Discovery of substituted N′-(2-oxoindolin-3-ylidene)benzohydrazides as new apoptosis inducers using a cell- and caspase-based HTS assay

Article abstract of DOI:10.1016/j.bmcl.2009.03.121

We report the discovery of a series of substituted N′-(2-oxoindolin-3-ylidene)benzohydrazides as inducers of apoptosis using our proprietary cell- and caspase-based ASAP HTS assay. Through SAR studies, N′-(4-bromo-5-methyl-2-oxoindolin-3-ylidene)-3,4,5-tr

Full text of DOI:10.1016/j.bmcl.2009.03.121

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2710–2713
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of substituted N0-(2-oxoindolin-3-ylidene)benzohydrazides
as new apoptosis inducers using a cell- and caspase-based HTS assay
*
Nilantha Sirisoma, Azra Pervin, John Drewe, Ben Tseng, Sui Xiong Cai
EpiCept Corporation, 6650 Nancy Ridge Drive, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
We report the discovery of a series of substituted N0-(2-oxoindolin-3-ylidene)benzohydrazides as induc-
ers of apoptosis using our proprietary cell- and caspase-based ASAP HTS assay. Through SAR studies,
N0-(4-bromo-5-methyl-2-oxoindolin-3-ylidene)-3,4,5-trimethoxybenzohydrazide (3g) was identified as
Article history:
Received 4 March 2009
Revised 23 March 2009
Accepted 25 March 2009
Available online 28 March 2009
a potent apoptosis inducer with an EC50 value of 0.24 lM in human colorectal carcinoma HCT116 cells,
more than a 40-fold increase in potency from the initial screening hit N0-(5-bromo-2-oxoindolin-3-yli-
dene)-3,4,5-trimethoxybenzohydrazide (2a). Compound 3g also was found to be highly active in a
Keywords:
growth inhibition assay with a GI50 value of 0.056
ous soluble analogs were prepared and found to be highly active. Among them, compound 4e incorporat-
ing a methyl piperazine moiety was found to have EC50 values of 0.17, 0.088 and 0.14 M in human
lM in HCT116 cells. A group of potentially more aque-
Apoptosis inducer
Anticancer agent
HTS
l
colorectal carcinoma cells HCT116, hepatocellular carcinoma cancer SNU398 cells and human colon can-
cer RKO cells, respectively. Compounds 3g and 4e were found to function as inhibitors of tubulin
polymerization.
SAR
Tubulin inhibitor
Ó 2009 Elsevier Ltd. All rights reserved.
Apoptosis, or program cell death, plays a crucial role in normal
cell development and tissue homeostasis.1 Apoptosis is used by
organisms to control their cell numbers and to eliminate unneeded
or damaged cells.2 Inappropriate apoptosis induction is known to
result in excessive cell death, and could cause degenerative dis-
eases.3 Inadequate apoptosis, however, could lead to over prolifer-
ation of cells and cancer.4 In addition, it is known that the anti-
tumor efficacy of many chemotherapeutical agents is correlated
to their apoptosis inducing ability.5 Identification of compounds
that promote or induce apoptosis in cancer cells, therefore, is an
attractive approach for anticancer research.6
We have been interested in the discovery and development of
apoptosis inducers as potential anticancer agents.7 Applying our
novel caspase-3 substrates,8 we have developed a caspase- and
cell-based, high throughput screening technology, termed Apopto-
sis Screening and AntiCancer Platform (ASAP), for the identification
of apoptosis inducers.9 We have reported the discovery of several
novel series of apoptosis inducers, including 4-aryl-4H-chromenes
(1a),10 gambogic acid (1b),11 3-aryl-5-aryl-1,2,4-oxadiazoles (1c),12
N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines (1d),13 4-anilino-
quinazolines (1e)14,15 and 4-aryl-3-(3-aryl-1-oxo-2-propenyl)-
2(1H)-quinolinones (1f)16 (Chart 1). Herein we report the discovery
of substituted N0-(2-oxoindolin-3-ylidene)-benzohydrazide (2a),
an isatin derivative, as an apoptosis inducer using our HTS assay.
SAR study of 2a led to the discovery of N0-(4-bromo-5-methyl-2-
oxoindolin-3-ylidene)-3,4,5-trimethoxybenzohydrazide (3g) and
analogs as potent apoptosis inducers.
Many isatins and isatin derivatives have been synthesized and
reported to have a variety of biological activities, including as SARS
coronavirus 3C-like protease inhibitors,17 caspase-3 inhibitors,18
and as inhibitors of Src homology-2 domain containing protein
tyrosine phosphatase-2.19 More recently, N-alkyl isatin acylhyd-
razone derivatives such as 7a (Chart 2) have been reported to be
potent and selective cannabinoid receptor 2 inverse agonists for
the potential treatment of neuropathic pain.20 In addition, N-
substituted isatins such as 7b have been reported to be cytotoxic
with a mode of action that includes inhibition of tubulin polymer-
ization, induction of G2/M cell cycle arrest and activation of cas-
pase-3 and -7.21
Substituted N0-(5-bromo-2-oxoindolin-3-ylidene)-benzohydraz-
ides 2a–2f were obtained from ChemDiv and Asinex, and their
1
structures were confirmed by H NMR and MS. Substituted N0-(2-
oxoindolin-3-ylidene)-3,4,5-trimethoxybenzohydrazides
3a–3m
were prepared from condensation of the corresponding substituted
isatin (5)19 with substituted 3,4,5-trimethoxybenzohydrazide (6)
according to reported procedures.20,22 The N-substituted analogs
4a–4h were prepared from condensation of 2a, 3a, 3g and 3h with
formaldehyde and an amine following literature procedures
(Scheme 1).23,24
The apoptosis inducing activity of substituted N0-(2-oxoindolin-
3-ylidene)-benzohydrazides was measured using our cell- and cas-
* Corresponding author. Tel.: +1 858 202 4006; fax: +1 858 202 4000.
E-mail address: scai@epicept.com (S.X. Cai).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.121

N. Sirisoma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2710–2713
2711
O
OMe
OH
MeO
Br
O
Cl
O
O
O
Cl
O
N
CN
S
N
O
N
Me2N
O
NH2
OH
1a
1b
1c
O
OMe
F
O
HN
N
Br
NO2
N
N
N
N
N
H
O
N
Me
1d
1e
1f
Chart 1.
colon cancer RKO cells, and the results are summarized in Tables
1–3. Compound 2a was found to have EC50 values of 4–10 M in
the three cell lines tested. By maintaining the 5-bromo group in
the isatin, we explored replacement of the 3,4,5-trimethoxy groups
in the benzoyl group of 2a by other groups. Table 1 showed that,
except for compound 2b, all these compounds (2c–2f) were inac-
O
l
O
NH
N
Br
O
O
N
N
MeO
Br
I
tive up to 20 lM in all the three cell lines, indicating that the
3,4,5-trimethoxy group is preferred.
7a
7b
By maintaining the 3,4,5-trimethoxybenzoyl group, we then ex-
plored substitutions in the 4- to 7-positions of isatin ring (Table 2).
The 5-methoxy (3a) and 5-iodo (3b) analogs were similar or
slightly more active than 2a. The 5-trifluoromethoxy (3c), 5-amino
(3d) and 5-acetylamino (3e) analogs were less active or inactive in
Chart 2.
pase-based HTS assay7 in human colorectal carcinoma cells
HCT116, hepatocellular carcinoma cancer SNU398 cells and human
OMe
OMe
OMe
OMe
HCHO/HNR1R2
OMe
O
O
MeO
OMe
O
MeOH
AcOH
NH
NH
O
N
OMe
N
OMe
R
O+
H2O/EtOH
N
R
R
O
H
HN
NH2
O
N
N
NR1R2
H
3
6
4
5
Scheme 1.
Table 1
Activity of substituted N0-(5-bromo-2-oxoindolin-3-ylidene)-benzohydrazides in the caspase activation assay
R4
R3
O
R2
NH
N
R1
Br
O
N
H
R1
R2
R3
R4
EC50 (lM)
a
Compound #
HCT116
SNU398
RKO
2a
2b
2c
2d
2e
2f
H
H
OMe
H
H
OMe
OMe
OMe
H
H
H
H
10.7 0.5
>20
>20
>20
>20
8.9 0.2
>20
>20
>20
>20
4.4 0.5
9.7 0.6
>20
>20
>20
OCH2O
H
F
Br
H
H
H
H
NO2
H
H
>20
>20
>20
a
Cells were treated with the test compounds for 48 h, and data are the mean of three or more experiments and are reported as mean standard error of the mean (SEM).

2712
N. Sirisoma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2710–2713
Table 2
the three cell lines. These data suggested that strong electron with-
drawing, or hydrophilic, or large groups are not favored at the 5-
position. The 4-chloro analog 3f was >2-fold more potent than 2a
in all the three cell lines, indicating that a small group at the 4-pos-
tion might increase potency. Combination of substitutions at both
the 4- and 5-positions led to the 4-bromo-5-methyl analog 3g that
was highly potent with an EC50 value of 0.24 lM in HCT116 cells,
>40-fold more potent than 2a. The 4,5-dichloro analog 3h also
was highly active. Interestingly, 5-methyl-4-phenyl analog 3i was
inactive up to 20 lM, indicating that a large group is not tolerated
at 4-position. 5,6-Di-substituted analogs (3j and 3k) and 5,7-di-
substituted analogs (3l and 3m) were found to have low activity
or inactive, suggesting that substitutions at the 6- and 7-positions
may not be preferred.
To explore the SAR further and to improve the aqueous solubil-
ity, we introduced an N-morpholinomethyl group into the nitrogen
of the isatin of compounds 2a, 3a, 3g and 3h. Table 3 showed that
compounds 4a–4d had activities similar to the corresponding non-
N-substituted analogs. Several compounds with various amino-
methyl groups were prepared from 3g and found to be highly ac-
tive. Compounds 4e and 4f were the most potent ones, both with
Activity of substituted N0-(2-oxoindolin-3-ylidene)-3,4,5-trimethoxybenzohydrazides
in the caspase activation assay
OMe
O
OMe
OMe
R1
R4
NH
N
R2
R3
O
N
H
Compound #
R1
R2
R3
R4
EC50 (l
M)a
HCT116
6.6 0.8
10.5 0.2
>20
>20
>20
3.8 0.3
0.24 0.03
0.64 0.02
>20
8.4 1.1
>20
SNU398
RKO
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
H
H
H
OMe
I
OCF3
NH2
AcNH
H
Me
Cl
Me
Me
OMe
Cl
H
H
H
H
H
H
H
H
H
Br
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Br
4.1 0.8
4.1 0.1
>20
>20
>20
2.1 0.2
0.13 0.01
0.57 0.11
>20
3.7 0.7
>20
4.0 0.5
3.6 0.4
8.3 1.5
>20
H
>20
Cl
Br
Cl
Ph
H
H
H
H
2.0 0.2
0.23 0.03
0.73 0.12
>20
4.6 0.8
>20
3j
3k
3l
EC50 values of 0.17 lM. These data indicated that substitution at
the nitrogen of isatin is tolerated and it could be used to introduce
aqueous solubility enhancing groups.
>20
10.6 0.1
>20
>20
>20
4.9 0.4
3m
Me
Overall, the apoptosis inducing activities of these compounds in
human colon cancer HCT116 cells were similar to that observed in
hepatocellular carcinoma cancer SNU398 cells and human colon
cancer RKO cells (Tables 1–3). Compound 3g, 4e and 4f, three of
a
Cells were treated with the test compounds for 48 h, and data are the mean of
three or more experiments and are reported as mean standard error of the mean
(SEM).
Table 3
Activity of N-substituted N0-(2-oxoindolin-3-ylidene)-3,4,5-trimethoxybenzohydrazides in the caspase activation assay
OMe
O
OMe
OMe
R1
NH
N
R2
O
N
R3
R1
R2
R3
EC50 (lM)
a
Compound #
HCT116
SNU398
RKO
4a
4b
4c
4d
4e
4f
H
Br
>20
>20
5.1 0.8
N
N
N
N
N
N
O
H
OMe
Cl
>20
>20
>20
O
O
O
N
Cl
Br
Br
Br
Br
Br
0.49 0.09
0.31 0.04
0.17 0.02
0.17 0.03
0.25 0.01
0.25 0.02
0.47 0.11
0.12 0.01
0.088 0.010
0.087 0.016
0.14 0.01
0.13 0.01
0.66 0.10
0.20 0.02
0.13 0.02
0.14 0.04
0.21 0.03
0.23 0.03
Me
Me
Me
Me
Me
4g
N
N
N
4h
N
a
Cells were treated with the test compounds for 48 h, and data are the mean of three or more experiments and are reported as mean standard error of the mean (SEM).

N. Sirisoma et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2710–2713
2713
Table 4
utilizing our ASAP assay. Through SAR studies, potent compound
3g and significantly more aqueous soluble compound 4e were
identified with EC50 values in HCT116 cells >40-fold more potent
than the screening hit 2a. The mode of action for the potent com-
pounds 3g and 4e was found to be inhibition of tubulin
polymerization.
Growth inhibition activity of substituted N0-(2-oxoindolin-3-ylidene)-3,4,5-
trimethoxybenzohydrazides
a
Compound #
GI50
(l
M)
HCT116
7.1 1.1
SNU398
RKO
2.7 1.2
2a
3g
3h
4d
4e
4f
2.7 0.1
0.056 0.015
0.32 0.14
0.062 0.008
0.051 0.018
0.088 0.043
0.086 0.008
0.022 0.008
0.14 0.04
0.026 0.011
0.023 0.008
0.024 0.006
0.029 0.014
0.019 0.003
0.054 0.012
0.019 0.003
0.018 0.004
0.036 0.012
0.043 0.016
References and notes
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the most active compounds in HCT116 cells, also were the most ac-
tive ones in SNU398 and RKO cells, suggesting that these com-
pounds most probably will be broadly active against many
cancer cell lines.
Representative compounds were assayed in
a traditional
growth inhibition (GI50) assay to confirm that the active com-
pounds in the caspase induction assay also inhibit tumor cell
growth. The growth inhibition assays in T47D, HCT116 and
SNU398 cells were run in a 96-well microtiter plate as described
previously7 and the data are summarized in Table 4. Compound
3g had GI50 values of 0.056, 0.022 and 0.019 lM in HCT116,
SNU398 and RKO cells, respectively, which are >100-fold more ac-
tive than the original hit compound 2a. Compounds 4d–4f and 4h
were also highly active with GI50 values similar to that of 3g. Com-
pound 3h was less active than 3g. These data confirmed that the
cell-based caspase activation HTS assay is not only useful for the
identification of inducers of apoptosis, but also for subsequent
optimization and SAR studies.
The potent compounds in this series of substituted N0-(2-oxoin-
dolin-3-ylidene)-benzohydrazides, such as compounds 3g and 4e,
were tested by cell cycle analysis10 and found to arrest HCT116
cells in G2/M followed by apoptosis, which is similar to what was
reported for compound 7b (Chart 2).21 We suspected that com-
pounds 3g and 4e might be tubulin inhibitors. In a tubulin poly-
merization assay,25 compounds 3g and 4e were found to inhibit
tubulin polymerization with IC50 values of 0.97 and 0.19
comparison, the IC50 values for vinblastine and colchicine were
0.5 M. These data suggest that inhibition of tubulin polymeriza-
lM. In
l
tion might be the main mechanism of action for these compounds
as apoptosis inducers.
In conclusion, a series of substituted N0-(2-oxoindolin-3-yli-
dene)-benzohydrazides were identified as apoptosis inducers