Structure-activity relationship in the antitumor activity of 6-, 8- or 6,8-substituted 3-benzylamino-β-carboline derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.03.077

We synthesized 47 kinds of 3-amino- or 3-benzylamino-β-carboline derivatives with a substituent on the 6-, 8-, or 6,8-carbon atoms and evaluated their antitumor activities for Hela S-3 and Sarcoma 180 cell lines using a 3-(4,5-dimethylthiazol-2-yl)-2,5-di

Full text of DOI:10.1016/j.bmcl.2012.03.077

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3506–3515
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship in the antitumor activity of 6-, 8- or
6,8-substituted 3-benzylamino-b-carboline derivatives
Reiko Ikeda a,b, Takanori Kimura a, Tatsuya Tsutsumi a, Syunsuke Tamura a, Norio Sakai a,
Takeo Konakahara a,b,
⇑
a Department of Pure and Applied Chemistry, Faculty of Science and Technology, Tokyo University of Science (RIKADAI), Noda, Chiba 278-8510, Japan
b 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:
We synthesized 47 kinds of 3-amino- or 3-benzylamino-b-carboline derivatives with a substituent on the
6-, 8-, or 6,8-carbon atoms and evaluated their antitumor activities for Hela S-3 and Sarcoma 180 cell lines
using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Consequently, we suc-
ceeded to develop 3-benzylamino-8-methylamino-b-carboline (17a) and 8-methylamino-3-(3-phen-
Received 16 January 2012
Revised 21 March 2012
Accepted 22 March 2012
Available online 30 March 2012
oxybenzyl)amino-b-carboline (17c) with antitumor activity with IC50 values of 0.046, 0.032
respectively, against HeLa S-3 cell line, which are higher than that of previously reported 3-(3-phenoxyb-
enzyl)amino-b-carboline (10e) of 0.074 M. Furthermore, effects of Cl group at 6-carbon atom on the type
lM,
Keywords:
b-Carboline
l
of cell death was evaluated using 3-benzylamino-6-chloro-b-carboline (10b), 3-benzylamino-b-carboline
(10d), N-(3-benzylamino)-6-chloro-9H-b-carbolin-8-yl)benzamide (14g), and N-(3-benzylamino-9H-b-
carbolin-8-yl)benzamide (17b) to show no effect. Hoechst 33342 staining and DNA fragmentation assay
suggested that these compounds induced cell death by apoptosis. In addition, using flow cytometry anal-
ysis, we established that the cell death pathway was through the arrest of the cell cycle in the G2/M phase.
Ó 2012 Elsevier Ltd. All rights reserved.
Antitumor drug
Apoptosis
Cell cycle in the G2/M phase
Structure–activity relationship
Natural products have 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. Chemotherapeu-
tic drugs such as etoposide and taxol are widely used in clinical
oncology. b-Carboline is made up of planar aromatic tricyclic struc-
tures, and its derivatives are widely distributed in nature: in plants,
marine life, human tissues, and body fluids.1 b-Carboline and its
derivatives have antitumor and anti-cancer properties.2–5 The
interest in b-carboline and its derivatives for clinical purposes is
due to multiple physiological effects, such as the inhibition of
cyclin-dependent kinase (CDK),1 IkappaB kinase (IKK), and topoiso-
merase I.6
Unfortunately, 6,8-dichloro-b-carboline had poor aqueous solubil-
ity and therefore could not be further evaluated in secondary cell
assays.6 Therefore, we synthesized 3-benzylamino-b-carboline
derivatives with substituents at the 6- and/or 8-positions, and eval-
uated their substituent effects on biological activities.
We planned to synthesize 8-amino-3-benzylamino-6-chloro-b-
carboline (1) as a precursor compound of 3-benzylamino-b-carbo-
line derivatives with various kinds of substituent on the N atom of
the 8-amino group (Scheme 1).
The b-carboline framework was constructed by three-step reac-
tions containing the Pictet–Spengler reaction of
L-tryptophan
(2).10–12 After introducing a chloro group on the 6-position of ethyl
b-carboline-3-carboxylate (5),13 3-amino-6-chloro-b-carboline (9)
was synthesized via three-step reactions containing the Curtius
rearrangement. The 3-amino group of 9 was protected withan acetyl
group, and then the 8-position was nitrated using sodium nitrate in
TFA. After removal of the acetyl group, the amino group was benzy-
lated by the reductive amination of benzaldehyde in an 83% yield.
The total synthesis of 8-amino-3-benzylamino-6-chloro-b-carbo-
line (1) was completed in 12 steps with a 14% overall yield after
reduction of the nitro group of 13 in a 72% yield.
Previously, we reported that 3-benzylamino-b-carboline
showed high antitumor activity, with an IC50 value of 0.11
against HeLa S-3,7,8 and that 3-(3-phenoxybenzyl)amino-b-carbo-
line had extremely-high activity (IC50 0.074 M) against Hela S-3-
induced apoptosis and SubG1 cell population after G2/M cell cycle
arrest interacting with
-tubulin protein.9 However, Castro et al. re-
lM
l
a
ported that the IC50 value of 6,8-dichloro-b-carboline was 0.20 lM
against HeLa S-3.6 Furthermore, the selectivity for IKK over Casein
kinase II of this compound had improved by more than 20-fold.
We synthesized 3-benzylamino- and 3-(3-phenoxyben-
zyl)amino-6-chloro-b-carboline (10b,c) from 9 by the reductive
amination of benzaldehyde (or 3-phenoxybenzaldehyde), as shown
in Scheme 2.
⇑
Corresponding author.
E-mail address: konaka@rs.noda.tus.ac.jp (T. Konakahara).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.077

R. Ikeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3506–3515
3507
O
HCHO aq
(1.1 eq)
COOH
NH2
SOCl2 (1.5 eq)
2.5 M NaOH aq
OH
NH
N
H
2
MeOH, rt, 4 h
rt, 2 h and then
reflux, 3 h
N
H
and reflux, 20 h
3: 92%
O
O
TCAA (1.1 eq)
TEA (2.6 eq)
NCS (1.5 eq)
O
O
AcOEt, reflux,
18 h
DMF, 0 °C, 2 h
N
NH
N
N
H
H
4: 79%
5: 99%
O
NH2NH2.H2O
(25 eq)
O
Cl
Cl
NHNH2
O
1-pentanol,
reflux, 7 h
N
N
N
H
N
H
7: 87%
6: 83%
O
NaNO2 (3 eq)
2N-HCl aq (2 eq)
Cl
50% AcOH aq
reflux, 1 h
N3
N
H2O, 0 °C, 2 h
N
H
8: 85%
1) RCHO (2 eq),
AcOH (pH 4)
H
N
Cl
NH2
Cl
2) NaBH3CN (2 eq)
N
N
N
O
MeOH, rt, 4 h and
then 2 h
N
H
H
10a: 93%
9: 83%
H
N
Cl
Cl
NH2
NaNO3 (5 eq)
TFA (excess)
1N-HCl aq
N
N
O
N
H
N
H
H2O, reflux, 12 h
rt, 18 h
NO2
NO2
Cl
12: 88%
11: 79%
H
1) PhCHO (1.2 eq)
AcOH (pH 4)
2) NaBH3CN (2 eq)
N
SnCl2.2H2O (3.5 eq)
12N-HCl aq ( 50 eq)
N
N
H
MeOH, rt, 48 h
MeOH, rt, 4 h
and then 1 h
NO2
13: 83%
H
N
Cl
N
N
H
NH2
1: 72%
Scheme 1. Synthesis of 1.
H
N
Cl
Cl
NH2
1) RCHO (2eq)
R
AcOH (pH=4)
N
N
2) NaBH3CN (2eq)
N
H
N
H
MeOH, rt, 4 h
and then 2 h
10b: 80% (R = H)
9
10c: 76% (R = OPh)
Scheme 2. Synthesis of 10b and 10c.
We synthesized b-carboline derivatives 14a–m, possessing an
8-amido group, from 1 by the methods shown in Scheme 3. In most
cases, we synthesized 14 by a reaction of 1 with the corresponding
carboxylic acid anhydride in the presence of DMAP (Scheme 3,
Method A) in quantitative yield (see Table 1, 14a, b, d, e, k–m). If
Method A was not applicable to the reaction, we used the
corresponding carboxylic acid with a combination of DCC and
DMAP (Method B; see Table 1, 14c, h–j) or acyl chloride (Method

3508
R. Ikeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3506–3515
H
H
N
Ph
Ph
N
Cl
Cl
Method A:
N
N
N
H
(RCO)2O (1.0 eq),
DMAP (1.2 eq),
CHCl3, rt, 1-24 h
N
H
NH2
O
NH
1
14a-m
Method B:
R
RCOOH (1 eq), DCC (1.2 eq),
DMAP (1.2 eq)/CH2Cl2,
rt, 7-12 h
Method C:
RCOCl (1 eq)/CHCl3,
rt, 0.5 h
Scheme 3. Synthesis of 14a–m.
Table 1
Next, we synthesized 8-alkylamnio-6-chloro-3-benzylamino-b-
carbolines 15a–k by the methods shown in Scheme 5; the yields
are shown in Table 2. First, we synthesized the 8-methylamino
derivative 15a by a reaction of 1 with paraformaldehyde in the
presence of sodium methoxide followed by sodium cyanoborohy-
dride reduction in a 79% yield (Scheme 5, Method D-a). In the reac-
tion with acetaldehyde, however, instead of 15b, we obtained the
corresponding diethylamino derivative 15b0 in a 88% yield. There-
fore, we reduced the acetamido group of 14a by lithium aluminum
hydride to produce 15b in a 69% yield (Scheme 5, Method E). We
also synthesized the compounds 15c,e from 14b,c by the same
method. We synthesized the other derivatives 15d,f–k by Method
D-b (reductive amination) with high yields, as shown in Table 2.
Oxidative de-benzylation of 14e,g was performed by DDQ in
dioxane at room temperature to form 16a and 16c in 84% and
75% yields, respectively (Scheme 6). Contrary to our expectations,
the reductive de-benzylation of 14e, 14g and 15a with hydrazine
in the presence of 10% Pd/C in methanol caused elimination of
the 6-chloro group together with de-benzylation to produce 16b,
16d and 16e in 94%, 77% and 85% yields, respectively. In order to
examine the effects of the 6-chloro group on antitumor activity,
16e and 16b was treated with benzaldehyde to form the corre-
sponding 3-benzylamino derivatives 17a and 17b in 60% and 79%
yields, and with 3-phenoxybenzaldehyde to form 17c in a 57%
yield.
Synthesis of b-carboline derivatives 14a–m
Compd
R
Conditions
Yield (%)
Methoda
Time (h)
14a
14b
14c
14d
14e
14f
Me
Et
n-Pr
iso-Pr
n-Bu
n-Pentyl
A
A
B
A
A
C
1
2
8
6
6
0.5
87
84
74
94
94
58
14g
14h
14i
C
B
B
0.5
12
7
58
76
72
N
N
14j
B
12
88
N
14k
14l
14m
CF3
CF2CF3
CF2CF2CF3
A
A
A
18
3
99
91
91
We synthesized b-carboline derivatives 19, 20, and 21 having a
dimethylamino group on the 8-carbon atom from 11 as shown in
Scheme 7. We reduced the nitro group of 11 with 10% hydrazine
monohydrate and palladium on activated carbon to produce 8-ami-
no-3-acetoamido-b-carboline (18) in a 44% yield, accompanied
with de-chlorination. We completed the total synthesis of 8-di-
methyl-amino-3-(3-phenoxy-benzyl)amino-b-carboline (21) in 13
3
a
Reagents and conditions: Method A: (RCO)2O (1.0 equiv), DMAP (1.2 equiv),
CHCl3, room temperature, 1–24 h; Method B: RCOOH (1 equiv), DCC (1.2 equiv),
DMAP (1.2 equiv)/CH2Cl2, room temperature, 7–12 h; Method C: RCOCl (1 equiv)/
CHCl3, room temperature, 0.5 h.
steps from L-tryptophan in a 4% overall yield after methylation of
C; see Table 1, 14f, g). However, the yields decreased to 58% in
Method C.
We synthesized b-carboline derivatives having a sulfonamido
group as shown in Scheme 4. We obtained the compounds 14n,o
from 1 by the reaction with the corresponding alkanesulfonyl chlo-
ride in the presence of triethylamine and DMAP in dichloromethane.
the 8-amino group of 18 (19, 62% yield), de-acetylation on the 3-
amino-nitrogen atom of 19 (20, 70% yield), and benzylation of the
3-amino-nitrogen atom of 20 (68% yield). We synthesized 3-ben-
zylamino- and 3-(3-phenoxybenzyl)-amino-b-carboline (10d,e)
analogously by reductive amination of benzaldehyde or 3-phen-
oxybenzaldehyde with 3-amino-b-carboline (24).
H
H
Cl
Cl
N
N
R3SO2Cl (1.2 eq)
TEA (10 eq)
N
N
N
H
N
H
DMAP (0.2 eq)
R
O
NH
O
NH2
S
1
14n: 47% (R3 = Me)
14o: 56% (R3 = Et)
CH2Cl2, rt, 24 h
Scheme 4. Synthesis of 14n and 14o.

R. Ikeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3506–3515
3509
Method D:
a: 1) (CH2O)n (5eq),
NaOMe (5 eq),
MeOH, reflux,
H
N
H
Cl
2 h
N
Cl
2) NaBH4 (5 eq)/
MeOH,
N
N
N
H
reflux, 1 h
N
H
NH
R1
b: 1) aldehyde or
NH2
ketone (1 eq),
1
15a-k
AcOH (pH= 4)/MeOH
rt, 5 h
2) NaBH3CN (1.5 eq),
MeOH, rt, 1 h
Method E:
H
N
Cl
O
LiAlH4 (2 eq)
THF, reflux, 2 h
N
N
H
NH
14a, 14b, 14c
R
Scheme 5. Synthesis of 15a–k.
Table 2
on the 3-amino-nitrogen atom by reductive amination to produce
28 in a 50% yield. Finally, we reduced 28 by SnCl2/HCl leading to
the formation of the objective compound 29 in an 81% yield
(Scheme 8).
Synthesis of b-carboline derivatives 15a–k
H
Cl
N
We modified of the 6-amino group of 29 using three methods: F,
G and H (Scheme 9 and Table 3). In Method F, we reductively ami-
nated aldehyde with 29 and sodium cyanoborohydride to produce
the corresponding compounds 30a,b in 80% and 65% yields. In
Method G, we treated 29 with alkanesulfonyl chloride, carboxylic
acid anhydride, or carboxylic acid chloride in the presence of
triethylamine and DMAP in THF to produce the corresponding
compounds 30c–e,g in 25%, 50%, 74%, and 70% yields, respectively.
In Method H, we reductively methylated 29 with paraformalde-
hyde and sodium cyanoborohydride to produce 30f in 60% yield.
Next, we evaluated the antitumor activity of b-carboline deriv-
atives, thus obtained, by MTT assay for HeLa S-3, Sarcoma 180, and
293T cell lines. First, we applied a series of 8-carboxamino deriva-
tives 14a–m, and the results are summarized in Table 4. The com-
pound 14e showed the highest antitumor activity against HeLa S-3,
Sarcoma 180, and 293T; the IC50 values were 0.99, 0.53, and
N
N
H
NH
R1
Compd
R1
Methoda
Yield (%)
15a
15b
15c
15d
5e
Me
D-a
Eb
E
D-b
E
79
69
53
82
83
Et
n-Pr
iso-Pr
n-Bu
15f
D-b
D-b
99
97
N
15g
N
15h
15i
15j
D-b
D-b
D-b
85
93
53
0.50 lM, respectively. These results suggested that the introduc-
tion of an alkyl group promotes antitumor activity, because in-
creased hydrophobicity of a compound enhances cell membrane
permeability. However, the introduction of the acyl–alkyl group,
including more than four carbons, inhibited its activity. In addition,
sarcoma 180 cells tend to be more sensitive to 14a–m than HeLa S-
3 cells. The toxicity for 293T cells was affected to the same degree.
Secondly, we evaluated the antitumor activities of 8-amino-
N
O
S
15k
D-b
64
and 8-alkylamino-3-benzylamino-6-chloro-b-carbolines
1 and
a
Reagents and conditions: Method D-a: (CH2O)n (5 equiv), NaOMe (5 equiv),
15a–k, and the results are shown in Table 5. Generally speaking,
the activities of 15a–k were higher than those of 14a–m. In partic-
ular, the antitumor activity of the 8-methylamino derivative 15a
was highest against HeLa S-3, Sarcoma 180 and 293T cells; the
MeOH, reflux, 2 h, and then NaBH4 (5 equiv)/MeOH, reflux, 1 h; Method D-b:
aldehyde or ketone (1 equiv), AcOH (pH = 4)/MeOH, room temperature, 5 h, and
then NaBH3CN (1.5 equiv), MeOH, room temperature, 1 h; Method E: LiAlH4
(2 equiv), THF, reflux, 2 h.
b
By Method D-a: the corresponding N,N-diethylamino derivative 5b0 was
IC50 values were 0.20, 0.25 and 0.35 lM, respectively (Table 5).
obtained with 88% yield.
Subsequently, we evaluated the antitumor activities of the com-
pounds 9, 10b–e, 14n,o, 16a–e, and 17a–c, with various substitu-
ents at R3, R1 and/or R7, and those of the corresponding mother
compound 24 against HeLa S-3 and Sarcoma 180 cells using the
same method (Table 6). 3-(3-Phenoxybenzyl)amino-9H-b-carbo-
line (10e) is indicating the highest antitumor activity among the
b-carboline derivatives that we have reported previously.9 In this
study, we tried to develop b-carboline derivatives having higher
antitumor activity than 10e. Initially, we examined the effect of
the R3 group on activity. A benzyl group on the 3-amino-nitrogen
In order to synthesize the 3,6-diamino-b-carboline derivative
29, we synthesized 3-amino-b-carboline (24) from 5 by a method
reported previously8 (Scheme 8). After protecting the 3-amino
group by an acetyl group (25, 78% yield), we nitrated 25 to afford
the corresponding 6-nitro compound in a 93% yield. Then we re-
moved the acetyl group (73% yield) and introduced a benzyl group

3510
R. Ikeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3506–3515
H
DDQ (1.2 eq)
Cl
N
NH2
Cl
N
1, 4-dioxane
rt, 30 min
N
N
H
N
H
R'
R'
14e: R' = NHCOn-Bu
14g: R' = NHCOPh
15a: R' = NHMe
16a: 84% (R' = NHCOPh)
16c: 75% (R' = NHCOn-Bu)
NH2NH2 . H2O (20 eq)
10% Pd/C (0.5 eq)
MeOH, reflux, 5 h
1) aldehyde (1 eq)
AcOH (pH = 4)
H
N
NH2
2) NaBH3CN (1.5 eq)
R''
N
N
MeOH, rt, 4 h and
then 1 h
N
H
N
H
R'
R'
17a: 60% (R' = NHMe, R'' = H)
16b: 94% (R' = NHCOPh)
17b: 79% (R' = NHCOPh, R'' = H)
17c: 57% (R' = NHMe, R'' = OPh)
16d: 77% (R' = NHCOn-Bu)
16e: 85% (R' = NHMe)
Scheme 6. Synthesis of 17a–c.
NH2NH2 . H2O (10 eq)
10% Pd/C (0.5 eq)
H
N
H
N
Cl
O
O
N
N
MeOH, reflux, 5 h
N
H
N
H
NO2
NH2
11
18: 44%
H
N
1) HCHO aq (5 eq)
AcOH (pH=4)
O
N
N
H
1N-HCl aq (10 eq)
reflux, 12 h
2) NaBH3CN (10 eq)
N
19: 62%
DMF, 0 °C, 2 h
1)
H
N
NH2
O
O
CHO
N
N
AcOH (pH=4)
N
H
N
H
2) NaBH3CN (1.5 eq)
N
N
MeOH, rt, 4 h
and then 1 h
20: 70%
21: 68%
Scheme 7. Synthesis of 21.
atom also strikingly increased the antitumor activity of 6-chloro-b-
carboline (9),7–9 as shown by the IC50 values of 9 and 10b. In addi-
tion, the lower activities of 16a–e, which have no benzyl group on
the amino-nitrogen atom, supported this effect. The effect of the
R6 group on the activity was strange. The 6-chloro group of 10b,c
decreased the antitumor activity of the corresponding un-chlori-
nated derivatives 10d,e (Table 6). In contrast, the 6-chloro substitu-
ent of 16a,c increased the antitumor activity of the corresponding
un-chlorinated derivatives 16b,d. It is not clear why or how these
substituent effects occurred. The results, however, were quite inter-
esting, and they will be made clear in the near future.
phenoxybenzyl)-amino-b-carboline 10d,e (0.11 and 0.074 lM,
respectively). As a result, we succeeded in the development of
the compounds 17a and 17c with higher activity than that of 10e.
Finally, we evaluated the antitumor activity of 3-benzylamino-
b-carboline derivatives with substitutes other than a chloro group
at R6, 28, 29, and 30a–g, and the results are shown in Table 7,
which shows that antitumor activities were lower than those of
10d; IC50 values were 0.11 0.01 lM against HeLa S-3 cells and
0.44 0.04 lM against Sarcoma 180 cells. Acceleration of antitu-
mor activity by the methylamino group was not observed at R6,
which was different from R8.
Next, we discuss the effect of the R8 group on the activity. The
8-methane- or 8-ethanesulfonamido group of 14n,o slightly de-
creased (or had little effect on) the antitumor activity of the corre-
sponding mother compound 10b. At last, we succeeded in finding
two highly potent antitumor compounds: 8-methyl-amino-3-ben-
zylamino-b-carboline 17a and 8-methyl-amino-3-(3-phenoxyben-
zyl)amino-b-carboline 17c. The IC50 values of these two
In order to determine whether a change in the cell death path-
way depending on the presence or absence of Cl group at R6, we
investigated the type of cell death using two pairs of compounds
10b, 10d, and 14g, 17b. Both chromatin condensation and frag-
mented nuclei reportedly are the classic characteristics of apopto-
sis. Hoechst 33342 staining showed significant morphological
changes in the nuclear chromatin. After treatment with 10b, 10d,
14g, or 17b, we observed the nuclear chromatin (Fig. 1). Compared
with the compound-treated cells, untreated cells exhibited less
compounds were 0.046 and 0.032
lM against HeLa S-3, respec-
tively, which are smaller than those of 3-benzylamino- and 3-(3-

R. Ikeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3506–3515
3511
O
O
O
NHNH
2
.
NH
N
NH NH H O (25 eq)
2
2
2
N
H
N
H
1-pentanol, reflux, 7 h
O
22: 83%
5: 69%
NaNO (3 eq)
2
50% AcOH aq
reflux, 1 h
0.2 N-HCl aq (2eq)
N
3
N
H O, 0 °C, 2 h
2
N
H
23: 89%
H
N
NaNO (5 eq)
3
NH
2
Ac O (5 eq)
2
TFA (20 eq)
O
N
N
N
H
rt, 18 h
N
H
CHCl3, reflux, 1 h
25: 78%
24: 78%
H
O N
2
NH
2
N
O N
2
1 N HCl aq
reflux, 12 h
O
N
N
N
H
N
H
27: 73%
26: 93%
H
N
1) PhCHO (1.0 eq), AcOH (pH=4)
O N
2
2) NaBH CN (1.5 eq)
3
N
N
H
MeOH, rt, 4 h and then 1 h
28: 50%
H
N
H N
2
.
SnCl 2H O (3.5 eq)
2
2
N
12 N HCl aq (50 eq)
N
H
MeOH, rt, 48 h
29: 81%
Scheme 8. Synthesis of 29.
H
N
H2N
N
N
H
Method F:
1) aldehyde (1.0 eq), AcOH (pH = 4)/MeOH,
rt, 4 h
29
2) NaBH3CN (1.5 eq)/MeOH, rt, 1 h
Method G:
R'SO2Cl or(R'CO2)O or R'COCl (1 eq),
TEA (1.5 eq), DMAP (0.2 eq)/THF, rt, 1-18 h
Method H:
1) NaOMe (5eq), (CH2O)n(5 eq)/MeOH,
reflux, 2 h
2) NaBH4 (5 eq)/MeOH, reflux, 1 h
H
N
H
N
R
N
N
H
30
Scheme 9. Synthesis of 30
intense nuclear staining. However, a large proportion of 10b-, 10d-,
14g-, and 17b-treated cells exhibited fragmented nuclei.
To verify induced cell death types, we performed a DNA frag-
mentation assay. DNA fragmentation is well known as the typical
biochemical index of cell apoptosis. After exposure to 10d, 10b,
14g, and 17b for 48 h, we extracted and detected the genomic
DNA by electrophoresis (Fig. 2). We detected DNA fragmentation
in cells treated with either compound. Together with fluorescence

3512
R. Ikeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3506–3515
Table 3
Table 5
Synthesis of b-carboline derivatives 30a–g
Antitumor activity of 8-amino-b-carboline derivatives against HeLa S-3 cells
Compd
R
Methoda
F
Yield (%)
80
H
N
Cl
30a
N
N
H
30b
30c
F
65
25
NH
R
O
Compd
R
IC50, l
Ma
S
G
O
HeLa S-3
Sarcoma 180
293T
O
1
H
Me
Et
n-Pr
iso-Pr
n-Bu
6.09 0.09
0.200 0.004
0.27 0.02
2. 9 0.1
1.1 0.1
2.60 0.06
3.24 0.2
0.25 0.05
0.24 0.03
1.36 0.07
—
9.8 0.2
0.35 0.01
0.36 0.01
2.0 0.2
0.7 0.1
2.1 0.2
S
30d
G
50
15a
15b
15c
15d
15e
O
O
30e
30f
G
H
74
60
1.6 0.2
O
Me
15f
15g
15h
15i
4.2 0. 1
1. 5 0.1
1.2 0.1
1.4 0.2
2.6 0.1
1.0 0.1
0.76 0.05
1.0 0.1
3.8 0.4
1.1 0.2
1.0 0.2
1.4 0.1
30g
G
75
O
N
a
Reagents and conditions: Method F: aldehyde (1.0 equiv), AcOH (pH = 4)/MeOH,
room temperature, 4 h, and then NaBH3CN (1.5 equiv)/MeOH, room temperature,
1 h; Method G: R0SO2Cl or (R0CO2)O or R0COCl (1 equiv), TEA (1.5 equiv), DMAP
(0.2 equiv)/THF, room temperature, 1–18 h; Method H: NaOMe (5 equiv), (CH2O)n
(5 equiv)/MeOH, reflux, 2 h, and then NaBH4 (5 equiv)/MeOH, reflux, 1 h.
N
Table 4
Antitumor activity of 8-carboxamino-b-carboline derivatives against HeLa S-3 cells
N
H
N
Cl
15j
3.8 0.2
4.2 0.1
—
3.1 0.4
4.0 0.2
N
N
H
O
S
O
NH
R
15k
1.69 0.09
Compd
R
IC50, l
Ma
HeLa S-3
21
9.9 0.3
2.6 0.1
Sarcoma 180
293T
a
Drug toxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
14a
14b
14c
14d
14e
14f
Me
Et
n-Pr
iso-Pr
n-Bu
1
16.2 0.4
5.4 0.3
1.35 0.02
8.0 0.3
15
1
tetrazolium bromide (MTT) assay. IC50 is drug concentration inhibited the 50% cell
growth. Data represent the mean S.D. from four independent experiments per-
formed in triplicate experiments.
5.8 0.5
2.3 0.2
9.0 0.4
8
1
0.99 0.04
2.13 0.04
0.53 0.02
0.92 0.07
0.50 0.05
1.4 0.3
n-Pentyl
microscopy analysis, these observations demonstrated that the
compounds 10d, 10b, 14g, and 17b cause apoptotic cell death.
These results showed that the presence or absence of Cl group at
R6 does not change the type of cell death.
14g
14h
14i
4.60 0.04
3.7 0.3
2.8 0.2
1.9 0.1
8.3 0.5
4.8 0.4
4.0 0.3
To examine the effects on cell cycle progression, we exposed the
cells to 10d, 10b, 14g, and 17b for 9–48 h. As shown in Table 8, we
detected the G2/M phase population in the cells that were
compound-treated for 9 h. The average G2/M population in the
control cells and in 10d-, 10b-, 14g-, and 17b-treated cells were
16.21, 57.00, 46.93, 49.09, and 65.56%, respectively. In addition,
the population for 24 and 48 h had a tendency to shift from the
G2/M phase to the mean apoptotic population sub-G1 phase. These
results may suggest that 10d, 10b, 14g, and 17b induced cell death
through G2/M cell cycle arrest.
The antitumor activity of many anti-cancer drugs results mostly
from interference with cell cycle progression, inhibition of
activation of cell cycle regulation, inhibition of DNA replication,
induction of DNA damage, or disruption of mitototic spindle for-
mation.14,15 In the present study, we also determined the cell death
type and the effects on cell cycle progression in 10b-, 10d-, 14g-,
N
15
13
4
2
10
8
3
N
14j
7.9 0.5
1
N
14k
14l
14m
CF3
6.3 0.5
9.2 0.1
7.0 0.4
5.1 0.5
6.1 0.2
5.2 0.5
—
—
—
CF2CF3
CF2CF2CF3
a
Drug toxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay. IC50 is drug concentration inhibited the 50% cell
growth. Data represent the mean S.D. from four independent experiments per-
formed in triplicate experiments.

R. Ikeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3506–3515
3513
Table 6
Antitumor activity of R3-, R6-or/and R8-substituted b-carbolines derivatives against HeLa S-3 cells
H
R6
N
R3
N
N
H
R8
10b-e, 14n, 14o, 15a-e, 17a-c
Compd
R6
R8
R3
IC50, l
Ma
HeLa S-3
71
Sarcoma180
29
9
Cl
Cl
H
H
H
7
2
10b
2.8 0.1
1.8 0.1
1.1 1.1
O
10c
Cl
H
H
H
H
H
1.36 0.07
0.44 0.04
0.03 0.01
10db
0.11 0.01
0.074 0.07
O
b
10e
NH
14n
14o
Cl
Cl
8.0 0.1
—
—
S
O
S
O
NH
4.84 0.06
O
O
O
O
NH
16a
Cl
H
26.1 0.8
12.4 0.9
NH
16b
16c
H
H
H
118
27
8
1
98
7
O
O
NH
NH
Cl
18.1 0.3
16d
16e
H
H
H
H
56
12
4
1
38
—
2
NH
NH
17a
17b
H
H
0.046 0.001
0.141 0.003
0.024 0.001
O
NH
—
(continued on next page)

3514
R. Ikeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3506–3515
Table 6 (continued)
Compd
R6
R8
R3
IC50, l
Ma
HeLa S-3
Sarcoma180
—
O
NH
17c
H
H
0.032 0.001
24
H
H
66
3
41
1
a
Drug toxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. IC50 is drug concentration inhibited the 50% cell growth.
Data represent the mean S.D. from 4 independent experiments performed in triplicate.
b
The synthesis method for 10d, 10e has been previously reported.9
Table 7
Antitumor activity of 6-substituted 3-benzylamino-b-carbolines against HeLa S-3 cells
H
R6
N
N
N
H
28, 29, 30a-g
Compd
R6
IC50, l
Ma
28
29
NO2
NH2
28
11
1
1
30a
30b
21
15
1
1
NH
O
NH
30c
30d
19
1
S
O
O
NH
3.4 0.3
S
O
O
Figure 2. Effects of 10d, 10b, 14g, and 17b on apoptosis induction in HeLa S-3 cells.
We treated cells with each compound for 48 h. We detected DNA fragmentation by
electrophoresis on a 2% agarose gel. Marker; 100 bp ladder.
NH
30e
30f
14
10
1
1
O
MeNH
NH
30g
10
1
and 17b-treated HeLa S-3 cells. In conclusion, 10b, 10d, 14g, and
17b are a new class of drugs for cell-cycle arrest and induction of
apoptosis. The present study explored the molecular mechanisms
of action for a b-carboline derivative and further characterized its
potential as an anti-cancer agent.
O
a
We determined drug toxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) assay. IC50 is drug concentration inhibited the 50% cell
growth. Data represent the mean S.D. from four independent experiments per-
formed in triplicate.
Figure 1. HeLa S-3 cells which were treated with the compounds 10d, 10b, 14g, and 17b for 48 h. Apoptosis was detected by Hoechst 33342 staining.

R. Ikeda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3506–3515
3515
Table 8
2004–2006 (No. 16550148) and 2009–2011 (No. 21590025); a
Grant for the ‘‘High-Tech Research Center’’ Project for Private
Universities, a matching fund subsidy from MEXT, 2000–2004
and 2005–2007; a Grant from the Japan Private School Promotion
Foundation (2008–2009); and a Program for Development of
Strategic Research Center in Private Universities supported by
MEXT, Center for Technologies against Cancer (CTC), 2009–2013.
Effects of 10d, 10b, 14g, and 17b on the distribution of cell-cycle phase in HeLa S-3a
Treatment
SubG1
G1
S
G2/M
10d
Control
9 h
24 h
48 h
10b
2.41
6.08
16.18
18.82
75.29
22.64
6.93
6.36
14.28
9.18
16.21
57.00
67.71
52.75
12.21
16.22
Control
9 h
24 h
48 h
14g
Control
9 h
24 h
48 h
17b
2.41
9.14
14.59
21.40
75.29
33.02
8.20
6.36
10.91
7.74
16.21
46.93
69.47
49.04
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.
077.
18.25
11.31
2.41
6.59
14.76
21.96
75.29
33.91
32.43
11.69
6.36
10.41
12.72
11.06
16.21
49.09
40.09
55.29
References and notes
Control
9 h
24 h
48 h
2.41
3.89
5.87
75.29
21.15
12.82
9.36
6.36
9.40
10.18
9.05
16.21
65.56
71.13
63.42
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We would like to thank Dr. Okada (Institute for Biological Re-
sources and Functions, National Institute of Advanced Industrial
Science and Technology) for kindly providing the HeLa S-3 cells.
This work was partially supported by a Grant-in-Aid for Scientific
15. Jordan, M. A. Curr. Med. Chem. Anti-Cancer Agents 2002, 2, 1.
Research from MEXT,
a matching fund subsidy from MEXT