Synthesis and biological evaluation of novel N9-heterobivalent β-carbolines as angiogenesis inhibitors

Article abstract of DOI:10.1080/14756366.2018.1497619

A series of novel N⁹-heterobivalent β-carbolines has been synthesized. All the novel compounds were tested for their anticancer activity against six tumour cell lines in vitro. Among these molecules, compounds 5b, and 5w exhibited strong cytotoxic activities with IC₅₀ value of lower than 20 μM. Acute toxicities and antitumor efficacies of the selected compounds in mice were also evaluated, compounds 5b and 5w exhibited that tumour inhibition rate of over 40% in the Sarcoma 180 and Lewis lung cancer animal models. Preliminary structure–activity relationships (SARs) analysis indicated that: (1) C¹-methylation and C⁷-methoxylation were favorable for increased activities; (2) 3-Pyridyl or 2-thienyl group substituent into position-1 of the β-carboline core, and the aryl substituent into another β-carboline ring might be detrimental to cytotoxic effects of this class compounds. Investigation of the preliminary mechanism of action demonstrated that compound 5b had obvious angiogenesis inhibitory effects in the chicken chorioallantoic membrane (CAM) assay.

Full text of DOI:10.1080/14756366.2018.1497619

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
9
Synthesis and biological evaluation of novel N -
heterobivalent β-carbolines as angiogenesis
inhibitors
Liang Guo, Qin Ma, Wei Chen, Wenxi Fan, Jie Zhang & Bin Dai
To cite this article: Liang Guo, Qin Ma, Wei Chen, Wenxi Fan, Jie Zhang & Bin Dai (2019)
9
Synthesis and biological evaluation of novel N -heterobivalent β-carbolines as angiogenesis
inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 375-387, DOI:
10.1080/14756366.2018.1497619
To link to this article: https://doi.org/10.1080/14756366.2018.1497619
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2018 The Author(s). Published by Informa
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
019, VOL. 34, NO. 1, 375–387
2
https://doi.org/10.1080/14756366.2018.1497619
RESEARCH PAPER
9
Synthesis and biological evaluation of novel N -heterobivalent b-carbolines as
angiogenesis inhibitors
a
b
b
b
a
a
Liang Guo , Qin Ma , Wei Chen , Wenxi Fan , Jie Zhang and Bin Dai
a
School of Chemistry and Chemical Engineering/Key Laboratory for Green Processing of Chemical Engineering of XinJiang Bingtuan, Shihezi
b
University, Shihezi, China; XinJiang Huashidan Pharmaceutical Research Co. Ltd., Urumqi, China
ABSTRACT
ARTICLE HISTORY
9
A series of novel N -heterobivalent b-carbolines has been synthesized. All the novel compounds were
tested for their anticancer activity against six tumour cell lines in vitro. Among these molecules, com-
pounds 5b, and 5w exhibited strong cytotoxic activities with IC50 value of lower than 20 lM. Acute toxic-
ities and antitumor efficacies of the selected compounds in mice were also evaluated, compounds 5b and
Received 19 March 2018
Revised 21 June 2018
Accepted 3 July 2018
KEYWORDS
5
w exhibited that tumour inhibition rate of over 40% in the Sarcoma 180 and Lewis lung cancer animal
Heterobivalent b-carboline;
cytotoxic activities;
angiogenesis inhibitors;
structure–activity
relationship
1
models. Preliminary structure–activity relationships (SARs) analysis indicated that: (1) C -methylation and
7
C -methoxylation were favorable for increased activities; (2) 3-Pyridyl or 2-thienyl group substituent into
position-1 of the b-carboline core, and the aryl substituent into another b-carboline ring might be detri-
mental to cytotoxic effects of this class compounds. Investigation of the preliminary mechanism of action
demonstrated that compound 5b had obvious angiogenesis inhibitory effects in the chicken chorioallan-
toic membrane (CAM) assay.
1
7–20
Introduction
the corresponding monomers)
. Therefore, bivalent b-carbo-
lines were expected to exhibit more potent antitumor efficacies
than monomers. Inspired by this information, our group reported
the synthesis, in vitro evaluation, in vivo efficacies and structure-
activity relationships for the novel homobivalent b-carbolines with
an alkyl spacer or alkylamino spacer in positon-1, 3, 7, and 9 of
The population growth and aging associated with some risk fac-
tors leveraged the incidence of new cases of cancer and related
deaths in developed countries and in developing countries .
Cancer resistance to therapy is becoming a common phenomenon
that threatens the current strategies adopted against this disease.
For that reason we need to discover new anticancer agents. One
of the successful and effective methods for the discovery of new
anticancer drugs from natural products is to synthesize novel
compounds through chemical structural modifications on the
basis of leading compounds.
1
21–26
the b-carboline nucleus, respectively (Figure 1)
. In these com-
pounds, 1-Methyl-9-[4–(1-methyl-b-carboline-9-yl)butyl]-b-carboline
2
1,27
(B-9–3)
exhibited potent antitumor activity. It was a symmetric
dimeric b-carboline compound that contains two molecules of
harman bound to each other by a tetramethylene group. The
pharmacological mechanisms showed that the angiogenesis
inhibitor B-9–3 selectively induces apoptosis of endothelial cells,
Peganum harmala L. have been traditionally used for hundreds
of years to treat the alimentary tract cancers and malaria in
2
8
Northwest China. Harmine, originally isolated from the seeds of in part through disruption of VEGF-A/VEGFR2 signaling .
Peganum harmala L. in 1847, is the most representative natural
Our strategy was based on the modification of the prototype
occurring b-carboline alkaloid, having a common tricyclic pyrido B-9–3, following this previous work, we have continued our search
3,4-b] indole ring structure. In the past several decades, it has for novel antitumor agents endowed with better antitumor activ-
confirmed that harmine was the important active ingredients to ities, and we provide detailed studies of structure–activity relation-
[
2
,3
treat the alimentary tract cancers . Recent reports demonstrated ships (SARs) on the antitumor efficacies in vitro and in vivo of this
that harmine and its derivatives had remarkable antitumor class of compounds. Here, we designed and synthesized a series
2–5
9
activities, together with potential neurotoxicity . Moreover, it has of novel N -heterobivalent b-carboline derivatives as potent anti-
been reported that harmine and its derivatives can exert tumor agents.
antitumor activities through multiple mechanisms, such as DNA
6
–8
9,10
binding , inhibition topoisomerases I and II , CDK (cyclin-
1
1,12
13
14,15
dependent kinase)
and IjB kinases .
Previous investigations has shown that some dimer antitumor
, PLK1 (polo-like kinase) , lipoxygenase
Materials and methods
1
6
Chemistry
agents via an appropriate linker could lead to significantly All reagents were purchased from commercial suppliers and were
improved antitumor activities (100- to 500-fold improvement over dried and purified when necessary. The following intermediates
CONTACT Bin Dai
db_tea@shzu.edu.cn
School of Chemistry and Chemical Engineering/Key Laboratory for Green Processing of Chemical Engineering of
XinJiang Bingtuan, Shihezi University, Shihezi 832000, China
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

3
76
L. GUO ET AL.
N
N
6
O
O
N
N
N
C H
N
C H
CH3
N
N
C
H C
3
CH2
5NHCH2
NH(CH )
2
4
9
4
9
C H
H
4
9
4
9
N
CH2
5NHCH2
2
NH(CH )
CH3
N
N
N
(CH
2
)
4
N
N
N
CH3
CH3
H C
3
N
OCH3
H CO
3
B-9-3
Figure 1. The chemical structure of the representative reported symmetric bivalent b-carbolines.
2
9
21
30
31
32
33
3
a-3b , 3c-3e , 3f , 3g , 3h , 4g-4h were prepared as previ- 9–(4-bromobutyl)-1-methyl-b-carboline (4b)
ꢀ
1
ously described.
Colorless crystals, yield 88%, m.p. 213.9–215.6 C.
H NMR
Melting points were determined in capillary tubes on an elec- (400 MHz, DMSO-d
6
): d 8.71 (dd, J ¼ 6.4, 1.2 Hz, 1H), 8.54 (dd,
trothermal X-5 apparatus and without correction. NMR spectra J ¼ 8.0, 1.2 Hz, 1H), 8.49 (dd, J ¼ 6.4, 2.0 Hz, 1H), 8.00 (d, J ¼ 8.4 Hz,
were recorded at room temperature on a Bruker Avance III HD 1H), 7.83–7.87 (m, 1H), 7.46–7.50 (m, 1H), 4.75 (t, J ¼ 7.6 Hz, 2H),
1
13
13
4
00 instrument at 400 MHz for H NMR and 100 MHz for C NMR. 3.70 (t, J ¼ 6.4 Hz, 2H), 3.27 (s, 3H), 2.04–1.77 (m, 4H). C NMR
Chemical shifts (d) are reported in ppm relatively to the residual (100 MHz, DMSO-d ): d 144.33, 139.00, 133.74, 133.26, 132.07,
6
1
2
29.29, 123.86, 122.14, 119.67, 116.15, 111.78, 45.35, 44.31, 29.63,
8.29, 18.31.
solvent peak and the multiplicity of each signal is designated by
the following abbreviations: s, singlet; d, doublet; t, triplet; q, quar-
tet; m, multiplet. Coupling constants (J) are quoted in Hz. High
resolution mass spectra (HRMS) were recorded on Bruker
ultrafleXtreme MALDI-TOF/TOF-MS and HCCA (alpha-cyano-4-
hydroxycinnamic acid) is used as matrix. Elemental analyses (C, H,
and N) were carried out on an Elementar Vario ELIII CHNS
Elemental Analyzer. Column chromatography was performed with
9
–(5-bromopentyl)-1-methyl-b-carboline (4c)
ꢀ
1
Colorless crystals, yield 90%, m.p. 187.7–189.5 C.
H NMR
(
400 MHz, CDCl ): d 8.42–8.33 (m, 2H), 8.29 (dt, J ¼ 8.0, 1.2 Hz, 1H),
3
7
4
2
1
3
.82–7.86 (m, 1H), 7.66 (dt, J ¼ 8.4, 0.8 Hz, 1H), 7.48–7.51 (m, 1H),
.69 (t, J ¼ 7.6 Hz, 2H), 3.52 (s, 3H), 1.82–1.98 (m, 4H), 1.60–1.68 (m,
silica gel (200–300 mesh) and analytical TLC on silica gel 60-F254
.
13
H). C NMR (100 MHz, CDCl ): d 144.30, 137.12, 133.87, 133.76,
3
32.22, 128.57, 123.11, 122.38, 119.70, 115.13, 110.68, 45.35, 44.43,
1.86, 30.29, 24.04, 18.07.
General procedure for the preparation of compounds 4a-h
9
–(6-bromohexyl)-1-methyl-b-carboline (4d)
A mixture of 3a (1.68 g, 10mmol) and anhydrous DMF (50 ml) was
stirred at room temperature for 0.5h, then NaH (0.50g, 20mmol)
and the 1,4-dibromobutane (20mmol) were added. The mixture
was stirred at room temperature. After that period, the mixture was
monitored via TLC and at the end of the reaction the mixture was
ꢀ
1
Colorless crystals, yield 69%, m.p. 73.9–74.8 C. H NMR (400 MHz,
CDCl
): d 8.33 (d, J ¼ 5.2 Hz, 1H), 8.12 (d, J ¼ 7.6 Hz, 1H), 7.84 (d,
J ¼ 5.2 Hz, 1H), 7.56–7.60 (m, 1H), 7.45 (d, J ¼ 8.4 Hz, 1H), 7.25–7.29
m, 1H), 4.54 (t, J ¼ 7.6 Hz, 2H), 3.38 (t, J ¼ 6.4 Hz, 2H), 3.05 (s, 3H),
3
(
13
1
.81–1.89 (m, 4H), 1.40–1.55 (m, 4H). C NMR (100 MHz, CDCl
141.51, 141.13, 137.94, 135.09, 129.14, 128.17, 121.55, 121.33,
19.66, 112.99, 109.67, 44.76, 33.56, 30.70, 27.90, 26.13, 23.55.
3
): d
poured into H O (150 ml) and extracted with ethyl acetate. The
2
organic phase was collected and washed with water and brine,
then dried under anhydrous sodium sulfate, filtered, and evapo-
rated. The resulting oil was crystallized from ethyl ether or ethyl
1
ether-petroleum ether to afford the compound 4a. Products 4b-h 9–(4-bromobutyl)-1–(3-pyridyl)-b-carboline (4e)
ꢀ
1
were prepared according to the same method of 4a.
Yellow crystals, yield 71%, m.p. 135.0–136.7 C. H NMR (400 MHz,
CDCl
3
): d 8.67 (d, J ¼ 6.0 Hz, 1H), 8.53 (d, J ¼ 6.0 Hz, 1H), 8.37 (d,
J ¼ 8.0 Hz, 1H), 7.87–7.81 (m, 2H), 7.68–7.64 (m, 2H), 7.54–7.49 (m,
1
6
H), 7.36–7.33 (m, 1H), 4.19 (t, J ¼ 8.0 Hz, 2H), 3.32 (dt, J ¼ 55.2,
13
.4 Hz, 2H), 1.77–1.69 (m, 2H), 1.60–1.44 (m, 2H).
C NMR
9
–(4-bromobutyl)-b-carboline (4a)
Colorless crystals, yield 87%, m.p. 285.3–286.8 C.
400 MHz, DMSO-d
): 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),
.60–7.64 (m, 1H), 7.27–7.31 (m, 1H), 4.56 (t, J ¼ 6.8 Hz, 2H), 3.56 (t,
(
100 MHz, CDCl
3
): d 149.60, 149.24, 142.21, 139.90, 138.50, 137.27,
ꢀ
1
H
NMR
1
1
31.21, 129.04, 128.23, 125.31, 123.53, 121.85, 121.25, 120.45,
14.54, 110.17, 43.77, 32.59, 29.44, 27.35.
(
6
7
1
3
J ¼ 6.4 Hz, 2H), 1.90–1.97 (m, 2H), 1.81–1.88 (m, 2H). C NMR 9–(4-bromobutyl)-1–(2-thienyl)-b-carboline (4f)
ꢀ
1
(
100 MHz, DMSO-d ): d 141.13, 138.88, 136.47, 133.13, 128.77, Light yellow crystals, yield 60%, m.p. 197.6–199.8 C. H NMR
6
1
3
27.66, 122.44, 120.80, 119.92, 115.04, 110.62, 42.15, 35.09, (400 MHz, CDCl ): d 8.55 (d, J ¼ 5.2 Hz, 1H), 8.19 (d, J ¼ 7.6 Hz, 1H),
3
0.23, 27.87.
8.03 (d, J ¼ 5.2 Hz, 1H), 7.62–7.54 (m, 3H), 7.54–7.51 (m, 1H),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
377
7
.49–7.41 (m, 3H), 7.30 (t, J ¼ 7.2 Hz, 1H), 4.03–3.78 (m, 2H), 3.20 (d, J ¼ 8.4 Hz, 1H), 7.29–7.33 (m, 3H), 7.23–7.25 (m, 1H), 4.35 (t,
13
(dt, J ¼ 54.8, 6.4 Hz, 2H), 1.71–1.49 (m, 2H), 1.44–1.24 (m, 2H).
C
J ¼ 7.6 Hz, 2H), 3.95 (t, J ¼ 7.6 Hz, 2H), 2.96 (s, 3H), 1.47–1.55 (m,
13
NMR (100 MHz, CDCl ): d 141.58, 140.89, 138.69, 138.31, 134.19, 2H), 1.35–1.42 (m, 2H), 0.86–0.94 (m, 2H). C NMR (100 MHz,
3
1
1
31.47, 130.09, 129.47, 128.58, 126.95, 121.70, 121.26, 119.96, CDCl ) d: 150.12, 149.51, 142.03, 141.61, 140.69, 140.38, 138.84,
3
14.31, 109.76, 43.23, 32.60, 29.77, 27.94.
137.29, 136.56, 135.99, 134.87, 134.27, 130.85, 129.51, 128.80,
1
1
2
4
28.52, 123.01, 121.76, 121.67, 121.30, 121.16, 120.27, 119.98,
14.26, 113.15, 110.01, 109.69, 44.48, 44.24, 30.29, 28.56, 23.87,
General procedure for the preparation of compounds 5a-x
þ
3.07. HRMS calcd for
96.2503. Anal. calcd for C33
C
33
H
30
N
5
[M þ H] 496.2496, found
H
29
N
5
: C, 79.97; H, 5.90; N, 14.13;
A solution of compound 3c (2 mmol) in anhydrous DMF (6 ml) was
added slowly with stirring to a solution of 4a (3 mmol), NaH
found C 79.11, H 5.88, N 13.90.
(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
1
–(3-pyridyl)-9–(6-(1-methyl-b-carboline-9-yl)hexyl)-b-carbo-
line (5d)
This compound was obtained as yellow crystals in 66% yield, m.p.
(
3 ꢁ 30 ml), washed with water and brine, dried over anhydrous
Na SO , and filtered. Purification by column chromatography
DCM/MeOH 100:1 as the eluent) furnished the dimeric b-carboline
ꢀ
1
1
0
8
41.3–142.9 C. H NMR (400 MHz, CDCl
3
) d: 8.89 (dd, J ¼ 2.0,
2
4
.8 Hz, 1H), 8.72 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.56 (d, J ¼ 5.2 Hz, 1H),
.31 (d, J ¼ 5.2 Hz, 1H), 8.17–8.19 (m, 1H), 8.08–8.10 (m, 1H), 8.02
(
5a. Compounds 5b-x were synthesized using similar procedure as
(d, J ¼ 5.2 Hz, 1H), 7.93–7.96 (m, 1H), 7.82 (d, J ¼ 5.2 Hz, 1H),
compound 5a.
7
.53–7.60 (m, 2H), 7.30–7.41 (m, 4H), 7.24–7.28 (m, 1H), 4.39 (t,
J ¼ 7.2 Hz, 2H), 3.97 (t, J ¼ 7.2 Hz, 2H), 2.97 (s, 3H), 1.58–1.66 (m,
13
1
–(3-pyridyl)-9–(4-(b-carboline-9-yl)butyl)-b-carboline (5a)
2H), 1.29–1.37 (m, 2H), 1.07–1.15 (m, 2H), 0.81–0.90 (m, 2H).
) d: 150.16, 149.54, 142.11, 141.45, 141.02,
) d: 8.77 (dd, J ¼ 2.4, 140.44, 138.79, 137.84, 136.65, 136.07, 134.98, 134.35, 130.86,
C
This compound was obtained as colourless crystals in 65% yield, NMR (100 MHz, CDCl
3
ꢀ
1
m.p. 173.3–175.1 C. H NMR (400 MHz, CDCl
3
0
8
8
3
4
.8 Hz, 1H), 8.65 (d, J ¼ 0.8 Hz, 1H), 8.61 (dd, J ¼ 4.8, 1.6 Hz, 1H), 129.13, 128.73, 128.15, 123.07, 121.72, 121.53, 121.31, 121.26,
.50 (d, J ¼ 5.2 Hz, 1H), 8.47 (d, J ¼ 5.2 Hz, 1H), 8.15–8.18 (m, 2H), 120.19, 119.66, 114.23, 112.98, 110.10, 109.60, 44.54, 44.39, 30.59,
þ
.00 (dd, J ¼ 5.2, 0.8 Hz, 1H), 7.98 (d, J ¼ 5.2 Hz, 1H), 7.56–7.63 (m, 28.66, 26.33, 23.44. HRMS calcd for C H N [M þ H] 510.2652,
3
4 32 5
H), 7.30–7.34 (m, 3H), 7.24 (d, J ¼ 8.4 Hz, 1H), 7.11–7.15 (m, 1H), found 510.2656. Anal. calcd for C H N : C, 80.13; H, 6.13; N,
3
4 31 5
.14 (t, J ¼ 7.2 Hz, 2H), 3.96 (t, J ¼ 7.2 Hz, 2H), 1.38–1.41 (m, 4H). 13.74; found C 79.72, H 6.14, N 13.32.
3
1
3
C NMR (100 MHz, CDCl ) d: 150.00, 149.59, 142.09, 141.14,
1
1
1
40.25, 138.92, 138.05, 136.35, 136.06, 135.77, 134.09, 130.89,
28.85, 128.80, 123.04, 122.11, 121.81, 121.26, 120.90, 120.33,
1
–(2-thienyl)-9–(4-(b-carboline-9-yl)butyl)-b-carboline (5e)
This compound was obtained as yellow crystals in 56% yield, m.p.
20.02, 114.80, 114.22, 109.90, 109.37, 44.08, 42.65, 26.53, 26.01.
ꢀ
1
þ
1
8
8
1
7
41.6–143.1 C. H NMR (400 MHz, CDCl
3
) d: 8.72 (d, J ¼ 0.8 Hz, 1H),
HRMS calcd for C H N [M þ H] 468.2183, found 468.2183. Anal.
3
1 26 5
.49 (d, J ¼ 5.2 Hz, 1H), 8.46 (d, J ¼ 5.2 Hz, 1H), 8.15–8.16 (m, 1H),
.13–8.14 (m, 1H), 7.97 (dd, J ¼ 5.2, 0.8 Hz, 1H), 7.95 (d, J ¼ 5.2 Hz,
H), 7.51–7.71 (m, 3H), 7.33–7.35 (m, 1H), 7.30–7.32 (m, 1H),
.28–7.29 (m, 1H), 7.24–7.28 (m, 2H), 7.05 (dd, J ¼ 4.0, 1.2 Hz, 1H),
calcd for C H N : C, 79.63; H, 5.39; N, 14.98; found C 79.16, H
3
1 25 5
5
.49, N 14.38.
1
–(3-pyridyl)-9–(4-(1-methyl-b-carboline-9-yl)butyl)-b-carbo-
6.94 (dd, J ¼ 5.2, 3.6 Hz, 1H), 4.14 (t, J ¼ 7.2 Hz, 2H), 4.13 (t,
13
line (5b)
J ¼ 7.2 Hz, 2H), 1.47–1.58 (m, 4H). C NMR (100 MHz, CDCl
) d: 8.79 (dd, 131.55, 130.86, 130.80, 128.78, 128.68, 128.54, 128.49, 128.21,
3
3
) d:
This compound was obtained as slight yellow crystals in 54% 141.95, 141.05, 140.84, 138.62, 136.84, 136.22, 134.60, 132.44,
yield, m.p. 201.2–202.2 C. H NMR (400 MHz, CDCl
ꢀ
1
J ¼ 2.4, 0.8 Hz, 1H), 8.65 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.52 (d, J ¼ 5.2 Hz, 127.03, 126.58, 122.04, 121.73, 121.28, 121.02, 120.17, 119.79,
1
1
1
H), 8.33 (d, J ¼ 5.2 Hz, 1H), 8.17–8.19 (m, 1H), 8.11 (d, J ¼ 7.6 Hz, 114.70, 114.23, 109.90, 109.34, 43.91, 42.68, 26.98, 26.28. HRMS
þ
H), 7.98 (d, J ¼ 5.2 Hz, 1H), 7.84 (d, J ¼ 5.2 Hz, 1H), 7.69–7.71 (m, calcd for C H N S [M þ H] 473.1794, found 473.1792. Anal.
3
0 25 4
H), 7.52–7.61 (m, 2H), 7.27–7.35 (m, 3H), 7.18–7.24 (m, 2H), 4.23 calcd for C H N S: C, 76.24; H, 5.12; N, 11.86; S, 6.78; found C,
3
0 24 4
(
t, J ¼ 7.2 Hz, 2H), 3.96 (t, J ¼ 7.2 Hz, 2H), 2.81 (s, 3H), 1.37–1.45 (m, 76.01; H, 5.61; N, 11.46; S, 6.34.
13
2
1
1
1
1
3 :
H), 1.28–1.35 (m, 2H). C NMR (100 MHz, CDCl ) d 150.06,
49.64, 142.18, 141.40, 140.66, 140.31, 139.05, 136.41, 135.83,
34.70, 134.12, 130.97, 129.35, 128.86, 128.39, 123.11, 121.85,
21.63, 121.34, 121.17, 120.38, 119.91, 114.23, 113.07, 109.95,
09.55, 44.13, 43.90, 29.70, 27.71, 26.07. HRMS calcd for C H N
2 28 5
1
–(2-thienyl)-9–(4-(1-methyl-b-carboline-9-yl)butyl)-b-carboline (5f)
This compound was obtained as yellow crystals in 47% yield, m.p.
ꢀ
1
2
8
14.8–216.0 C. H NMR (400 MHz, CDCl ) d: 8.50 (d, J ¼ 5.2 Hz, 1H),
3
3
þ
.32 (d, J ¼ 5.2 Hz, 1H), 8.14–8.17 (m, 1H), 8.12–8.06 (m, 1H), 7.96
[
7
M þ H] 482.2339, found 482.2342. Anal. calcd for C H N : C,
3
2 27 5
(
7
d, J ¼ 5.2 Hz, 1H), 7.83 (d, J ¼ 5.2 Hz, 1H), 7.56–7.60 (m, 1H),
9.81; H, 5.65; N, 14.54; found C 79.39, H 5.37, N 14.75.
.51–7.55 (m, 1H), 7.35–7.37 (m, 1H), 7.33–7.34 (m, 1H),
.28–7.31(m, 1H), 7.24–7.26 (m, 1H), 7.20–7.23 (m, 1H), 7.12 (dd,
7
1
–(3-pyridyl)-9–(5-(1-methyl-b-carboline-9-yl)pentyl)-b-carbo-
J ¼ 4.0, 1.2 Hz, 1H), 6.99 (dd, J ¼ 5.2, 3.6 Hz, 1H), 4.26 (t, J ¼ 7.2 Hz,
13
line (5c)
2H), 4.13 (t, J ¼ 7.2 Hz, 2H), 2.89 (s, 3H), 1.41–1.55 (m, 4H). C NMR
) d: 142.02, 141.36, 140.95, 140.86, 138.72, 137.86,
) d: 8.82 (d, J ¼ 2.0 Hz, 1H), 136.90, 134.85, 134.59, 130.89, 129.24, 128.70, 128.22, 127.07,
.55 (d, J ¼ 5.2 Hz, 1H), 8.51 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.34 (d, 126.67, 121.76, 121.60, 121.36, 121.25, 120.22, 119.79, 114.23,
This compound was obtained as yellow crystals in 62% yield, m.p. (100 MHz, CDCl
3
ꢀ
1
189.4–190.5 C. H NMR (400 MHz, CDCl
8
3
J ¼ 5.2 Hz, 1H), 8.19 (d, J ¼ 7.6 Hz, 1H), 8.14 (d, J ¼ 8.4 Hz, 1H), 8.02 113.03, 109.94, 109.55, 44.04, 43.94, 27.94, 26.52, 23.35. HRMS
(d, J ¼ 5.2 Hz, 1H), 7.85–7.89 (m, 2H), 7.57–7.61 (m, 2H), 7.37 calcd for C31
H
N
27 4
S [M þ H]þ 487.1951, found 487.1950. Anal.

3
78
L. GUO ET AL.
26 4
calcd for C31H N S: C, 76.51; H, 5.39; N, 11.51; S, 6.59; found C, 1–(2-chlorophenyl)-9–(5-(1-methyl-b-carboline-9-yl)pentyl)-b-carbo-
7
6.27; H, 5.59; N, 10.93; S, 6.38.
line (5j)
This compound was obtained as yellow crystals in 70% yield, m.p.
ꢀ
1
1
8
69.1–171.2 C. H NMR (400 MHz, CDCl
3
) d: 8.52 (d, J ¼ 5.2 Hz, 1H),
.36 (d, J ¼ 5.2 Hz, 1H), 8.17–8.19 (m, 1H), 8.13–8.15 (m, 1H), 8.01
1
–(2-thienyl)-9–(5-(1-methyl-b-carboline-9-yl)pentyl)-b-carbo-
(d, J ¼ 5.2 Hz, 1H), 7.87 (d, J ¼ 5.2 Hz, 1H), 7.55–7.59 (m, 2H), 7.41
(dd, J ¼ 7.6, 1.6 Hz, 1H), 7.36 (d, J ¼ 8.4 Hz, 1H), 7.30–7.33 (m, 3H),
line (5g)
This compound was obtained as yellow crystals in 62% yield, m.p. 7.24–7.28 (m, 1H), 7.09–7.13 (m, 1H), 6.98–7.02 (m, 1H), 4.37 (t,
ꢀ
1
J ¼ 7.6 Hz, 2H), 3.76–3.93 (m, 2H), 2.97 (s, 3H), 1.35–1.56 (m, 4H),
1
8
58.4–159.2 C. H NMR (400 MHz, CDCl ) d: 8.51 (d, J ¼ 5.2 Hz, 1H),
3
1
3
0
1
1
1
1
.88–1.02 (m, 2H). C NMR (100 MHz, CDCl ) d: 141.58, 141.41,
.34 (d, J ¼ 5.2 Hz, 1H), 8.15–8.17 (m, 1H), 8.11–8.14 (m, 1H), 7.97
3
40.97, 140.86, 138.72, 138.27, 137.92, 134.99, 134.09, 133.94,
31.43, 130.06, 129.85, 129.30, 129.17, 128.54, 128.23, 126.56,
21.71, 121.59, 121.29, 121.22, 119.91, 119.82, 114.30, 113.04,
(
(
7
d, J ¼ 5.2 Hz, 1H), 7.86 (d, J ¼ 5.2 Hz, 1H), 7.54–7.60 (m, 2H), 7.40
d, J ¼ 8.4 Hz, 1H), 7.27–7.32 (m, 3H), 7.21 (dd, J ¼ 5.2, 1.2 Hz, 1H),
.16 (dd, J ¼ 3.2, 1.2 Hz, 1H), 6.91 (dd, J ¼ 5.2, 3.6 Hz, 1H), 4.37 (t,
09.68, 109.65, 44.52, 43.80, 30.20, 28.98, 24.03, 23.50. HRMS calcd
J ¼ 7.6 Hz, 2H), 4.12 (t, J ¼ 7.6 Hz, 2H), 2.96 (s, 3H), 1.53–1.60 (m,
þ
13
for C H ClN [M þ H] 529.2154, found 529.2161. Anal. calcd for
3
4
30
4
2
H), 1.42–1.50 (m, 2H), 0.98–1.06 (m, 2H). C NMR (100 MHz,
CDCl ) d: 142.08, 141.45, 141.00, 140.94, 138.55, 137.76, 136.96,
34.97, 134.66, 130.82, 129.21, 128.62, 128.29, 128.23, 126.85,
C H ClN : C, 77.19; H, 5.53; N, 10.59; found C 76.74, H 5.32,
3
4
29
4
3
N 10.82.
1
1
1
26.51, 121.67, 121.55, 121.27, 121.25, 120.09, 119.75, 114.16,
13.02, 110.02, 109.66, 44.54, 44.06, 30.22, 28.89, 23.98, 23.40 .
1
–(4-methoxyphenyl)-9–(4-(1–(3-pyridyl)-b-carboline-9-yl)butyl)-
b-carboline (5k)
þ
HRMS calcd for C H N S [M þ H] 501.2107, found 501.2113.
3
2 29 4
Anal. calcd for C H N S: C, 76.71; H, 6.24; N, 10.84; S, 6.20; found
3
2 28 4
This compound was obtained as yellow solid in 57% yield, m.p.
C, 76.24; H, 5.92; N, 10.45; S, 6.07.
ꢀ
1
2
8
41.2–242.4 C. H NMR (400 MHz, CDCl
3
) d: 8.67 (d, J ¼ 2.0 Hz, 1H),
.62 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.51 (d, J ¼ 5.2 Hz, 1H), 8.47 (d,
J ¼ 5.2 Hz, 1H), 8.17 (m, 2H), 7.97 (d, J ¼ 5.2 Hz, 1H), 7.91 (d,
J ¼ 5.2 Hz, 1H), 7.54–7.59 (m, 3H), 7.29–7.35 (m, 2H), 7.13–7.23 (m,
1–(2-thienyl)-9–(6-(1-methyl-b-carboline-9-yl)hexyl)-b-carbo-
5
3
1
H), 6.87 (s, 1H), 6.84 (s, 1H), 3.78 (s, 3H), 3.70–3.75 (m, 2H),
1
3
line (5h)
.61–3.67 (m, 2H), 0.78–0.85 (m, 4H). C NMR (100 MHz, CDCl ) d:
59.68, 149.96, 149.45, 143.69, 142.07, 141.98, 140.22, 138.83,
138.42, 136.39, 135.75, 133.95, 133.70, 132.04, 130.74, 130.44,
3
This compound was obtained as yellow crystals in 57% yield, m.p.
ꢀ
1
2
8
38.5–239.6 C. H NMR (400 MHz, CDCl ) d: 8.52 (d, J ¼ 5.2 Hz, 1H),
3
.32 (d, J ¼ 5.2 Hz, 1H), 8.15–8.17 (m, 1H), 8.09–8.11 (m, 1H), 7.97 130.40, 128.73, 128.40, 122.96, 121.75, 121.65, 121.34, 121.19,
(
(
1
d, J ¼ 5.2 Hz, 1H), 7.83 (d, J ¼ 5.2 Hz, 1H), 7.53–7.59 (m, 2H), 7.45 120.20, 119.95, 114.16, 113.43, 113.32, 110.01, 109.95, 55.34, 43.73,
þ
dd, J ¼ 5.2, 1.2 Hz, 1H), 7.40 (d, J ¼ 8.4 Hz, 1H), 7.35 (d, J ¼ 8.4 Hz, 43.41, 25.96, 25.80. HRMS calcd for C H N O [M þ H] 574.2601,
3
8 32 5
H), 7.24–7.30 (m, 3H), 7.13 (dd, J ¼ 5.2, 3.6 Hz, 1H), 4.39 (t, found 574.2606. Anal. calcd for C H N O: C, 79.56; H, 5.45; N,
3
8 31 5
J ¼ 7.6 Hz, 2H), 4.14 (t, J ¼ 7.6 Hz, 2H), 2.98 (s, 3H), 1.61–1.68 (m, 12.21; found C 79.51, H 5.66, N 11.72.
1
3
2
H), 1.38–1.46 (m, 2H), 1.13–1.21 (m, 2H), 0.91–0.98 (m, 2H).
C
NMR (100 MHz, CDCl ) d: 142.18, 141.47, 141.21, 141.01, 138.54,
3
1
–(3,4-dimethoxyphenyl)-9–(4-(1–(3-pyridyl)-b-carboline-9-yl)bu-
1
1
1
2
37.75, 137.09, 134.98, 134.77, 130.86, 129.14, 128.57, 128.27,
28.16, 126.98, 126.65, 121.66, 121.53, 121.34, 121.23, 120.06,
tyl)-b-carboline (5l)
This compound was obtained as yellow crystals in 63% yield, m.p.
19.66, 114.12, 112.99, 110.20, 109.62, 44.58, 44.17, 30.61, 28.96,
ꢀ
1
þ
239.8–240.5 C. H NMR (400 MHz, CDCl ) d: 8.68 (d, J ¼ 2.0 Hz, 1H),
3
6.43, 26.34, 23.40 . HRMS calcd for C33
H
N
31
N
4
S [M þ H] 515.2264,
8
.61 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.51 (d, J ¼ 5.2 Hz, 1H),8.48 (d,
J ¼ 5.2 Hz, 1H), 8.18 (d, J ¼ 7.6 Hz, 1H), 8.16 (d, J ¼ 7.6 Hz, 1H),7.97
d, J ¼ 5.2 Hz, 1H),7.95 (d, J ¼ 5.2 Hz, 1H), 7.55–7.60 (m, 3H),
.31–7.35 (m, 2H), 7.13–7.20 (m, 3H), 6.96 (d, J ¼ 2.0 Hz, 1H), 6.85
found 515.2268. Anal. calcd for C33
H
30
4
S: C, 77.01; H, 5.88; N,
1
0.89; S, 6.23; found C, 76.80; H, 5.76; N, 10.72; S, 5.97.
(
7
(
d, J ¼ 8.0 Hz, 1H), 6.79 (d, J ¼ 8.0 Hz, 1H), 3.84 (s, 3H), 3.80 (s, 3H),
3
.76 (t, J ¼ 6.4 Hz, 2H), 3.66 (t, J ¼ 6.4 Hz, 2H), 0.82–0.92 (m, 4H).
1–(2-chlorophenyl)-9–(4-(1-methyl-b-carboline-9-yl)butyl)-b-carbo-
1
3
C NMR (100 MHz, CDCl ) d: 149.95, 149.44, 149.20, 148.54,
3
line (5i)
1
1
1
1
2
6
43.62, 142.00, 141.87, 140.20, 138.83, 138.28, 136.36, 135.75,
33.98, 133.77, 132.17, 130.74, 130.46, 128.79, 128.49, 122.97,
21.73, 121.68, 121.34, 121.19, 120.23, 120.04, 114.19, 113.52,
12.45, 110.61, 110.09, 109.89, 55.99, 55.96, 43.82, 43.46, 26.05,
5.89. HRMS calcd for C39
This compound was obtained as yellow crystals in 51% yield, m.p.
ꢀ
1
2
8
(
7
32.0–232.8 C. H NMR (400 MHz, CDCl
3
) d: 8.52 (d, J ¼ 5.2 Hz, 1H),
.34 (d, J ¼ 5.2 Hz, 1H), 8.21–8.16 (m, 1H), 8.12–8.09 (m, 1H), 8.01
d, J ¼ 5.2 Hz, 1H), 7.85 (d, J ¼ 5.2 Hz, 1H), 7.60–7.51 (m, 2H),
.35–7.31 (m, 4H), 7.30–7.26 (m, 2H), 7.24–7.23 (m, 1H), 7.17–7.13
m, 1H), 4.34–4.22 (m, 2H), 3.94–3.78 (m, 2H), 2.90 (s, 3H),
þ
H
H
34
N
5
O
2
[M þ H] 604.2707, found
04.2705. Anal. calcd for C39
33
N
5
O
2
: C, 77.59; H, 5.51; N, 11.60;
(
found C 77.36, H 5.57, N 11.51.
1
3
1
1
1
1
1
.63–1.34 (m, 4H). C NMR (100 MHz, CDCl
40.87, 140.83, 138.62, 138.44, 137.97, 134.89, 134.02, 133.93,
31.34, 130.13, 130.05, 129.45, 129.30, 128.63, 128.27, 126.72, 1–(3,4,5-trimethoxyphenyl)-9–(4-(1–(3-pyridyl)-b-carboline-9-yl)bu-
21.81, 121.64, 121.31, 120.05, 119.86, 114.34, 113.08, 109.62, tyl)-b-carboline (5m)
09.58, 44.06, 43.78, 28.23, 26.73, 23.45. HRMS calcd for This compound was obtained as yellow crystals in 49% yield, m.p.
3
) d: 141.57, 141.41,
þ
ꢀ
1
C H ClN [M þ H] 515.1997, found 515.2003. Anal. calcd for 231.0–232.3 C. H NMR (400 MHz, CDCl ) d: 8.71 (dd, J ¼ 2.0,
3
3
28
4
3
C H ClN : C, 76.96; H, 5.28; N, 10.88; found C 76.39, H 5.37, 0.8 Hz, 1H), 8.60 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.51 (d, J ¼ 0.8 Hz, 1H),
3
3
27
4
N 10.57.
8.49 (d, J ¼ 0.8 Hz, 1H), 8.18 (d, J ¼ 8.0 Hz, 1H), 8.14 (d, J ¼ 8.0 Hz,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
379
1
3
1
H), 7.98 (d, J ¼ 5.2 Hz, 1H), 7.96 (d, J ¼ 5.2 Hz, 1H), 7.57–7.62 (m, 121.68, 121.46, 121.23, 120.10, 119.98, 114.20, 113.57, 112.47,
H), 7.30–7.36 (m, 2H), 7.21 (d, J ¼ 8.4 Hz, 1H), 7.17 (d, J ¼ 8.4 Hz, 110.63, 110.11, 109.88, 55.99, 55.97, 43.66, 43.59, 26.29, 26.22.
þ
H), 7.11–7.15 (m, 1H), 6.67 (s, 2H), 3.80 (s, 3H), 3.78 (s, 6H), 3.76 HRMS calcd for C H N O S [M þ H] 609.2319, found 609.2314.
3
8 33 4 2
1
3
(t, J ¼ 6.4 Hz, 2H), 3.68 (t, J ¼ 7.2 Hz, 2H), 0.95–0.98 (m, 4H).
C
38 32 4 2
Anal. calcd for C H N O S: C, 74.98; H, 5.30; N, 9.20; S, 5.27;
NMR (100 MHz, CDCl ) d: 153.03, 149.94, 149.43, 143.58, 141.94, found C, 74.47; H, 5.24; N, 9.36; S, 5.02.
3
1
1
1
1
41.68, 140.15, 138.78, 138.28, 138.24, 136.34, 135.79, 135.24,
33.98, 133.74, 130.72, 130.39, 128.97, 128.57, 122.98, 121.69,
21.67, 121.31, 121.11, 120.28, 120.11, 114.20, 113.79, 110.13,
09.86, 106.54, 60.93, 56.24, 43.91, 43.54, 26.09, 25.94. HRMS calcd
1
–(3,4,5-trimethoxyphenyl)-9–(4-(1–(2-thienyl)-b-carboline-9-yl)bu-
þ
for C40
H
36
N
O
5 3
[M þ H] 634.2813, found 634.2817. Anal. calcd for
tyl)-b-carboline (5q)
This compound was obtained as yellow crystals in 65% yield, m.p.
C H N O : C, 75.81; H, 5.57; N, 11.05; found C 75.79, H 5.56,
N 11.32.
4
0 35 5 3
ꢀ
1
2
8
1
2
1
3
26.5–227.8 C. H NMR (400 MHz, CDCl
3
) d: 8.50 (d, J ¼ 5.2 Hz, 1H),
.46 (d, J ¼ 5.2 Hz, 1H), 8.19 (d, J ¼ 7.6 Hz, 1H), 8.12 (d, J ¼ 7.6 Hz,
H), 7.99 (d, J ¼ 5.2 Hz, 1H), 7.92 (d, J ¼ 5.2 Hz, 1H), 7.56–7.60 (m,
H), 7.28–7.36 (m, 2H), 7.17–7.24 (m, 3H), 6.96 (dd, J ¼ 7.6, 1.2 Hz,
H), 6.88 (dd, J ¼ 5.2, 3.6 Hz, 1H), 6.71 (s, 2H), 3.80–3.87 (m, 4H),
1
–(2-chlorophenyl)-9–(4-(1–(3-pyridyl)-b-carboline-9-yl)butyl)-b-car-
boline (5n)
This compound was obtained as yellow crystals in 58% yield, m.p.
1
3
1
.82 (s, 3H), 3.79 (s, 6H), 1.03–1.12 (m, 4H). C NMR (100 MHz,
ꢀ
1
0
8
7
7
77.6–178.9 C. H NMR (400 MHz, CDCl
3
) d: 8.73 (dd, J ¼ 2.4,
CDCl
3
) d: 153.07, 143.44, 141.91, 141.87, 140.71, 138.45, 138.40,
.8 Hz, 1H), 8.66 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.53 (d, J ¼ 5.2 Hz, 1H),
.50 (d, J ¼ 5.2 Hz, 1H), 8.17–8.21 (m, 2H), 8.00 (d, J ¼ 2.4 Hz, 1H),
.99 (d, J ¼ 2.4 Hz, 1H), 7.64 (d, J ¼ 7.6 Hz, 1H), 7.60–7.63 (m, 1H),
.57–7.59 (m, 1H), 7.26–7.37 (m, 4H), 7.22–7.25 (m, 2H), 7.13–7.20
1
1
1
2
37.94, 136.61, 134.50, 133.86, 130.70, 130.66, 128.83, 128.62,
28.05, 126.82, 126.48, 121.80, 121.64, 121.39, 121.15, 120.17,
20.15, 114.22, 113.88, 110.19, 109.84, 106.65, 60.95, 56.24, 43.68,
6.36, 26.27. HRMS calcd for C H N O S [M þ H] 639.2424,
þ
3
9 35 4 3
(
m, 3H), 3.72 (t, J ¼ 7.2 Hz, 2H), 3.48–3.64 (m, 2H), 0.84–0.93 (m,
1
3
39 34 4 3
found 639.2422. Anal. calcd for C H N O S: C, 73.33; H, 5.37; N,
4
1
1
1
1
H). C NMR (100 MHz, CDCl ) d: 149.99, 149.55, 142.03, 141.51,
3
8
.77; S, 5.02; found C, 73.37; H, 5.59; N, 8.65; S, 4.81.
40.52, 140.19, 138.80, 138.34, 138.14, 136.48, 135.71, 134.02,
34.00, 133.71, 131.32, 130.86, 130.04, 129.92, 129.23, 128.76,
28.62, 126.60, 123.02, 121.81, 121.72, 121.24, 121.09, 120.31,
20.02, 114.31, 114.24, 109.97, 109.71, 43.72, 43.19, 26.30, 26.08.
þ
1
–(2-chlorophenyl)-9–(4-(1–(2-thienyl)-b-carboline-9-yl)butyl)-b-car-
HRMS calcd for C37
Anal. calcd for C H ClN : C, 76.87; H, 4.88; N, 12.11; found C boline (5r)
H
29ClN
5
[M þ H] 578.2106, found 578.2111.
3
7
28
5
7
6.38, H 4.85, N 11.75.
This compound was obtained as yellow crystals in 52% yield, m.p.
ꢀ
1
2
1
1
25.4–227.2 C. H NMR (400 MHz, CDCl ) d: 8.52 (s, 1H), 8.50 (s,
3
H), 8.16–8.20 (m, 2H), 8.01 (d, J ¼ 5.2 Hz, 1H), 7.97 (d, J ¼ 5.2 Hz,
1
–(4-methoxyphenyl)-9–(4-(1–(2-thienyl)-b-carboline-9-yl)butyl)-
H), 7.56–7.62 (m, 2H), 7.27–7.38 (m, 7H), 7.19–7.25 (m, 2H), 7.06
b-carboline (5o)
(
dd, J ¼ 3.6, 1.2 Hz, 1H), 7.03 (dd, J ¼ 5.2, 3.2 Hz, 1H), 3.88 (t,
13
This compound was obtained as yellow crystals in 64% yield, m.p.
J ¼ 7.2 Hz, 2H), 3.53–3.71 (m, 2H), 1.01–1.13 (m, 4H). C NMR
ꢀ
1
2
8
1
7
6
32.5–233.3 C. H NMR (400 MHz, CDCl ) d: 8.50 (d, J ¼ 5.2 Hz, 1H),
3
(100 MHz, CDCl ) d: 141.97, 141.52, 140.78, 140.60, 138.51, 138.41,
3
.49 (d, J ¼ 5.2 Hz, 1H), 8.16 (t, J ¼ 7.6 Hz, 2H), 7.96 (d, J ¼ 5.2 Hz,
H), 7.94 (d, J ¼ 5.2 Hz, 1H), 7.55–7.59 (m, 2H), 7.29–7.38 (m, 5H),
.23 (d, J ¼ 8.4 Hz, 1H), 7.18 (d, J ¼ 8.4 Hz, 1H), 7.00–7.02 (m, 1H),
.96–6.98 (m, 1H), 6.90–6.94 (m, 2H), 3.74–3.84 (m, 7H), 0.95–1.01
1
1
1
1
[
7
38.11, 136.77, 134.53, 134.06, 133.87, 131.34, 130.86, 130.07,
30.05, 129.33, 128.62, 128.54, 128.25, 127.03, 126.66, 126.63,
21.75, 121.29, 121.18, 120.19, 119.96, 114.33, 114.22, 109.97,
09.68, 43.59, 43.29, 26.42, 26.40. HRMS calcd for C36
H
28ClN
4
S
1
3
(
m, 4H). C NMR (100 MHz, CDCl
3
) d: 159.87, 142.11, 141.94,
þ
M þ H] 583.1718, found 583.1720. Anal. calcd for C36
H
27ClN S: C,
4
1
1
1
4
40.82, 138.52, 136.77, 134.54, 133.87, 130.72, 130.55, 128.60,
28.52, 128.10, 126.92, 126.55, 121.77, 121.69, 121.38, 121.25,
20.10, 120.03, 114.18, 113.58, 113.48, 110.04, 109.93, 55.37, 43.60,
3.59, 26.18, 26.10. HRMS calcd for C37
4.15; H, 4.67; N, 9.61; S, 5.50; found C, 73.73; H, 4.53; N, 9.63;
S, 5.19.
þ
H
31
N
4
OS [M þ H] 579.2213,
found 579.2209. Anal. calcd for C H N OS: C, 76.79; H, 5.23; N,
3
7 30 4
9
.68; S, 5.54; found C, 76.10; H, 5.50; N, 9.29; S, 5.16.
7-methoxy-1-methyl-9–(4-(1-methyl-b-carboline-9-yl)butyl)-b-carbo-
line (5s)
This compound was obtained as colorless crystals in 74% yield,
1
–(3,4-dimethoxyphenyl)-9–(4-(1–(2-thienyl)-b-carboline-9-yl)bu-
ꢀ
1
m.p. 188.7–189.4 C. H NMR (400 MHz, DMSO-d
.21 (s, 1H),8.16 (d, J ¼ 5.2 Hz, 1H), 8.08 (d, J ¼ 8.8 Hz, 1H), 7.99 (d,
J ¼ 5.2 Hz, 1H), 7.87 (d, J ¼ 5.2 Hz, 1H), 7.70 (d, J ¼ 8.8 Hz, 1H),
6
) d: 8.23 (s, 1H),
tyl)-b-carboline (5p)
This compound was obtained as yellow crystals in 49% yield, m.p.
8
ꢀ
1
2
8
1
2
17.2–218.4 C. H NMR (400 MHz, CDCl ) d: 8.51 (d, J ¼ 5.2 Hz, 1H),
3
7
.54–7.58 (m, 1H), 7.22–7.26 (m, 1H), 7.20 (d, J ¼ 2.0 Hz, 1H), 6.86
.47 (d, J ¼ 5.2 Hz, 1H), 8.18 (d, J ¼ 7.6 Hz, 1H), 8.14 (d, J ¼ 7.6 Hz,
H), 7.96 (d, J ¼ 5.2 Hz, 1H), 7.94 (d, J ¼ 5.2 Hz, 1H), 7.55–7.59 (m,
H), 7.22 (d, J ¼ 8.4 Hz, 1H), 7.19 (d, J ¼ 8.4 Hz, 1H), 7.03 (d,
(dd, J ¼ 8.8, 2.0 Hz, 1H), 4.58–4.62 (m, 4H), 3.87 (s, 3H), 2.94 (s, 3H),
1
3
2
6
.89 (s, 3H), 1.82–1.86 (m, 4H). C NMR (100 MHz, DMSO-d ) d:
J ¼ 2.0 Hz, 1H), 6.98 (dd, J ¼ 3.2, 1.2 Hz, 1H), 6.96 (dd, J ¼ 8.0, 160.38, 142.59, 141.14, 140.96, 140.39, 137.69, 137.49, 134.37,
2
3
.0 Hz, 1H), 6.92 (dd, J ¼ 5.2, 3.2 Hz, 1H), 6.85 (d, J ¼ 8.4 Hz, 1H), 134.23, 128.33, 127.97,127.94, 122.27, 121.36, 120.36, 119.36,
13
.86 (s, 3H), 3.82(s, 3H),3.78–3.85 (m, 4H), 0.98–1.08 (m, 4H).
C
114.09, 112.83, 112.12, 110.27, 108.91, 93.74, 55.48, 43.67, 43.57,
þ
NMR (100 MHz, CDCl
1
1
3
) d: 149.24, 148.63, 143.72, 141.92, 141.90, 27.55, 27.37, 22.98, 22.86. HRMS calcd for C29
40.81, 138.53, 138.26, 136.75, 134.54, 133.98, 132.30, 130.69, 449.2336, found 449.2340. Anal. calcd for C29
30.51, 128.65, 128.41, 128.04, 126.86, 126.50, 121.77, 121.73, 6.29; N, 12.49; found C 77.39, H 5.97, N 12.38.
H
29
N
4
O [M þ H]
H N O: C, 77.65; H,
28 4

3
80
L. GUO ET AL.
7
-methoxy-1-methyl-9–(5-(1-methyl-b-carboline-9-yl)pentyl)-b-car-
7.55–7.60 (m, 1H), 7.34–7.38 (m, 2H), 7.29–7.33 (m, 1H), 7.13 (dd,
J ¼ 3.6, 1.2 Hz, 1H), 7.02 (dd, J ¼ 5.2, 3.6 Hz, 1H), 6.88 (dd, J ¼ 8.4,
boline (5t)
This compound was obtained as colorless crystals in 67% yield, 2.0 Hz, 1H), 6.66 (d, J ¼ 2.0 Hz, 1H), 4.21 (t, J ¼ 7.2 Hz, 2H), 4.17–4.08
1
1
3
ꢀ
m.p. 176.8–177.6 C.
H
NMR (400 MHz, CDCl
3
)
d: 8.33 (d, (t, J ¼ 7.2 Hz, 2H), 3.90 (s, 3H), 2.86 (s, 3H), 1.42–1.54 (m, 4H).
C
J ¼ 5.2 Hz, 1H), 8.29 (d, J ¼ 5.2 Hz, 1H), 8.11 (d, J ¼ 7.6 Hz, 1H), 7.97 NMR (100 MHz, CDCl
) d: 160.86, 142.92, 142.05, 140.94, 140.18,
3
(
7
(
d, J ¼ 8.4 Hz, 1H), 7.84 (d, J ¼ 5.2 Hz, 1H), 7.74 (d, J ¼ 5.2 Hz, 1H), 138.72, 138.18, 136.91, 135.10, 134.60, 130.91, 129.54, 128.69,
.52–7.56 (m, 1H), 7.36 (d, J ¼ 8.4 Hz, 1H), 7.25–7.29 (m, 1H), 6.89 128.26, 127.12, 126.70, 122.49, 121.75, 121.35, 120.22, 115.24,
dd, J ¼ 8.4, 2.0 Hz, 1H), 6.77 (d, J ¼ 2.0 Hz, 1H), 4.49 (t, J ¼ 7.2 Hz, 114.21, 112.33, 109.94, 108.45, 93.68, 55.75, 43.94, 29.69, 27.70,
þ
2
1
1
1
1
3
4
6
H), 4.42 (t, J ¼ 7.2 Hz, 2H), 3.89 (s, 3H), 2.99 (s, 3H), 2.97 (s, 3H), 26.51, 23.28. HRMS calcd for C32
29
H N
4
OS [M þ H] 517.2057, found
13
.81–1.91 (m, 4H), 1.41–1.50 (m, 2H). C NMR (100 MHz, CDCl ) d: 517.2063. Anal. calcd for C32
H
N
28 4
OS: C, 74.39; H, 5.46; N, 10.84; S,
3
60.92, 143.09, 141.45, 141.04, 140.26, 138.15, 138.09, 135.17, 6.21; found C, 74.21; H, 5.42; N, 10.65; S, 6.17.
35.01, 129.58, 129.14, 128.21, 122.48, 121.53, 121.29, 119.73,
15.21, 112.97, 112.31, 109.56, 108.60, 93.51, 55.72, 44.55, 30.83,
þ
7-methoxy-1-methyl-9–(5-(1–(2-thienyl)-b-carboline-9-yl)pentyl)-
b-carboline (5x)
This compound was obtained as yellow crystals in 71% yield, m.p.
0.56, 24.42, 23.58, 23.33. HRMS calcd for C30
63.2492, found 463.2497. Anal. calcd for C30
.54; N, 12.11; found C, 77.42; H, 6.33; N, 12.07.
H
31
N
4
O [M þ H]
H N O: C, 77.89; H,
30 4
ꢀ
1
1
8
96.1–197.4 C. H NMR (400 MHz, CDCl
3
) d: 8.50 (d, J ¼ 5.2 Hz, 1H),
.30 (d, J ¼ 5.2 Hz, 1H), 8.13–8.16 (m, 1H), 7.94–7.98 (m, 2H), 7.74
7
-methoxy-1-methyl-9–(4-(1–(3-pyridyl)-b-carboline-9-yl)butyl)-
(d, J ¼ 5.2 Hz, 1H), 7.55–7.59 (m, 1H), 7.39 (d, J ¼ 8.4 Hz, 1H), 7.30
(m, 1H), 7.23 (dd, J ¼ 5.2, 1.2 Hz, 1H), 7.16 (dd, J ¼ 3.6, 1.2 Hz, 1H),
b-carboline (5u)
This compound was obtained as colorless crystals in 70% yield, 6.92 (dd, J ¼ 5.2, 3.6 Hz, 1H), 6.89 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.72 (d,
ꢀ
1
m.p. 163.8–164.5 C.
H
NMR (400 MHz, CDCl
3
)
d: 8.79 (d, J ¼ 2.0 Hz, 1H), 4.28 (t, J ¼ 7.6 Hz, 2H), 4.10 (t, J ¼ 7.6 Hz, 2H), 3.90
J ¼ 2.0 Hz, 1H), 8.65 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.51 (d, J ¼ 5.2 Hz, 1H), (s, 3H), 2.92 (s, 3H), 1.50–1.58 (m, 2H), 1.40–1.48 (m, 2H), 0.96–1.04
1
3
8
(
(
.29 (d, J ¼ 5.2 Hz, 1H), 8.15–8.17 (m, 1H), 7.94–7.96 (m, 2H), 7.71 (m, 2H). C NMR (100 MHz, CDCl
) d: 160.86, 143.01, 142.10,
3
d, J ¼ 5.2 Hz, 1H), 7.65–7.68 (m, 1H), 7.56–7.60 (m, 1H), 7.30–7.34 141.02, 140.33, 138.56, 138.18, 136.98, 135.20, 134.67, 130.84,
m, 2H), 7.23–7.25 (m, 1H), 6.89 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.60 (d, 129.48, 128.66, 128.33, 126.89, 126.56, 122.44, 121.67, 121.28,
J ¼ 2.0 Hz, 1H), 4.16 (t, J ¼ 7.2 Hz, 2H), 3.95 (t, J ¼ 7.2 Hz, 2H), 3.90 120.12, 115.23, 114.18, 112.32, 110.04, 108.52, 93.62, 55.73, 44.54,
1
3
(
(
s, 3H), 2.77 (s, 3H), 1.48–1.35 (m, 2H), 1.35–1.27 (m, 2H). C NMR 44.09, 29.72, 28.94, 23.97, 23.38. HRMS calcd for C33
H
31 4
N OS
þ
100 MHz, CDCl ) d: 160.85, 150.02, 149.66, 142.77, 142.20, 140.30, [M þ H] 531.2213, found 531.2213. Anal. calcd for C33
30 4
H N OS: C,
3
1
1
1
2
5
40.13, 139.02, 138.36, 136.42, 135.80, 135.01, 134.09, 130.92, 74.69; H, 5.70; N, 10.56; S, 6.04; found C, 74.06; H, 6.08; N, 10.49;
29.43, 128.81, 123.15, 122.45, 121.79, 121.30, 120.33, 115.16, S, 6.44.
14.17, 112.29, 109.94, 108.54, 93.55, 55.73, 44.12, 43.84, 27.37,
6.02, 23.30. HRMS calcd for C33
12.2453. Anal. calcd for C33
þ
H
30
N
5
O [M þ H] 512.2445, found
Biological evaluation
H
29
N
5
O: C, 77.47; H, 5.71; N, 13.69;
found C, 76.67; H, 5.75; N, 13.05.
In vitro cell growth inhibition assay
Target compounds were assayed by the MTT method for cytotoxic
5
activity, as described previously . The panel of cell lines included
7
-methoxy-1-methyl-9–(5-(1–(3-pyridyl)-b-carboline-9-yl)pentyl)-
gastric carcinoma (BGC-823), liver carcinoma (HepG2), breast carcin-
oma (MCF-7), colon carcinoma (HT-29), esophageal carcinoma (Eca-
109), and Lewis lung carcinoma (LLC). Growth inhibition rates were
b-carboline (5v)
This compound was obtained as colorless crystals in 62% yield,
m.p. 183.6–184.9 C. H NMR (400 MHz, CDCl
ꢀ
1
3
) d: 8.82 (dd, J ¼ 2.0,
calculated with the following equitation: Inhibition ratio (%)¼
0
8
1
1
7
1
.8 Hz, 1H), 8.54 (d, J ¼ 5.2 Hz, 1H), 8.53 (dd, J ¼ 5.2, 1.6 Hz, 1H)
ODcompdꢂODblank
ꢁ
100%. Half maximal inhibitory concentration (IC50) of
.30 (d, J ¼ 5.2 Hz, 1H), 8.18 (d, J ¼ 7.6 Hz, 1H), 8.02 (d, J ¼ 5.2 Hz,
H), 7.98 (d, J ¼ 8.4 Hz, 1H), 7.85–7.88 (m, 1H), 7.75 (d, J ¼ 5.2 Hz,
H), 7.56–7.60 (m, 1H), 7.37 (d, J ¼ 8.4 Hz, 1H), 7.30–7.34 (m, 1H),
.24–7.28 (m, 1H), 6.90 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.72 (d, J ¼ 2.0 Hz,
H), 4.27 (t, J ¼ 7.6 Hz, 2H), 3.94 (t, J ¼ 7.6 Hz, 2H), 3.91 (s, 3H), 2.90
ODDMSOꢂODblank
each compound was calculated using software Graph-Pad Prism
(version 6.0).
Assay of acute toxicities
1
3
(s, 3H), 1.45–1.53 (m, 2H), 1.33–1.42 (m, 2H), 0.87–0.90 (m, 2H).
C
NMR (100 MHz, CDCl ) d: 160.95, 150.08, 149.51, 143.06, 142.01, Specific pathogen-free KM mice (6–8 weeks old) weighing 19–22 g
3
ꢀ
1
1
1
3
5
5
40.36, 140.19, 138.81, 138.05, 136.60, 135.99, 135.11, 134.25, were housed in a mouse room at 24 ± 2 C and 60–70% humidity
30.83, 129.58, 128.81, 123.05, 122.48, 121.74, 121.26, 120.26, with 12 h light/dark cycles. The mice were provided rodent labora-
15.15, 114.25, 112.34, 110.00, 108.66, 93.56, 55.76, 44.43, 44.25, tory chow pellets and tap water for a week to adapt to the envir-
þ
0.09, 28.61, 23.82, 23.22 . HRMS calcd for C34
26.2601, found 526.2609. Anal. calcd for C34
.94; N, 13.32; found C, 77.76; H, 5.95; N, 13.74.
H
32
5
N O [M þ H]
onment of the mouse room. The experimental protocol was
H
31
N
5
O: C, 77.69; H, 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.
7
-methoxy-1-methyl-9–(4-(1–(2-thienyl)-b-carboline-9-yl)butyl)-
Various doses of the asymmetric dimeric b-carboline derivatives,
ranging from 5.0 to 500 mg kg dissolved in 0.5% carboxymethyl
ꢂ1
b-carboline (5w)
This compound was obtained as colorless crystals in 68% yield, cellulose sodium (CMC-Na) salt solution, were given intraperito-
ꢀ
1
m.p. 189.3–190.1 C.
3
H NMR (400 MHz, CDCl ) d: 8.50 (d, neally to different groups of healthy KM mice, and each group
J ¼ 5.2 Hz, 1H), 8.28 (d, J ¼ 5.2 Hz, 1H), 8.14–8.16 (m, 1H), 7.96 (d, contained 10 mice (5 males and 5 females). After the administra-
J ¼ 1.6 Hz, 1H), 7.95 (d, J ¼ 2.0 Hz, 1H), 7.72 (d, J ¼ 5.2 Hz, 1H), tion of the compounds, the mice were observed continuously for

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
381
the first 2 h for any gross behavioral changes and deaths, then eggs were obtained from a local hatchery. We injected 5 ml of
intermittently for the next 24h, and occasionally thereafter for albumin, and the eggs were incubated horizontally to allow the
1
4days, and for the onset of any delayed effects. All of the animals CAM to detach from the shell to produce a sham chamber.
were killed on the 14th day after drug administration, and they Compounds 5b and 5w were prepared in gelatin sponge discs
3
were checked macroscopically for possible damage to the heart, (5 ꢁ 5 ꢁ 5 mm ) at the concentration of 0.5, 5.0, and 50 mM/disc,
liver, and kidneys. Mice that experienced immediate death following respectively. CA4P was used as the positive control drug. Discs
drug administration were also examined for any possible organ
damage. LD50 values were calculated graphically as described .
containing the vehicle only (DMSO) were used as the negative
control. A small window opening was made in the shell, and the
discs were directly applied onto the CAM. The square opening
was covered with sterilized surgical tape and the embryos were
34
In vivo antitumor activity
ꢀ
incubated for 48 h at 38.5 C. The CAMs were photographed under
Sarcoma180 and Lewis lung cancer cell lines were provided by a dissecting microscope and blood vessels in each CAM were
Shanghai Institute of Pharmaceutical Industry. Mice were inocu- counted. The results are presented as a mean percentage of inhib-
lated with Sarcoma180 and Lewis lung cancer tumour cells. After ition to the control ± SD, (n ¼ 3).
7
days, the tumours were removed and the cells were harvested.
Mice received subcutaneous injections of viable tumour cells
6
Results and discussion
(
2 ꢁ 10 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 consecu-
tive days. This dose was the maximum tolerated dose for most of
Chemistry
9
The synthesis of the desired N -heterobivalent b-carbolines (5a-r)
were performed in four steps starting from L-tryptophan as out-
lined in Scheme 1–2. The tetrahydro-b-carbolines 2a-h was pre-
the compounds based on our preliminary studies. CTX at 30 mg
ꢂ1
kg
was used as the positive control and the vehicle as the pared by condensation of L-tryptophan with appropriate aldehyde
negative control. The weight of the animals was recorded every via the Pictet-Spengler condensation, and followed by oxidation
three days. All of the animals were killed on the 21st day after and decarboxylation to afford the intermediate 1-substituted-
2
1,29
9
tumour inoculation, and the tumours were excised and weighed. b-carbolines 3a-h
. Then the N of 3a-d was alkylated by the
The inhibition rate was calculated as follows:
action of sodium hydride (NaH) in N,N-dimethylformamide (DMF)
followed by the addition of the appropriate dibromo alkane to
obtain intermediates 4a-f. Finally, 4a-f with appropriate 1-substi-
ðC ꢂ TÞ=C ꢁ 100
where T is the average tumor weight of the treated group and C tuted-b-carbolines 3c-h in the presence of sodium hydride in DMF
is the average tumor weight of the negative control group.
at room temperature to afford the target compounds 5a-r in
4
7–74% yield.
In Scheme 3, the intermediates 4g-f were synthesized from
harmine and 1,4-dibromobutane or 1,5-dibromopentane using the
CAM assay in vivo
Anti-angiogenic activity of the selected compounds 5b and 5w same method of compound 4a, and finally compounds 5s-x were
were investigated in vivo using a CAM assay. Five-day-old fertilized obtained from the similar method of 5a.
Scheme 1. Synthetic route to compounds 3a-h.

3
82
L. GUO ET AL.
9
Scheme 2. Synthesis of the N -heterobivalent b-carbolines 5a-r.
9
Scheme 3. Synthesis of the N -heterobivalent b-carbolines 5s-x.
9
The chemical structures of all the novel N -heterobivalent
As illustrated in Table 1, compounds 5b and 5w displayed a
1
13
b-carbolines were characterized by H NMR, C NMR, HRMS and broad spectrum of cytotoxic activities with IC50 value of lower
the elemental analysis.
than 20 lM against the tested six tumor cell lines, while com-
pounds 5c, 5s, 5t and 5v only exhibited strong cytotoxic effects
with IC50 value of lower than 20 lM against three or four tumour
cell lines. Interestingly, compounds 5d and 5j were selectively
In vitro cytotoxicity and structure–activity relationships
9
All the 24 novel synthesized N -heterobivalent b-carbolines (5a-x) active against Eca-109 and LLC cell lines with IC50 value of lower
were screened for their in vitro cytotoxic activities against six dif- than 20 lM but fail to show cytotoxic effects in other cell lines.
ferent cancer cell lines, namely BGC-823 (gastric carcinoma), Similarly, compound 5u displayed selective activities against HT-
HepG2 (liver carcinoma), MCF-7 (breast carcinoma), HT-29 (colon 29 and LLC cell lines. Moreover, compound 5f only exhibited
carcinoma), Eca-109 (esophageal carcinoma) and LLC (Lewis lung strong cytotoxic effects with IC50 value of lower than 20 lM
carcinoma). Cisplatin and B-9–3 were used as the reference control against LLC cell lines. compounds 5g and 5i showed weak cyto-
and the results were expressed as IC50 values and summarized in toxic activities with IC50 values in the range of 18.8–97.6 lM.
Table 1. The IC50 values were the average of at least three inde- Unfortunately, compounds 5k–5r were weak or inactive against all
pendent experiments.
tumour cell lines tested.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
383
9
Table 1. Cytotoxic activity of N -heterobivalent b-carbolines in vitro
a
IC50 (lM) ±SD
b
Compd.
R
1
R
1
’
R
7
’
N
BGC
HepG2
MCF7
HT-29
Eca-109
LLC
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
H
H
3
>100
12.1 ± 2.3
22.6 ± 1.9
>100
39.1 ± 5.4
>100
>100
>100
>100
CH
CH
CH
H
3
3
3
H
H
H
H
H
H
H
H
3
4
5
3
3
4
5
3
15.9 ± 1.4
10.5 ± 2.1
>100
8.4 ± 1.7
15.3 ± 2.6
>100
12.6 ± 1.2
13.3 ± 2.4
>100
10.5 ± 1.3
25.3 ± 3.2
18.9 ± 3.1
>100
12.4 ± 2.1
5.7 ± 1.2
14.5 ± 0.9
32.3 ± 4.5
6.1 ± 0.3
22.7 ± 1.4
30.2 ± 1.5
44.7 ± 3.6
>100
>100
>100
18.1 ± 3.6
56.1 ± 4.2
32.3 ± 5.3
47.8 ± 6.5
28.3 ± 1.6
CH
CH
CH
CH
3
3
3
3
38.8 ± 2.9
97.6 ± 12.4
>100
>100
46.6 ± 4.8
75.5 ± 10.7
75.9 ± 9.4
95.5 ± 14.8
64.1 ± 7.6
30.8 ± 3.2
30.2 ± 3.4
23.4 ± 4.1
g
h
i
18.8 ± 2.3
>100
37.9 ± 4.2
23.9 ± 3.7
5
5
j
CH
3
H
H
4
3
>100
>100
>100
>100
46.8 ± 3.7
24.1 ± 2.2
18.6 ± 2.1
16.6 ± 3.1
k
>100
>100
>100
>100
5
l
H
3
>100
79.5 ± 12.3
>100
91.1 ± 10.6
93.2 ± 7.1
46.8 ± 6.4
5
5
5
5
m
H
H
H
H
3
3
3
3
>100
>100
>100
>100
85.1 ± 11.6
>100
>100
86.9 ± 15.3
>100
>100
72.2 ± 5.2
>100
70.9 ± 8.4
>100
>100
>100
n
o
>100
>100
96.8 ± 9.8
>100
p
>100
>100
>100
>100
5
5
q
r
H
H
3
3
>100
>100
>100
>100
>100
>100
>100
>100
>100
60.7 ± 4.8
96.2 ± 5.1
96.7 ± 11.9
5
5
s
t
CH
CH
3
CH
CH
3
3
OCH
OCH
3
3
4
17.9 ± 1.1
42.7 ± 2.9
37.6 ± 5.8
31.6 ± 2.2
36.4 ± 2.1
17.6 ± 3.4
11.1 ± 0.8
4.5 ± 0.6
11.3 ± 2.7
51.9 ± 7.4
6.2 ± 0.6
3.1 ± 0.4
3
3
5
5
5
5
u
v
CH
CH
CH
CH
CH
3
3
3
3
3
OCH
OCH
OCH
OCH
H
3
3
3
3
3
4
3
4
3
39.1 ± 2.4
23.2 ± 1.5
16.5 ± 1.9
>100
26.8 ± 2.8
31.1 ± 3.4
15.4 ± 2.7
>100
23.9 ± 0.7
18.7 ± 0.5
14.1 ± 1.4
41.6 ± 5.3
15.1 ± 3.1
13.5 ± 2.2
13.1 ± 3.5
>100
25.2 ± 5.5
13.2 ± 3.6
15.4 ± 1.9
46.7 ± 6.9
12.2 ± 2.3
11.8 ± 1.8
13.1 ± 2.4
12.2 ± 3.7
w
x
B-9-3
Cisplatin
a
CH
3
22.3 ± 2.9
11.6 ± 0.7
>100
14.8 ± 0.4
13.2 ± 1.5
12.4 ± 0.7
40.6 ± 5.8
26.8 ± 1.4
14.5 ± 2.2
8.9 ± 0.6
6.1 ± 0.9
7.6 ± 0.4
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. The data represent the mean values ±SD of at least three independent determinations. Values >100 lM indicate less than 50% growth inhibition at >100 lM.
b
Cell lines include gastric carcinoma (BGC), liver carcinoma (HepG2), breast carcinoma (MCF-7), colon carcinoma (HT-29), esophageal carcinoma (Eca-109), and Lewis
lung carcinoma (LLC).

3
84
L. GUO ET AL.
9
When N -heterobivalent b-carbolines had the same linker, we tested tumor cell lines than 5u (except for HepG-2 cell line).
examined the influence of the substituents in position-1 of the Similarly, comparing the structure 5w with 5x, compound 5w dis-
b-carboline core on the cytotoxic activities. In order to enhance played the higher cytotoxic activities against the tested tumor cell
the range of substituents, we designed compounds have methyl lines than 5x (except for LLC cell line). These results suggested
and different pattern of substitution with an aryl ring substituted that the length of the spacer had no obvious relationship with
by electron withdrawing (Cl) and donating (OCH
ition-1 of b-carboline. For example, compound 5a, 5b, and 5k–5n,
3
) groups in pos- the cytotoxic activities against the tumor cell lines.
An overview of the cytotoxic activities data of all new synthe-
all have a 3-pyridyl group in R of one b-carboline ring, while in sized N -heterobivalent b-carbolines. 11 compounds were found
9
1
another b-carboline core, unlike compound 5a, the substituted to exhibit selective activity against LLC cell lines with IC50 value of
group of the position-1’ were methyl (5b), 4-methoxyphenyl (5k), lower than 20 lM. Moreover, Compound 5b, 5s displayed higher
3
,4-dimethoxyphenyl (5l), 3,4,5-trimethoxyphenyl (5m), 2-chloro- cytotoxic activities against at least four tumour cell lines than the
phenyl (5n), respectively. Of these six dimeric b-carbolines, the prototype B-9–3, and compound 5c, 5t, 5v, 5w exhibited higher
compound 5b displayed higher cytotoxic activities against BGC- cytotoxic effects against three tumour cell lines than B-9–3.
8
1
23, HepG2, MCF-7, HT-29, Eca-109 and LLC with IC50 values of
2.1, 15.9, 8.4, 12.6, 10.5, and 12.4 lM, respectively. Additionally, N -heterobivalent b-carbolines in vitro clearly suggest that: (1)
In summary, a total analysis of the cytotoxic activities of
9
9
1
7
among these six N -heterobivalent b-carbolines 5e, 5f, and 5o–5r C -methylation and C -methoxylation were favorable for increased
bearing a 2-thienyl group in R
1
of one b-carboline core, com- activities; (2) 3-Pyridyl or 2-thienyl group substituent into position-
pound 5f (R ’ ¼ methyl) showed the highest cytotoxic activities 1 of the b-carboline core, and the aryl (electron withdrawing and
1
against the test cell lines except HepG2. These results suggested donating groups) substituent into position-1’ of another b-carbo-
that 3-pyridyl or 2-thienyl group substituent into position-1 of the line ring might be detrimental to cytotoxic effects of this class
b-carboline core, and in another b-carboline ring, the methyl sub- of compounds.
stituent into R
stituent into R
Next, we examined the influence of the spacer length of N -
1
’ facilitated cytotoxic potency, and the aryl sub-
1
’ might be detrimental to cytotoxic effects.
9
Assessment of acute toxicity
9
heterobivalent b-carbolines on cytotoxic activities. According the The LD50 values of the selected N -heterobivalent b-carbolines in
9
previous investigation, the synthesized N -heterobivalent b-carbo- mice after intraperitoneal (i.p.) administration are shown in
lines were connected at the indolo-N by an alkyl chain, which Table 2. All of the tested dimeric b-carbolines resulted in acute
ranged from 4 to 6 carbon atoms. Comparing the structure of toxic manifestation but they did not cause any obvious neurotoxic
5
b–5d, which bearing different carbon atoms linker and 3-pyridyl effects, including tremors, twitch, jumping, and supination. The
group in position-1 of the b-carboline core, compound 5b bearing animals showed a decrease in locomotive activity after the admin-
-carbon atoms showed a broad spectrum of cytotoxic activities istration of various bivalent b-carbolines. Death occurred mostly in
4
with IC50 value of lower than 20 lM against six tumor cell lines, the high dosage group within 4–8 h after injection. All of the sur-
and all the three compounds show selective cytotoxicities with viving animals returned to normal within the next day. Autopsies
IC50 value of lower than 20 lM against LLC. Similarly, further intro- of the animals that died during the course of experiment and the
duction of 2-thienyl group in position-1 of the b-carboline ring necropsy findings in the surviving animals at the end of the
resulted in dimeric derivatives 5f–5h. Among these derivatives, experimental period (14 days) revealed no obvious changes in any
the IC50 values of compound 5f against BGC, MCF7, and LLC cells of the organs.
is 38.8, 46.6, and 6.1 lM, respectively, compound 5 g displayed
Of all of the investigated asymmetric dimeric b-carbolines,
IC50 values for HepG2 and HT-29 of 18.8 and 32.3 lM, respectively, compounds 5b and 5c, which had no substituent in position-7,
and compound 5h only exhibited higher cytotoxic effects with and bearing different carbon atoms linker and 3-pyridyl group in
IC50 value of 30.2 lM against Eca-109 than other two compounds. position-1 of the b-carboline core, demonstrated weaker acute
ꢂ1
ꢂ1
Comparing the structure of 5i with 5j, compound 5i showed toxicities with the LD50 values of 150 mg kg and 175 mg kg
,
weak or moderate cytotoxic activities against all tumor cell lines respectively. Compounds 5t and 5w, which have a harmine
tested, and compound 5j was selectively active against Eca-109 molecular linked with another 1-substituted b-carboline ring, dis-
and LLC cell lines with IC50 value of lower than 20 lM but fail to played remarkable acute toxicity with the LD values of 35 mg
50
ꢂ1
ꢂ1
show cytotoxic effects in BGC and HepG2 at the concentration of kg and 50 mg kg , respectively. These results suggested that
00 lM. Comparing the structure of 5u with 5v, there is an extra the methoxy substituent in position-7 of b-carboline nucleus
1
methoxyl group attached to position-7 of the b-carboline, com- played
a
vital role in determining the remarkable neuro-
pound 5v demonstrated the higher cytotoxic activities against the toxic effects.
9
Table 2. Acute toxic effects of N -heterobivalent b-carbolines in mice and antitumor activities of these
compounds against mice bearing Sarcoma 180 and Lewis lung cancer
a
Acute toxicity
Tumor inhibition rate (%)
ꢂ1
ꢂ1
Comp.
LD50(mg kg
)
Neurotoxic effect Dosage (mg kg
)
Sarcoma 180 Lewis lung cancer
b
5
5
5
5
b
c
t
150
175
35
50
200
–
–
þ
þ
–
30
35
7
43.6 ± 8.9
33.6 ± 9.2
37.2 ± 4.4
47.1 ± 6.2
56.2
41.9 ± 5.3
ND
ND
42.3 ± 5.9
40.4
c
w
10
40
30
2
0
B-9-3
CTX
a
82.5 ± 3.4
80.7 ± 2.1
Data are expressed as mean ± SD.
b
Acute neurotoxic manifestation were denoted by “þ” and “–”. “þ” represents toxic responses including
tremble, twitch, jumping and supination, while “–” means no such reaction.
c
ND ¼ not determined.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
385
Figure 2. Compounds 5b and 5w inhibited angiogenesis. (A) In vivo anti-angiogenic effect of compounds 5b and 5w in chick chorioallantoic membrane (CAM) assay.
positive control (CA4P, 0.5–50 mg/mL) and vehicle control (0.1% DMSO). (B) Quantification graphs of the inhibitory effects of compounds 5b and 5w on angiogenesis
ꢃ
ꢃꢃ
and migration. p < 0.05, p < 0.01.
9
Evaluation of antitumor activity of N -heterobivalent
b-carbolines in vivo
same substituent and different carbon atoms linker. Compound
5b, which bearing 4-carbon atoms, exhibited the potent antitumor
agent with the tumor inhibition rate of 43.6 and 41.9% against
Sarcoma 180-bearing mice and Lewis lung cancer-bearing mice,
respectively, and compound 5c displayed moderate antitumor
Based on the in vitro assay results, we further tested the antitumor
activity of four N -heterobivalent b-carbolines in vivo against mice
9
bearing Sarcoma 180 and Lewis lung cancer, respectively, and the
reference drugs Cyclophosphamide (CTX). Our previous investiga-
tion demonstrated that mice bearing Lewis lung cancer were
more susceptible to b-carbolines than other animal models; there-
fore, these animal models were selected and evaluated in the pre-
activity with the tumor inhibition rate of 33.6% against Sarcoma
ꢂ1
1
80-bearing mice at doses of 35 mg kg . Compound 5t, the har-
mine and harman linked with five methylene units, showed mod-
erate antitumor activity against mice with Sarcoma 180 with the
ꢂ1
5
tumour inhibition rate of 37.2% at doses of 7 mg kg
.
sent investigation. The tumor inhibition rates of all of the
Particularly, compound 5w was found to be the remarkable anti-
tumor agent with the tumor inhibition rate of 47.1 and 42.3%
against mice bearing Sarcoma 180 and Lewis lung cancer, respect-
investigated asymmetric dimeric b-carbolines were summarized in
Table 2.
9
As shown in Table 2, all the tested N -heterobivalent b-carbo-
lines displayed moderate to strong antitumor activities in animal ively. These results implied that the harmine or harman linked
model. Interestingly, compounds 5b and 5c, which having the with another of b-carboline nucleus (3-pyridyl or 2-thienyl sub-
stituent into position-1) enhanced their antitumor activities.

3
86
L. GUO ET AL.
In vivo anti-angiogenic effect of compounds 5b and 5w
4. Cao RH, Guan XD, Shi BX, et al. Design, synthesis and 3D-
QSAR of beta-carboline derivatives as potent antitumor
agents . Eur J Med Chem 2010; 45:2503–15.
The most potent compounds, 5b and 5w, were selected to evaluate
anti-angiogenic activity by chicken chorioallantoic membrane (CAM)
assay. The inhibitory effects of compounds 5b and 5w on the angio-
genesis of CAM are shown in Figure 2(A). The anti-angiogenetic activ-
ity of compounds 5b and 5w were semi-quantitatively analyzed
using Graph Pad Prism 5.0 (shown in Figure 2(B)). The results showed
that compound 5b (p< 0.05) could inhibit the angiogenesis of CAM.
The anti-angiogenetic activity of compound 5b was comparable to
the CA4P in vivo CAM assay at the same dose (50 lM).
5
.
Cao RH, Fan WX, Guo L, et al. Synthesis and structure-activ-
ity relationships of harmine derivatives as potential antitu-
mor agents. Eur J Med Chem 2013; 60:135–43.
6
.
Shankaraiah N, Siraj KP, Nekkanti S, et al. DNA-binding affin-
ity and anticancer activity of b-carboline–chalcone conju-
gates as potential DNA intercalators: molecular modelling
and synthesis. Bioorg Chem 2015; 59:130–9.
7
8
9
.
.
.
TAIRA Z, KANZAWA S, DOHARA C, et al. Intercalation of six
b-carboline derivatives into DNA. J Toxicol Environ Health
1
997; 43:83–91.
Cao RH, Peng WL, Chen HS, et al. DNA binding properties of
-substituted harmine derivatives. Biochem Biophys Res
Conclusions
9
In this study, we synthesized 24 new, N -heterodimeric b-carboline
9
derivatives and focused on compounds with 4 ꢂ 6 carbon linkers
between the indole nitrogen. All of the compounds were screened
for their in vitro cytotoxic activity against BGC-823, HepG2, MCF-7,
HT-29, Eca-109 and LLC cancer cell lines. The results showed that
compounds 5b, and 5w exhibited strong cytotoxic activities with
IC50 value of lower than 20 lM against the six tested tumor cell
lines. In addition, four asymmetric dimeric b-carbolines were
selected for evaluation in vivo against mice bearing Sarcoma 180
and Lewis lung cancer, compounds 5b and 5w exhibited potent
antitumor efficacies with tumour inhibition rate of over 40% in
the tested animal models. Moreover, the pharmacological mecha-
nisms showed that compound 5b could retard in the CAM assay,
and anti-angiogenetic potency was more potent than the refer-
ence drug CA4P. Preliminary structure-activity analysis indicated
Commun 2005; 338:1557–63.
Kamal A, Sathish M, Nayak VL, et al. Design and synthesis of
dithiocarbamate linked b-carboline derivatives: DNA topo-
isomerase II inhibition with DNA binding and apoptosis
inducing ability. Bioorg Med Chem 2015; 23:5511–26.
Figueiredo PO, Perdomo RT, Garcez FR, et al. Further
constituents of Galianthe thalictroides (Rubiaceae) and
inhibition of DNA topoisomerases I and IIa by its cytotoxic
b-carboline alkaloids. Bioorg Med Chem Lett 2014;
10.
2
4:1358–61.
1
1
1. Song YC, Kesuma D, Wang J, et al. Specific inhibition of cyc-
lin-dependent kinases and cell proliferation by harmine.
Biochem Biophys Res Commun 2004; 317:128–32.
2. Li Y, Liang FS, Jiang W, et al. DH334, a beta-carboline anti-
cancer drug, inhibits the CDK activity of budding yeast .
Cancer Biol Ther 2007; 6:1193–9.
1
7
that: (1) C -methylation and C -methoxylation were favorable for
increased activities; (2) 3-Pyridyl or 2-thienyl group substituent
into position-1 of the b-carboline core, and the aryl (electron with- 13. Zhang J, Li Y, Guo L, et al. DH166, a beta-carboline deriva-
drawing and donating groups) substituent into position-1’ of
tive, inhibits the kinase activity of PLK1. Cancer Biol Ther
another b-carboline ring might be detrimental to cytotoxic effects
of this class of compounds. Although most N -heterodimeric 14. Insaf F, Mohamed AB, Aymen BN, et al. Synthesis, cytotoxic,
2009; 8:2374–83.
9
b-carbolines presented here showed modest cytotoxic activities,
the investigations of these structural modifications and preliminary
SARs would be helpful to further design and develop more
potent compounds.
anti-lipoxygenase and anti-acetylcholinesterase capacities of
novel derivatives from harmine. J Enzyme Inhib Med Chem
2016;31:23–33.
15. Insaf F, Jalloul B, Mansour Z, et al. Synthesis of new isoxazo-
line derivatives from harmine and evaluation of their anti-
Alzheimer, anti-cancer and anti-inflammatory activities. J
Enzyme Inhib Med Chem 2015; 30:371–6.
Disclosure statement
1
6. Castro AC, Dang LC, Soucy F, et al. Novel IKK inhibitors:
b-carbolines. Bioorg Med Chem Lett 2003; 13:2419–22.
No potential conflict of interest was reported by the authors.
1
7. Posner GH, D'Angelo J, O'Neill PM, et al. Anticancer activity
of artemisinin-derived trioxanes. Expert Opin Ther Pat 2006;
Funding
1
6:1665–72.
This work was supported by the scientific research innovation pro-
ject in Xinjiang under Grant XJGRI2016052; and the Program for
Outstanding Youth Science and Technology Innovation Talents
Training in Xinjiang Uygur Region under Grant qn2015jc067.
1
8. Alagbala AA, McRiner AJ, Borstnik K, et al. Biological
mechanisms of action of novel C-10 non-acetal trioxane
dimers in prostate cancer cell lines. J Med Chem 2006;
4
9:7836–42.
1
9. Posner GH, McRiner AJ, Paik IH, et al. Anticancer and anti-
malarial efficacy and safety of artemisinin-derived trioxane
dimers in rodents. J Med Chem 2004; 47:1299–301.
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