Design and synthesis of N-methylmaleimide indolocarbazole bearing modified 2-acetamino acid moieties as Topoisomerase I inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.11.061

A novel series of N-methylmaleimide indolocarbazole derivatives bearing modified 2-acetamino acid moieties are first reported. The cytotoxic effects of these compounds were tested in five human tumor cell lines. The potent compounds 9a, 9b, 9d, and 9e have been further evaluated for their effect on Topoisomerase I (TOPO I) and cancer cell cycle. It is concluded that the indolocarbazoles with alkyl piperazine or morpholine substituent groups instead of esters or glycosyl residues would have better activities against tumors.

Full text of DOI:10.1016/j.bmcl.2008.11.061

Bioorganic & Medicinal Chemistry Letters 19 (2009) 406–409
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of N-methylmaleimide indolocarbazole bearing
modified 2-acetamino acid moieties as Topoisomerase I inhibitors
Zhiyu Li a, Fuming Zhai a, Li Zhao b, Qinglong Guo b, Qidong You a,b,
*
a Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China
b Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, Jiangsu 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 July 2008
Revised 12 November 2008
Accepted 18 November 2008
Available online 21 November 2008
A novel series of N-methylmaleimide indolocarbazole derivatives bearing modified 2-acetamino acid
moieties are first reported. The cytotoxic effects of these compounds were tested in five human tumor
cell lines. The potent compounds9a, 9b, 9d, and 9e have been further evaluated for their effect on Topo-
isomerase I (TOPO I) and cancer cell cycle. It is concluded that the indolocarbazoles with alkyl piperazine
or morpholine substituent groups instead of esters or glycosyl residues would have better activities
against tumors.
Keywords:
Cancer cell cycle
Cytotoxicities
Ó 2008 Elsevier Ltd. All rights reserved.
Indolocarbazole derivatives
Topoisomerase I
The indolocarbazole alkaloids are appealing molecules with
broad spectrum of biological activities including anti-microbial,1
anti-hypotension,2 cytotoxic,3 as well as the inhibition of platelet
aggregation.4 The most significant biological profile of these com-
pounds is their potential anti-tumor effects. The indolocarbazole
derivatives have shown complicated and distinct anti-tumor
mechanisms including Topoisomerase I (TOPO I) poisoning, Protein
Kinase C (PKC), Protein Kinase A (PKA), CDK1/cyclin B, and CDK5/
p25 inhibition.5 Their different mechanisms of action are owing
to their structural diversity at some important substituted posi-
tions. For example, Rebeccamycin and NB-506 can form ternary
complex with the DNA/TOPO I duplex via intercalation between
the base pairs at the site of DNA cleavage, 6,7 while their analog Sat-
urosporine exhibits high levels of PKC inhibition with no effect to-
ward TOPO I (Fig. 1).3,8
Concerning for their multi-targeted anti-tumor properties,
numerous attempts including adding other functional groups to
the indolocarbazole framework as well as the modification of the
carbohydrate moiety have been launched in the hope of obtaining
analogs with improved pharmacological profiles.9 Besides, it is well
acknowledged that the introduction of amino acid and peptide-like
groups are general strategies to improve the solubility and increase
the capacity of molecules to the cellular targets,10 which has al-
ready been applied in the modification of indolocarbazole deriva-
tives.11 In this letter, to improve the solubility of indolocarbazole
scaffolds and to increase the binding affinity of the compounds
with TOPO I, a series of N-methylmaleimide indolocarbazole deriv-
atives bearing modified amino acids through 2-acetyl linker were
synthesized and evaluated.
Indolocarbazole nucleus was synthesized from N-methyl pyr-
role. Bromination and oxidation of N-methyl pyrrole gave N-
methyl-dibromomaleimide 1. Indolylmagnesium bromide was
coupled with N-methyl-dibromomaleimide 1 to afford the bis-
indolylmaleimides 2. Oxidative cyclization of the bis-indolylmalei-
mides 2 in the presence of DDQ led to the indolocarbazole 3. N-
bromoacetyl amino acid methyl esters (6a–e) were prepared from
the corresponding amino acids by esterification, neutralization and
acylation of the amino group with bromoacetyl bromide. Alkyl-
ation of the indolocarbazole 3 with the esters 6a–e in the presence
of NaH gave the indolocarbazole amino acid ester derivatives 7a–e.
Hydrolysis of the methyl esters with NaH in DMF with small
amount of water followed by the amidation of the indolocarbazole
8a–e with the piperazine or morpholine derivatives to give the
amide derivatives 9a–e (Scheme 1). The positive control JDC-108
(Fig. 1) was synthesized according to the literature procedures.6b
The cytotoxic evaluation of compounds were carried out on five
human cancer cell lines by MTT assay: HT-29 (human colon carci-
noma), HCT-8 (human ileocecal adenocarcinoma), Bel-7402 (hu-
man hepatoma cells), A549 (non-small cell lung carcinoma), and
MCF-7 (human breast carcinoma). As shown in Table 1, most of
the tested compounds were able to induce cell death in five human
cancer cell lines. Among them, compound 7a, 9a, 9b, 9d, and 9e
(one ester and four imide derivatives) showed comparable anti-tu-
mor activity with JDC-108 which also bearing N-methyl indolocar-
bazole scaffold. Taking whole views of indolocarbazole analogs we
* Corresponding author. Tel./fax: +86 25 83271351.
E-mail addresses: youqidong@gmail.com, youqd@163.com (Q. You).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.061

Z. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 406–409
407
H
N
NHCHO
H
CH3
N
O
O
N
N
O
O
H
O
O
O
N
N
H
N
N
H
N
N
N
N
Cl
HO
Cl
H
O
HO
HO
OH
OH
O
H3C
H
O
OH
OH
HO
H3CO
H3CO
HO
Rebeccamycin
NHCH3
HO
HO
HO
HO
JDC-108
Saturosporine
NB-506
Figure 1. Structure of the mentioned indolocarbazole derivatives.
CH3
N
CH3
N
CH3
O
O
O
O
CH3
N
N
c
O
O
b
a
N
H
N
H
Br
Br
N
N
H
H
1
3
2
H
N
H2N
COOH
d
HCl
COOCH3
H2N
COOCH3
e,f
g
Br
R1
4
R1
O
R1
6
5
CH3
N
CH3
N
CH3
N
O
O
O
O
O
O
h
i
N
N
N
N
H
N
N
H
H
O
O
O
O
COOH
R1
COOCH3
R1
R2
HN
HN
HN
R1
9a R1=CH(CH3)2
9b R1=CH2Ph
9c R1=CH3
R2=
8a R1=H
N
O
N
7a R1=H
8b R1=CH2PH
8c R1=CH2CH(CH3)2
8d R1=CH(CH3)2
8e R1=CH3
7b R1=CH2PH
R2=
R2=
N
C2H5
7c R1=CH2CH(CH3)2
7d R1=CH(CH3)2
7e R1=CH3
N
N
N
O
9d R1=CH2CH(CH3)2 R2=
9e R1=CH2CH(CH3)2 R2=
N
C2H5
CH3
N
Scheme 1. The Synthesis of indolocarbazole analogs. Reagents and conditions: (a) Br2, À78 °C, HNO3; (b) CH3CH2Br, Mg, THF, indole, toluene; (c) DDQ, toluene; (d) Dry HCl,
CH3OH; (e) 10%NaOH, CH2Cl2; (f) BrCH2COBr, TEA; (g) NaH, DMF; (h) NaH, DMF; (i) CDI, R2H,THF.
synthesized, it is suggested that the amide analogs (9a–e) are more
potential than those acid (8a–e) and ester (7a–e) analogs. Consid-
ering their better anti-proliferation activities, compounds 9a, 9b,
9d, and 9e were selected for further evaluation of their effect on
DNA–TOPO I complex (Fig. 2) and their influence on TOPO I expres-
sion in HT-29 cells (Fig. 3).
The TOPO I inhibition assay was carried out on the TOPO I Drug
Screening Kit (Cat. No. 1018, TopoGEN Corporation), following the
protocol established by Trask et al.12 According to the mechanism
of TOPO I poison, compound like Camptothecin (CPT) is able to
bind and stabilize the DNA/TOPO I binary complex which would
result in an increase in the nicked DNA product (open-circular
DNA). As shown in Figure 2, the conversions of supercoiled DNA
(Form I) to open circular (OC DNA) is observed after treatment with
CPT, 9a, 9b, 9d, and 9e, separately. At equivalent concentration,
these indolocarbazole compounds showed comparable activities
with CPT as TOPO I inhibitors. The changes of TOPO I expression
in HT-29 cells induced by 9a, 9b, 9d, and 9e were also evaluated

408
Z. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 406–409
Table 1
The in vitro MTT assay of 15 indolocarbazole analogs
CH3
N
O
O
N
N
H
O
O
R2
HN
R1
Compound
R1
R2
IC50 (lM)
HT-29
HCT-8
Bel-7402
A549
MCF-7
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
H
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
OH
2.88
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.13
>10
>10
>10
>10
>10
>10
>10
>10
>10
2.23
>10
>10
>10
>10
>10
>10
>10
>10
>10
CH2Ph
CH2CH(CH3)2
CH(CH3)2
CH3
H
CH2Ph
CH2CH(CH3)2
CH(CH3)2
CH3
9a
9b
9c
9d
CH(CH3)2
CH2Ph
2.66
2.88
>10
>10
1.09
>10
>10
>10
1.91
>10
>10
5.11
1.86
>10
>10
1.91
>10
2.44
N
N
N
N
O
N
C2H5
CH3
O
CH2CH(CH3)2
3.48
7.94
N
C2H5
CH3
9e
CH2CH(CH3)2
—
1.26
7.44
>10
>10
2.23
6.68
1.84
>10
1.94
>10
N
N
JDC-108
—
Figure 2. The effect of 9a, 9b, 9d, and 9e on DNA–TOPO I complex.
I on HT-29 cells were decreased compared to the control group,
which were correlated with their cytotoxic IC50 values. Further-
more, the apoptosis-inducing activity of 9a, 9b, 9d, and 9e were
evaluated using flow cytometry. As shown in Figure 4, incubation
with 20 lM of 9a, 9b, 9d, and 9e for 24 h resulted in the increased
percentage of apoptotic cells in Sub-G1 phase (2.49%, 10.3%, 8.86%,
14.33% compared to 0.65% in control group).
Figure 3. The influence of TOPO I expression in HT-29 cells.
In conclusion, we have synthesized 15 novel N-methylmalei-
mide indolocarbazole analogs bearing modified 2-acetamino acid
moieties and have tested their cell growth inhibitory activities.
Compounds with the alkyl amide chains showed potent anti-prolif-
eration activities against different tumor cell lines. Further studies
following the common protocols with the commercial available
TOPO I antibody.13 As shown in Figure 3, after incubated with
20 lM of 9a, 9b, 9d, and 9e for 24 h, the expression levels of TOPO

Z. Li et al. / Bioorg. Med. Chem. Lett. 19 (2009) 406–409
409
Figure 4. Compound 9a, 9b, 9d, and 9e could induce apoptosis in HT-29 cells.
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comparable TOPO I inhibition activities with CPT. Moreover, the
anti-tumor activities of 9a, 9b, 9d, and 9e were partly attributed
to their abilities to modulate the expression levels of TOPO I pro-
teins and to induce apoptosis in cancer cells. All the above results
demonstrated that these compounds could be promising lead com-
pounds of novel antitumor drugs. Further studies about the cellular
mechanism of the potent compounds are in progress.
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Acknowledgments
This work was supported in part by the Natural Science Founda-
tion of Jiangsu Province in China (Grant No. BK2005102). The
author gratefully acknowledged the unknown reviewers for their
fair and essential comments and suggestions to this letter.
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Supplementary data
Supplementary data of synthesis and identification of the 15
compounds in this article can be found in the online version at
www.sciencedirect.com and doi:10.1016/j.bmcl.2008.11.061.
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