3-Benzylamino-β-carboline derivatives induce apoptosis through G 2/M arrest in human carcinoma cells HeLa S-3

Article abstract of DOI:10.1016/j.ejmech.2010.11.044

β-Carboline derivatives are known as the lead compounds for anti-tumor agents. To examine an optimal structure for anti-tumor activity, we synthesized a variety of β-carboline derivatives, possessing a variety of substituents on the nitrogen atom of the amino group of 3-amino-β-carboline, and evaluated their anti-tumor activity for HeLa S-3 cell line. 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that an optimal structure for anti-tumor activity was 3-cyclohexylmethylamino (1e) or 3-benzylamino-β-carboline (1f). An optimal counter anion of 2-methyl-3-benzylamino- β-carbolinium salts was a triflate anion 2c. In addition, the introduction of a hydroxyl group on the meta-position of the benzyl group of 3-benzylamino-β-carboline (3e) enhanced its anti-tumor activity. Hoechst 33342 staining and DNA fragmentation assay suggested that 1f, 2c and 3e induced cell death by apoptosis unlike 1e. Flow cytometry analysis showed that 1f, 2c and 3e induced cell apoptosis through arrest of the cell cycle in the G₂/M phase.

Full text of DOI:10.1016/j.ejmech.2010.11.044

European Journal of Medicinal Chemistry 46 (2011) 636e646
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
3-Benzylamino-
b-carboline derivatives induce apoptosis through G2/M arrest
in human carcinoma cells HeLa S-3
,
Reiko Ikeda a c, Toshie Iwaki a, Tomoko Iida a, Takasumi Okabayashi a, Eishiro Nishi a, Masaki Kurosawa a,
,b,c,
Norio Sakai a, Takeo Konakahara a
*
a Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science (RIKADAI), Noda, Chiba 278-8510, Japan
b Research Institute for Science and Technology (RIST), Tokyo University of Science (RIKADAI), Noda, Chiba, 278-8510, Japan
c Center for Technologies against Cancer, Tokyo University of Science (RIKADAI), Noda, Chiba, 278-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
b
-Carboline derivatives are known as the lead compounds for anti-tumor agents. To examine an optimal
structure for anti-tumor activity, we synthesized a variety of -carboline derivatives, possessing a variety
of substituents on the nitrogen atom of the amino group of 3-amino- -carboline, and evaluated their
Received 9 March 2010
Received in revised form
22 November 2010
Accepted 27 November 2010
Available online 8 December 2010
b
b
anti-tumor activity for HeLa S-3 cell line. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay showed that an optimal structure for anti-tumor activity was 3-cyclohexylmethylamino (1e)
or 3-benzylamino-
salts was a triflate anion 2c. In addition, the introduction of a hydroxyl group on the meta-position of the
benzyl group of 3-benzylamino- -carboline (3e) enhanced its anti-tumor activity. Hoechst 33342
staining and DNA fragmentation assay suggested that 1f, 2c and 3e induced cell death by apoptosis
unlike 1e. Flow cytometry analysis showed that 1f, 2c and 3e induced cell apoptosis through arrest of the
cell cycle in the G2/M phase.
b-carboline (1f). An optimal counter anion of 2-methyl-3-benzylamino- b-carbolinium
Keywords:
b
b-Carboline
Anti-tumor activity
Apoptosis
Chromatin condensation
DNA ladder
Ó 2010 Elsevier Masson SAS. All rights reserved.
G2/M cell cycle arrest
1. Introduction
effects, such as the inhibition of cyclin-dependent kinase (CDK)
[10], IkappaB kinase (IKK) [11,12] and topoisomerase I [8]. We have
Natural products have long proven to be a bountiful resource for
the identification of bioactive compounds used in the treatment of
a variety of ailments and diseases, including cancer. Chemothera-
peutic drugs, such as etoposide and taxol, are widely used in clinical
reported the anti-tumor activity of 3-benzylamino-
[13]. However, the optimization of the structure of
derivatives has not been defined. In this study, a variety of b-car-
b
b
-carboline
-carboline
boline derivatives 1e3 were synthesized and their biological
oncology.
and its derivatives are widely distributed in nature in plants,
marine life, human tissues and body fluids [1]. -Carboline and its
b
-Carboline is made up of planar tricyclic ring structures,
activities were evaluated.
b
2. Results and discussions
derivatives are endowed with anti-tumor and anti-cancer proper-
ties [2e5]. The best-known members of the class are harman and
normarman. These are DNA intercalating molecules with benzo-
diazepine receptors, and their high DNA binding affinity is thought
to be responsible, in part, for these pharmacological properties
2.1. Chemistry
To determine an optimal structure for anti-tumor activity,
a variety of substituent was introduced onto the amino- or pyridyl-
[6e9]. The main reason for the interest in
b-carboline and its
nitrogen atom of 3-amino-
position of the benzyl group of 3-benzylamino-
according to the known methods (Schemes 1e4). The construction
of -carboline framework was accomplished by PicteteSpengler
reaction of -tryptophan (4) with formaldehyde. After esterification
of the carboxyl group with thionyl chloride and methanol, the
1,2,3,4-tetrahydro- -carboline-3-carboxylic acid methyl ester (7)
was dehydrogenated by trichloroisocyanuric acid, TCCA, leading to
b
-carboline, or onto the meta- or para-
derivatives for clinical purposes are their multiple physiological
b-carboline
b
L
* Corresponding author. Department of Pure and Applied Chemistry, Faculty of
Science and Technology, Tokyo University of Science (RIKADAI), Noda, Chiba
278-8510, Japan. Tel.: þ81 4 7122 9502; fax: þ81 4 7123 9326.
b
E-mail address: konaka@rs.noda.tus.ac.jp (T. Konakahara).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.044

R. Ikeda et al. / European Journal of Medicinal Chemistry 46 (2011) 636e646
637
Scheme 1. Synthesis of 11.
the formation of
b-carboline-3-carboxylic acid methyl ester (7). The
the same way, 1beh was synthesized by LAH reduction of the cor-
total synthesis of 3-amino-
b
-carboline (11) was completed in 7
respondingamidesinmoderatetohighyields(Scheme2). Inaddition,
steps and 33% overall yield, after Curtius rearrangement of the
corresponding carbonylazide 9 followed by hydrolysis of the benzyl
carbamate 10 (Scheme 1).
the synthesis of2-methyl-3-benzylamino-b-carboliniumsalts (2aec)
was illustrated in Scheme 3. The solution of 1f in dichloromethane
was treated with methyl trifluoromethanesulfonate to quanternize
the N-2 atom of the pyridine ring to afford the tri-
fluoromethanesulfonate salt 2c. The counter anion of 2c was changed
As shown in Scheme 2, 3-methylamino-b-carboline (1a) was
prepared by LAH reduction of the benzyl carbamate 10 in 70% yield. In
Scheme 2. Synthesis of 1a-h.

638
R. Ikeda et al. / European Journal of Medicinal Chemistry 46 (2011) 636e646
Scheme 3. Synthesis of 2a-c.
to chloride and acetate anions using Amberlite IRA-900 anion
exchange resin to form 2a and 2b in 43, 45% yields, respectively
(Scheme 3).
22 and 21
of 3-ethylamino-
boline (1d), 17 M. That is to say, the extended carbon chain at the
m
M, respectively. These values are almost same as those
b
-carboline (1b), 19 M, and 3-isopentyl- -car-
m
b
m
The meta- or para-substituted 3-benzylamino derivatives 3a-e
were synthesized from 11 by a convenient reductive amination with
the corresponding meta- or para- substituted benzaldehydes and
sodium cyanoborohydride in high yields, as shown in Scheme 4.
3-position of 1f had markedly decreased its anti-tumor activity.
Surprisingly, the observed IC50 value of 3-(cyclohexylmethyl)
amino-b-carboline (1e) is 5.2 mM, which is smaller than the
expected from those of 1bed. Norharman without any substituent
was used as a negative control. These results showed that intro-
ducing a substituent on the nitrogen atom of the amino group was
important to increase anti-tumor activity, and that the benzyl
group was appropriate for the substituent.
2.2. Anti-tumor activity
In order to determine the anti-tumor effects of synthesized
Next, the anti-tumor activities of 2-methyl-3-benzylamino-b-
b-carboline derivatives on the human cervical cancer cell line (HeLa
carbolinium salts (2aec) with a different counter anion were
examined. Surprisingly, these salts showed different anti-tumor
activities. The compound 2a with a chloride counter anion is less
S-3), anti-tumor evaluation was performed by 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Table 1).
Initially, the 3-alkylamino-
(1aeh). Most of their anti-tumor activities were higher than that of
1a. Especially, 3-benzylamino- -carboline (1f) showed the highest
anti-tumor activity; the IC50 value is 0.12 M [12], which is 10-fold
higher than that of 3-isobutylamino- -carboline (1c), 13 M. It is
b-carboline derivatives were examined
active against tumor cells; the value of IC50 was 99 mM. In contrast,
the anti-tumor activity of 2b with an acetate anion is 10-fold higher
than that of 2a. Furthermore, 2c with a triflate counter anion
showed the highest anti-tumor activity among the three 2-methyl-
b
m
b
m
3-benzylamino-
b-carbolinium salts; the value of IC50 of 2c is
notable that 1f demonstrates high anti-tumor activity. In order to
0.54 M. Generally, it is said that such phenomena should be
m
clarify the effect of the methylene group, we evaluated the anti-
observed in pharmacokinetic studies. Although the reason is not
tumor activities of 3-(2-phenylethyl)amino-
b-carboline (1g) and
clear, it is a sufficiently interesting and valuable fact that 2-methyl-
3-(3-phenylpropyl)amino- -carboline (1h); their IC50 values were
b
Scheme 4. Synthesis of 1f and 3a-e.

R. Ikeda et al. / European Journal of Medicinal Chemistry 46 (2011) 636e646
639
Table 1
of IC50 had detectable fragmented nuclei in cells treated with any
compound (Fig. 1CeE, right panel).
Anti-tumor activity of 3-alkylamino- and 3-benzylamino-b-carboline derivatives in
HeLa S-3 cells.
2.3.2. DNA fragmentation
To verify induced cell death types, a DNA fragmentation assay
was performed. DNA fragmentation is well known as the typical
biochemical index of cell apoptosis. After exposure to 1e, 1f, 2c and
3e for 12e48 h, the genomic DNA was extracted and detected by
electrophoresis (Fig. 2). The DNA fragmentation was readily
detected in the cells treated with 1f and 3e for 24 h, and was
enhanced with time. The DNA fragmentation in the 2c-treated cells
was undetectable at 24 h, but was detectable at 48 h. The 1e-treated
cells did not have detectable DNA fragmentation, even after treat-
ment for 48 h. The DNA fragmentation assay demonstrated that 1f,
2c and 3e, but not 1e, induced DNA fragmentation in a time-
dependent manner. Together with fluorescence microscopy anal-
ysis, these observations demonstrated that 1f, 2c and 3e caused
apoptotic cell death, whereas 1e had a cell death pathway other
than apoptosis.
Compd. R1
X
R2
R3
IC50 (m
M)a
1a
1b
1c
1d
1e
1f
CH3
101 ꢀ 11
19 ꢀ 2
CH2CH3
C4Hꢁ9 iso
13 ꢀ 1
CH2C4Hꢁ9 tert
CH2C6H11
CH2C6H5
17 ꢀ 1
5.2 ꢀ 0.3
0.12 ꢀ 0.06
22 ꢀ 2
1g
1h
2a
2b
2c
3a
3b
3c
3d
3e
(CH2)2C6H5
(CH2)3C6H5
21 ꢀ 1
Cl
99 ꢀ 12
9.9 ꢀ 0.4
0.54 ꢀ 0.07
45 ꢀ 3
CH3COO
CF3SO3
H
COOCH3
H
OC2H4NHBoc
H
H
2.3.3. Activated caspase-3 of execution factor for apoptosis
After determining that 1f, 2c and 3e induced the chromatin
condensation and the DNA fragmentation, we further investigated
the activation of caspase-3 in these cells. It is well known that
caspase-3 is the execution factor of apoptosis. Therefore, if caspase-
3 is activated, these cells were induced apoptosis. As shown in
Fig. 3, activated caspase-3 was detected in 1f-, 2c- and 3e-treated
cells, but not 1e. In addition, activation levels of caspase-3 were
dependent on IC50 value in anti-tumor activity. These observations
are closely connected with results of the chromatin condensation
and the DNA fragmentation, and are suggesting that 1f, 2c and 3e
induced apoptosis.
COOCH3
H
OC2H4NHBoc
OH
17 ꢀ 2
42 ꢀ 3
16 ꢀ 1
0.92 ꢀ 0.1
>100
norharman
a
Drug toxicity was determined by 3-(4,4-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) assay (see Experimental). IC50 is drug concentration
inhibited the 50% cell growth. Data represent the mean ꢀ S.D. from four indepen-
dent experiments performed in triplicate experiments.
3-benzylamino-b-carbolinium chloride, acetate, and triflate (2aec)
showed different anti-tumor activities each other. Johansson et al.
have reported that human antibacterial protein LL-37, a natively
unfolded protein with extremely basic net charge, was shown to be
essentially folded in the presence of several anions [14]. Schulman
has also reported the influence of surface forces by various kinds of
salts in membrane permeability [15]. In addition, Sauvant et al.
(2003) have reported that stimulator of organic anion excretion
was important mediator of inflammation, and uptake of organic
anion was very important in cell maintenance and repair [16]. Thus,
counter anions or cations affect on the function of biomolecules to
a greater or lesser degree. Finally, the anti-tumor activity of the
compounds 3aee, with a substituent at the para- or meta-position
2.3.4. Flow cytometry
To examine the effects on cell cycle progression, the cells were
exposed to either 1
shown in Fig. 4, the G2/M phase populations in 1
treated cells were enhanced with time, but not those in 1e-treated
cells. After treatment with each 3-amino- -carboline derivative for
mM or 5
mM of 1e, 1f, 2c, and 3e for 6e24 h. As
mM of 1f- and 2c-
b
12 h, the percentages of average G2/M population in the control
cells and in 1e-, 1f-, 2c- and 3e-treated cells were 23.1, 28.9, 84.3,
73.5 and 31.3%, respectively. In addition, the populations in 1f-, 2c-
and 3e-treated cells for 24 h, had a tendency to shift from the G2/M
phase to the mean apoptotic population sub-G1 phase. These
results may suggest that 1f, 2c and 3e, but not 1e, induced cell
death through G2/M cell cycle arrest.
of the benzyl group of 3-benzylamino-b-carboline, was examined.
Both 3b and 3d with a substituent at the meta-position showed
higher anti-tumor activity than either 3a or 3c with a substituent at
the para-position. In addition, 3e with a hydroxyl group at the
meta-position showed the highest anti-tumor activity among
2.3.5. Analyses of expression of proteins during progression of the
cell cycle
3aee; the value of IC50 of 3e was 0.92
mM.
Entry and exit of mitosis are regulated by the cyclin-B-cdc2
complex, also known as the M-phase-promoting factor. To obtain
insight into the molecular mechanisms underlying the cell-cycle
profiles in 1f-, 2c- and 3e-treated cells, expression patterns of cdc2
and cdk2 protein were detected using western blot assay. The
conditions of the experimental groups (Fig. 5) were identical to
those in cell-cycle distribution assays (Fig. 4) but with one time
point (24 h). The protein levels of cdc2 were considerably increased
in 1f-, 2c- and 3e-treated cells, however, the expression of cdk2
proteins remained unchanged. The changes were according to flow
cytometric results showing stationary S-phase and increased G2/M
phase arrest, as the expression of cdc2 was raised and the protein
levels of cdk2 were constant.
2.3. Pharmacology
2.3.1. Hoechst 33258 staining
After the determination, using MTT assay, that 1e, 1f, 2c and 3e
had high anti-tumor activity, the induced cell-death type was
investigated. Chromatin condensation and fragmented nuclei are
known as the classic characteristics of apoptosis. Hoechst 33342
staining showed significant morphological changes in the nuclear
chromatin. After treatment with or without 5- or 10-fold concen-
tration of IC50 for each compound, the nuclear chromatin was
observed (Fig. 1). In the untreated group, the nuclei were stained
less bright (Fig. 1A). With 5-fold concentration of IC50, 1f-, 2c- and
3e-treated cells exhibited numerous cells with fragmented nuclei
(Fig. 1CeE, left panel), which was not the case with 1e-treated cells
(Fig. 1B, left panel). However, the cells with a 10-fold concentration
The anti-tumor activity of many anti-cancer drugs results
mostly from interference with cell cycle progression, by inhibition
of activation of cell cycle regulation, inhibition of DNA replication,

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R. Ikeda et al. / European Journal of Medicinal Chemistry 46 (2011) 636e646
Fig. 1. Identification of 1e-, 1f-, 2c- or 3e-induced an anti-proliferative effect and apoptosis. HeLa S-3 cells were treated with 1e (B), 1f (C), 2c (D) and 3e (E) for 48 h. The
concentration were either 26 M (B, left panel) or 52 M (B, right panel) 1e, 0.6 M (C, left panel) or 1.6 M (C, right panel) 1f, 2.7 M (D, left panel) or 5.4 M (D, right panel) 2c,
4.6 M (E, left panel) or 9.2 M (E, right panel) 3e, and non-treated group (A). Apoptosis was detected by Hoechst 33342 staining.
m
m
m
m
m
m
m
m
induction of DNA damage, or disruption of mitotic spindle forma-
tion [17e19]. In particular, since the discovery of taxol, spindle
microtubules have been popular for use in the discovery of new
anti-cancer drugs [20,21]. These drugs inhibit cell proliferation
either by increasing microtubule polymerization, as taxol does, or
by promoting microtubule depolymerization, as vinca alkaloids do,
thus impeding cell division and inducing cell death at relatively
high concentrations [19]. These reports indicate that a delay in
progression through mitosis by the induction of aberrant spindle
formation might be another mechanism for anti-cancer drugs to
alter cancer cell growth. In this study, flow cytometric analysis
demonstrated G2/M-arrest of the cell cycle with 1f, 2c and 3e.

R. Ikeda et al. / European Journal of Medicinal Chemistry 46 (2011) 636e646
641
the extract was dried over Na2SO4, and concentrated under reduced
pressure to give the title compound [23]. Yield 91%; colorless solid,
mp 187.2e188.8 ꢂC. 1H NMR (500 MHz, DMSO-d6)
d
2.72 (dd, J ¼ 9.0,
14.5 Hz, 1H), 2.75e2.76 (br, 1H), 2.91 (dd, J ¼ 5.0, 14.5Hz, 1H), 3.33
(s, 3H), 3.71 (dd, J ¼ 5.0, 9.0 Hz,1H), 3.90 (d, J ¼ 16.5 Hz, 1H), 4.00 (d,
J ¼ 16.5 Hz,1H), 6.92 (dd, J ¼ 6.5, 7.5 Hz,1H), 7.00 (dd, J ¼ 6.5, 8.0 Hz,
1H), 7.25 (d, J ¼ 7.5 Hz, 1H), 7.36 (d, J ¼ 8.0 Hz, 1H), 10.71 (br s, 1H);
MS (FAB) m/z 231 (M þ 1, 55%), 169 (100%).
4.1.3.
b-Carboline-3-carboxylic acid methyl ester (7)
To N,N-dimethylformamide (DMF) (50 ml) solution of 6 (7.95 g,
34.5 mmol) and triethylamine (9.09 g, 89.8 mmol), a DMF solution
of trichloroisocyanuric acid (8.0 g, 34.5 mmol) was added slowly at
ꢁ20 ꢂC. After the addition was completed, the reaction mixture was
allowed to warm slowly up to 0 ꢂC with stirring for 2 h. The
resulting product was precipitated from ice water, filtrated, washed
with ice water and dried in vacuo. The crude product was purified
by silica gel column chromatography (AcOEt as the eluent),
affording the title compound [24]. Yield 77%; colorless solid, mp
Fig. 2. Effect of 1e, 1f, 2c or 3e on apoptosis induction in HeLa S-3 cells by DNA
fragment assay. Cells were treated with or without 10-fold concentrations of IC50 in
each compound for 12 h, 24 h or 48h. DNA fragmentation was detected by electro-
phoresing on a 2% agarose gel. M: 100 bp ladder marker; C: non-treated cells.
242.3e243.9 ꢂC. 1H NMR (300 MHz, DMSO-d6)
d 3.90 (s, 3H), 7.31
(dd, J ¼ 8.1, 9.0 Hz, 1H), 7.60 (dd, J ¼ 8.1, 9.0 Hz, 1H), 7.67 (d,
J ¼ 8.1 Hz, 1H), 8.38 (d, J ¼ 8.1 Hz, 1H), 8.91 (s, 1H), 8.96 (s, 1H),
11.1e11.2 (br, 1H); MS (FAB) m/z 227 (M þ 1, 80%), 195 (100%).
3. Conclusions
We concluded that 1f, 2c and 3e are new class of drugs for cell
4.1.4.
b-Carboline-3-carboxylic acid hydrazide (8)
cycle arrest and induction of apoptosis. This study explored the
molecular mechanisms of action for a b-carboline derivative and
further characterized its potential as an anti-cancer agent.
Hydrazine monohydrate (5.76 g, 115 mmol) was added to the
suspension of 7 (1.05 g, 4.64 mmol) in 1-pentanol (42.7 ml) and the
mixture was stirred for 7 h at 150 ꢂC. Then, the solution was
evaporated and the crude compound was washed with hexane,
dried in vacuo [24]. Yield 89%; yellow solid, mp >350 ꢂC. 1H NMR
4. Experimental
(500 MHz, DMSO-d6)
d
3.9e5.0 (br, 2H), 7.28 (dd, J ¼ 7.5, 7.5 Hz, 1H),
7.51 (dd, J ¼ 7.5, 8.0 Hz, 1H), 7.64 (d, J ¼ 7.5 Hz, 1H), 8.39 (d,
J ¼ 8.0 Hz,1H), 8.81 (s, 1 H), 8.87 (s, 1H), 9.6e9.8 (br s,1H),11.5e12.0
(br, 1H); MS (FAB) m/z 227 (M þ 1, 100%).
4.1. Chemistry
4.1.1. 1,2,3,4-Tetrahydro-b-carboline-3-carboxylic acid (5)
A mixture of -tryprophan (4) (817 mg, 4 mmol) and 2.5 M NaOH
L
4.1.5.
b-Carboline-3-carbonyl azide (9)
(1.6 ml) was stirred until the mixture became clear at room
temperature, and then 37% formalin (4.87 ml) was added. The
mixture was stirred for 2 h at this temperature, refluxed for 3 h and
then neutralized (pH 5) with 2 M HCl. The precipitate was filtered,
washed with H2O, MeOH and dried in vacuo to give the title
compound [22]. Yield 99%; colorless solid, mp 302.2e304.6 ꢂC. MS
(FAB) m/z 217 (M þ 1, 100%).
The acid hydrazide 8 (5.0 g, 22.1 mmol) was dissolved in 0.2 M
HCl aq. solution (65.4 ml) and the mixture was cooled to 0 ꢂC and
then neutralized with Na2CO3 solution. The precipitate was fil-
trated, washed with ice water and dried in vacuo. Crude yield 90%;
yellow solid. 1H NMR (500 MHz, DMSO-d6)
d
7.33 (dd, J ¼ 7.5, 8.0 Hz,
1H), 7.61 (dd, J ¼ 8.0, 8.0 Hz, 1H), 7.67 (d, J ¼ 8.0 Hz, 1H), 8.42 (d,
J ¼ 7.5 Hz,1H), 8.96 (s,1H), 8.98 (s,1H),11.17 (br s,1H); MS (FAB) m/z
238 (M þ 1, 100%).
4.1.2. 1,2,3,4-Tetrahydro-b-carboline-3-carboxylic acid methyl ester
(6)
4.1.6. (b-Carboline-3-yl)carbamic acid benzyl ester (10)
To a methanol solution (500 ml) of 5 (43 mg, 0.2 mmol), SOCl2
(16 ml) was added slowly at ꢁ10 ꢂC, and the reaction mixture was
stirred for 4 h at 30 ꢂC, refluxed for 20 h and concentrated under
reduce pressure. After adding water, the residue was neutralized
(pH 8.0) with saturated Na2CO3, extracted with ethyl acetate, and
The carbonyl azide 9 (154.9 mg, 0.653 mmol), benzyl alcohol
(141.7 mg, 1.31 mmol) were added to o-xylene (3.6 ml) and the
suspension was stirred for 1 h at 160 ꢂC. The hot solution was
filtered. Hexane was added to the filtrate and the solution was
cooled to 5 ꢂC for 24 h. The deposited crystal was filtered, recrys-
tallized from ethanol. Yield 55%; brown solid; mp 210e211 ꢂC
(dec.). IR (KBr/cmꢁ1) 3416, 1720; 1H NMR (500 MHz, DMSO-d6)
d
5.21 (s, 2H), 7.19 (t, 1H), 7.32 (dd, J ¼ 6.0, 8.0 Hz, 1H), 7.39 (dd,
J ¼ 7.5, 8.0 Hz, 2H), 7.45 (d, J ¼ 8.0 Hz, 2H), 7.51 (dd, J ¼ 6.8, 8.0 Hz,
1H), 7.55 (d, J ¼ 8.0 Hz, 1H), 8.16 (d, J ¼ 8.0 Hz, 1H), 8.45 (s, 1H), 8.60
(s, 1H), 10.08 (br s, 1H), 11.42 (br s, 1H); 13C NMR (500 MHz, DMSO-
d6)
d 65.5, 103.1, 111.9, 119.0, 120.7, 121.7, 127.8, 127.9, 128.3, 128.4,
129.9, 131.2, 133.2, 136.9, 141.6, 143.1, 153.8; MS (FAB) m/z 317 (Mþ,
100%), HRMS (FAB): Calcd for C19H15N3O2: 317.1164, Found:
317.1162.
Fig. 3. Western blot analysis of activated caspase-3 in 1e-, 1f-, 2c- and 3e-treated cells.
Cells were treated with or without 5-fold concentrations of IC50 in each compound for
4.1.7. 3-Amino-b-carboline (11)
A mixture of 10 (99.9 mg, 0.315 mmol) and 6 M potassium
hydroxide in aqueous alcohol (3.7 ml 6 M KOH aq and 4.5 ml EtOH)
24 h. The proteins were detected by western blot analysis.
internal control. Control, non-treated cells.
b-actin was used as an

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R. Ikeda et al. / European Journal of Medicinal Chemistry 46 (2011) 636e646
Fig. 4. Effect of 1e, 1f, 2c or 3e on the distribution of cell-cycle phase in HeLa S-3 cells. Cells were exposed to 1 mM or 5 mM of each 3-benzylamino-b-carboline derivatives for 6 h,
12 h or 24 h. The cells were then fixed and stained with propidium iodide to analyze DNA content by flow cytometric analysis. M1: subG1-phase; M2: G1-phase; M3: S-phase; M4:
G2/M-phase.

R. Ikeda et al. / European Journal of Medicinal Chemistry 46 (2011) 636e646
643
212.1188, Found 212.1163. HRMS (ESI): C15H9N3 (M þ H) 212.1187,
Found 212.1189.
4.1.9.2. (
b
-Carbolin-3-yl)isobutanecarboxamide (12c). Yield: 92%;
1.31 (d, 6H,
colorless solid, mp 192.8e193.6 ꢂC 1H NMR (CDCl3):
d
J ¼ 7.0 Hz), 2.61 (septet, 1H, J ¼ 7.0 Hz), 7.23 (t, 1H, J ¼ 8.0 Hz), 8.07
(d, 1H, J ¼ 8.0 Hz), 8.30 (s, 1H), 8.51 (s, 1H), 8.57 (s, 1H), 8.92 (s, 1H).
13C NMR (CDCl3):
d 19.6, 36.8, 104.9, 111.4, 120.1, 121.8, 121.9, 122.3,
128.8, 130.5, 131.7, 133.6, 141.5, 143.5, 175.3. MS (FAB): m/z 254
(M þ H). HRMS (FAB): Calcd for C15H16N3O (M þ H) 254.1293, Found
254.1304.
4.1.9.3. 3-Isobutylamino-
b-carboline (1c). Yield: 65%; yellow solid,
mp 145.1e145.8 ꢂC 1H NMR (CDCl3):
d
1.04 (t, 3H, J ¼ 7.5 Hz), 1.72
Fig. 5. Cell cycle-related expression of proteins in HeLa S-3 cells with 1e, 1f, 2c or 3e.
Cells were treated with or without 5-fold concentrations of IC50 in each compound for
(sex, 2H, J ¼ 7.5 Hz), 3.35 (t, 2H, J ¼ 7.5 Hz), 4.40 (br s, 1H), 7.00 (s,
1H), 7.18 (t, 1H, J ¼ 7.5 Hz), 7.36 (d, 1H, J ¼ 7.5 Hz), 7.46 (t, 1H,
J ¼ 7.5 Hz), 8.03 (d, 1H, J ¼ 7.5 Hz), 8.33 (s, 1H), 9.16 (s, 1H). 13C NMR
24 h. The proteins were detected by western blot analysis.
internal control. Control, non-treated cells..
b-actin was used as an
(CDCl3): d 20.4, 28.1, 51.5, 94.9, 111.4, 118.7, 121.2, 121.6, 128.2, 130.6,
130.9, 132.6, 142.1, 153.2. MS (FAB): m/z 240 (M þ H). HRMS (FAB):
was stirred for 2 h under reflux. After the solvent was removed, the
resulting residure was cooled to 5 ꢂC for 24 h. The deposited crystal
was filtered, washed with water, and dried in vacuo. The product
was recrystallized from EtOH/hexane. Yield 85%; light-brownish
Calcd for C16H20N3 (M þ H) 240.1501, Found 240.1521.
4.1.9.4. 3-(2,2-Dimethylpropyl)amino-
b
-carboline (1d). Yield: 88%;
1.02 (s, 9H), 3.10
white solid, mp 169.5e170.4 ꢂC 1H NMR (CDCl3):
d
solid; mp 287e292 ꢂC 1H NMR (500 MHz, DMSO-d6)
d
5.30 (s, 2H),
(s, 2H), 7.02 (s, 1H), 7.16 (t, 1H, J ¼ 7.5 Hz), 7.33e7.473 (m, 2H), 8.01
7.06 (d, J ¼ 8.0 Hz, 1H), 7.07 (s, 1H), 7.41 (s, 2H), 7.99 (d, J ¼ 8.0 Hz,
(d, 1H, J ¼ 7.5 Hz), 8.34 (s, 1H), 8.52 (s, 1H). 13C NMR (CDCl3):
d 27.6,
1H), 8.29 (s, 1H), 10.87 (br s, 1H) [24].
31.8, 55.6, 94.8, 111.4, 119.0, 121.5, 121.7, 128.4, 130.4, 130.7, 132.8,
142.1, 153.8. MS (FAB): m/z 268 (M þ H). HRMS (FAB): C16H20N3
(M þ H) 254.1657, Found 254.1664.
4.1.8. 3-Methylamino-b-carboline (1a)
To a rapidly stirred suspension of the 10 (200 mg, 0.63 mmol) in
anhydrous THF (12 ml) was added LAH (119 mg, 3.15 mmol) and the
mixture was refluxed for 2 h. A piece of ice (1 g) was added, and the
mixture was stirred at room temperature for 2 h and then filtered.
The filtrate was concentrated in vacuo and the residue was purified
by column chromatography on silica gel. Yield: 70%, colorless solid,
4.1.9.5. (
b
-Carbolin-3-yl)cyclohexanecarboxamide (12e). Yield: 83%;
1.26e1.37 (m,
colorless solid, mp 220.3e220.8 ꢂC 1H NMR (CDCl3):
d
4H), 1.54e1.62 (m, 2H), 1.84e2.36 (m, 5H), 7.23e7.28 (m, 1H),
7.44e7.56 (m, 2H), 8.07e8.13 (m, 2H), 8.25 (s, 1H), 8.51 (s, 1H), 8.92
(s, 1H). 13C NMR (CDCl3):
d 25.7, 25.7, 29.7, 46.6, 104.8, 111.4, 120.1,
mp 219.5e220.2 ꢂC 1H NMR (DMSO-d6):
d
2.82 (d, 3H, J ¼ 4.5 Hz),
121.9, 122.3, 128.8, 130.5, 131.7, 133.4, 141.5, 143.6, 174.4. MS (FAB):
m/z 294 (M þ H). HRMS (FAB): Calcd for C18H20N3O (M þ H)
294.1606, Found 294.1587.
5.80 (d, 1H, J ¼ 4.5 Hz), 7.03e7.07 (m, 2H), 7.40e7.42 (m, 2H), 8.05
(d, 1H, J ¼ 7.8 Hz), 8.35 (s, 1H), 10.85 (s, 1H). 13C NMR (DMSO-d6):
d
29.6, 94.4, 111.5, 118.0, 120.6, 121.7, 127.9, 130.4, 130.7, 131.3, 141.9,
153.8. MS (FAB): m/z 198 (M þ H). HRMS (FAB): Calcd for C12H12N3
4.1.9.6. 3-Cyclohexylmethylamino-
b
-carboline (1e). Yield: 50%;
(M þ H) 198.1031, Found 198.1035.
yellow solid, mp 137.0e137.8 ꢂC 1H NMR (CDCl3):
d
0.99e1.26 (m,
5H), 1.70e1.87 (m, 6H), 3.14 (d, 2H, J ¼ 6.9 Hz), 4.49 (br s, 1H), 6.98
(s, 1H), 7.16 (t, 1H, J ¼ 7.5 Hz), 7.32e7.47 (m, 2H), 8.03 (d, 1H,
4.1.9. General procedure for the synthesis of 3-alkylamino-b-
carboline (1bee)
J ¼ 7.5 Hz), 8.35 (s, 1H), 8.52 (s, 1H). 13C NMR (CDCl3):
d 25.9, 26.5,
A solution of 11 (50 mg, 0.27 mmol) and carbxylic anhydride
(0.54 mmol) in anhydrous pyridine (5 ml) was stirred at room
temperature for 5 h. Saturated aqueous Na2CO3 solution was added
and the mixture was stirred at room temperature for 1 h and
extracted twice with ethyl acetate. The extracts were dried with
anhydrous Na2SO4 and evaporated under reduced pressure. The
residue was purified by flash chromatography on silica gel to afford
31.3, 37.7, 50.4, 94.9, 111.4, 119.0, 121.5, 121.8, 128.3, 130.7, 130.8,
132.7, 142.0, 142.0, 153.5. MS (FAB): m/z 280 (M þ H). HRMS (FAB):
Calcd for C18H22N3 (M þ H) 280.1814, Found 280.1803.
4.1.10. 3-Benzylamino-b-carboline (1f)
Acetic acid (528 l, 8.82 mmol) and benzaldehyde (289 mg,
m
2.73 mmol) were added to a solution of 11 (100 mg, 0.55 mmol) in
5 ml methanol and the mixture was stirred for 1.5 h at room
temperature. NaBH3CN (343 mg, 5.46 mmol) was then added and
the mixture was stirred for an additional 5 h. The reaction was
quenched by 6 N HCl (5 ml) and the reaction mixture was cooled to
0 ꢂC, neutralized with NH3 aq, and extracted with ethyl acetate. The
combined organic extract was washed with brine, dried with
Na2SO4, and concentrated in vacuo. The residue was purified by
silica gel chromatography to give pure 1f. Yield: 81%; yellow solid,
(b-carbolin-3-yl)alkanecarboxamide (12bee). To 12 in anhydrous
THF (5 ml) was added LiAlH4 (25 mg, 0.66 mmol), and the mixture
was refluxed for 2 h. A piece of ice (1 ml) was added, and the
mixture was stirred at room temperature for 2 h and then filtered.
The filtrate was concentrated in vacuo and the residue was purified
by column chromatography on silica gel to afford the title
compounds (1bee).
4.1.9.1. 3-Ethylamino-
b
-carboline (1b). Yield: 51%; yellow solid, mp
mp 199.9e200.5 ꢂC 1H NMR (CDCl3):
d
4.51 (d, 2H, J ¼ 6.0 Hz), 6.42
138.2e139.5 ꢂC 1H NMR (CDCl3):
d
1.32 (t, 3H, J ¼ 7.0 Hz), 3.36 (q,
(d, 1H, J ¼ 6.0 Hz), 7.06 (t, 1H, J ¼ 7.5 Hz), 7.10 (s, 1H), 7.18 (t, 1H,
2H, J ¼ 7.0 Hz), 4.33 (br s, 1H), 7.16 (s, 1H), 7.18 (t, 1H, J ¼ 7.5 Hz),
7.37 (d, 1H, J ¼ 8.0 Hz), 7.46 (t, 1H, J ¼ 7.5 Hz), 8.03 (d, 1H,
J ¼ 7.5 Hz), 7.28 (t, 2H, J ¼ 7.5 Hz), 7.39e7.43 (m, 4H), 7.99 (d, 1H,
J ¼ 7.5 Hz), 8.33 (s, 1H), 10.84 (s, 1H). 13C NMR (DMSO-d6):
d 45.9,
J ¼ 8.0 Hz), 8.26 (s, 1H), 8.38 (s, 1H). 13C NMR (CDCl3):
d
15.0, 38.4,
95.5, 111.4, 117.9, 120.5, 121.5, 126.2, 127.2, 127.8, 128.6, 130.5, 130.6,
131.2, 141.3, 141.9, 152.5. MS (FAB): m/z 274 (M þ H). HRMS (ESI):
Calcd for C18H16N3 (M þ H) 274.13442, Found 274.13555.
95.0, 111.3, 119.1, 121.6, 121.8, 128.4, 130.8, 130.9, 132.7, 141.9, 153.4.
MS (FAB): m/z 212 (M þ H). HRMS (FAB): C13H14N3 (M þ H)

644
R. Ikeda et al. / European Journal of Medicinal Chemistry 46 (2011) 636e646
4.1.11. 3-(2-Phenylethyl)amino-
b-carboline (1g)
methyl trifluoromethanesulfonate (4.8 ml, 0.22 mmol) was added
(
b
-Carbolin-3-yl)-2-phenylacetamide (12g) was obtained from
and then stirred for 12 h. The precipitated product was filtrated,
washed with cold dichloromethane, and dried in vacuo. Yield: 83%;
11 and phenylacetyl chloride using a procedure similar to that of
12b. Yield: 93%; colorless solid, mp 137.8e139.0 ꢂC 1H NMR (CDCl3):
yellow solid. 1H NMR (CD3OD, 500 MHz):
d 4.09 (s, 3H), 4.73 (s, 2H),
d
3.95 (s, 2H), 7.29 (t, 1H, J ¼ 7.5 Hz), 7.40e7.55 (m, 5H), 7.67 (d, 1H,
J ¼ 7.5 Hz), 7.78 (t,1H, J ¼ 7.5 Hz), 8.15 (s, 1H), 8.33 (d, 1H, J ¼ 7.5 Hz),
8.69 (s, 1H). 13C NMR (CDCl3):
45.0, 104.8, 111.4, 120.1, 122.3, 127.6,
7.19 (t, 1H, J ¼ 7.5 Hz), 7.28 (t, 1H, J ¼ 7.5 Hz), 7.38 (t, 2H, J ¼ 7.5 Hz),
7.45 (d, 1H, J ¼ 7.5 Hz), 7.49 (t, 3H, J ¼ 7.5 Hz), 7.60 (t, 1H, J ¼ 7.5 Hz),
8.02 (d, 1H, J ¼ 7.5 Hz), 8.39 (s, 1H). 13C NMR (CD3OD, 125 MHz):
d
128.8, 129.2, 130.4, 131.5, 133.5, 134.3, 141.4, 143.1, 169.1. MS (FAB):
m/z 320 (M þ H). HRMS (FAB): Calcd for C19H16N3O (M þ H)
302.1293, Found 302.1300. From 12g thus obtained, the title
compound 1g was obtained using a procedure similar to that of 1b.
d 43.3, 47.8,100.0,113.0,120.5,121.5,124.3,125.5,128.2,128.7,129.9,
131.2, 133.4, 138.5, 139.3, 147.3, 148.9. 19F NMR (CD3OD, 470 MHz), A
single peak was detected; MS (FAB): m/z 288 (M ꢁ CF3SO3); HRMS
(FAB): Calcd for M ꢁ CF3SO3: C19H18N3 288.1501, Found 288.1511.
Yield: 50%; yellow solid, mp 164.3e165.1 ꢂC 1H NMR (CDCl3):
d 3.00
(t, 2H, J ¼ 6.5 Hz), 3.61 (t, 2H, J ¼ 6.5 Hz), 4.43 (s, 1H), 7.00 (s, 1H),
4.1.16. 4-Methoxycarbonyl-3-benzylamino-b-carboline (3a)
7.17e7.46 (m, 7H), 7.47 (t, 1H, J ¼ 7.5 Hz), 8.00e8.02 (m, 2H), 8.39 (s,
Methyl 4-formylbenzoate was synthesized from 4-for-
mylbenzoic acid. The solution of dry 4-formylbenzoic acid (3 g,
20.00 mmol) in methanol (80 ml) was cooled to 0 ꢂC under argon
atmosphere, and thionyl chloride (2.1 ml, 30.00 mmol) was added
drop-by-drop to the solution. The mixture was stirred for 1.5 h, and
then heated under reflux for 20 h. The reaction mixture was
concentrated in vacuo, dissolved in water, neutralized with sodium
carbonate aqueous solution, and extracted with chloroform. The
combined organic phase was dried with Na2SO4 to afford methyl
4-formylbenzoate. To 11 (50 mg, 0.27 mmol) in 35 ml methanol was
added acetic acid (4.41 mmol) under argon atmosphere. The
mixture was then treated with methyl 4-formylbenzoate (134 mg,
0.82 mmol), obtained above, and stirred for 1.5 h at room
temperature. Subsequently, NaBH3CN (172 mg, 2.73 mmol) was
added and the mixture was stirred for 12 h. The reaction was
quenched by 6 N HCl (33 ml) and the reaction mixture was cooled
to 0 ꢂC. NH3 aq was added to adjust the reaction mixture to pH 10,
and then extracted with chloroform. The combined organic fraction
was washed with brine, dried with Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel chromatography to
afford pure 3a. Yield: 85%; yellow solid. Mp 171.2e173.8 ꢂC 1H NMR
1H). 13C NMR (CDCl3):
d 35.8, 44.9, 95.4, 111.4, 119.2, 121.6, 126.4,
128.4, 128.6, 128.9, 130.8, 130.9, 132.7, 139.5, 141.8, 153.0. MS (FAB):
m/z 288 (M þ H). HRMS (FAB): Calcd for C19H17N3 (M þ H) 288.1501,
Found 288.1520.Anal. Calcd for C19H17N3: C, 79.41; H, 5.96; N, 14.61;
Found C, 79.33; H, 6.21; N, 14.81.
4.1.12. 3-(3-Phenylpropyl)amino-
-carbolin-3-yl)-3-phenylpropionamide (12h) was obtained
from 3-amino- -carboline and phenylpropionyl chloride using
a procedure similar to that of 12b. Yield: 83%; colorless solid, mp
112.4e113.3 ꢂC 1H NMR (CDCl3):
b-carboline (1h)
(b
b
d
2.69 (t, 2H, J ¼ 7.5 Hz), 3.07 (t, 2H,
J ¼ 7.5 Hz), 7.16e7.28 (m, 6H), 7.38e7.50 (m, 2H), 8.00 (d, 1H,
J ¼ 7.8 Hz), 8.60 (s, 1H), 8.75 (s, 1H), 8.80e8.85 (m, 2H). 13C NMR
(CDCl3):
d 31.4, 39.2, 105.1, 111.4, 120.7, 122.7, 128.8, 128.5, 128.8,
130.4, 131.5, 133.5, 140.6, 141.5, 143.1, 170.5. MS (FAB): m/z 316
(M þ H). HRMS (FAB): Calcd for C20H18N3O (M þ H) 316.1450, Found
316.1455. From the (b-carbolin-3-yl)-3-phenylpropionamide thus
obtained, the title compound was obtained using a procedure
similar to that of 1b. Yield: 54%; yellow solid, mp 137.8e138.4 ꢂC 1H
NMR (CDCl3):
d
1.96 (quin, 2H, J ¼ 6.9 Hz), 2.70 (t, 2H, J ¼ 6.9 Hz),
3.30 (t, 2H, J ¼ 6.9 Hz), 4.43 (br s,1H), 6.92 (s, 1H), 7.11e7.30 (m, 6H),
(CDCl3, TMS, 500 MHz): d 3.88 (s, 3H), 4.60 (s, 2H), 6.94 (s, 1H), 7.14
7.41 (t,1H, J ¼ 7.5 Hz), 7.70 (t,1H, J ¼ 7.5 Hz), 8.31 (s,1H), 8.78 (s,1H).
(t, 1H, J ¼ 7.5 Hz), 7.34 (d, 1H, J ¼ 8.5 Hz), 7.45 (m, 3H), 7.92 (d, 1H,
13C NMR (CDCl3):
d 31.1, 33.2, 43.1, 95.0, 111.3, 118.9, 121.3, 121.7,
J ¼ 7.5 Hz), 7.97 (d,1H, J ¼ 8.5 Hz), 8.37 (s,1H), 8.53 (br,1H). 13C NMR
125.8, 128.3, 128.4, 130.7, 130.9, 132.6, 141.6, 141.7, 142.0, 153.0. MS
(FAB): m/z 302 (M þ H). HRMS (FAB): C20H20N3 (M þ H) 302.1657,
Found 302.1658.
(CDCl3, TMS, 125 MHz): d 47.3, 52.0, 95.6, 111.4, 119.2, 121.2, 121.8,
127.0, 127.1, 128.7, 128.9, 129.9, 130.1, 131.0, 133.0, 135.5, 142.1, 145.1,
152.2, 167.0. MS (FAB): m/z 332 (M þ H); HRMS (FAB): Calcd for
C20H18N3O2:333.1399, Found 332.1404.
4.1.13. 2-Methyl-3-benzylamino-
b
-carbolinium chloride (2a)
-carbolinium tri-
To the solution of 2-methyl-3-benzylamino-
b
4.1.17. 3-Methoxycarbonyl-3-benzylamino-b-carboline (3b)
fluoromethanesulfonate (2c) (50 mg, 0.183 mmol) in methanol
(10 ml), Amberlite IRA-900 (Clꢁ form) was added and then stirred
for 24 h at room temperature. The solution was filtrated, concen-
trated and dried in vacuo. Yield: 95%; 19F NMR (CD3OD, 470 MHz),
no peak was detected.
Methyl 3-formylbenzoate was synthesized as described previ-
ously [24]. The title compound 3b was synthesized in the same way
as that of 3a. Yield: 86%; yellow solid. Mp 170e173 ꢂC 1H NMR
(CD3OD, TMS, 500 MHz):
d 3.82 (s, 3H, eOCH3), 4.53 (s, 2H,
eOCH2e), 4.82 (b, 1H, eNHe), 6.91 (s, 1H, eNHe), 7.10 (dd, 1H, 6-H,
J ¼ 7.5, 8 Hz), 7.31e7.40 (m, 3H, ePh-4-H and 7-H, J ¼ 8.5, 8, 7.5, 14,
7, 7.4 Hz), 7.58 (d, 1H, ePhe, J ¼ 8 Hz), 7.82 (b, 1H, NH indole), 7.89
(dd, 2H, ePhe and 8-H, J ¼ 8.75 Hz), 8.06 (s, 1H, 5-H), 8.35 (s, 1H, 1-
4.1.14. 2-Methyl-3-benzylamino-
b
-carbolinium acetate (2b)
-carbolinium tri-
To the solution of 2-methyl-3-benzylamino-
b
fluoromethanesulfonate (2c) (50 mg, 0.183 mmol) in methanol
(10 ml), Amberlite IRA-900 (CH3COOꢁ form) was added and then
stirred for 24 h at room temperature. The solution was filtrated,
concentrated and dried in vacuo. Yield: 90%; 1H NMR (CD3OD,
H). 13C NMR (CDCl3, 125 MHz):
d 45.2, 52.0, 79.1, 95.7, 111.4, 118.0,
120.4, 121.6, 127.1, 127.9, 128.5, 129.5, 130.5, 131.3, 132.2, 141.9, 142.4,
152.3, 166.3. 19F NMR (CD3OD, 470 MHz), MS (FAB): m/z 288
(M ꢁ CF3SO3); HRMS (FAB): Calcd for M ꢁ CF3SO3: C19H18N3
288.1501, Found 288.1511. MS (FAB): m/z 331 (M þ H); Anal. Calcd
for C20H17N3O2: C, 72.49; H, 5.17; N, 12.68; Found C, 72.26; H, 5.26;
N, 12.63.
300 MHz)
1H, J ¼ 7.5 Hz), 7.37e7.51 (m, 6H), 7.57 (t,1H, J ¼ 7.5 Hz), 7.99 (s, 1H),
8.37 (s, 1H). 13C NMR (CD3OD, 75 MHz):
24.1, 43.3, 47.7, 99.8, 113.0,
d
1.93 (s, 3H), 4.07 (s, 3H), 7.20 (t, 1H, J ¼ 7.5 Hz), 7.31 (d,
d
120.4, 121.4, 124.2, 125.5, 128.2, 129.9, 131.2, 133.3, 138.6, 139.1,
147.3, 148.8, 180.1.
4.1.18. 3-[4-[2-(N-tert-Butoxycarbonyl)amino]ethoxybenzyl]
amino-b-carboline (3c)
4.1.15. 2-Methyl-3-benzylamino-
trifluoromethanesulfonate (2c)
The title compound 2c was obtained from 1f. To the stirred
solution of 1f (50 mg, 0.18 mmol) in dichloromethane (10 ml),
b
-carbolinium
4-[2-(N-tert-Butoxycarbonyl)amino]ethoxybenzaldehyde was
synthesized as described previously [13]. The title compound 3c
was synthesized in the same way as that of 3a. Yield: 66%; yellow
solid. Mp 167.8e168.0 ꢂC 1H NMR (CD3OD, TMS, 500 MHz):
d 1.41

R. Ikeda et al. / European Journal of Medicinal Chemistry 46 (2011) 636e646
645
(s, 9H), 3.38 (s, 2H), 3.96 (s, 2H), 4.44 (s, 2H), 6.88 (d, 2H, J ¼ 8.5 Hz),
4.3. Pharmacology
7.09 (t,1H, J ¼ 7.5 Hz), 7.13 (s,1H), 7.32 (d, 2H, J ¼ 8.0 Hz), 7.38 (d,1H,
J ¼ 8.5 Hz), 7.41 (d, 1H, J ¼ 7.5 Hz), 7.97 (d, 1H, J ¼ 8.0 Hz), 8.26 (s,
4.3.1. Hoechst 33342 staining
1H). 13C NMR (CDCl3, 125 MHz):
d
28.7, 41.0, 47.9, 67.9, 80.2, 97.6,
The cells were treated with the vehicle alone (0.1% DMSO) and
with various concentrations of either compounds for 48 h and
112.4, 115.6, 119.6, 122.1, 122.7, 129.5, 129.7, 130.7, 132.7, 133.6, 134.1,
144.1, 154.0, 158.5, 159.3. MS (FAB): m/z 433 (M þ H); Anal Calcd for
C25H28N4O3: C, 69.42; H, 6.53; N, 12.95; Found C, 68.84; H, 6.76; N,
12.90.
stained with Hoechst 33342 (1 m
g/ml) for 40 min at 37 ꢂC. The cells
were examined for apoptosis under a confocal laser microscopic
system (IX71, Olympus), and evaluated by morphological changes
of the hallmarks of apoptotic cells. The cells were treated with the
4.1.19. 3-[3-[2-(N-tert-Butoxycarbonyl)amino]ethoxybenzyl]
vehicle alone (0.1% DMSO) and with each 3-aminobenzyl-
b-car-
amino-b-carboline (3d)
boline derivative for 48 h. The concentrations of either 1e, 1f, 2c or
3-[2-(N-tert-Butoxycarbonyl)amino]ethoxybenzaldehyde was
synthesized as described previously [13]. The title compound 3d
was synthesized in the same way as that of 3a. Yield: 95%; yellow
3e were 26 or 52 mM, 0.6 or 1.2 mM, 2.7 or 5.4 mM and 4.6 or 9.2 mM,
respectively.
solid. Mp 163.8e164.2 ꢂC 1H NMR (CD3OD, TMS, 500 MHz):
d
1.28
4.3.2. DNA extraction and agarose-gel electrophoresis
(s, 9H), 3.26 (s, 2H), 3.81 (s, 2H), 4.36 (s, 2H), 6.64 (d, 2H, J ¼ 8.0 Hz),
6.88 (s, 2H), 6.96 (m, 2H), 7.08 (t, 1H, J ¼ 8.0 Hz), 7.26 (d, 1H,
J ¼ 8.0 Hz), 7.30 (d, 1H, J ¼ 7.0 Hz), 7.81 (d, 1H, J ¼ 8.0 Hz), 8.15 (s,
The cells were treated with the vehicle alone (0.1% DMSO) and
either compounds for various incubation times of 10, 24 or 48 h,
and were then collected by centrifugation. The pellets were washed
with PBS, lysed with DNA fragmentation lysis buffer (0.6% Sarco-
sine, 20 mM TriseHCl pH7.4 and 20 mM EDTA pH 8.0) and incu-
bated with RNase A (2 mg/ml) for 2 h at 50 ꢂC. Proteinase K (1.2 mg/
ml) was subsequently added to the samples overnight at 50 ꢂC. DNA
was isolated with neutral-phenol, phenol/chloroform (1:1), and
chloroform/isoamyl alcohol (24:1), and subsequently centrifuged at
14,000 rpm for 30 min at 4 ꢂC. The supernatants were electro-
phoresed on a 2% agarose gel in TAE buffer (40 mM Tris base,
20 mM EDTA and 20 mM acetic acid) at 50 V. The gel was stained
with ethidium bromide and photographed with UV illumination.
1H). 13C NMR (CDCl3, TMS, 125 MHz):
d 28.9, 41.1, 43.9, 61.7, 62.2,
68.0, 80.3, 97.6, 112.6, 114.8, 119.8, 121.1, 122.3, 129.7, 130.6, 131.0,
132.8, 134.3, 143.4, 144.3, 154.1, 158.6, 160.6. MS (FAB): m/z 433
(M þ H); Anal Calcd for C25H29N3O5: C, 68.42; H, 6.53; N, 12.95;
Found C, 68.22; H, 6.76; N, 12.77.
4.1.20. 3-Hydroxy-3-benzylamino-b-carboline (3e)
The title compound 3e was synthesized in the same way as that
of 3a. Yield: 74%; brown solid. Mp 204.2e206.8 ꢂC 1H NMR (CD3OD,
TMS, 500 MHz):
d 4.37 (s, 2H), 6.56 (s, 1H), 6.80 (s, 2H), 7.00e7.05
(m, 3H), 7.29 (d, 1H, J ¼ 8.0 Hz), 7.33 (d, 1H, J ¼ 7.0 Hz), 7.87 (d, 1H,
J ¼ 8.0 Hz), 8.17 (s, 1H). 13C NMR (CD3OD, TMS, 125 MHz):
d
97.4,
4.3.3. Western blot analysis
112.4, 114.8, 115.2, 119.6, 122.2, 129.6, 130.5, 130.7, 132.7, 134.3, 143.1,
144.2, 151.4, 154.0, 158.7. MS (FAB): m/z 290 (M þ H); HRMS (FAB):
Calcd for M þ H C18H16N3O1; 290.1215, Found 290.1302.
The cells were treated with either 1e, 1f, 2c or 3e. After treat-
ment, the cells were rinsed in ice-cold PBS and lysed in RIPA buffer
consisting of 50 mM TriseHCl (pH8.0), 150 mM NaCl, 1%NP-40, 0.5%
sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS) and
Protease Inhibitor Cocktail (Sigma). The lysate was centrifuged at
12,000 rpm for 5 min to obtain whole cell extract. Subsequently, the
whole cell extract were applied to electrophoresis on a poly-
acrylamide gel containing 0.1% SDS. After electrophoresis, poly-
peptides were transferred onto the polyviniylidene difluoride
(PVDF) membranes (Millipore, Bedford, MA, USA) at 200 mA for 1 h.
The membrane was blocked with 5% skim milk in Tris-buffer saline
containing 0.05% Tween 20 (TTBS) and then incubated with specific
4.2. Anti-tumor activity
4.2.1. Cell lines and culture
HeLa S-3 cells were provided by Dr. Okada (Institute for Bio-
logical Resources and Functions, National Institute of Advanced
Industrial Science and Technology). The cells were maintained in
MEM (Nissui) supplemented with 10% FBS (GIBCO), 2% HEPES, 3%
primary polyclonal/monoclonal antibodies, including
b-actin
NEAA and 2 mM
L
-glutamine in a water-saturated atmosphere of 5%
(1:5000; AC-15, Abcam), cdc2 (1:1000; C12720, Transduction
Laboratories), cdk2 (1:1000; SC-163, Santa Cruz) and cleaved cas-
pase-3 antibodies (1:1000; Asp175, Cell Signaling). The membrane
was washed with TTBS and then incubated with appropriate
secondary antibodies conjugated with horseradish peroxidase (Bio-
Rad Laboratories, Tokyo, Japan). After washing with TTBS, the
antigeneantibody complex was visualized with the ECL kit
(Amersham Pharmacia Biotech, Buckinghamshire, England).
CO2 at 37 ꢂC.
4.2.2. Cell viability assay
Cell viability was determined by 3-[4,5-dimethylthiazol-2-y]-
2,5-diphenyltetrazolium bromide (MTT) assay,
a method for
determining cell viability by measuring the mitochondrial dehy-
drogenase action. Cells were seeded in a 96-well cell culture cluster
(Becton Dickinson) at a density of 2 ꢃ 104 cells/ml and cultured 3 h
prior to drug treatment. Cells were exposed to 3-benzylamino-
b
-
4.3.4. Cell cycle analysis
carboline derivatives at 37 ꢂC for 24e72 h. The MTT reagent (Nacalai
Tesuque) was prepared at a concentration of 2 mg/ml in Dulbecco’s
PBS without calcium and magnesium, and stored at 4 ꢂC. After
treatment for the indicated times, cells were incubated with MTT
reagent for 4 h at 37 ꢂC. The plate was centrifuged at 3000 rpm for
10 min, and the medium was removed. To solubilize the resulting
The cells were treated with either 1e, 1f, 2c or 3e. After treat-
ment, the cells were washed twice with ice-cold PBS, collected by
centrifugation, and fixed in ice-cold 70% (v/v) ethanol, washed with
PBS, re-suspended with 0.1 mg/ml RNase, stained with 40 mg/ml PI,
and analyzed by flow cytometry using FACScalibur (Becton Dick-
inson). The cell cycle distributions were calculated using Cell Quest
software (Becton Dickinson).
MTT-formazan, 200
mL/well of dimethyl sulfoxide (DMSO) was
added to each well, followed by thorough mixing with a mechan-
ical plate mixer. Absorbance at 540 nm was measured on a micro-
plate reader (MTP-500, CORONA), and the percentage of cell
Acknowledgements
viability was taken as the percentage absorbance at 540 nm of
carboline-treated cells against control cells.
b
-
We would like to thank Dr. Okada (Institute for Biological
Resources and Functions, National Institute of Advanced Industrial

646
R. Ikeda et al. / European Journal of Medicinal Chemistry 46 (2011) 636e646
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