3,3-Diaryl-1,3-dihydroindol-2-ones as Antiproliferatives Mediated by Translation Initiation Inhibition

Article abstract of DOI:10.1021/jm0499716

A series of substituted 3,3-diphenyl-1,3-dihydroindol-2-ones was synthesized from the corresponding isatins. The compounds were studied for cell growth inhibition mediated by partial depletion of intracellular Ca ²⁺ stores that leads to phosphorylation of eIF2α. The diphenyloxindole (1) showed mechanism-specific antiproliferative activity that was comparable to known translation initiation inhibitors such as clotrimazole or troglitazone. SAR studies identified m'-tert-butyl and o-hydroxy substituted diphenyloxindole (25) as a lead compound for Ca²⁺-depletion-mediated inhibition of translation initiation.

Full text of DOI:10.1021/jm0499716

1882
J . Med. Chem. 2004, 47, 1882-1885
3,3-Dia r yl-1,3-d ih yd r oin d ol-2-on es a s
An tip r olifer a tives Med ia ted by
Tr a n sla tion In itia tion In h ibition
Amarnath Natarajan, Yun-Hua Fan, Han Chen,
Yuhong Guo, J ulia Iyasere, Frederick Harbinski,
William J . Christ, Huseyin Aktas, and
J ose A. Halperin*
F igu r e 1. Clotrimazole and 3,3-diphenyloxindole Ca2+-deplet-
ing translation initiation inhibitors.
Laboratory for Translational Research, Harvard Medical
School, One Kendall Square, Building 600, 3rd Floor,
Cambridge, Massachusetts 02139
Sch em e 1. General Synthetic Approach to
Diaryloxindole Analoguesa
Received J anuary 8, 2004
Abstr a ct: A series of substituted 3,3-diphenyl-1,3-dihydro-
indol-2-ones was synthesized from the corresponding isatins.
The compounds were studied for cell growth inhibition medi-
ated by partial depletion of intracellular Ca2+ stores that leads
to phosphorylation of eIF2R. The diphenyloxindole (1) showed
mechanism-specific antiproliferative activity that was com-
parable to known translation initiation inhibitors such as
clotrimazole or troglitazone. SAR studies identified m′-tert-
butyl and o-hydroxy substituted diphenyloxindole (25) as a
lead compound for Ca2+-depletion-mediated inhibition of trans-
lation initiation.
a
(a) Chloral hydrate, NH2OH‚HCl, HCl, Na2SO4, H2O, reflux,
In tr od u ction . Initiation of mRNA translation, a
complex cellular process generally known as translation
initiation, plays a critical role in the expression of
oncogenic, prometastatic, and growth regulatory pro-
teins.1 Translation initiation is regulated by changes in
the expression and/or phosphorylation status of the
various translation initiation factors that configure the
translation initiation machinery; for example, phos-
phorylation of eIF2R on its serine 51 residue reduces
the overall rate of translation.2 Translation initiation
begins with the formation of a ternary complex between
eIF2, GTP, and initiator methionine tRNA (met-tRNA).
Phosphorylation of the R subunit of eIF2 prevents the
formation of the eIF/GTP/met-tRNA complex, limits the
rate of translation, and thereby inhibits protein syn-
thesis.3 We have demonstrated that clotrimazole (CLT)
exerts a strong anticancer effect in cancer cells and
tumors and that these anticancer properties are medi-
ated by inhibition of translation initiation (Figure 1).4
CLT exerts a distinct effect on the intracellular Ca2+
homeostasis; it releases Ca2+ from intracellular Ca2+
stores and at the same time inhibits restorative Ca2+
store-regulated Ca2+ influx through the plasma mem-
brane. This results in a sustained partial depletion of
the intracellular Ca2+ stores.4,5 Depletion of intracellular
Ca2+ stores activates eIF2 kinases (PKR and/or PERK)
resulting in phosphorylation inactivation of eIF2R on
serine 51. Inactivation of eIF2R primarily inhibits
translation initiation resulting in the preferential down-
regulation of oncogenes and growth-promoting proteins
such as cyclin D1, ras, and c-myc.6 More recently we
have shown that Ca2+-release-mediated inhibition of
translation initiation also mediates the anticancer
activity of other small molecules such as the n-3
10 min; (b) concentrated H2SO4, heat, 1 h; (c) ArMgBr, 0 °C to
room temperature; (d) ArR3, p-TsOH, DCE, 85 °C or TfOH, DCM,
room temperature.
polyunsaturated fatty acid eicosapentaenoic acid (EPA)7
and troglitazone (TRO),8 hereon termed Ca2+-depleting
translation initiation inhibitors. Because Ca2+-depleting
translation initiation inhibitors preferentially modulate
the synthesis of oncoproteins over “housekeeping” pro-
teins, analogues of this type of compound have the
potential to make robust anticancer drugs.
As part of an ongoing project aimed at developing
novel mechanism-specific anticancer agents, we recently
identified 3,3-diphenyloxindole 1 (Figure 1) as a Ca2+
-
depleting translation initiation inhibitor. In this letter,
we report the synthesis of 3,3-diphenyloxindole ana-
logues and their functional evaluation in translation
initiation specific assays.
Ch em istr y. The general synthetic approach to the
3,3-diaryloxindole compounds is outlined in Scheme 1.
The appropriate isatins A were either commercially
available or synthesized starting from appropriate
anilines.10 As an example, 9 and 10 (Table 1) were
synthesized starting from 3-bromoaniline, which upon
condensation with hydroxylamine and chloral hydrate
followed by concentrated sulfuric acid resulted in an
inseparable regioisomeric mixture of 4- and 6-bromo-
isatins. The addition of the two phenyl groups to the
3-position of the oxindole allowed easy chromatographic
separation of the 4- and 6-bromo-3,3-diphenyloxindoles
9 and 10. A bioassay-guided iterative approach was
undertaken in the study of the diaryloxindole com-
pounds. The compounds synthesized addressed the
electronics and sterics by substituting the three phenyl
rings and the nitrogen with various functional groups.
The symmetric diphenyl compounds were obtained
using TfOH following previously reported procedures,11
* To whom correspondence should be addressed. Phone: 617-621-
6136. Fax: 617-621-6148. E-mail: jose_halperin@hms.harvard.edu.
10.1021/jm0499716 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/09/2004

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 1883
Ta ble 1. 3,3-Diaryloxindole Analoguesa Assayed for Intracellular Ca2+ Depletion, eIF2R Phosphorylation, and Growth Inhibition
compd
R1
R2
R3
R4
R5
intracellular Ca2+ depletion
eIF2R phosphorylationb
GI50 (µM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
tosyl
Ph
Me
H
H
H
H
H
H
H
H
H
H
H
yes
yes
yes
yes
yes
yes
yes
no
yes
yes
no
no
no
1
13
5-F
5-Cl
5-Br
5-I
5-Et
5-NO2
5-SO3H
4-Br
6-Br
7-Br
H
H
H
H
H
H
H
H
H
0.44
0.61
0.79
1.31
0.85
1.2
8
>20
14
9
0.84
0.89
no
yes
no
>20
o-OMe
m-OMe
p-OMe
o-OH
p-OH
m-t-Bu
p-t-Bu
o-OMe
o-OH
p-OH
H
0.48
no
yes
yes
yes
yes
no
yes
yes
yes
yes
no
0.56
0.86
1.04
0.95
0.93
1.33
2.01
2
4
8
H
H
H
H
H
5-I
H
p-t-Bu
p-t-Bu
p-t-Bu
m′-t-Bu
m′-t-Bu
m′-CF3
7
14
3
4
>20
OH
OH
OH
H
H
H
H
1.4
a
b
All compounds were characterized and purity was assessed using 1H NMR and LCMS. Normalized to 1.
while the unsymmetric compounds were synthesized
using a two-step procedure. The first step involved a
Grignard addition to isatin using appropriately substi-
tuted arylmagnesium halide, and the key second step
involved the generation of a quaternary center at the
3-position of the oxindole ring.12 This was accomplished
by an acid-catalyzed Friedel-Crafts type condensation
of B (Scheme 1) with the appropriate aromatic ring to
generate diaryloxindoles C. For activated ring systems
that contained an electron-donating group, p-toluene-
sulfonic acid in dichloroethane allowed the condensation
to occur smoothly, while the unactivated ring systems
required the use of triflic acid (a much stronger acid).13
Biology. All compounds synthesized were first
screened for their ability to release Ca2+ from internal
stores using FURA-2AM loaded cells assayed in Ca2+
free media, as previously described.6-8 Compounds that
release Ca2+ in this assay were further evaluated for
their ability to phosphorylate eIF2R and were quantified
by Western blot analysis using both a phospho-specific
and a total anti-eIF2R antibody.9 The compounds that
released intracellular Ca2+ and phosphorylated eIF2R
were further tested for anticancer activity in vitro using
a human lung cancer cell line, as previously described.6-8
The endoplasmic reticulum (ER) is a main site of
cellular Ca2+ storage. To formally demonstrate that the
3,3-diaryloxindoles compounds partially deplete the ER-
calcium stores, we tested our lead compound, 25, in an
ER-Ca2+ specific assay that uses stable cells lines
carrying ER-targeted Ca2+-sensitive cameleon pro-
teins.14 These proteins emit light, and the Ca2+ content
is measured by fluorescent resonance energy transfer
(FRET), which is a function of the Ca2+ content of the
ER.
Resu lts a n d Discu ssion . To develop Ca2+-depleting
translation initiation inhibitors, we took a bioassay-
guided iterative approach. Ca2+-depleting translation
initiation inhibitors are those compounds that induce
Ca2+ release from intracellular stores, phosphorylation
of eIF2R, and growth inhibition, as summarized in Table
1.
Several 5-substituted isatins are commercially avail-
able; hence, the first batch of compounds involved the
variation of the functional groups at the 5-position on
the oxindole phenyl ring (2-8) to explore the size and
electronic effects of the substituents on the biological
activity. Interestingly, all the substitutions except a
sulfonic acid group generated compounds that released
intracellular Ca2+. Given the short time course of our
Ca2+ release assay (minutes), the absence of Ca2+
depletion activity in the sulfonic acid analogue could be
due in part to poor cell permeability. The apparently
high growth inhibitory effect of 8 may reflect increased
cellular toxicity. Comparison of eIF2R phosphorylation
among 1-8 showed a good correlation between the size
of the substituent at the 5-position and the eIF2R
phosphorylation activity of the compounds. The 5-iodo
substitution increased eIF2R phosphorylation by 20%

1884 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8
Letters
compared to the parent compound 1 and also exhibited
stronger inhibitory activity on cancer cells growth. In
contrast, an ethyl substitution at the 5-position of the
oxindole ring (6) resulted in a 15% reduction in eIF2R
phosphorylation, which was also reflected in a reduced
inhibition of cancer cells growth.
synthesized 26 to explore the effect of combining a
hydroxy at the ortho position and a t-Bu at the meta′
position of the 3-phenyl ring of 25, with the iodo group
at the 5-position of the phenyl ring of the oxindole, as
in 5. Contrary to our expectation, these combined
substitutions did not improve the biological activity.
Interestingly, replacing the t-Bu on 25 with a trifluoro-
methyl group (27) completely abolished the Ca2+-deplet-
ing and the growth inhibitory activity observed in the
parent compounds.
In conclusion, we have identified 3,3-diaryloxindoles
as Ca2+-depleting translation initiation inhibitors. Using
a bioassay-guided modification approach, we have iden-
tified a new mechanism-specific lead, 25, which induced
partial depletion of ER-Ca2+ stores, is more potent than
its parent compound 1 in inducing eIF2R phosphoryl-
ation, and is also 4 times more potent in inhibiting
cancer cells growth. We are currently using 25 as a lead
compound in our efforts to identify the molecular target
for ER Ca2+ depletion. We are also conducting experi-
ments to evaluate 25 preclinically in animal models of
experimental cancer.
The positional effect (4, 5, 6, and 7) of a substituent
on the phenyl ring of oxindole on the biological activity
was investigated by synthesizing 9, 4, 10, and 11 with
a bromo group at positions 4, 5, 6, and 7, respectively.
The 7-Br substitution in 11 abolished the Ca2+ depletion
activity, and the other substitutions did not result in
any improvement of the biological activities. Interest-
ingly, substituting the nitrogen (12-14) with alkyl, aryl,
and electron-withdrawing groups abolished the Ca2+
depletion activity.
We then turned our attention to substituting the
phenyl rings at the 3-position of the oxindole ring. We
placed electron-releasing groups such as methoxy, hy-
droxy, and tert-butyl at different positions on one of the
phenyl rings (15-21). We observed a loss of Ca2+
depletion activity with meta- and para-substituted
methoxy groups (16 and 17). However, the ortho-
substituted methoxy, hydroxyl (15, 18), and the para-
substituted hydroxyl showed Ca2+ depletion but with
diminished eIF2R phosphorylation activity compared to
1. It is interesting to note that both methoxy and
hydroxyl substitutions at the ortho position (15 and 18)
showed Ca2+ depletion but not the para-substituted
methoxy compound (17); this result suggests that the
biological activity of these compounds is mediated by a
nonelectronic effect. Most rewarding in this monosub-
stituted series was the t-Bu substitution at meta and
para positions, (20 and 21, respectively), which caused
Ca2+ depletion and eIF2R phosphorylation with im-
proved growth inhibitory activity. These results prompted
us to differentially substitute the two phenyl rings (22-
24). Although, the m-t-Bu substituted compound 20
shows better activity compared to the p-t-Bu substituted
compound 21, we decided to use the p-t-Bu for the
differential substitution because the p-t-BuPhMgBr is
commercially available. As predicted, ortho and para
hydroxyl substitutions on one ring and a para tert-butyl
on the other (23 and 24) resulted in a significant
improvement of biological activity compared to the
parent compound 1. Interestingly, the o-OMe substitu-
tion (22) resulted in an inactive compound.
Ack n ow led gm en t. We thank Drs. Daniel C. Toste-
son, Magdalena Tosteson, and Michael Chorev for com-
ments and continuous support. This work was supported
in part by NIH Grant NCDDG 5 U19 CA 87427.
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ring might result in a better Ca2+-depleting translation
initiation inhibitor. To test this hypothesis, we synthe-
sized and assayed 25, which has an o-hydroxy and a
m′-t-Bu on the same ring.15 This compound showed very
good Ca2+-depleting activity, a 2-fold increase in the
eIF2R phosphorylation, and a 4-fold improvement in the
growth inhibition assay. Furthermore, 25 induced a
depletion of ER-Ca2+, as determined in cells carrying
ER targeted Ca2+-sensitive cameleon protein. Combined,
these results indicate that the growth inhibitory activity
of 25 is most likely mediated by partial depletion of ER-
Ca2+ stores, phosphorylation of eIF2R, and its down-
stream effects on cell cycle regulatory proteins.
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Since the 5-iodo substituted compound 5 was the most
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a Combinatorial
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Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 8 1885
(12) Hewawasam, P.; Gribkoff, V. K.; Pendri, Y.; Dworetzky, S. I.;
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Fluorooxindoles as Openers of Maxi-K Potassium Channels.
Bioorg. Med. Chem. Lett. 2002, 12, 1023-1026.
washed with water followed by brine, dried over Na2SO4, and
filtered. The filterate was concentrated in vacuo to yield 3-
hydroxy-3-phenyl-1,3-dihydroindol-2-one (20.1 g, 90%) as a pale-
yellow solid. A mixture of the above solid (2.5 g, 10 mmol), p-t-
Bu-phenol (1.5 g, 10 mmol) and p-toluenesulfonic acid (3 g) in
40 mL of dichloroethane, was heated to 95 °C for 6 h. The
mixture was cooled and filtered, and the filterate was concen-
trated in vacuo. Flash chromatography of the resulting solid
yielded 25 (2.4 g, 66%) as a colorless solid: 1H NMR (400 MHz,
DMSO-d6) δ 10.41 (s, 1H), 9.24 (s, 1H), 7.23-7.34 (m, 5H), 7.18
(t, J ) 8.0 Hz, 1H), 7.08 (dd, J ) 8.0, 1.6 Hz, 1H), 7.01 (d, J )
7.2 Hz, 1H), 6.93 (t, J ) 8.0 Hz, 1H), 6.86 (d, J ) 7.2 Hz, 1H),
6.74 (d, J ) 1.6 Hz, 1H), 6.59 (m, 1H), 1.07 (s, 9H); 13CNMR
(125 MHz, DMSO-d6) δ 180.22, 153.56, 143.14, 140.92, 140.28,
133.49, 129.08, 128.94, 128.60, 128.47, 127.88, 127.42, 126.30,
125.43, 121.79, 115.73, 110.01, 60.70, 34.36, 31.99 ppm; MS
(APCI+) m/z 358.23 (MH), 302.18 (loss of t-Bu), 208.14 (loss of
tert-butylphenol).
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Snyder, S. A. Total Synthesis of Diazonamide A. Angew. Chem.,
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(14) Miyawaki, A.; Mizuno, H.; Llopis, J .; Tsien, R. Y.; J alink, K.
Cameleons as Cytosolic and Intra-Organellar Calcium Probes;
Oxford University Press: Oxford, U.K., 2001; pp 3-16.
(15) (5-tert-Butyl-2-hydroxyphenyl)-3-phenyl-1,3-dihydroindol-2-
one (25): To isatin (15 g, 0.1 mole) in 60 mL THF at 0 °C was
added dropwise a 1 M solution of phenylmagnesium bromide
(0.33 mol). The resulting mixture was allowed to warm to room
temperature and was spun at room temperature for 12 h. The
reaction was quenched with a saturated solution of NH4Cl (100
mL), and the mixture was diluted with 100 mL of dichloro-
methane. The layers were separated, and the organic layer was
J M0499716