2-Arylidenedihydroindole-3-ones: Design, synthesis, and biological activity on bladder carcinoma cell lines

Article abstract of DOI:10.1016/j.bmcl.2006.09.057

2-Arylidenedihydroindole-3-ones were assayed for their antiproliferative and apoptotic abilities as potential drug candidates to treat bladder tumor. These compounds were tested on cell lines obtained from bladder tumors of various stages [superficial (pT

Full text of DOI:10.1016/j.bmcl.2006.09.057

Bioorganic & Medicinal Chemistry Letters 17 (2007) 208–213
2-Arylidenedihydroindole-3-ones: Design, synthesis,
and biological activity on bladder carcinoma cell lines
a
b,
b
c
*
`
Bastien Gerby, Ahcene Boumendjel, Madeleine Blanc, Pierre Paul Bringuier,
a
b
a
a
´
Pierre Champelovier, Antoine Fortune, Xavier Ronot and Jean Boutonnat
aLaboratoire de Dynamique Cellulaire, EPHE, UMR-CNRS 5525, IFRT 130, Universite´ Joseph Fourier,
Pavillon Taillefer, 38706 La Tronche Cedex, France
bDe´partement de Pharmacochimie Mole´culaire, UMR-CNRS 5063, Faculte´ de Pharmacie de Grenoble,
5, avenue de Verdun, BP 138, 38243 Meylan, France
c
ˆ
Laboratoire d’Anatomie pathologique, Hopital Edouard Herriot, 69437 Lyon, France
Received 29 August 2006; revised 19 September 2006; accepted 20 September 2006
Available online 16 October 2006
Abstract—2-Arylidenedihydroindole-3-ones were assayed for their antiproliferative and apoptotic abilities as potential drug candi-
dates to treat bladder tumor. These compounds were tested on cell lines obtained from bladder tumors of various stages [superficial
(pTa and pT1) vs. invasive (PpT2)]. The most active compound (3c) inhibited the proliferation, induced apoptosis, and decreased
the expression of p-Stat5 and p-Pyk2 in DAG-1 and RT112 lines in which the FGFR3 is either mutated or overexpressed. Knowing
that FGFR3 is involved in cell proliferation, differentiation, and migration through cell signaling pathways including p-Stat5 way
via p-Pyk2, let us assume that compound 3c may probably act through FGFR3 pathway.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Bladder cancer is the fourth most common malignancy
in men and the ninth most common in women in the
Western world. More than 90% of bladder tumors are
urothelial cell carcinomas (UCC).1 The carcinoma can
be classified in two development stages [superficial
(pTa and pT1) vs invasive (PpT2)] and three grades
(G1 to G3).2 The clinical course of superficial (pTa
and pT1) papillary urothelial cell carcinoma is charac-
terized by a high risk of recurrence (>70%) and a pro-
pensity to progress in invasive tumors (10–15%).
Muscle-invasive tumors (PpT2) have a poorer progno-
sis because 50% of these patients will relapse with meta-
static disease within 2 years of treatment.3–5
opment. Depending on the cellular context, these effects
are activated through various signal transduction path-
ways.6 FGFR3 can interact with various proteins such
as SH2-B, p-Stat5 through p-Pyk2 (a member of the fo-
cal adhesin kinase family), and PI3kinase.7–9 FGFR3
mutations and surexpression of wild type FGFR3 are
consequently frequent events in low stage UCC and
superficial bladder tumors.10
Currently, drugs available for treating UCC-related can-
cer need to be taken in high doses to be efficient. In this
context, molecules targeting cells where FGFR3 is
mutated or overexpressed can be of high interest as po-
tential drug candidates for treating bladder carcinoma.
In this regard, several molecules acting as FGFR3 inhib-
itors have been recently reported. Such inhibitors
Fibroblast growth factor receptors (FGFRs) are tyro-
sine kinase receptors that integrate many different intra-
cellular signals affecting cell growth, differentiation,
migration, and angiogenesis. Activation of FGFR3
induces phosphorylation residues in the intracellular do-
main and causes cellular proliferation and tumor devel-
include:
PKC412,
benzimidazole
quinolinones,
derivatives of arylaminooxazole, CHIR-258, and
imidazolidinediones.11–15
The naturally occurring flavonoids are known to either
induce apoptosis or to modulate cell proliferation of
UCC.16 Flavonoid-rich nutrition is highly recommended
for preventing bladder cancer. In earlier studies, it was
demonstrated that aurones, a flavonoid-subgroup, were
able to interact with ATP-binding site of P-glycoprotein
Keywords: 2-Arylidenedihydroindole-3-ones; Bladder carcinoma; Cell
signaling; Antiproliferative.
*
Corresponding author. Tel.: +33 4 76 04 10 06; fax: +33 4 76 04 10
07; e-mail: Ahcene.Boumendjel@ujf-grenoble.fr
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.057

B. Gerby et al. / Bioorg. Med. Chem. Lett. 17 (2007) 208–213
209
as agents for UCC treatment. Especially, we studied 2-
arylidene-2,3-dihydro-1H-indole-3-ones (azaaurones) as
structural analogs of the naturally occurring aurones
(Fig. 1). In this series, we maintained the 4,6-dime-
thoxy-2,3-dihydro-1H-indole-3-one moiety and varied
the arylidene group. The arylidene moieties were chosen
on the basis of literature reports, where halogenated ar-
yls and imidazoles were used as structural features in a
number of kinase inhibitors.19–22
H
N
1
MeO
Ar
R2
X
6
R1
3
4
O
OMe
O
X = O: aurones
X = N-H: azaaurones
2-arylidenedihydroindole-
-3-ones
Figure 1. Structures of investigated molecules.
Azaaurones 3 were prepared from indolin-3-one 2 by
base-catalyzed condensation with an arylaldehyde
(Scheme 1). The condensation affords exclusively Z-aza-
MeO
NH2
MeO
MeO
NH2
a
1
b, c
aurones as confirmed by H NMR.23 Oxindole 2 was
Cl
81%
prepared starting from 3,5-dimethoxyaniline and chlo-
roacetonitrile as previously reported.24
80%
1
OMe O
OMe
OMe
Ac
N
H
N
In this study, we have used three bladder tumour cell
lines that mimic the bladder tumour progression pro-
cess. These lines were obtained from bladder tumors
of various grades and various stages (RT112 was from
a pT1G2, DAG-125 from a pT1G3 and J82 from a
pT2G3 tumors). DAG-1 cells featured the S249C muta-
tion on exon 7 and J82 cells the K650E mutation on
exon 15, whereas RT112 line overexpressed the wild
type FGFR3.26 Flow cytometry analysis showed a more
important FGFR3 expression in RT112 (39 2 a.u.)
cells than in DAG-1 (28 3 a.u.) and J82 (19 4 a.u.)
cells (data not shown). A preliminary screening using
an in vitro test to assess the ability of a set of aurones
MeO
R-CHO
d
R
11 - 80%
O
O
OMe
3
2
Scheme 1. Reactions and conditions: (a) Cl–CH2–CN, ZnCl2/BCl3,
CH2Cl2 (reflux) then HCl (2 N), reflux; (b) CH3CO–Cl, 85 ꢁC; (c)
K2CO3, acetone, reflux; (d) KOH, H2O/MeOH, 50 ꢁC.
and to inhibit kinases.17,18 Therefore, we tested aurone
family members for their antiproliferative effect on
UCC, because no prior studies had investigated them
Table 1. The effect of azaaurones (at 10 lM) on cell viability in DAG-1, RT112, and J82 cell lines after 72 h incubation
H
N
MeO
Ar
O
OMe
Compound (10 lM)
Ar
Cell lines
RT112
DAG-1
J82
99
3a
3b
3c
98
99
58
3
4
2
97
93
48
3
3
3
4
4
4
Et
Cl
94
97
F
Cl
3d
3e
91
90
3
5
93
92
5
3
95
89
4
4
F
HN
N
Me
N
3f
89
4
3
92
4
2
90
3
2
N
H
Control
Without compound
100
100
100
Results are expressed in percentage of viable cells.

210
B. Gerby et al. / Bioorg. Med. Chem. Lett. 17 (2007) 208–213
analogs to effect cell viability on DAG-1, RT112, and
J82 cell lines allowed us to retain 2-arylidene-4,6-dime-
thoxy-2,3-dihydro-1H-indole-3-ones (azaaurones) as po-
tential inhibitors of FGFR3 pathway (Table 1).
pound able to decrease cell viability in DAG-1 and
RT112 cells, but not in J82 line. These results indicate
that on one hand, the effect of 3c was not related to
either grade or stage of bladder tumors and on the
other hand, this effect was not simply a direct conse-
quence of the increase of FGFR3 expression but likely
to be related to the type of mutation. The results
shown in Table 1 for compound 3c were confirmed
The preliminary test aimed to evaluate the effect of
our compounds on the cell viability shows clearly that
the fluorobenzylidene analog (3c) is the only com-
A
B
100
90
80
70
60
50
40
30
20
10
0
100
90
Control
80
3c at 10-6 M
70
60
50
40
30
20
10
0
3c at 10-5 M
Control
3c at 10-6 M
3c at 10-5 M
DAG-1R
T112
J82
DAG-1
RT112
J82
Figure 2. Effect of 3c on cell viability and cell proliferation. DAG-1, RT112, and J82 cells were incubated for 72 h at 10ꢀ5 and 10ꢀ6 M 3c. Then, cells
were trypsinized and counted. Cell viability (A) and the inhibition of proliferation percentage (B) were calculated using the formula [(Nc ꢀ Nf)/
Nc] · 100%.
DAG-1 C
RT112 C
A
(a.u)
(a.u)
DAG-1 + 3c
RT112 + 3c
B
(a.u)
(a.u)
Figure 3. Effect of 3c on cell division. DAG-1 and RT112 cells were labeled with PKH67 and treated or not with 3c at 10ꢀ5 M. The cell proliferation
index was determined by calculating the ratio of the daily means of fluorescence intensities compared with Day 0 (MIFD0/MIFDx). The cell
proliferation index was compared at D4 and D7 for DAG-1 cells (A), and at D2 and D5 for RT112 cells (B).

B. Gerby et al. / Bioorg. Med. Chem. Lett. 17 (2007) 208–213
211
DAG-1 C
DAG-1 + 3c
A
DNA content (a.u.)
RT112 C
DNA content (a.u.)
B
RT112 + 3c
DNA content (a.u.)
DNA content (a.u.)
J82 C
C
J82 + 3c
DNA content (a.u.)
DNA content (a.u.)
Figure 4. Flow cytometry analysis of apoptosis. DAG-1 (A), RT112 (B), and J82 (C) cells (5 · 105 cells/ml) were cultured without (C) or with 3c at
10ꢀ5 M for 48 h. Cultures were then trypsinized, cells stained using either the DNA cycle test kit and analyzed by flow cytometry (FACScalibur).
at lower concentration 1 lM (Fig. 2). The position of
the fluorine atom is crucial, because no significant
activity was observed when this atom was moved to
30 or 40 positions (results not shown). In attempting
to study the 20,60-difluoroanalog, we met with difficul-
ties in synthesizing it because one of the two fluoro
atoms was lost and we end up with 3c.
We also observed that 3c induced apoptosis on FGFR-
3-expressing cell lines. After treatment of DAG-1 and
RT112 cells with 10 lM of 3c for 48 h, adherent cells
progressively detached, showing signs of an apoptotic
process. The apoptosis of DAG-1 and RT112 cells was
analyzed with the DNA cycle test kit. Flow cytometry
showed that in presence of 3c, the percentage of apopto-
tic and necrotic cells (subdiploid cells) increased up to
86 4% and 20 3%, respectively, in DAG-1 and
RT112 cells (Fig. 4). The compound did not induce an
apoptotic process on the J82 cells.
In order to confirm the effect of 3c on cell division, we
proceeded for PKH67 labeling. The three cell lines were
labeled with PKH67 and incubated with or without 3c at
10 lM for various durations (D0, D4, and D7) for
DAG-1 cells; (D0, D2, and D5) for RT112 and J82 cells.
As shown in Figure 3, the cell proliferation index in-
creased sensibly in DAG-1 and RT112 untreated cells
but not in J82 (results not shown).
The effect of 3c on the p-Stat5 and p-Pyk2 antigen level
was examined. We found that the inhibition of cellular
growth and induction of apoptosis were associated with
the inhibition of the FGFR3 cell signaling pathway. At

212
B. Gerby et al. / Bioorg. Med. Chem. Lett. 17 (2007) 208–213
A
Isotypic Control
Isotypic Control
3c
3c
Isotypic
Control
3c
Untreated
Untreated
Untreated
p-Stat5 Fluorescence (a.u.)
p-Stat5 Fluorescence (a.u.)
p-Stat5 Fluorescence (a.u.)
B
Isotypic
Control
Isotypic
Control
3c
Isotypic
Control
3c
Untreated
Untreated
Untreated
3c
p-Pyk2 Fluorescence (a.u.)
p-Pyk2 Fluorescence (a.u.)
p-Pyk2 Fluorescence (a.u.)
Figure 5. Flow cytometry analysis of p-STAT5 and p-Pyk2 antigens expression. DAG-1, RT112, and J82 cells were treated with 3c (10ꢀ5 M) for 6 h.
Then, p-Stat5 (A) and p-Pyk2 (B) antigen level expression was analyzed by flow cytometry.
10 lM, it inhibited the expression of phosphory-
lated Stat5 (p-Stat5) and phosphorylated Pyk2
(p-Pyk2) in both DAG-1 and RT112 cells in a time
dependent manner. The compound caused a transient
(from 0 to 6 h) decrease in the intracellular levels of both
p-Stat5 and p-Pyk2 antigen expressions (9.5-fold for
p-Stat5 and 2-fold for p-Pyk2 in DAG-1 cells) and
(2.5-fold for p-Stat5 and 1.3-fold for p-Pyk2 in
RT112 cells) (Fig. 5). However, no effect on the
p-Stat5 and p-Pyk2 antigen levels in the J82 cells was
observed.
Stat5 cell-signaling pathway. This family of compounds
may open a new and selective way to propose potential
drug candidates for UCC treatment.
Acknowledgment
This work was supported in part by grants from la Ligue
´
Nationale contre le Cancer (Comite de la Savoie) and
´ ´ `
GEFLUC (Delegation de l’Isere).
We showed that the title compound (3c) modulates cell
proliferation by decreasing FGFR3 protein signaling
expression (p-Stat5 and p-Pyk2) in DAG-1 and RT112
cells. The fact that there is no modification of the expres-
sion of p-Pyk2 and p-Stat5 in J82 cells confirms that the
FGFR3 signal could be mediated through p-Stat5 and
p-Pyk2. In this study, we showed that FGFR3 acts by
using Pyk2 and Stat5 signaling pathways like cytokines
which can modulate cell proliferation by using several
signaling pathways.8
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2006.09.057.
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