2668 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Brief Articles
Ta ble 3. Growth Inhibition, Cytostatic and Cytotoxic Activity of Compounds 3e-i on the 60-Cell Panela
3eb
3fb
3gb
3h b
3ib
vincristine sulfatec
GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50 GI50 TGI LC50 GI50
TGI LC50
leukemia
NSCLC
colon
-7.2 -4.7 -4.1 -6.6 -4.6 -4.0 -6.3 -5.1 -4.2 -6.3 -4.8 -4.2 -5.6 -4.4 -4.0 -7.0 -4.8 -3.2
-6.9 -4.7 -4.3 -6.1 -5.0 -4.1 -5.6 -4.8 -4.2 -5.5 -4.8 -4.2 -5.5 -4.5 -4.1 -6.6 -4.8 -3.6
-7.1 -5.1 -4.6 -6.5 -5.0 -4.2 -6.0 -5.0 -4.4 -5.4 -4.7 -4.2 -5.6 -4.7 -4.2 -7.0 -5.4 -4.1
-7.1 -5.6 -4.4 -6.7 -5.6 -4.5 -6.1 -5.2 -4.7 -6.2 -5.3 -4.6 -5.5 -4.7 -4.2 -6.9 -5.2 -3.7
CNS
melanoma -6.9 -5.0 -4.6 -6.2 -4.6 -4.0 -6.0 -5.0 -4.5 -5.5 -4.8 -4.3 -5.5 -4.9 -4.3 -6.8 -5.1 -3.6
ovarian
renal
-7.1 -5.2 -4.4 -6.5 -5.2 -4.3 -6.1 -5.3 -4.3 -5.7 -4.9 -4.4 -5.6 -4.8 -4.3 -6.5 -4.7 -3.5
-6.7 -4.7 -4.3 -5.7 -4.2 -4.0 -5.7 -4.8 -4.3 -5.6 -4.8 -4.3 -5.4 -4.4 -4.1 -6.5 -4.7 -3.6
-6.9 -4.8 -4.4 -6.4 -4.7 -4.0 -5.9 -5.1 -4.4 -5.9 -4.9 -4.4 -5.5 -4.9 -4.0 -6.9 -5.2 -3.5
-7.0 -5.3 -4.2 -6.8 -5.1 -4.0 -6.0 -5.0 -4.2 -5.6 -4.7 -4.1 -5.6 -5.0 -4.1 -6.5 -5.1 -3.5
prostate
breast
MG_MIDd -7.0 -5.0 -4.3 -6.4 -4.9 -4.1 -6.0 -5.0 -4.3 -5.7 -4.8 -4.3 -5.5 -4.7 -4.1 -6.7 -5.0 -3.6
a
b
All values are log of the molar concentration. Highest concentration tested is 10-4 M. c Highest concentration tested is 10-3 M.
d
MG_MID ) calculated mean panel.
1
270 °C (dec). IR, νmax (cm-1): 3165, 1671, 1194, 861, 683. H
Con clu sion s
NMR, δ (ppm): 2.07 (3H, s, CH3), 3.33 (2H, s, CH2), 6.51 (1H,
s, ind), 6.66 (1H, s, ind), 8.79 (1H, s, OH), 10.02 (1H, s, NH).
Compounds 3e-i showed good growth inhibition
activity with GI50 values ranging from -5.4 to -7.2. In
particular, E-3-(2-chloro-5-methoxy-6-methyl-3-indolyl-
methylene)-1,3-dihydroindol-2-one 3e was as active as
vincristine in prostate cancer and more potent in all the
other lines. The substituents at the 5 and 6 positions of
the 2-chloroindole moiety are critical because all the
disubstituted compounds (3e-i) were active on the 60
lines; the lack of even one of these substituents or their
presence in the 2-indolinone moiety results in loss of
activity. Also the N substitution in the 2-indolinone
moiety is important because the most active compounds
are unsubstituted (3e) or bear a small group (3f)
whereas the introduction of a bulky group (3i) results
in a 10- to 15-fold reduction of activity.
2-Ch lor o-5-h ydr oxyin dol-3-car boxaldeh yde 1c. The Vils-
meier reagent was prepared at 0-5 °C by dropping POCl3 (100
mM) into a stirred solution of DMF (50 mM) in CHCl3 (10 mL).
5-Hydroxindol-2-one 2c (20 mM), dissolved in a mixture of
CHCl3 (70 mL) and DMF (10 mL), was added dropwise to the
Vilsmeier reagent while maintaining stirring and cooling. The
reaction mixture was kept for 24 h at room temperature,
chloroform was removed under reduced pressure, and the
resulting residue was treated with ice-water. The mixture was
neutralized with NaHCO3, and the crude aldehyde thus
obtained was collected by filtration and crystallized from
ethanol with a yield of 65%. C9H6ClNO2 (195.6): mp 230-231
°C (dec). IR, νmax (cm-1): 3180, 1640, 1250, 1195, 855. 1H NMR,
δ (ppm): 6.75 (1H, dd, ind-6, J ) 2.3, 8.7), 7.23 (1H, d, ind-7,
J ) 8.7), 7.47 (1H, d, ind-4, J ) 2.3), 9.23 (1H, s, OH), 9.91
(1H, s, CH), 12.84 (1H, s, NH)
As a second attempt to investigate the mechanism of
the antitumor activity, compounds 3e-g (i.e., the most
active considering the calculated mean panel of Table
3) have been analyzed by means of COMPARE.11 This
program compared the patterns evinced by the test
compounds with the patterns produced by the standard
anticancer agents in the NCI database12 and determined
that the test compounds most likely were tubulin
binders. Among these drugs, we chose vincristine be-
cause it bears two indole rings and was present in all
the compounds tested (Table S2 in Supporting Informa-
tion). The available antitumor activity data of vincris-
tine are reported in Table 3 for comparison purposes
(the highest dose tested was 10-3 instead of 10-4).
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
3. The appropriate 2-chloroaldehyde 1 (10 mM) was dissolved
in methanol (100 mL) and treated with the equivalent of the
appropriate indolinone 2 and piperidine (1 mL). The reaction
mixture was refluxed for 3-5 h (according to a TLC test), and
the precipitate formed on cooling was collected by filtration
and crystallized. The yield was 60-70% for compounds
3a ,f,h -l and 40-50% for compounds 3b-e,g,m -o. In some
cases the precipitation was favored by partial evaporation of
the solvent and/or acidification.
The spectroscopic data for the 2-chloroderivatives are
reported in Table S1. In an attempt to synthesize compound
3b in the presence of piperidine, the following derivative was
isolated: E-3-(2-piperidinyl-3-indolylmethylene)-1-phenyl-1,3-
dihydroindol-2-one. C28H25N3O (419.5): mp 175-179 °C (dec,
MeOH). IR, νmax (cm-1): 3250, 1660, 1570, 1210, 1160. 1H
NMR, δ (ppm): 1.56 (6H, s, piperidine), 3.38 (4H, s, piperidine),
6.81 (2H, m, ar), 6.89 (1H, m, ar), 7.00 (3H, m, ar), 7.10 (1H,
t, ar, J ) 7.2), 7.29 (1H, d, ar, J ) 7.2), 7.42 (1H, m, ar), 7.52
(4H, m, ar), 7.92 (1H, s, CH), 11.46 (1H, s, NH).
Exp er im en ta l Section
(a ) Ch em istr y. The melting points are uncorrected. Analy-
ses (C, H, N) were within (0.4% of the theoretical values.
Bakerflex plates (silica gel IB2-F) were used for TLC; the
eluent was petroleum ether/acetone in various proportions.
Kieselgel 60 (Merck) was used for column chromatography.
The IR spectra were recorded in Nujol on a Nicolet Avatar
320 E.S.P.; νmax is expressed in cm-1. The 1H NMR spectra
were recorded in (CD3)2SO on a Varian Gemini (300 MHz);
the chemical shift (referenced to solvent signal) is expressed
in δ (ppm) and J in Hz. (Table S1 in Supporting Information).
The synthesis of 2d and 1c is reported below; all other starting
compounds are commercially available or previously described.
5-Hyd r oxy-6-m eth yl-2-in d olin on e 2d . 5-Methoxy-6-meth-
yl-2-indolinone 2b (6 mM) was dissolved in 60 mL of xylene
and treated with 2 g of anhydrous AlCl3. The mixture was
refluxed for 2 h, the solvent was evaporated under reduced
pressure, and the residue was treated with ice-water. The
resulting precipitate was collected by filtration and crystallized
from ethanol with a yield of 90%. C9H9NO2 (163.2): mp 269-
For experimental information on (b) antitumor activity,8 (c)
positive inotropic activity,5 and (d) CDK1 inhibitory activity,9
see their references and also the Supporting Information.
Ack n ow led gm en t. We are grateful to the National
Cancer Institute (Bethesda, MD) for the antitumor
tests and to CNRS, Station Biologique, Roscoff, France
(Laurent Meijer and Matthieu Garnier) for the CDK1
test. This work was supported by a grant from MURST
and is part of the Ph.D. thesis by M. Granaiola.
Su p p or tin g In for m a tion Ava ila ble: Experimental sec-
tion (NOE and biology), Table S1 (IR and NMR data), and
Table S2 (PCC values from COMPARE). This material is