Synthesis and in vitro antitumor activity of novel bivalent β-carboline-3-carboxylic acid derivatives with DNA as a potential target

Article abstract of DOI:10.3390/ijms19103179

A series of novel bivalent β-carboline derivatives were designed and synthesized, and in vitro cytotoxicity, cell apoptosis, and DNA-binding affinity were evaluated. The cytotoxic results demonstrated that most bivalent β-carboline derivatives exhibited stronger cytotoxicity than the corresponding monomer against the five selected tumor cell lines (A549, SGC-7901, Hela, SMMC-7721, and MCF-7), indicating that the dimerization at the C³ position could enhance the antitumor activity of β-carbolines. Among the derivatives tested, 4B, 6i, 4D, and 6u displayed considerable cytotoxicity against A549 cell line. Furthermore, 4B, 6i, 4D, and 6u induced cell apoptosis in a dose-dependent manner, and caused cell cycle arrest at the S and G2/M phases. Moreover, the levels of cytochrome C in mitochondria, and the expressions of bcl-2 protein, decreased after treatment with β-carbolines, which indicated that 6i and 6u could induce mitochondria-mediated apoptosis. In addition, the results of UV-visible spectral, thermal denaturation, and molecular docking studies revealed that 4B, 6i, 4D, and 6u could bind to DNA mainly by intercalation.

Full text of DOI:10.3390/ijms19103179

International Journal of
Molecular Sciences
Article
Synthesis and In Vitro Antitumor Activity of Novel
Bivalent β-Carboline-3-carboxylic Acid Derivatives
with DNA as a Potential Target
Hongling Gu 1, Na Li 1, Jiangkun Dai 1, Yaxi Xi 1, Shijun Wang 1 and Junru Wang 1,2,
*
1
College of Chemistry and Pharmacy, Northwest A&F University, No. 22 Xinong Road,
Yangling 712100, China; honglingguw@163.com (H.G.); lnuk@nwsuaf.edu.cn (N.L.);
daijkun@hotmail.com (J.D.); mysteryxi@163.com (Y.X.); 444795415@nwafu.edu.cn (S.W.)
State key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China
2
*
Correspondence: wangjunru@nwafu.edu.cn; Tel.: +86-29-8709-2662
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 22 August 2018; Accepted: 25 September 2018; Published: 15 October 2018
Abstract: A series of novel bivalent
and in vitro cytotoxicity, cell apoptosis, and DNA-binding affinity were evaluated. The cytotoxic
results demonstrated that most bivalent -carboline derivatives exhibited stronger cytotoxicity
β-carboline derivatives were designed and synthesized,
β
than the corresponding monomer against the five selected tumor cell lines (A549, SGC-7901, Hela,
SMMC-7721, and MCF-7), indicating that the dimerization at the C3 position could enhance the
antitumor activity of
β
-carbolines. Among the derivatives tested, 4B, 6i, 4D, and 6u displayed
considerable cytotoxicity against A549 cell line. Furthermore, 4B
,
6i, 4D, and 6u induced cell apoptosis
in a dose-dependent manner, and caused cell cycle arrest at the S and G2/M phases. Moreover,
the levels of cytochrome C in mitochondria, and the expressions of bcl-2 protein, decreased after
treatment with β-carbolines, which indicated that 6i and 6u could induce mitochondria-mediated
apoptosis. In addition, the results of UV-visible spectral, thermal denaturation, and molecular
docking studies revealed that 4B, 6i, 4D, and 6u could bind to DNA mainly by intercalation.
Keywords: bivalent β-carbolines; antitumor; apoptosis; DNA-binding affinity; bcl-2
1. Introduction
β
-carboline alkaloids, originally isolated from Peganum harmala, are a class of natural and
synthetic indole alkaloids with a tricyclic pyrido[3,4-b]indole ring system [ ]. -carbolines have
multiple biological and pharmacological properties [ ], of which antitumor activity is the most
widely studied [ ]. Previous studies have shown that -carbolines exerted their antitumor activity
mainly through intercalating into DNA [ 13], inducing apoptosis, and inhibiting topoisomerase I
and II (Top I and II) [13 15], cyclin-dependent kinases (CDKs) [16 17], mitogen-activated protein kinase
(MAPK) [18], and I-Kappa-B kinase (IKK) [19]. Particularly, parent carbolines can be inserted into
DNA, which can further cause cell apoptosis [20 21]. The interaction between -carboline alkaloids and
DNA could cause changes in DNA conformation, which further affects DNA replication, transcription,
and repair [22 23]. Furthermore, the existing studies have demonstrated that -carboline could induce
HepG2 cells apoptosis by down-regulating bcl-2 expression [24].
1,
2
β
2–4
5–8
β
7,9–
–
,
,
β
,
β
DNA-targeted antitumor drugs, such as anthracyclines, acridines, and quinones, which exert
antitumor activity through DNA insertion, are considered to be one of the most effective drugs
in clinical applications [25]. These drugs exert antitumor activity mainly through non-covalent
and covalent interactions with the minor groove, major groove, or base pairs (intercalation) of the
DNA double helix. The discovery of antitumor drugs has focused on the development of new
Int. J. Mol. Sci. 2018, 19, 3179; doi:10.3390/ijms19103179
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2018, 19, 3179
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DNA-intercalating scaffolds, such as
β
-carboline derivatives [
7
]. Therefore, we expect to discover and
develop new antitumor agents by modifying these bioactive scaffolds with appropriate substituents.
Previous structure-activity relationships (SARs) have shown that the introduction of appropriate
substituents at the C1, C3, and N9 positions of the
activity and DNA-binding affinity [ 10 26
substituted at the C1 and C3 positions of
-carbolines, respectively, have exhibited potential antitumor
activity [12 28
30]. The introduction of methyl and benzyl substituents at the N9 position of
-carbolines could enhance the DNA affinity [10]. Further studies have demonstrated that dimerization
β
-carboline scaffolds could enhance antitumor
5
,7
,
,
,27]. It has also been reported that phenyl and heterocyclic
β
,
–
β
of small molecules by appropriate linkers could significantly improve the DNA-binding affinity. It has
been found that the dimerized compounds could bind to DNA by a bis-intercalation mode, causing
pronounced changes in DNA structure [31
position have been synthesized, and were found to be potential anti-Alzheimer agents [33]. In addition,
the synthesis and evaluation of bivalent
-carbolines linked by 3–10 methylene units at the N9 position
,32]. Bivalent β
-carbolines linked at the C6 position or N9
β
as antitumor agents have also been reported [34]. Our group has synthesized 25 bivalent β-carbolines
modified at the C1 (-CH3), C7 (-OCH3), and N9 positions (-CH3 and -CH2Ph2), and dimerized at the C2
position, and the results showed that the dimerization could significantly increase antitumor activity
of 6d, and 6e, which exhibit good antitumor activity, have
β
-carbolines. Moreover, compounds 4A
,
6b
,
been reported [5,35]. However, the SARs of the antitumor activity in vitro of bivalent β-carbolines
linked at the C3 position were rarely reported.
In this study, we designed and synthesized a series of novel β-carboline derivatives modified
at the N9 position and linked at the C3 position, and further investigated the antitumor activity and
DNA-binding affinity in vitro. We expected to discover novel
antitumor activity and DNA-binding affinity.
β-carboline derivatives with promising
2. Results
2.1. Chemistry
Using L-tryptophan as a raw material, N9 substituted
(monomers, 4A 4B 4C, and 4D) were synthesized mainly through the Pictet–Spengler (P–S) reaction,
esterification reaction, and 9-substitution reaction, as previously described [
36]. The N9 substituted
-carboline-3-carboxylic acid methyl esters prepared in the above steps were hydrolyzed and then
β-carboline-3-carboxylic acid methyl esters
,
,
5
,
β
reacted with dibromoalkane to obtain a series of bivalent
β
-carboline derivatives (dimers, 6a-6f
-carboline monomers
, 6m-6r, and 6s-6x) are shown in Schemes 1 and 2,
,
6g-6l
,
6m-6r, and 6s-6x) [37]. The synthetic routes and reaction conditions of
4C, and 4D) and dimers (6a-6f 6g-6l
β
(
4A 4B
,
,
,
respectively. In this study, we synthesized 3 novel monomers and 21 novel dimers, and evaluated their
antitumor activity.
Scheme 1. Synthesis of N9-substituted
Reagents and conditions: (
) H+, HCHO, room temperature (r.t.); (
xylene, reflux; (d) DMF, NaH, r.t., R1-Br.
β
-carboline-3-carboxylic acid methyl esters (4A
,
4B
,
4C, and 4D).
) Pd/C,
a
b
) SOCl2, CH3OH, reflux; (c

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Scheme 2. Synthesis of bivalent
β-carboline-3-carboxylic acid derivatives (6a-6f, 6g-6l, 6m-6r,
and 6s-6x). Reagents and conditions: (a) THF/CH3OH, OH–, r.t.; (b) DMF, K2CO3, BrR2Br, heat.
2.2. In Vitro Cytotoxicity Assay
The antitumor activity of novel β
-carboline derivatives modified at the N9 position and linked
at the C3 position have been evaluated in vitro against A549, Hela, SGC-7901, SMMC-7721, MCF-7,
and MRC5 cell lines using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay,
with the results expressed as IC50 Values. Previous reports have evidenced that β-carbolines substituted
with an alkoxycarbonyl or carboxyl at the C3 position, and with a short alkyl or benzyl at the N9
position, exhibited more significant antitumor activities against Hela cells [5]. In the present research
(Table 1), for all the cell lines tested, only the
(4D) at N9 position displayed hihest antitumor activity. For A549 cell line, among all the substituents at
the N9 position, it was found that the antitumor activity of p-methylbenzyl (4C, 28.06
M) substituent
was better than that of the o-methylbenzyl (4B, 39.20 M) substituent. Next, the o-fluorobenzyl side
chain with the lowest IC50 value (4D, 13.94 M) was considered to be the most beneficial to increase
antitumor activity at the N9 position. But the antitumor activities of bivalent
-carboline derivatives
(dimer, 6i, 6.93 M; 6u, 5.61 M) were apparently better than that of the corresponding monomer
4B, 39.20 M; 4D, 13.94 M), indicating that the dimerization was an effective modification for
improving the antitumor activity of -carbolines. The IC50 values of bivalent -carboline derivatives
were relatively lower when the linkers were short in length and odd in carbon numbers (6s, 6.12 M;
6u, 5.61
M). These results indicated that the introduction of o-fluorobenzyl at the N9 position and
the dimerization at the C3 position could significantly increase the antitumor activity of
-carbolines,
and they have better antitumor activity when the length of the linkers was four to six methylene units.
β-carboline monomer substituted with o-fluorobenzyl
µ
µ
µ
β
µ
µ
(
µ
µ
β
β
µ
µ
β

Int. J. Mol. Sci. 2018, 19, 3179
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Moreover, most compounds exhibited low cytotoxicities against the MRC5 cell line. Because of
the modest cytotoxicity of 6i (6.93 M) and 6u (5.61 M) against the A549 cell line, and in order to
µ
µ
compare the antitumor mechanisms between monomers and dimers, 4B, 6i, 4D, and 6u, the A549 cells
were selected to do the further investigation.
Table 1. The IC50 values (
derivatives (6a-6f 6g-6l
and MRC5 cell lines.
µM) of β-carboline monomers (4A, 4B, 4C, and 4D) and bivalent β-carboline
,
,
6m-6r, and 6s-6x) against A549, SGC-7901, Hela, SMMC-7721, MCF-7,
Substituents
R1 R2
IC50 (µM) Mean ± SD a
Compounds
A549 b
SGC-7901 b
Hela b
SMMC-7721 b
MCF-7 b
MRC5
4A
4B
4C
-
-
-
>80
>80
>80
>80
>80
35.60 ± 0.30
51.78 ± 0.10
39.20 ± 1.18
28.06 ± 0.13
42.31 ± 2.13
47.98 ± 0.09
31.28 ± 2.33
42.55 ± 0.98
>80
>80
>80
>80
35.35 ± 0.30
35.38 ± 0.01
32.57 ± 0.10
4D
6a
-
13.94 ± 0.07
24.27 ± 0.12
17.84 ± 0.66
17.76 ± 0.17
26.36 ± 1.33
17.89 ± 0.03
35.61 ± 1.33
25.73 ± 0.32
53.92 ± 2.02
73.45 ± 0.88
-(CH2)3-
-(CH2)4-
-(CH2)5-
-(CH2)6-
-(CH2)8-
6b
6c
6d
6e
6f
12.26 ± 0.04
17.65 ± 0.09
40.03 ± 0.15
50.32 ± 3.15
29.04 ± 0.06
18.33 ± 0.28
8.09 ± 0.25
6.93 ± 0.33
12.18 ± 0.06
37.36 ± 2.08
30.13 ± 0.37
15.48 ± 0.32
18.72 ± 0.07
58.41 ± 0.72
69.97 ± 0.13
51.24 ± 3.22
29.42 ± 1.03
17.75 ± 0.93
7.18 ± 0.01
26.26 ± 1.03
35.00 ± 2.09
39.26 ± 1.22
18.22 ± 0.52
18.15 ± 0.03
32.36 ± 0.02
53.97 ± 0.56
66.3 ± 1.02
17.99 ± 0.15
45.75 ± 0.55
9.88 ± 0.04
18.05 ± 1.03
36.69 ± 2.02
59.68 ± 1.52
66.53 ± 0.31
55.15 ± 0.03
33.49 ± 0.01
75.36 ± 0.89
57.35 ± 1.13
73.49 ± 8.06
24.71 ± 0.5
14.22 ± 0.28
16.68 ± 0.03
33.93 ± 0.52
69.66 ± 0.30
70.88 ± 2.33
76.85 ± 3.01
75.47 ± 2.32
>80
64.44 ± 0.09
39.34 ± 0.11
66.99 ± 0.10
>100
-CH2-C6H4-
CH2-
>80
>100
6g
6h
6i
-(CH2)3-
-(CH2)4-
-(CH2)5-
-(CH2)6-
-(CH2)8-
53.14 ± 2.28
76.73 ± 5.0
59.29 ± 5.86
64.49 ± 3.31
44.93 ± 2.99
70.76 ± 2.02
32.61 ± 0.10
50.78 ± 0.09
37.99 ± 0.10
41.0 ± 0.07
65.85 ± 0.07
37.66 ± 0.13
6j
6k
6l
-CH2-C6H4-
CH2-
6m
-(CH2)3-
19.84 ± 0.04
18.41 ± 0.31
17.95 ± 0.02
33.32 ± 0.03
28.43 ± 0.28
49.05 ± 0.12
6n
6o
6p
6q
6r
-(CH2)4-
-(CH2)5-
-(CH2)6-
-(CH2)8-
13.36 ± 0.35
11.23 ± 0.61
19.28 ± 0.02
23.65 ± 0.05
46.61 ± 0.03
37.79 ± 1.02
17.18 ± 0.03
17.84 ± 0.22
35.78 ± 0.82
65.93 ± 1.18
27.96 ± 0.99
18.76 ± 0.46
15.66 ± 0.33
45.21 ± 1.66
58.25 ± 2.98
42.72 ± 2.18
36.62 ± 0.12
26.75 ± 0.51
29.82 ± 0.93
>80
49.06 ± 2.23
49.37 ± 1.65
55.04 ± 2.22
43.05 ± 2.01
68.26 ± 1.25
26.39 ± 0.01
31.28±0.07
47.60 ± 0.02
42.06 ± 0.09
56.11 ± 0.01
-CH2-C6H4-
CH2-
6s
6t
-(CH2)3-
-(CH2)4-
-(CH2)5-
-(CH2)6-
6.12 ± 0.03
12.39 ± 0.59
5.61 ± 0.04
7.95 ± 0.06
8.06 ± 0.08
9.01 ± 0.19
5.86 ± 0.08
8.60 ± 0.13
13.77 ± 0.11
15.20 ± 0.32
14.63 ± 0.36
22.85 ± 1.36
14.71 ± 0.08
18.26 ± 0.18
16.68 ± 0.11
24.65 ± 0.06
25.20 ± 1.12
25.69 ± 1.21
36.03 ± 2.99
42.41 ± 2.08
22.82 ± 0.02
>100
6u
6v
39.89 ± 0.05
67.75 ± 0.11

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Table 1. Cont.
Substituents
IC50 (µM) Mean ± SD a
Compounds
A549 b
SGC-7901 b
Hela b
SMMC-7721 b
MCF-7 b
R1
R2
MRC5
6w
6x
-(CH2)8-
16.54 ± 0.33
29.98 ± 0.07
31.34 ± 1.88
44.57 ± 0.02
76.32 ± 3.36
60.78 ± 0.16
-CH2-C6H4-
CH2-
34.27 ± 0.05
15.39 ± 0.09
45.00 ± 0.16
39.61 ± 2.59
>80
>80
63.28 ± 0.10
16.33 ± 0.30
5-FU c
C4H3FN2O2
>80
>80
52.32 ± 2.06
57.69 ± 1.39
a
IC50 was the concentration when cell viability was reduced by 50% after treatment with
β
-carbolines for 48 h,
b
and all data were the mean values of three independent experiments. A549: lung carcinoma, SGC-7901: gastric
carcinoma, Hela: cervical carcinoma, SMMC-7721: liver carcinoma, MCF-7: breast carcinoma, MRC5: normal
lung cell. 5-FU (5-fluorouracil, which could insert into DNA, interfering with DNA synthesis) was used as a
c
positive control.
2.3. The Morphological Observation of Cell Apoptosis
Apoptotic morphology is one of the most intuitive and reliable ways to determine whether cells
are apoptotic [38]. The typical features of apoptosis include membrane blebbing, cell contraction,
chromatin condensation, and formation of apoptotic bodies [39]. To identify the typical features of
apoptosis, we performed Hoechst 33342/propidium iodide (PI) dual staining assay. As shown in
Figure 1, A549 cells in the control group were evenly dyed weak blue with clear edge. However,
after incubation with 6i
were stained with strong blue fluorescence and strong red fluorescence, and shrinkage, condensation,
or fragmentation was observed (Figure 1). These results indicated that the second series of dimer (6i
, 4D, and 6u for 48 h, the cell density decreased significantly, and the cells
)
and the fourth series (containing fluorine atom) of monomer (4D) and dimer (6u) could induce cell
apoptosis and further cell necrosis, while the second series of monomer (4B) could only induce cell
apoptosis in a dose-dependent manner.
Figure 1. Morphological observation was performed by Hoechst 33342/propidium iodide (PI) dual
staining. A549 cells were ◦treated with 4B
, 6i, 4D, and 6u at 8 µM for 48 h, and then stained with
Hoechst 33342 and PI at 37 C in dark conditions. The stained cells were observed under a fluorescence
microscope (Leica DM6B, Leica, Nussloch, Germany). Hoechst 33342 was used to stain the living and
apoptotic nuclei, and emitted blue fluorescence (upper). PI was used to stain the dead nuclei and
emitted red fluorescence (lower).
2.4. Cell Cycle Assay
In order to determine the cell cycle arrest induced by
iodide (PI) staining using flow cytometry. As shown in Figure 2 and Table 2, when the concentration of
4B 6i 4D, and 6u was 4 M, the percentages of cells in the G2/M phase increased to 46.96%, 42.40%,
35.46%, and 40.34% (control group 12.25%), respectively, indicating that 4B 6i 4D, and 6u could
induce cell cycle arrest at the G2/M phase in A549 cells. At 8 M, the percentages of cells in the S
phase of 4B and 6i increased to 45.81% and 53.30% (control group 27.6%), respectively, indicating
that 4B and 6i could exert antitumor activity by causing S and G2/M phase arrest. At 8 M of
β-carbolines, we performed propidium
,
,
µ
,
,
µ
µ

Int. J. Mol. Sci. 2018, 19, 3179
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4D and 6u, the percentages of cells in the G1 phase increased to 88.01% and 81.62% (control group
60.15%), respectively, indicating that they could exert antitumor activity by causing G1 and G2/M
phase arrest. Moreover, at 8 µM, the percentages of cells in sub-G1 by 4B, 6i, 4D, and 6u increased to
4.15%, 21.32%, 46.29%, and 44.92% (control group 1.78%), respectively, indicating that they could also
induce apoptosis.
Figure 2. The cell cycle was detected by flow cytometry using PI single staining. A549 cells were
◦
treated with 4B
,
6i,
4D, and 6u at 2, 4, and 8
µ
M for 48 h, and then stained with PI for 30 min at 37 C.
The proportions of cells containing different DNA content were analyzed by FCS Express 5.0 (De Novo
Software, Thornhill, Canada).
Table 2. The percentage of cell cycles of A549 cells at indicated compound concentrations (4B
, 6i, 4D,
and 6u at 2, 4, and 8 µM).
Concentration
Percentage of Cell Cycles (%)
Compounds
4B
(µM)
SubG1
G1
S
G2/M
2
4
8
6.56
10.49
4.15
59.38
43.06
52.09
25.16
9.98
45.81
15.46
46.96
2.1
2
4
8
6.86
26.33
21.32
57.38
45.19
46.64
32.08
12.41
53.30
10.54
42.40
0.06
6i
2
4
8
6.41
39.03
46.29
56.89
47.21
88.01
24.17
17.33
11.69
18.94
35.46
0.3
4D
2
4
8
6.84
46.25
44.92
56.24
47.06
81.62
23.79
12.60
0.08
19.97
40.34
18.30
6u
Control
1% DMSO
1.78
60.15
27.6
12.25
2.5. Cell Apoptosis Assay
To better understand the apoptosis induced by β-carboline derivatives, we further performed
Annexin V-FITC/PI dual staining assay using flow cytometry. The transfer of phosphatidylserine from
the cell inner membrane to the cell outer membrane is considered as a potential marker of cell apoptosis.

Int. J. Mol. Sci. 2018, 19, 3179
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Moreover, Annexin V is a cellular protein which selectively binds phosphatidylserine [40
,
41]. As shown
in Figure 3 and Table 3, after treatment with 8
of early apoptosis were 8.40%, 4.24%, 5.28%, and 7.65%, respectively. And the percentages of late
apoptosis or necrosis were 69.65%, 83.24%, 77.09%, and 83.52%, respectively, indicating that 4B 6i 4D,
µM of 4B, 6i, 4D, and 6u for 48 h, the percentages
,
,
and 6u could induce significant apoptosis compared with the control group (early apoptosis: 4.22%;
late apoptosis or necrosis: 7.44%). To sum up, the results of cell cycle and apoptosis experiments
demonstrated that 4B, 6i, 4D, and 6u could exert antitumor activity by inducing cell cycle arrest at the
S or G2/M phase and significant cell apoptosis.
Figure 3. The cell apoptosis was detected by flow cytometry using Annexin V-FITC/PI dual staining.
A549 cells were treated with 4B
V-FITC and PI for 15 min at 37 C. The proportions of the living cells, the early apoptosis cells, and the
late apoptotic cells were analyzed by FCS Express 5.0.
,
6i
,
4D, and 6u at 2, 4, and 8
µ
M for 48 h, and then stained with Annexin
◦
Table 3. The percentage of cell apoptosis of A549 cells at indicated compound concentrations (4B
4D, and 6u at 2, 4, and 8 µM).
, 6i,
Percentage of Cell Apoptosis (%)
Concentration
Comp.
Normal Living Cell
Early Apoptosis
Late Apoptosis and Necrosis
(µM)
2
4
8
75.93
48.59
21.95
3.18
15.35
8.40
20.89
36.06
69.65
4B
6i
2
4
8
72.12
44.94
12.52
6.85
12.65
4.24
21.03
42.41
83.24
2
4
8
73.94
47.93
17.63
7.27
6.80
5.28
18.78
45.26
77.09
4D
2
4
8
66.05
32.53
8.83
12.14
6.54
7.65
21.81
60.93
83.52
6u
Control
1% DMSO
88.34
4.22
7.44

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2.6. The Detection of Apoptosis-Related Protein (Cytochrome C, bcl-2)
A large number of studies have found that the release of cytochrome C (Cyt C) from mitochondria
to cytoplasm, and the decrease in expression of bcl-2 protein, are important features of apoptosis [42 43].
,
As shown in Figure 4, the levels of Cyt C (in mitochondria) and the expression of bcl-2 protein decreased
in a dose-dependent manner after treatment with 6i and 6u. The results demonstrated that 6i and 6u
could induce mitochondria-mediated apoptosis.
Figure 4. Western blot analysis of cytochrome C and bcl-2 proteins. A549 cells were treated with with
6i and 6u at 2, 4, and 8 µM for 48 h. β-actin was used as an internal control.
2.7. DNA Binding Studies
The binding mode and binding strength of β-carboline derivatives (4B, 6i, 4D, and 6u) to DNA
were studied through UV-visible spectral, thermal denaturation, and molecular docking studies.
2.7.1. UV-Visible Spectral Study
In order to reveal the binding mode of
spectra of the interactions of -carbolines (4B
investigated (Figure 5). The absorbance at 325 nm of 4B
with red shift (4B-15 nm, 6i-18 nm, and 6u-20 nm), indicating that 4B
β
-carboline derivatives to DNA, the UV absorption
4D, and 6u) with calf thymus DNA (CT DNA) were
6i 4D, and 6u was gradually decreased
6i, and 6u could bind to DNA
β
, 6i,
,
,
,
by intercalation. Among them, the intercalation ability of 4D was very weak (may be a reasonable
experimental error), and it may interact with DNA through other intermolecular forces.
Figure 5. UV-visible spectroscopy study of β-carbolines with calf thymus (CT) DNA. UV-visible
absorption titrations were done by adding 200 nM CT DNA solution each time to the quartz cuvette
containing 10 µM 4B, 6i, 4D, and 6u. The absorption spectra were recorded from 200 nm to 350 nm.

Int. J. Mol. Sci. 2018, 19, 3179
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2.7.2. Thermal Denaturation Study
The binding affinity of
β
-carboline derivatives (4B
,
6i
,
4D, and 6u) to DNA were investigated
through thermal denaturation experiments. Small molecules interact with DNA through the
intercalation mode, which makes the DNA double helix more stable, thereby increasing the melting
temperature (Tm) [44]. Therefore, the thermal denaturation experiment provides a simple and effective
method for detecting DNA-binding affinity. As shown in Figure 6, the
were 7.5, 8.5, 3.5, and 9.0 ◦C, respectively, indicating that they could bind to DNA by intercalation,
and the binding strength of 4B 6i, and 6u was greater than that of 4D. Thus, thermal denaturation
results indicated that 4B, 6i, 4D, and 6u could exhibit significant DNA-binding affinity.
∆Tm values of 4B, 6i, 4D, and 6u
,
Figure 6. Thermal denaturation study of the complexes of β-carbolines (4B, 6i, 4D, and 6u) and
CT-DNA. Melting temperatures were measured in PBS-EDTA buffer (1 mM Na2HPO4, 0.1 mM EDTA,
4B,6i,4D, or 6u + DNA
PH 7.4) with a
∆TmDNA).
β
-carboline (4B
,
6i
,
4D, and 6u)/DNA ratio of 0.5.
∆
Tm = (
∆
Tm
−
2.7.3. Molecular Docking Study
In order to further predict the binding mode of
the Sybyl-X 2.0 package (Tripos, Princeton, NJ, USA) was used to perform molecular docking studies
between -carboline derivatives and DNA, to explore possible mechanisms of antitumor activity.
The structures and docking scores of the DNA- -carboline complex are shown in Figure 7. The total
scores of 4B 6i 4D, and 6u were 7.5936, 8.9892, 7.6925, and 8.6512, respectively, indicating that 4B 6i
β
-carboline to DNA, the Surflex-Dock program in
β
β
,
,
,
,
4D, and 6u could well insert into the DNA. Moreover, the scores of dimers (6i and 6u) were higher than
that of monomers (4B and 4D), indicating that the dimers have stronger DNA-binding ability than the
corresponding monomers, thereby exerting better antitumor activity. In addition, the planar structure
of
bond (non-classical hydrogen bond), and the
fluorine bond between DNA and 6u might support its higher antitumor activity.
β
-carbolines (4B
,
6i, and 4D) could interact with DNA through
π-π stacking, the carbon-hydrogen
π
-alkyl bond (hydrophobic force). The presence of the

Int. J. Mol. Sci. 2018, 19, 3179
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Figure 7. Three-dimensional conformations of the complexes of β-carbolines (4B, 6i, 4D, and 6u)
docked with DNA.
3. Discussion
β
-carboline alkaloids are a class of natural and synthetic indole alkaloids with a wide range of
pharmacological activities, including antitumor, antiviral, antiparasitic, and antibacterial activities [ ].
Particularly, previous studies have shown that -carboline alkaloids exerted antitumor activity through
insertion into DNA [10 12], inducing apoptosis [13 45], inhibiting CDKs [16], and topoisomerase I
and II activity [13 -carbolines could significantly
15]. The substitution at the C3 and N9 positions of
increase their DNA insertion ability [ ]. Moreover, the existing studies have found that dimerization
of small molecules could significantly increase [26].
DNA binding affinity [31 32]. Therefore, designing and synthesizing DNA-targeted
derivatives is an important way to find potentially effective antitumor drugs. Here, we synthesized a
series of bivalent
-carboline derivatives modified at the N9 position and dimerized at the C3 position,
5,6
β
–
,
,
β
7
,
β-carboline
β
and evaluated their antitumor activity and DNA binding affinity in vitro. The IC50 values of 4B
, 6i,
4D, and 6u against A549 were 39.20, 6.39, 13.94, and 5.61 µM, respectively.
Previous studies have shown that the antitumor activity of
β
-carboline could be significantly
35]. In this
improved with benzyl substituted at the N9 position and dimerized at the C3 position [
5
,
study, the monomers exhibited good-to-strong antitumor activity with the tendency of o-fluorobenzyl >
p-methylbenzyl > o-methylbenzyl > benzyl. Furthermore, for the dimers of o-methylbenzyl substituted
at the N9 position, most dimers were generally more active than the corresponding monomers,
following the tendency of 5 > 4 > 6 > 3 > 8 methylene units. For the dimers of o-fluorobenzyl
substituted at the N9 position, most dimers were generally more active than the corresponding
monomers, following the tendency of 5 > 3 > 6 > 4 > 8 methylene units. These results indicated that
N9-benzyl substitution and C3-dimerizition with 5–6 methylene units were favorable for increasing
antitumor activity, which was consistent with the results of previous studies.
Previous studies have shown that cell cycle arrest and cell apoptosis are the main mechanisms
used by
the introduction of substituents (a methyl or benzyl group) at the N9 position and the dimerization
of -carbolines could significantly increase the antitumor activity, and could cause cell cycle arrest
at the S or G2/M phase and induce apoptosis in a dose-dependent manner [36 37]. In the present
study, we found that the cell cycle arrested at the S and G2/M phases with sub G1 peaks, and induced
β-carboline derivatives as antitumor drugs [4,46]. Our previous studies have shown that
β
,

Int. J. Mol. Sci. 2018, 19, 3179
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apoptosis after treatment with 4B
,
6i
,
4D, and 6u. Furthermore, the reduction of cytochrome C
(Cyt C) in mitochondria, and the decrease in expression of bcl-2 protein, are the typical features of
mitochondria-mediated apoptosis [47]. Additionally, the results of western blot showed that the levels
of Cyt C (in mitochondria) and the expression of bcl-2 protein decreased in a dose-dependent manner
after incubation with 6i and 6u. Taken together, these results revealed that 6i and 6u could induce cell
apoptosis through a mitochondria-mediated pathway.
To date, DNA intercalators have been effective antitumor drugs in clinical application. After the
drugs are inserted into DNA, they can cause conformational changes of the double helix, which
disrupts DNA replication, transcription, and repair [23]. The antitumor activity of a large number
of natural and synthetic
been discovered and evaluated. Furthermore, the interactions between these derivatives (4B
and 6u) and DNA were investigated by UV-visible spectroscopy, thermal denaturation, and molecular
β
-carboline derivatives that could interact with DNA by intercalation have
,
6i, 4D,
modeling studies. The absorbance of 4B
,
6i
,
4D, and 6u decreased with red shift, and the
∆
T
m values of
◦
4B 6i
,
,
4D, and 6u were 7.5, 8.5, 3.5, and 9.0 C, respectively, indicating that 4B
,
6i, and 6u could stably
bind to DNA by intercalation, which is in agreement with the results of molecular docking studies.
In conclusion, a series of novel bivalent -carboline-3-carboxylic acid derivatives were synthesized,
β
and antitumor activity and DNA-binding affinity were preliminarily evaluated in vitro. The SARs
studies revealed that the introduction of appropriate substituents at the N9 position and dimerization
at the C3 position of
β
-carbolines could significantly increase the antitumor activity against A549, Hela,
SGC-7901, SMMC-7721, and MCF-7 cell lines. Furthermore, 4B 6i 4D, and 6u could further induce
tumor cell cycle arrest and apoptosis, and could interact with DNA through intercalation. In summary,
further studies of 4B 6i 4D, and 6u could be performed to develop potential DNA-targeted agents in
clinical applications.
,
,
,
,
4. Experimental Section
4.1. Materials and Methods
All chemicals and solvents used in the synthesis were purchased from suppliers. They were dried
and purified when needed. Melting points were determined on a capillary melting apparatus and were
uncorrected (SGW X-4, Shanghai Shenguang Instrument Co., Ltd., Shanghai, China). The 1H-NMR
and 13C-NMR spectra were recorded on a Bruker Avance III at 500 MHz using tetra methyl silane
(TMS) as the internal standard. The column chromatography and analytical thin layer chromatography
(TLC) were performed with silica gel (100–200 mesh) and silica gel GF254 (Qingdao Marine Chemical
Company, Qingdao, China).
All cell lines (A549, SGC-7901, Hela, SMMC-7721, and MCF-7) were purchased from the American
Type Culture Collection (Manassas, VA, USA), and were cultured in Dulbecco’s modified Eagle’s
medium (DMEM, HycloneLaboratories Inc., Logan, Utah, USA) supplemented with 10% fetal bovine
serum (FBS, Gibco&Invitrogen, Carlsbad, CA, USA) at 37 ◦C and 5% CO2.
4.2. Chemistry
4.2.1. General Synthesis Procedure for N9 Substituted β-Carboline-3-carboxylic Acid Methyl Esters
(4A, 4B, 4C, and 4D)
Using L-tryptophan as the raw material, β-carboline monomers (4A, 4B, 4C, and 4D) were
synthesized through the Pictet–Spengler reaction (P–S reaction), esterification reaction, oxidation
reaction, and N9 substitution reaction, as previously described [
5,26,36]. Reaction routes and conditions
are shown in Scheme 1.

Int. J. Mol. Sci. 2018, 19, 3179
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4.2.2. General Reaction of Synthesis of Bivalent β-Carboline Derivatives (6a-6f, 6g-6l, 6m-6r,
and 6s-6x)
The N9 substituted
in the above steps were hydrolyzed and reacted with dibromoalkane to obtain a series of
bivalent -carboline-3-carboxylic acid derivatives (6a-6f 6g-6l 6m-6r, and 6s-6x), as previously
β-carboline-3-carboxylic acid methyl esters (4A, 4B, 4C, and 4D) prepared
β
,
,
described [34,35,37,48]. Reaction routes and conditions are shown in Scheme 2.
4.3. MTT Assay
The cytotoxic activities of
method, as previously described [49]. Cells (1
Guangzhou, China) were incubated with DMEM containing various concentrations (2.5, 5, 10, 20,
40, and 80 M) of -carbolines (4A 4B 4C 4D 6a-6f 6g-6l
6m-6r, and 6s-6x) for 48 h at 37 ◦C and
5% CO2. Then, 10 L of 5 mg/mL MTT reagent dissolved in PBS (Phosphate buffered saline) was
added per well, and plates were incubated at 37 ◦C for 4 h. The medium was carefully removed and
100 L dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance
β
-carboline-3-carboxylic acid derivatives were investigated by the MTT
×
104 cells per well) seeded into 96-well plates (JET,
µ
β
,
,
,
,
,
,
µ
µ
was measured at 490 nm with a microplate reader (ThermoFisher Scientific Inc., Waltham, MA, USA).
◦
The
β
-carbolines were dissolved in DMSO as stock solutions (10 mM) at
−
20 C, and diluted in
the final experimental solution using DMEM before use. A549 (lung carcinoma), SGC-7901 (gastric
carcinoma), Hela (cervical carcinoma), SMMC-7721 (liver carcinoma), and MCF-7 (breast carcinoma)
cell lines were used for the antitumor activity screening. The IC50 values were the mean values of three
independent experiments.
4.4. Hoechst 33342/PI Dual Staining Assay
The morphological observation of apoptosis was performed by Hoechst 33342 and propidium
iodide (PI) dual staining according to the manufacturer’s instructions. A549 cells (2
×
104 cells per
well) seeded into 24-well plates (JET, Guangzhou, China) were incubated with DMEM containing
4
µ
M of
β
-carbolines (4B
,
6i
,
4D◦, and 6u) for 48 h at 37 ◦C and 5% CO2. Then, the cells were stained
by Hoechst 33342 and PI at 37 C for 30 min in dark conditions. Finally, cells were observed and
photographed with a fluorescence microscope (LeicaDM6B, Leica, Nussloch, Germany).
4.5. Cell Cycle Assay
The cell cycle was determined by PI single staining. A549 cells (1
6-well plates (JET, Guangzhou, China) were incubated with DMEM containing various concentrations
×
106 cells per well) seeded into
◦
(2, 4, and 8
µ
M) of
β
-carbolines (4B
,
6i
,
4D, and 6u) for 48 h at 37 C and 5% CO2. The cells
were harvested, washed with pre-cooled PBS (4 ◦C), and fixed with pre-cooled 90% ethanol (4 ◦C).
Then, the cells were incubated with 20 µL RNAse (30 µg/mL) and 20 µL PI (50 µg/mL) (Sigma Aldrich,
Saint Louis, MO, USA) for 30 min at 37 ◦C. The samples were analyzed using flow cytometry (BD
FACSCalibur, San Jose, CA, USA).
4.6. Cell Apoptosis Assay
The cell apoptosis was evaluated using Annexin V-FITC/PI dual staining (Beyotime, China).
A549 cells (1
×
106 cells per well) seeded into 6-well plates (JET, Guangzhou, China) were incubated
with DMEM containing various concentrations (2, 4, and 8
µM) of β-carbolines (4B, 6i, 4D, and 6u) for
48 h at 37 ◦C and 5% CO2. The cells in the supernatant and adherent were collected and washed with
PBS, and then the cells were stained with Annexin V-FITC and PI for 20 min at r.t. The samples were
analyzed using flow cytometry (Becton Dickinson, San Jose, CA, USA).

Int. J. Mol. Sci. 2018, 19, 3179
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4.7. Western Blot Assay
The level of cytochrome C (Cyt C) in mitochondria, and the expression of bcl-2 protein, were
evaluated by western blot. A549 cells (1
106 cells per well) seeded into 6-well plates (JET, Guangzhou,
×
China) were incubated with DMEM containing various concentrations (2, 4, and 8
(
µ
M) of
β
-carbolines
◦
6i and 6u) for 48 h at 37 C and 5% CO2. Then, the cells were lysed with RIPA lysis buffer for
30 min on ice (containing 1% PMSF), centrifuged at 12,000
◦
×
g for 5 min at 4 C, and then the
supernatants were transferred to 1.5 mL EP tubes. Proteins were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on polyvinylidene difluoride
(PVDF) membranes (Millipore, Burlington, MA, USA), as previously described [50]. The membranes
were blocked for 2 h at r.t. with 5% non-fat milk in TBST buffer (20 mM Tris-HCl,150 mM NaCl
and 0.05% Tween-20, pH7.4), and then incubated with the mouse anti-bcl-2 monoclonal antibody
(Sigma, Saint Louis, MO, USA), anti-cytochrome C monoclonal antibody (Beyotime, Haimen, China),
◦
and anti-
β
-actin antibody (1:4000 dilution; Abcam, Cambridge, MA, USA) overnight at 4 C, and finally
incubated with the peroxidase conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories,
West Grove, PA, USA) at 37 ◦C for 1 h. Immunostained proteins were visualized using ECL reagent
according to the manufacturer’s instructions (Pierce, Rockford IL, CA, USA).
internal control.
β-actin was used as an
4.8. UV-Visible Spectral Study
UV-visible spectroscopy was carried out to determine the binding mode of
to DNA (CT DNA) using UV-2500 spectrophotometer (Shimadzu, Kyoto, Japan) at 25 C. Stock
β
-carboline derivatives
◦
solutions of 10 mM β-carboline derivatives were dissolved in DMSO, and 10 µM CT DNA was
prepared in 100 mM KBPES buffer (30 mM Potassium Phosphate with 100 mM KCl, pH 7.0) [13].
UV-visible absorption titrations were performed by adding 200 nM CT DNA solution to the quartz
cuvette containing approximately 10
recorded from 200 nm to 350 nm.
µM derivative solutions. UV-Visible absorption spectra were
4.9. Thermal Denaturation Study
The DNA-binding modes of the
β-carboline derivatives (4B, 6i, 4D, and 6u) with CT DNA were
examined by DNA thermal denaturation experiments, as previously described [10]. The solutions
containing complexes of CT DNA and 4B
and the absorbance was measured in 1 C steps from 30 C to 95 C at 260 nm (At) by a UV-Vis
spectrophotometer (A is the absorbance of the solution at 260 nm at different temperatures, t is the
,
◦ 6i
,
4D, or 6u wer◦e heated using a thermostatic water bath,
◦
temperature). The experiments were performed in PE buffer (pH = 7.4) with a
β
-carboline/DNA ratio
(DNA alone)
of 0.5. The ∆Tm value of DNA alone is 56.8 ◦C. ∆Tm = ∆Tm
− ∆Tm
.
(DNA + 4B, 6i, 4D, or 6u)
4.10. Molecular Modeling Study
Molecular docking software (Sybyl-X 2.0 Tripos, Princeton, NJ, USA) and Discovery Studio 2017
client (BIOVIA, San Diego, CA, USA) was used to investigate the interaction of 4B 6i 4D, and 6u
,
,
with DNA. The DNA was retrieved from the Protein Data Bank (PDB entry code: 1Z3F). The crystal
structure of the DNA molecule was prepared by adding all the hydrogen atoms, and the charge was
added using the AMBER7 FF99 method. The structures of 4B, 6i, 4D, and 6u were drawn in the Sybyl-X
2.0 package (Tripos, Princeton, NJ, USA). Hydrogen atoms were added, and then the energy was
optimized using the Tripos force field and the Gasteiger–Hückel method. The Surflex-Dock scoring
function is a weighted sum of non-linear functions based on the binding affinities of DNA-ligand
complexes and their crystallographic structures.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/10/
3179/s1.

Int. J. Mol. Sci. 2018, 19, 3179
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Author Contributions: J.W. conceived and designed the experiments; H.G., N.L., J.D., Y.X. performed the
experiments; H.G. analyzed data and drafted the manuscript; S.W. edited and revised the manuscript. All authors
approved the final version of the paper.
Funding: This work was financially supported by the National Natural Science Foundation of China (Grant No.
81773603), the Open Foundation of Key Laboratory of Synthetic and National Foundation Molecule Chemistry of
the Ministry of Education (Northwest University, China), the State Key Laboratory of Drug Research (Grant No.
SIMM1705KF-09), the National Training Program on Undergraduate Innovation and Entrepreneurship (Grant No.
201803018, Northwest A&F University, China).
Acknowledgments: Chengbao Wang, from the college of veterinary medicine in the university provided some
technical support and materials in the biochemical experiments. H.W., an undergraduate from the innovative
experimental college in the university participated in some experiments within the present research.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Bluefarb, S.M. Peganum harmala. Int. J. Dermatol. 1980, 19, 535. [CrossRef]
Allen, J.R.F.; Holmstedt, B.R. The simple -carboline alkaloids. Phytochemistry 1980, 19, 1573–1582. [CrossRef]
Dai, J.; Dan, W.; Li, N.; Wang, J. Computer-aided drug discovery: Novel 3,9-disubstituted eudistomin U
β
derivatives as potent antibacterial agents. Eur. J. Med. Chem. 2018, 157, 333–338. [CrossRef] [PubMed]
4.
5.
Dai, J.; Dan, W.; Schneider, U.; Wang, J. β-Carboline alkaloid monomers and dimers: Occurrence, structural
diversity, and biological activities. Eur. J. Med. Chem. 2018, 157, 622–656. [CrossRef] [PubMed]
Cao, R.; Chen, Q.; Hou, X.; Chen, H.; Guan, H.; Ma, Y.; Peng, W.; Xu, A. Synthesis, acute toxicities, and
antitumor effects of novel 9-substituted
[CrossRef] [PubMed]
β-carboline derivatives. Bioorg. Med. Chem. 2004, 12, 4613–4623.
6.
Cao, R.; Chen, H.; Peng, W.; Ma, Y.; Hou, X.; Guan, H.; Liu, X.; Xu, A. Design, synthesis and in vitro
and in vivo antitumor activities of novel
β
-carboline derivatives. Eur. J. Med. Chem. 2005, 40, 991–1001.
[CrossRef] [PubMed]
7.
8.
Guan, H.; Chen, H.; Peng, W.; Ma, Y.; Cao, R.; Liu, X.; Xu, A. Design of β-carboline derivatives as
DNA-targeting antitumor agents. Eur. J. Med. Chem. 2006, 41, 1167–1179. [CrossRef] [PubMed]
Shen, Y.-C.; Chang, Y.-T.; Lin, C.-L.; Liaw, C.-C.; Kuo, Y.H.; Tu, L.-C.; Yeh, S.F.; Chern, J.-W. Synthesis of
1-Substituted Carbazolyl-1,2,3,4-tetrahydro-and Carbazolyl-3,4-dihydro-β-carboline Analogs as Potential
Antitumor Agents. Mar. Drugs 2011, 9, 256–277. [CrossRef] [PubMed]
9.
Taira, Z.; Kanzawa, S.; Dohara, C.; Ishida, S.; Matsumoto, M.; Sakiya, Y. Intercalation of Six β-Carboline
Deriyatives into DNA. Jpn. J. Toxicol. Environ. Health 1997, 42, 83–91. [CrossRef]
10. Cao, R.H.; Peng, W.L.; Chen, H.S.; Ma, Y.; Liu, X.D.; Hou, X.R.; Guan, H.J.; Xu, A.L. DNA binding properties
of 9-substituted harmine derivatives. Biochem. Biophys. Res. Commun. 2005, 338, 1557–1563. [CrossRef]
[PubMed]
11. Chen, Z.Y.; Cao, R.H.; Yu, L.A.; Shi, B.X.; Sun, J.; Guo, L.A.; Ma, Q.; Yi, W.; Song, X.A.; Song, H.C. Synthesis,
cytotoxic activities and DNA binding properties of beta-carboline derivatives. Eur. J. Med. Chem. 2010
45, 4740–4745. [CrossRef] [PubMed]
,
12. Shankaraiah, N.; Siraj, K.P.; Nekkanti, S.; Srinivasulu, V.; Sharma, P.; Senwar, K.R.; Sathish, M.;
Vishnuvardhan, M.V.P.S.; Ramakrishna, S.; Jadala, C.; et al. DNA-binding affinity and anticancer activity
of β-carboline–chalcone conjugates as potential DNA intercalators: Molecular modelling and synthesis.
Bioorg. Chem. 2015, 59, 130–139. [CrossRef] [PubMed]
13. Kamal, A.; Sathish, M.; Nayak, V.L.; Srinivasulu, V.; Kavitha, B.; Tangella, Y.; Thummuri, D.; Bagul, C.;
Shankaraiah, N.; Nagesh, N. Design and synthesis of dithiocarbamate linked β-carboline derivatives: DNA
topoisomerase II inhibition with DNA binding and apoptosis inducing ability. Bioorg. Med. Chem. 2015
23, 5511–5526. [CrossRef] [PubMed]
,
14. Deveau, A.M.; Labroli, M.A.; Dieckhaus, C.M.; Barthen, M.T.; Smith, K.S.; Macdonald, T.L. The Synthesis
of Amino-Acid Functionalized β-Carbolines as Topoisomerase II Inhibitors. Bioorg. Med. Chem. Lett. 2001,
11, 1251–1255. [CrossRef]
15. Sathish, M.; Kavitha, B.; Nayak, V.L.; Tangella, Y.; Ajitha, A.; Nekkanti, S.; Alarifi, A.; Shankaraiah, N.;
Nagesh, N.; Kamal, A. Synthesis of podophyllotoxin linked -carboline congeners as potential anticancer
agents and DNA topoisomerase II inhibitors. Eur. J. Med. Chem. 2018, 144, 557–571. [CrossRef] [PubMed]
β

Int. J. Mol. Sci. 2018, 19, 3179
15 of 16
16. Li, Y.; Liang, F.S.; Jiang, W.; Yu, F.S.; Cao, R.H.; Ma, Q.H.; Da, X.Y.; Jiang, J.D.; Wang, Y.C.; So, S. DH334,
a beta-carboline anti-cancer drug, inhibits the CDK activity of budding yeast. Cancer Biol. Ther. 2007
6, 1193–1199. [CrossRef] [PubMed]
,
17. He, L.; Liao, S.-Y.; Tan, C.-P.; Ye, R.-R.; Xu, Y.-W.; Zhao, M.; Ji, L.-N.; Mao, Z.-W.
Ruthenium–Arene– -Carboline Complexes as Potent Inhibitors of Cyclin-Dependent Kinase 1: Synthesis,
β
Characterization and Anticancer Mechanism Studies. Chem. Eur. J. 2013, 19, 12152–12160. [CrossRef]
[PubMed]
18. Trujillo, J.I.; Meyers, M.J.; Anderson, D.R.; Hegde, S.; Mahoney, M.W.; Vernier, W.F.; Buchler, I.P.; Wu, K.K.;
Yang, S.; Hartmann, S.J.; et al. Novel tetrahydro-β-carboline-1-carboxylic acids as inhibitors of mitogen
activated protein kinase-activated protein kinase 2 (MK-2). Bioorg. Med. Chem. Lett. 2007, 17, 4657–4663.
[CrossRef] [PubMed]
19. Castro, A.C.; Dang, L.C.; Soucy, F.; Grenier, L.; Mazdiyasni, H.; Hottelet, M.; Parent, L.; Pien, C.; Palombella, V.;
Adams, J. Novel IKK inhibitors: Beta-carbolines. Bioorg. Med. Chem. Lett. 2003, 13, 2419–2422. [CrossRef]
20. Hayashi, K.; Nagao, M.; Sugimura, T. Interactions of norharman and harman with DNA. Nucleic Acids Res.
1977, 4, 3679–3685. [CrossRef] [PubMed]
21. Uezono, T.; Maruyama, W.; Matsubara, K.; Naoi, M.; Shimizu, K.; Saito, O.; Ogawa, K.; Mizukami, H.;
Hayase, N.; Shiono, H. Norharman, an indoleamine-derived β-carboline, but not Trp-P-2, a γ-carboline,
induces apoptotic cell death in human neuroblastoma SH-SY5Y cells. J. Neural Transm. 2001, 108, 943–953.
[CrossRef] [PubMed]
22. Taira, Z.; Kanzawa, S.; Dohara, C.; Ishida, S.; Matsumoto, M.; Sakiya, Y. Intercalation of Six β-Carboline
Derivatives into DNA. Eisei kagaku 1997, 43, 83–91. [CrossRef]
23. Xiao, S.; Lin, W.; Wang, C.; Yang, M. Synthesis and biological evaluation of DNA targeting flexible side-chain
substituted β-carboline derivatives. Bioorg. Med. Chem. Lett. 2001, 11, 437–441. [CrossRef]
24. Chen, Q.; Chao, R.; Chen, H.; Hou, X.; Yan, H.; Zhou, S.; Peng, W.; Xu, A. Antitumor and neurotoxic effects
of novel harmine derivatives and structure-activity relationship analysis. Int. J. Cancer 2005, 114, 675–682.
[CrossRef] [PubMed]
25. Georgi, M.; Adriana, B.; Margarita, K. Novel Approaches Towards Development of Non-Classical
Platinum-Based Antineoplastic Agents: Design of Platinum Complexes Characterized by an Alternative
DNA-Binding Pattern and/or Tumor-Targeted Cytotoxicity. Curr. Med. Chem. 2005, 12, 2177–2191.
26. Cao, R.H.; Peng, W.L.; Chen, H.S.; Hou, X.R.; Guan, H.J.; Chen, Q.; Ma, Y.; Xu, A.L. Synthesis and in vitro
cytotoxic evaluation of 1,3-bisubstituted and 1,3,9-trisubstituted beta-carboline derivatives. Eur. J. Med. Chem.
2005, 40, 249–257. [CrossRef] [PubMed]
27. Chen, Z.Y.; Cao, R.H.; Shi, B.X.; Guo, L.; Sun, J.; Ma, Q.; Fan, W.X.; Song, H.C. Synthesis and biological
evaluation of 1,9-disubstituted beta-carbolines as potent DNA intercalating and cytotoxic agents. Eur. J.
Med. Chem. 2011, 46, 5127–5137. [CrossRef] [PubMed]
28. Formagio, A.S.N.; Tonin, L.T.D.; Foglio, M.A.; Madjarof, C.; de Carvalho, J.E.; da Costa, W.F.; Cardoso, F.P.;
Sarragiotto, M.H. Synthesis and antitumoral activity of novel 3-(2-substituted-1,3,4-oxadiazol-5-yl) and
3-(5-substituted-1,2,4-triazol-3-yl)
[PubMed]
β-carboline derivatives. Bioorg. Med. Chem. 2008, 16, 9660–9667. [CrossRef]
29. Savariz, F.C.; Foglio, M.A.; de Carvalho, J.E.; Ruiz, A.L.; Duarte, M.C.; da Rosa, M.F.; Meyer, E.;
Sarragiotto, M.H. Synthesis and evaluation of new beta-carboline-3-(4-benzylidene)-4H-oxazol-5-one
derivatives as antitumor agents. Molecules 2012, 17, 6100–6113. [CrossRef] [PubMed]
30. Shankaraiah, N.; Nekkanti, S.; Chudasama, K.J.; Senwar, K.R.; Sharma, P.; Jeengar, M.K.;
Naidu, V.G.M.; Srinivasulu, V.; Srinivasulu, G.; Kamal, A. Design, synthesis and anticancer evaluation
of tetrahydro-
[PubMed]
β-carboline-hydantoin hybrids. Bioorg. Med. Chem. Lett. 2014, 24, 5413–5417. [CrossRef]
31. Gaugain, B.; Barbet, J.; Capelle, N.; Roques, B.P.; Le Pecq, J.B.; Le Bret, M. DNA Bifunctional Intercalators.
2. Fluorescence Properties and DNA Binding Interaction of an Ethidium Homodimer and an Acridine
Ethidium Heterodimer. Biochemistry 1978, 17, 5078–5088. [CrossRef] [PubMed]
32. Capelle, N.; Barbet, J.; Dessen, P.; Blanquet, S.; Roques, B.P.; Le Pecq, J.B. Deoxyribonucleic acid bifunctional
intercalators: Kinetic investigation of the binding of several acridine dimers to deoxyribonucleic acid.
Biochemistry 1979, 18, 3354–3362. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2018, 19, 3179
16 of 16
33. Rook, Y.; Schmidtke, K.-U.; Gaube, F.; Schepmann, D.; Wünsch, B.; Heilmann, J.; Lehmann, J.; Winckler, T.
Bivalent -Carbolines as Potential Multitarget Anti-Alzheimer Agents. J. Med. Chem. 2010, 53, 3611–3617.
β
[CrossRef] [PubMed]
34. Shi, B.; Cao, R.; Fan, W.; Guo, L.; Ma, Q.; Chen, X.; Zhang, G.; Qiu, L.; Song, H. Design, synthesis and in vitro
and in vivo antitumor activities of novel bivalent
[PubMed]
β-carbolines. Eur. J. Med. Chem. 2013, 60, 10–22. [CrossRef]
35. Wu, Q.F.; Bai, Z.S.; Ma, Q.; Fan, W.X.; Guo, L.; Zhang, G.X.; Qiu, L.Q.; Yu, H.J.; Shao, G.; Cao, R.H. Synthesis
and biological evaluation of novel bivalent beta-carbolines as potential antitumor agents. MedChemComm
2014, 5, 953–957. [CrossRef]
36. Du, H.; Tian, S.; Chen, J.; Gu, H.; Li, N.; Wang, J. Synthesis and biological evaluation of N9-substituted
harmine derivatives as potential anticancer agents. Bioorg. Med. Chem. Lett. 2016, 26, 4015–4019. [CrossRef]
[PubMed]
37. Du, H.; Gu, H.; Li, N.; Wang, J. Synthesis and biological evaluation of bivalent β-carbolines as potential
anticancer agents. MedChemComm 2016, 7, 636–645. [CrossRef]
38. Kerr, J.F.R. Morphological criteria for identifying apoptosis. Cell Biol. Lab. Handb. 1994, 1, 319–329.
39. Escrich, L.; Grau, N.; Meseguer, M.; Pellicer, A.; Escribá, M.-J. Morphologic indicators predict the stage of
chromatin condensation of human germinal vesicle oocytes recovered from stimulated cycles. Fertil. Steril.
2010, 93, 2557–2564. [CrossRef] [PubMed]
40. Weir, N.M.; Selvendiran, K.; Kutala, V.K.; Tong, L.; Vishwanath, S.; Rajaram, M.; Tridandapani, S.; Anant, S.;
Kuppusamy, P. Curcumin induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer
cells by modulating akt and p38 mAPK. Cancer Biol. Ther. 2007, 6, 178–184. [CrossRef] [PubMed]
41. Wlodkowic, D.; Skommer, J.; Darzynkiewicz, Z. Cytometry in cell necrobiology revisited. Recent advances
and new vistas. Cytom. Part A 2010, 77, 591–606. [CrossRef] [PubMed]
42. Ray, S.K.; Fidan, M.; Nowak, M.W.; Wilford, G.G.; Hogan, E.L.; Banik, N.L. Oxidative stress and Ca2+ influx
upregulate calpain and induce apoptosis in PC12 cells. Brain Res. 2000, 852, 326–334. [CrossRef]
43. Chiu, W.-T.; Chang, H.-A.; Lin, Y.-H.; Lin, Y.-S.; Chang, H.-T.; Lin, H.-H.; Huang, S.-C.; Tang, M.-J.; Shen, M.-R.
Bcl-2 regulates store-operated Ca2+ entry to modulate ER stress-induced apoptosis. Cell Death Discov. 2018
4, 37–50. [CrossRef] [PubMed]
,
44. Silva, M.M.; Savariz, F.C.; Silva-Júnior, E.F.; de Aquino, T.M.; Sarragiotto, M.H.; Santos, J.C.C.; Figueiredo, I.M.
Interaction of -Carbolines with DNA: Spectroscopic Studies, Correlation with Biological Activity and
β
Molecular Docking. J. Braz. Chem. Soc. 2016, 27, 1558–1568. [CrossRef]
45. Zhang, J.; Zhang, Z.; Shu, B.; Cui, G.; Zhong, G. Cytotoxic and Apoptotic Activity of the Novel Harmine
Derivative ZC-14 in Sf9 Cells. Int. J. Mol. Sci. 2018, 19, 811. [CrossRef] [PubMed]
46. Cao, R.H.; Peng, W.L.; Wang, Z.H.; Xu, A.L. beta-Carboline alkaloids: Biochemical and pharmacological
functions. Curr. Med. Chem. 2007, 14, 479–500. [CrossRef] [PubMed]
47. Zhang, X.-F.; Sun, R.-Q.; Jia, Y.-F.; Chen, Q.; Tu, R.-F.; Li, K.-K.; Zhang, X.-D.; Du, R.-L.; Cao, R.-H. Synthesis
and mechanisms of action of novel harmine derivatives as potential antitumor agents. Sci. Rep. 2016
6, 33204–33219. [CrossRef] [PubMed]
,
48. Chen, W.; Zhang, G.; Guo, L.; Fan, W.; Ma, Q.; Zhang, X.; Du, R.; Cao, R. Synthesis and biological evaluation
of novel alkyl diamine linked bivalent β-carbolines as angiogenesis inhibitors. Eur. J. Med. Chem. 2016,
124, 249–261. [CrossRef] [PubMed]
49. Van Meerloo, J.; Kaspers, G.J.L.; Cloos, J. Cell Sensitivity Assays: The MTT Assay. In Cancer Cell Culture:
Methods and Protocols; Cree, I.A., Ed.; Series 731; Humana Press: New York, NY, USA, 2011; pp. 237–245.
50. Chen, Q.; Shao, X.; Ling, P.; Liu, F.; Shao, H.; Ma, A.; Wu, J.; Zhang, W.; Liu, F.; Han, G.; et al. Low molecular
weight xanthan gum suppresses oxidative stress-induced apoptosis in rabbit chondrocytes. Carbohydr. Polym.
2017, 169, 255–263. [CrossRef] [PubMed]
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