Scaffold hybridization in generation of indenoindolones as anticancer agents that induce apoptosis with cell cycle arrest at G2/M phase

Article abstract of DOI:10.1016/j.bmcl.2012.02.007

Scaffold hybridization of several natural and synthetic anticancer leads led to the consideration of indenoindolones as potential novel anticancer agents. A series of these compounds were prepared by a diversity-feasible synthetic method. They were found

Full text of DOI:10.1016/j.bmcl.2012.02.007

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2474–2479
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Scaffold hybridization in generation of indenoindolones as anticancer
agents that induce apoptosis with cell cycle arrest at G2/M phase
Maneesh Kashyap a, Dipon Das b, Ranjan Preet b, Purusottam Mohapatra b, Shakti Ranjan Satapathy b,
a,
Sumit Siddharth c, Chanakya N. Kundu b, , Sankar K. Guchhait
⇑
⇑
a National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar, Mohali, Punjab 160062, India
b School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024, India
c Institute of Life Sciences, Nalco Square, Bhubaneswar, Orissa 751023, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Scaffold hybridization of several natural and synthetic anticancer leads led to the consideration of inden-
oindolones as potential novel anticancer agents. A series of these compounds were prepared by a diver-
sity-feasible synthetic method. They were found to possess anticancer activities with higher potency
compared to etoposide and 5-fluorouracil in kidney cancer cells (HEK 293) and low toxicity to corre-
sponding normal cells (Vero). They exerted apoptotic effect with blocking of cell cycle at G2/M phase.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 13 December 2011
Revised 16 January 2012
Accepted 3 February 2012
Available online 11 February 2012
Keywords:
Indenoindolones
Hybrid
Anticancer
Cell cycle
Apoptosis
Cancer is the second leading cause of mortality worldwide after
cardiovascular diseases.1 Chemotherapeutic treatment, although
major choice, suffers from increasing resistance responses2 among
patients to existing drugs, lack of wider activities and selectivity.
To overcome these, the development of novel anticancer agents
is of immense demand. Towards generation of new chemical enti-
ties (NCEs), the structural simplification/modification of bioactive
natural product’s complex molecular architecture3 and the gener-
ation of analogue/mimicking scaffold and hybrid4 of chemotypes
of known drugs or therapeutic agents have gained importance. In
the context of generation of motif-hybrid as lead, an excellent
example is azatoxin derived from topoisomerase II-targeted anti-
cancer drugs etoposide and ellipticine.5 Azatoxin with relevant
substitution was found to possess higher inhibitory activities of
topoisomerase II than etoposide or ellipticine. Recently, reported
examples of scaffold-hybrids, which have been identified as prom-
ising anticancer leads, include indole-barbituric acid6 and stilbene-
coumarin.7 As a part of our research on anticancer drug discovery,
we focused at scaffold hybridization design approach. Indolo[2,3-a]
carbazole (e.g., natural occurring rebeccamycin, arcyriaflavins A–D,
staurosporine), neo-tanshinlactone (a component of Chinese tradi-
tional medicine Tanshen), indenoisoquinolines, and 3-arylindoles
are known to possess anticancer activities (Fig. 1).8 Based on these
chemotypes, we considered indenoindolone as important hybrid
scaffold for potential anticancer activity. Compound comprising
of indenoindolone-oxime template and terminal alkyne moiety
was reported to possess cytotoxicity.9 It is noteworthy that inden-
oindole derivatives are known to possess various pharmaceutical
activities such as inhibitors of protein kinase CK2 and antioxidant
activity.10
In this communication, we present indenoindolones as antican-
cer agents that possess activities of higher potency and lower tox-
icity than clinically used drugs etoposide and 5-fluorouracil (5-FU)
in kidney cancer cells (HEK 293). Most of the conventional antican-
cer drugs/agents often arrest the cell cycle in a specific phase
depending on their mechanism of action. Recently, we developed
N-fused imidazole as novel anticancer agents that induced apopto-
sis in G1/S phase.11 The present studies of indenoindolones with
experiments of nuclear staining using 40,6-diamidino-2-phenylin-
dole (DAPI), expression of apoptotic markers and fluorescence acti-
vated cell sorter (FACS) analysis indicated their apoptotic effect
with cell cycle arrest at G2/M phase.
The organic synthesis plays important role in drug discovery
process. Particularly, the convenient new synthetic methods and
technologies especially towards rapid generation of molecular
diversity available to synthetic and medicinal chemists help the
lead generation.12 Recently, we developed an efficient route of
Friedel–Crafts 3-(2-bromo)benzoylation of indoles and intramolec-
ular direct arylation towards the synthesis of indenoindolones.13
⇑
Corresponding authors. Tel.: +91 (0)6742725466; fax : +91 (0)6742725732
(C.N. Kundu), tel.: +91 (0)172 2214683; fax: +91 (0)172 2214692 (S.K. Guchhait).
E-mail addresses: cnkundu@gmail.com (C.N. Kundu), skguchhait@niper.ac.in
(S.K. Guchhait).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.007

M. Kashyap et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2474–2479
2475
15, and 22
cells for compounds 2, 5, 6 and 10, were found to be 53, 39,
48, and 54 M, respectively. Interestingly, compound 14 even
at 100 M did not cause 50% cell death in Vero cells (Fig. 3). Eto-
poside, a widely used anticancer drug, showed LC50 at 18 M for
HEK 293 cells and at 28 M for Vero cells. The LC50 values of all
these five investigational compounds at various time periods in
both the Vero and HEK 293 cell lines are depicted in Table 1.
It was observed that anti-cell proliferative effect of tested inden-
oindolones did not change significantly after 48 h of treatment.
To compare the anti-proliferation potential of the investiga-
tional compounds with a clinically used common anticancer drug
5-FU, an experiment using HEK 293 cells was carried out (Fig. 4).
The cells were treated with various concentrations of the investiga-
tional compounds and also that of 5-FU for 48 h and then the cell
survivility was measured by MTT assay.11,14,15 The LC50 values for
the compounds 2, 5, 6, 10 and 14 were 17, 17, 15, 15, and
lM, respectively. However, the LC50 values for Vero
H
N
O
O
O
l
l
O
l
N
O
Cl
l
NH
OH
OH
Cl
O
Me
Neo-tanshinlactone
OH
Rebeccamycin
OMe
OMe
OMe
OMe
O
O
H3CO
MeO
H
N
H3CO
N
N
OH
O
H
O
NSC 706744
22
25
l
l
M, respectively, whereas 5-FU caused 50% cell death at
M. Thus, these data indicate that the investigational com-
R1
pounds possess higher anti-cell proliferation potential than 5-FU
in HEK 293 cells.
R3
N
To determine the effect of compounds 2, 5, 6, 10 and 14 on apop-
tosis in HEK 293 cells, DAPI nuclear staining was performed
(Fig. 5A).11,14,15 Cells when 70–80% confluent, were treated with
investigational indenoindolones for 48 h. After treatment cells were
washed with 1X PBS and fixed with acetone–methanol (1:1) and
then DAPI reagent was added. Finally, images were taken under fluo-
rescence microscope (Nikon, Japan) at 40ꢀ magnification. It was ob-
served that the punchate, bubble shape, shrinkage (apoptotic) nuclei
increased in HEK 293 cells on treatment with investigational com-
pounds at their respective LC50 concentrations (Fig. 5A). The relative
number of apoptotic/non-apoptotic nuclei generated in HEK 293
cells is presented as bar diagram in Figure 5B (P <0.05).
R2
Indenoindolone
Figure 1. Natural/synthetic anticancer agents.
This protocol afforded the convenient preparation of diverse
substituted/functionalized indenoindolones from easily accessible
starting materials in reduced number (two only) of reaction steps.
Following this process, a number of suitably relevant substituted
indenoindolones were prepared (Scheme 1; Fig. 2).
Cytotoxicity of all synthesized compounds (Fig. 2) was mea-
sured by using MTT assay in human embryonic kidney cancer
cells (HEK 293, cat. # CRL-1573) and normal monkey kidney
cells (Vero, cat. # CCL-81).11,14,15 HEK 293 and Vero cells were
purchased from American Tissue Culture Collection, VA, USA.
According to DNA profiling and cytogenetic analysis, HEK 293
is hypotriploid and its nodal chromosome number is 64 occur-
ring 30% of cells, but Vero cell is hypodiploid and its nodal chro-
mosome number is 58 occurring 66% of cells. Approximately
10,000 cells were plated in a 96-well tissue culture plate and
were treated with investigational compounds for 48 h. The cells
were washed with 1X PBS and then MTT was added. It was incu-
bated for 6 h at 37 °C. Detergent solution (10% NP-40 with 4 mM
HCl) was added to each well of the culture plate and the color
intensity was measured at 570 nm using the microplate reader
(Berthold, Germany). Among all indenoindolones, compounds 2,
5, 6, 10 and 14 showed relatively higher cytotoxic activities in
comparison to others. With increasing concentration of these
compounds, the cell death of HEK 293 significantly increased
in comparison to Vero cells (Fig. 3). Compounds 2, 5, 6, 10 and
14 caused 50% cell death (LC50) of HEK 293 cells at 17, 17, 15,
The regulation of cell cycle profile was investigated for under-
standing the mechanism by which these compounds can induce
apoptosis. The kidney cancer cells after treatment with compounds
(with respective LC50 concentrations) for 48 h were studied using
FACS by staining with propidium iodide (Fig. 5C).11,14,15 Cells were
cultured in 60 mm tissue culture disc and were treated with the
investigational compounds for 48 h. After the end of the treatment,
cells were washed with PBS containing RNase-A. The cells were then
fixed with ethanol and incubated at ꢁ20 °C overnight and later they
were stained with PI. The cells were then sorted by FACS (Becton and
Dickinson, CA) and DNA content of the cells at various phases of the
cell cycle was measured by Cell Quest Software (Becton and Dickin-
son, CA). The effect of compounds 2, 5, 6, 10 and 14 resulted in the
accumulation of cells at G2/M phase of cell cycle. The exact biologi-
cal target(s) for the anticancer activities of these indenoindolones
are yet unknown in this preliminary investigation. The possible tar-
gets can be those proteins responsible for G2/M phase of the cell cy-
cle, such as different cyclins, cyclin dependent kinase (CDKs)
families, p21 and p53.
To further confirm the apoptotic effect of these five investiga-
tional compounds inside the cell, a western blot analysis with
O
O
C
O
Intramolecular
Friedel-Crafts
R3
Cl
indole-C2-arylation R2
Step 2
R2
indole-3-aroylation
Step 1
R2
R3
N
R3
Br
Br
N
N
R1
R1 = H, alkyl
R1
R1
Scheme 1. Reaction conditions13b: For step 1; indole (1.3 mmol), 2-bromobenzoyl chloride (1 mmol), ZrCl4 (1.5 mmol), DCE, addition at 0 °C, 0 to 30 °C, then continuation at
30 °C. For step 2; compounds 1–5: NH-protected 3-aroylindole (1 mmol), Pd(dppf)2Cl2ꢂCH2Cl2 (5 mol %), K2CO3 (2 equiv), DMF, 130 °C; For step 2; compounds 6–15: NH-
unprotected 3-aroylindole (1 mmol), Pd(OAc)2 (5 mol %), Ph3P (10 mol %), CsOAc (2 equiv), DMA, 130 °C.

2476
M. Kashyap et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2474–2479
O
O
O
O
H3CO
Ph
N
N
N
N
N
(4)
(1)
(2)
O
(3)
Ph
O
O
O
H3CO
OCH3
OCH3
OCH3
OCH3
N
H
N
H
N
H
(6)
(7)
(8)
(5)
H3CO
O
O
H3CO
O
O
H3CO
OCH3
OCH3
OCH3
OCH3
OCH3
N
H
N
H
(9)
N
H
OCH3
OCH3
N
H
(10)
(12)
(11)
O
O
O
H3CO
H3CO
F
OCH3
OCH3
N
H
N
H
N
H
(13) H3CO
(14)
(15)
Figure 2. Synthesized indenoindolones.
Figure 3. MTT cell survival assay. Cells were treated with investigational compounds and etoposide of various concentrations for 48 h and the experiment was carried out
according to the known protocol.11,14,15 The sign —ꢀ—, —j—, –ꢂ–N–ꢂ– and —d— represent for Vero/etoposide, HEK 293/etoposide, Vero/investigational compound and HEK
293/investigational compound, respectively. Data is the mean SD of three different experiments.
Table 1
LC50 values of the investigational compounds at different time periods
Compound
24 h
48 h
72 h
VERO (
l
M)
HEK 293 (
lM)
VERO (
lM)
HEK 293 (lM)
VERO (lM)
HEK 293 (lM)
2
5
6
10
14
57
42
56
47
2
2
2
2
50
39
50
16
18
2
2
2
2
2
53
39
48
54
2
2
2
2
17
17
15
15
22
2
2
2
2
2
45
35
40
55
65
2
2
2
2
2
16
16
14
12
15
2
2
2
2
2
>100
>100

M. Kashyap et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2474–2479
2477
contrast to BCL-XL, the expression level of BAX increased. The
cleaved products of Caspase 3 (antibody cat #9962, cell signaling,
CA, USA) increased in comparison to control. The level of house-
keeping protein glyceraldehyde 3-phosphate dehydrogenase (GAP-
DH) was measured to confirm the same amount of protein loaded
in each well. The increased levels of BAX/BCL-XL ratio, and cleaved
product of Caspase 3 and PARP indicate that the cells underwent
apoptosis when exposed to these investigational compounds (2,
5, 6, 10 and 14).
Experiment for studying the expression of Caspase 3 level was
also done by immunocytochemistry analysis of HEK 293 cells
after exposure to investigational compounds by Caspase 3 anti-
body (cat #9662, cell signaling) (Fig. 7).16,17 The images showed
the increased accumulation of Caspase 3 protein expression in
treated cells compared to control. All together, these results indi-
cate that the investigational compounds caused apoptosis in HEK
293 cells.
In conclusion, the indenoindolones have been found to possess
anticancer activities with higher potency compared to etoposide
and 5-FU in kidney cancer cells and low toxicity to normal cells.
The anticancer activities of these compounds were due to their
apoptotic effect with cell cycle arrest at G2/M phase. These studies
indicate that this class of compounds with further studies of bio-
chemical pathways and related structural modulation by facile
diversity-feasible synthesis may find their potential applications
in anticancer drug discovery.
Figure 4. Comparison of cell survival activities of compounds (2, 5, 6, 10, and 14)
with 5-FU in HEK 293 cells by MTT assay. Data is the mean SD of three different
experiments. The symbols —d—, —j—, – – –– – –, –ꢂ –ꢂꢂ, —ꢀ—, and –ꢂ–N–ꢂ–
represent 5-FU, compounds 2, 5, 6, 10 and 14, respectively.
apoptotic markers was carried out (Fig. 6).11,14,15 The level of
cleaved product of PARP (86 KD) was higher in comparison to con-
trol when treated with the investigational compounds for 48 h. The
expression of BCL-XL reduced compared to control. However, in
Figure 5. (A) DAPI nuclear staining. HEK 293 cells were treated with indenoindenoindolones (2, 5, 6, 10 and 14) of their LC50 concentrations. Images were taken using a
fluorescent microscope (Nikon-Eclipse, Japan) at 40ꢀ magnification. Data is the representation of one of the replicates of three different experiments. (B) A graphical
representation of apoptotic nuclei of (A) (P <0.05). (C) FACS analysis was done and then the DNA content of the cell was measured by Cell Quest Software (Becton and
Dickinson, CA).

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M. Kashyap et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2474–2479
Figure 6. Western blot analysis with effect of indenoindolones on apoptotic markers in HEK 293 cells after treated for 48 h. Lower panel (GAPDH) represent the equal loading
of proteins in each lane.
Figure 7. Immunocytochemistry analysis of Caspase 3 expression: HEK 293 cells were treated with the investigational compounds for 48 h. After their fixation and
permeabilization, the nuclei were stained with DAPI (upper panel) followed by incubation with caspase 3 antibodies (cat #9662 from cell signaling) and FITC labeled
secondary antibodies (middle panel). The photographs were taken of the same fields in both the upper and middle panels. Lower panel represents the merged images of FITC
and DAPI staining.
5. (a) Cline, S. D.; Macdonald, T. L.; Osheroff, N. Biochemistry 1997, 36, 13095; (b)
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Werbovetz, K. A.; Lehnert, E. K.; Macdonald, T. L. Bioorg. Med. Chem. 1997, 5,
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We gratefully acknowledge financial support from CSIR, DBT,
ICMR, New Delhi for this investigation.
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