Synthesis and?antiproliferative activities of?indolin-2-one?derivatives bearing amino acid moieties

Article abstract of DOI:10.1016/j.ejmech.2006.03.021

A convenient synthesis of indolin-2-ones substituted in the 3 position by an aminomethylene group bearing different amino acid moieties is described. Their antiproliferative activities were evaluated toward a panel of human solid tumor cell lines (PC 3, DLD-1, MCF-7, M4 Beu, A549, PA 1) and healthy cell lines (a murine fibroblast L929 and a human fibroblast primary culture).

Full text of DOI:10.1016/j.ejmech.2006.03.021

European Journal of Medicinal Chemistry 41 (2006) 709–716
http://france.elsevier.com/direct/ejmech
Original article
Synthesis and antiproliferative activities
of indolin-2-one derivatives bearing amino acid moieties
Mathieu Sassatelli a, Éric Debiton b, Bettina Aboab a, Michelle Prudhomme a, Pascale Moreau a,*
a Laboratoire SEESIB, université Blaise-Pascal, UMR 6504 du CNRS, 24, avenue des Landais, 63177 Aubière, France
b Laboratoire de pharmacognosie–biotechnologies, UMR Inserm 484, université d’Auvergne,
centre Jean-Perrin, rue Montalembert, BP 184, 63005 Clermont-Ferrand cedex, France
Received 26 October 2005; received in revised form 16 February 2006; accepted 9 March 2006
Available online 03 May 2006
Abstract
A convenient synthesis of indolin-2-ones substituted in the 3 position by an aminomethylene group bearing different amino acid moieties is
described. Their antiproliferative activities were evaluated toward a panel of human solid tumor cell lines (PC 3, DLD-1, MCF-7, M4 Beu, A549,
PA 1) and healthy cell lines (a murine fibroblast L929 and a human fibroblast primary culture).
© 2006 Elsevier SAS. All rights reserved.
Keywords: Indolin-2-ones; Amino acids; Antiproliferative activities
1. Introduction
one was evaluated for its potency toward cyclin-dependent pro-
tein kinases [9] and diversely substituted 3-arylaminomethy-
lene-indolin-2-ones were described as VEGFR [10], Trk A
[11] or CDK inhibitors [12,13]. However, none of the de-
scribed compounds was functionalized with an amino acid
group (Fig. 2).
In this paper, the synthesis of indolin-2-ones substituted in
the 3 position with an aminomethylene group bearing different
amino acid moieties is described (compounds 4, 12 and 13;
Fig. 2). The amino acid moiety could reinforce the hydrogen
bond net inside the active site of the target(s) enzyme(s). To
get an insight into the substitution pattern required for the best
biological potencies, the side chains are either flexible (com-
pound 4) or more rigid and differently oriented (compounds
12, 13).
As part of our ongoing studies concerning the preparation of
potential antitumor compounds, we were interested in the
synthesis of indolin-2-one derivatives [1,2]. 3-Substituted indo-
lin-2-one derivatives are usually known as ATP competitive
receptor tyrosine kinase inhibitors. Alterations in receptor tyr-
osine kinases pathways have been implicated in oncogenic ac-
tivation, tumor angiogenesis and mitogenic stimulation. Ac-
cordingly, receptor tyrosine kinases are attractive targets for
the development of novel anticancer agents. Among a large
number of receptor tyrosine kinase antagonists, several oxi-
ndole derivatives (ATP competitive inhibitors) are in phase I
to III clinical development [3–6] (Fig. 1). Moreover, some imi-
dazo[2,1-b]thiazolylmethylene-2-indolinones such as com-
pound A or indolylmethylene-2-indolinones such as compound
B have been described as CDK1/cyclin B inhibitors [7]
(Fig. 1). Recently, SU9516 (Fig. 1) was described as an ATP
competitive CDK inhibitor [8].
2. Chemistry
The synthesis of indolin-2-one derivative 4 bearing a lysine
moiety in the 3 position was achieved in three steps from the
commercially available isatine (Scheme 1). 3-Chloromethy-
lene-indolin-2-one 1 was prepared as a mixture of Z,E isomers
in 51% yield by a Wittig reaction performed with (chloro-
methyl)triphenylphosphonium iodide in THF in the presence
of n-butyllithium. The dichlorinated product 2, which could
The synthesis and biological activities of some amino-
methylene-indolin-2-one have already been described in the lit-
erature. For example, 3-N-methylaminomethylene-indolin-2-
* Corresponding author. Tel.: +33 4 73 40 79 63; fax: +33 4 73 40 77 17.
E-mail address: Pascale.MOREAU@univ-bpclermont.fr (P. Moreau).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.03.021

710
M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 709–716
Fig. 1.
Fig. 2.
formed by the use of a phosphonium salt excess (3 equiva-
lents), was also isolated in 4% yield (Scheme 1). Compounds
1 and 2 have already been described in the literature via other
pathways [14–17]. Compound 1 Z/E isomeric ratio (70/30) was
nol, the single product identified was the “dimer” 3 resulting
from the displacement of the vinylic chlorine atoms of two in-
dolin-2-one derivatives 1 by both lysine NH ε and NH2 α func-
1
tions (Scheme 1). The complexity of the H NMR spectrum of
1
determined from the H NMR spectrum on the H6 signals at
3 is due to the presence of four diastereoisomers: (3Z, 3′Z),
(3Z, 3′E), (3E, 3′E) and (3E, 3′Z) (Scheme 1). All NHindolic
as well as two Hvinylic signals could be easily distinguished
on the spectrum. The integral values of these two Hvinylic sig-
nals were identical to those of the major NHindolic signals at
10.17 and 10.23 ppm. On this basis, the diastereoisomeric ratio
was 35/35/15/15. The dimeric structure was confirmed by
HRMS. To limit the formation of this by-product, the reaction
was carried out in the same conditions with 2 equivalents of L-
7.24 and 7.32 ppm, respectively, since the signals of the
vinylic protons were superposed upon other signals. Despite
numerous purification trials (crystallization and chromato-
graphic methods including HPLC), the isomers could not be
cleanly separated, only small amounts of 1Z were obtained
by crystallization in EtOAc. When the synthesis of compound
4 was tried by reaction of 1 equivalent of L-lysine and 1
equivalent of compound 1 in the presence of NaOH in metha-

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 709–716
711
Scheme 1.
lysine and 1 equivalent of compound 1. Under these condi-
tions, compound 4 was only obtained as traces. Compound 4
was finally obtained in 41% yield by reaction of 1 and N-(α)-
tert-butoxycarbonyl-L-lysine in the presence of NaOH in
methanol, followed by the removal of the tert-butoxycarbonyl
protective group in an acidic medium. Compound 4 was ob-
tained as a mixture (70/30) of Z/E isomers. The Z/E isomeric
compound 8. However, in contrast with what was described in
the literature [20], the required p-nitro-L-phenylalanine was
never obtained. When the nitration step was carried out on D-
phenylalanine, p-nitro-D-phenylalanine 6 was obtained in 16%
yield. The nitration leading to 6 and 9 derivatives was per-
formed in the presence of a mixture of concentrated HNO3 and
H2SO4. The α-amino function was further protected using di-
tert-butyl dicarbonate in the presence of saturated aqueous
NaHCO3. The amino intermediates 8 and 11 were finally ob-
tained by catalytic hydrogenation of the corresponding pro-
tected nitro derivatives 7 and 10. The physical properties, in-
cluding [α]D values and spectral data of 6, 7, 8 were identical
to those described in the literature for their enantiomers [18,
20]. Several batches of these compounds were prepared; how-
ever, in all the cases, the [α]D values were opposite to those
expected on the basis of the literature data. For compounds 9,
10 and 11, the physical properties and spectral data were in
accordance with those already described in the literature for
the corresponding enantiomer (compound 9) [21] or racemate
(compound 10 and 11) [22].
1
ratio was determined from the H NMR spectrum on the sig-
nals of the vinylic protons respectively at 7.93 and 7.43 ppm.
The coupling reaction was also carried out in the same condi-
tions with a small quantity of 1Z (Scheme 1). Surprisingly, the
only product obtained was the (E)-3-methoxymethylene-indo-
lin-2-one 5 [15] formed by a side reaction with the solvent
MeOH (Scheme 1). In contrast with what was obtained with
the mixture of Z/E isomers of 1, when starting from the Z iso-
mer no traces of compound 4 was detected. It could be possible
that, in the case of the Z/E mixture, only the E isomer has
reacted with the N-(α)-tert-butoxycarbonyl-L-lysine to give 4
as a mixture of Z/E isomers. This result could also be ex-
plained by isomerization of vinyl chloride and/or the resulted
enamine.
For the synthesis of compounds 12 and 13 (Fig. 2), the re-
quired intermediates 8 and 11 were prepared respectively from
D-phenylalanine and L-phenylglycine according to the proce-
dure described for the synthesis of p-amino-L-phenylalanine
(Scheme 2) [18,19]. First, different conditions for the nitration
of L-phenylalanine have been tried to prepare the enantiomer of
Compounds 12 and 13, substituted in the 3 position by ami-
no acid moieties containing a phenyl ring, were obtained using
the same procedure as described above for compound 4
(Scheme 3).
In these conditions, compound 12 was obtained as a mixture
(85/15) of Z/E isomers in 29% yield whereas compound 13
was obtained as a mixture (80/20) of Z/E isomers in 52% yield.

712
M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 709–716
Scheme 2.
Scheme 3.
The Z/E isomeric ratio for compound 12 was determined from
the H NMR spectrum on the signals of the vinylic protons
ergy difference between the reactants and the transition state)
of Z and E isomers for compounds 4, 12 and 13 showed that
the Z configuration is the major one (Table 1). The results
showed that, for these three compounds, the Z isomer was sta-
bilized by an intramolecular hydrogen bond (black lines,
Fig. 3) between the NH hydrogen and the oxygen of the oxi-
ndole carbonyl group. For compound 4, the folding of the flex-
ible side chain was stabilized by an additional hydrogen bond
between the α-amino function and the oxygen atom of the oxi-
1
respectively at 8.63 and 7.79 ppm and for compound 13, on
the signals of the vinylic protons at 8.65 and 7.74 ppm. The
Z/E isomeric ratios of compounds 4, 12 and 13 were evaluated
by quantum semi-empirical calculations, using Ampac8.0 pro-
gram [23], on both isomers global minima geometries found
previously by conformational analysis using Macromodel 7.0
program [24]. The comparison of the Ea activation energy (en-

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 709–716
713
Table 1
metric assays: Resazurin reduction test (RRT) and Hoechst
33342 dye DNA assay [26] in a panel of human solid cancer
cell lines (PC 3: prostatic adenocarcinoma, DLD-1: colon car-
cinoma, MCF-7: breast adenocarcinoma, M4Beu: melanoma
teratocarcinoma, A549: lung carcinoma, PA 1: ovarian carcino-
ma), and healthy cell lines (a murine fibroblast cell line L929
and a human fibroblast primary culture). Compound 12 and 13
were inactive against the cell lines tested. Only compound 4,
bearing a flexible side chain, showed antiproliferative activ-
ities. This compound was selective toward some of the cell
lines tested since it exhibited cytotoxicity only against DLD-1
(IC50 = 10 μM), PA-1 (IC50 = 15 μM), L929 (IC50 = 17 μM)
and fibroblast (IC50 = 14 μM) cell lines.
In conclusion, a convenient method for the synthesis of sub-
stituted indolin-2-ones bearing different amino acid moieties in
the 3 position is described. The antiproliferative activities of
these compounds have shown that a better pharmaceutical pro-
file is obtained with a flexible side chain which allows the
folding of this chain. The inhibitory activities of compound 4
toward various kinases and the synthesis of compound 4 ana-
logues are currently under investigation in our laboratory.
Computational results of Z/E isomers for compounds 4, 12 and 13
4Z
20.0
4E
23.0
Ea activational energy (kcal mol–1
)
)
)
Ea (Z) – Ea (E) (kcal mol–1
Theoretical ratio Z/E
)
3.0
3.0
3.0
99.0
70.0
12Z
63.0
1.0
Experimental ratio Z/E
30.0
12E
66.0
Ea activational energy (kcal mol–1
Ea (Z) – Ea (E) (kcal mol–1
Theoretical ratio Z/E
)
99.0
85.0
13Z
72.0
1.0
Experimental ratio Z/E
15.0
13E
75.0
Ea activational energy (kcal mol–1
Ea (Z) – Ea (E) (kcal mol–1
Theoretical ratio Z/E
)
99.0
80.0
1.0
20.0
Experimental ratio Z/E
4. Experimental
4.1. Chemistry
IR spectra were recorded on a Perkin-Elmer 881 spectro-
meter (n in cm–1). NMR spectra were performed on a Bruker
AVANCE 400 (1H: 400 MHz, 13C: 100 MHz) (chemical shifts
δ in ppm, the following abbreviations are used: singlet (s),
doublet (d), triplet (t), doubled triplet (dt), doubled doublet
(dd), doubled doubled doublet (ddd), multiplet (m), broad sig-
nal (br s), tertiary carbons (C tert), quaternary carbons (C
quat.). Mass spectra (ES) were determined on a high resolution
Waters Micro Q-toff apparatus. Chromatographic purifications
were performed by flash silica gel Geduran SI 60 (Merck)
0.040–0.063 mm column chromatography. For purity tests,
TLC was performed on fluorescent silica gel plates (60 F254
from Merck).
4.1.1. (Z/E)-3-chloromethylene-indolin-2-one 1
and 3,3-dichloromethylene-indolin-2-one 2
Fig. 3.
A solution of butyllithium (1.6 M in hexane, 1.8 ml,
2.9 mmol) was added to a suspension of (chloromethyl)triphe-
nylphosphonium iodide (1.3 g, 3.06 mmol) in THF (20 ml).
The mixture was stirred at room temperature for 1 h before
cooling to –10 °C and addition of a solution of isatine
(150 mg, 1.02 mmol) in THF (20 ml). The mixture was stirred
at –10 °C for 1 h before addition of brine. After extraction with
EtOAc, the combined organic phases were dried over MgSO4,
evaporated and the residue was purified by chromatography
(eluent cyclohexane/EtOAc 80:20) to give two products. Com-
pound 2 (9 mg, 0.04 mmol, 4% yield) and a mixture of Z/E 1
(93 mg, 0.52 mmol, 51% yield) were isolated as yellow pow-
ders. These compounds have already been described in the lit-
erature [14–17].
ndole moiety. Moreover, the conformation around the CH
vinylic-NH bond was also characterized by H NMR spectro-
1
scopy. Arising from restricted rotation around this bond, the
signals of the vinylic protons were doublets (compounds 4,
12 and 13) and the NH signals were doublets for compounds
12 and 13 and a doubled triplet for compound 4 with a JCH–NH
coupling constant of 12.0–13.5 Hz, which is typical of an anti
conformation [25].
3. Biological activities
A preliminary evaluation of the antiproliferative activities of
compounds 4, 12 and 13 was performed in vitro by two fluoro-

714
M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 709–716
1
Compound 1: H NMR spectrum of the diastereoisomeric
on the spectrum. 13C NMR (100 MHz, DMSO-d6): 21.3, 29.5,
30.2, 47.9 (CH2 lys), 51.7 (CHlys), 94.3, 125.3, 135.5 (Carom.),
108.6, 114.9, 119.7, 121.9, 147.8 (CHarom.), 169.5, 170.9
(C=O).
mixture (400 MHz, DMSO-d6): 6.83 (d, J = 7.5 Hz, 1H, Z iso-
mer), 6.88 (d, J = 7.5 Hz, 1H, E isomer), 6.96 (t, J = 7.5 Hz,
1H, Z isomer), 7.03 (t, J = 7.5 Hz, 1H, E isomer), 7.24 (dt, J1
= 7.5 Hz, J2 = 1.0 Hz, 1H, Z isomer), 7.32 (dt, J1 = 7.5 Hz, J2
= 1.0 Hz, 1H, E isomer), 7.58 (s, 1H, Hvinylic, E isomer), 7.60
(d, J = 7.5 Hz, 1H, Z isomer), 7.88 (d, J = 7.5 Hz, 1H, E iso-
mer), 7.88 (s, 1H, Hvinylic, Z isomer), 10.64 (s, 1H, NH, Z iso-
mer), 10.67 (s, 1H, NH, E isomer).
4.1.4. m-Nitro-L-phenylglycine 9
At 0 °C, (S)-(+)-phenylglycine (1 g, 6.6 mmol) was added
to a mixture of concentrated H2SO4 (1 ml) and fuming HNO3
(1 ml). The reaction mixture was stirred for 1 h at 0 °C then
30 min. at room temperature before hydrolysis with cold water
(20 ml). The pH was adjusted to pH 9 by addition of an aqu-
eous solution of 4 M NH4OH. The resulting mixture was stir-
red at 4 °C for 96 h, compound 9 was isolated by filtration.
After washing with cold water, compound 9 was isolated
(647 mg, 3.3 mmol, 50% yield) as a yellow powder: m.p.
4.1.2. Diastereoisomeric mixture of dimer 3
To a solution of 1 (90 mg, 0.5 mmol) in MeOH (5 ml) were
added successively, L-lysine (73 mg, 0.5 mmol) and an aqu-
eous 1N NaOH solution (0.5 ml). After stirring for 4 hours at
room temperature, the reaction mixture was neutralized by ad-
dition of HCl (1 N) before extraction with EtOAc. The organic
phases were dried over MgSO4 and concentrated under va-
cuum to give 3 (34 mg, 0.079 mmol, 32% yield) as a green
amorphous solid. IR (NaCl) υNH, OH 3671–3044 cm–1 υC=O
1730 cm–1 υC=O 1671 cm–1 υC=C 1606 cm–1. HRMS (ES) calcd
for C24H25N4O4 [M + H]+: 433.1876, found 433.1889. 1H
NMR spectrum of the mixture of four diastereoisomers
(400 MHz, DMSO-d6): 1.35–1.46 (m, CH2 lys), 1.55–1.70 (m,
CH2 lys), 1.78–1.98 (m, CH2 lys), 3.35–3.48 (m, CH2 lys), 4.06–
4.15 (m, CHα), 6.76–7.01 (m), 7.26–7.35 (m), 7.38–7.50 (m),
7.55–7.68 (m), 7.92 (d, J = 13.5 Hz, Hvinylic), 7.93 (d,
J = 13.5 Hz, Hvinylic), 8.82 (dt, J1 = 13.0 Hz, J2 = 6.0 Hz,
NHvinylic), 8.99–9.06 (m, NHvinylic), 9.91 (s, NHindolic), 9.96 (s,
NHindolic), 10.17 (br s, NHindolic), 10.23 (s, NHindolic). The sig-
nal of the carboxylic acid proton is not visible on the spectrum.
25
160 °C. ½αꢀD = +100 (c = 0.66, HCl 1 M), Literature value
25
for the corresponding enantiomer ½αꢀD = –89.5 (c = 0.8, aq.
HCl) [21]. IR (KBr) υNH,
3639–3267 cm–1 υC=O
OH
1682 cm−1 υC=C 1623 cm–1. H (400 MHz, DMSO-d6): 4.50
(s, 1H, Hα), 7.65 (t, J = 8.0 Hz, 1H). 7.87 (d, J = 7.5 Hz, 1H),
8.16 (d, J = 8.0 Hz, 1H), 8.34 (s, 1H). Due to the insolubility
of compound 9, the 13C NMR spectrum could not be recorded.
1
4.1.5. Nα-Boc-m-nitro-L-phenylglycine 10
At 0 °C, saturated aqueous NaHCO3 (1.16 ml) and Boc2O
(0.43 ml, 2.04 mmol) were added to a solution of 9 (200 mg,
1.02 mmol) in 1,4-dioxane (3.4 ml) and water (2.3 ml). The
mixture was stirred at room temperature for 24 h. The pH
was adjusted to pH 2–3 by addition of 3 M HCl. After
extraction with EtOAc, the organic phases were dried over
MgSO4 and the solvent was removed to give a residue which
was purified by flash chromatography (eluent EtOAc/MeOH,
from 10:0 to 8:2). Compound 10 was isolated (299 mg ,
1.01 mmol, 99% yield) as a yellow powder: m.p. 115 °C
(dec.). ½αꢀ2D5 = +90 (c = 1, MeOH). IR (KBr) υOH 3396 cm–1
υNH 2980 cm–1 υC=O 1683 cm–1 υC=C 1605 cm–1. The spectral
data (1H, 13C NMR and HRMS) of the corresponding racemate
were already described in the literature [22].
4.1.3. Typical coupling and deprotection procedure
for the preparation of compounds 4, 12 and 13
An aqueous 1 N NaOH solution (0.35 ml) was added to a
solution of N-(α)-Boc-amino acids (0.35 mmol) in MeOH
(3 ml) before adding a solution of (Z/E)-3-chloromethylenein-
dolin-2-one 1 (0.35 mmol) in MeOH (3 ml). After stirring at
room temperature for 24 hours, the reaction mixture was neu-
tralized by addition of HCl (1 N) before extraction with
EtOAc. The organic phases were dried over MgSO4 and con-
centrated under vacuum. EtOAc (4.2 ml) and HCl 3 M
(1.26 ml) were added to the residue. The mixture was stirred
for 4 hours at 50 °C. The required compounds were isolated by
filtration of the solid obtained after evaporation and addition of
EtOAc.
4.1.6. Nα-Boc-m-amino-L-phenylglycine 11
A mixture of 7 (310 mg, 1.05 mmol), MeOH (30 ml) and
10% Pd/C was hydrogenated at room temperature under 50 psi
for 24 h. After filtration over Celite and washing with MeOH,
the filtrate was evaporated to give compound 11 (209 mg,
25
0.79 mmol, 75% yield) as a yellow powder: ½αꢀD = +75
(c = 1, MeOH). IR (KBr) υOH 3410 cm–1 υNH 2990 cm–1
υC=O 1680 cm–1 υC=C 1589 cm–1. The spectral data (1H NMR
and HRMS) of the corresponding racemate were already de-
scribed in the literature [22].
(Z/E, S)-3-(5-amino-5-carboxypentylaminomethylene)-indo-
lin-2-one 4 : yellow solid. IR (KBr) nOH NH
3660–3170 cm–1, n
C=O
,
1735 cm–1, nC=O 1653 cm–1 nC=C 1615 cm–1. HRMS (ES)
calcd for C15H19N3NaO3 [M + Na]+: 312.1324, found 312.1342.
1
NMR of the major Z isomer: H NMR (400 MHz, DMSO-d6):
4.1.7. (Z/E, R)-3-[(4-aminoethyl-4-carboxyethyl)
phenylaminomethylene]-indolin-2-one 12
1.33–1.55 (m, 2H, CH2 lys), 1.58–1.69 (m, 2H, CH2 lys), 1.78–
1.92 (m, 2H, CH2 lys), 3.34–3.44 (m, 2H, CH2 lys), 3.92–3.99
(m, 1H, CHα), 6.78–6.93 (m, 3H), 7.31 (d, J = 8.0 Hz, 1H), 7.93
(d, J = 13.5 Hz, 1H, Hvinylic), 8.29–8.41 (br s, 3H, NH3 ), 8.79
(dt, J1 = 13.5 Hz, J2 = 6.5 Hz, 1H, NHvinylic), 10.20 (s, 1H,
Prepared according to the procedure described above
+
for compound 4. Yellow solid, IR (KBr) nNH,
OH
3664–3147 cm–1, nC=O 1737, 1661 cm–1, nC=C 1610 cm–1.
NHindolic). The signal of the carboxylic acid proton is not visible
HRMS (ES) calcd for C18H18N3O3 [M + H]+: 324.1348, found

M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 709–716
715
1
324.1364. NMR of the major Z isomer: H NMR (400 MHz,
DMSO-d6): 3.06–3.17 (m, 2H, CH2), 4.23 (br s, 1H, Hα), 6.88
(d, J = 7.0 Hz, 1H), 6.97 (dt, J1 = 7.5 Hz, J2 = 1.0 Hz, 1H),
7.05 (dt, J1 = 7.5 Hz, J2 = 1.0 Hz, 1H), 7.31 (d, J = 9.0 Hz,
2H), 7.43 (d, J = 9.0 Hz, 2H), 7.62 (d, J = 7.5 Hz, 1H), 8.20–
a humidified atmosphere containing 5% CO2. Fluorescence
was then measured on an automated 96-well plate reader
(Fluoroscan Ascent FL, Labsystem) using an excitation wave-
length of 530 nm and an emission wavelength of 590 nm. Un-
der the conditions used, fluorescence was proportional to the
number of living cells in the well. The IC50, defined as the
drug concentration required to inhibit cell proliferation by
50%, was calculated from the curve of concentration-depen-
dent survival percentage, defined as fluorescence in experimen-
tal wells compared with fluorescence in control wells, after
subtraction of the blank values.
+
8.38 (br s, 3H, NH3 ), 8.63 (d, J = 12.5 Hz, 1H, Hvinylic), 10.54
(s, 1H, NHindolic), 10.76 (d, J = 12.5 Hz, 1H, NHvinylic). The
signal of the carboxylic proton is not visible on the spectrum.
13C NMR (100 MHz, DMSO-d6): 35.0 (CH2), 53.1 (CHα),
99.7, 124.1, 129.3, 137.0, 139.1 (Carom.), 109.1, 115.9 (2C),
117.0, 120.3, 124.0, 130.6 (2C), 137.7 (CHarom.), 169.8,
170.3 (C=O).
After reading, cells were prepared for cellular DNA quanti-
tation with Hoechst dye 3342. They were rinsed with PBS,
resazurin solution was then eliminated and plates were stored
at –80 °C.
4.1.8. (Z/E, S)-3-[(3-aminomethyl-3-carboxymethyl)
phenylaminomethylene]-indolin-2-one 13
Prepared according to the procedure described above for
compound 4. Yellow solid, IR (KBr) nNH,
OH
3665–3136 cm–1, nC=O 1732, 1668 cm–1, nC=C 1594 cm–1.
4.2.4. Hoechst dye 3342 test
On the day of assay, plates were thawed at room tempera-
ture for 10 min. 100 μl of a 0.01% (m/v) SDS solution in dis-
tilled water was then distributed into each well, the plates were
incubated for 1 h at room temperature and frozen again at
–80 °C for 1 h. After thawing, 100 μl of Hoechst dye 33342
solution at 30 μg ml–1 in a hypersaline buffer (10 mM Tris–
HCl, pH 7.4, 1 mM EDTA and 2 M NaCl) were added to each
well. The plates protected from light were incubated in this
solution at room temperature for 1 h on a plate shaker. Fluor-
escence was then measured at 360/460 nm on a microplate
fluorescence reader.
HRMS (ES) calcd for C17H16N3O3 [M+H]+ 310.1192, found
1
310.1205. NMR of the major Z isomer: H NMR (400 MHz ,
DMSO-d6): 5.13 (br s, 1H, Hα), 6.88–6.95 (m, 1H), 6.97–7.00
(m, 1H), 7.06–7.10 (m, 1H), 7.20–7.22 (m, 1H), 7.50–7.55 (m,
2H), 7.58–7.63 (m, 2H), 8.65 (d, J = 12.0 Hz, 1H, Hvinylic),
+
8.81–8.92 (m, 4H, NH3 , OH), 10.61 (s, 1H, NHindolic), 10.86
(d, J = 12.5 Hz, 1H, NHvinylic). 13C NMR (100 MHz, DMSO-
d6): 55.3 (CHα), 100.3, 123.9, 134.6, 137.1, 140.3 (Carom.),
109.2, 115.4, 116.1, 117.2, 120.4, 122.3, 124.3, 130.2, 137.4
(CHarom.), 169.3, 169.8 (C=O).
Under the conditions used, fluorescence was proportional to
the amount of biomass and the IC50 was calculated as above.
4.2. Antiproliferative activities
4.2.1. Cell cultures
Acknowledgements
Stock cell cultures were maintained as monolayers in 75-
cm2 culture flasks in Glutamax Eagle’s minimum essential
medium (MEM) with Earle’s salts supplemented with 10% fe-
tal calf serum, 5 ml 100 mM sodium pyruvate, 5 ml of 100×
non-essential amino acids and 2 mg gentamicin base. Cells
were grown at 37 °C in a humidified incubator under an atmo-
sphere containing 5% CO2.
The authors are grateful to Professor C. Barthomeuf (labor-
atoire de pharmacognosie–biotechnologies, UMR Inserm 484,
université d’Auvergne, centre Jean-Perrin, Clermont-Ferrand)
for allowing the biological assays in her laboratory.
References
4.2.2. Survival assays
[1] S. Messaoudi, M. Sancelme, V. Polard-Housset, B. Aboab, P. Moreau,
M. Prudhomme, Eur. J. Med. Chem. 39 (2004) 453–458.
[2] M. Sassatelli, E. Saab, F. Anizon, M. Prudhomme, P. Moreau, Tetrahe-
dron Lett. 45 (2004) 4827–4830.
[3] A.D. Laird, J.M. Cherrington, Expert Opin. Investig. Drugs 12 (2003)
51–64.
[4] L. Sun, C. Liang, S. Shirazian, Y. Zhou, T. Miller, J. Cui, J.Y. Fukuda,
J.-Y. Chu, A. Nematalla, X. Wang, H. Chen, A. Sistla, T.C. Luu, F.
Tang, J. Wei, C. Tang, J. Med. Chem. 46 (2003) 1116–1119.
[5] P. Haluska, A.A. Adjei, Curr. Opin. Investig. Drugs 2 (2001) 280–286.
[6] L.M. Toledo, N.B. Lydon, D. Elbaum, Curr. Med. Chem. 6 (1999) 775–
805.
Cells were plated at a density of 5 × 103 cells in 190 μl
culture medium in each well of 96-well microplates and were
allowed to adhere for 16 h before treatment with tested drug. A
stock solution 20 mM of each tested drug was prepared in
DMSO and kept at –20 °C until use. Then 50 μl of each tested
solution were added to the cultures. A 48 h continuous drug
exposure protocol was used. The antiproliferative effect of the
tested drug was assessed by both the resazurin reduction test
and determination of DNA cellular content after cell lysis.
[7] A. Andreani, A. Cavalli, M. Granaiola, A. Leoni, A. Locatelli, R. Morigi,
M. Rambaldi, M. Recanatini, M. Garnier, L. Meijer, Anticancer Drug
Des. 15 (2000) 447–452.
[8] D.J. Moshinsky, C.R. Bellamacina, D.C. Boisvert, P. Huang, T. Hui, J.
Jancarik, S.-H. Kim, A.G. Rice, Biochem. Biophys. Res. Commun. 310
(2003) 1026–1031.
4.2.3. Resazurin reduction test
Plates were rinsed with 200 μl PBS at 37 °C and emptied by
overturning on absorbent toweling. Then 150 μl of a 25 μg
ml–1 solution of resazurin in MEM without phenol red was
added to each well. Plates were incubated for 1 h at 37 °C in

716
M. Sassatelli et al. / European Journal of Medicinal Chemistry 41 (2006) 709–716
[9] C.L. Woodard, Z. Li, A.K. Kathcart, J. Terrell, L. Gerena, M. Lopez-
Sanchez, D.E. Kyle, A.K. Bhattacharjee, D.A. Nichols, W. Ellis, S.T.
Prigge, J.A. Geyer, N.C. Waters, J. Med. Chem. 46 (2003) 3877–3882.
[10] S.W. Andrews, J.A. Wurster, C.E. Hull, E.M. Wang, T. Malone, WO
Patent (2003) WO 03027102 Chem. Abstr. 138:287525.
[17] D.R. Long, C.G. Richard, M.S.F. Ross, J. Heterocyclic Chem. 15 (1978)
633–634.
[18] G.J. Landis, G. Lui, J.E. Shook, H.I. Yamamura, T.F. Burks, V. Hruby,
J. Med. Chem. 32 (1989) 638–643.
[19] R.N. Atkinson, L. Moore, J. Tobin, S. Bruce King, J. Org. Chem. 64
(1999) 3467–3475.
[20] J.S. Davies, A.K.A. Mohammed, J. Chem. Soc. Perk. Trans. II (1984)
1723–1727.
[21] P. Friis, A. Kjaer, Acta Chem. Scand. 17 (1963) 2391–2396.
[22] Y. Lee, A.M. Marletta, P. Martasek, L.J. Roman, B.S.S. Masters, R.B.
Silverman, Bioorg. Med. Chem. 7 (1999) 1097–1104.
[23] Ampac8.0, © 2000 Semichem, 7128 Summit, Shawnee, KS 66216,
USA.
[24] Macromodel 7.0, Shroedinger Inc, 1500 SW First Ave. Suite 1180, Port-
land, OR 97201, USA.
[25] R. Quintanilla-Licea, J.F. Colunga-Valladares, A. Caballero-Quintero, C.
Rodriguez-Padilla, R. Tamez-Guerra, R. Gomez-Flores, N. Waksman,
Molecules 7 (2002) 662–673.
[11] E.R. Wood, L. Kuyper, K.G. Petrov, R.N. Hunter III, P.A. Harris, K.
Lackey, Bioorg. Med. Chem. Lett. 14 (2004) 953–957.
[12] R. Walter, W. Grell, A. Heckel, F. Himmelsbach, W. Eberlein, G. Roth,
et al., WO Patent (2000) 265083 WO 0018734 Chem. Abstr. 132:
265083.
[13] H.N. Bramson, J. Corona, S.T. Davis, S.H. Dickerson, M. Edelstein, S.V.
Frye, R.T. Gampe Jr., P.A. Harris, A. Hassel, W.D. Holmes, R.N. Hun-
ter, K.E. Lackey, B. Lovejoy, M.J. Luzzio, V. Montana, W.J. Rocque, D.
Rusnak, L. Shewchuk, J.M. Veal, D.H. Walker, L.F. Kuyper, J. Med.
Chem. 44 (2001) 4339–4358.
[14] H. Behringer, H. Weissauer, Chem. Ber. 85 (1952) 743–749.
[15] M.S. Morales-Rios, M. Garcia-Velgara, H. Cervantes-Cuevas, C. Alvar-
ez-Cisneros, P. Joseph-Nathan, Magn. Reson. Chem. 38 (2000) 172–176.
[16] D. Borrmann, R. Wegler, Chem. Ber. 102 (1969) 64–70.
[26] E. Debiton, J.-C. Madelmont, J. Legault, C. Barthomeuf, Cancer Che-
mother. Pharmacol. 51 (2003) 474–482.