Synthesis and structure-activity relationships of asymmetric dimeric β-carboline derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2018.02.003

A series of newly asymmetric dimeric β-carbolines with a spacer of 4–6 methylene units between the indole nitrogen and the harmine oxygen were synthesized. Structures of all the novel synthesized compounds were confirmed by their spectral and analytical studies. All of the synthesized compounds were screened for their in vitro cytotoxic activity against nine cancer cell lines. The results revealed that compounds 7c, 7o and 7s exhibited the highest cytotoxic activities with IC₅₀ values of less than 20 μM against the tumor cell lines tested. Acute toxicities and antitumor efficacies of the selected compounds in mice were also evaluated, and compound 7o exhibited potent antitumor activities with the tumor inhibition rate of over 40%. The wound healing assay displayed a specific impairment in the motility of the HT-29 cells, which suggested the anti-metastatic potential of compound 7o. Moreover, compound 7o had obvious angiogenesis inhibitory effects in the chicken chorioallantoic membrane (CAM) assay. Preliminary structure-activity relationship (SAR) analysis indicated that: (1) 3-phenylpropyl substituent at the N⁹-position of the indole ring was the most suitable group giving rise to potent cytotoxic agents; (2) the spacer length affected the antitumor potencies, and four methylene units were more favorable.

Full text of DOI:10.1016/j.ejmech.2018.02.003

European Journal of Medicinal Chemistry 147 (2018) 253e265
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and structure-activity relationships of asymmetric dimeric
-carboline derivatives as potential antitumor agents
b
,
,
, *
Liang Guo a 1, Wei Chen c 1, Rihui Cao b, Wenxi Fan c, Qin Ma c, Jie Zhang a, Bin Dai a
a School of Chemistry and Chemical Engineering, Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, Shihezi University,
Shihezi 832003, PR China
b School of Chemistry and Chemical Engineering, Sun Yat-sen University, 135 Xin Gang West Road, Guangzhou 510275, PR China
c Xinjiang Huashidan Pharmaceutical Research 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 newly asymmetric dimeric b-carbolines with a spacer of 4e6 methylene units between the
Received 8 November 2017
Received in revised form
2 February 2018
Accepted 2 February 2018
Available online 7 February 2018
indole nitrogen and the harmine oxygen were synthesized. Structures of all the novel synthesized
compounds were confirmed by their spectral and analytical studies. All of the synthesized compounds
were screened for their in vitro cytotoxic activity against nine cancer cell lines. The results revealed that
compounds 7c, 7o and 7s exhibited the highest cytotoxic activities with IC50 values of less than 20 mM
against the tumor cell lines tested. Acute toxicities and antitumor efficacies of the selected compounds in
mice were also evaluated, and compound 7o exhibited potent antitumor activities with the tumor in-
hibition rate of over 40%. The wound healing assay displayed a specific impairment in the motility of the
HT-29 cells, which suggested the anti-metastatic potential of compound 7o. Moreover, compound 7o had
obvious angiogenesis inhibitory effects in the chicken chorioallantoic membrane (CAM) assay. Pre-
liminary structure-activity relationship (SAR) analysis indicated that: (1) 3-phenylpropyl substituent at
the N9-position of the indole ring was the most suitable group giving rise to potent cytotoxic agents; (2)
the spacer length affected the antitumor potencies, and four methylene units were more favorable.
© 2018 Elsevier Masson SAS. All rights reserved.
Keywords:
Harmine
Asymmetric dimeric
Cytotoxic
Antitumor
b-carboline
Structure-activity relationship
1. Introduction
reports [12e15] have demonstrated that harmine and its de-
rivatives have remarkable antitumor activities, together with po-
Peganum harmala L. is a perennial, glabrous plant that is
distributed in the Xinjiang Uyghur and Inner Mongolia Autono-
mous Regions of China. The extracts of Peganum harmala seeds have
been traditionally used for hundreds of years in these areas. Pega-
num harmala L. has been regarded as a traditional herb to that
possesses a wide spectrum of pharmacological actions with
different applications, including nervous system [1e3], antimicro-
bial [4e6], and antineoplasm treatments [2,7e10], and it is effective
in the treatment of dermatoses [11].
tential neurotoxicity. Moreover, it has been reported that harmine
and its derivatives can exert antitumor activities through multiple
mechanisms, such as DNA binding [16e18], inhibition top-
oisomerases I and II [19,20], CDK (cyclin-dependent kinase) [21,22],
PLK1 (polo-like kinase) [23], kinesin-like protein Eg5 [24] and I
kB
kinases [25].
For more than a decade, our group [15,26e31] has focused on
incorporating substituents into positions-1, 2, 3, 7 and 9 of the
b
-
carboline nucleus as antitumor agents. Structure-activity relation-
ship (Fig. 1) analysis has demonstrated that: (1) the methoxy group
substituent at position-7 of harmine might play a crucial role in
determining their remarkable neurotoxic effects; (2) prolonged or
enlarged alkoxy substituents at position-7 led to enhanced cyto-
toxic activities and eliminated completely neurotoxic effects; and
Harmine, originally isolated from Peganum harmala seeds in
1847, is the most representative naturally occurring
b-carboline
alkaloid, having a core indole structure and a pyridine ring. In the
last several decades, harmine has been confirmed as an important
active ingredient to treatalimentary tract cancers [12,13]. Recent
(3) the substituents in position-9 of the b-carboline nucleus played
a vital role in the modulation of their antitumor activities.
Previous literature [32e35] has shown that some dimer anti-
tumor agents via an appropriate linker could lead to significantly
improved antitumor activities (100- to 500-fold improvement over
* Corresponding author. Tel.: þ86 993 2058176; fax: þ86 993 2058176.
E-mail address: db_tea@shzu.edu.cn (B. Dai).
Contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2018.02.003
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

254
L. Guo et al. / European Journal of Medicinal Chemistry 147 (2018) 253e265
Fig. 1. The reported structure-activity relationships of b-carbolines against tumor cells.
the corresponding monomers). Therefore, our group reported the
2. Chemistry
synthesis, in vitro evaluation, in vivo efficacies and structure-
activity relationships for the novel symmetric bivalent
lines with an alkyl spacer or alkylamino spacer in position-1, 3, 7
and 9 of the -carboline nucleus, respectively (Fig. 2) [36e40,44].
Some of bivalent -carbolines have exhibited more potent anti-
tumor efficacies than monomers, and the others had limited utility
for cancer therapy because of their poor water solubility. The
conclusion of structure-activity relationship information revealed
that: (1) the length of the spacer affected the cytotoxic activities
in vitro and 4e6 methylene units were more favorable; and (2) the
b
-carbo-
The overall synthetic routes that were used to design the
asymmetric dimeric
b-carbolines are shown in Schemes 1e3. The
b
starting material -tryptophan was reacted with the corresponding
L
b
aldehyde via the Pictet-Spengler condensation followed by oxida-
tion and decarboxylation to afford the intermediate 1-substituted-
-carbolines 3a-i [36,41], and the result of yield in Table 1. Then, 3a-
b
b further reacted with the appropriate dibromoalkane by N9-
alkylation to obtain the intermediates 4a-f. Harmine, which we
extracted from Peganum harmala L., was N9-alkylated by treatment
with sodium hydride (NaH) and 1,4-dibrombutane or an alkyl
halide in dimethylformamide (DMF) at room temperature, to yield
the harmine derivatives 4g, and 5a-c [13]. The preparation of
compounds 6a-c followed a common synthetic scheme, which was
characterized by the demethylation of compounds 5a-c using acetic
acid and hydrobromic acid as the reaction solvent [14]. The reaction
of compounds 6a-c with the corresponding intermediates 4a-f, and
4g readily took place at room temperature to provide the target
introduction of substituents into position-1 of the b-carboline ring
might be detrimental to antitumor potency in vivo models.
We have continued our search for novel antitumor agents
endowed with better antitumor activities and less neurotoxicities,
and we provide detailed studies of structure-activity relationships
(SARs) on the antitumor efficacies in vitro and in vivo of this class of
compounds. Here, we designed and synthesized a series of meth-
ylene units linked with asymmetric dimeric b-carboline derivatives
as potent antitumor agents. These compounds were expected to
asymmetric dimeric b-carbolines 7a-m, and 7u in a 51e87% yield.
exhibit significantly improved cytotoxic activities. We report herein
After considering the drawbacks of the previous synthesis, we
sought to explore an alternative synthetic strategy for these com-
pounds (Scheme 3). The reaction of compound 6c with 1,4-
the preparation of the novel asymmetric dimeric
their biological evaluation as antitumor agents.
b-carbolines and
Fig. 2. The chemical structure of the representative reported symmetric bivalent b-carbolines.

L. Guo et al. / European Journal of Medicinal Chemistry 147 (2018) 253e265
255
Scheme 1. Synthesis of the 1-substituted b-carboline derivatives 3a-3i.
Reagents and conditions: (i) a: NaOH, H2O, formaldehyde, reflux, 3 h; b: H2SO4, H2O, acetaldehyde, room temperature, 3 h; c-i: HOAc, appropriate aldehyde, reflux, 3 h; (ii) H2O,
K2CrO7, HOAc, stirred at 100 ꢀC, 20 min (iii) NaHSO3, NaOH.
Scheme 2. Synthesis of the asymmetric dimeric b-carboline derivatives 7a-m.
Scheme 3. Synthesis of the asymmetric dimeric b-carboline derivatives 7n-u.

256
L. Guo et al. / European Journal of Medicinal Chemistry 147 (2018) 253e265
Table 1
Similarly, dimers 7e-f, 7k-m, employing n-butyl in position-9 of
carboline nucleus, showed the same tendency. It was interesting to
note that the spacer length in ethyl substituent dimeric -carbolines
b-
Substrate scope of primary 1-substituted
b
-carboline derivatives.
Product
b
Entry
R1
Yield(%)
has little influence on their cytotoxic activities (7a vs. 7b, 7i vs. 7j).
These results suggested that the length of the spacers affected the
cytotoxic activities and four methylene units were more favorable.
Next, we examined the influence of the substituents in position-
1
2
3
4
5
6
7
8
9
H
CH3
3a
3b
3c
3d
3e
3f
3g
3h
3i
75
81
73
69
82
84
65
67
71
Isopropyl
C6H4(p-OCH3)
3,4-dimethoxyphenyl
3,4,5-trimethoxyphenyl
2-chlorophenyl
3-pyridyl
1 of the b-carboline ring on cytotoxic potencies. In comparison with
compound 7g, 7c, and 7n-t, bearing an additional methyl, isopropyl,
4-methoxyphenyl, 3,4-dimethoxyphenyl, 3,4,5-trimethoxyphenyl,
2-chlorophenyl, 2-thienyl, and 3-pyridyl group in position-1 of the
2-thienyl
b
-carboline core, respectively. Of all these dimeric
compound 7o having a 4-methoxyphenyl in position-1 of the
carboline ring displayed strong cytotoxic activities against 769-P,
SK-OV-3, Eca-109 and LLC with IC50 values of 4.9, 4.2, 4.5, and 3.4 M,
b-carbolines, the
b
-
dibrombutane by the action of NaH in anhydrous DMF furnished
intermediate 8, and then the dimeric -carbolines 7n-t were pre-
b
m
pared by reaction of compounds 3c-i with intermediate 8 in
respectively. Meanwhile, 7c, 7g, 7p-t only showed significant cyto-
toxic activities against one or two cell lines with IC50 values lower
anhydrous DMF. The chemical structures of all the novel asym-
metric dimeric b
-carbolines were confirmed by 1H NMR, 13C NMR
than 5
of cytotoxic activities against the tested tumor cell lines with IC50
values lower than 10 M (except for HT-29 cell line). Compound 7n
with an isopropyl in position-1 showed moderate cytotoxic activ-
ities with IC50 values ranging from 10.8 to 46.2 M. In addition, in the
LLC cell line assay, most compounds exhibited selective activities
against it with IC50 values lower than 10 M (except 7n and 7r). On
mM, and compound 7p demonstrated the broader spectrum
and HRMS. By contrasting the 1H NMR spectrum data of com-
pounds 7n-t, we found an interesting phenomenon that com-
m
pounds 7n-q, 7s-t show a triplet peak at
d 4.3 and a group of
multiplets at 1.5e1.8, respectively. But compound 7r in the region
d
m
have two sets of multiple peaks.
m
3. Results and discussion
the other hand, most compounds displayed moderate cytotoxic
activities against the HT-29 cell line with IC50 values ranging from
3.1. In vitro cell cytotoxicity assay
11.2 to 22.7 mM (except 7q).
All of the synthesized newly asymmetric dimeric
b-carbolines
3.2. Assessment of acute toxicity
were evaluated to invitro cytotoxic potencies using the MTTassay in a
panel of human tumor cell lines, and they were compared with the
The LD50 values of the selected asymmetric dimeric b-carbolines
reference drug cisplatin and the reported symmetric bivalent
b
-car-
inmiceafterintraperitoneal(i.p.)administrationareshowninTable 3.
All of the tested asymmetric dimeric -carbolines resulted in acute
toxic manifestation but they did not cause any obvious neurotoxic
effects, including tremors, twitch, jumping, and supination. The ani-
mals showed a decrease in locomotive activity after the administra-
bolines (B-1, B-3, B-4, B-5). The humantumorcelllinepanelconsisted
of gastric carcinoma (BGC-823), liver carcinoma (HepG2), breast
carcinoma (MCF-7), malignant melanoma (A375), colon carcinoma
(HT-29), renal carcinoma (769-P), ovarian carcinoma (SK-OV-3),
esophageal carcinoma (Eca-109), and Lewis lung carcinoma (LLC). In
order to enhance the solubility in aqueous solution, all of the com-
pounds were prepared in the form of hydrochloride salts before use.
The activitywasexpressed asthe concentration (IC50) that causes 50%
inhibition of cancer cell growth and is summarized in Table 2.
As shown in Table 2, compounds 7c, 7d, 7g, 7h and 7o-u dis-
played significant and selective cytotoxicities with IC50 values
b
tion of various bivalent b-carbolines. Death occurred mostly in the
high dosage group within 4e8 h after injection. All of the surviving
animals returned to normal within the next day. Autopsies of the
animals that died during the course of experiment and the necropsy
findings in the surviving animals at the end of the experimental
period (14 days) revealed no obvious changes in any of the organs.
Of all of the investigated asymmetric dimeric
compound 7u, with a methoxy group in position-7 of the b
b
-carbolines,
-car-
lower than 20
compounds, 7o exhibited good activity against 769-P, SK-OV-3, Eca-
109 and LLC with IC50 values of 4.9, 4.2, 4.5 and 3.4 M, respectively.
mM against at least six tumor cell lines. Of these
boline ring, displayed remarkable acute toxicity with the LD50 value
m
of 8 mg kgꢁ1, while compound 7c, which had no substituent in
On the other hand, compounds 7a, 7b, 7i, 7j, 7k, 7l and 7m
exhibited weak to inactive cytotoxic activities.
position-7 of the b-carboline ring, demonstrated weaker acute
toxicities with the LD50 value of 150 mg kgꢁ1. Replacement of the 3-
phenylpropyl group in position-9 of compound 7c with an n-butyl
substituent gave compound 7e, which also exhibited higher acute
toxicity with the LD50 value of 50 mg kgꢁ1. In addition, compound
When asymmetric dimeric
b-carbolines had the same linker, we
examined the influence of the substituents in position-9 of the
b-
carboline core on the cytotoxic activities. In the series of compounds
having an ethyl, n-butyl, or 3-phenylpropyl substituent at the N9-
position of the indole ring. Compounds 7c, 7d, 7g and 7h with a 3-
phenylpropyl displayed higher cytotoxic activities against human
tumor cell lines tested (7c > 7e, 7d > 7b, 7g > 7i and 7k, 7h > 7j and
7m). The results implied that the 3-phenylpropyl group represented
the most optimal structure for these compounds class to exhibit
remarkable cytotoxicity. Table 2 also showed that the different
7g, with no substituent in position-1 of the
exhibited same acute toxicity with the LD50 value of 50 mg kgꢁ1
Compounds 7o and 7s, which have the 3-phenylpropyl group in
position-9 and a different group in position-1 of the -carboline
b-carboline ring,
.
b
ring, displayed lower acute toxicity with the LD50 values of
41.5 mg kgꢁ1 and 37.5 mg kgꢁ1, respectively. These results sug-
gested that the introduction of the substituent into position-1 and
spacer lengths of the asymmetric dimeric
effect on the cytotoxic activities. Of all asymmetric dimeric
bolines 7a-m, compounds 7c-d, 7g-h bearing a 3-phenylpropyl
substituent in position-9 of -carboline nucleus and with a spacer
of four or six methylene units, dimeric -carbolines with four
b
-carbolines had a great
the methoxy group in position-7 of the b-carboline ring might play
an important role in determining acute toxicity.
b-car-
b
3.3. Evaluation of antitumor activity of asymmetric dimeric
carbolines in vivo
b-
b
methylene units as linkers had stronger cytotoxic activities than
compounds with a spacer of six methylene units (7c > 7d, 7g > 7h).
Based on the in vitro assay results, we further tested the

L. Guo et al. / European Journal of Medicinal Chemistry 147 (2018) 253e265
257
Table 2
Cytotoxic activities of derivatives in vitroc (IC50 Ma).
, m
Comp.
R1
R7
R9
n
IC50
BGCb
HepG2
MCF7
A375
HT-29
769-P
SK-OV-3
Eca-109
LLC
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
CH3
CH3
CH3
CH3
CH3
CH3
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
ethyl
ethyl
phenylpropyl
phenylpropyl
n-butyl
5
6
4
6
4
5
4
6
4
6
4
5
6
4
4
4
4
4
4
4
4
35.4
44.7
7.4
12.5
33.6
>100
8.5
11.2
41.5
40.7
41.5
>100
>100
37.8
12.1
9.7
18.2
12.4
11.4
4.2
111.9
74.6
9.1
14.1
15.7
>100
9.7
12.1
>100
>100
29.5
65.1
51.7
46.2
14.1
2.4
24.9
14.4
8.1
10.7
5.6
13.0
9.66
NR
56.3
75.2
4.0
11.7
11.2
>100
4.5
31.5
74.6
8.4
14.9
36.2
14.1
28.1
16.4
10.1
15.9
16.2
37.0
60.3
21.8
32.6
>100
12.1
13.3
22.7
3.9
20.2
18.1
11.2
21.6
6.1
11.3
NR
NR
70.6
75.2
9.9
30.8
11.2
13.1
19.4
21.7
14.3
13.8
76.4
19.8
>100
17.3
65.1
>100
28.1
4.2
46.6
61.4
9.9
15.1
12.2
>100
9.5
12.6
37.0
17.4
21.8
>100
>100
16.2
4.5
>100
51.1
4.4
14.1
13.3
12.4
9.6
28.2
16.5
21.8
38.0
>100
46.1
10.8
3.4
6.2
8.7
10.1
4.2
9.4
14.4
NR
7.68
NR
NR
7.6
15.8
14.1
50.8
33.4
37.1
30.7
34.7
72.4
>100
78.3
23.8
13.6
6.7
16.1
15.3
11.6
10.7
13.1
27.4
>100
22.9
31.1
>100
18.5
4.9
n-butyl
phenylpropyl
phenylpropyl
ethyl
ethyl
n-butyl
8.5
19.4
19.0
28.8
16.7
>100
14.4
8.5
7.2
11.7
5.9
n-butyl
n-butyl
H
isopropyl
phenylpropyl
phenylpropyl
phenylpropyl
phenylpropyl
phenylpropyl
phenylpropyl
phenylpropyl
phenylpropyl
4-methoxyphenyl
3,4-dimethoxyphenyl
3,4,5-trimethoxyphenyl
2-chlorophenyl
2-thienyl
8.2
8.8
9.3
9.7
7q
7r
7s
7t
7.1
3.9
14.1
21.9
11.9
7.2
8.5
NR
12.7
>100
6.5
10.3
22.9
11.2
22.9
30
18.4
4.9
12.9
18.4
NR
14.3
NR
NR
12
14.6
18.9
15.3
NR
13.0
18.2
13.1
9.4
3-pyridyl
CH3
29.8
23.4
>100
7.16
NR
7u
CH3O
4.7
B-1 [38]
B-3 [44]
B-4 [37]
B-5 [36]
Cisplatin
NRd
10.8
43.2
22.3
11.6
NR
16.0
>100
4.8
NR
14.8
NR
12.4
26.8
19.2
5.6
8.9
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 gastric carcinoma (BGC), liver carcinoma (HepG2), breast carcinoma (MCF-7), malignant melanoma (A375), colon carcinoma (HT-29), renal carcinoma
(769-P), ovarian carcinoma (SK-OV-3), esophageal carcinoma (Eca-109), Lewis lung carcinoma (LLC).
c
Data represent the mean values of three independent determinations.
NR ¼ not reported.
d
Table 3
Acute toxic effects of asymmetric dimeric b-carbolines in mice and antitumor activities of these compounds against mice bearing Sarcoma 180 and Lewis lung cancer.
Comp.
Acute toxicity
Dosage (mg kgꢁ1
)
Tumor inhibition rate (%)
Sarcoma 180
LD50(mg kgꢁ1
)
Neurotoxic effect
Lewis lung cancer
7c
7e
150
50
50
41.5
37.5
8
ea
e
e
e
e
þ
30
10
10
8.3
7.5
1.6
30
36.7
53.3
33.5
54.6
47.3
49.9
82.5
NDb
27.2
ND
48.1
31.6
38.2
80.7
7g
7o
7s
7u
CTX
a
Acute neurotoxic manifestation were denoted by “þ” and “-”. “þ” represents toxic responses including tremble, twitch, jumping and supination, while “-” means no such
reaction.
b
ND ¼ not determined.
antitumor activity of six asymmetric dimeric
b-carbolines in vivo
As shown in Table 3, all of the tested asymmetric dimeric b-
against mice bearing Sarcoma 180 and Lewis lung cancer, respec-
tively, and the positive control Cyclophosphamide (CTX). Our pre-
vious investigation demonstrated that mice bearing Lewis lung
cancer were more susceptible to b-carbolines than other animal
models; therefore, these animal models were selected and evalu-
ated in the present investigation [15]. The tumor inhibition rates of
carbolines displayed moderate-to-strong antitumor activities in the
animal model. Compounds 7e, 7o, 7s, and 7u showed remarkable
antitumor activity with the tumor inhibition rate of over 40%
against Sarcoma 180-bearing mice at doses of 10, 8.3, 7.5, and
1.6 mg kgꢁ1, respectively. However, the four compounds showed
moderate antitumor activity against mice with Lewis lung cancer
with the tumor inhibition rate ranging from 27.2% to 48.1% at the
same dose. In particular, compound 7o, which has a 3-phenylpropyl
all of the investigated asymmetric dimeric
trated in Table 3.
b-carbolines are illus-

258
L. Guo et al. / European Journal of Medicinal Chemistry 147 (2018) 253e265
group in position-9 of the
b
-carboline ring, exhibited the most
compound 7o was semi-quantitatively analyzed using Graph Pad
Prism 5.0 (shown in Fig. 5). The results showed that compound 7o
(p < 0.05) could inhibit the angiogenesis of CAM. The anti-
angiogenetic activity of compound 7o was comparable to the
potent antitumor agent with the tumor inhibition rate of 54.6% and
48.1% against Sarcoma 180-bearing mice and Lewis lung cancer-
bearing mice, respectively. Compounds 7c and 7g displayed mod-
erate antitumor activity with the tumor inhibition rate of 36.7% and
33.5% against Sarcoma 180-bearing mice at doses of 30 and
10 mg kgꢁ1, respectively. Interestingly, the Sarcoma 180 mice were
more susceptible to all of the tested compounds than the Lewis
lung cancer-bearing mice. These results suggested that introducing
CA4P in vivo CAM assay at the same dose (50 mM).
4. Conclusions
We designed and synthesized a novel series of asymmetric
dimeric -carboline derivatives with a spacer of 4e6 methylene
the 3-phenylpropyl group in position-9 of the b-carboline nucleus
significantly improved the antitumor activity.
b
units between the indole nitrogen and the harmine oxygen atom.
All of the compounds were screened for their in vitro cytotoxic
activity against BGC-823, HepG2, MCF-7, A375, HT-29, 769-P, SK-
OV-3, Eca-109 and LLC cancer cell lines. The results demonstrated
that compounds 7c, 7o and 7s exhibited prominent cytotoxic ac-
3.4. Inhibitory effect of 7o on tumor cell migration
Cell migration plays an important role in tumor formation and
cancer metastasis. It is relevant for angiogenesis to ensure tumor
nutrition as well as for the formation of metastases, in which tumor
cells leave the primary tumor site and spread to other tissues. In the
present study, the effect of 7o on the migration ability of HT-29 and
LLC cells was examined by the wound healing technique. Fig. 3
shows that the two cancer cell lines were treated with different
concentrations of positive control Combretastatin A4 phosphate
(CA4P) and 7o after 24 h. Surprisingly, 7o showed an inhibitory
tivity with IC50 values of lower than 20
Antitumor evaluation of the selected asymmetric dimeric
bolines in animal models, revealed that compound 7o, which has a
3-phenylpropyl group in position-9 of the -carboline ring,
mM against the test cell lines.
b
-car-
b
exhibited the most potent antitumor agent with the tumor inhi-
bition rate of 54.6% and 48.1% against mice both bearing Sarcoma
180 and Lewis lung cancer, respectively. Moreover, the pharmaco-
logical mechanisms showed that compound 7o has a certain
impairment in the motility of LLC cells, which suggests the anti-
metastatic potential. The in vivo study indicated that compound
7o could retard in the CAM assay, and anti-angiogenetic potency
was more potent than the reference drug CA4P. Preliminary SARs
analysis indicated that: (1) 3-phenylpropyl substituent at the N9-
position of the indole ring was the most suitable group giving rise
to potent cytotoxic agents; (2) the spacer length affected antitumor
potencies, and four methylene units were more favorable.
effect on cell migration on the LLC cells, especially at 50 mM.
3.5. Anti-angiogenic activity in vivo of compound 7o
The most potent compound, 7o, was selected to evaluate anti-
angiogenic activity by chicken chorioallantoic membrane (CAM)
assay. The inhibitory effects of compound 7o on the angiogenesis of
CAM are shown in Fig. 4. The anti-angiogenetic activity of
Fig. 3. Wound healing migration assay of HT29 (A and B) and LLC (C and D) cells after 24 h of treatment with 7o.

L. Guo et al. / European Journal of Medicinal Chemistry 147 (2018) 253e265
259
Fig. 4. Inhibitory effects of compound 7o on the angiogenesis of CAM.
5. Experimental section
All reagents were purchased from commercial suppliers and
were dried and purified when necessary. The following in-
5.1. Reagents and general methods
termediates,
isopropyl- -carboline 3c [36], 1-(4-methoxyphenyl)-
3d [13], 1-(3,4-dimethoxyphenyl)- -carboline 3e [13], 1-(3,4,5-
trimethoxyphenyl)- -carboline 3f [42], 1-(2-chlorophenyl)- -car-
boline 3g [36], 1-(pyridyl-3-yl)- - carboline 3h [36],1-(thiophen-2-
yl)- -carboline 3i [36],. 7-methoxy-9-ethyl-1-methyl- -carboline
5a [13], 7-methoxy-9-n-butyl-1-methyl- -carboline 5b [13], 7-
methoxy-9-(3-phenylpropyl)-1-methyl- -carboline 5c [13], 9-
ethyl-1-methyl- -carboline7-ol 6a [14], 9-n-butyl-1-methyl- -car-
boline-7-ol 6b [14], and 1-methyl-9-(3-phenyl-propyl)- -carbo-
b
-carboline 3a [41], 1-methyl-
b
-carboline 3b [41], 1-
b
b-carboline
Melting points were determined in capillary tubes on an elec-
trothermal WRS-3 apparatus and without correction. NMR spectra
were recorded at room temperature on a Bruker Avance III HD 400
instrument at 400 MHz for 1H NMR and 100 MHz for 13C NMR.
HRMS were measured on Bruker ultrafleXtreme MALDI-TOF/TOF-
MS and HCCA (alpha-cyano-4-hydroxycinnamic acid) is used as
matrix. Column chromatography was performed with silica gel
b
b
b
b
b
b
b
b
b
b
(200e300 mesh) and analytical TLC on silica gel 60-F254
.
b
line-7-ol 6c [14], were synthesized according to published
procedures.
5.2. General procedure for the preparation of compounds 4a-f
A mixture of 3a-b (10 mmol) and anhydrous DMF (50 mL) was
stirred at room temperature for 0.5 h, then NaH (0.50 g, 20 mmol)
and the appropriate dibromoalkane (20 mmol) were added. The
mixture was stirred at room temperature. 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
washed with water and brine, then dried over anhydrous sodium
sulfate, filtered and evaporated. The resulting oil was crystallized
from ethyl ether or ethyl ether-petroleum ether, giving white
crystals.
5.2.1. 9-(4-bromobutoxyl)-
White crystals (2.65 g, 87%) were obtained. 1H NMR (400 MHz,
DMSO-d6):
b-carboline (4a)
d
9.10 (s, 1H), 8.40 (d, J ¼ 5.2 Hz, 1H), 8.28 (d, J ¼ 8.0 Hz,
1H), 8.14 (dd, J ¼ 5.2, 0.8 Hz, 1H), 7.75 (d, J ¼ 8.4 Hz, 1H), 7.60e7.64
(m, 1H), 7.27e7.31 (m, 1H), 4.56 (t, J ¼ 6.8 Hz, 2H), 3.56 (t, J ¼ 6.4 Hz,
2H), 1.90e1.97 (m, 2H), 1.81e1.88 (m, 2H). 13C NMR (100 MHz,
Fig. 5. Anti-angiogenic activity of compound 7o.
DMSO-d6): d 141.13, 138.88, 136.47, 133.13, 128.77, 127.66, 122.44,

260
L. Guo et al. / European Journal of Medicinal Chemistry 147 (2018) 253e265
120.80, 119.92, 115.04, 110.62, 42.15, 35.09, 30.23, 27.87.
5.3.1. 9-Ethyl-1-methyl-7-((5-(1-methyl-
oxy)- -carboline (7a)
White crystals (1.42 g, 74%) were obtained, m.p. 155.4e156.8 ꢀC.
1H NMR (400 MHz, CDCl3):
J ¼ 5.2 Hz,1H), 8.12 (d, J ¼ 7.6 Hz,1H), 7.96 (d, J ¼ 8.8 Hz,1H), 7.85 (d,
J ¼ 5.2 Hz, 1H), 7.73 (d, J ¼ 5.2 Hz, 1H), 7.55e7.59 (m, 1H), 7.48 (d,
J ¼ 8.4 Hz, 1H), 7.25e7.28 (m, 1H), 6.82e6.87 (m, 2H), 4.59 (t,
J ¼ 7.6 Hz, 2H), 4.53 (q, J ¼ 7.2 Hz, 2H), 4.08 (t, J ¼ 6.0 Hz, 2H), 3.07 (s,
3H), 3.03 (s, 3H), 1.88e2.01 (m, 4H), 1.63e1.71 (m, 2H), 1.43 (t,
b-carboline-9-yl)pentyl)
b
5.2.2. 9-(5-bromopentyl)-
White crystals (2.72 g, 86%) were obtained. 1H NMR (400 MHz,
CDCl3):
b-carboline (4b)
d
8.33 (d, J ¼ 5.2 Hz, 1H), 8.28 (d,
d
9.45 (d, J ¼ 5.2 Hz, 1H), 8.53 (t, J ¼ 5.2 Hz, 1H), 8.44 (d,
J ¼ 6.0 Hz, 1H), 8.32 (dt, J ¼ 8.0, 1.0 Hz, 1H), 7.84e7.88 (m, 1H), 7.67
(d, J ¼ 8.4 Hz,1H), 7.49e7.54 (m, 1H), 4.63 (t, J ¼ 7.2 Hz, 2H), 3.45 (dt,
J ¼ 54.0, 6.4 Hz, 2H), 1.96e2.04 (m, 2H), 1.80e1.95 (m, 2H),
1.56e1.64 (m, 2H). 13C NMR (100 MHz, CDCl3):
d 144.19, 135.44,
J ¼ 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3):
d 160.24, 142.68, 141.45,
133.92, 132.44, 128.57, 124.18, 123.42, 122.24, 119.70, 116.69, 110.66,
44.45, 32.03, 28.57, 25.63, 24.40.
141.14, 140.41, 138.01, 135.10, 135.04, 129.43, 129.08, 128.11, 122.44,
121.52, 121.33, 119.62, 115.31, 112.97, 112.26, 109.65, 109.05, 93.78,
67.97, 44.79, 39.46, 30.66, 29.15, 23.63, 23.60, 23.15, 15.53. HRMS
(ESI) calcd for C31H33N4O 477.2649 [MþH]þ, found 477.2647.
5.2.3. 9-(6-bromohexyl)-
Yellow oil (2.31 g, 70%) were obtained. 1H NMR (400 MHz,
CDCl3):
b-carboline (4c)
d
8.83 (s, 1H), 8.43 (d, J ¼ 5.6 Hz, 1H), 8.05 (d, J ¼ 7.6 Hz, 1H),
5.3.2. 9-Ethyl-1-methyl-7-((6-(1-methyl-
oxy)- -carboline (7b)
White crystals (1.38 g, 70%) were obtained, m.p. 127.1e128.3 ꢀC.
1H NMR (400 MHz, CDCl3):
b-carboline-9-yl)hexyl)
7.86 (d, J ¼ 5.2 Hz, 1H), 7.52 (t, J ¼ 7.6 Hz, 1H), 7.36 (d, J ¼ 8.4 Hz, 1H),
7.22 (t, J ¼ 7.6 Hz, 1H), 4.22 (t, J ¼ 7.2 Hz, 2H), 3.27 (t, J ¼ 6.8 Hz, 2H),
1.84e1.76 (m, 2H), 1.74e1.67 (m, 2H), 1.41e1.26 (m, 4H). 13C NMR
b
d
8.34 (d, J ¼ 5.2 Hz, 1H), 8.28 (d,
(100 MHz, CDCl3):
d 141.02, 138.71, 136.35, 131.91, 128.30, 128.17,
J ¼ 5.2 Hz,1H), 8.15 (d, J ¼ 8.0 Hz,1H), 7.95 (d, J ¼ 8.8 Hz,1H), 7.84 (d,
J ¼ 5.2 Hz, 1H), 7.73 (d, J ¼ 5.2 Hz, 1H), 7.55e7.59 (m, 1H), 7.50 (d,
J ¼ 8.4 Hz, 1H), 7.27e7.31 (m, 1H), 6.85 (dd, J ¼ 8.8, 2.0 Hz, 1H), 6.81
(d, J ¼ 2.0 Hz, 1H), 4.68 (t, J ¼ 8.0 Hz, 2H), 4.42 (t, J ¼ 8.0 Hz, 2H), 4.12
(t, J ¼ 6.0 Hz, 2H), 3.08 (s, 3H), 3.02 (s, 3H), 2.08e2.16 (m, 2H),
1.94e2.00 (m, 2H), 1.75e1.82 (m, 2H), 1.38e1.47(m, 2H), 0.96 (t,
121.83, 120.93, 119.54, 114.51, 109.40, 43.06, 33.71, 32.44, 28.92,
27.78, 26.33.
5.2.4. 9-(4-bromobutoxyl)-1-methyl-
White crystals (2.81 g, 88%) were obtained. 1H NMR (400 MHz,
DMSO-d6):
b-carboline (4d)
d
8.71 (dd, J ¼ 6.2, 1.2 Hz, 1H), 8.54 (dd, J ¼ 8.0, 1.2 Hz,
J ¼ 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3):
d 159.91, 143.05, 141.45,
1H), 8.49 (dd, J ¼ 6.2, 2.0 Hz, 1H), 8.00 (d, J ¼ 8.4 Hz, 1H), 7.83e7.87
(m, 1H), 7.46e7.50 (m, 1H), 4.75 (t, J ¼ 7.6 Hz, 2H), 3.70 (t, J ¼ 6.4 Hz,
2H), 3.27 (s, 3H), 2.04e1.77 (m, 4H). 13C NMR (100 MHz, DMSO-d6):
141.17, 140.49, 138.10, 138.01, 135.28, 135.08, 129.35, 129.13, 128.12,
122.42, 121.54, 121.38, 119.67, 115.32, 112.97, 112.24, 109.68, 108.78,
94.10, 67.73, 44.67, 44.59, 32.72, 27.80, 26.69, 23.59, 23.30, 20.17,
13.90. HRMS (ESI) calcd for C32H35N4O 491.2805 [MþH]þ, found
491.2813.
d
144.33, 139.00, 133.74, 133.26, 132.07, 129.29, 123.86, 122.14,
119.67, 116.15, 111.78, 45.35, 44.31, 29.63, 28.29, 18.31.
5.2.5. 9-(5-bromopentyl)-1-methyl-
White crystals (2.97 g, 90%) were obtained. 1H NMR (400 MHz,
CDCl3):
b-carboline (4e)
5.3.3. 1-Methyl-7-(4-(1-methyl-
phenylpropyl- -carboline (7c)
White crystals (1.57 g, 71%) were obtained, m.p. 147.7e148.7 ꢀC.
1H NMR (400 MHz, CDCl3):
b-carboline-9-yl)butoxy)-9-
b
d
8.42e8.33 (m, 2H), 8.29 (dt, J ¼ 8.0, 1.2 Hz, 1H), 7.82e7.86
(m, 1H), 7.66 (dt, J ¼ 8.4, 0.8 Hz, 1H), 7.48e7.51 (m, 1H), 4.69 (t,
d
8.35 (d, J ¼ 5.2 Hz, 1H), 8.27 (d,
J ¼ 7.6 Hz, 2H), 3.52 (s, 3H), 3.48 (dt, J ¼ 54.0, 6.4 Hz, 1H), 1.82e1.98
J ¼ 5.2 Hz, 1H), 8.12e8.14 (m, 1H), 7.94 (d, J ¼ 8.4 Hz, 1H), 7.86 (d,
J ¼ 5.2 Hz, 1H), 7.73 (d, J ¼ 5.2 Hz, 1H), 7.56e7.60 (m, 1H), 7.51 (d,
J ¼ 8.4 Hz, 1H), 7.24e7.30 (m, 3H), 7.14e7.19 (m, 3H), 6.84 (dd,
J ¼ 8.4, 2.0 Hz, 1H), 6.60 (d, J ¼ 2.0 Hz, 1H), 4.69 (t, J ¼ 7.6 Hz, 2H),
4.43 (t, J ¼ 7.6 Hz, 2H), 4.01 (t, J ¼ 6.0 Hz, 2H), 3.09 (s, 3H), 2.90 (s,
(m, 4H), 1.60e1.68 (m, 2H). 13C NMR (100 MHz, CDCl3):
d 144.30,
137.12, 133.87, 133.76, 132.22, 128.57, 123.11, 122.38, 119.70, 115.13,
110.68, 45.35, 44.43, 31.86, 30.29, 24.04, 18.07.
3H), 2.74 (t, J ¼ 7.2 Hz, 2H), 2.08e2.17 (m, 4H), 1.93e1.98(m, 2H). 13
C
5.2.6. 9-(6-bromohexyl)-1-methyl-
White crystals (2.37 g, 69%) were obtained. 1H NMR (400 MHz,
CDCl3):
b-carboline (4f)
NMR (100 MHz, CDCl3):
d 160.07, 143.10, 141.51, 141.14, 140.58,
d
8.33 (d, J ¼ 5.2 Hz, 1H), 8.12 (d, J ¼ 7.6 Hz, 1H), 7.84 (d,
140.27, 138.02, 135.14, 135.09, 129.63, 129.22, 128.58, 128.41, 128.20,
126.31, 122.53, 121.59, 121.39, 119.74, 115.21, 113.02, 112.32, 109.70,
109.34, 93.74, 67.67, 44.62, 44.06, 32.89, 31.70, 27.82, 26.61, 23.53,
22.76. HRMS (ESI) calcd for C37H37N4O 553.2962 [MþH]þ, found
553.2953.
J ¼ 5.2 Hz, 1H), 7.56e7.60 (m, 1H), 7.45 (d, J ¼ 8.4 Hz, 1H), 7.25e7.29
(m, 1H), 4.54 (t, J ¼ 7.6 Hz, 2H), 3.38 (t, J ¼ 6.4 Hz, 2H), 3.05 (s, 3H),
1.81e1.89 (m, 4H), 1.40e1.55 (m, 4H). 13C NMR (100 MHz, CDCl3):
d
141.51, 141.13, 137.94, 135.09, 129.14, 128.17, 121.55, 121.33, 119.66,
112.99, 109.67, 44.76, 33.56, 30.70, 27.90, 26.13, 23.55.
5.3.4. 1-Methyl-7-((6-(1-methyl-
phenylpropyl- -carboline (7d)
White crystals (1.79 g, 77%) were obtained, m.p. 126.1e127.4 ꢀC.
1H NMR (400 MHz, CDCl3):
8.33 (d, J ¼ 5.2 Hz, 1H), 8.26 (d,
b-carboline-9-yl)hexyl)oxy)-9-
5.3. General procedure for the preparation of compounds 7a-m
b
A solution of compound 6a-c (4 mmol) in anhydrous DMF
(10 mL) was added slowly with stirring to a solution of 4a-f
(6 mmol), NaH (0.25 g, 10 mmol), potassium iodide (1.68 g,
10 mmol) in anhydrous DMF (25 mL). The mixture was stirred at
room temperature until the reaction is completed. Then the
mixture was poured into ice-cold water. The reaction mixture was
extracted with ethyl acetate (3 ꢂ 70 mL), washed with water and
brine, dried over anhydrous Na2SO4 and evaporated to give the
d
J ¼ 5.2 Hz, 1H), 8.15e8.09 (m, 1H), 7.93 (d, J ¼ 8.4 Hz, 1H), 7.85 (d,
J ¼ 5.2 Hz, 1H), 7.71 (d, J ¼ 5.2 Hz, 1H), 7.55e7.59 (m, 1H), 7.47 (d,
J ¼ 8.4 Hz, 1H), 7.25e7.31 (m, 3H), 7.18e7.22 (m, 3H), 6.83 (dd,
J ¼ 8.4, 2.0 Hz, 1H), 6.61 (d, J ¼ 2.0 Hz, 1H), 4.57 (t, J ¼ 7.6 Hz, 2H),
4.43 (t, J ¼ 7.6 Hz, 2H), 3.95 (t, J ¼ 6.0 Hz, 2H), 3.07 (s, 3H), 2.89 (s,
3H), 2.75 (t, J ¼ 7.2 Hz, 2H), 2.10e2.17 (m, 2H), 1.87e1.98 (m, 2H),
1.80e1.85 (m, 2H), 1.52e1.61 (m, 4H). 13C NMR (100 MHz, CDCl3):
dimeric
b
-carbolines 7a-m as crude product, which was purified
d 160.29, 143.03, 141.49, 141.17, 140.65, 140.41, 138.00, 135.19, 135.12,
using silica gel (200e300 mesh) and using CH2Cl2:CH3OH (100:1)
as the eluent. Then the compounds were dissolved in hydrochloric
acid alcohol, removed by evaporation and the residue was the 7a-m
hydrochloride salts.
129.49, 129.08, 128.58, 128.41, 128.09, 126.31, 122.37, 121.53, 121.34,
119.61, 115.08, 112.97, 112.24, 109.66, 109.36, 93.69, 68.03, 44.82,
44.06, 32.93, 31.71, 30.83, 29.24, 26.80, 26.04, 23.61, 23.02. HRMS
(ESI) calcd for C39H41N4O 581.3275 [MþH]þ, found 581.3273.

L. Guo et al. / European Journal of Medicinal Chemistry 147 (2018) 253e265
261
5.3.5. 9-Butyl-1-methyl-7-(4-(1-methyl-
-carboline (7e)
White crystals (1.42 g, 72%) were obtained, m.p. 130.1e131.5 ꢀC.
1H NMR (400 MHz, CDCl3):
8.33 (d, J ¼ 5.2 Hz, 1H), 8.28 (d,
b
-carboline-9-yl)butoxy)-
[MþH]þ, found 567.3112.
b
5.3.9. 1-Methyl-7-(4-(b-carboline-9-yl)butoxy)-9-ethyl-b-
d
carboline (7i)
J ¼ 5.2 Hz, 1H), 8.10e8.12 (m, 1H), 7.95 (d, J ¼ 8.8 Hz, 1H), 7.83 (d,
J ¼ 5.2 Hz, 1H), 7.71 (d, J ¼ 5.2 Hz, 1H), 7.54e7.59 (m, 1H), 7.49 (d,
J ¼ 8.4 Hz, 1H), 7.26e7.30 (m, 1H), 7.25 (d, J ¼ 0.8 Hz, 1H), 6.84 (dd,
J ¼ 8.4, 2.0 Hz, 1H), 6.80 (d, J ¼ 2.0 Hz, 1H), 4.66 (t, J ¼ 7.6 Hz, 2H),
4.41 (t, J ¼ 8.0 Hz, 2H), 4.11 (t, J ¼ 6.0 Hz, 2H), 3.07 (s, 3H), 3.01 (s,
3H), 2.07e2.15 (m, 2H), 1.93e1.99 (m, 2H), 1.74e1.82 (m, 2H),
1.37e1.46 (m, 2H), 0.96 (t, J ¼ 7.6 Hz, 3H). 13C NMR (100 MHz,
White crystals (1.46 g, 81%) were obtained, m.p. 186.2e186.7 ꢀC.
1H NMR (400 MHz, CDCl3):
d
8.96 (s, 1H), 8.48 (d, J ¼ 5.2 Hz, 1H),
8.28 (d, J ¼ 5.2 Hz, 1H), 8.18e8.13 (m, 1H), 8.00e7.92 (m, 2H), 7.74
(d, J ¼ 5.2 Hz, 1H), 7.58e7.62 (m, 1H), 7.52 (d, J ¼ 8.4 Hz, 1H),
7.28e7.32 (m, 1H), 6.84 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.81 (d, J ¼ 2.0 Hz,
1H), 4.52 (dt, J ¼ 8.0, 7.2 Hz, 4H), 4.10 (t, J ¼ 6.4 Hz, 2H), 3.04 (s, 3H),
2.17e2.24 (m, 2H), 1.90e1.97 (m, 2H), 1.42 (t, J ¼ 7.2 Hz, 3H). 13C
CDCl3):
d 160.24, 142.68, 141.45, 141.14, 140.41, 138.01, 135.10,
NMR (100 MHz, CDCl3):
d 160.13, 142.73, 141.12, 140.28, 138.97,
135.04, 129.43, 129.08, 128.11, 122.44, 121.52, 121.33, 119.62, 115.31,
112.97, 112.26, 109.65, 109.05, 93.78, 67.97, 44.79, 39.46, 30.66,
29.68, 29.15, 23.63, 23.60, 23.15, 15.53. HRMS (ESI) calcd for
137.71, 136.47, 134.99, 132.03, 129.55, 128.36, 122.52, 121.96, 121.13,
119.65, 115.33, 114.60, 112.30, 109.43, 109.06, 93.79, 67.79, 43.13,
39.48, 27.01, 26.14, 22.97, 15.51. HRMS (ESI) calcd for C29H29N4O
449.2336[MþH]þ, found 449.2332.
C
32H35N4O 491.2805 [MþH]þ, found 491.2807.
5.3.6. 9-Butyl-1-methyl-7-((5-(1-methyl-
oxy)- -carboline (7f)
White crystals (1.21 g, 61%) were obtained, m.p. 118.9e120.2 ꢀC.
1H NMR (400 MHz, CDCl3):
8.33 (d, J ¼ 5.2 Hz, 1H), 8.28 (d,
b-carboline-9-yl)pentyl)
b
5.3.10. 9-Ethyl-1-methyl-7-((6-(b-carboline-9-yl)hexyl)oxy)-b-
carboline (7j)
d
White crystals (1.66 g, 87%) were obtained, m.p. 150.0e151.1 ꢀC.
J ¼ 5.2 Hz, 1H), 8.09e8.12 (m, 1H), 7.95 (d, J ¼ 8.4 Hz, 1H), 7.83 (d,
J ¼ 5.2 Hz, 1H), 7.72 (d, J ¼ 5.2 Hz, 1H), 7.54e7.58 (m, 1H), 7.46 (d, J ¼
8.4 Hz, 1H), 7.24e7.28 (m, 1H), 6.80e6.85 (m, 2H), 4.57 (t, J ¼ 7.6 Hz,
2H), 4.41 (t, J ¼ 7.6 Hz, 2H), 4.07 (t, J ¼ 6.0 Hz, 2H), 3.06 (s, 3H), 3.01
(s, 3H), 1.87e1.99 (m, 4H), 1.74e1.82 (m, 2H), 1.62e1.70 (m, 2H),
1.38e1.47 (m, 2H), 0.97 (t, J ¼ 7.6 Hz, 3H). 13C NMR (100 MHz,
1H NMR (400 MHz, CDCl3):
d
8.92 (s, 1H), 8.47 (d, J ¼ 5.2 Hz, 1H),
8.28 (d, J ¼ 5.2 Hz,1H), 8.14e8.17 (m,1H), 7.94e7.98 (m, 2H), 7.75 (d,
J ¼ 5.2 Hz, 1H), 7.57e7.61 (m, 1H), 7.49 (d, J ¼ 8.4 Hz, 1H), 7.27e7.31
(m, 1H), 6.83e6.86 (m, 2H), 4.54 (q, J ¼ 7.2 Hz, 2H), 4.43 (t, J ¼
7.2 Hz, 2H), 4.05 (t, J ¼ 6.4 Hz, 2H), 3.06 (s, 3H), 1.95e2.02 (m, 2H),
1.79e1.86 (m, 2H), 1.55e1.62 (m, 2H), 1.47e1.53 (m, 2H), 1.44 (t, J ¼
CDCl3):
d
160.13, 143.06, 141.43, 141.15, 140.50, 138.05, 135.29,
7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3):
d 160.48, 142.88, 141.14,
135.09, 129.34, 129.05, 128.09, 122.34, 121.51, 121.34, 119.61, 115.22,
112.95, 112.22, 109.64, 108.86, 94.12, 67.96, 44.77, 44.65, 32.72,
30.67, 29.17, 23.63, 23.35, 20.17, 13.91. HRMS (ESI) calcd for
140.13, 138.82, 137.48, 136.51, 134.94, 132.01, 129.71, 128.30, 122.48,
121.93, 121.07, 119.55, 115.10, 114.58, 112.31, 109.42, 109.32, 93.70,
68.11, 43.29, 39.49, 29.15, 27.05, 25.96, 22.83, 15.53. HRMS (ESI)
calcd for C31H33N4O 477.2649 [MþH]þ, found 477.2646.
C
33H37N4O 505.2962 [MþH]þ, found 505.2957.
5.3.7. 1-Methyl-7-(4-(b-carboline-9-yl)butoxy)-9-phenylpropyl-b-
5.3.11. 9-Butyl-1-methyl-7-(4-(b-carboline-9-yl)butoxy)-b-
carboline (7g)
carboline (7k)
White crystals (1.56 g, 73%) were obtained, m.p. 154.5e155.2 ꢀC.
White crystals (1.62 g, 85%) were obtained, m.p. 124.4e124.7 ꢀC.
1H NMR (400 MHz, CDCl3):
d
8.96 (s, 1H), 8.49 (d, J ¼ 5.2 Hz, 1H),
1H NMR (400 MHz, CDCl3):
d
8.96 (s, 1H), 8.48 (d, J ¼ 5.2 Hz, 1H),
8.26 (d, J ¼ 5.2 Hz, 1H), 8.15e8.17 (m, 1H), 7.97 (d, J ¼ 5.2 Hz, 1H),
7.92 (d, J ¼ 8.4 Hz, 1H), 7.71 (d, J ¼ 5.2 Hz, 1H), 7.58e7.63 (m, 1H),
7.52 (d, J ¼ 8.4 Hz, 1H), 7.24e7.32 (m, 3H), 7.13e7.18 (m, 3H), 6.81
(dd, J ¼ 8.4, 2.0 Hz, 1H), 6.57 (d, J ¼ 2.0 Hz, 1H), 4.53 (t, J ¼ 7.2 Hz,
2H), 4.41 (t, J ¼ 8.0 Hz, 2H), 3.97 (t, J ¼ 6.4 Hz, 2H), 2.88 (s, 3H), 2.73
(t, J ¼ 7.2 Hz, 2H), 2.17e2.23 (m, 2H), 2.08e2.16 (m, 2H), 1.87e1.94
8.28 (d, J ¼ 5.2 Hz, 1H), 8.16 (d, J ¼ 7.6 Hz, 1H), 7.97 (dd, J ¼ 5.2,
1.2 Hz, 1H), 7.94 (d, J ¼ 8.4 Hz, 1H), 7.73 (d, J ¼ 5.2 Hz, 1H), 7.58e7.62
(m, 1H), 7.50 (d, J ¼ 8.4 Hz, 1H), 7.28e7.32 (m, 1H), 6.84 (dd, J ¼ 8.4,
2.0 Hz, 1H), 6.80 (d, J ¼ 2.0 Hz, 1H), 4.53 (t, J ¼ 7.2 Hz, 2H), 4.43 (t,
J ¼ 7.6 Hz, 2H), 4.10 (t, J ¼ 6.4 Hz, 2H), 3.02 (s, 3H), 2.17e2.24 (m,
2H),1.90e1.97 (m, 2H),1.75e1.82 (m, 2H),1.38e1.47 (m, 2H), 0.96 (t,
(m, 2H). 13C NMR (100 MHz, CDCl3):
d 159.99, 142.96, 141.11, 140.62,
J ¼ 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3):
d 160.00, 143.09, 141.11,
140.40, 139.02, 137.95, 136.48, 135.17, 132.07, 129.44, 128.56, 128.41,
128.36, 126.28, 122.44, 121.98, 121.14, 119.66, 115.19, 114.62, 112.27,
109.44, 109.21, 93.71, 67.69, 44.03, 43.14, 32.89, 31.70, 26.95, 26.18,
22.98. HRMS (ESI) calcd for C36H35N4O 539.2805 [MþH]þ, found
539.2807.
140.40, 139.00, 137.85, 136.47, 135.25,132.06, 129.42, 128.35,122.42,
121.97, 121.13, 119.64, 115.25, 114.61, 112.27, 109.44, 108.85, 94.10,
67.78, 44.67, 43.13, 32.72, 27.04, 26.18, 23.21, 20.16, 13.90. HRMS
(ESI) calcd for C31H33N4O 477.2649 [MþH]þ, found 477.2660.
5.3.8. 7-((6-(
phenylpropyl-
White crystals (1.26 g, 56%) were obtained, m.p. 122.2e123.3 ꢀC.
1H NMR (400 MHz, CDCl3):
b
-carboline-9-yl)hexyl)oxy)-1-methyl-9-
-carboline (7h)
5.3.12. 9-Butyl-1-methyl-7-((5-(b-carboline-9-yl)pentyl)oxy)-b-
b
carboline (7l)
White crystals (1.67 g, 85%) were obtained, m.p. 127.2e127.9 ꢀC.
d
8.92 (s, 1H), 8.47 (d, J ¼ 5.2 Hz, 1H),
1H NMR (400 MHz, CDCl3):
d
8.93 (s, 1H), 8.47 (d, J ¼ 5.2 Hz, 1H),
8.26 (d, J ¼ 5.2 Hz, 1H), 8.14e8.16 (m, 1H), 7.97 (d, J ¼ 5.2 Hz, 1H),
7.92 (d, J ¼ 8.4 Hz, 1H), 7.71 (d, J ¼ 5.2 Hz, 1H), 7.56e7.61 (m, 1H),
7.48 (d, J ¼ 8.4 Hz, 1H), 7.26e7.30 (m, 3H), 7.17e7.21 (m, 3H), 6.81
(dd, J ¼ 8.4, 2.0 Hz, 1H), 6.60 (d, J ¼ 2.0 Hz, 1H), 4.40e4.44 (m, 4H),
3.92 (t, J ¼ 6.4 Hz, 2H), 2.89 (s, 3H), 2.74 (t, J ¼ 7.2 Hz, 2H), 2.11e2.17
(m, 2H), 1.95e2.02 (m, 2H), 1.76e1.81 (m, 2H), 1.53e1.59 (m, 2H),
8.27 (d, J ¼ 5.2 Hz,1H), 8.15 (d, J ¼ 7.6 Hz,1H), 7.96 (d, J ¼ 5.2 Hz,1H),
7.93 (d, J ¼ 8.4 Hz, 1H), 7.71 (d, J ¼ 5.2 Hz, 1H), 7.57e7.61 (m, 1H),
7.48 (d, J ¼ 8.4 Hz, 1H), 7.26e7.30 (m, 1H), 6.78e6.82 (m, 2H),
4.39e4.44 (m, 4H), 4.03 (t, J ¼ 6.0 Hz, 2H), 3.01 (s, 3H), 1.99e2.07
(m, 2H), 1.85e1.92 (m, 2H), 1.74e1.81 (m, 2H), 1.58e1.65 (m, 2H),
1.37e1.46 (m, 2H), 0.98 (t, J ¼ 7.6 Hz, 3H). 13C NMR (100 MHz,
1.46e1.52 (m, 2H). 13C NMR (100 MHz, CDCl3):
d
160.32, 143.06,
CDCl3): d 160.11, 143.06, 141.10, 140.46, 138.92, 137.99, 136.50,
141.14, 140.63, 140.31, 138.86, 137.83, 136.51, 135.13, 132.04, 129.55,
128.57, 128.40, 128.30, 126.30, 122.36, 121.93, 121.08, 119.55, 114.98,
114.59, 112.24, 109.43, 93.63, 68.01, 44.03, 43.30, 32.91, 31.71, 29.69,
29.15, 27.10, 25.99, 22.90. HRMS (ESI) calcd for C38H39N4O 567.3118
135.26, 132.05, 129.36, 128.31, 128.28, 122.32, 121.93, 121.09, 119.58,
115.14, 114.58, 112.23, 109.43, 108.88, 94.07, 67.94, 44.64, 43.27,
32.72, 29.16, 29.06, 23.96, 23.33, 20.17, 13.92. HRMS (ESI) calcd for
C
32H35N4O 491.2805 [MþH]þ, found 491.2805.

262
L. Guo et al. / European Journal of Medicinal Chemistry 147 (2018) 253e265
5.3.13. 9-Butyl-1-methyl-7-((6-(
b
-carboline-9-yl)hexyl)oxy)-
b
-
2H), 1.48 (d, J ¼ 6.4 Hz, 6H). 13C NMR (100 MHz, CDCl3):
d 159.96,
carboline (7m)
150.47, 142.93, 141.78, 140.61, 140.48, 138.36, 138.09, 135.20, 133.36,
129.75, 129.40, 128.56, 128.41, 128.10, 126.28, 122.44, 121.54, 121.36,
119.62, 115.25, 112.50, 112.26, 109.63, 109.18, 93.65, 67.62, 45.12,
44.03, 32.90, 31.70, 31.37, 27.37, 26.63, 23.03, 22.67. HRMS (ESI)
calcd for C39H41N4O 581.3275 [MþH]þ, found 581.3275.
White crystals (1.52 g, 75%) were obtained, m.p. 154.4e155.1 ꢀC.
1H NMR (400 MHz, CDCl3):
d
8.91 (s, 1H), 8.47 (d, J ¼ 5.2 Hz, 1H),
8.27 (d, J ¼ 5.2 Hz, 1H), 8.15 (d, J ¼ 7.6 Hz, 1H), 7.92e7.96 (m, 2H),
7.71 (d, J ¼ 5.2 Hz, 1H), 7.56e7.60 (m, 1H), 7.47 (d, J ¼ 8.4 Hz, 1H),
7.26e7.30 (m, 1H), 6.81e6.84 (m, 2H), 4.38e4.44 (m, 4H), 4.04 (t,
J ¼ 6.4 Hz, 2H), 2.99 (s, 3H), 1.94e2.01 (m, 2H), 1.75e1.85 (m, 4H),
1.54e1.61 (m, 2H), 1.38e1.52 (m, 4H), 0.96 (t, J ¼ 7.2 Hz, 3H). 13C
5.5.2. 7-(4-(1-(4-methoxyphenyl)-
methyl-9-phenylpropyl - -carboline (7o)
White crystals (1.79 g, 69%) were obtained, m.p. 117.2e117.9 ꢀC.
1H NMR (400 MHz, CDCl3):
8.52 (d, J ¼ 5.2 Hz, 1H), 8.26 (d, J ¼
b-carboline-9-yl)butoxy)-1-
b
NMR (100 MHz, CDCl3):
d 160.20, 143.09, 141.11, 140.45, 138.87,
138.00, 136.50, 135.26, 132.05,129.37, 128.28, 128.26,122.30,121.92,
121.06, 119.53, 115.09, 114.57, 112.22, 109.42, 108.89, 94.07, 68.08,
44.65, 43.28, 32.72, 29.19, 29.14, 27.08, 25.97, 23.33, 20.17, 13.91.
d
5.2 Hz, 1H), 8.17 (d, J ¼ 7.6 Hz, 1H), 7.95 (d, J ¼ 5.2 Hz, 1H), 7.90 (d,
J ¼ 8.4 Hz, 1H), 7.69 (d, J ¼ 5.2 Hz, 1H), 7.56e7.60 (m, 3H), 7.50 (d,
J ¼ 8.4 Hz, 1H), 7.29e7.32 (m, 1H), 7.22e7.26 (m, 2H), 7.13e7.17 (m,
3H), 6.99e7.02 (m, 2H), 6.73 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.48 (d,
J ¼ 2.0 Hz, 1H), 4.39 (t, J ¼ 8.0 Hz, 2H), 4.14 (t, J ¼ 8.0 Hz, 2H), 3.79 (s,
3H), 3.68 (t, J ¼ 6.4 Hz, 2H), 2.88 (s, 3H), 2.72 (t, J ¼ 7.2 Hz, 2H),
2.07e2.14 (m, 2H), 1.58e1.66 (m, 2H), 1.34e1.41 (m, 2H). 13C NMR
HRMS (ESI) calcd for
505.2962.
C
33H37N4O 505.2962 [MþH]þ, found
5.4. Synthesis of 7-(4-bromobutoxyl)-1-methyl-9-(3-
phenylpropyl)- -carboline (8)
b
(100 MHz, CDCl3):
d 159.93, 159.76, 144.12, 142.90, 142.09, 140.62,
Compound 6c (3.16 g, 10 mmol) and NaH (0.50 g, 20 mmol) were
dissolved in anhydrous DMF (50 mL). 1,4-dibromobutane
(20 mmol) was added and then stirred at room temperature until
the reaction is completed. Then the reaction solution was poured
into cool water (20 mL), the organic compounds were extracted
with ethyl acetate and washed sequentially with water and brine,
then dried over anhydrous sodium sulfate, filtered and evaporated.
The residue was crystallized from ethyl ether-petroleum ether to
afford white crystals (0.36 g, 80%).
140.46, 138.54, 138.12, 135.17, 134.23, 132.55, 130.55, 130.45, 129.37,
128.56, 128.41, 128.33, 126.28, 122.33, 121.65, 121.59, 119.88, 115.11,
113.67, 113.40, 112.22, 110.21, 109.13, 93.66, 67.43, 55.35, 44.14,
43.99, 32.89, 31.68, 26.32, 25.84, 23.08. HRMS (ESI) calcd for
C
43H41N4O2 645.3224 [MþH]þ, found 645.3234.
5.5.3. 1-Methyl-9-phenylpropyl-7-(4-(1-(3,4-dimethoxyphenyl)-
carboline-9-yl)-butoxy)- -carboline (7p)
White crystals (1.89 g, 70%) were obtained, m.p. 76.5e77.4 ꢀC. 1H
NMR (400 MHz, CDCl3):
8.53 (d, J ¼ 5.2 Hz, 1H), 8.26 (d, J ¼ 5.2 Hz,
b-
b
1H NMR (400 MHz, CDCl3):
d
8.26 (d, J ¼ 5.2 Hz, 1H), 7.95 (d,
d
J ¼ 8.4 Hz, 1H), 7.73 (d, J ¼ 5.2 Hz, 1H), 7.21e7.35 (m, 5H), 6.85 (dd,
J ¼ 8.4, 2.0 Hz, 1H), 6.61 (d, J ¼ 2.0 Hz, 1H), 4.45 (t, J ¼ 6.4 Hz, 2H),
4.01 (t, J ¼ 6.0 Hz, 2H), 3.53 (t, J ¼ 6.4 Hz, 2H), 2.91 (s, 3H), 2.77 (t,
J ¼ 7.2 Hz, 3H), 2.10e2.19 (m, 4H), 1.98e2.05 (m, 2H). 13C NMR
1H), 8.20 (d, J ¼ 7.6 Hz, 1H), 7.98 (d, J ¼ 5.2 Hz, 1H), 7.91 (d,
J ¼ 8.8 Hz, 1H), 7.71 (d, J ¼ 5.2 Hz, 1H), 7.57e7.61 (m, 1H), 7.49 (d,
J ¼ 8.4 Hz, 1H), 7.30e7.33 (m, 1H), 7.12e7.26 (m, 7H), 6.96 (d,
J ¼ 8.0 Hz, 1H), 6.73 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.51 (d, J ¼ 2.0 Hz, 1H),
4.42 (t, J ¼ 8.0 Hz, 2H), 4.15 (t, J ¼ 8.0 Hz, 2H), 3.91 (s, 3H), 3.89 (s,
3H), 3.72 (t, J ¼ 6.0 Hz, 2H), 2.88 (s, 3H), 2.73 (t, J ¼ 7.2 Hz, 2H),
2.08e2.15 (m, 2H), 1.64e1.71 (m, 2H), 1.35e1.43 (m, 2H). 13C NMR
(100 MHz, CDCl3):
d 160.32, 143.20, 140.62, 140.19, 139.78, 135.13,
129.73, 128.65, 128.47, 126.41, 122.51, 115.08, 112.34, 109.61, 93.66,
67.24, 44.06, 33.46, 32.92, 31.73, 29.49, 27.87, 22.70.
(100 MHz, CDCl3):
d 159.94, 149.26, 148.76, 144.06, 142.94, 142.07,
5.5. General procedure for the preparation of compounds 7n-u
140.60, 140.44, 138.41, 138.05, 135.17, 134.19, 132.72, 130.49, 129.41,
128.55, 128.38, 126.27, 122.37, 121.89, 121.67, 121.56, 119.92, 115.14,
113.54, 112.48, 112.22, 110.82, 110.24, 109.10, 93.70, 67.48, 65.84,
56.01, 44.17, 44.02, 32.89, 31.68, 26.36, 26.00, 22.99, 15.26. HRMS
(ESI) calcd for C44H43N4O3 675.3330 [MþH]þ, found 675.3330.
A mixture of compound 8 (6 mmol) and potassium iodide
(1.68 g, 10 mmol) in anhydrous DMF (25 mL), followed by stirring
for 2 h at room temperature. Then NaH (0.25 g,10 mmol) was added
at 0 ꢀC, and harmine or compound 3c-i (4 mmol) in anhydrous DMF
(10 mL) was added slowly. The reaction mixture was then allowed
to reach room temperature and stirred. The progress of the reaction
was monitored by TLC. After completion of the reaction, the
mixture was poured into ice-cold water and extracted with ethyl
acetate (3 ꢂ 70 mL). The organic layer was washed with water and
brine, dried over Na2SO4 and filtered. The filtrate was evaporated at
reduced pressure. The residue obtained was purified by flash col-
umn chromatography using CH2Cl2:CH3OH (100:1) as the eluent.
Then the compounds were dissolved in hydrochloric acid alcohol,
removed by evaporation and the residue was the 7n-u hydrochlo-
ride salts.
5.5.4. 1-Methyl-9-phenylpropyl-7-(4-(1-(3,4,5-trimethoxyphenyl)-
b
-carboline-9-yl)-butoxy)-
White crystals (1.97 g, 70%) were obtained, m.p. 91.3e91.8 ꢀC. 1H
NMR (400 MHz, CDCl3):
8.52 (d, J ¼ 5.2 Hz, 1H), 8.25 (d, J ¼ 5.2 Hz,
b-carboline (7q)
d
1H), 8.18e8.21 (m, 1H), 8.00 (d, J ¼ 5.2 Hz, 1H), 7.89 (d, J ¼ 8.8 Hz,
1H), 7.69 (d, J ¼ 8.8 Hz, 1H), 7.57e7.61 (m, 1H), 7.49 (d, J ¼ 8.4 Hz,
1H), 7.29e7.33 (m,1H), 7.22e7.27 (m, 2H), 7.12e7.17 (m, 3H), 6.87 (s,
2H), 6.73 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.53 (d, J ¼ 2.0 Hz, 1H), 4.41 (t,
J ¼ 8.0 Hz, 2H), 4.13 (t, J ¼ 8.0 Hz, 2H), 3.94 (s, 3H), 3.87 (s, 6H), 3.76
(t, J ¼ 6.0 Hz, 2H), 2.87 (s, 3H), 2.72 (t, J ¼ 7.2 Hz, 2H), 2.07e2.14 (m,
2H), 1.71e1.78 (m, 2H), 1.41e1.48 (m, 2H). 13C NMR (100 MHz,
CDCl3):
d 159.91, 153.13, 143.94, 142.89, 141.98, 140.61, 140.48,
5.5.1. 1-Methyl-9-phenylpropyl-7-(4-(1-isopropyl-
yl)butoxy)- -carboline (7n)
White crystals (1.58 g, 68%) were obtained, m.p. 100.2e100.9 ꢀC.
1H NMR (400 MHz, CDCl3):
8.44 (d, J ¼ 5.2 Hz, 1H), 8.27 (d, J ¼
b
-carboline-9-
138.24, 138.13, 135.64, 135.18, 133.98, 130.41, 129.33, 128.54, 128.49,
128.37, 126.27, 122.34, 121.68, 121.45, 119.99, 115.17, 113.83, 112.20,
110.20, 109.10, 106.52, 93.68, 67.53, 65.83, 61.01, 56.21, 44.27, 32.89,
31.70, 26.44, 26.27, 23.06. HRMS (ESI) calcd for C45H45N4O4
705.3435 [MþH]þ, found 705.3424.
b
d
5.2 Hz,1H), 8.11 (d, J ¼ 8.0 Hz,1H), 7.93 (d, J ¼ 8.4 Hz,1H), 7.83 (d, J ¼
5.2 Hz, 1H), 7.71 (d, J ¼ 5.2 Hz, 1H), 7.55e7.59 (m, 1H), 7.50 (d,
J ¼ 8.4 Hz, 1H), 7.24e7.29 (m, 3H), 7.13e7.18 (m, 3H), 6.83 (dd,
J ¼ 8.4, 2.0 Hz, 1H), 6.59 (d, J ¼ 2.0 Hz, 1H), 4.65 (t, J ¼ 8.0 Hz, 2H),
4.41 (t, J ¼ 8.0 Hz, 2H), 4.00 (t, J ¼ 6.0 Hz, 2H), 3.73e3.83 (m, 1H),
2.88 (s, 3H), 2.73 (t, J ¼ 7.2 Hz, 2H), 2.08e2.16 (m, 4H), 1.92e1.98 (m,
5.5.5. 1-Methyl-9-phenylpropyl-7-(4-(1-(2-chlorophenyl)-
carboline-9-yl)butoxy)- -carboline (7r)
White crystals (1.83 g, 71%) were obtained, m.p. 172.8e173.4 ꢀC.
1H NMR (400 MHz, CDCl3):
8.55 (d, J ¼ 5.2 Hz, 1H), 8.27 (d, J ¼
b-
b
d

L. Guo et al. / European Journal of Medicinal Chemistry 147 (2018) 253e265
263
5.2 Hz, 1H), 8.19e8.21 (m, 1H), 8.03 (d, J ¼ 5.2 Hz, 1H), 7.93 (d,
J ¼ 8.8 Hz, 1H), 7.71 (d, J ¼ 5.2 Hz, 1H), 7.57e7.61 (m, 2H), 7.45e7.50
(m, 2H), 7.35e7.40 (m, 2H), 7.29e7.33 (m, 1H), 7.23e7.27 (m, 2H),
7.13e7.18 (m, 3H), 6.77 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.53 (d, J ¼ 2.0 Hz,
1H), 4.41 (t, J ¼ 8.0 Hz, 2H), 3.91e4.14 (m, 2H), 3.72e3.77 (m, 2H),
2.89 (s, 3H), 2.73 (t, J ¼ 7.2 Hz, 2H), 2.08e2.16 (m, 2H), 1.58e1.80 (m,
26.55, 23.27, 22.94. HRMS (ESI) calcd for C38H39N4O2 583.3068
[MþH]þ, found 583.3076.
5.6. Cytotoxicity assays in vitro
All of the synthesized compounds were evaluated against nine
different cancer cell lines using MTT assay in vitro. The target tumor
cell lines were cultured to log phase in RPMI-1640 medium sup-
2H), 1.35e1.49 (m, 2H). 13C NMR (100 MHz, CDCl3):
d 159.96, 142.94,
141.67, 140.98, 140.60, 140.45, 138.78, 138.29, 138.07, 135.19, 134.18,
134.10, 131.45, 130.09, 130.01, 129.47, 129.42, 128.57, 128.52, 128.39,
126.80, 126.30, 122.36, 121.71, 121.29, 119.90, 115.16, 114.31, 112.25,
109.87, 109.15, 93.73, 67.47, 44.04, 43.91, 32.90, 31.71, 26.53, 26.27,
23.03. HRMS (ESI) calcd for C42H38ClN4O 649.2729 [MþH]þ, found
649.2729.
plemented with 10% (v/v) fetal bovine serum (FBS), 100
m
g$mLꢁ1
penicillin, and 100
m
g$mLꢁ1 streptomycin. After diluting to
approximate 105 cells per mL with complete medium,100 mL of the
obtained cell suspension was plated onto each well of a 96-well
culture plate and incubated in 5% CO2 at 37 ꢀC for 24 h. Cell lines
were obtained from Shanghai Institute of Biochemistry and Cell
Biology, Chinese Academy of Science. DMSO was used as the so-
lution for the drugs. The final concentration of DMSO in the growth
medium was 2% (v/v) or lower without an effect on cell replication.
Cisplatin was the positive control. The optical density (OD) was
read at 490 nm. Three replicate wells were used for each drug
concentration in all of the experiments. Each assay was carried out
at least three times. The results that were expressed as IC50
(inhibitory concentration 50%) values were calculated using the
Logit method.
5.5.6. 1-Methyl-9-phenylpropyl-7-(4-(1-(2-thienyl)-
yl)butoxy)- -carboline (7s)
White crystals (1.89 g, 76%) were obtained, m.p. 132.7e134.4 ꢀC.
1H NMR (400 MHz, CDCl3):
8.53 (d, J ¼ 5.2 Hz, 1H), 8.26 (d, J ¼
b-carboline-9-
b
d
5.2 Hz, 1H), 8.17e8.19 (m, 1H), 7.98 (d, J ¼ 5.2 Hz, 1H), 7.92 (d,
J ¼ 8.8 Hz, 1H), 7.71 (d, J ¼ 5.2 Hz, 1H), 7.58e7.62 (m, 1H), 7.51 (d,
J ¼ 8.4 Hz, 1H), 7.45 (dd, J ¼ 5.2, 1.2 Hz, 1H), 7.29e7.34 (m, 2H),
7.23e7.27 (m, 2H), 7.11e7.18 (m, 4H), 6.76 (dd, J ¼ 8.4, 2.0 Hz, 1H),
6.51 (d, J ¼ 2.0 Hz,1H), 4.40 (t, J ¼ 8.0 Hz, 2H), 4.27 (t, J ¼ 8.0 Hz, 2H),
3.75 (t, J ¼ 6.4 Hz, 2H), 2.88 (s, 3H), 2.73 (t, J ¼ 7.2 Hz, 2H), 2.08e2.16
(m, 2H), 1.67e1.75 (m, 2H), 1.44e1.51 (m, 2H). 13C NMR (100 MHz,
5.7. Assay of acute toxicities
CDCl3):
d 159.95, 142.91, 142.19, 141.10, 140.61, 140.43, 138.60,
138.05, 137.10, 135.17, 134.82, 130.88, 129.41, 128.63, 128.57, 128.41,
128.28, 127.03, 126.73, 126.30, 122.35, 121.71, 121.39, 120.12, 115.10,
114.21, 112.25, 110.27, 109.21, 93.65, 67.43, 44.16, 44.01, 32.90, 31.71,
26.43, 26.08, 23.06. HRMS (ESI) calcd for C40H37N4OS 621.2683
[MþH]þ, found 621.2689.
Specific pathogen-free KM mice (6e8 weeks old) weighing
19e22 g were housed in a mouse room at 24 2 ꢀC and 60e70%
humidity with 12 h light/dark cycles. The mice were provided ro-
dent laboratory chow pellets and tap water for a week to adapt to
the environment of the mouse room. The experimental protocol
was approved by the Institutional Animal Ethical Committee, and
all of the animals were provided by Laboratory Animal Center of
Xinjiang Uygur Autonomous Region. Prior to each experiment, mice
were fastened overnight and allowed free access to water. Various
5.5.7. 1-Methyl-9-phenylpropyl-7-(4-(1-(3-pyridyl)-
yl)butoxy)- -carboline (7t)
White crystals (1.97 g, 80%) were obtained, m.p. 124.6e125.5 ꢀC.
1H NMR (400 MHz, CDCl3):
8.94 (dd, J ¼ 2.0, 0.8 Hz, 1H), 8.71 (dd,
b-carboline-9-
b
d
doses of the asymmetric dimeric b-carboline derivatives, ranging
J ¼ 4.8, 1.6 Hz, 1H), 8.57 (d, J ¼ 5.2 Hz, 1H), 8.27 (d, J ¼ 5.2 Hz, 1H),
8.19e8.22 (m, 1H), 8.03 (d, J ¼ 5.2 Hz, 1H), 7.99e8.02 (m, 1H), 7.92
(d, J ¼ 8.8 Hz, 1H), 7.71 (d, J ¼ 5.2 Hz, 1H), 7.59e7.64 (m, 1H), 7.50 (d,
J ¼ 8.4 Hz, 1H), 7.38e7.41 (m, 1H), 7.32e7.36 (m, 1H), 7.24e7.27 (m,
2H), 7.13e7.19 (m, 3H), 6.74 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.52 (d,
J ¼ 2.0 Hz, 1H), 4.43 (t, J ¼ 8.0 Hz, 2H), 4.12 (t, J ¼ 8.0 Hz, 2H), 3.71 (t,
J ¼ 6.0 Hz, 2H), 2.88 (s, 3H), 2.74 (t, J ¼ 7.2 Hz, 2H), 2.09e2.16 (m,
2H), 1.60e1.67 (m, 2H), 1.36e1.42 (m, 2H). 13C NMR (100 MHz,
from 5.0 to 500 mg kgꢁ1 dissolved in 0.5% carboxymethyl cellulose
sodium (CMC-Na) salt solution, were given intraperitoneally to
different groups of healthy KM mice, and each group contained 10
mice (5 males and 5 females). After the administration of the
compounds, the 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 of the animals were killed on the
14th day after drug administration, and they were checked
macroscopically for possible damage to the heart, liver, and kid-
neys. Mice that experienced immediate death following drug
administration were also examined for any possible organ damage.
LD50 values were calculated graphically as described [43].
CDCl3):
d 159.82, 150.09, 149.57, 142.88, 142.10, 140.59, 140.46,
138.79, 138.08, 136.59, 136.00,135.16, 134.36, 130.86, 129.34, 128.74,
128.51, 128.37, 126.23, 123.11, 122.32, 121.72, 121.35, 120.21, 115.13,
114.23, 112.20, 110.17, 109.14, 93.61, 67.29, 44.35, 44.01, 32.86, 31.68,
26.23, 25.75, 23.02. HRMS (ESI) calcd for C41H38N5O 616.3071
[MþH]þ, found 616.3074.
5.8. In vivo antitumor activity
5.5.8. 1-Methyl-9-phenylpropyl-7-(4-(1-methyl-7-methoxy-
carboline-9-yl)butoxy)- -carboline (7u)
White crystals (1.80 g, 77%) were obtained, m.p. 127.4e128.1 ꢀC.
1H NMR (400 MHz, CDCl3):
8.30 (d, J ¼ 5.2 Hz, 1H), 8.27 (d, J ¼
b-
b
Sarcoma180 and Lewis lung cancer cell lines were provided by
Shanghai Institute of Pharmaceutical Industry. Mice were inocu-
lated with Sarcoma180 and Lewis lung cancer tumor cells. After 7
days, the tumors were removed and the cells were harvested. Mice
received subcutaneous injections of viable tumor cells
(2 ꢂ 106 cells/mouse) in the armpit. Each compound was adminis-
tered via i.p. injection to different groups of mice (each group
contained 10 female mice) 24 h after the inoculation at a dosage
about one-fifth of the LD50 value once a day for seven consecutive
days. This dose was the maximum tolerated dose for most of the
compounds based on our preliminary studies. CTX at 30 mg kgꢁ1
was used as the positive control and the vehicle as the negative
control. The weight of the animals was recorded every three days.
d
5.2 Hz, 1H), 7.98 (d, J ¼ 8.8 Hz, 1H), 7.93 (d, J ¼ 8.8 Hz, 1H), 7.75 (d,
J ¼ 5.2 Hz, 1H), 7.71 (d, J ¼ 5.2 Hz, 1H), 7.21e7.27 (m, 2H), 7.10e7.18
(m, 3H), 6.88e6.90 (m, 2H), 6.83 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.59 (d,
J ¼ 2.0 Hz, 1H), 4.61 (t, J ¼ 8.0 Hz, 2H), 4.41 (t, J ¼ 8.0 Hz, 2H), 4.01 (t,
J ¼ 6.0 Hz, 2H), 3.89 (s, 3H), 3.05 (s, 3H), 2.88 (s, 3H), 2.73 (t,
J ¼ 7.2 Hz, 2H), 2.04e2.17 (m, 4H), 1.91e1.98 (m, 2H). 13C NMR
(100 MHz, CDCl3):
d 160.94, 159.92, 143.12, 142.94, 140.60, 140.47,
140.41, 138.17, 138.03, 135.25, 135.20, 129.57, 129.42, 128.56, 128.50,
128.40, 128.25, 126.28, 122.47, 122.45, 115.25, 112.35, 112.29, 109.16,
108.70, 93.71, 93.52, 67.70, 55.65, 44.60, 44.02, 32.88, 31.69, 27.66,

264
L. Guo et al. / European Journal of Medicinal Chemistry 147 (2018) 253e265
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This work was supported by the scientific research innovation
project in Xinjiang (No. XJGRI2016052), the Program for
Outstanding Youth Science and Technology Innovation Talents
Training in Xinjiang Uygur Region, China (No. qn2015jc067) and
Xinjiang Huashidan Pharmaceutical Research Co. Ltd.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at
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