Synthesis and biological evaluation of novel 3,9-substituted β-carboline derivatives as anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2015.07.058

Abstract In our previous studies on 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) analogs, we synthesised numerous substituted carbazole and α-carboline derivatives, which exhibited anticancer activity. In this study, we designed and synthesised a series of 3,9-substituted β-carbolines, by replacing the tricyclic rings of carbazole and α-carboline derivatives with isosteric β-carboline, and evaluated anticancer activity. We observed that 9-(2-methoxybenzyl)-β-carboline-3-carboxylic acid (11a) inhibited the growth of HL-60 cells by inducing apoptosis, with a half maximal inhibitory concentration of 4.0 μM. Our findings indicate that β-carboline derivatives can be used as lead compounds for developing novel antitumor agents.

Full text of DOI:10.1016/j.bmcl.2015.07.058

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel 3,9-substituted
b-carboline derivatives as anticancer agents
Yi-Fong Chen a,b, , Yi-Chien Lin b, , Jeng-Pang Chen b, Hsu-Chin Chan c, Mei-Hua Hsu b, Hui-Yi Lin b,
Sheng-Chu Kuo a,b, Li-Jiau Huang a,b,
⇑
a The Ph.D. Program for Cancer Biology and Drug Discovery, China Medical University and Academia Sinica, Taichung 40402, Taiwan, ROC
b School of Pharmacy, China Medical University, Taichung 40402, Taiwan, ROC
c Department of Biochemistry, School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
In our previous studies on 1-benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) analogs, we synthesised
Received 28 April 2015
Revised 3 July 2015
Accepted 18 July 2015
Available online xxxx
numerous substituted carbazole and
study, we designed and synthesised a series of 3,9-substituted b-carbolines, by replacing the tricyclic
rings of carbazole and -carboline derivatives with isosteric b-carboline, and evaluated anticancer activ-
ity. We observed that 9-(2-methoxybenzyl)-b-carboline-3-carboxylic acid (11a) inhibited the growth of
HL-60 cells by inducing apoptosis, with a half maximal inhibitory concentration of 4.0 M. Our findings
a-carboline derivatives, which exhibited anticancer activity. In this
a
l
Keywords:
b-Carboline derivatives
Anticancer activity
indicate that b-carboline derivatives can be used as lead compounds for developing novel antitumor
agents.
Ó 2015 Elsevier Ltd. All rights reserved.
Apoptosis
Structure–activity relationships (SARs)
1-Benzyl-3-(5-hydroxymethyl-2-
furyl)indazole (YC-1) analogs
Cancer is the primary cause of human death worldwide.1
Numerous anticancer agents have been designed, several of which
exert antitumor effects by inducing apoptosis.2,3 However, the
majority of these anticancer drugs have limitations; therefore,
the development of novel anticancer drugs is urgently required.
1-Benzyl-3-(5-hydroxymethyl-2-furyl)indazole (YC-1) (Fig. 1)
is a compound associated with various biological activities, includ-
ing antiplatelet,4 neuroprotective,5 antiinflammatory,6 and apopto-
sis-inducing7,8 activities. The core structure of YC-1 is a bicyclic
ring of indazole. In previous studies, we synthesised bicyclic YC-1
analogs, such as furo[3,2-c]pyrazole (1),9 selenolo[3,2-c]pyrazole
(2)10 and 3-phenylindazole (3)11 derivatives. We also synthesised
tricyclic YC-1 analogs, such as furo[3,2-b]indole (4),12 carbazole
investigated b-carboline-containing compounds and reported var-
ious pharmacological properties,25 including sedative,26,27 vasodi-
latatory,28
antiinflammatory,19
antimicrobial,29
antiviral,30
antithrombin31 and antitumor32–40 activities. Studies have also
shown that introducing appropriate substituents into positions 3
and 9 of the b-carboline nucleus augments antitumor activity
and reduces acute toxicity.33,34 In this study, we aimed to develop
novel b-carbolines with improved pharmacological profiles and to
elucidate their antitumor structure–activity relationships (SARs).
We designed various 3,9-substituted b-carboline derivatives and
evaluated their anticancer activities.
We prepared the key intermediate, ethyl b-carboline-3-car-
boxylate by using methods described previously.41 Scheme 1 illus-
trates the overall synthesis of the 3,9-substituted b-carboline
derivatives. We synthesised 1,2,3,4-tetrahydro-b-carboline (7)
from L-tryptophan by using the Pictet-Spengler reaction,41,42 in
the presence of formaldehyde and sodium hydroxide solution.
We reacted 1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid (7)
with thionyl chloride in dry ethanol to convert it to ethyl 1,2,3,4-
tetrahydro-b-carboline-3-carboxylate (8). We then used man-
ganese dioxide in refluxing toluene to oxidize ethyl 1,2,3,4-te-
trahydro-b-carboline-3-carboxylate to produce the corresponding
b-carboline (9). We produced the target compounds of 3,9-substi-
tuted b-carboline derivatives (10–20) through alkylation or benzy-
lation from the intermediate ethyl b-carboline-3-carboxylate (9)
(5)13 and
a
-carboline (6)14–16 derivatives. In this study, we applied
-carboline nucleus of
the concept of bioisosterism17 to replace the
a
YC-1 analogs with a b-carboline nucleus (10–20, 10a, 11a, 19a,
20a). We then evaluated the synthesised compounds for use as
anticancer agents.
b-Carbolines are widely distributed in various plants,18–20
fungi21 and marines.22–24 Previous studies have extensively
⇑
Corresponding author. Tel.: +886 4 22053366x5609.
E-mail address: ljhuang@mail.cmu.edu.tw (L.-J. Huang).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2015.07.058
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chen, Y.-F.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.058

2
Y.-F. Chen et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 1. Previous studies and design of YC-1 analogs.
O
O
O
O
OH
NH2
O
OH
NH
O
(a)
(b)
NH
N
(c)
N
H
N
H
7
N
H
8
N
H
9
-Tryptophan
L
O
O
OH
O
N
N
(d)
(e)
N
N
X
Ar
Ar
Ar
X = Cl or Br
10-20
10a, 11a, 19a, 20a
Ar
:
OCH3
O
18 =
19 =
10 =
11 =
12 =
14 =
OH
OH
9
N
OCH3
N
(e)
OCH3
15 =
N
H
9a
H3CO
F
OCH3
OCH3
16 =
17 =
N
20 =
Cl
N
OCH3
(f)
N
H
9b
13 =
N
OCH3
Scheme 1. Reagents and conditions : (a) NaOH(aq), 35% formalin, 37 °C; (b) SOCl2, dried ethanol; (c) MnO2, toluene; (d) substituted arylmethyl halide, KOH, THF; (e) 50%
ethanol, NaOH; (f) LiAlH4, THF.
by using various arylmethyl halides, and reduced compound 9 (an
ester) in lithium aluminium hydride to produce the corresponding
carbinol, compound 9b. We hydrolyzed the ethyl b-carboline-3-
carboxylate derivatives (9–11, 19 and 20) in alkaline solution to
obtain the corresponding carboxylic acid derivatives (9a–11a,
19a and 20a). We confirmed the chemical structures of all of the
new synthetic products by using IR, 1H and 13C NMR spectroscopy,
and mass spectroscopy.
concentration (IC50) >100
N9-methylaryl moiety (10–20) exhibited moderate cytotoxic activ-
ities, ranging from 2.9 to 50.4 M. Compound 10, which contains
an N9-benzyl substitution, exhibited anticancer activity against
lM). The compounds containing the
l
SK-OV-3 cells (IC50 = 3.0 lM). Compound 11, which contains an
N9-(2-methoxybenzyl) substitution exerted potent inhibitory
effects against HL-60 and Hep-3B cells (IC50 = 3.9 and
IC50 = 8.9
inhibitory effects against SK-OV-3 cells (IC50 = 3.0 and
IC50 = 2.9 M, respectively). Compounds 9a–11a, 19a and 20a,
lM, respectively). Compounds 10 and 20 exerted potent
We evaluated the anticancer activities of the novel compounds
against several human tumor cell lines by using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazoliun bromide (MTT)
assay.43 Table 1 lists our results. Previous studies have shown that
substituting the benzyl groups at the N9-position of the b-carboline
ring system increases cytotoxic activity.33,34 As shown in Table 1,
l
which contain a free carboxylic acid group at position 3, were asso-
ciated with anticancer activities marginally more potent than
those of compounds containing the ethoxycarbonyl substituent.
However, compound 9b (a carbinol) exerted marginal or no cyto-
toxic effects on any cell line. These results suggested that 3-car-
boxylic acid-containing compounds exert more potent anticancer
compound 9,
exerted minimal cytotoxic activity (half maximal inhibitory
a
N9-nonsubstituted b-carboline-3-carboxylate,
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Y.-F. Chen et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
Table 1
Cytotoxicity of 3,9-substituted b-carbolines
R3
N
N
R9
9-20, 9a, 9b, 10a-11a, 19a-20a
Compd
R3
R9
IC50 (l
M)a
b
b
b
b
b
b
HL-60b
Hep 3B
H460
A498
COLO 205b
2774
SK-OV-3
Detroit 551
7
8
9
—
—
—
—
H
—
—
>100
>100
3.9
—
—
>100
>100
8.9
>50
>50
>100
>100
>100
—
>50
>50
>50
>50
—
—
—
—
>100
3.0
—
—
>100
50
>100
—
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOEt
COOH
CH2OH
COOH
COOH
COOH
COOH
—
>100
48.2
41.3
21.6
38.5
>100
>100
46.3
>100
>100
50.4
>100
>100
>100
9.2
>100
>100
>100
26.7
36.9
>100
>100
38.7
>100
>100
>100
>100
49.6
>100
31
>100
26.8
>100
>100
>100
>100
>100
—
>100
>100
>100
16.4
>100
>100
—
10
11
12
13
14
15
16
17
18
19
20
9a
9b
10a
11a
19a
20a
YC-1
Benzyl
2-Methoxybenzyl
3-Methoxybenzyl
4-Methoxybenzyl
3,5-Dimethoxybenzyl
3,4,5-Trimethoxybenzyl
2-Picolyl
3-Picolyl
4-Picolyl
4-Fluorobenzyl
4-Chlorobenzyl
H
>100
>100
>100
24.4
>100
—
>100
>100
>100
2.9
>100
>100
—
21.4
27.7
—
—
—
50
39.0
>100
21.9
>100
>100
—
>100
>100
75
>100
5.0
4.0
—
3.9
50
50
>100
>100
>100
>100
—
>100
>100
75
>100
4.4
6.7
—
1.2
>100
>100
>100
>100
—
>100
>100
70.5
>100
18.5
8.0
>100
>100
>100
>100
—
>100
>100
>100
>100
—
H
Benzyl
2-Methoxybenzyl
4-Fluorobenzyl
4-Chlorobenzyl
—
7.5
7.6
9.9
0.3
32.5
33.5
26.5
—
12.6
23.5
—
>100
—
—
—
15.1
—
25.27
—
—
—
—
a
Human tumor cells were treated with different concentrations of samples for 48 h. The data represented the mean values of three independent determinations and
showed as IC50 M, the concentration of 50% proliferation-inhibitory effect).
(
l
b
Cell lines include leukemia (HL-60), liver carcinoma (Hep3B), lung carcinoma (H460), renal carcinoma (A498), colon carcinoma (COLO205), ovarian carcinoma (2774 and
SK-OV-3) and normal skin fibroblast (Detroit 551).
activity than compounds containing the 3-ethoxycarbonyl sub-
stituent do. The 3,9-substituted b-carboline derivatives exhibited
diverse anticancer properties. Our preliminary biological studies
indicated that of the evaluated compounds, compound 11a exhib-
ited the most potent anticancer activity against the tested cell lines
(IC50 ranging from 4.0 to 32.5 lM).
Morphological analysis confirmed the cytotoxic effects of com-
pound 11a. As shown in Figure 2, the morphological changes
observed included nuclear shrinkage and the appearance of apop-
totic bodies (black arrowheads indicates an apoptotic nuclei) after
24 h incubation with 5 lM compound 11a.
Flow cytometric analysis indicated the cell cycle distribution of
the tumor cells (Fig. 3). We treated the HL-60 cells with various
concentrations (1 lM, 5 lM and 10 lM) of compound 11a. After
48 h treatment, we investigated the effects on cell cycle distribu-
tion using propidium iodide (PI) staining. The results indicated that
compound 11a induced a significant and dose-dependent increase
in the percentage of HL-60 cells in the sub-G1 phase (an index of
apoptotic DNA fragmentation). After 48 h incubation, compound
Figure 2. Morphological changes induced by 11a in HL-60 cells. HL-60 cells were
treated with 1 M, 5 M, and 10 M of compound 11a for 24 h and 48 h. The black
arrowhead indicates an apoptotic nucleus. Scale bar = 50 m.
l
l
l
l
significantly increased as the incubation time grew longer. The
numbers of advanced apoptotic cells stained by positive annexin
V-FITC and PI (Q2) also increased significantly with incubation
time. These data demonstrate that compound 11a induced cell
apoptosis in HL-60 cells.
After identifying that compound 11a induces apoptosis in HL-
60 cells, we evaluated the expression of selected apoptosis-associ-
ated proteins. To investigate the potential involvement of com-
pound 11a-induced apoptosis in the activation of caspase
11a (1 lM, 5 lM and 10 lM) induced an increase in the sub-G1
fraction (9.83%, 14.55% and 17.79%, respectively) compared with
the control cells (2.5%). These results indicated that the majority
of the cells treated with compound 11a had undergone apoptosis.
Determination of apoptotic cells by fluorescent staining was
carried out as described previously.44 The annexin V/propidium
iodide stainings45 were also used to confirm the compound 11a
on apoptotic characteristics in HL-60 cells. As shown in Figure 4,
cells incubated in the absence of compound 11a for 24 h and
48 h were undamaged and were negative for both annexin V-
cascades, we exposed HL-60 cells to 5 lM compound 11a for 24
and 48 h, and evaluated caspase-9 and caspase-3 activities by
using western blotting. Poly(ADP-ribose) polymerase (PARP) cleav-
age (by caspase-3) is a key marker of apoptosis. Western blot assay
results indicated that compound 11a upregulated caspase-3 and
caspase-9 expression, and induced PARP cleavage (Fig. 5). Our
FITC and PI staining (Q3). Treatments with 5 lM of compound
11a for 24 h to 48 h, the numbers of advanced apoptotic cells
stained by positive annexin V-FITC and negative PI (Q4) were
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Y.-F. Chen et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Figure 6. Effects of 11a on the protein levels of mitochondrial protein in HL-60
cells. HL-60 cells were assayed for AIF, Endo G, Apaf-1, cytochrome c by western
blotting analysis. b-Actin was used as a loading control.
Figure 3. Effects of 11a on the cell cycle in HL-60 cells. HL-60 cells were incubated
with 1 lM, 5 lM, and 10 lM of 11a for 48 h. They were then harvested and
analyzed using flow cytometry.
Figure 7. Effects of 11a on the protein levels of Bcl-2 family protein in HL-60 cells.
HL-60 cells were assayed for Bax and Bcl-2 by western blotting analysis. b-Actin
was used as a loading control.
reduced the levels of antiapoptotic Bcl-2 and increased the levels
of proapoptotic Bax, thus altering the Bax:Bcl-2 ratio. These results
indicated the involvement of a mitochondrial-dependent pathway
in compound 11a-induced apoptosis in HL-60 cells.
Upon binding to ligands, death receptors trigger apoptosis by
stimulating caspase-8-mediated caspase cascades.48,49 In this
study, we detected several death receptors (Fas, DR4 and DR5)
and their ligands (FasL and TRAIL) in HL-60 cells (Fig. 8).
However, treatment with compound 11a did not upregulate the
expression of these death receptors and their ligands. These results
suggested that death receptor pathways do not play major roles in
compound 11a-mediated apoptosis in HL-60 cells.
Figure 4. Confirmation of 11a-induced apoptosis was assessed using annexin V/PI
staining and flow cytometry. The fraction of annexin V-positive HL-60 cells was 0%
prior to treatment and 37.9% and 46.6% after treatment with 5
and 48 h, respectively.
lM of 11a for 24 h
In this study, we synthesised a series of novel 3,9-substituted b-
carbolines and evaluated their cytotoxic activities against various
human tumor cell lines in vitro. Some of the compounds exhibited
potent antitumor activities, particularly compound 11a. SARs anal-
ysis indicated that the substituted benzyl moiety at the N9-position
Figure 5. Effects of 11a on the protein levels of caspase-9, caspase-3, and PARP in
HL-60 cells. HL-60 cells were assayed for caspase-9, caspase-3, and PARP by
western blotting analysis. b-Actin was used as a loading control.
results also indicated the release of mitochondrial apoptosis-in-
ducing factor (AIF), endonuclease G (Endo G), apoptotic protease
activating factor 1 (Apaf-1) and cytochrome c into the cytosol dur-
ing compound 11a-induced apoptosis (Fig. 6).
The Bcl-2 family proteins are key regulators of mitochondrial-
related apoptotic pathways.46 An increase in the ratio of Bax:Bcl-
2 within a cell predisposes it to selected apoptotic stimuli.47 We
treated HL-60 cells with 5 lM compound 11a for 24 and 48 h to
establish the involvement of Bcl-2 protein activity in compound
11a-induced apoptosis. As shown in Figure 7, compound 11a
Figure 8. Effects of 11a on the protein levels of death receptor and their ligands in
HL-60 cells. HL-60 cells were assayed for Fas, DR4, DR5, FasL, and TRAIL by western
blotting analysis. b-Actin was used as a loading control.
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Y.-F. Chen et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
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Acknowledgements
This investigation was supported by research grants from the
National Science Council of the Republic of China awarded to L. J.
Huang (NSC 98-2628-B-039-018-MY3; NSC 101-2320-B-039-009-
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Please cite this article in press as: Chen, Y.-F.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2015.07.058