Design, synthesis and in vitro and in vivo antitumor activities of novel bivalent β-carbolines

Article abstract of DOI:10.1016/j.ejmech.2012.11.033

A series of bivalent β-carbolines with a spacer of three to ten methylene units between the indole nitrogen was synthesized and evaluated as antitumor agents. The results demonstrated that compounds 18c, 21b, 25a and 31b exhibited strong cytotoxic activities with IC₅₀ value of lower than 20 μM against four tumor cell lines. Acute toxicities and antitumor efficacies of the selected compounds in mice were also evaluated, compounds 18b, 21b, 26a and 31b exhibited potent antitumor activities with tumor inhibition rate of over 40% in animal models. Preliminary structure-activity relationships analysis indicated that (1) the spacer length affected antitumor potencies, and four to six methylene units were more favorable; (2) the introduction of appropriate substituent into position-1 of β-carboline facilitated antitumor potencies.

Full text of DOI:10.1016/j.ejmech.2012.11.033

European Journal of Medicinal Chemistry 60 (2013) 10e22
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and in vitro and in vivo antitumor activities of novel bivalent
-carbolines
b
,
b
b
b
b
a
a
Buxi Shi a, Rihui Cao a , Wenxi Fan , Liang Guo , Qin Ma , Xuemei Chen , Guoxian Zhang , Liqin Qiu ,
*
,
Huacan Song a
*
a School of Chemistry and Chemical Engineering, Sun Yat-sen University, 135 Xin Gang Xi Road, Guangzhou 510275, PR China
b 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:
A series of bivalent
b-carbolines with a spacer of three to ten methylene units between the indole
Received 10 September 2012
Received in revised form
20 November 2012
Accepted 22 November 2012
Available online 29 November 2012
nitrogen was synthesized and evaluated as antitumor agents. The results demonstrated that compounds
18c, 21b, 25a and 31b exhibited strong cytotoxic activities with IC50 value of lower than 20 M against
four tumor cell lines. Acute toxicities and antitumor efficacies of the selected compounds in mice were
also evaluated, compounds 18b, 21b, 26a and 31b exhibited potent antitumor activities with tumor
inhibition rate of over 40% in animal models. Preliminary structureeactivity relationships analysis
indicated that (1) the spacer length affected antitumor potencies, and four to six methylene units were
m
Keywords:
Synthesis
more favorable; (2) the introduction of appropriate substituent into position-1 of b-carboline facilitated
antitumor potencies.
Bivalent
b-carbolines
Ó 2012 Elsevier Masson SAS. All rights reserved.
Antitumor
Structureeactivity relationships
1. Introduction
causes much more pronounced structural changes of DNA [35].
Consequently, bivalent b-carbolines were expected to exhibit more
The
b
-carboline alkaloids are a large group of natural and
potent antitumor efficacies than monomers and exert tumor cell
synthetic indole alkaloids with a broad spectrum of biochemical
effects and pharmaceutical functions [1]. Many previous reports
killing effects through similar mechanisms of action.
Bivalent
these cases, bivalent
-carboline ring (Fig. 1). Such compounds were found to be
potential anti-Alzheimer agents. In addition, the synthesis and
evaluation of bivalent -carbolines with a spacer of four to ten
methylene units at position-1 of -carboline nucleus as antitumor
b
-carbolines have been synthesized before [36e38]. In
focused on the effects of
b-carbolines on the central nervous system
b-carbolines were linked at postion-6 or 9 of
(CNS) [1], however, there have been intense research efforts in
b
recent years in the design and development of
b-carbolines as
a new class of antitumor agents [2e21].
b
b-Carbolines are initially discovered to exert their antitumor
b
effects by intercalating into DNA [22,23]. Subsequently, Top I and II
(topoisomerase I and II) [24e26], CDK (cyclin-dependent kinase)
[4,27,28], MK-2 (mitogen activated protein kinase-activated protein
kinase 2) [29], PLK1 (polo-like kinase) [30], kinesin-like protein Eg5
[31] and IKK (I-Kappa-B kinase) [32] were also found to be the
pharmacological targets of this class of compounds.
Previous studies indicated that dimerization of various inter-
calating agents by an appropriate linker could lead to a dramatic
increase in the DNA binding affinity [33,34]. Such compounds can
bind to DNA by bis-intercalation mode, which produces binding
sites that are at least twice as potent as monointercalation sites and
agents were also reported [35] (Fig. 1). However, there was no
information of systematic and detailed studies of structuree
activity relationships on antitumor efficacies in vitro and in vivo
of this class of compounds. In the present investigation, we re-
ported the synthesis, in vitro evaluation, in vivo efficacies and
preliminary structureeactivity relationships for the new bivalent
carbolines with a spacer of three to ten methylene units between
the indole nitrogen. To the best of our knowledge, all bivalent
b-
b
-
carbolines except 18b are novel compounds, and this is the first
time to report the acute toxicities and antitumor efficacies of this
class of compounds in mice.
2. Chemistry
* Corresponding authors. Tel.: þ86 20 84110918; fax: þ86 20 84112245.
E-mail addresses: caorihui@mail.sysu.edu.cn (R. Cao), yjhchx@mail.sysu.edu.cn
(H. Song).
The overall synthetic routes of monovalent and bivalent
b-car-
bolines are outlined in Schemes 1e4. Monovalent -carbolines 2, 3,
b
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.033

B. Shi et al. / European Journal of Medicinal Chemistry 60 (2013) 10e22
11
Fig. 1. The chemical structure of the representative reported and newly synthesized
bivalent -carbolines.
b
Scheme 2. Synthesis of the monovalent b-carbolines 10e14.
6 and 9 were synthesized according to already published methods
[2,39]. The preparation of monovalent -carbolines 1, 4e5, 7e8 and
b
16e17 have been already described as antitumor agents in our
previous reports [5e7,12].
and 7 and the reference drugs cisplatin. The human tumor cell
line panel consisted of gastric carcinoma (BGC-823), malignant
melanoma (A375), renal carcinoma (769-P and OS-RC-2),
epidermoid carcinoma of the nasopharynx (KB). In order to
enhance the solubility in aqueous solution, compounds 18e34
were converted into their water-soluble hydrochloride salt by
the usual methods before use. The results were summarized in
Table 1.
7-Methoxy-1-methyl-
-carboline-7-ol 8a by using acetic acid and hydrobromic acid as
demethylation solvent, and then reacted with isopropyl bromide by
b-carboline 7 was converted to 1-methyl-
b
the action of anhydrous K2CO3 in acetone to give monovalent
carboline 8 (Scheme 1). The 1-substituted 1,2,3,4-tetrahydro-
b
b
-
-
carboline-3-carboxylic acids 10e14a were prepared by the
condensation of -tryptophan with appropriate aldehydes via well-
L
As shown in Table 1, compounds 18c, 21b, 25a and 31b displayed
a broad spectrum of cytotoxic activities with IC50 value of lower
known PicteteSpengler condensation in acid media, and were
subsequently oxidated and decarboxylated in a single step through
the reaction of potassium dichromate according to the method
previously described by Snyder et al. [40] to afford monovalent b-
carbolines 10e14 (Scheme 2).
than 20
24b, 24e, 26a, 32a and 33b only exhibited strong cytotoxic effects
with IC50 value of lower than 20 M against three tumor cell lines.
mM against four tumor cell lines, while compounds 24a,
m
Interestingly, compounds 27b, 30a and 33a were selectively active
against A375 and 769-P cell lines with IC50 value of lower than
Esterification of the intermediate 14a with ethanol in the
presence of SOCl2 by heating yielded the corresponding ethyl
20
mM but fail to show cytotoxic effects in other cell lines at the
1,2,3,4-tetrahydro-
by dehydrogenation with sulfur in refluxing xylene to afford
monovalent -carboline 15 (Scheme 3).
The symmetrical bivalent -carbolines 18e34 were prepared by
reaction of appropriate dibromoalkane with monovalent -carbo-
b-carboline-3-carboxylate 14b, and followed
concentration of 200
mM. Similarly, compounds 19 and 21h dis-
played selective activities against 769-P and SK-OV-3 cell lines but
were inactive against BGC, A375 and KB cell lines. Moreover,
compounds 21c, 21e and 24c were found to exhibit selective
activity against SK-OV-3 cell lines. Unfortunately, compounds 20,
21g, 23b, 25b, 28a, 32b, 34a and 34b were almost inactive against
all tumor cell lines tested.
b
b
b
lines 1e17 in anhydrous DMF. The chemical structures of all the
newly synthesized compounds were characterized by EI-MS, 1H
NMR and 13C NMR.
We examined the influence of the spacer length of bivalent b-
carbolines on cytotoxic activities. The data collected in Table 1
showed that compounds 18aec with a spacer of four to six meth-
ylene units exhibited moderate to strong cytotoxic activities, and
compound 18c with a spacer of six methylene units was found to be
3. Results and discussion
3.1. Cytotoxicity in vitro
the highest cytotoxic agent with all IC50 value of lower than 40 mM
The cytotoxic potencies of all the newly synthesized bivalent
b
-carbolines against a panel of human tumor cell lines were
investigated and compared with the monovalent -carbolines 4
b
Scheme 3. Synthesis of the monovalent b-carboline 15.
Scheme 1. Synthesis of the monovalent b-carboline 8.

12
B. Shi et al. / European Journal of Medicinal Chemistry 60 (2013) 10e22
introducing an appropriate substituent into position-1 of b-carbo-
line ring facilitated cytotoxic potency.
3.2. Preliminary assessment of acute toxicity
The LD50 values and scores for neurotoxicity of the selected
bivalent -carbolines in mice after administration by i.p. route were
summarized in Table 2. All the tested bivalent -carbolines resulted
b
b
in acute toxic manifestation but caused no obvious neurotoxic effects
including tremor, twitch, jumping and supination just like mono-
valent
b-carboline 7 (Harmine). Animals showed a decrease in
Scheme 4. Synthesis of the bivalent b-carbolines 18e34.
locomotor activity after the administration of various bivalent
b-
carbolines. Death occurred mostly in the high dosage group with
30 min after injection. All surviving animals returned to normal in
the next day. Autopsy of the animals that died in the course of
experimentandthenecropsyfindings insurviving animals attheend
of experimental period (14 days) revealed no obvious changes in any
organs.
against all tumor cell lines tested. While compounds 18d,e with
a spacer of seven to eight methylene units only had marginal or no
cytotoxic effect in any cell lines. Similarly, compounds 21aeh with
a spacer of three to ten methylene units showed low to high
cytotoxic effects against tumor cell lines, compound 21b with
a spacer of four methylene units displayed the highest cytotoxic
Of all investigated bivalent
b-carbolines, compound 24a having
effects with IC50 value of lower than 30
m
M against all tumor cell
a methoxy group in position-7 of b
-carboline ring displayed
lines tested, and compounds 21a and 21d with a spacer of three and
six methylene units, respectively, showed moderate cytotoxic
activities, while other compounds only exhibited selective activities
against one or two tumor cell lines. These results suggested that the
length of the spacer affected cytotoxic activities and four to six
methylene units were more favorable.
remarkable acute toxicity with LD50 value of 50 mg kgꢀ1. Replace-
ment of the methoxy group of compound 24a in postion-7 with an
isopropoxy substituent gave compound 26a which exhibited lower
acute toxicity with LD50 value of 220 mg kgꢀ1. In addition,
compounds 18aec, 21b, 21d, 31b and 33a having no substituent in
position-7 of
toxicities with LD50 value of over 150 mg kgꢀ1. These results sug-
gested that (1) the methoxy group in position-7 of -carboline ring
b-carboline ring also demonstrated weaker acute
In comparison with compounds 21aeh, compounds 24aee had
an additional methoxy group appended to position-7 of
ring. It was interesting to note that the spacer length in these
bivalent -carbolines has little influence on their cytotoxic activi-
ties. These results suggested that the electron-donating group of
b-carboline
b
might play an important role in determining acute toxicity; (2) the
acute toxicity decreased remarkably by replacing the methoxy
substituent with a bulky alkoxy group or removing the methoxy
substituent.
b
b
-
carboline ring might be facilitated their cytotoxic potencies.
Moreover, the A375 and SK-OV-3 cell lines were more sensitive to
these compounds than other cell lines.
3.3. Evaluation of antitumor activity
Next, we evaluated the effect of various substituents in b-car-
boline ring on cytotoxic activities in these bivalent compounds.
Table 1 showed that compound 18c with a spacer of six methylene
units showed strong cytotoxic activities with all IC50 value of lower
Nine bivalent b-carbolines were selected for evaluation in vivo
against mice bearing CT-26 colon cancer, Lewis lung cancer,
Sarcoma 180, H22 liver cancer and B16 melanoma and compared
than 40
m
M against all tumor cell lines. Introduction of a bromo
with monovalent
drugs Cyclophosphamide (CTX). The tumor inhibition rates of all
investigated bivalent -carbolines were summarized in Table 2.
As shown in Table 2, all the tested bivalent -carbolines dis-
played moderate to strong antitumor activities in animal model.
Interestingly, compounds 18b and 21b exhibited remarkable anti-
tumor activities with the tumor inhibition rate of over 40% against
mice bearing CT-26 colon cancer, Lewis lung cancer, Sarcoma 180
and H22 liver cancer at dose 30 and 40 mg kgꢀ1, respectively.
Similarly, compounds 26a and 31b also exhibited excellent anti-
tumor activities with the tumor inhibition rate of over 40% against
CT-26 colon cancer, Lewis lung cancer and Sarcoma 180 at dose 40
and 50 mg kgꢀ1, respectively.
Particularly, compound 21b was found to be the most potent
antitumor agent with the tumor inhibition rate of 50.8 and 56.2%
against mice bearing CT-26 colon cancer and Sarcoma 180, respec-
tively. Moreover, compounds 26a and 31b exhibited good antitumor
activities with the tumor inhibition rate of 51.2 and 50.6% against
mice bearing CT-26 colon cancer. It is interesting to note that CT-26
colon cancer was more susceptible to all tested compounds than
Lewis lung cancer, Sarcoma 180, H22 liver cancer and B16
melanoma.
b-carbolines 4 and 7 (harmine) and the reference
group in position-6 of compound 18c led to compound 19, which
only exhibited selective cytotoxic activities against 769-P and SK-
OV-3 cell lines. Replacement of the hydrogen atom in position-8
of compound 18c by a bromo group to yield compound 20 failed
to show cytotoxic effects in other cell lines at the concentration of
b
b
200
activities, while compound 23b bearing a bromo group in position-
6 of -carboline ring was almost inactive. These results suggested
mM. Similarly, compound 21d displayed moderate cytotoxic
b
that the bromo group in position-6 or 8 of
detrimental to their cytotoxic activities.
b-carboline ring was
Compound 21d having a methyl group in position-1 displayed
moderate cytotoxic activities. The cytotoxic potency of compound
27a bearing an isopropyl group in position-1 was comparable to
compound 21d. Replacement of the methyl group in position-1 of
compound 21d by a 2-chloro-5-nitrophenyl group (compound 30a)
and a 3,4,5-trimethoxyphenyl substituent (compound 33a) resul-
ted in some enhancement in the activity against A375 and 769-P
cell lines but loss of activity against BGC, KB and SK-OV-3 cell
lines. Interestingly, replacement of the methyl group in position-1
of compound 21d by a pyridin-3-yl group to yield compound 31b
showed significant enhance the activity with IC50 value of 14.1, 5.3,
13.3 and 14.9
m
M against BGC, A375, KB and SK-OV-3 cell lines,
Compounds 18a and 18c with a spacer of 4 and 6 methylene
units, respectively, showed moderate antitumor activities in all
animal models with the tumor inhibition rate of lower than 40%,
whereas compound 18b with a spacer of 5 methylene units
exhibited strong antitumor activities against mice bearing CT-26
respectively. Unfortunately, replacement of the methyl group in
position-1 of compound 21d by a thiophen-3-yl group (compound
28a) and a 2-chlorophenyl group (compound 29a) resulted in
significant loss of cytotoxic activities. These results suggested that

B. Shi et al. / European Journal of Medicinal Chemistry 60 (2013) 10e22
13
Table 1
Cytotoxic activities of bivalent b-carbolines in vitro.
R3
R6
R7
N
N
R1
R8
(CH2)n
R1
N
R8
R7
R6
N
R3
Compounds
R1
R3
R6
R7
R8
n
IC50
BGCb
(m
Ma)
A375
769-P
199
15.3
12.8
53.0
>200
3.1
>200
145
6.5
>200
149
51.7
>200
>200
16.2
>200
4.3
>200
65.6
81.0
>200
114
65.6
>200
56.0
KB
SK-OV-3
18a
18b
18c
18d
18e
19
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
Br
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4
5
6
7
8
4
6
3
4
5
6
7
8
9
10
6
5
6
4
5
6
7
8
59.7c
23.5
13.8
>200
>200
>200
>200
>200
10.2
10.8
>200
>200
>200
>200
69.5
13.1
>200
38.0
>200
>200
67.2
>200
>200
>200
>200
13.9
38.9
>200
33.5
8.9
>200
26.4
4.8
18.8
16.9
96.8
>200
61.9
8.5
>200
>200
63.4
>200
>200
>200
>200
>200
160
6.4
>200
87.9
>200
>200
>200
>200
>200
>200
>200
2.8
5.0
155
>200
>200
>200
>200
22.3
>200
134
>200
>200
>200
>200
>200
>200
>200
75.7
58.6
>200
104
75.7
8.5
>200
20
21a
21b
21c
21d
21e
21f
21g
21h
22
23a
23b
24a
24b
24c
24d
24e
25a
25b
26a
26b
27a
27b
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH(CH3)2
CH(CH3)2
COOC2H5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OC4H9
OC4H9
OCH(CH3)2
OCH(CH3)2
H
6.2
7.1
1.7
11.7
10.2
7.1
<1.4
35.3
113
6.2
3.8
>200
18.5
43.3
53.5
19.6
38.6
2.8
6.8
4
6
6
9
6
10
5.5
>200
19.8
112
>200
>200
>200
17.1
101
31.8
>200
194
>200
>200
>200
95.4
40.0
18.5
4.6
H
28a
28b
H
H
H
H
H
H
H
H
6
8
>200
>200
>200
>200
>200
>200
>200
>200
S
32.1
7.0
>200
S
Cl
Cl
Cl
29a
H
H
H
H
6
>200
3.8
>200
>200
NO2
29b
30a
H
H
H
H
H
H
H
H
9
6
>200
>200
>300
1.8
>200
>200
>200
>200
NO2
23.2
>200
(continued on next page)

14
B. Shi et al. / European Journal of Medicinal Chemistry 60 (2013) 10e22
Table 1 (continued )
Compounds
R1
R3
R6
R7
R8
n
IC50
BGCb
(m
Ma)
A375
769-P
KB
SK-OV-3
Cl
30b
H
H
H
H
7
>200
>200
10.4
82.1
>200
>200
N
31a
31b
32a
32b
H
H
H
H
H
H
H
H
H
H
H
H
H
4
6
5
6
85.3
5.3
>200
>200
132
>200
36.8
14.9
N
N
N
H
14.1
13.3
COOC2H5
COOC2H5
24.1
13.0
15.7
>200
68.3
>200
>200
>200
>200
OCH3
OCH3
33a
33b
34a
34b
H
H
H
H
H
H
H
H
H
H
H
H
H
6
7
6
9
>200
>200
>200
5.9
4.0
16.8
>200
>200
>200
>200
>200
OCH3
OCH3
H
3.3
7.6
OCH3
OCH3
OCH3
COOC2H5
>200
>200
>200
OCH3
OCH3
OCH3
COOC2H5
>200
>200
63.2
17.3
>200
>200
72.5
39.2
>200
>200
>200
57.8
13.1
OCH3
OCH3
4
7
175
48.9
11.6
92.6
74.6
4.2
Cisplatin
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
c
Cell lines include gastric carcinoma (BGC-823), malignant melanoma (A375), renal carcinoma (769-P and OS-RC-2), epidermoid carcinoma of the nasopharynx (KB).
Data represent the mean values of three independent determinations.
colon cancer, Lewis lung cancer, Sarcoma 180, H22 liver cancer with
the tumor inhibition rate of 48.6, 42.5, 43.8 and 40.4%. These results
indicated that the length of spacer played a vital role in deter-
mining antitumor activities in animal models.
substituent into position-1 of b-carboline nucleus, respectively,
increased the antitumor activities of these compounds. These results
implied that introducing an appropriate substituent into position-1
of b-carboline nucleus enhanced their antitumor activities.
In comparison with compound 21b, methoxy substituent in
position-7 (24a) and replacement of methyl group inposition-1 with
hydrogen atom (18a) reduced the antitumor activities of the
4. Conclusions
compounds. Of all bivalent
b-carbolines witha spacer of 6 methylene
In this study, a series of bivalent b-carbolines with a spacer of
units, compound 18c having no substituents in
showed moderate antitumor activities, while introducing a methyl
(21d), a pyridin-3-yl (31b) and a 3,4,5-trimethoxyphenyl (33a)
b
-carboline ring
three to ten methylene units between the indole nitrogen was
designed, synthesized and evaluated as potential antitumor agents.
The results demonstrated that compounds 18c, 21b, 25a and 31b

B. Shi et al. / European Journal of Medicinal Chemistry 60 (2013) 10e22
15
Table 2
Acute toxic effects of bivalent b-carbolines in mice and antitumor activities of these compounds against mice bearing CT-26 colon cancer, Lewis lung cancer, Sarcoma 180, H22
liver cancer and B16 melanoma.
Compounds
Acute toxicity
Dosage
% Maximum
weight loss
Tumor inhibition rate (%)
(mg kgꢀ1
)
LD50
Neurotoxic
effect
CT-26 colon
cancer
Lewis lung
cancer
Sarcoma
180
H22 liver
cancer
B16
melanoma
(mg kgꢀ1
)
18a
18b
18c
21b
21d
24a
26a
31b
200
150
200
200
200
50
220
250
200
32.6
59.0
ea
40
30
40
40
40
10
40
50
40
10
7.5
50
þ14.5
þ10.0
þ15.0
þ21.5
þ5.1
39.7
48.6
32.4
50.8
39.6
45.5
51.2
50.6
45.8
ND
34.2
42.5
37.7
40.4
42.1
38.1
49.6
43.2
43.3
ND
NDb
43.8
ND
56.2
ND
ND
ND
e
40.4
31.2
42.3
ND
26.1
24.8
29.9
ND
e
e
e
e
þ7.7
ND
ND
ND
e
þ10.2
þ11.9
þ7.2
40.6
40.5
ND
35.2
15.3
85.6
39.7
35.0
ND
ND
ND
20.5
24.3
ND
ND
ND
e
33a
e
4c
e
þ11.8
þ8.5
7 (Harmine)d
CTX
þþ
ND
97.2
34.1
97.8
þ7.7
83.8
86.5
a
Acute neurotoxic manifestation were denoted by “þ” and “ꢀ”. A “þ” represents toxic responses including tremble, twitch, jumping and supination, while “e” means no
such reaction.
b
c
ND ¼ not determined.
See Ref. [7].
d
See Ref. [22].
exhibited strong cytotoxic activities with IC50 value of lower than
20 M against four tumor cell lines. Acute toxic assays of the
selected bivalent -carbolines in mice indicated that all compounds
caused no obvious neurotoxic effects including tremor, twitch,
jumping and supination just like harmine. In addition, nine bivalent
determined in capillary tubes on an electrothermal PIF YRT-3
apparatus and without correction. ESI-MS spectra were obtained
from VG ZAB-HS spectrometer. 1H NMR and 13C NMR spectra were
recorded on a Mercury-Plus 300 spectrometer at 300 MHz and
75 MHz and a Varian INOVA 500NB spectrometer at 500 MHz and
125 MHz, respectively, using TMS as internal standard and CDCl3 or
m
b
b
-carbolines were selected for evaluation in vivo against mice
bearing CT-26 colon cancer, Lewis lung cancer, Sarcoma 180, H22
liver cancer and B16 melanoma, compounds 18b, 21b, 26a and 31b
exhibited remarkable antitumor efficacies with tumor inhibition
rate of over 40% in three animal models, and the CT-26 colon cancer
model was more susceptible to all tested compounds than other
animal models. Preliminary structureeactivity analysis indicated
that (1) the spacer length affected antitumor activities, and four to
six methylene units were more favorable; (2) the introduction of
DMSO-d6 as solvent and chemical shifts (d) were expressed in ppm.
Silica gel F254 were used in analytical thin-layer chromatography
(TLC) and silica gel were used in column chromatography
respectively.
The preparation of the following monovalent
was described earlier: -carboline 1 [5], 6-bromo-
[39], 8-bromo- -carboline 3 [39], 1-methyl- -carboline 4 [7], ethyl
1-methyl- -carboline-3-carboxylate 5 [6], 6-bromo-1-methyl-
carboline 6 [39], 7-methoxy-1-methyl- -carboline 7 [5], 7-butoxy-
1-methyl- -carboline 9 [2], 1-(3,4,5-trimethoxyphenyl)- -carbo-
-carboline-3-
b-carbolines
b
b-carboline 2
b
b
b
b-
appropriate substituent into position-1 of
tated antitumor potencies.
b-carboline ring facili-
b
b
b
Although our current investigation described a preliminary
structureeactivity analysis between bivalent -carbolines and
line 16 [12], ethyl 1-(3,4,5-trimethoxyphenyl)-
b
b
carboxylate 17 [12].
antitumor activities, while the antitumor efficacies of the studied
compounds presented here remained relatively moderate. More-
over, the low water solubility of bivalent b-carbolines in this study
limited their in vitro and in vivo efficacies. Therefore, further design
and synthesize more potent antitumor agents together with
improved water solubility is needed.
5.2. Synthesis of 7-isopropoxy-1-methyl-
b
-carboline (8)
-carboline 7 (2.12 g,
A
mixture of 7-methoxy-1-methyl-
b
10 mmol), acetic acid (100 ml) and 40% hydrobromic acid (50 ml)
was refluxed for 12 h. After completion of the reaction as indicated
by TLC, the mixture was cooled and poured onto ice. The aqueous
mixture, made basic with sodium hydroxide. The precipitate
formed was collected by filtration, washed well with water and
dried in vacuum to give the intermediate 1-methyl-b-carboline-7-
ol 8a. The intermediate 8a prepared in these ways could be used
directly in the next step without further purification.
Our previous studies indicated that intercalation into DNA is
a major cellular event of monovalent b-carbolines [26] and these
compounds can pass through cell membrane and penetrate into
nucleus quickly resulting in intercalating into DNA in cells [41].
Moreover, previous reports showed that dimerization of various
intercalating agents could lead to a very large increase in the DNA
binding affinity by bis-intercalation [33,34]. Consequently, such
A mixture of 1-methyl-b-carboline 7-ol 8a (1.98 g, 10 mmol) and
bivalent b-carbolines were expected to exert their tumor cell killing
acetone (60 ml) was stirred for 10 min, and then anhydrous K2CO3
(2.76 g, 20 mmol) and isopropyl bromide (20 mmol) were added.
The mixture was stirred and refluxed. 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 crystallized 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 to give white solids
effects through similar mechanisms of action. Further investigation
to elucidate the pharmacological mechanisms of this class of
compounds are underway in our laboratory, and the data will be
published elsewhere.
5. Experimental section
5.1. Reagents and general methods
All reagents were purchased from commercial suppliers and
were dried and purified when necessary. Melting points were

16
B. Shi et al. / European Journal of Medicinal Chemistry 60 (2013) 10e22
(2.1 g, 87%). 1H NMR (300 MHz, CDCl3):
J ¼ 5.4 Hz), 7.94 (1H, d, J ¼ 8.7 Hz), 7.71 (1H, d, J ¼ 5.4 Hz), 6.95 (1H,
d, J ¼ 2.1 Hz), 6.87 (1H, dd, J ¼ 2.1 Hz, 8.7 Hz), 4.58e4.70 (1H, m),
2.81 (3H, s), 1.41 (3H, s), 1.39 (3H, s); 13C NMR (75 MHz, CDCl3):
d
8.58 (1H, s), 8.29 (1H, d,
4.5 Hz), 8.61 (1H, d, J ¼ 5.4 Hz), 8.38e8.42 (1H, m), 8.18 (1H, d,
J ¼ 7.8 Hz), 8.04 (1H, d, J ¼ 5.4 Hz), 7.52e7.61 (3H, m), 7.30e7.36
(1H, m); 13C NMR (75 MHz, DMSO):
149.9, 149.5, 141.7, 139.8,
d
139.2,136.5,134.6,133.8,130.1, 129.1, 124.5,122.3,121.3, 120.4,115.1,
d
158.8, 142.5, 141.8, 138.2, 135.1, 128.0, 123.3, 115.4, 112.6, 110.9,
113.0; ESI-MS m/z: 246 [M þ H]þ.
97.4, 70.3, 22.6, 21.0; ESI-MS m/z: 241 [M þ H]þ.
5.4. Synthesis ofethyl 1-(pyridin-3-yl)-b-carboline-3-carboxylate (15)
5.3. Generalprocedure for thepreparationofmonovalent b-carbolines
(10e14)
A mixture of the 14a (30 mmol), anhydrous ethanol (500 ml)
and SOCl2 (20 ml) was heated at reflux for 4 h, and then evaporated
in reduced pressure. The resulting mixture was poured into H2O
(300 ml) and neutralized with sodium hydrogen carbonate. The
solution was extracted with ethyl acetate (3 ꢁ 300 ml). The organic
phase was washed with water and brine, then dried over anhydrous
sodium sulfate, filtered and evaporated. The residue was crystal-
lized from ethyl acetate to afford white solid 14b. Further purifi-
cation was not necessary and used directly for the next steps.
A suspension of compound 14b (30 mmol) and sulfur (4.8 g,
150 mmol) in xylene (250 ml) was heated at reflux for 8 h. The
solution was cooled and stored at 4 ꢂC for 3 h, and then filtered and
washed generously with petroleum ether, the solid was dried and
recrystallized from ethyl acetate to afford white crystals (8.2 g,
A mixture of L-tryptophan (20.4 g,100 mmol), acetic acid (300 ml)
and the appropriate aldehydes (100 mmol) was refluxed for 3 h, then
cooled and adjusted pH to 5 with concentrated ammonium
hydroxide, the precipitated product was collected by filtration and
washed well with water and dried to provide the intermediates
1,2,3,4-tetrahydro-
purification was not necessary and used directly for the next steps.
1,2,3,4-Tetrahydro- -carboline-3-carboxylic acid 10ae14a
(10 mmol) was diluted to 100 ml with water, and then heated to
boiling. To the hot solution was added potassium dichromate (15 g)
and acetic acid (20 ml). The brown suspension was heated for
20 min and then cooled under the tap. After treating the cold
solution with sodium sulfite to remove the excess oxidizing agent,
the mixture was made definitely alkaline with sodium hydroxide.
The solution was extracted exhaustively with ethyl acetate
(1000 ml), the extracts dried over anhydrous sodium sulfate, and
the solvent removed. The residue was crystallized from anhydrous
ethanol, and compounds 10e14 were obtained.
b-carboline-3-carboxylic acid 10ae14a. Further
b
86%), 1H NMR (300 MHz, CDCl3):
d 11.6 (1H, s), 9.48 (1H, s), 8.91 (1H,
s), 8.52e8.54 (1H, m), 8.42e8.45 (1H, m), 8.23 (1H, d, J ¼ 7.8 Hz),
7.58 (2H, d, J ¼ 3.9 Hz), 7.43e7.48 (1H, m), 7.35e7.40 (1H, m), 4.55
(2H, q, J ¼ 7.2 Hz), 1.52 (3H, t, J ¼ 7.2 Hz); 13C NMR (75 MHz, DMSO):
d
165.9, 150.3, 149.6, 142.0, 139.8, 137.7, 136.9, 135.2, 133.9, 130.0,
129.5, 124.6, 122.6, 121.6, 121.2, 117.7, 113.3, 61.5, 15.2; ESI-MS m/z:
318 [M þ H]þ.
5.3.1. 1-Isopropyl-b-carboline (10)
Afforded white crystals (1.41 g, 67%). 1H NMR (300 MHz, CDCl3):
5.5. General procedure for the preparation of bivalent b-carbolines
d
8.50 (1H, s), 8.43 (1H, d, J ¼ 5.4 Hz), 8.12 (1H, d, J ¼ 7.8 Hz), 7.82
18e34
(1H, d, J ¼ 5.4 Hz), 7.52e7.54 (2H, m), 7.27e7.30 (1H, m), 3.45e3.59
(1H, m), 1.52 (3H, s), 1.49 (3H, s); 13C NMR (75 MHz, CDCl3):
d
150.8,
To a stirred solution of monovalent b-carbolines 1e17
140.7, 138.5, 133.8, 129.1, 128.4, 122.2, 121.9, 120.1, 113.2, 111.9, 32.4,
(2.0 mmol) in anhydrous DMF (30 ml) was added 60% NaH (0.4 g,
10 mmol). After stirring for 20 min at room temperature, the
appropriate dibromoalkane (1.0 mmol) was added. And then the
reaction mixture was stirred at room temperature for 8e20 h. After
completion of the reaction as indicated by TLC, the solution was
poured into ice-water and extracted with CH2Cl2. The combined
organic layer was washed with water and brine, dried over anhy-
drous Na2SO4, filtered and evaporated in vacuum. The residue was
purified by column chromatography with CH2Cl2/MeOH (50:1) to
successfully afford the desirable target products.
22.0; ESI-MS m/z: 211 [M þ H]þ.
5.3.2. 1-(Thiophen-3-yl)-b-carboline (11)
Afforded yellow solids (2.1 g, 83%). 1H NMR (300 MHz, CDCl3):
d
8.77 (1H, s), 8.47 (1H, d, J ¼ 5.1 Hz), 8.11 (1H, d, J ¼ 8.1 Hz), 7.87
(2H, d, J ¼ 5.1 Hz), 7.74e7.75 (1H, m), 7.53e7.55 (2H, m), 7.47e7.49
(1H, m), 7.28e7.33 (1H, m), 7.19e7.22 (1H, m); 13C NMR (75 MHz,
DMSO):
d 144.2, 141.7, 138.7, 137.1, 131.4, 130.4, 129.1 (2C), 128.6,
126.4, 122.1, 121.4, 120.6, 114.5, 113.2; ESI-MS m/z: 251[M þ H]þ.
5.3.3. 1-(2-Chlorophenyl)-
b
-carboline (12)
5.5.1. 9-[4-(
Starting from
(1.0 mmol), compound 18a was obtained as white solid (0.18 g,
46%), mp >270 ꢂC. 1H NMR (500 MHz, DMSO-d6):
9.03 (2H, s),
b
-Carboline-9-yl)butyl]-
b-carboline (18a)
Afforded white solids (1.38 g, 50%). 1H NMR (300 MHz, CDCl3):
b
-carboline 1 (2.0 mmol) and 1,4-dibromobutane
d
8.55 (1H, d, J ¼ 5.1 Hz), 8.29 (1H, s), 8.14e8.19 (1H, m), 8.00 (1H,
dd, J ¼ 0.6 Hz, 5.4 Hz), 7.51e7.62 (3H, m), 7.39e7.46 (3H, m), 7.27e
d
7.33 (1H, m); 13C NMR (75 MHz, DMSO):
d
141.9, 141.4, 138.4, 137.8,
8.36 (2H, d, J ¼ 5.0 Hz), 8.23 (2H, d, J ¼ 8.0 Hz), 8.09 (2H, d,
J ¼ 5.0 Hz), 7.65 (2H, d, J ¼ 8.5 Hz), 7.56 (2H, t, J ¼ 7.5 Hz), 7.25 (2H, t,
J ¼ 7.5 Hz), 4.49 (4H, t, J ¼ 7.5 Hz), 1.87e1.92 (4H, m); 13C NMR
134.4, 133.1, 132.5, 130.9, 130.4, 128.9 (2C), 128.0, 122.4, 121.3, 120.1,
115.1, 112.8; ESI-MS m/z: 279 [M þ H]þ.
(125 MHz, CDCl3):
d 141.0, 139.0, 136.4, 131.8, 128.5, 122.0, 121.1,
5.3.4. 1-(2-Chloro-5-nitrophenyl)-
b
-carboline (13)
119.8, 114.7, 109.3, 43.0, 27.0. ESI-MS m/z: 391.3 [M þ H]þ.
Afforded yellow solids (1.62 g, 50%). 1H NMR (300 MHz, CDCl3):
d
8.57 (1H, d, J ¼ 5.1 Hz), 8.51 (1H, d, J ¼ 2.7 Hz), 8.25 (1H, dd,
5.5.2. 9-[5-(
b
-Carboline-9-yl)pentyl]-
b-carboline (18b)
J ¼ 2.7 Hz, 8.7 Hz), 8.18 (1H, d, J ¼ 7.8 Hz), 8.08 (1H, d, J ¼ 5.1 Hz),
7.72 (1H, d, J ¼ 8.7 Hz), 7.55e7.61 (1H, m), 7.50 (1H, d, J ¼ 7.8 Hz),
Starting from
b
-carboline 1 (2.0 mmol) and 1,5-dibromopentane
(1.0 mmol), compound 18b was obtained as white solid (0.21 g,
7.32e7.37 (1H, m); 13C NMR (75 MHz, CDCl3):
d 147.0, 141.4, 140.3,
54%), mp 191e192 ꢂC (lit. [37], mp 174 ꢂC). 1H NMR (500 MHz,
139.5, 138.8, 138.6, 134.4, 132.0, 129.5, 129.3, 127.2, 125.5, 122.5,
CDCl3):
d
8.82 (2H, s), 8.47 (2H, d, J ¼ 5.0 Hz), 8.13 (2H, d, J ¼ 7.5 Hz),
121.2, 120.3, 115.8, 112.6; ESI-MS m/z: 324 [M þ H]þ.
7.95 (2H, d, J ¼ 5.0 Hz), 7.56 (2H, t, J ¼ 7.5 Hz), 7.35 (2H, d, J ¼ 8.0 Hz),
7.28 (2H, t, J ¼ 7.5 Hz), 4.30 (4H, t, J ¼ 7.0 Hz), 1.88e1.94 (4H, m),
5.3.5. 1-(Pyridin-3-yl)-
b
-carboline (14)
1.42e1.47 (2H, m); 13C NMR (125 MHz, CDCl3):
d 141.0, 138.9, 136.3,
Afforded yellow solids (1.97 g, 80%). 1H NMR (300 MHz, CDCl3):
131.8, 128.3, 128.2, 121.8, 121.0, 119.6, 114.5, 109.2, 43.0, 28.9, 25.1;
d
10.11 (1H, s), 9.39 (1H, d, J ¼ 1.8 Hz), 8.65 (1H, dd, J ¼ 1.8 Hz,
ESI-MS m/z: 404.9 [M þ H]þ.

B. Shi et al. / European Journal of Medicinal Chemistry 60 (2013) 10e22
17
5.5.3. 9-[6-(
Starting from
(1.0 mmol), compound 18c was obtained as white solid (0.22 g,
52%), mp 114e115 ꢂC. 1H NMR (300 MHz, CDCl3):
8.83 (2H, s), 8.45
b
-Carboline-9-yl)hexyl]-
b
-carboline (18c)
5.5.9. 1-Methyl-9-[4-(1-methyl-
(21b)
b
-carboline-9-yl)butyl]-
b-carboline
b-carboline 1 (2.0 mmol) and 1,6-dibromohexane
Starting from 1-methyl-
b-carboline 4 (2.0 mmol) and 1,4-
d
dibromobutane (1.0 mmol), compound 21b was obtained as
(2H, d, J ¼ 5.0 Hz), 8.12 (2H, d, J ¼ 8.0 Hz), 7.94 (2H, d, J ¼ 5.0 Hz),
7.53e7.56 (2H, m), 7.36 (2H, d, J ¼ 8.0 Hz), 7.26e7.28 (2H, m), 4.31
(4H, t, J ¼ 7.5 Hz), 1.82e1.87 (4H, m), 1.35e1.38 (4H, m); 13C NMR
white solid (0.26 g, 62%), mp 232e233 ꢂC. 1H NMR (300 MHz,
CDCl3)
d
8.35 (2H, d, J ¼ 3.9 Hz), 8.13 (2H, d, J ¼ 5.7 Hz), 7.85 (2H, d,
J ¼ 3.9 Hz), 7.55e7.57 (2H, m), 7.28e7.35 (4H, m), 4.53 (4H, t,
(100 MHz, CDCl3):
d 140.9, 138.8, 136.3, 131.9, 128.4, 128.2, 121.8,
J ¼ 6.9 Hz), 2.97 (6H, s), 1.94e1.97 (4H, m). 13C NMR (75 MHz,
120.9, 119.4, 114.4, 109.2, 43.0, 28.9, 26.9; ESI-MS m/z: 418.9
CDCl3): d 141.4, 141.0, 138.3, 135.0, 129.3, 128.3, 121.6, 121.4, 119.8,
[M þ H]þ.
113.0, 109.5, 44.4, 28.1, 23.5. ESI-MS m/z: 419.3 [M þ H]þ.
5.5.4. 9-[7-(
Starting from
(1.0 mmol), compound 18d was obtained as white solid (0.22 g,
51%), mp 151e152 ꢂC. 1H NMR (500 MHz, CDCl3):
8.85 (2H, s), 8.46
b
-Carboline-9-yl)heptyl]-
b
-carboline (18d)
5.5.10. 1-Methyl-9-[5-(1-methyl-
(21c)
Starting from 1-methyl-
b
-carboline-9-yl)pentyl]-
b-carboline
b
-carboline 1 (2.0 mmol) and 1,7-dibromoheptane
b
-carboline 4 (2.0 mmol) and 1,5-
d
dibromopentane (1.0 mmol), compound 21c was obtained as
(2H, d, J ¼ 5.0 Hz), 8.13 (2H, d, J ¼ 7.5 Hz), 7.95 (2H, d, J ¼ 5.0 Hz),
7.55e7.57 (2H, m), 7.41 (2H, d, J ¼ 8.0 Hz), 7.27e7.28 (2H, m), 4.32
(4H, t, J ¼ 7.5 Hz), 1.81e1.88 (4H, m), 1.28e1.37 (6H, m); 13C NMR
white solid (0.17 g, 40%), mp 213e214 ꢂC. 1H NMR (500 MHz,
CDCl3):
d
8.33 (2H, d, J ¼ 5.0 Hz), 8.11 (2H, d, J ¼ 8.0 Hz), 7.83 (2H, d,
J ¼ 5.0 Hz), 7.53e7.55 (2H, m), 7.36 (2H, d, J ¼ 8.0 Hz), 7.27 (2H, d,
(125 MHz, CDCl3):
d 141.0, 138.7, 136.3, 131.9, 128.2, 121.8, 120.9,
J ¼ 8.0 Hz), 4.48 (4H, t, J ¼ 7.5 Hz), 2.99 (6H, s), 1.83e1.89 (4H, m),
119.4, 114.4, 109.3, 43.1, 29.0, 28.9, 26.9; ESI-MS m/z: 433.3
1.44e1.49 (2H, m); 13C NMR (125 MHz, CDCl3):
d 141.3, 141.0, 138.1,
[M þ H]þ.
134.9, 129.0, 128.1, 121.4, 121.2, 119.6, 112.8, 109.5, 44.4, 30.6, 24.3,
23.5; ESI-MS m/z: 433.3 [M þ H]þ.
5.5.5. 9-[8-(
Starting from
(1.0 mmol), compound 18e was obtained as white solid (0.20 g,
44%), mp 180e181 ꢂC. 1H NMR (500 MHz, CDCl3):
8.87 (2H, s), 8.45
b
-Carboline-9-yl)octyl]-b-carboline (18e)
b-carboline 1 (2.0 mmol) and 1,8-dibromooctane
5.5.11. 1-Methyl-9-[6-(1-methyl-
(21d)
Starting from 1-methyl-
b
-carboline-9-yl)hexyl]-b-carboline
d
b-carboline 4 (2.0 mmol) and 1,6-
(2H, d, J ¼ 5.0 Hz), 8.13 (2H, d, J ¼ 8.0 Hz), 7.94 (2H, d, J ¼ 5.0 Hz),
7.55e7.59 (2H, m), 7.43 (2H, d, J ¼ 8.5 Hz), 7.26e7.28 (2H, m), 4.32
(4H, t, J ¼ 7.0 Hz), 1.81e1.87 (4H, m), 1.24e1.32 (8H, m); 13C NMR
dibromohexane (1.0 mmol), compound 21d was obtained as
white solid (0.26 g, 58%), mp 220e221 ꢂC. 1H NMR (500 MHz,
DMSO-d6)
d
8.19 (4H, d, J ¼ 4.5 Hz), 7.95 (2H, d, J ¼ 5.0 Hz), 7.64 (2H,
(125 MHz, CDCl3):
d
141.0, 138.7, 136.4, 131.9, 128.1 (4C), 121.7, 120.9,
d, J ¼ 8.5 Hz), 7.54e7.57 (2H, m), 7.22e7.25 (2H, m), 4.54 (4H, t,
119.4,114.4,109.3, 43.1, 29.0, 28.9, 26.9; ESI-MS m/z: 447.1 [M þ H]þ.
J ¼ 8.0 Hz), 2.93 (6H, s), 1.69e1.74 (4H, m), 1.36e1.43 (4H, m). 13
C
NMR (125 MHz, CDCl3):
d 141.6, 141.0, 137.8, 135.0, 129.2, 128.2,
5.5.6. 6-Bromo-9-[4-(6-bromo-
(19)
b
-carboline-9-yl)butyl]-
b
-carboline
121.6, 121.3, 119.7, 113.0, 109.6, 44.6, 30.8, 26.8, 23.4. ESI-MS m/z:
447.4 [M þ H]þ.
Starting from 6-bromo-
b-carboline 2 (2.0 mmol) and 1,4-
dibromobutane (1.0 mmol), compound 19 was obtained as white
5.5.12. 1-Methyl-9-[7-(1-methyl-
(21e)
b-carboline-9-yl)heptyl]-b-carboline
solid (0.28 g, 51%), mp 316e317 ꢂC. 1H NMR (300 MHz, CDCl3):
d
8.78 (2H, s), 8.46 (2H, d, J ¼ 5.1 Hz), 8.20 (2H, d, J ¼ 1.8 Hz), 7.87
Starting from 1-methyl-b-carboline 4 (2.0 mmol) and 1,7-
dibromoheptane (1.0 mmol), compound 21e was obtained as
white solid (0.12 g, 26%), mp 189e190 ꢂC. 1H NMR (500 MHz,
(2H, dd, J ¼ 5.1 Hz, 0.9 Hz), 7.55 (2H, dd, J ¼ 8.7 Hz, 1.8 Hz), 7.07 (2H,
d, J ¼ 8.7 Hz), 4.28e4.32 (4H, m), 1.92e1.98 (4H, m); ESI-MS m/z:
549.1 [M þ H]þ.
CDCl3):
d
8.31 (2H, d, J ¼ 5.5 Hz), 8.10 (2H, d, J ¼ 7.5 Hz), 7.83 (2H, d,
J ¼ 5.5 Hz), 7.53e7.55 (2H, m), 7.41 (2H, d, J ¼ 8.0 Hz), 7.27 (2H, t,
J ¼ 8.0 Hz), 4.48 (4H, t, J ¼ 8.0 Hz), 3.02 (6H, s), 1.77e1.84 (4H, m),
5.5.7. 8-Bromo-9-[6-(8-bromo-b-carboline-9-yl)hexyl]-b-carboline
(20)
1.36e1.42 (6H, m); 13C NMR (100 MHz, CDCl3):
d 141.4, 141.1, 138.0,
Starting from 8-bromo-
dibromohexane (1.0 mmol), compound 20 was obtained as
yellow oil (0.21 g, 37%). 1H NMR (500 MHz, CDCl3):
8.89 (2H, s),
b
-carboline 3 (2.0 mmol) and 1,6-
135.0, 129.0, 128.0, 121.4, 121.3, 119.5, 112.9, 109.5, 44.7, 30.6, 29.1,
26.7, 23.5; ESI-MS m/z: 460.9 [M þ H]þ.
d
8.49 (2H, d, J ¼ 5.5 Hz), 8.09 (2H, dd, J ¼ 1.0 Hz, 7.5 Hz), 7.91 (2H, d,
J ¼ 5.5 Hz), 7.72 (2H, dd, J ¼ 1.0 Hz, 7.5 Hz), 7.11 (2H, t, J ¼ 7.5 Hz),
4.79 (4H, t, J ¼ 7.5 Hz), 1.89e1.93 (4H, m), 1.46e1.49 (4H, m); 13C
5.5.13. 1-Methyl-9-[8-(1-methyl-b-carboline-9-yl)octyl]-b-carboline
(21f)
Starting from 1-methyl-b-carboline 4 (2.0 mmol) and 1,8-
NMR (125 MHz, CDCl3):
d
139.5, 137.3, 133.7, 132.6, 127.8, 124.4,
dibromooctane (1.0 mmol), compound 21f was obtained as white
120.9, 120.7, 114.2, 103.4, 44.3, 30.7, 26.5; ESI-MS m/z: 577.8
solid (0.19 g, 41%), mp 141e142 ꢂC. 1H NMR (500 MHz, DMSO-d6)
[M þ H]þ.
d
8.19 (2H, d, J ¼ 5.0 Hz), 8.17 (2H, d, J ¼ 8.0 Hz), 7.93e7.94 (2H, d,
J ¼ 5.0 Hz), 7.63 (2H, d, J ¼ 8.0 Hz), 7.54e7.57 (2H, m), 7.21e7.24 (2H,
m), 4.52 (4H, t, J ¼ 7.5 Hz), 2.94 (6H, s), 1.65e1.69 (4H, m), 1.22e1.31
5.5.8. 1-Methyl-9-[3-(1-methyl-b-carboline-9-yl)propyl]-b-carboline
(21a)
(8H, m); 13C NMR (125 MHz, CDCl3):
d 141.3, 141.0, 137.7, 134.9,
Starting from 1-methyl-
b
-carboline 4 (2.0 mmol) and 1,3-
128.8, 127.9, 121.3, 121.1, 119.4, 112.7, 109.5, 44.6, 30.5, 29.0, 26.6,
dibromopropane (1.0 mmol), compound 21a was obtained as
23.3. ESI-MS m/z: 475.4 [M þ H]þ.
white solid (0.18 g, 46%), mp 239e240 ꢂC. 1H NMR (500 MHz,
CDCl3):
d
8.31 (2H, d, J ¼ 5.0 Hz), 8.09 (2H, d, J ¼ 8.0 Hz), 7.81 (2H, d,
5.5.14. 1-Methyl-9-[9-(1-methyl-
(21g)
b
-carboline-9-yl)nonyl]-
b-carboline
J ¼ 5.0 Hz), 7.49e7.51 (2H, m), 7.24e7.27 (2H, m), 7.20 (2H, d,
J ¼ 8.0 Hz), 4.54 (4H, t, J ¼ 7.5 Hz), 2.74 (6H, s), 2.31e2.37 (2H, m);
Starting from 1-methyl-
b-carboline 4 (2.0 mmol) and 1,9-
13C NMR (125 MHz, CDCl3):
d
141.0, 140.8, 138.4, 134.8, 129.1, 128.3,
dibromononane (1.0 mmol), compound 21g was obtained as
121.6121.4, 119.9, 112.9, 109.1, 41.9, 31.3, 23.1; ESI-MS m/z: 404.9
white solid (0.26 g, 53%), mp 155e156 ꢂC. 1H NMR (500 MHz,
[M þ H]þ.
CDCl3):
d
8.32 (2H, d, J ¼ 5.0 Hz), 8.11 (2H, d, J ¼ 7.5 Hz), 7.83 (2H, d,

18
B. Shi et al. / European Journal of Medicinal Chemistry 60 (2013) 10e22
J ¼ 5.0 Hz), 7.54e7.57 (2H, m), 7.44 (2H, d, J ¼ 8.5 Hz), 7.26e7.28
(2H, m), 4.51 (4H, t, J ¼ 7.5 Hz), 3.04 (6H, s), 1.78e1.85 (4H, m),
5.5.20. 7-Methoxy-1-methyl-9-[5-(7-methoxy-1-methyl-
9-yl)pentyl]- -carboline (24b)
Starting from 7-methoxy-1-methyl-b-carboline 7 (2.0 mmol)
b-carboline-
b
1.25e1.40 (10H, m); 13C NMR (125 MHz, CDCl3):
d
141.4, 141.1, 137.9,
135.0, 128.9, 127.9, 121.4, 121.2, 119.5, 112.8, 109.6, 44.8, 30.7, 29.1,
and 1,5-dibromopentane (1.0 mmol), compound 24b was obtained
26.8, 23.5; ESI-MS m/z: 488.9 [M þ H]þ.
as white solid (0.26 g, 52%), mp 180e181 ꢂC. 1H NMR (500 MHz,
CDCl3):
d
8.29 (2H, d, J ¼ 5.5 Hz), 7.97 (2H, d, J ¼ 8.5 Hz), 7.73 (2H, d,
5.5.15. 1-Methyl-9-[10-(1-methyl-
(21h)
Starting from 1-methyl-
b
-carboline-9-yl)decyl]-b-carboline
J ¼ 5.5 Hz), 6.88 (2H, dd, J ¼ 8.5, 2.0 Hz), 6.77 (2H, d, J ¼ 2.0 Hz), 4.43
(4H, t, J ¼ 7.5 Hz), 3.88 (6H, s), 2.96 (6H, s), 1.83e1.89 (4H, m), 1.43e
b
-carboline 4 (2.0 mmol) and 1,10-
1.49 (4H, m); 13C NMR (125 MHz, CDCl3):
d 160.8,142.9,140.3,138.3,
dibromodecane (1.0 mmol), compound 21h was obtained as
135.1, 129.4, 122.3, 115.1, 112.2, 108.5, 93.4, 55.6, 44.4, 30.5, 24.3,
white solid (0.28 g, 56%), mp 152e154 ꢂC. 1H NMR (500 MHz,
23.4; ESI-MS m/z: 492.9 [M þ H]þ.
DMSO-d6)
d
8.18e8.20 (4H, m), 7.94 (2H, d, J ¼ 4.5 Hz), 7.66 (2H, d,
J ¼ 8.5 Hz), 7.55e7.58 (2H, m), 7.21e7.24 (2H, m), 4.56 (4H, t,
5.5.21. 7-Methoxy-1-methyl-9-[6-(7-methoxy-1-methyl-
9-yl)hexyl]- -carboline (24c)
Starting from 7-methoxy-1-methyl-
and 1,6-dibromohexane (1.0 mmol), compound 24c was obtained
as yellow oil (0.23 g, 45%). 1H NMR (500 MHz, CDCl3):
8.28 (2H, d,
b-carboline-
J ¼ 7.5 Hz), 2.96 (6H, s), 1.68e1.73 (4H, m), 1.15e1.32 (12H, m), 13
C
b
NMR (125 MHz, CDCl3):
d
141.5, 141.2, 137.9, 135.1, 129.0, 128.0,
b-carboline 7 (2.0 mmol)
121.4, 121.3, 119.5, 112.9, 109.7, 44.8, 30.8, 29.3, 29.2, 26.8, 23.5. ESI-
MS m/z: 503.4 [M þ H]þ.
d
J ¼ 5.0 Hz), 7.96 (2H, d, J ¼ 8.5 Hz), 7.72 (2H, d, J ¼ 5.5 Hz), 6.88 (2H,
dd, J ¼ 8.5, 2.0 Hz), 6.81 (2H, d, J ¼ 2.0 Hz), 4.43 (4H, t, J ¼ 7.5 Hz),
3.92 (6H, s), 2.98 (6H, s), 1.79e1.82 (4H, m), 1.41e1.44 (4H, m); 13C
5.5.16. 3-Ethoxycarbonyl-1-methyl-9-[6-(3-ethoxycarbonyl-1-met-
hyl-
b
-carboline-9-yl)hexyl]-
b
-carboline (22)
-carboline-3-carboxylate
Starting from ethyl 1-methyl-
b
5
NMR (125 MHz, CDCl3): d 160.8, 143.0, 140.4, 138.2, 135.2, 129.4,
(2.0 mmol) and 1,6-dibromohexane (1.0 mmol), compound 22 was
122.4,115.2, 112.2,108.4, 93.5, 55.6, 44.6, 30.6, 26.8, 23.3; ESI-MS m/
obtained as white solid (0.32 g, 54%), mp 268e269 ꢂC. 1H NMR
z: 507.1 [M þ H]þ.
(300 MHz, CDCl3):
d
8.73 (2H, s), 8.15 (2H, d, J ¼ 7.8 Hz), 7.55e7.60
(2H, m), 7.29e7.42 (4H, m), 4.48e4.56 (8H, m), 2.99 (6H, s), 1.77e
5.5.22. 7-Methoxy-1-methyl-9-[7-(7-methoxy-1-methyl-
9-yl)heptyl]- -carboline (24d)
Starting from 7-methoxy-1-methyl-b-carboline 7 (2.0 mmol)
b-carboline-
1.87 (4H, m), 1.49 (6H, t, J ¼ 7.2 Hz), 1.38e1.44 (4H, m); 13C NMR
b
(75 MHz, CDCl3):
d
166.2, 141.9, 141.4, 137.1, 136.7, 129.1, 128.7, 121.9,
121.8, 120.8, 116.5, 110.2, 61.7, 45.1, 31.2, 27.1, 24.5, 14.9; ESI-MS m/z:
and 1,7-dibromoheptane (1.0 mmol), compound 24d was obtained
590.1 [M]þ.
as white solid (0.27 g, 53%), mp 199e200 ꢂC. 1H NMR (500 MHz,
CDCl3):
d
8.28 (2H, d, J ¼ 5.0 Hz), 7.96 (2H, d, J ¼ 8.5 Hz), 7.72 (2H, d,
5.5.17. 6-Bromo-1-methyl-9-[5-(6-bromo-1-methyl-
yl)pentyl]- -carboline (23a)
Starting from 6-bromo-1-methyl-
b
-carboline-9-
J ¼ 5.5 Hz), 6.87 (2H, dd, J ¼ 8.5, 2.0 Hz), 6.81 (2H, d, J ¼ 2.0 Hz), 4.41
b
(4H, t, J ¼ 7.5 Hz), 3.91 (6H, s), 2.98 (6H, s), 1.78e1.81 (4H, m), 1.36e
b
-carboline 6 (2.0 mmol) and
1.44 (6H, m); 13C NMR (125 MHz, CDCl3):
d 160.7, 142.9, 140.4, 138.3,
1,5-dibromopentane (1.0 mmol), compound 23a was obtained as
135.2, 129.3, 122.3, 115.2, 112.2, 108.4, 93.4, 55.6, 44.7, 30.5, 29.2,
white solid (0.21 g, 35%), mp 208e209 ꢂC. 1H NMR (300 MHz,
26.8, 23.4; ESI-MS m/z: 520.9 [M þ H]þ.
CDCl3):
d
8.33 (2H, d, J ¼ 5.4 Hz), 8.20 (2H, d, J ¼ 1.8 Hz), 7.76 (2H, d,
J ¼ 5.4 Hz), 7.58 (2H, dd, J ¼ 1.8 Hz, 8.7 Hz), 7.18 (2H, d, J ¼ 8.7 Hz),
5.5.23. 7-Methoxy-1-methyl-9-[8-(7-methoxy-1-methyl-
9-yl)octyl]- -carboline (24e)
Starting from 7-methoxy-1-methyl-b-carboline 7 (2.0 mmol)
b-carboline-
4.45 (4H, t, J ¼ 7.5 Hz), 2.98 (6H, s), 1.78e1.88 (4H, m), 1.32e1.42
b
(2H, m); 13C NMR (75 MHz, CDCl3):
d
141.5, 140.0, 138.6, 135.3,
131.0, 128.1, 124.4, 123.0, 113.1, 112.6, 111.3, 44.9, 31.1, 24.8, 24.0; ESI-
and 1,8-dibromooctane (1.0 mmol), compound 24e was obtained as
MS m/z: 605.5 [M]þ.
white solid (0.38 g, 71%), mp 181e182 ꢂC. 1H NMR (500 MHz,
CDCl3):
d
8.27 (2H, d, J ¼ 5.0 Hz), 7.97 (2H, d, J ¼ 8.5 Hz), 7.76 (2H, d,
5.5.18. 6-Bromo-1-methyl-9-[6-(6-bromo-1-methyl-
yl)hexyl]- -carboline (23b)
Starting from 6-bromo-1-methyl-
b
-carboline-9-
J ¼ 5.0 Hz), 6.89 (2H, dd, J ¼ 8.5, 2.0 Hz), 6.83 (2H, d, J ¼ 2.0 Hz), 4.44
b
(4H, t, J ¼ 7.5 Hz), 3.93 (6H, s), 3.04 (6H, s), 1.76e1.82 (4H, m), 1.31e
b
-carboline 6 (2.0 mmol) and
1.39 (8H, m); 13C NMR (125 MHz, CDCl3):
d 160.8, 143.1, 140.3, 137.9,
1,6-dibromohexane (1.0 mmol), compound 23b was obtained as
135.2, 129.4, 122.3, 115.2, 112.2, 108.5, 93.5, 55.6, 44.8, 30.4, 29.2,
white solid (0.41 g, 67%), mp >270 ꢂC. 1H NMR (500 MHz, CDCl3):
26.8, 23.2; ESI-MS m/z: 535.0 [M þ H]þ.
d
8.33 (2H, d, J ¼ 5.0 Hz), 8.22 (2H, d, J ¼ 2.0 Hz), 7.77 (2H, d,
J ¼ 5.0 Hz), 7.62 (2H, dd, J ¼ 8.5, 2.0 Hz), 7.24 (2H, d, J ¼ 8.5 Hz), 4.47
(4H, t, J ¼ 7.5 Hz), 2.99 (6H, s), 1.76e1.83 (4H, m), 1.35e1.41 (4H, m);
5.5.24. 7-Butoxy-1-methyl-9-[4-(7-butoxy-1-methyl-
9-yl)butyl]- -carboline (25a)
Starting from 7-butoxy-1-methyl-b-carboline 9 (2.0 mmol) and
b-carboline-
b
13C NMR (125 MHz, CDCl3):
d
141.5, 140.0, 138.5, 135.3, 130.8, 128.0,
124.2, 123.0, 113.0, 112.4, 111.1, 44.8, 30.7, 26.8, 23.5; ESI-MS m/z:
1,4-dibromobutane (1.0 mmol), compound 25a was obtained as
604.0 [M]þ.
white solid (0.22 g, 62%), mp 193e194 ꢂC. 1H NMR (300 MHz,
CDCl3):
d
8.26 (2H, d, J ¼ 5.1 Hz), 7.92 (2H, d, J ¼ 8.4 Hz), 7.68 (2H, d,
5.5.19. 7-Methoxy-1-methyl-9-[4-(7-methoxy-1-methyl-
9-yl)butyl]- -carboline (24a)
Starting from 7-methoxy-1-methyl-
b
-carboline-
J ¼ ¼ 5.1 Hz), 6.85 (2H, dd, J ¼ 1.8 Hz, 8.4 Hz), 6.71 (2H, d, J ¼ 1.8 Hz),
4.41 (4H, t, J ¼ 6.9 Hz), 4.01 (4H, t, J ¼ 6.6 Hz), 2.91 (6H, s), 1.79e1.89
(8H, m), 1.48e1.61 (4H, m), 1.02 (6H, t, J ¼ 7.5 Hz); 13C NMR
b
b
-carboline 7 (2.0 mmol)
and 1,4-dibromobutane (1.0 mmol), compound 24a was obtained as
(100 MHz, CDCl3): d 160.6, 143.1, 140.3, 138.6, 135.3, 129.8, 122.6,
white solid (0.23 g, 49%), mp >270 ꢂC. 1H NMR (300 MHz, CDCl3):
115.3,112.5, 109.4, 94.3, 68.5, 44.6, 31.8, 28.2, 23.7, 19.7, 14.3; ESI-MS
d
8.27 (2H, d, J ¼ 5.1 Hz), 7.93 (2H, d, J ¼ 8.4 Hz), 7.70 (2H, d,
m/z: 563.4 [M þ H]þ.
J ¼ 5.1 Hz), 6.86 (2H, dd, J ¼ 8.4, 2.1 Hz), 6.70 (2H, d, J ¼ 2.1 Hz), 4.48
(4H, t, J ¼ 7.2 Hz), 3.87 (6H, s), 2.93 (6H, s), 1.90e1.92 (4H, m); 13C
5.5.25. 7-Butoxy-1-methyl-9-[6-(7-butoxy-1-methyl-
9-yl)hexyl]- -carboline (25b)
Starting from 7-butoxy-1-methyl-
1,6-dibromohexane (1.0 mmol), compound 25b was obtained as
b-carboline-
NMR (100 MHz, CDCl3):
d
160.9, 142.9, 140.3, 138.6, 135.2, 129.6,
b
122.5, 115.3, 112.3, 108.7, 93.4, 55.7, 44.3, 27.7, 23.4. ESI-MS m/z:
b-carboline 9 (2.0 mmol) and
479.6 [M þ H]þ.

B. Shi et al. / European Journal of Medicinal Chemistry 60 (2013) 10e22
19
white solid (0.23 g, 65%). 1H NMR (500 MHz, CDCl3):
d
8.27 (2H, d,
7.08e7.10 (2H, m), 4.03 (4H, t, J ¼ 7.8 Hz), 1.21e1.30 (4H, m), 0.69e
0.74 (4H, m); 13C NMR (75 MHz, CDCl3):
142.3, 141.3, 138.7, 137.3,
J ¼ ¼ 5.0 Hz), 7.95 (2H, d, J ¼ 8.5 Hz), 7.71 (2H, d, J ¼ 5.0 Hz), 6.87
(2H, dd, J ¼ 8.5, 2.0 Hz), 6.81 (2H, d, J ¼ 2.0 Hz), 4.42 (4H, t,
J ¼ 7.5 Hz), 4.06 (4H, t, J ¼ 7.0 Hz), 2.97 (6H, s), 1.79e1.83 (8H, m),
1.49e1.57 (4H, m), 1.41e1.44 (4H, m), 0.99 (6H, t, J ¼ 6.0 Hz); 13C
d
134.9,131.0,128.8,128.5,127.2,126.9,121.9,121.5,120.3,114.4,110.5,
44.5, 29.2, 26.5; ESI-MS m/z: 583.4 [M þ H]þ.
NMR (125 MHz, CDCl3):
d
160.3, 143.0, 140.3, 138.2, 135.2, 129.4,
5.5.31. 1-(Thiophen-3-yl)-9-[8-[1-(thiophen-3-yl) -
yl]octyl]- -carboline (28b)
Starting from 1-(thiophen-3-yl)-b-carboline 11 (2.0 mmol) and
b-carboline-9-
122.2, 115.1, 121.1, 108.8, 94.2, 68.1, 44.6, 31.3, 30.6, 26.8, 23.3, 19.2,
b
13.8; ESI-MS m/z: 590.4 [M þ H]þ.
1,8-dibromooctane (1.0 mmol), compound 28b was obtained as
5.5.26. 7-Isoproxy-1-methyl-9-[6-(7-isoproxy-1-methyl-
9-yl)hexyl]- -carboline (26a)
Starting from 7-isopropoxy-1-methyl-
b-carboline-
yellow solid (0.43 g, 70%), mp 161e162 ꢂC. 1H NMR (300 MHz,
b
CDCl3):
d
8.50 (2H, d, J ¼ 5.1 Hz), 8.15 (2H, d, J ¼ 7.2 Hz), 7.96 (2H, d,
b
-carboline 8 (2.0 mmol)
J ¼ 5.1 Hz), 7.56e7.61 (2H, m), 7.41e7.44 (4H, m), 7.22e7.32 (4H, m),
and 1,6-dibromohexane (1.0 mmol), compound 26a was obtained
7.08e7.11 (2H, m), 4.09 (4H, t, J ¼ 7.5 Hz), 1.32e1.42 (4H, m), 0.80e
as white solid (0.25 g, 71%), mp 155e156 ꢂC. 1H NMR (300 MHz,
0.88 (8H, m); 13C NMR (75 MHz, CDCl3):
d 142.4, 141.3, 138.6, 137.3,
CDCl3):
d
8.25 (2H, d, J ¼ 5.4 Hz), 7.95 (2H, d, J ¼ 8.7 Hz), 7.71 (2H, d,
135.0,131.0,128.8,128.4,127.2,126.8,121.9,121.5,120.2,114.4,110.5,
J ¼ 5.4 Hz), 6.86 (2H, dd, J ¼ 1.8 Hz, 8.7 Hz), 6.82 (2H, d, J ¼ 1.8 Hz),
44.7, 29.3, 29.2, 26.9; ESI-MS m/z: 611.4 [M þ H]þ.
4.62e4.74 (2H, m), 4.41 (4H, t, J ¼ 7.5 Hz), 2.98 (6H, s), 1.75e1.85
(4H, m), 1.39e1.41 (16H, m); 13C NMR (100 MHz, CDCl3):
d
159.2,
5.5.32. 1-(2-Chlorophenyl)-9-[6-[1-(2-chlorohenyl)-
yl]hexyl]- -carboline (29a)
Starting from 1-(2-chlorophenyl)-b-carboline 12 (2.0 mmol) and
b-carboline-9-
143.3, 140.5, 138.3, 135.4, 129.7, 122.6, 115.4, 112.5, 110.1, 96.4, 70.9,
b
45.0, 31.0, 27.3, 23.7, 22.6; ESI-MS m/z: 562.5 [M þ H]þ.
1,6-dibromohexane (1.0 mmol), compound 29a was obtained as
5.5.27. 7-Isoproxy-1-methyl-9-[9-(7-isoproxy-1-methyl-
9-yl)nonyl]- -carboline (26b)
Starting from 7-isopropoxy-1-methyl-
b-carboline-
white solid (0.57 g, 90%), mp 210e211 ꢂC. 1H NMR (300 MHz,
b
CDCl3):
d
8.52 (2H, d, J ¼ 5.1 Hz), 8.18 (2H, 7.8 Hz), 8.02 (2H, d,
b
-carboline 8 (2.0 mmol)
J ¼ 5.1 Hz), 7.54e7.59 (2H, m), 7.43e7.49 (4H, m), 7.27e7.39 (8H, m),
and 9-dibromononane (1.0 mmol), compound 26b was obtained as
3.67e3.89 (4H, m), 1.17e1.39 (4H, m), 0.56e0.69 (4H, m); 13C NMR
white solid (0.4 g, 67%), mp 161e162 ꢂC. 1H NMR (300 MHz, CDCl3):
(75 MHz, CDCl3): d 141.8,141.1,139.0,138.5,134.4,134.3,131.7,130.2,
d
8.25 (2H, d, J ¼ 5.1 Hz), 7.94 (2H, d, J ¼ 9.3 Hz), 7.70 (2H, d,
130.1, 129.6, 128.7, 126.9, 121.9, 121.4, 120.1, 114.5, 110.0, 44.3, 29.4,
J ¼ 5.1 Hz), 6.84e6.87 (4H, m), 4.63e4.75 (2H, m), 4.41 (4H, t,
26.6; ESI-MS m/z: 639.3 [M]þ.
J ¼ 7.8 Hz), 3.00 (6H, s), 1.75e1.85 (4H, m), 1.26e1.45 (22H, m); 13
C
NMR (100 MHz, CDCl3):
d
159.1, 143.2, 140.6, 138.4, 135.5, 129.5,
5.5.33. 1-(2-Chlorophenyl)-9-[7-[1-(2-chlorohenyl)-
yl]heptyl]- -carboline (29b)
Starting from 1-(2-chlorophenyl)-
1,7-dibromoheptane (1.0 mmol), compound 29b was obtained as
yellow oil (0.49 g, 75%). 1H NMR (300 MHz, CDCl3):
8.53 (2H, d,
b-carboline-9-
122.5, 115.4, 112.4, 110.0, 96.3, 70.8, 45.1, 31.0, 29.8, 29.6, 27.3, 23.8,
b
22.5; ESI-MS m/z: 605.5 [M þ H]þ.
b-carboline 12 (2.0 mmol) and
5.5.28. 1-Isopropyl-9-[6-(1-isopropyl-
b-carboline-9-yl)hexyl]-
b
-
d
carboline (27a)
J ¼ 5.1 Hz), 8.18 (2H, d, J ¼ 7.8 Hz), 8.02 (2H, d, J ¼ 5.1 Hz), 7.55e7.60
(2H, m), 7.46e7.52 (4H, m), 7.27e7.39 (8H, m), 3.70e3.93 (4H, m),
1.32e1.42 (4H, m), 0.56e0.85 (6H, m); 13C NMR (75 MHz, CDCl3):
Starting from 1-isopropyl-b-carboline 10 (2.0 mmol) and 1,6-
dibromohexane (1.0 mmol), compound 27a was obtained as
white solid (0.14 g, 28%), mp 241e242 ꢂC. 1H NMR (300 MHz,
d
141.9, 141.1, 139.0, 138.3, 134.4, 134.3, 131.7, 130.2, 130.1, 129.6,
CDCl3):
d
8.41 (2H, d, J ¼ 4.5 Hz), 8.09 (2H, d, J ¼ ¼ 7.5 Hz), 7.82 (2H,
128.7, 126.9, 121.9, 121.4, 120.1, 114.6, 110.1, 44.4, 29.4, 28.9, 26.9;
d, J ¼ 4.5 Hz), 7.51e7.56 (2H, m), 7.37 (2H, d, J ¼ 8.4 Hz), 7.22e7.27
(2H, m), 4.46 (4H, t, J ¼ 7.5 Hz), 3.62e3.73 (2H, m), 1.76e1.86 (4H,
m), 1.48 (6H, s), 1.46 (6H, s), 1.22e1.34 (4H, m); 13C NMR (75 MHz,
ESI-MS m/z: 653.3 [M]þ.
5.5.34. 1-(2-Chloro-5-nitrophenyl)-9-[6-[1-(2-chloro-5-nitrophenyl)-
CDCl3):
d
150.6, 142.0, 138.5, 133.5, 129.9, 128.3, 121.7, 121.6, 119.8,
b-carboline-9-yl]-hexyl]-b-carboline (30a)
112.7, 109.8, 45.5, 31.7, 30.7, 27.2, 23.1, 23.0; ESI-MS m/z: 503.4
Starting from 1-(2-chloro-5-nitrophenyl)-b-carboline 13
[M þ H]þ.
(2.0 mmol) and 1,6-dibromohexane (1.0 mmol), compound 30a was
obtained as yellow oil (0.40 g, 55%). 1H NMR (300 MHz, CDCl3):
5.5.29. 1-Isopropyl-9-[10-(1-isopropyl-
b
-carboline-9-yl)decyl]-
b
-
d 8.52e8.54 (2H, m), 8.38e8.40 (2H, m), 8.16e8.27 (4H, m), 8.05e
carboline (27b)
8.08 (2H, m), 7.57e7.67 (4H, m), 7.29e7.37 (4H, m), 3.78e3.91
(2H, m), 3.52e3.67 (2H, m), 1.09e1.27 (4H, m), 0.44e0.65 (4H,
Starting from 1-isopropyl-
dibromodecane (1.0 mmol), compound 27b was obtained as
yellow oil (0.18 g, 36%). 1H NMR (300 MHz, CDCl3):
8.42 (2H, d,
b-carboline 10 (2.0 mmol) and 1,10-
m); 13C NMR (75 MHz, CDCl3):
d 146.3, 141.9, 141.5, 140.4, 138.6,
d
138.3,134.1,131.0,130.7,129.2,126.5,124.7,122.0,121.3,120.5,115.4,
J ¼ 5.1 Hz), 8.09 (2H, d, J ¼ 7.8 Hz), 7.81 (2H, 5.1 Hz), 7.53e7.58 (2H,
m), 7.43 (2H, d, J ¼ 8.4 Hz), 7.22e7.27 (2H, m), 4.48 (4H, t, J ¼ 7.8 Hz),
3.68e3.81 (2H, m),1.79e1.89 (4H, m),1.52 (6H, s),1.49 (6H, s),1.28e
110.2, 44.3, 29.5, 26.8; ESI-MS m/z: 729.3 [M]þ.
5.5.35. 1-(2-Chloro-5-nitrophenyl)-9-[9-[1-(2-chloro-5-nitrophenyl)-
1.42 (12H, m); 13C NMR (75 MHz, CDCl3):
d
150.7, 142.0, 138.4, 133.6,
b-carboline-9-yl]-nonyl]-b-carboline (30b)
129.9, 128.2, 121.7, 121.5, 119.7, 112.7, 109.9, 45.8, 31.7, 30.6, 29.7,
Starting from 1-(2-chloro-5-nitrophenyl)-b-carboline 13
29.6, 27.3, 23.1; ESI-MS m/z: 559.5 [M þ H]þ.
(2.0 mmol) and 1,9-dibromononane (1.0 mmol), compound 30b
was obtained as yellow oil (0.32 g, 41%). 1H NMR (300 MHz, CDCl3):
5.5.30. 1-(Thiophen-3-yl)-9-[6-[1-(thiophen-3-yl) -
yl]hexyl]- -carboline (28a)
Starting from 1-(thiophen-3-yl)-
b
-carboline-9-
d
8.52e8.54 (2H, m), 8.46 (2H, d, J ¼ 2.7 Hz), 8.26e8.32 (2H, m), 8.19
b
(2H, d, J ¼ 7.8 Hz), 8.07 (2H, dd, J ¼ 1.8 Hz, 5.1 Hz), 7.68e7.73 (2H,
m), 7.58e7.64 (2H, m), 7.41e7.45 (2H, m), 7.29e7.35 (2H, m), 3.91e
4.02 (2H, m), 3.60e3.75 (2H, m),1.24e1.34 (4H, m), 0.68e0.90 (10H,
b
-carboline 11 (2.0 mmol) and
1,6-dibromohexane (1.0 mmol), compound 28a was obtained as
white solid (0.37 g, 64%), mp 198e199 ꢂC. 1H NMR (300 MHz,
m); 13C NMR (75 MHz, CDCl3):
d 146.4, 142.0, 141.5, 140.5, 138.5,
CDCl3):
d
8.50 (2H, d, J ¼ 5.1 Hz), 8.15 (2H, d, J ¼ 7.8 Hz), 7.96 (2H, d,
138.4, 134.2, 130.8, 130.7, 129.1, 126.6, 124.8, 121.9, 121.2, 120.4,
J ¼ 5.1 Hz), 7.55e7.60 (2H, m), 7.27e7.43 (6H, m), 7.19e7.21 (2H, m),
115.3, 110.2, 44.6, 29.6, 29.3, 27.1; ESI-MS m/z: 771.3 [M]þ.

20
B. Shi et al. / European Journal of Medicinal Chemistry 60 (2013) 10e22
5.5.36. 1-(Pyridin-3-yl)-9-[4-[1-(pyridine-3-yl)-
butyl]- -carboline (31a)
Starting from 1-(pyridin-3-yl)-
b
-carboline-9-yl]
5.5.41. 1-(3,4,5-Trimethoxyphenyl)-9-[7-[1-(3,4,5-trimethoxyphen-
yl)]- -carboline-9-yl]-heptyl]- -carboline (33b)
Starting from 1-(3,4,5-trimethoxyphenyl)-b-carboline 16
b
b
b
b
-carboline 14 (2.0 mmol) and
1,4-dibromobutane (1.0 mmol), compound 31a was obtained as
(2.0 mmol) and 1,7-dibromoheptane (1.0 mmol), compound 33b
yellow solid (0.26 g, 46%), mp 224e225 ꢂC. 1H NMR (300 MHz,
was obtained as yellow oil (0.56 g, 62%). 1H NMR (500 MHz, CDCl3):
CDCl3):
d
8.60e8.62 (4H, m), 8.50 (2H, d, J ¼ 5.1 Hz), 8.17 (2H, d,
d
8.46 (2H, d, J ¼ 5.1 Hz), 8.14 (2H, d, J ¼ 7.8 Hz), 7.95 (2H, d,
J ¼ 7.8 Hz), 7.95 (2H, d, J ¼ 5.1 Hz), 7.52e7.62 (4H, m), 7.32e7.37 (2H,
J ¼ 5.1 Hz), 7.54e7.59 (2H, m), 7.39 (2H, d, J ¼ 7.8 Hz), 7.25e7.29 (2H,
m), 6.75 (4H, s), 3.91 (4H, t, J ¼ 7.8 Hz), 3.83 (6H, s), 3.80 (12H, s),
1.25e1.38 (4H, m), 0.72e0.86 (6H, m); 13C NMR (125 MHz, CDCl3):
m), 7.18e7.24 (4H, m), 3.67 (4H, t, J ¼ 7.2 Hz), 0.76e0.84 (4H, m); 13
C
NMR (75 MHz, CDCl3): d 150.1, 149.6,142.2,140.3,139.1,136.6,135.9,
133.9, 130.9, 129.0, 123.2, 122.0, 121.3, 120.5, 114.4, 110.2, 43.9, 26.1;
d 153.1, 144.0, 142.1, 138.3, 135.7, 134.0, 130.4, 128.6, 121.8, 121.5,
ESI-MS m/z: 545.5 [M þ H]þ.
120.1, 114.0, 110.4, 106.7, 61.2, 56.4, 44.7, 29.5, 29.1, 26.8; ESI-MS m/
z: 765.4 [M þ H]þ.
5.5.37. 1-(Pyridin-3-yl)-9-[6-[1-(pyridine-3-yl)-
hexyl]- -carboline (31b)
Starting from 1-(pyridin-3-yl)-
1,6-dibromohexane (1.0 mmol), compound 31b was obtained as
yellow solid (0.32 g, 56%). 1H NMR (300 MHz, CDCl3):
8.81e8.82
b-carboline-9-yl]
b
5.5.42. 3-Ethoxycarbonyl-1-(3,4,5-trimethoxyphenyl)-9-[6-[3-eth-
b
-carboline 14 (2.0 mmol) and
oxycarbonyl-1-(3,4,5-trimethoxyphenyl)-
b
-carboline-9-yl]hexyl]-
b-
carboline (34a)
d
Starting from ethyl 1-(3,4,5-trimethoxyphenyl)-b-carboline-3-
(2H, m), 8.67 (2H, d, J ¼ 3.9 Hz), 8.52 (2H, d, J ¼ 5.1 Hz), 8.16 (2H,
d, J ¼ 7.8 Hz), 7.99 (2H, d, J ¼ 5.1 Hz), 7.87e7.91 (2H, m), 7.55e7.60
(2H, m), 7.28e7.39 (6H, m), 3.84 (4H, t, J ¼ 7.8 Hz), 1.08e1.18 (4H,
carboxylate 17 (2.0 mmol) and 1,6-dibromohexane (1.0 mmol),
compound 34b was obtained as white solid (0.6 g, 67%), mp 264e
265 ꢂC. 1H NMR (300 MHz, CDCl3):
d 8.83 (2H, s), 8.21 (2H, d,
m), 0.52e0.57 (4H, m); 13C NMR (75 MHz, CDCl3):
d
150.1, 149.6,
J ¼ 7.8 Hz), 7.58e7.64 (2H, m), 7.33e7.40 (4H, m), 6.73 (4H, s), 4.50
(4H, q, J ¼ 7.2 Hz), 3.83e3.89 (10H, m), 3.73 (12H, s), 1.23e1.28 (4H,
m), 1.46 (6H, t, J ¼ 7.2 Hz), 0.65e0.73 (4H, m); 13C NMR (75 MHz,
142.2, 140.5, 138.9, 136.8, 136.1, 134.4, 131.0, 128.9, 123.3, 121.9,
121.4, 120.4, 114.5, 110.4, 44.6, 28.8, 26.3; ESI-MS m/z: 573.5
[M þ H]þ.
CDCl3):
d 166.1, 153.2, 143.8, 142.4, 138.6, 137.1, 135.5, 134.9, 130.3,
129.1,122.0,121.7, 121.0, 116.9,110.7,107.2, 61.8, 61.1, 56.4, 44.7, 29.6,
5.5.38. 3-Ethoxycarbonyl-1-(pyridin-3-yl)-9-[5-[3-ethoxycarbonyl-
26.6, 14.9; ESI-MS m/z: 895.5 [M þ H]þ.
1-(pyridine-3-yl)-b-carboline-9-yl]pentyl]-b-carboline (32a)
Starting from ethyl 1-(pyridin-3-yl)-
b
-carboline-3-carboxylate
5.5.43. 3-Ethoxycarbonyl-1-(3,4,5-trimethoxyphenyl)-9-[9-[3-eth-
15 (2.0 mmol) and 1,5-dibromopentane (1.0 mmol), compound
oxycarbonyl-1-(3,4,5-trimethoxyphenyl)-
-carboline (34b)
Starting from ethyl 1-(3,4,5-trimethoxyphenyl)-b-carboline-3-
b-carboline-9-yl]nonyl]-
32a was obtained as yellow solid (0.3 g, 43%), mp 172e174 ꢂC. 1H
b
NMR (300 MHz, CDCl3):
d
8.90 (2H, s), 8.76 (2H, d, J ¼ 2.1 Hz), 8.46e
8.48 (2H, dd, J ¼ 1.5, 5.1 Hz), 8.26 (2H, d, J ¼ 8.1 Hz), 7.83e7.87 (2H,
m), 7.61e7.66 (2H, m), 7.33e7.43 (4H, m), 7.21e7.26 (2H, m), 4.53
(4H, q, J ¼ 6.9 Hz), 3.81 (4H, t, J ¼ 7.8 Hz), 1.49 (6H, t, J ¼ 6.9 Hz),
1.01e1.11 (4H, m), 0.31e0.39 (2H, m); 13C NMR (75 MHz, CDCl3):
carboxylate 17 (2.0 mmol) and 1,9-dibromononane (1.0 mmol),
compound 34b was obtained as yellow oil (0.25 g, 53%). 1H NMR
(300 MHz, CDCl3):
d
8.85 (2H, s), 8.23 (2H, d, J ¼ 7.8 Hz), 7.58e7.63
(2H, m), 7.32e7.47 (4H, m), 6.82 (2H, s), 6.79 (2H, s), 4.51 (4H, q,
J ¼ 7.2 Hz), 3.84e4.00 (22H, m), 1.37e1.58 (10H, m), 1.24e1.28 (4H,
d
165.9, 150.3, 149.9, 142.4, 140.3, 137.9, 137.1, 135.8, 135.5, 131.0,
129.4, 123.2, 122.1, 121.7, 121.4, 117.3, 110.6, 62.0, 44.5, 28.6, 23.5,
m), 0.84e0.97 (6H, m); 13C NMR (75 MHz, CDCl3):
d 166.1, 153.2,
14.8; ESI-MS m/z: 703.5 [M þ H]þ.
143.8, 142.4, 138.6, 136.9, 135.6, 135.0, 130.3, 129.0, 121.9, 121.8,
120.9, 116.9, 110.7, 107.0, 61.7, 61.2, 56.5, 45.0, 29.8, 29.6, 29.4, 27.0,
14.9; ESI-MS m/z: 937.5 [M þ H]þ.
5.5.39. 3-Ethoxycarbonyl-1-(pyridin-3-yl)-9-[6-[3-ethoxycarbonyl-
1-(pyridine-3-yl)-b-carboline-9-yl]hexyl]-b-carboline (32b)
Starting from ethyl 1-(pyridin-3-yl)-
b
-carboline-3-carboxylate
5.6. Cytotoxicity assays in vitro
15 (2.0 mmol) and 1,6-dibromohexane (1.0 mmol), compound
32b was obtained as yellow solid (0.35 g, 49%), mp 221e222 ꢂC. 1H
Cytotoxicity assays in vitro were carried out using 96 microtitre
plate cultures and MTT staining according to the procedures
described by Cao et al. [5]. Briefly, cells were grown in RPMI-1640
NMR (300 MHz, CDCl3):
d
8.88 (2H, s), 8.83 (2H, d, J ¼ 1.5 Hz), 8.67
(2H, dd, J ¼ 1.5 Hz, 5.1 Hz), 8.23 (2H, d, J ¼ 7.8 Hz), 7.91e7.95 (2H, m),
7.59e7.65 (2H, m), 7.35e7.41 (6H, m), 4.51 (4H, q, J ¼ 7.2 Hz), 3.87
(4H, t, J ¼ 7.8 Hz), 1.48 (6H, t, J ¼ 7.2 Hz), 1.11e1.21 (4H, m), 0.51e
medium containing 10% (v/v) fetal calf serum and 100
m
g mlꢀ1
penicillin and 100
m
g mlꢀ1 streptomycin. Cultures were propagated
0.60 (4H, m); 13C NMR (75 MHz, CDCl3):
d
165.9, 150.4, 149.9,
at 37 ꢂC in a humidified 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.
142.5, 140.4, 137.8, 137.2, 135.8, 135.6, 131.1, 129.3, 123.3, 122.1, 121.7,
121.3, 117.2, 110.7, 61.9, 44.8, 29.0, 26.2, 14.8; ESI-MS m/z: 717.5
[M þ H]þ.
5.5.40. 1-(3,4,5-Trimethoxyphenyl)-9-[6-[1-(3,4,5-trimethoxyphen-
yl)]-b-carboline-9-yl]-hexyl]-b-carboline (33a)
Starting from 1-(3,4,5-trimethoxyphenyl)-
b
-carboline 16
(2.0 mmol) and 1,6-dibromohexane (1.0 mmol), compound 33a was
5.7. Assay of acute toxicities
obtained as white solid (0.57 g, 76%), mp 199e200 ꢂC. 1H NMR
(500 MHz, CDCl3):
d
8.47 (2H, d, J ¼ 5.5 Hz), 8.16 (2H, d, J ¼ 8.0 Hz),
Acute toxicity assay was performed according to the method
described by Cao et al. [5]. Briefly, healthy C57BL/6 mice (9e12
weeks) weighing 18e22 g were housed in rooms where the
temperature was approximately 24 ꢃ 2 ꢂC, with a relative humidity
60e70%, andin 12 hlightedark cycle. The sterilefood andwater were
provided according to institutional guidelines. All animals were
provided by Shanghai Laboratory Animal Center of Chinese Academy
7.96 (2H, d, J ¼ 5.5 Hz), 7.55e7.59 (2H, m), 7.36 (2H, d, J ¼ 8.5 Hz),
7.28e7.31 (2H, m), 6.73 (4H, s), 3.88 (4H, t, J ¼ 7.5 Hz), 3.84 (6H, s),
3.74 (12H, s), 1.22e1.32 (4H, m), 0.67e0.70 (4H, m); 13C NMR
(125 MHz, CDCl3):
d 152.9, 143.8, 141.8, 138.1, 138.0, 135.4, 133.7,
130.2, 128.4, 121.5, 121.2, 119.8, 113.6, 110.0, 106.4, 60.8, 56.0, 44.2,
28.9, 26.1; ESI-MS m/z: 750.6 [M]þ.

B. Shi et al. / European Journal of Medicinal Chemistry 60 (2013) 10e22
21
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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
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performed as described by Cao et al. [5] with a slightly modification.
Briefly, CT-26 colon cancer, Lewis lung cancer, Sarcoma 180, H22 liver
cancer and B16 melanoma cell lines were provided by Shanghai
Institute of Pharmaceutical Industry. Tumor cells of CT-26 colon
cancer, Lewis lung cancer, Sarcoma 180, H22 liver cancer and B16
melanoma were inoculated to mice. After 7 days, tumors were taken
out andcellsharvested. Viable tumorcells(2 ꢁ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 inocu-
lation at a dosage about one fifth of LD50 value once a day for
consecutive 7 days. Cyclophosphamide (CTX) at 30 mg kgꢀ1 was used
as a positive control and vehicle as negative control. The weights of
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Acknowledgments
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This work was supported by MEGA-Project (2009ZX09102-004)
and the Fundamental Research Funds for the Central Universities
and Xinjiang Huashidan Pharmaceutical Co. Ltd.
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