Structure-activity relationship of N-methyl-bisindolylmaleimide derivatives as cell death inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.04.015

A series of N-methyl-bisindolylmaleimide derivatives was synthesized and evaluated as cell death inhibitors. N-Methyl-2-[1-(3-aminopropyl)-1H-indol-3-yl] -3-(1H-indol-3-yl)maleimide (21) was the most potent inhibitor of H ₂O₂-induced necrotic death of human leukemia HL60 cells among them.

Full text of DOI:10.1016/j.bmcl.2005.04.015

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3109–3113
Structure–activity relationship of
N-methyl-bisindolylmaleimide derivatives as cell death inhibitors
a,b
Miho Katoh, Kosuke Dodo, Mikako Fujita and Mikiko Sodeoka
a,c
b
a,b,c,
*
a
Institute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku University, 2-1-1 Katahira, Aoba, Sendai,
Miyagi 980-8577, Japan
b
Sagami Chemical Research Center, Kanagawa, Japan
RIKEN (The Institute of Physical and Chemical Research), 2-1, Hirosawa, Wako, Saitama 351-0198, Japan
c
Received 26 March 2005; revised 9 April 2005; accepted 11 April 2005
Available online 17 May 2005
This paper is dedicated to Prof. Iwao Ojima on his 60th birthday
Abstract—A series of N-methyl-bisindolylmaleimide derivatives was synthesized and evaluated as cell death inhibitors. N-Methyl-2-
1-(3-aminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide (21) was the most potent inhibitor of H -induced necrotic death of
human leukemia HL60 cells among them.
2005 Elsevier Ltd. All rights reserved.
[
2 2
O
ꢀ
5
Cell death signaling is currently one of the hottest topics
in biological research. A great deal of knowledge has
been accumulated on apoptosis, a naturally occurring
process of cell suicide, which plays a crucial role in the
development and maintenance of multicellular organ-
stroke and neurodegenerative disorders. Recently, Asa-
kai et al. reported that bisindolylmaleimide I (BM I or
GF109203X) inhibited necrotic cell death induced by
oxidative stress, such as H O or sodium nitroprusside
2
2
(SNP, in vivo NO donor), in a variety of primary-cul-
6
1
isms. Signals of typical physiological apoptosis are
tured cells (Fig. 1). They also reported that necrotic cell
death induced by H O was inhibited by a well-known
mediated by a cascade of proteases, called caspases,
and the use of low-molecular-weight caspase inhibitors
has made a major contribution to apoptosis re-
2
2
chemical antioxidant, N-acetylcysteine (NAC), but not
by a caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-
Asp(OMe)-fluoromethyl ketone (Z-VAD). Since BM I
was originally reported as a strong protein kinase C
1
b,1c
search.
pase-independent cell death signaling pathways has
Recently, evidence for the existence of cas-
2
7
been accumulating. Such caspase-independent path-
ways are suggested to be involved in some types of
necrosis, which were classically regarded as passive,
(PKC) inhibitor, PKC inhibition was considered likely
to be a mechanism of action. However, the experimental
fact that bisindolylmaleimide V (BM V), a bisindolyl-
maleimide derivative that has no PKC-inhibitory activ-
ity, showed weaker but significant inhibition of cell
death, suggested that PKC inhibition is not essential
for the cytoprotective effect. Moreover BM I and V
did not inhibit caspase-dependent apoptotic cell death.
Thus, it is likely that these compounds inhibit a novel
3
unregulated cell death. The molecular basis of cas-
pase-independent cell death pathways, however, remains
to be clarified, despite its importance, for example, in
ischemia-reperfusion injury and neurodegenerative dis-
4
orders. Small molecules, which specifically inhibit cas-
pase-independent cell death pathways would be
powerful biological probes. Furthermore, a specific cell
death inhibitor that did not affect physiologically essen-
tial apoptosis could be a lead compound for developing
therapeutic agents for the treatment of diseases such as
Keywords: Cell death; Necrosis; Apoptosis; Bisindolylmaleimide.
*
Corresponding author. Tel./fax: +81 22 217 5601; e-mail:
sodeoka@tagen.tohoku.ac.jp
Figure 1. Structures of BM I and BM V.
0
960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.015

3110
M. Katoh et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3109–3113
caspase-independent signaling pathway, and it is of
interest to clarify their molecular mechanism of action.
BM I itself is, however, not suitable for this purpose, be-
cause it inhibits not only PKC, but also several other ki-
8
nases, and is cytotoxic at high concentration. BM V has
a negligible inhibitory effect on several kinases including
8
PKC, but its cell death-inhibitory activity is not potent
enough for mechanistic studies. Therefore, a highly spe-
cific and potent derivative is required. Herein, we report
the structure–activity relationship of bisindolylmaleim-
ide derivatives and the development of a novel cell death
inhibitor which is more potent than BM I and lacks
strong PKC-inhibitory activity.
Figure 3. Cell death inhibition by BM I, BM V, Z-VAD, and NAC.
TM
Cell viability was determined by AlamarBlue assay.
First, we established an assay system not involving pri-
mary-cultured cells, which are unsuitable for mechanis-
tic study. After several trials, human leukemia HL60
cells were found to be suitable. As shown in Figure 2,
morphologically typical necrotic cell death was induced
examined for cell death inhibitory activity and cytotox-
icity at 3 lM (Fig. 4). Succinimide derivatives 1 and 2
showed negligible cell death inhibition. The maleic
anhydride derivative 3 and amide derivative 4 exhibited
some inhibition, but were much less potent than BM V.
These results suggest that the maleimide ring is essential
for cell death inhibition. Next, we examined the effects
of various substituents on the indole rings. Since the
maleimide N–H group of bisindolylmaleimide deriva-
tives and indolocarbazole derivatives such as stauro-
sporine is critical for the inhibition of various
9
by 100 lM H O in HL60 cells. As in the case of the cul-
2
2
tured cells reported by Asakai et al., a high concentra-
tion of NAC strongly inhibited the H O -induced
2
2
death of HL60 cells, whereas the effect of a general cas-
pase inhibitor, Z-VAD, was quite limited even at a high
concentration (Fig. 3). BM I and V significantly inhib-
1
0
ited cell death (Fig. 3) even at 1 lM. These results im-
ply that the cell death induced in this assay system is not
mediated by caspases, but rather, similar molecular
mechanisms to those of the necrotic cell death of pri-
mary-cultured cells reported by Asakai may be involved.
However, the effects of BM I and BM V were limited in
this system. At lower concentrations, dose-dependent
improvement of the viability of HL60 cells was ob-
served, but at higher concentrations the viability reached
a plateau (Fig. 3), probably due to cytotoxicity at these
concentrations. In fact BM I showed some cytotoxicity
to HL60 cells (with 10 lM BM I, viability was reduced
to 81% without H O treatment). The observed cytotox-
1
2
kinases, we decided to retain the N-methyl-maleimide
structure to avoid undesired interaction with PKC and
other kinases. As shown in Table 1, the N-methyl bisin-
dolylmaleimide derivatives 6–14 were synthesized from
1
1a,13
5a or 5b in good yields.
Figure 4a and b shows
the cell death-inhibitory activity and cytotoxicity of
these compounds at 3 lM, respectively. Among the
methyl substituted derivatives 6–10, the 2- and 4-substi-
tuted derivatives 6 and 7 had very weak activity, whereas
the 5-, 6-, and 7-methyl derivatives 8–10 inhibited cell
death much more potently than did BM V. BM V
(and also 8–10) can take a conformation in which one
of the indole rings is coplanar with the maleimide ring
and the p-electrons can be delocalized over the indole
and maleimide rings. However, the 2- and 4-substituted
derivatives 6 and 7 cannot readily take such a coplanar
conformation due to the severe steric repulsion between
the methyl group and the carbonyl oxygen or the other
indole ring. The observed drastic difference depending
on the position of the substituent may suggest the
2
2
icity may be induced by the inhibition of kinases. There-
fore, by using this assay system, both cytoprotective and
cytotoxic activities of the bisindolylmaleimide deriva-
tives could be evaluated in terms of the viability of
H O @treated and untreated HL60 cells.
2
2
First, compounds 1–4, BM V derivatives modified on
1
the maleimide ring, were synthesized (Scheme 1) and
1
2 2
Figure 2. Phase-contrast micrographs of HL60 cells. (a) Intact cells; (b) typical necrotic cell death induced by H O (100 lM, 3 h).

M. Katoh et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3109–3113
3111
Scheme 1. Conversion of maleimide ring. Reagents and conditions: (a) Mg, MeOH (84%); (b) H
N HClaq (71%); (d) LiAlH , THF (60%).
2
, Pd/C, DMF (92%); (c) 10% KOHaq, reflux, then
2
4
importance of such a coplanar structure for the cell
death-inhibitory activity. Among the methyl-substituted
derivatives, the 6-methyl derivative 9 had the strongest
activity, but unfortunately it showed some cytotoxicity.
Cytotoxicity was also observed for the 7-methyl deriva-
tive 10, but the 5-methyl substituted derivative 8 had
strong activity without cytotoxicity. Thus, we examined
other 5-substituted derivatives 11–14, but none of them
were superior to 8.
Next, we examined the effects of the substitution at the
7
,14
indole nitrogen. Compounds 17–20 were synthesized
from BM V or 15 (Scheme 2) and tested for activity (Fig.
). Methyl-substituted derivatives 17 and 18 showed not
4
only improved cell death inhibition, but also showed
cytotoxicity. Introduction of the dimethylaminopropyl
group(s), which is the same substituent as in BM I, in-
creased the inhibitory activity, and the mono-substituted
derivative 19 was more potent than the di-substituted
derivative 20. Finally, the mono-aminopropyl derivative
Figure 4. Activities of various bisindolylmaleimide derivatives. (a) Cell
death inhibition. Viability of HL60 cells treated with H (100 lM,
h) in the absence (control) or presence of 3 lM bisindolylmaleimide
derivative. (b) Cytotoxicity. Viability of HL60 cells treated with 3 lM
bisindolylmaleimide derivative.
2 2
O
21 was found to be the best compound. It has the high-
3
est cell death-inhibitory activity among the bisindolyl-
maleimide derivatives synthesized and showed no
cytotoxicity.
The dose–response curve of 21 is shown in Figure 5.
Although, as with BM I and BM V, the activity curve
reaches a plateau at high concentration, 21 is much
more potent than BM I and BM V under our assay con-
ditions. Finally, we also synthesized compounds 22 and
Table 1. Synthesis of bisindolylmaleimide derivatives
23, which have an aminopropyl group on one indole
nitrogen and methyl groups at the 5- or 6-position of
the two indole rings, in the expectation that both substit-
uents would contribute to improve the cell death inhibi-
tion (Scheme 2). However, greatly decreased cell
viability was observed at 10 lM for these compounds
Compound
X
R
Solvent
Yield (%)
(
Fig. 5).
BMV
Cl
Br
Cl
Cl
Br
Br
Cl
Cl
Cl
Cl
H
Toluene
THF
98
62
92
92
88
97
85
91
82
91
6
7
8
9
2-Me
4-Me
5-Me
6-Me
7-Me
5-F
Finally, we examined the inhibitory activities of 21
toward several kinases, which are reported to be inhib-
ited by BM I, to confirm that N-methylation of the
maleimide ring indeed completely suppresses the kinase
inhibitory activity. Table 2 summarizes the activities of
various kinases in the presence of 1 lM BM I (data cited
from Ref. 8) or 50 lM 21. Generally, as expected, the
N-methyl derivative 21 did not inhibit the kinases, except
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
8
1
1
1
1
1
0
1
2
3
4
5-CI
5-Br
5-OMe
1
5

3112
M. Katoh et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3109–3113
Scheme 2. Synthesis of N-substituted bisindolylmaleimide derivatives. Reagents and conditions: (a) MeI, K
toluene (93%); (c) MeI, K CO , DMF (99%); (d) NaH, Cl(CH NMe ÆHCl, DMF (80%); (e) NaH, Cl(CH
Cl(CH NH ÆHCl, DMF (21: 69%, 22: 81%, 23: 10%).
2
CO
3
, DMF (98%); (b) indolyl–MgBr,
ÆHCl, DMF (23%); (f) NaH,
2
3
2
)
3
2
2
)
3
NMe
2
2
)
3
2
for PKCa and S6K1. Considering the high dose of 21
compared to BM I, the weak inhibition of PKCa by 21
should have had little effect on the activity in our assay
system. However, the activity of S6K1 was reduced to
2
8% in the presence of 50 lM 21, and IC50 value of 21
for this kinase was determined to be 25 lM. Since
S6K1 is suggested to be one of the prosurvival and pro-
1
6
liferative kinases, inhibition of this enzyme may con-
tribute to the cytotoxicity at high concentrations of 21.
In conclusion, we have synthesized various N-methyl-
bisindolylmaleimide derivatives and elucidated their
basic structure–activity relationships as cell death inhib-
itors. The data should provide critical information for
the future design of more potent and specific cell death
inhibitors. Among these compounds, 21 was more po-
tent than BM I as a cell death inhibitor, with lower tox-
icity. Inhibition of various kinases by 21 was also tested.
In contrast to BM I, 21 showed negligible inhibition of
the tested kinases, except for S6K1, even at high concen-
tration. Removal of this S6K1 inhibitory activity may be
the key to the development of more advanced analogs.
Figure 5. Dose–response curves of bisindolylmaleimide derivative for
ability to improve the viability of HL60 cells treated with H
100 lM, 3 h).
2
O
2
(
Table 2. Kinase inhibition by bisindolylmaleimide derivatives
Kinase
Activity (% of control)
a
BMI (1 lM)
21 (50 lM)
PKCa
RSK2
MSK1
S6K1
4 ± 0
9 ± 2
66 ± 3
106 ± 1
90 ± 6
28 ± 0
94 ± 8
88 ± 1
93 ± 5
109 ± 14
Acknowledgments
21 ± 1
32 ± 8
50 ± 5
54 ± 0
60 ± 2
63 ± 6
We thank Professor Y. Hashimoto for the generous gift
of cell lines. We also thank Professor R. Asakai for dis-
cussions. This work was supported in part by a Grant-
in-Aid for Creative Scientific Research (No.
13NP0401), a Grant-in-Aid for Scientific Research from
the JSPS, and a Grant-in-Aid for Scientific Research on
GSK3b
AMPK
CHK1
SGK
a
Data cited from Ref. 8.

M. Katoh et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3109–3113
3113
Priority Areas (A) from The Ministry of Education,
Culture, Sports, Science, and Technology. K.D. was
supported by a JSPS Research Fellowship for Young
Scientists and by the Special Postdoctoral Researchers
Program in RIKEN.
test compounds (DMSO solution, 0.5 lL/well) for 1 h and
then H (in medium, 4 lL/well, 100 lM at final
2
O
2
concentration) was added (final volume 100 lL/well). In
all experiments, the final DMSO concentration was the
TM
same (0.5%). After 3 h, 10 lL of AlamarBlue (Biosource
International) was added to each well. The cell viability
was determined based on the increase of fluorescence
excitation 560 nm/emission 590 nm) during 4–5 h of
(
References and notes
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4
5
TM
15. KinaseProfiler
inhibition assays were performed using the KinasePro-
(Upstate USA, Inc.): Protein kinase
TM
filer
service (Upstate USA, Inc.). Briefly, protein
kinases were assayed for their ability to phosphorylate
the appropriate peptide/protein substrates in the presence
of 50 lM compound 21 and 100 lM ATP. Activities are
given as mean percentages of those in control incubations
(averages of duplicate determinations). Abbreviations of
kinase are as follows: AMPK, AMP-activated protein
kinase; CHK1, checkpoint kinase 1; GSK3b, glycogen
synthase kinase 3b; MSK1, mitogen- and stress-activated
protein kinase 1; PKC, protein kinase C; RSK2,
ribosomal S6 kinase 2; SGK, serum- and glucocorti-
coid-induced kinase; S6K1, p70 ribosomal protein S6
kinase 1.
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2
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. Cell culture: HL60 cells were maintained in RPMI 1640
medium supplemented with 5% heat-inactivated fetal
bovine serum (FBS), 100 U/mL penicillin, and 100 lg/
mL streptomycin. Cells were grown in a humidified
8
9
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incubator at 37 ꢁC under 5% CO
2
/95% air.
TM
4
1
0. AlamarBlue assay: HL60 cells (4 · 10 cells/well) were
suspended in fresh medium in a 96-well plate (95.5 lL/
well). After 2 h of incubation, the cells were treated with