þ 1) 325; IR
(500 MHz, CDCl3)
d
8.27 (1H, d, J ¼ 5.5 Hz); 7.95 (1H, d, J ¼ 8.5 Hz);
(KBr) 3412, 2959, 2873, 2529, 1620, 1570, 1462, 1336, 1242, 1197,
7.72 (1H, d, J ¼ 5.5 Hz); 7.20e7.31 (5H, m); 6.83e6.89 (2H, m); 4.43
(2H, t, J ¼ 7.5 Hz); 4.10 (2H, t, J ¼ 8.0 Hz); 3.02 (3H, s); 2.86e2.89
(2H, m); 2.17e2.20 (2H, m); 1.78e1.84 (2H, m); 1.42e1.51 (2H,
1140, 1111, 1035, 980, 954, 822 cmꢁ1 1H NMR (500 MHz, CDCl3)
;
d
8.26 (1H, d, J ¼ 5.0 Hz); 7.94 (1H, d, J ¼ 9.0 Hz); 7.71 (1H, d,
J ¼ 5.0 Hz); 6.86e6.88 (2H, m); 4.43 (2H, t, J ¼ 7.5 Hz); 4.24e4.29
(1H, m); 3.01 (3H, s); 1.73e1.82 (6H, m); 1.42e1.47 (2H, m);
m); 0.97 (3H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz, CDCl3)
d 160.4, 143.2,
141.6, 140.7, 138.4, 135.4, 129.4, 128.7(2C), 126.2, 122.4, 115.4, 112.4,
109.2, 94.4, 67.7, 44.8, 33.1, 32.6, 31.3, 23.8, 20.6, 14.3. Anal. Calcd for
C25H28N2O: C, 80.61; H, 7.58; N, 7.52. Found: C, 80.50; H, 7.55; N,
7.56.
0.97e1.03 (9H, m). 13C NMR (75 MHz, CDCl3)
d 160.6, 143.4, 140.3,
137.8, 135.3, 129.8, 122.4, 115.2, 112.4, 109.5, 94.3, 75.2, 44.9, 33.0,
28.8, 23.3, 20.5, 19.7, 14.2.
5.2.9. 7-(1,1,1-Trifluoro-2-hydroxyl-propoxy)-9-n-butyl-1-methyl-
5.2.14. 7-Isobutoxy-9-isobutyl-1-methyl-b-carboline (4u)
b-carboline (4n)
Yellow solid (1.27 g, 82%) was obtained, mp 93e95 ꢀC. FAB-MS
m/z (M þ 1) 311; IR (KBr) 2956, 2869, 2480, 1624, 1575, 1470,
White crystals (1.24 g, 68%) were obtained, mp 162e164 ꢀC. FAB-
MS m/z (M þ 1) 367; IR (KBr) 3061, 2961, 2932, 2837, 1624, 1568,
1497, 1453, 1410, 1351, 1241, 1134, 1048, 810 cmꢁ1 1H NMR
(500 MHz, CDCl3)
1432, 1337, 1256, 1204, 1138, 1043, 806 cmꢁ1 1H NMR (500 MHz,
;
;
CDCl3)
d
8.28 (1H, d, J ¼ 5.5 Hz); 7.97 (1H, d, J ¼ 8.5 Hz); 7.80 (1H, d,
d
8.24 (1H, d, J ¼ 5.0 Hz); 7.93 (1H, d, J ¼ 8.5 Hz);
J ¼ 5.5 Hz); 6.86e6.92 (2H, m); 4.29 (2H, d, J ¼ 8.5 Hz); 3.85 (2H, d,
7.72 (1H, d, J ¼ 5.0 Hz); 6.87e6.89 (2H, m); 6.64 (1H, s); 4.50e4.53
(1H, m), 4.35e4.37 (1H, m), 4.20e4.28 (3H, m); 2.94 (3H, s); 1.66e
1.73 (2H, m); 1.36e1.41 (2H, m); 0.98 (3H, t, J ¼ 7.5 Hz). 13C NMR
J ¼ 7.5 Hz); 3.08 (3H, s); 2.13e2.29 (2H, m); 1.10 (6H, d, J ¼ 7.0 Hz);
0.94 (6H, d, J ¼ 6.5 Hz). 13C NMR (75 MHz, CDCl3)
d 160.6, 143.9,
140.3, 137.7, 135.5, 129.8, 122.2, 114.9, 112.3, 109.6, 95.0, 75.1, 51.9,
30.8, 28.7, 23.5, 20.4, 19.7.
(75 MHz, CDCl3) d 159.7, 143.2, 140.7, 137.5, 135.4, 129.7, 122.5, 115.7,
115.6, 112.6, 109.4, 94.6, 69.7, 69.3, 67.7, 44.9, 33.0, 22.7, 22.6, 20.4,
14.1. Anal. Calcd for C19H21F3N2O2: C, 62.29; H, 5.78; N, 7.65. Found:
C, 62.23; H, 5.76; N, 7.74.
5.2.15. 7-Dceyloxy-9-isobutyl-1-methyl-b-carboline (4v)
White crystals (1.48 g, 75%) were obtained, mp 62e64 ꢀC. FAB-
MS m/z (M þ 1) 395; IR (KBr) 3431, 2950, 2922, 2849, 1625, 1567,
1447, 1335, 1254, 1202, 1142, 809 cmꢁ1; 1H NMR (500 MHz, CDCl3)
5.2.10. 7-Octyloxy-9-n-butyl-1-methyl-b-carboline (4o)
White crystals (1.37 g, 75%) were obtained, mp 75e76 ꢀC. FAB-
MS m/z (M þ 1) 367; IR (KBr) 2927, 2855, 1622, 1563, 1446, 1410,
1373, 1244, 1198, 1140, 1042, 806 cmꢁ1; 1H NMR (500 MHz, CDCl3)
d
8.27 (1H, d, J ¼ 5.0 Hz); 7.96 (1H, d, J ¼ 8.5 Hz); 7.75 (1H, d,
J ¼ 5.5 Hz); 6.86e6.89 (2H, m); 4.27 (2H, d, J ¼ 8.5 Hz); 4.08 (2H, t,
J ¼ 7.5 Hz); 3.03 (3H, s); 2.23e2.29 (1H, m); 1.82e1.88 (2H, m);
1.48e1.54 (2H, m); 1.28e1.40 (12H, m); 0.93 (6H, d, J ¼ 6.5 Hz); 0.88
d
8.27 (1H, d, J ¼ 5.5 Hz); 7.96 (1H, d, J ¼ 8.5 Hz); 7.74 (1H, d,
J ¼ 5.5 Hz); 6.85e6.90 (2H, m); 4.46 (2H, t, J ¼ 7.5 Hz); 4.10 (2H, t,
J ¼ 8.0 Hz); 3.05 (3H, s); 1.80e1.89 (4H, m); 1.29e1.53 (12H, m);
0.99 (3H, t, J ¼ 7.5 Hz); 0.89 (3H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz,
(3H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz, CDCl3)
d 160.3, 143.7, 140.7,
138.4, 135.8, 129.6, 122.2, 115.2, 112.3, 109.2, 95.2, 68.8, 52.1, 32.3,
30.8, 29.9(2C), 29.8(2C), 29.7, 26.5, 24.0, 23.0, 20.5, 14.5. Anal. Calcd
for C26H38N2O: C, 79.14; H, 9.71; N, 7.10. Found: C, 79.28; H, 9.76; N,
7.15.
CDCl3)
d 160.6, 143.3, 140.3, 137.9, 135.3, 129.6, 122.3, 115.1, 112.3,
109.3, 94.3, 68.7, 44.8, 33.0, 32.2, 30.1, 29.8 (2C), 29.6, 26.5, 23.4,
23.0, 20.5, 14.5, 14.2. Anal. Calcd for C24H34N2O: C, 78.64; H, 9.35; N,
7.64. Found: C, 78.58; H, 9.33; N, 7.68.
5.2.16. 7-(3-Phenylpropoxy)-9-isobutyl-1-methyl-b-carboline (4x)
White crystals (1.54 g, 83%) were obtained, mp 94e95 ꢀC. FAB-
MS m/z (M þ 1) 373; IR (KBr) 3041, 2958, 1621, 1567, 1448, 1405,
5.2.11. 7-Benzyloxy-9-n-butyl-1-methyl-b-carboline (4q)
White crystals (1.43 g, 83%) were obtained, mp 121e122 ꢀC. FAB-
MS m/z (M þ 1) 345; IR (KBr) 3424, 3036, 2957, 2929, 2866, 1622,
1565, 1495, 1448, 1408, 1377, 1349, 1240, 1192, 1139, 1009, 813,
1339, 1253, 1196, 1139, 1050, 977, 817 cmꢁ1 1H NMR (500 MHz,
;
CDCl3)
d
8.28 (1H, d, J ¼ 5.5 Hz); 7.97 (1H, d, J ¼ 8.5 Hz); 7.77 (1H, d,
J ¼ 5.5 Hz); 7.19e7.31 (5H, m); 6.88e6.91 (1H, m); 6.84e6.85 (1H,
m); 4.26 (2H, d, J ¼ 7.5 Hz); 4.10 (2H, t, J ¼ 8.0 Hz); 3.04 (3H, s); 2.88
(2H, t, J ¼ 7.5 Hz); 2.21e2.28 (1H, m), 2.16e2.20 (2H, m); 0.92 (6H,
731 cmꢁ1; 1H NMR (500 MHz, CDCl3)
d
8.27 (1H, d, J ¼ 5.5 Hz); 7.98
(1H, d, J ¼ 8.5 Hz); 7.76 (1H, d, J ¼ 5.5 Hz); 7.26e7.50 (5H, m); 6.92e
6.99 (2H, m); 5.22 (2H, s); 4.43 (2H, t, J ¼ 8.0 Hz); 3.05 (3H, s); 1.75e
1.78 (2H, m); 1.38e1.43 (2H, m); 0.95 (3H, t, J ¼ 8.0 Hz). 13C NMR
d, J ¼ 6.5 Hz). 13C NMR (75 MHz, CDCl3)
d 160.2, 143.7, 141.6, 140.8,
138.5, 135.8, 129.6, 128.7(2C), 126.2, 122.4, 115.3, 112.4, 109.3, 95.2,
67.7, 52.1, 32.6, 31.3, 30.9, 24.1, 20.6. Anal. Calcd for C25H28N2O: C,
80.61; H, 7.58; N, 7.52. Found: C, 80.48; H, 7.63; N, 7.58.
(75 MHz, CDCl3) d 160.0,143.2,140.8,138.4,137.1,135.6,129.5,128.9,
128.2, 127.7, 122.5, 115.7, 112.4, 109.4, 95.3, 70.9, 45.1, 33.0, 23.7,
20.5, 14.2. Anal. Calcd for C23H24N2O: C, 80.20; H, 7.02; N, 8.13.
Found: C, 80.16; H, 6.99; N, 8.19.
5.2.17. 7-n-Butoxy-9-(3-phenylpropyl)-1-methyl-b-carboline (4z)
White crystals (1.52 g, 82%) were obtained, mp 92e93 ꢀC. FAB-
MS m/z (M þ 1) 373; IR (KBr) 3024, 2955, 2868, 1622, 1565, 1497,
5.2.12. 7-(Perfluorobenzyloxy)-9-n-butyl-1-methyl-
(4r)
b-carboline
1447, 1409, 1368, 1241, 1161, 810, 738 cmꢁ1 1H NMR (500 MHz,
;
White crystals (1.58 g, 73%) were obtained, mp 121e122 ꢀC. FAB-
MS m/z (M þ 1) 435; IR (KBr) 3424, 3036, 2957, 2929, 2866, 1622,
1565, 1495, 1448, 1408, 1377, 1349, 1240, 1192, 1139, 1009, 813,
CDCl3)
d
8.25 (1H, d, J ¼ 5.5 Hz); 7.93 (1H, d, J ¼ 8.5 Hz); 7.72 (1H, d,
J ¼ 5.5 Hz); 7.20e7.33 (5H, m); 6.86 (1H, dd, J ¼ 2.0 Hz, 8.5 Hz); 6.64
(1H, d, J ¼ 2.0 Hz); 4.44 (2H, t, J ¼ 8.0 Hz); 3.99 (2H, t, J ¼ 8.5 Hz);
2.90 (3H, s); 2.76 (2H, t, J ¼ 7.5 Hz); 2.12e2.19 (2H, m); 1.80e1.86
731 cmꢁ1; 1H NMR (500 MHz, CDCl3)
d
8.29 (1H, d, J ¼ 5.5 Hz); 8.00

R. Cao et al. / European Journal of Medicinal Chemistry 60 (2013) 135e143
141
(2H, m); 1.52e1.59(2H, m); 1.03 (3H, t, J ¼ 7.5 Hz). 13C NMR
5.3. General procedure for the preparation of
bromides
b-carbolinium
(75 MHz, CDCl3)
d 160.5, 143.1, 140.9, 140.6, 138.4, 135.4, 129.5,
128.8, 128.6, 126.5, 122.4, 115.2, 112.3, 109.6, 93.9, 68.4, 44.3, 33.3,
32.0, 31.8, 23.5, 19.8, 14.4. Anal. Calcd for C25H28N2O: C, 80.61; H,
7.58; N, 7.52. Found: C, 80.68; H, 7.63; N, 7.56.
A mixture of b-carboline (2 mmol) and benzyl bromide (30e
50 mmol) in ethyl acetate (50 ml) was refluxed for 5e10 h. After
completion of the reaction as indicated by TLC, the solution was
cooled and filtered to afford yellow solid. The solid was recrystal-
lized from ethanol.
5.2.18. 7-Isobutoxy-9-(3-phenylpropyl)-1-methyl-b-carboline
(4aa)
White crystals (1.34 g, 76%) were obtained, mp 123e124 ꢀC.
FAB-MS m/z (M þ 1) 373; IR (KBr) 3415, 2961, 2934, 2869, 1622,
1565, 1495, 1447, 1411, 1365, 1242, 1205, 1161, 1039, 817,
5.3.1. 7-Butoxy-2-benzyl-9-ethyl-1-methyl-b-carbolinium bromide
(5b)
744 cmꢁ1
;
1H NMR (500 MHz, CDCl3)
d
8.27 (1H, d, J ¼ 5.5 Hz);
Yellow crystals (0.68 g, 75%) were obtained, mp 215e216 ꢀC.
FAB-MS m/z 373; IR (KBr) 3386, 3045, 2958, 2931, 2869, 1622,
7.94e7.97 (1H, d, J ¼ 8.5 Hz); 7.72e7.73 (1H, d, J ¼ 5.5 Hz); 7.23e
7.34 (5H, m); 6.87e6.90 (1H, m); 6.66e6.67 (1H, m); 4.46 (2H, t,
J ¼ 7.5 Hz); 3.77 (2H, d, J ¼ 8.0 Hz); 2.91 (3H, s); 2.77e2.81 (2H,
m); 2.16e2.20 (3H, m); 1.11 (6H, d, J ¼ 7.0 Hz). 13C NMR (75 MHz,
1577, 1454, 1374, 1256, 1221, 1132, 1032, 809, 735 cmꢁ1 1H NMR
;
(500 MHz, DMSO-d6)
d
8.80 (1H, d, J ¼ 6.5 Hz); 8.62 (1H, d,
J ¼ 6.5 Hz); 8.38 (1H, d, J ¼ 8.5 Hz); 7.36e7.44 (4H, m); 7.20 (2H, d,
J ¼ 7.0 Hz); 7.08e7.10 (1H, m); 6.06 (2H, s); 4.70e4.75 (2H, q,
J ¼ 7.0 Hz); 4.22 (2H, t, J ¼ 7.5 Hz); 3.11 (3H, s); 1.78e1.83 (2H, m);
1.48e1.55 (2H, m); 1.39 (3H, t, J ¼ 8.0 Hz); 0.98 (3H, t, J ¼ 7.5 Hz). 13C
CDCl3)
d 160.8, 143.2, 140.8, 140.5, 138.1, 135.3, 129.7, 128.8, 128.6,
126.5, 122.4, 115.1, 112.4, 109.8, 93.9, 75.0, 44.2, 33.2, 32.0, 28.9,
23.3, 19.8. Anal. Calcd for C25H28N2O: C, 80.61; H, 7.58; N, 7.52.
Found: C, 80.54; H, 7.55; N, 7.53.
NMR (75 MHz, DMSO-d6)
d 163.6, 147.5, 139.5, 135.9, 135.1, 134.9,
133.7, 129.9, 129.2, 127.3, 125.1, 114.8, 114.0, 113.1, 94.4, 69.0, 60.6,
41.1, 31.3,19.5,16.7,15.9,14.4. Anal. Calcd for C25H29BrN2O: C, 66.22;
H, 6.45; N, 6.15. Found: C, 66.35; H, 6.49; N, 6.13.
5.2.19. 7-(Pentan-3-yloxy)-9-(3-phenylpropyl)-1-methyl-b-
carboline (4ab)
White crystals (1.21 g, 63%) were obtained, mp 93e94 ꢀC. FAB-
MS m/z (M þ 1) 387; IR (KBr) 2967, 2934, 2874, 1621, 1564, 1494,
5.3.2. 7-Isobutoxy-2-benzyl-9-ethyl-1-methyl-b-carbolinium
1449, 1409, 1238, 1202, 1158, 974, 815 cmꢁ1
;
1H NMR (500 MHz,
bromide (5c)
CDCl3)
d
8.25 (1H, d, J ¼ 5.5 Hz); 7.93 (1H, d, J ¼ 8.5 Hz); 7.72 (1H,
Yellow crystals (0.72 g, 79%) were obtained, mp 222e224 ꢀC.
FAB-MS m/z 373; IR (KBr) 3420, 2959, 1622, 1454, 1372, 1259,
d, J ¼ 5.5 Hz); 7.20e7.33 (5H, m); 6.86e6.88 (1H, m); 6.71e6.72
(1H, m); 4.44 (2H, t, J ¼ 7.5 Hz); 4.19e4.21 (1H, m); 2.90 (3H,
s); 2.76 (2H, t, J ¼ 7.5 Hz); 2.12e2.18 (2H, m); 1.71e1.76 (4H, m);
1223, 1134, 1034, 1010, 826, 736 cmꢁ1 1H NMR (500 MHz, DMSO-
;
d6)
d
8.78 (1H, d, J ¼ 6.5 Hz); 8.61e8.62 (1H, d, J ¼ 6.5 Hz); 8.38 (1H,
1.01 (6H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz, CDCl3)
d
160.1, 143.2,
d, J ¼ 9.0 Hz); 7.35e7.43 (4H, m); 7.20 (2H, d, J ¼ 7.0 Hz); 7.09e7.11
(1H, m); 6.05 (2H, s); 4.74 (2H, t, J ¼ 7.5 Hz); 4.00 (2H, d, J ¼ 7.5 Hz);
3.11 (3H, s); 2.11e2.14 (1H, m); 1.39 (3H, t, J ¼ 7.5 Hz); 1.06 (6H, d,
140.9, 140.7, 138.5, 135.5, 129.6, 128.8, 128.6, 126.5, 122.5, 115.4,
112.3, 110.5, 96.1, 81.0, 44.5, 33.4, 32.1, 26.6,23.6, 10.1. Anal. Calcd
for C26H30N2O: C, 80.79; H, 7.82; N, 7.25. Found: C, 80.68; H, 7.89;
N, 7.28.
J ¼ 6.5 Hz). 13C NMR (75 MHz, DMSO-d6)
d 168.4, 152.3, 144.3, 140.6,
140.0, 139.7, 138.5, 134.6, 134.0, 132.0, 129.8, 119.5, 118.8, 117.9, 99.3,
73.8, 65.4, 36.0, 24.2, 21.5, 20.7, 19.1. Anal. Calcd for C25H29BrN2O: C,
66.22; H, 6.45; N, 6.18. Found: C, 66.38; H, 6.42; N, 6.23.
5.2.20. 7-(3-Methylbut-2-enyloxy)-9-(3-phenylpropyl)-1-methyl-
b
-carboline (4ac)
White crystals (1.25 g, 65%) were obtained, mp 95e96 ꢀC. FAB-
5.3.3. 7-Benzyloxy-2-benzyl-9-ethyl-1-methyl-b-carbolinium
MS m/z (M þ 1) 385; IR (KBr) 2962, 2927, 2856, 1622, 1567, 1446,
bromide (5d)
1409, 1239, 1159, 988, 817, 753 cmꢁ1 1H NMR (500 MHz, CDCl3)
;
Yellow crystals (0.81 g, 82%) were obtained, mp 240e242 ꢀC.
FAB-MS m/z 407; IR (KBr) 3437, 3005, 2969, 1620, 1577, 1452,
1335, 1260,1210,1133,1034, 994, 823, 731 cmꢁ1. 1H NMR (500 MHz,
d
8.25 (1H, d, J ¼ 5.5 Hz); 7.96 (1H, d, J ¼ 8.5 Hz); 7.73 (1H, d,
J ¼ 5.5 Hz); 7.19e7.33 (5H, m); 6.88e6.90 (1H, m); 6.70e6.71 (1H,
m); 5.52e5.56 (1H, m); 4.55 (2H, d, J ¼ 8.0 Hz); 4.44 (2H, t,
J ¼ 6.5 Hz); 2.91 (3H, s); 2.76 (2H, t, J ¼ 7.5 Hz); 2.13e2.19 (2H, m);
DMSO-d6)
d
8.81 (1H, J ¼ 7.0 Hz); 8.64 (1H, d, J ¼ 6.5 Hz); 8.41 (1H,
d, J ¼ 9.0 Hz); 7.53e7.56 (3H, m); 7.35e7.45 (6H, m); 7.17e7.21 (3H,
1.80 (6H, d, J ¼ 13.5 Hz). 13C NMR (75 MHz, CDCl3)
d 160.3, 143.1,
m); 6.06 (2H, s); 5.36 (2H, s); 4.73 (2H, q, t, J ¼ 6.5 Hz); 3.11 (3H, s);
140.8, 140.6, 138.4, 138.3, 135.4, 129.6, 128.8, 128.6, 126.5, 122.5,
119.8, 115.4, 112.4, 109.7, 94.4, 65.6, 44.5, 33.4, 32.1, 26.2, 23.4, 18.7.
Anal. Calcd for C26H28N2O: C, 81.21; H, 7.34; N, 7.29. Found: C, 81.28;
H, 7.43; N, 7.23.
1.38 (3H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz, DMSO-d6)
d 163.1, 147.4,
139.6, 136.7, 135.8, 135.3, 134.7, 133.7, 129.7, 129.0, 128.9, 128.5,
128.2, 127.1, 125.0, 114.7, 114.0, 113.4, 95.3, 70.7, 60.4, 40.9, 16.3, 15.3.
Anal. Calcd for C28H27BrN2O: C, 68.99; H, 5.58; N, 5.75. Found: C,
68.86; H, 5.61; N, 5.80.
5.2.21. 7-(3-Phenylpropoxyl)-9-(3-phenylpropyl)-1-methyl-b-
carboline (4ag)
5.3.4. 7-(3-Phenylpropoxy)-2-benzyl-9-ethyl-1-methyl-b-
White crystals (1.84 g, 85%) were obtained, mp 118e119 ꢀC.
FAB-MS m/z (M þ 1) 435; IR (KBr) 2932, 2867, 1623, 1567, 1495,
carbolinium bromide (5f)
Yellow crystals (0.8 g, 78%) were obtained, mp 189e191 ꢀC. FAB-
MS m/z 435; IR (KBr) 3410, 2989, 2933, 2876, 1623, 1453, 1370, 1341,
1260, 1220, 1134, 1035, 824, 732 cmꢁ1; 1H NMR (500 MHz, DMSO-
1449, 1411, 1366, 1240, 1161, 1042, 813, 752 cmꢁ1 1H NMR
;
(500 MHz, CDCl3)
d
8.25 (1H, d, J ¼ 5.5 Hz); 7.96 (1H, d, J ¼ 8.5 Hz);
7.77 (1H, d, J ¼ 5.5 Hz); 7.18e7.32 (10H, m); 6.89e6.92 (1H, m);
6.24e6.28 (1H, m); 4.44 (2H, t, J ¼ 8.0 Hz); 3.99 (2H, t, J ¼ 6.5 Hz);
2.92 (3H, s); 2.88 (2H, t, J ¼ 8.0 Hz); 2.76 (2H, t, J ¼ 7.0 Hz); 2.12e
d6)
d
8.81 (1H, d, J ¼ 6.5 Hz); 8.63 (1H, d, J ¼ 6.5 Hz); 8.40 (1H, d,
J ¼ 9.0 Hz); 7.11e7.43 (12H, m); 6.06 (2H, s); 4.71 (2H, q, J ¼ 7.5 Hz);
4.25 (2H, t, J ¼ 6.5 Hz); 3.11 (3H, s); 2.82 (2H, t, J ¼ 7.5 Hz); 2.10e2.16
(2H, m); 1.38 (3H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz, DMSO-d6)
2.20 (4H, m). 13C NMR (75 MHz, CDCl3)
d 160.4, 143.1, 141.7, 140.9,
140.7, 138.5, 135.5, 129.6, 128.8 (2C), 128.7, 128.6, 126.5, 126.2,
122.5, 115.4, 112.4, 109.6, 94.1, 67.6, 44.4, 33.3, 32.6, 32.1, 31.3, 23.6.
Anal. Calcd for C30H30N2O: C, 82.91; H, 6.96; N, 6.45. Found: C,
82.78; H, 7.01; N, 6.48.
d 163.5, 147.5, 141.8, 139.5, 135.9, 135.1, 134.9, 133.7, 130.0, 129.2,
128.9, 128.8, 127.3, 126.5, 125.1, 114.8, 113.9, 113.2, 94.5, 68.5, 60.7,
41.1, 32.2, 30.9, 16.8, 15.9. Anal. Calcd for C30H31BrN2O: C, 69.90; H,
6.06; N, 5.43. Found: C, 70.03; H, 6.12; N, 5.47.

142
R. Cao et al. / European Journal of Medicinal Chemistry 60 (2013) 135e143
5.3.5. 7-Isobutoxy-2-benzyl-9-n-butyl-1-methyl-
bromide (5g)
b
-carbolinium
d, J ¼ 8.5 Hz); 4.21 (2H, t, J ¼ 7.5 Hz); 3.07 (3H, s); 2.04e2.10 (1H,
m); 1.77e1.83 (2H, m); 1.44e1.50 (2H, m); 1.26e1.36 (12H, m);
Yellow crystals (0.8 g, 78%) were obtained, mp 247e249 ꢀC. FAB-
MS m/z 401; IR (KBr) 3401, 3020, 2957, 2869, 1620, 1578, 1456, 1377,
1247, 1207, 1137, 1012, 821, 721 cmꢁ1; 1H NMR (500 MHz, DMSO-d6)
0.83e0.86 (9H, m). 13C NMR (75 MHz, DMSO-d6)
d 163.4, 148.6,
139.7, 136.1, 135.6, 134.7, 134.0, 129.7, 129.0, 127.1, 124.8, 114.8, 113.9,
113.0, 95.6, 69.1, 60.5, 52.2, 31.5, 30.5, 29.2, 29.1, 29.0, 28.8, 28.7,
25.8, 22.3, 19.8, 16.6, 14.0. Anal. Calcd for C33H45BrN2O: C, 70.07; H,
8.02; N, 4.95. Found: C, 70.21; H, 8.04; N, 4.90.
d
8.80 (1H, d, J ¼ 6.5 Hz); 8.62 (1H, d, J ¼ 6.5 Hz); 8.38 (1H, d,
J ¼ 9.5 Hz); 7.36e7.43 (4H, m); 7.18e7.20 (2H, d, J ¼ 7.5 Hz); 7.09e
7.11 (1H, m); 6.05 (2H, s); 4.67 (2H, t, J ¼ 7.5 Hz); 4.00 (2H, d,
J ¼ 7.5 Hz); 3.09 (3H, s); 2.10e2.15 (1H, m); 1.70e1.76 (2H, m);
1.30e1.38 (2H, m); 1.05e1.06 (6H, d, J ¼ 6.5 Hz); 0.87 (3H, t,
5.3.10. 7-(3-Phenylpropoxy)-2-benzyl-9-isobutyl-1-methyl-b-
carbolinium bromide (5n)
J ¼ 7.5 Hz). 13C NMR (75 MHz, DMSO-d6)
d
163.8, 148.1, 139.6, 135.9,
Yellow crystals (0.78 g, 72%) were obtained, mp 204e206 ꢀC.
FAB-MS m/z 463; IR (KBr) 3410, 3023, 2957, 2871, 1621, 1579,
135.5, 134.8, 133.9, 129.7, 129.1, 127.1, 125.0, 114.6, 113.8, 113.2, 95.0,
75.3, 60.5, 45.5, 32.4, 28.1, 19.6, 19.3, 16.5, 13.8. Anal. Calcd for
C27H33BrN2O: C, 67.35; H, 6.91; N, 5.82. Found: C, 67.49; H, 6.96; N,
5.79.
1454, 1253, 1216, 1137, 1032, 821, 728 cmꢁ1 1H NMR (500 MHz,
;
DMSO-d6)
d
8.82 (1H, d, J ¼ 6.5 Hz); 8.65 (1H, d, J ¼ 6.5 Hz); 8.40
(1H, d, J ¼ 9.0 Hz); 7.11e7.44 (12H, m); 6.05 (2H, s); 4.53 (2H, d,
J ¼ 7.5 Hz); 4.22 (2H, t, J ¼ 7.5 Hz); 3.06 (3H, s); 2.80e2.83 (2H, m);
2.10e2.14 (2H, m); 2.03e2.09(1H, m); 0.82e0.83 (6H, d, J ¼ 6.5 Hz).
5.3.6. 7-n-Octyloxy-2-benzyl-9-n-butyl-1-methyl-b-carbolinium
bromate (5h)
13C NMR (75 MHz, DMSO-d6)
d 163.2, 148.6, 141.8, 139.7, 136.2,
Yellow crystals (0.92 g, 86%) were obtained, mp 196e198 ꢀC.
FAB-MS m/z 457; IR (KBr) 2926, 2857, 1621, 1560, 1458, 1374,
135.3, 134.9, 133.9, 129.9, 129.2, 129.0, 128.9, 127.2, 126.5, 125.0,
114.8, 114.0, 112.9, 95.5, 68.3, 60.7, 52.2, 32.1, 31.1, 30.9, 20.1, 16.9.
Anal. Calcd for C32H35BrN2O: C, 70.71; H, 6.49; N, 5.15. Found: C,
70.63; H, 6.45; N, 5.20.
1349, 1247, 1136, 1034, 819, 727 cmꢁ1 1H NMR (500 MHz, DMSO-
;
d6)
d
8.80 (1H, d, J ¼ 6.5 Hz); 8.63 (1H, d, J ¼ 6.5 Hz); 8.38 (1H, d,
J ¼ 9.0 Hz); 7.36e7.44 (4H, m); 7.19e7.20 (2H, m); 7.08e7.10 (1H,
m); 6.06 (2H, s); 4.66 (2H, t, J ¼ 7.5 Hz); 4.21 (2H, t, J ¼ 7.5 Hz); 3.09
(3H, s); 1.71e1.82 (2H, m); 1.46e1.49 (2H, m); 1.26e1.37 (12H, m);
5.3.11. 7-n-Butoxy-9-(3-phenylpropyl)-2-benzyl-1-methyl-b-
carbolinium bromide (5o)
0.85e0.94 (6H, m). 13C NMR (75 MHz, DMSO-d6)
d
163.6, 148.1,
White crystals (0.88 g, 82%) were obtained, mp 204e205 ꢀC.
FAB-MS m/z 463; IR (KBr) 3401, 3024, 2931, 2868, 1621, 1579,
139.6, 136.2, 135.4, 134.9, 133.9, 129.9, 129.2, 127.3, 125.1, 114.8,
113.9, 113.1, 94.9, 69.2, 60.7, 45.7, 32.8, 31.9, 29.4, 29.2, 29.1, 26.2,
22.7, 20.0, 16.9, 14.6, 14.3. Anal. Calcd for C31H41BrN2O: C, 69.26; H,
7.69; N, 5.21. Found: C, 69.30; H, 7.72; N, 5.18.
1453, 1372, 1243, 1255, 1135, 1025, 827, 755 cmꢁ1 1H NMR
;
(500 MHz, DMSO-d6)
d
8.78 (1H, d, J ¼ 6.5 Hz); 8.60 (1H, d,
J ¼ 6.5 Hz); 8.36 (1H, d, J ¼ 8.5 Hz); 7.07e7.43 (12H, m); 6.03 (2H, s);
4.67 (2H, t, J ¼ 7.5 Hz); 4.15 (2H, t, J ¼ 7.5 Hz); 2.97 (3H, s); 2.68e
2.71 (2H, m); 2.04e2.11 (2H, m); 1.77e1.83 (2H, m); 1.48e1.56
(2H, m); 0.99 (3H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz, DMSO-d6)
5.3.7. 7-Benzyloxy-2-benzyl-9-n-butyl-1-methyl-b-carbolinium
bromide (5i)
Yellow crystals (0.84 g, 82%) were obtained, mp 229e230 ꢀC.
FAB-MS m/z 435; IR (KBr) 3423, 3028, 2957, 2927, 2868, 1622,
d 163.5, 147.9, 141.2, 139.6, 136.1, 135.4, 134.9, 134.2, 133.9, 129.9,
129.2, 128.9, 128.8, 127.2, 126.6, 125.1, 114.8, 114.1, 94.5, 68.9, 60.6,
45.3, 32.4, 31.9, 31.2, 19.5, 16.6, 14.4. Anal. Calcd for C32H35BrN2O: C,
70.71; H, 6.49; N, 5.15. Found: C, 70.85; H, 6.53; N, 5.21.
1579, 1495, 1454, 1373, 1247, 1200, 1135, 1028, 819, 733 cmꢁ1 1H
;
NMR (500 MHz, DMSO-d6)
d
8.80 (1H, d, J ¼ 6.5 Hz); 8.63 (1H, d,
J ¼ 6.5 Hz); 8.41 (1H, d, J ¼ 9.0 Hz); 7.17e7.55 (12H, m); 6.05 (2H, s);
5.37 (2H, s); 4.65 (2H, t, J ¼ 7.5 Hz); 3.09 (3H, s); 1.68e1.71 (2H, m);
1.30e1.34 (2H, m); 0.87 (3H, t, J ¼ 7.5 Hz). 13C NMR (75 MHz, DMSO-
5.3.12. 7-(3-Pentyloxy)-9-(3-phenylpropyl)-2-benzyl-1-methyl-b-
carbolinium bromide (5p)
d6)
d
162.8, 147.8, 139.8, 136.8, 136.0, 135.3, 134.9, 133.7, 129.9, 129.2,
Yellow crystals (0.86 g, 78%) were obtained, mp 216e217 ꢀC.
FAB-MS m/z 477; IR (KBr) 3405, 2964, 2874, 1620, 1579, 1456,
128.8, 128.5, 127.2, 125.2, 114.9, 114.3, 113.3, 95.6, 70.7, 60.7, 45.8,
32.8, 20.0, 16.8, 14.4. Anal. Calcd for C30H31BrN2O: C, 69.90; H, 6.06;
N, 5.43. Found: C, 69.82; H, 6.01; N, 5.49.
1374, 1247, 1223, 1137, 1107, 1030, 979, 935, 826 cmꢁ1 1H NMR
;
(500 MHz, DMSO-d6)
d
8.78 (1H, d, J ¼ 6.5 Hz); 8.60 (1H, d,
J ¼ 6.5 Hz); 8.37 (1H, d, J ¼ 9.0 Hz); 7.09e7.43 (12H, m); 6.03 (2H, s);
4.67 (2H, t, J ¼ 7.5 Hz); 4.57e4.61 (1H, m); 2.97 (3H, s); 2.70 (2H, t,
J ¼ 6.5 Hz); 2.03e2.10 (2H, m); 1.65e1.78 (4H, m); 0.96 (6H, t,
5.3.8. 7-Isobutoxy-2-benzyl-9-isobutyl-1-methyl-b-carbolinium
bromide (5k)
Yellow crystals (0.76 g, 79%) were obtained, mp 255e257 ꢀC.
FAB-MS m/z 401; IR (KBr) 3422, 2992, 2959, 2894, 1619, 1578,
J ¼ 7.5 Hz). 13C NMR (75 MHz, DMSO-d6)
d 163.4, 148.0, 141.0, 139.4,
136.0, 135.5, 134.8, 134.0, 129.7, 129.0, 128.7, 128.5, 127.1, 126.4,
125.2, 114.6, 113.2, 95.9, 80.8, 60.5, 45.1, 32.2, 31.5, 26.1, 16.3, 9.5.
Anal. Calcd for C33H37BrN2O: C, 71.09; H, 6.69; N, 5.02. Found: C,
70.98; H, 6.75; N, 5.06.
1454, 1376, 1252, 1212, 1135, 1003, 825 cmꢁ1 1H NMR (500 MHz,
;
DMSO-d6)
d
8.82 (1H, d, J ¼ 6.5 Hz); 8.65 (1H, d, J ¼ 6.5 Hz); 8.39
(1H, d, J ¼ 9.0 Hz); 7.37e7.44 (4H, m); 7.18 (2H, d, J ¼ 7.0 Hz); 7.09e
7.11 (1H, m); 6.05 (2H, s); 4.54 (2H, d, J ¼ 7.5 Hz); 3.99 (2H, d,
J ¼ 7.5 Hz); 3.07(3H, s); 1.98e2.14 (2H, m); 1.05 (6H, d, J ¼ 6.5 Hz);
0.83 (6H, d, J ¼ 6.5 Hz). Anal. Calcd for C27H33BrN2O: C, 67.35; H,
6.91; N, 5.82. Found: C, 67.43; H, 6.97; N, 5.90.
5.3.13. 7-(3-Phenylpropoxy)-9-(3-phenylpropyl)-2-benzyl-1-
methyl-b-carbolinium bromide (5r)
Yellow crystals (1.06 g, 87%) were obtained, mp 207e208 ꢀC.
FAB-MS m/z 525; IR (KBr) 3401, 3022, 2938, 1620, 1579, 1453,
5.3.9. 7-Decyloxy-2-benzyl-9-isobutyl-1-methyl-
b
-carbolinium
1373, 1348, 1248, 1135, 1028, 825, 737 cmꢁ1 1H NMR (500 MHz,
;
bromide (5l)
DMSO-d6)
d
8.80 (1H, d, J ¼ 6.0 Hz); 8.61 (1H, d, J ¼ 6.0 Hz); 8.38
Yellow crystals (0.92 g, 78%) were obtained, mp 202e203 ꢀC.
FAB-MS m/z 485; IR (KBr) 3409, 2957, 2924, 2852, 1621, 1579,
(1H, d, J ¼ 9.0 Hz); 7.30e7.43 (3H, m), 7.10e7.29 (14H, m); 6.04 (2H,
s); 4.65 (2H, t, J ¼ 7.5 Hz); 4.16 (2H, t, J ¼ 7.5 Hz); 2.98 (3H, s); 2.83
(2H, t, J ¼ 7.5 Hz); 2.69 (2H, t, J ¼ 7.5 Hz); 2.06e2.16 (4H, m). 13C
1456, 1375, 1252, 1219, 1137, 1030, 820, 726 cmꢁ1 1H NMR
;
(500 MHz, DMSO-d6)
d
8.83 (1H, d, J ¼ 6.5 Hz); 8.65 (1H, d,
NMR (75 MHz, DMSO-d6) d 163.5, 147.9, 141.7, 141.0, 139.5, 136.0,
J ¼ 6.5 Hz): 8.39 (1H, d, J ¼ 9.0 Hz); 7.37e7.43 (4H, m); 7.18e7.20
135.5,134.7,133.9,129.7,129.1,128.8,128.7,128.6,128.5,127.1,126.4,
126.3, 125.0, 114.6, 113.9, 113.3, 94.7, 68.2, 60.5, 45.1, 32.2, 31.8, 31.4,
(2H, d, J ¼ 7.5 Hz); 7.08 (1H, d, J ¼ 8.5 Hz); 6.06 (2H, s); 4.54 (2H,

R. Cao et al. / European Journal of Medicinal Chemistry 60 (2013) 135e143
143
30.4, 16.3. Anal. Calcd for C37H37BrN2O: C, 73.38; H, 6.16; N, 4.63.
Found: C, 73.28; H, 6.20; N, 4.67.
control and vehicle as negative control. The weights of animals
were recorded every 3 days. All animals were sacrificed at the 21st
day after tumor inoculation and the tumors were excised and
weighed. The inhibition rate was calculated as follows:
5.4. Cytotoxicity assays in vitro
ðC ꢁ TÞ=C ꢃ 100
T: average tumor weight of treated group; C: average tumor
weight of negative control group.
Cytotoxicity assays in vitro were carried out using 96 microtitre
plate cultures and MTT staining according to the procedures
described by Cao et al. [4]. Briefly, cells were grown in RPMI-1640
medium containing 10% (v/v) fetal calf serum and 100
m
g mlꢁ1
penicillin and 100
m
g mlꢁ1 streptomycin. Cultures were propagated
Acknowledgments
at 37 ꢀC in a humified atmosphere containing 5% CO2. Cell lines
were obtained from Shanghai Institute of Biochemistry and Cell
Biology, Chinese Academy of Science. DMSO was used as the
solution for drugs. Final concentration of DMSO in the growth
medium was 2% (v/v) or lower, concentration without effect on cell
replication. In all of these experiments, three replicate wells were
used to determine each point.
This work was supported by MEGA-Project (2009ZX09102-004)
and Xinjiang Huashidan Pharmaceutical Co. Ltd and the Funda-
mental Research Funds for the Central Universities.
References
[1] M. Mahmoudian, H. Jalilpour, P. Salehian, Toxicity of Pegamum harmala:
review and a case report, Iran. J. Pharmacol. Ther. 1 (2002) 1e4.
5.5. Assay of acute toxicities
[2] Q. Chen, R. Chao, H. Chen, X. Hou, H. Yan, S. Zhou, W. Peng, A. Xu, Antitumor
and neurotoxic effects of novel harmine derivatives and structure-eactivity
relationship analysis, Int. J. Cancer 114 (2004) 675e682.
Acute toxicity assay was performed according to the method
described by Cao et al. [4]. Briefly, healthy C57BL/6 mice (9e12
weeks) weighing 18e22 g were housed in rooms where the
temperature was approximately 24 ꢂ 2 ꢀC, with a relative humidity
60e70%, and in 12 h lightedark cycle. The sterile food and water
were provided according to institutional guidelines. All animals
were provided by Shanghai Laboratory Animal Center of Chinese
Academy of Science. All animal procedures were approved by the
Animal Ethical Committee of the Sun Yat-sen University. Prior to
each experiment, mice were fastened overnight and allowed free
access to water. Various doses of the harmine derivatives ranging
from 1.0 to 500 mg/kg dissolved in 0.5% carboxymethyl cellulose
sodium (CMC-Na) salt solution were given via intraperitoneal (i.p.)
to different groups of healthy C57BL/6 mice, and each group con-
tained 10 mice (5 males and 5 females). After the administration of
the compounds, mice were observed continuously for the first 2 h
for any gross behavioral changes and deaths, then intermittently for
the next 24 h and occasionally thereafter for 14 days, and for the
onset of any delayed effects. All animals were sacrificed at the 14th
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 [19].
[3] J. Ishida, H.K. Wang, K.F. Bastow, C.Q. Hu, K.H. Lee, Cytotoxicity of harmine and
b-carboline analogs, Bioorg. Med. Chem. Lett. 9 (1999) 3319e3324.
[4] R. Cao, Q. Chen, X. Hou, H. Chen, H. Guan, Y. Ma, W. Peng, A. Xu, Synthesis,
acute toxicities and antitumor effects of novel 9-substituted
b-carboline
derivatives, Bioorg. Med. Chem. 12 (2004) 4613e4623.
[5] G. Duportail, Linear and circular dichroism of harmine and harmaline inter-
acting with DNA, Int. J. Biol. Macromol. 3 (1981) 188e193.
[6] Z. Taira, S. Kanzawas, C. Dohara, S. Ishida, M. Matsumoto, Y. Sakiya, Interca-
lation of six beta-carboline derivatives into DNA, Jpn. J. Toxicol. Environ.
Health 43 (1997) 83e91.
[7] A.M. Sobhani, S.A. Ebrahimi, M. Mahmoudian, An in vitro evaluation of human
DNA topoisomerase I inhibition by Peganum harmala L. seeds extract and its
carboline alkaloids, J. Pharm. Pharm. Sci. 5 (2002) 19e23.
b-
[8] R. Cao, W. Peng, H. Chen, Y. Ma, X. Liu, X. Hou, H. Guan, A. Xu, DNA binding
properties of 9-substituted harmine derivatives, Biochem. Biophys. Res.
Commun. 338 (2005) 1557e1563.
[9] C. Meester, Genotoxic potential of beta-carbolines: a review, Mutat. Res. 339
(1995) 139e153.
[10] Y. Song, J. Wang, S.F. Teng, D. Kesuma, Y. Deng, J. Duan, J.H. Wang, R.Z. Qi,
M.M. Sim,
b-Carboline as specific inhibitors of cyclin-dependent kinases,
Bioorg. Med. Chem. Lett. 12 (2002) 1129e1132.
[11] Y. Song, D. Kesuma, J. Wang, Y. Deng, J. Duan, J.H. Wang, R.Z. Qi, Specific
inhibition of cyclin-dependent kinases and cell proliferation by harmine,
Biochem. Biophys. Res. Commun. 317 (2004) 128e132.
[12] H. Kim, S.O. Sablin, R.R. Ramsay, Inhibition of monoamine oxidase A by beta-
carboline derivatives, Arch. Biochem. Biophys. 337 (1997) 137e142.
[13] S. Manabe, Y. Kanai, S. Ishikawa, O. Wada, Carcinogenic tryptophan pyrolysis
products potent inhibitors of type A monoamine oxidase and the platelet
response to 5-hydroxytryptamine, J. Clin. Chem. Clin. Biochem. 26 (1988)
265e270.
[14] C. Naranjo, in: D.K. Efron, B. Holmstedt, N.S. Kline (Eds.), Ethnopharmacologic
Search for Psychoactive Drugs, US Government Printing Office, Washington,
DC, 1967, pp. 385e391.
5.6. Assay of antitumor activity
[15] J.B. Hudson, E.A. Graham, G.H.N. Towers, Antiviral effect of harmine, a pho-
Antitumor activity against Lewis lung cancer and Sarcoma 180
was performed as described by Cao et al. [4] with a slightly modi-
fication. Briefly, Lewis lung cancer and S180 sarcoma cell lines were
provided by Shanghai Institute of Pharmaceutical Industry. Tumor
cells of Lewis lung cancer and S180 sarcoma were inoculated to
mice. After 7 days, tumors were taken out and cells harvested.
Viable tumor cells (2 ꢃ 106 cells/mouse) were inoculated to the
armpit of 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
a dosage about one fifth of LD50 value once a day for consecutive 7
days. Cyclophosphamide (CTX) at 30 mg/kg was used as a positive
toactive,
[16] C. Di Giorgio, F. Delmas, E. Ollivier, R. Elias, G. Balansard, P. Timon-David,
In vitro activity of the -carboline alkaloids harmane, harmine and harmaline
b-carboline alkaloid, Photochem. Photobiol. 43 (1986) 21e26.
b
toward parasites of the species Leishmania infantum, Exp. Parasitol. 106 (2004)
67e74.
[17] R. Cao, X. Guan, B. Shi, Z. Chen, Z. Ren, W. Peng, H. Song, Design, synthesis and
3D-QSAR of b-carboline derivatives as potent antitumor agents, Eur. J. Med.
Chem. 45 (2010) 2503e2515.
[18] J. Duan, R. Zhou, S. Zhao, M. Wang, C. Che, Studies on the chemical constit-
uents of Peganum multisectum Maxim I. The alkaloids from seeds and anti-
tumor activity, J. China Pharm. Univ. 29 (1998) 21e23.
[19] J. Kassa, J. Vachek, A comparison of the efficacy of pyridostigmine alone and
the combination of pyridostigmine with anticholinergic drugs as pharmaco-
logical pretreatment of tabun-poisoned rats and mice, Toxicology 177 (2002)
179e185.