Inhibition of hydrogen peroxide-induced necrotic cell death with 3-amino-2-indolylmaleimide derivatives

Article abstract of DOI:10.1016/j.bmcl.2005.04.016

Novel analogs of indolylmaleimide derivatives (IM derivatives) were synthesized and tested for cell death-inhibitory activity. 2-(1H-Indol-3-yl)-3- pentylamino-maleimide IM-54 was the most effective cell death inhibitor among the compounds tested. IM-54 inhibited necrotic cell death induced by H ₂O₂, but not apoptotic cell death induced by etoposide. These results indicated that this novel cell death inhibitor is distinct from the well-known caspase inhibitor, Z-VAD, which can block apoptotic cell death, but not necrotic cell death. IM-54 is expected to be a powerful bioprobe for clarifying the unique signaling pathway of necrotic cell death.

Full text of DOI:10.1016/j.bmcl.2005.04.016

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3114–3118
Inhibition of hydrogen peroxide-induced necrotic cell death with
3-amino-2-indolylmaleimide derivatives
Kosuke Dodo,a,b Miho Katoh,a,c Tadashi Shimizu,a Masahiro Takahashia
and Mikiko Sodeokaa,b,c,
*
aInstitute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku University, 2-1-1 Katahira, Aoba, Sendai,
Miyagi 980-8577, Japan
bRIKEN (The Institute of Physical and Chemical Research), 2-1, Hirosawa, Wako, Saitama 351-0198, Japan
cSagami Chemical Research Center, Kanagawa, Japan
Received 26 March 2005; revised 9 April 2005; accepted 11 April 2005
Available online 4 May 2005
This paper is dedicated to Professor Iwao Ojima on his 60th birthday
Abstract—Novel analogs of indolylmaleimide derivatives (IM derivatives) were synthesized and tested for cell death-inhibitory
activity. 2-(1H-Indol-3-yl)-3-pentylamino-maleimide IM-54 was the most effective cell death inhibitor among the compounds tested.
IM-54 inhibited necrotic cell death induced by H2O2, but not apoptotic cell death induced by etoposide. These results indicated that
this novel cell death inhibitor is distinct from the well-known caspase inhibitor, Z-VAD, which can block apoptotic cell death, but
not necrotic cell death. IM-54 is expected to be a powerful bioprobe for clarifying the unique signaling pathway of necrotic cell
death.
Ó 2005 Elsevier Ltd. All rights reserved.
In the previous paper,1 we reported the structure–activ-
ity relationship of bisindolylmaleimide derivatives as
inhibitors of H2O2-induced necrotic cell death, and
established that compound 1 is a more potent cell death
inhibitor than the previously reported compound, BM I2
(Fig. 1). Although 1 was much less toxic than BM I, it
still had some toxicity at a very high concentration. Ki-
nase inhibition profiling suggested that this toxicity may
be due to the inhibition of S6K1. Thus, more specific
analogs lacking S6K1-inhibitory activity are desirable
for mechanistic studies. Here we report a novel indo-
lylmaleimide derivative (IM-54) with a strong cell
death-inhibitory activity but without inhibitory activity
for S6K1 or any examined subtype of PKCs.
Based on the SAR of the bisindolylmaleimides reported
in the previous paper,1 we hypothesized that the coplan-
arity of one indole ring with the maleimide ring is
important for the activity, and we expected that removal
of the second indole ring would favor such a coplanar
conformation. Thus, the mono-indolylmaleimide, IM-
1, was synthesized from 23 according to the reported
procedure4 (Scheme 1), and tested as an inhibitor of
necrotic cell death induced by H2O2 (100 lM) with the
assay system using HL60 cells described in the previous
paper.1 Contrary to our expectation, IM-1 (3 lM)
showed negligible inhibition, being similar in this respect
to the corresponding succinimide derivative 2 (Fig. 2a).
Interestingly, however, the N-methylated derivative IM-
2 showed a slightly greater inhibition (Fig. 2). Similar
activities were observed with 3-halo-2-indolylmaleimides
IM-3 and IM-4, although the 3-bromo derivative, IM-4,
showed strong cytotoxicity at 10 lM even in the absence
of H2O2 (Fig. 2b), probably due to its electrophilic nat-
ure. Next, various mono-indolylmaleimide (IM)
Figure 1. Structures of BM I, BM V, and 1.
Keywords: Cell death; Necrosis; Apoptosis; Indolylmaleimide.
*
Corresponding author. Tel./fax: +81 22 217 5601; e-mail:
sodeoka@tagen.tohoku.ac.jp
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.016

K. Dodo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3114–3118
3115
Scheme 1. Synthesis of indolylmaleimide derivatives. Reagents: (a) DDQ, dioxane (45%); (b) MeI, K2CO3, DMF (IM-2: 98%, IM-3: 88%, and IM-4:
95%); (c) PhCOCO2Et or (CO2Me)2, t-BuOK, DMF (4: 88% and 5: 90%); (d) MeI, NaH, DMF (IM-5: 97%, IM-6: 84%, IM-7: 90%, and IM-8: 16%);
(e) RCH2CONH2, t-BuOK, DMF (7: 77% and 8: 61%); (f) ROH, NaH, THF (IM-9: 90%, IM-10: 91%, and IM-11: 93%); (g) 40%MeNH2aq/THF
(2/1) (IM-20: 28%); (h) RNH2, CH2Cl2 (IM-12: 82%, IM-13: 76%, and IM-14: 88%) or THF (IM-54: 59% and IM-25: 83%).
derivatives having a methyl group on the indole nitrogen
were synthesized as shown in Scheme 1, based on the re-
ported procedure.5 The 3-phenyl derivative, IM-5, was
less active than IM-2, but the 3-butyl derivative, IM-6,
showed a comparable activity to that of IM-2. IM-7,
having a long hydrophobic chain, was less effective. Fi-
nally we were pleased to find that alkyloxy and alkyla-
mino derivatives showed a much stronger cell death
inhibition. No cytotoxicity was observed for these com-
pounds even at 10 lM. At the same length of methylene
chain, alkylamino derivatives IM-20, IM-12, and IM-13
showed a slightly higher activity than the alkyloxy deriv-
atives, IM-8–IM-10. Among the aminoalkyl derivatives,
the best activity was obtained with IM-54, having the C5
alkyl chain. The derivatives having a long hydrophobic
chain, IM-11 and IM-14, had a very weak activity.
The structure–activity relationship clearly suggests that
heteroatom substitution at the maleimide C3 position
would be important, so that the lone-pair electrons
can be delocalized on the maleimide ring. In BM V,
the lone pair of the indole nitrogen may also be distrib-
uted partly on the maleimide ring.
The dose–response curves of BM I, BM V, compound 1,
and IM-54 for ability to improve the viability of HL60
cells treated with H2O2 are shown in Figure 3a. The
cytoprotective effect of compound 1 plateaued at
3 lM, and a slight decrease in cell viability was observed
at 10 lM. In contrast, dose-dependent cell death inhibi-
tion was observed up to 10 lM for IM-54. Furthermore,

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K. Dodo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3114–3118
Figure 2. Structure and activities of various indolylmaleimide derivatives. (a) Cell death inhibition: viability of HL60 cells treated with H2O2
(100 lM, 3 h) in the absence (control) or the presence of 3 lM indolylmaleimide derivative. (b) Cytotoxicity: viability of HL60 cells treated with
10 lM indolylmaleimide derivative.
we examined the inhibitory activity of IM-54 toward
S6K1 (Table 1).1 As expected, IM-54 showed no inhibi-
tion of S6K1 even at 50 lM, in clear contrast to
compound 1. We also tested the inhibitory activity of
IM-54 and 1 at a high concentration (50 lM) with all
subtypes of PKCs. Significant inhibition of PKCe was
observed with the bisindolylmaleimide derivative 1,
but IM-54 did not inhibit any of the subtypes of PKC.
These results imply that the toxicity observed for 1 at
high concentrations may be caused by the inhibition of
kinases such as S6K1 and PKCe.
eral caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-
Asp(OMe)-fluoromethyl ketone (Z-VAD) (Figs. 4b
and 5a). The suicide-type chemical antioxidant NAC
had to be used at a much higher concentration (1 mM)
than H2O2 (100 lM), whereas IM-54 strongly inhibited
necrotic cell death at much lower concentrations
(ꢀ10 lM). These experimental results clearly indicate
that IM-54 is not a suicide-type antioxidant. In fact,
IM-54 was chemically stable upon treatment with
H2O2. We next examined the effect of IM-54 on the typ-
ical apoptotic cell death induced by etoposide (Figs. 4c
and 5b).6 The apoptotic cell death induced by etoposide
was completely inhibited by Z-VAD, indicating that this
apoptosis is caspase-dependent, whereas NAC was not
effective against this type of cell death.7 In contrast to
its strong inhibition of necrotic cell death, IM-54 was
not effective against etoposide-induced apoptotic cell
Finally we compared IM-54 with well-known cell death
inhibitors in two different models of cell death. As de-
scribed in the previous paper,1 H2O2-induced necrotic
cell death of HL60 cells was inhibited by the well-known
antioxidant N-acetylcysteine (NAC), but not by the gen-

K. Dodo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3114–3118
3117
Figure 5. Comparison of the effects of Z-VAD (100 lM), NAC
(1 mM), and IM-54 (10 lM) on different types of cell death. (a)
Necrotic cell death induced by H2O2 (100 lM, 3 h); (b) apoptotic cell
death induced by etoposide (100 lM, 4 h).
Figure 3. Dose–response curves of indolylmaleimide derivative for its
ability to improve the viability of HL60 cells treated with H2O2
(100 lM, 3 h).
Apoptosis and necrosis were classically distinguished on
the basis of morphological changes,8 and, until recently,
only apoptosis was believed to be a regulated cell death
process, whereas necrosis was taken to be a passive
process caused by overwhelming cell damage.8 Recent
studies, however, indicate the existence of signaling
pathways for necrotic cell death, and necrosis is now
suggested to be a kind of programmed cell death.9
Moreover, necrosis was found to play an important
role in pathological cell death, for example, in
ischemia-reperfusion injury9,10 and neurodegenerative
disorders.9,11 In contrast to our extensive knowledge of
apoptosis,12 however, the molecular basis of necrosis
remains to be elucidated. Since IM-54 effectively blocks
necrotic cell death at low concentrations, it is likely that
this compound selectively affects a signaling pathway,
which plays a critical role in necrotic cell death. There-
fore clarification of the molecular mechanism of action
of the novel cell death inhibitor IM-54 should contribute
to future necrosis research. Furthermore, IM-54 is
expected to be a useful probe for the classification of cell
death type. IM-54 could be a lead compound for
developing therapeutic agents for treatment of diseases
such as stroke and neurodegenerative disorders.
Table 1. Kinase inhibition by 1 and IM-54
Kinase
Activity (% of control)
1 (50 lM) IM-54 (50 lM)
96 15
S6K1
PKC
28
0
a
b1
b2
c
d
e
g
i
l
h
66
78
84
85
82
10
55
87
88
78
85
3
1
3
7
5
1
3
1
2
3
6
95
84
1
2
0
4
8
1
1
2
0
3
1
106
115
108
95
98
96
97
100
100
f
death. These results suggest that IM-54 does not affect
this caspase-dependent pathway, and has significant
selectivity for necrotic cell death. Overall, IM-54 has suf-
ficient potency and selectivity to be useful as a tool for
the further mechanistic study of this novel type of necro-
tic cell death inhibition.
In conclusion, we have succeeded in developing a novel
cell death inhibitor, which has unique activity compared
with other cell death inhibitors. The low inhibitory
Figure 4. Phase-contrast micrographs of HL60 cells. (a) Intact cells; (b) typical necrotic cell death induced by H2O2 (100 lM, 3 h); (c) typical
apoptotic cell death induced by etoposide (100 lM, 4 h).

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K. Dodo et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3114–3118
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cytoprotective effect up to 10 lM suggested that our
novel IM derivatives are much more suitable for
mechanistic studies than previously reported bisindolyl-
maleimide derivatives as regards potency and selectivity.
Biological studies to identify the target molecule of
IM-54 are in progress.
Acknowledgments
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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
Priority Areas (A) from The Ministry of Education,
Culture, Sports, Science, and Technology. K.D. was
the recipient of a JSPS Research Fellowship for Young
Scientists and a Special Postdoctoral Researchers Pro-
gram Fellowship from RIKEN.
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