Novel arylsulfoanilide-oxindole hybrid as an anticancer agent that inhibits translation initiation

Article abstract of DOI:10.1021/jm0496234

Structure-activity relationship studies of substituted arylsulfoanilides as antiproliferatives, which are mediated by the partial depletion of intracellular Ca²⁺ stores, resulted in the identification of compounds with micromolar activity against lung cancer cells in a growth inhibition assay. Incorporating the substitution pattern of the best arylsulfoanilides onto the 3-phenyloxindole scaffold resulted in a potent arylsulfoanilide-oxindole hybrid, 27. Compound 27 inhibits cancer cell growth by partial depletion of intracellular Ca²⁺ stores and phosphorylation of eIF2α.

Full text of DOI:10.1021/jm0496234

© Copyright 2004 by the American Chemical Society
Volume 47, Number 21
October 7, 2004
Letters
Novel Ar ylsu lfoa n ilid e-Oxin d ole Hybr id
a s a n An tica n cer Agen t Th a t In h ibits
Tr a n sla tion In itia tion
Amarnath Natarajan, Yuhong Guo,
Frederick Harbinski, Yun-Hua Fan, Han Chen,
Lia Luus, J ana Diercks, Huseyin Aktas,
Michael Chorev, and J ose A. Halperin*
F igu r e 1. Diaryloxindoles as Ca2+ depleting translation
initiation inhibitors.
Laboratory for Translational Research, Harvard Medical
School, One Kendall Square, Building 600, 3rd Floor,
Cambridge, Massachusetts 02139
Importantly, the low eIF2B/eIF2R molecular ratio sug-
gests that even partial phosphorylation of eIF2R will
suffice to scavenge eIF2B effectively and block transla-
tion initiation.8 Small-molecule translation initiation
inhibitors such as clotrimazole (CLT), the n-3 polyun-
saturated fatty acid eicosapentaenoic acid (EPA), and
troglitazone (TRO) cause phosphorylation of the key
translation initiation factor eIF2R, which mediates their
growth inhibitory activity in cancer cells and anticancer
activity in tumors.9-11 The anticancer effects of CLT,
EPA, and TRO are triggered by the partial depletion of
intracellular Ca2+ stores and inhibition of restorative
Ca2+ store-regulated Ca2+ influx through the plasma
membrane, thereby causing a sustained depletion of the
intracellular Ca2+ stores.12,13 This partial Ca2+ depletion
activates eIF2 kinases (PKR and/or PERK), resulting
in phosphorylation of eIF2R on serine 51 leading to its
inactivation. Inactivation of eIF2R inhibits primarily
ternary-complex-dependent translation initiation, re-
sulting in the preferential down-regulation of growth-
promoting proteins such as cyclin D1.10-12 We have
identified this Ca2+ depletion mediated translation
initiation inhibition in other small molecules including
the 3,3-diaryloxindole14 (Figure 1, 1 and 2) and TRO
analogues.15
Received May 21, 2004
Abstr a ct: Structure-activity relationship studies of substi-
tuted arylsulfoanilides as antiproliferatives, which are medi-
ated by the partial depletion of intracellular Ca2+ stores,
resulted in the identification of compounds with micromolar
activity against lung cancer cells in a growth inhibition assay.
Incorporating the substitution pattern of the best arylsulfo-
anilides onto the 3-phenyloxindole scaffold resulted in a potent
arylsulfoanilide-oxindole hybrid, 27. Compound 27 inhibits
cancer cell growth by partial depletion of intracellular Ca2+
stores and phosphorylation of eIF2R.
Translation initiation plays a vital role in the expres-
sion of oncogenic, prometastatic, and growth regulatory
proteins;1-3 inhibitors of translation initiation make an
attractive class of anticancer agents.4 The key step in
translation initiation is the formation of a ternary
complex between eukaryotic initiation factor 2 (eIF2),
guanosine 5′-triphosphate (GTP), and initiator methio-
nine transfer ribonucleic acid (tRNAimet).5 At the end of
each initiation cycle the GTP in the ternary complex is
hydrolyzed to guanosine 5′-diphosphate (GDP) that
must be exchanged for GTP by eIF2B to trigger a new
round of translation initiation.6 Phosphorylation of
eIF2R on serine 51 increases the affinity of eIF2R for
the scarce eIF2B and converts the phosphorylated eIF2R
into an inhibitor of the GDP-GTP exchange factor
eIF2B, and thereby inhibits translation initiation.7
During a high-throughput screen of the open reposi-
tory NCI library for translation initiation inhibitors, we
encountered several hits containing the sulfonamide
functionality. Sulfonamides are an important class of
compounds that are reported to have substantial anti-
cancer activity in vitro and in vivo.16,17 A p-substituted
* To whom correspondence should be addressed. Phone: 617-621-
6136. Fax: 617-621-6148. E-mail: jose_halperin@hms.harvard.edu.
10.1021/jm0496234 CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/04/2004

4980 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Letters
Ta ble 1. Arylsulfoanilide Analogues Assayed for Partial
Sch em e 1. General Synthetic Approach to Aryl
Sulfonanilide-Oxindole Hybridsa
Depletion Intracellular Ca2+ Stores and Growth Inhibition of
Lung Cancer Cells (A549)
GI50 ( <10% Ca2+ depletion
compd
R1
R2
R3
R4
(µM)
assaya
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
13
3
0.87
1.10
0
H
Br
I
t-Bu
Ph
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2-OH
3-OH
4-OH
2-Me
3-Me
4-Me
2-OH 4-Me
3-OH 4-Me
3-OH 4Me
3-OH 4-Me
3-OH 4-Me
3-OH 4-Me
3-OH 4-Me
3-OH 4-Me
3-OH 4-Me
H
>80
30
23
16
17
16
52
30
13
12
14
>80
80
11
8
H
H
H
H
H
H
H
H
H
H
H
H
H
0.38
0.87
1.44
1.37
0.63
0.13
0.14
1.17
0.55
0.33
0.49
0.95
1.11
1.44
1.12
0.14
0.12
0.19
0.27
0.45
0.72
1.10
a
Reagents and conditions: (a) pyridine, 0 °C, 1-12 h; (b)
p-TsOH, DCE, 85 °C.
OMe
NO2
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
t-Bu
NO2
OMe
NHAc
Br
diarylsulfonamide (E7070) that is in clinical trials
displays antitumor cell cycle in the G1 phase.18-20 Taken
together, this led us to investigate arylsulfoanilides as
calcium-depleting translation initiation inhibitors. Herein,
we report a structure-activity relationship study of a
series of arylsulfoanilides that led to the synthesis and
functional evaluation of a series of arylsulfoanilide-
oxindole hybrids (Figure 1, 27) using translation initia-
tion specific assays.
6
29
51
48
20
15
9
The general synthetic approach to the arylsulfoanil-
ides and the arylsulfoanilide-oxindole hybrids is out-
lined in Scheme 1. The appropriately substituted aryl-
amines were coupled to the corresponding sulfonyl
chlorides in pyridine at 0 °C.21,22 The sulfonanilides
3-25 synthesized were purified by recrystallization or
by flash silica gel chromatography. The different sub-
stitution patterns on the two phenyl rings and the
nitrogen with various functional groups represent a
wide range of electronic and steric effects. A bioassay
guided iterative approach using two cell-based assays
was undertaken in the study of the arylsulfoanilide
compounds. The structure-function relationship study
identified potent arylsulfoanilides as Ca2+ depleting
anticancer agents. The substitution pattern of potent
arylsulfoanilides was used to generate the arylsulfo-
anilide-oxindole hybrids. An electrophilic substitution
of the anilide portion of the appropriately substituted
sulfonanilides on the 3-hydroxy-3-phenyl-1,3-dihydroin-
dol-2-one (Scheme 1) was carried out in the presence of
p-toluenesulfonic acid (p-TsOH) to yield the arylsulfo-
anilide-oxindole hybrids.
All compounds were screened for their ability to
partially deplete intracellular Ca2+ stores using Fura-2
AM loaded cells (NIH 3T3) and cell growth inhibition
in a human lung cancer cell line (A549).23,24 To formally
demonstrate that the most active arylsulfoanilide-
oxindole hybrid, 27, targets ternary-complex-dependent
translation initiation, 27 was further evaluated in
translation initiation specific functional assays, namely,
phosphorylation of eIF2R and a dual luciferase ternary
complex assay.25 Additionally, the growth inhibitory
activity of the hit, lead, and lead-optimized diaryloxin-
doles 1, 2, and 27, respectively, was assessed against
two cancer cell lines (A549 and DU145) using the
sulforhodamine B (SRB) assay.24
I
Ph
OPh
7
a
Ratio of the peak heights of the compound normalized to the
peak of CLT as a reference measured in the same batch of cells.
The data presented are the average of two runs with a variability
of <10%.
and Ca2+ depletion assays of a series of arylsulfoanilides
are summarized in Table 1.
To explore the size and electronic effects of a sub-
stituent at the p-position on the partial depletion of
intracellular Ca2+ stores and growth inhibitory activity,
commercially available p-substituted sulfonyl chlorides
were condensed with aniline to generate the first set of
arylsulfoanilides (3-9). Both electron-donating and
-withdrawing groups at the p-position contributed to
enhanced growth inhibition and partial depletion of
intracellular Ca2+ stores activity (cf. 6, 8, and 9 to 3).
However, the improvement in the biological activity
contributed by the electron-withdrawing group (9) is
marginal compared to the contribution from electron-
donating groups (6 and 8).
The biological data also show that the 4-position
substituted with a hydrophobic group (cf. 5-7 to 3 and
9) results in better depletion of intracellular Ca2+ stores
and growth inhibitory activity. From this series of
compounds a tert-butyl group at the 4-position is the
most active arylsulfoanilide compound in both assays.
Unexpectedly, we observed that substituting the nitro-
gen of the sulfonanilide group (6 and 10) resulted in an
order of magnitude loss of activity in the Ca2+ depletion
assay and a 50% loss of activity in the growth inhibition
assay. This suggests that the -NH of sulfonamide group
is critical for its biological activity.
In our efforts to develop novel mechanism-specific
antiproliferatives, we explored the SAR of arylsulfo-
anilides and arylsulfoanilide-oxindole hybrids as anti-
cancer agents mediated by partial depletion of intra-
cellular stores. The results from the growth inhibition
Although 6 and 8 are equipotent in the growth
inhibition assay, 6 (which includes a p-tert-butyl sub-
stitution) was more potent than 8 (which includes a

Letters
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 4981
p-methoxy substitution) in the Ca2+ depletion assay.
Therefore, the next set of compounds (11-16) were
generated by condensing p-tert-butylphenylsulfonyl chlo-
ride with o-, m-, and p-hydroxylanilines (11-13) and
o-, m-, and p-tolylamines (14-16). These compounds
addressed the electronics and the positional effects on
the arylamino ring while maintaining the best substitu-
ent, p-tert-Bu, on the arylsulfonyl ring. The positional
effect of the hydroxyl group (11-13) was minimal with
regard to the growth inhibition and showed progressive
improvement: o-OH (11) > m-OH (12) > p-OH (13) in
the Ca2+ depletion assay. Interestingly, the reverse
order of potency was true with the methyl substitutions
(14-16), namely, the p-Me (16) > m-Me (15) > o-Me
(14) in the growth inhibition and the calcium depletion
assays. On the basis of these results we hypothesized
that doubly substituting the arylamino ring with an o-
or m-hydroxy and p-methyl groups (17 and 18) while
maintaining the p-tert-butyl substitution on the aryl-
sulfonyl ring would result in a more active Ca2+ deplet-
ing anticancer agent. As anticipated, 17 and 18 turned
out to be low micromolar Ca2+ depleting antiprolifera-
tives, suggesting that the substitutions on the arylamino
ring result in an additive effect.
Ta ble 2. Arylsulfoanilide-Oxindole Hybrids Assayed for
Partial Depletion Intracellular Ca2+ Stores and Growth
Inhibition of Lung Cancer Cells (A549)
GI50 ( <10% Ca2+ depletion
compd -NHSO2Ar R1
R2
(µM)
assaya
26
27
28
29
m
o
o
t-Bu o′-Me
t-Bu m′-Me
>20
0.8
4
0.44
1.06
0
Ph
m′-Me
o
OPh m′-Me
5
0
a
Ratio of the peak heights of the compound normalized to the
peak of CLT as a reference measured in the same batch of cells.
The data presented are the average of two runs with a variability
of <10%.
Ta ble 3. Comparison of Hit 1, Lead 2, and Lead-Optimized 27
in Cell-Based Translation Initiation Specific Assays
GI50 (µM)
compd
eIF2R-P
TCa
A549
DU145
1
2
27
1.00
2.01
2.14
1.00
2.15
4.98
13.12 ( 0.73
2.71 ( 0.15
0.7 ( 0.13
12.51 ( 0.99
2.49 ( 0.21
1.08 ( 0.09
We generated the next series of compounds (19-25),
maintaining its substitution pattern of 18, by condens-
ing 5-amino-o-cresol with a series of p-substituted
phenylsulfonyl chlorides. Upon testing this series in the
biological assays, we identified two additional low
micromolar compounds (24 and 25) that were good Ca2+
depletors. Again, substitution with large hydrophobic
substituents in the p-position of the arylsulfonyl ring
was favorable.
a
For details of the ternary complex assay (TC), see Supporting
Information.
is summarized in Table 3. The data from these assays
are normalized to the hit 1. Going from 1 to 27, we
observe a 2-fold increase in the phosphorylation and
about a 5-fold increase in the ternary complex assay,
which correlates well with an order of magnitude
improvement in the growth inhibition of lung (A549)
and prostate (DU145) cancer cells lines.
In conclusion, we used two cell-based assays to screen
a focused library of arylsulfoanilides. Structure-func-
tion analysis led to the identification of key substitution
patterns necessary for partial depletion of intracellular
Ca2+ stores mediated growth inhibition. Incorporating
these structural features into the diaryloxindole scaffold
resulted in a submicromolar mechanism specific trans-
lation initiation inhibitor 27 that is an order of magni-
tude more potent in growth inhibition assay than the
hit 1 in two different cancer cell lines. We are currently
conducting preclinical studies to evaluate the efficacy
of 27 in animal models of experimental cancer.
On the basis of our previous studies, we knew that 2
was a potent Ca2+ depleting translation initiation
inhibitor and that there was no activity-enhancing effect
by functionalizing the other two aromatic rings.14
Therefore, we hypothesized that incorporating the
structural features of the most active (GI50 < 10 µM)
arylsulfoanilide Ca2+ depletors (17, 18, 24, and 25) into
the 3-aryl-3-phenyl-1,3-dihydroindol-2-one scaffold would
result in potent Ca2+ depleting translation initiation
inhibitors. To test this hypothesis, we synthesized four
arylsulfoanilide-oxindole hybrids (26-29) by an elec-
trophillic substitution in the presence of p-toluene-
sulfonic acid. The biological data for this series are
summarized in Table 2. Indeed, incorporation of the (3-
hydroxy-4-methyl)-4′-tert-butylbenzenesulfonanilide (18)
resulted in a submicromolar 27 in a growth inhibition
assay with good Ca2+ depletion activity. Additionally,
moving the sulfonanilide moeity to the meta position
(26) results in reduced growth inhibitory (GI50 > 20 µM)
and Ca2+ depletion activity, suggesting that the orienta-
tion of the p-tert-butylphenylsulfonanilide group is
important for the activity. It is also interesting to note
that the -Ph (28) and -OPh (29) substitutions at R1
do not possess any Ca2+ depletion activity, suggesting
that their moderate growth inhibitory activity may not
be mediated by the Ca2+ depleting translation initiation
inhibitory pathway.
Ack n ow led gm en t. We thank Drs. Daniel C. Toste-
son and Magdalena Tosteson for their continuous
encouragement and support. This work was supported
in part by NIH Grant NCDDG 5 U19 CA87427.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for the biological assays and for all compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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