Synthesis and biological evaluation of a novel class of isatin analogs as dual inhibitors of tubulin polymerization and Akt pathway

Article abstract of DOI:10.1016/j.bmc.2011.08.044

A novel series of 5,7-dibromoisatin analogs were synthesized and evaluated for their cytotoxicities against four human cancer cell lines including colon HT29, breast MCF-7, lung A549 and melanoma UACC903. Analogs 6, 11 and 13 displayed good in vitro antic

Full text of DOI:10.1016/j.bmc.2011.08.044

Bioorganic & Medicinal Chemistry 19 (2011) 6006–6014
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a novel class of isatin analogs
as dual inhibitors of tubulin polymerization and Akt pathway
Gowdahalli Krishnegowda a,e, , A.S. Prakasha Gowda b, Hephzibah Rani S. Tagaram c,
⇑
Kevin F. Staveley-O’ Carroll c,e, Rosalyn B. Irby d,e, Arun K. Sharma a,e, Shantu Amin a,e
a Department of Pharmacology, Penn State Hershey College of Medicine, 500 University Drive, Hershey, PA 17033, USA
b Department of Biochemistry and Molecular Biology, Penn State Hershey College of Medicine, 500 University Drive, Hershey, PA 17033, USA
c Department of Surgery, Penn State Hershey College of Medicine, 500 University Drive, Hershey, PA 17033, USA
d Department of Medicine, Penn State Hershey College of Medicine, 500 University Drive, Hershey, PA 17033, USA
e Penn State Hershey Cancer Institute, Penn State Hershey College of Medicine, 500 University Drive, Hershey, PA 17033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 5,7-dibromoisatin analogs were synthesized and evaluated for their cytotoxicities
against four human cancer cell lines including colon HT29, breast MCF-7, lung A549 and melanoma
UACC903. Analogs 6, 11 and 13 displayed good in vitro anticancer activity on the HT29 human colon can-
Received 7 June 2011
Revised 15 August 2011
Accepted 20 August 2011
Available online 26 August 2011
cer cell line in the 1 lM range. Analogs 5, 9 and 12, containing a selenocyanate group in the alkyl chain
were the most promising compounds on the breast cancer MCF-7 cell line. Biological assays relating to
apoptosis were performed to understand the mechanism of action of these analogs. Compounds 5 and
6 were found to inhibit tubulin polymerization to the same extent as the anticancer drug vinblastine sul-
fate, but compounds 11 and 13 inhibited significantly better than vinblastine. Further western blot anal-
Keywords:
5,7-Dibromoisatin
Synthesis
Cytotoxicity
Apoptosis
ysis suggested that compound 6 at 2
lM reduced both levels and phosphorylation state of Akt.
Compounds 11 and 13 at 1 M caused reduced Akt protein levels and strongly suppressed the phosphor-
l
ylation of Akt. Therefore, 11 and 13 were demonstrated as efficient dual inhibitors of both tubulin poly-
merization and the Akt pathway and good candidates for further study. More importantly, the strategy of
microtubule and Akt dual inhibitors might be a promising direction for developing novel drugs for cancer.
Ó 2011 Elsevier Ltd. All rights reserved.
Tubulin polymerization
1. Introduction
Overexpression of Akt can result from inactivation of the tumor
suppressor PTEN and has been correlated with an increasing
The lack of selectivity of many anti-cancer agents and the occur-
rence of intrinsic or acquired resistance of tumors to chemotherapy
has been major obstacles in the treatment of cancer. Microtubules
have an important role in a variety of cellular process, including
mitosis and cell division.1,2 Various anti-mitotic agents interfering
with the natural dynamics of tubulin, the major protein component
of microtubules, inhibit cancer cell growth.3 Anti-mitotic agents
such as paclitaxel stabilize microtubules by preventing the depoly-
merization of tubulin. The vinca alkaloids and colchicines inhibit the
polymerization of tubulin. Anti-mitotic compounds have been used
clinically in the treatment of different cancers. Although several
antimitotic agents are available, due to the development of drug
resistance, side effects and the structural complexity of vinca alka-
loids and taxoids, there is still a need to identify novel anticancer
drugs that effectively target microtubules.4,5
number of human cancers.7 Akt is also responsible for promoting
survival signals that down regulate apoptotic pathways and con-
tribute to cancer progression. Correlation between resistance to
chemotherapy and Akt activation has also been observed in
prostate cancer cell lines and in human tumor tissue.8 Inhibition
of Akt alone or in combination with other standard cancer chemo-
therapeutics results in increased programmed death of cancer cells
leading to decreased tumor growth and tumor resistance to
chemotherapy.
The isatin (1H-indole-2,3-dione) 1 is found as an endogenous
molecule in humans and other mammals and its analogs display
diverse types of biological activities including anti-cancer activi-
ties.9–11 It is an oxidized derivative of an indole moiety; many
of the indole heterocycles are tubulin polymerization inhibi-
tors.12–14 In addition, many indole-based compounds appear to
act as inhibitors of various protein kinase families, particularly
receptor tyrosine kinases (RTKs) and serine/threonine-specific pro-
tein kinases such as the cyclic-dependent kinases (CDKs). SU11248
(Sutent), a 5-fluoro-3-substituted-2-oxoindole is approved by the
US FDA for the treatment of advanced renal carcinoma and
gastrointestinal stromal tumors.15,16 Recently, it has been reported
Protein kinase B, also known as Akt, is a 57-kDa serine/
threonine kinase plays a critical role in anti-apoptotic processes.6
⇑
Corresponding author. Tel.: +1 717 531 0003x285014; fax: +1 717 531 0244.
E-mail address: kxg21@psu.edu (G. Krishnegowda).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.044

G. Krishnegowda et al. / Bioorg. Med. Chem. 19 (2011) 6006–6014
6007
that 5,7-dibromoisatin 2 is significantly more potent in vitro as a
cytotoxic agent than the parent molecule 1 (Scheme 1) against
U937 (human monocyte-like histiocytic lymphoma) cells.11 More-
over, N-benzylation of 5,7-dibromoisatin 2 further increased the
cytotoxicity and targeting of microtubules in these lymphoma cells
and was potent against a range of human cancer cell lines includ-
ing a metastatic breast adenocarcinoma cell line (MDA-MB-231).17
In this context, it was of interest to investigate further the
cytotoxicity of N-alkylated 5,7-dibromoisatin analogs by altering
the chain length at N-1 to increase the lipophilicity and substitu-
tion of the functional groups containing isothiocyanate (ITCs), thio-
cyanate and selenocyanate in the alkyl chain. These functionalities
were chosen because of the well-known anti-cancer properties
shown by the agents having these moieties. For example, ITCs,
popular chemopreventive agents present in cruciferous vegetables
in the form of glucosinolates, provide growth-inhibiting and
apoptosis-inducing activities in cancer cell lines in vitro .18–20
Isothiocyanates are among the most effective naturally occurring
cancer chemopreventive agents in animal models.21,22 In addition,
epidemiological studies have demonstrated that the human
consumption of isothiocyanates in vegetables decreases cancer
risk.23,24 ITCs have been shown to exhibit the anticarcinogenic
effects through dual mechanisms occurring at the level of initiation
of carcinogenesis by blocking phase I enzymes (cytochrome P-450)
that activate procarcinogens and by inducing phase II enzymes
ITCs is inhibition of the PI3 kinase pathway.21,30 Our recent studies
have also shown that isothiocyanate/isoselenocyanate compounds
to be effective in inhibiting PI3K/Akt pathway.31–35 Therefore, the
use of this functional group was hoped to impart Akt inhibition
to the isatin compounds. Selenium is also an effective chemopre-
ventive agent and is known to modulate Akt activity.36–38 The
rationale for adding the selenocyanate group was that the
selenium compounds have been found to inhibit and/or retard
tumorigenesis in a variety of experimental animal models.39–41
Epidemiologic studies have reported an inverse association be-
tween the nutritional selenium status and cancer risk42, suggesting
that a relatively low Se status may be among the determinants of
cancer risk. We have also shown selenium compounds to be effec-
tive in inhibiting tumor growth in melanoma31–34 and colon43
xenograft models. Specifically, several synthetic alkyl and aryl sel-
enocyanates have been evaluated for anticarcinogenicity in animal
models. The more effective of these are benzylselenocyanate (BSC)
and 1,4-phenylenebis-(methylene)selenocyanate (p-XSC).44–47
From the combined literature survey of isatin derivatives, ITCs
and selenocyanates, we hypothesized that combination of indole
heterocycle with thiocyanate, isothiocyanate and selenocyanate
moieties would yield novel dual targeted inhibitors for cancer ther-
apy. Here, we provide a first report of the activity of such novel
agents, which are significantly toxic to cancer cells in culture by
inhibition of both tubulin polymerization and Akt phosphorylation
and expression. For these studies, we synthesized 12 derivatives of
5,7-dibromoisatin containing thiocyanate, isothiocyanate, and
that detoxify electrophilic metabolites generated by phase
I
enzymes.25–29 Certain studies suggest the mechanism of action of
Scheme 1. Synthesis of compounds 2–13 derivatives of 5,7-dibromoisatin. Reagents and conditions; (a) Br2/EtOH; (b) K2CO3/DMF, rt À80 °C, Cl(CH2)nBr, 4–18 h; (c) K2CO3/
DMF, rt, BrCH2C6H4CH2Br, 8 h; (d) K2CO3/DMF, rt À80 °C, Br(CH2)3NHBoc,12 h; (e) K2CO3/DMF, rt À80 °C, BrCH2C6H4CH2NHBoc, 16 h; (f) KI/CH3CN, KSCN or KSeCN; (g)
CF3COOH, CH2Cl2, K2CO3, CH2Cl2, CSCl2.

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G. Krishnegowda et al. / Bioorg. Med. Chem. 19 (2011) 6006–6014
selenocyanate groups in the alkyl chain. In vitro screening against
various cancer cell lines was carried out in order to establish a
more comprehensive structure–activity relationship (SAR).
dried under high vacuum. The purity of the compounds was tested
by HPLC, P99% pure compounds were used for biological assays
(data not shown).
2. Results and discussion
2.1. Synthesis
2.2. Biological characterization
2.2.1. Cytotoxicity studies
The cytotoxicity of a series of new N-alkyl derivatives of 5,7-
dibromoisatin was evaluated against a panel of four different hu-
man cancer cell lines including a colon (HT29), breast (MCF-7),
lung (A549) and melanoma (UACC903), after a continuous expo-
sure of 48 h. The results are summarized in Table 1. All the com-
pounds exhibited significant cytotoxicity with an IC50 values of
The general synthesis of N-propyl, N-butyl, and N-benzyl series
of 5,7-dibromoisatin listed in Table 1 is shown in Scheme 1.
Compounds 2–13 (Table 1) were prepared in good yield in two
or three steps. The first step consisted of alkylating 5,7-dibromoi-
satin with 1-bromo-3-chloropropane, l-bromo-4-chlorobutane
and 1,4-bis(bromomethyl)benzene to prepare 5,7-dibromo-N-(3’-
chloropropyl)isatin (3), 5,7-dibromo-N-(4’-chloropropyl)isatin (7)
and 5,7-dibromo-N-(p-bromomethylbenzyl)isatin (10). Alkylation
was accomplished by first converting 5,7-dibromoisatin to the
anionic species using the base, K2CO3 in DMF.17 Iodide-catalyzed
nucleophilic substitution of the N-propyl or -butyl chloride and
N-(p-methylbenzyl) bromide of 5,7-dibromoisatin with KSCN and
KSeCN by stirring in anhydrous acetonitrile at RT, afforded the
thiocyanates 4, 8, 11 and selenocyanates 5, 9, 12, respectively in
good yield (Scheme 1).
The isothiocyanate derivatives 6 and 13 (Scheme 1) were
synthesized by the treatment of 5,7-dibromoisatin with tert-butyl
3-bromopropylcarbamate or tert-butyl(4-bromomethyl benzyl)-
carbamate in the presence of K2CO3 in DMF, to afford
Boc-protected intermediates 14 and 15, respectively. The Boc
group in 14 and 15 was removed by trifluroacetic acid, followed
by a reaction with thiophosgene with K2CO3 in anhydrous methy-
lene chloride to give 6 and 13 in good yield. All of these compounds
were purified by column chromatography or recrystallization and
<5
l
M in HT29 cell line; compounds6, 11 and 13 showed relatively
M, respec-
higher potency with IC50 values of 1.56, 1.14 and 1.09
l
tively. The results showed that the cytotoxicity of compound 2 sig-
nificantly increased through N-alkylation, as reported previously
for the 5,7-dibromoisatin derivatives.17 Compounds 3–6, contain
a three-carbon linker, compounds 7–9 contain a four-carbon linker,
and compounds 10–13 contain an aromatic ring with a one-carbon
linker at the nitrogen N1. The findings showed that, by slightly
increasing the hydrophobicity, there is no significant change in
the inhibiting activity between N-butyl series (compounds 7–9)
and N-propyl series (compounds 3–6). Introduction of N-benzyl
with isothiocyanate/thiocyanate groups to the alkyl chain yielded
relatively more active compounds (11 and 13) in this series against
the growth of HT29 colon cancer cells.
The MCF-7 cell line was susceptible to compounds 5, 9 and 12
with IC50 values of 1.65, 1.53 and 1.45 lM, respectively. All other
compounds were less potent against MCF-7 cells. This effect may
be due in part to the expression of the anti-apoptotic Bcl-2 group
of proteins that have been identified in MCF-7 cells.48–51
Table 1
Cytotoxicity of 5,7-dibromoisatin derivatives 2–13, on HT29a, MCF-7b, A549c and UACC903d as calculated from dose response curvese
O
Br
O
N
R
Br
f
Compound
R
IC50 (lM) SD
HT29
48 h
>50
2.57 0.10
3.24
3.18 0.15
1.56 0.11
2.51 0.21
2.42 0.19
2.39 0.30
MCF-7
48 h
A549
48 h
UACC903
48 h
2
3
4
5
6
7
8
9
H
22.45 1.12
21.51 1.31
9.78 0.53
1.65 0.24
8.96 0.38
14.86 0.43
22.13 1.10
1.53 0.17
>50
26.5 1.8
8.57 0.94
2.89 0.18
21.64 1.09
4.37 0.30
3.74 0.26
2.06 0.15
9.03 0.34
–(CH2)3–Cl
–(CH2)3–SCN
–(CH2)3–SeCN
–(CH2)3–N@C@S
–(CH2)4–Cl
–(CH2)4–SCN
–(CH2)4–SeCN
5.27 0.32
3.53 0.18
3.46 0.27
2.13 0.19
3.81 0.22
5.53 0.18
3.15 0.26
0.25
10
11
12
H2C
H2C
H2C
H2C
CH2 Br
5.10 0.22
1.14 0.16
2.59 0.33
27.74 1.52
24.70 1.34
1.45 0.11
7.86 0.31
2.53 0.15
2.41 0.19
2.64 0.16
2.23 0.11
12.12 0.78
CH2 SCN
CH2 SeCN
CH2 N C S
13
1.09 0.09
0.55 0.08
12.01 0.89
NTg
3.82 0.27
NT
3.11 0.29
1.65 0.16
Vinblastine
a
b
c
d
e
f
HT29: human colorectal cancer cells.
MCF-7: human breast cancer (non-metastatic) cell line.
A549: human lung epithelial carcinoma cells.
UACC903: human melanoma cancer cells.
Sigmoidal dose response curves (variable slope) were generated using GraphPad Prism V. 4.02 (GraphPad Software Inc.).
Values are the mean of triplicates of at least two independent experiments.
NT = not tested.
g

G. Krishnegowda et al. / Bioorg. Med. Chem. 19 (2011) 6006–6014
6009
Substitution of SeCN group in the alkyl chain (5, 9 and 12), showing
high inhibitory activity compared to the other compounds against
MCF-7 cells, indicates that selenium might be playing a role in the
antiproliferative activity presumably through the inhibition of
anti-apoptotic Bcl-2 group of proteins. These selenium substituted
compounds also showed good inhibitory activity against HT29 and
A549 cell lines, but poor inhibitory activity in UACC903 cells. ITCs
and thiocyanates did not diminish the cytotoxic activity against
the MCF-7 cell lines, but were more potent in colon and lung can-
cer cell lines. Selenium compounds were more potent than corre-
sponding sulfur containing compounds in killing MCF-7 cells. All
the isatin compounds were 10–25 fold more potent than 2 against
A549 cell lines. In the presence of 6, 11, and 12 the growth of A549
cells was inhibited, with IC50 values of 2.13, 2.53 and 2.41 lM,
respectively. The thiocyanate substituted derivatives 4, 8, 11 show-
ing significant growth inhibitory activity against melanoma
UACC903 cells with IC50 values between 2.06 and 2.89 lM. To
examine whether isatin compounds are toxic to normal cells,
mouse embryonic fibroblasts (MEF) cells were treated with
compounds 6 and 11 for 48 h, later viability was evaluated by
MTS assay. Both compounds 6 and 11 exhibited IC50 >20 lM (data
not shown).
2.2.2. Apoptosis in cultured colon cancer cells
To assess the effect of isatins on cell growth inhibition and
apoptosis, we selected compounds 5, 6, 11 and 13 for an apoptosis
study using HT29 cells. HT29 cells were sensitive to these
compounds with IC50 values 3.18, 1.56, 1.14 and 1.09
tively (Table 1). We exposed HT29 cells grown in complete med-
ium with 0.1% Me2SO4, to 5, 6, 11 and 13 at 1.25, 2.5 and 5
lM respec-
lM
concentrations for 24 h. Cells were harvested, and the extent of
apoptosis was evaluated by the annexin V/7-Aminoactinomycin
D (7-AAD) method. Apoptosis was measured by quantifying the
PE annexin V and 7-AAD positive cells using flow cytometry (BD
FACScan). Within the apoptotic population, cells in the early stages
of apoptosis were annexin V-positive and 7-AAD-negative,
whereas those in the late stages were annexin V-positive and 7-
AAD–positive (Fig. 1). Approximately 6–7% of cells in the control
4
4
4
3
2
1
0
10
10
10
10
10
10
10
1.89
5.79
1.53
4.45
1.92
6.27
3
2
1
0
3
2
1
0
10
10
10
10
10
10
10
10
Compound 5
Compound 6
Compound 11
Compound 13
DMSO
48.6
43.8
1.94
89.6
92.1
2.25
0
1
2
3
4
10
10
10
10
10
0
1
2
3
4
0
1
2
3
4
10
10
10
10
10
10
10
10
10
Annexin V10
Annexin V
Annexin V
4
4
3
2
1
0
10
10
10
10
10
4
10
10
10
10
10
10
1.92
3.79
2.9
11.4
8.22
48.5
3
3
2
1
0
10
10
10
10
2
1
0
91.5
2.83
38.5
4.78
78.8
6.93
4
10
0
1
2
3
4
0
1
2
3
4
10
10
10
10
10
10
10
10
10
Annexin V10
0
1
2
3
10
10
10
10
Annexin V
Annexin V
4
3
2
1
0
4
10
10
10
10
10
4
10
10
10
10
10
10
10
10
10
10
11.4
52.9
2.59
4.46
7.76
21.1
3
2
1
0
3
2
1
0
60.6
33.1
2.68
88.5
4.42
10.6
0
1
2
3
4
0
1
2
3
4
0
1
2
3
4
10
10
10
10
10
10
10
10
10
10
10
10
10
10
Ann1e0xin V
Annexin V
Annexin V
4
4
4
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
6.56
23.3
4.29
8.59
10.8
47.5
3
2
1
0
3
2
1
0
3
2
1
0
3.5
2.93
83.6
38.8
63.4
6.78
4
10
0
1
2
3
4
0
1
2
3
4
10
10
10
10
10
10
10
10
10
10
0
1
2
3
10
10
10
Annexin V10
Annexin V
Annexin V
4
10
1.33
4.1
3
2
1
0
10
10
10
10
93.2
1.37
4
10
0
1
2
3
10
10
10
10
Annexin V
Figure 1. Quantitative analysis of cell apoptosis induced by 5,7-dibromoisatin derivatives. Apoptosis was measured by using the PE Annexin V and 7-AAD staining. Cells were
analyzed and quantified by using flow cytometry (BD FACScan). Human colon cancer cells HT29 treated with compounds 5, 6, 11 and 13 (1.25–5.0 lM) or DMSO as a control
for 24 h at 37 °C. At the end of incubation, cells were harvested and washed twice with cold PBS. Cells were resuspended in binding buffer at a concentration of 1 Â 106 cells/
ml and stained with 7-AAD and PE Annexin V and incubated for 15 min at rt in the dark.

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G. Krishnegowda et al. / Bioorg. Med. Chem. 19 (2011) 6006–6014
Table 2
Summary of
%
of HT29 cells undergoing apoptosis after
a 24 h treatment with
compounds 5, 6, 11 and 13 at 1.25, 2.5 and 5
l
M concentrations
Compound No.
1.25
—
lM
2.5
—
lM
5 lM
Control (DMSO)
6.8
5
6
11
13
7.92
10.44
21.23
39.46
36.64
56.28
61.5
66.98
61.23
8.54
11.47
16.38
Figure 3. Detection of in situ DNA fragmentation was analyzed by TUNEL staining
after 48 h treatment with the compounds. HT29 cells were grown for 24 h and
treated with 5 lM compound 6, and 11. The blue stain (a, b) represents the nuclear
Hoechst stain. The red stain (c, d) determines the extent of DNA fragmentation or
damage. The overlay figures (e, f) showing the number of DNA fragmented cells
(red) to the total number of cells (blue).
Figure 2. Isatin analogs induce apoptosis in cultured colon cancer cell lines by the
activation of caspase 3/7. Levels of caspase-3/7activity were measured in HT29 cells
exposed to compounds 5, 6, 11, and 13 using the Apo-ONE homogeneous caspase-3/
7 assay kit. Results show significant dose dependent increases in caspase-3/7
activity relative to DMSO vehicle treated cells. Data are means SD of one
representative experiment performed in triplicate.
6000
Paclitaxel
Control
population were undergoing spontaneous apoptosis. Treatment
with 5, 6, 11 and 13 induced apoptosis dose-dependently and at
5000
5 lM, 56, 61, 67 and 61% of cells underwent apoptosis, respec-
4000
tively. These populations are combined and presented in (Table
2). Thus growth inhibition appears to correlate well with the con-
centration of isatins that induce apoptosis in HT29 cells. As an
additional indication of apoptosis occurring in those cells, caspas-
es-3/7 activity, which plays a key role in apoptosis, was measured.
Caspases-3/7 activity was increased in a dose-dependent manner
to a maximum of 16 fold in response to isatins compared to control
(Fig. 2). To confirm that the observed reduction in viability of HT29
cells occurred via induction of apoptosis, we used TUNEL staining
to measure DNA fragmentation as an early hallmark of apoptotic
cell death in treated cells. HT29 cells were treated with compounds
Compound 5
3000
Compound 6
Vinblastine
2000
Compound 11
Compound 13
1000
0
1000
2000
Time (S)
3000
4000
Figure 4. The effect of compounds 5, 6, 11, 13, paclitaxel, and vinblastine sulfate at
10 M on the assembly of purified bovine neuronal tubulin. DMSO was used as a
l
control. A shift of the curve to the left or right of the control is indicative of either an
increase or a decrease, respectively, in the rate of tubulin polymerization. Changes
in fluorescence were measured at an excitation wavelength of 360 10 nm, and the
fluorescence was collected at 440 10 nm. Data points are the means of duplicate
experiments.
6 and 11, at 5 lM concentration for 48 h. Fragmented DNA of apop-
totic cells were stained using an Apop Tag Red In Situ Apoptosis
Detection Kit and visualized by fluorescence microscopy using
appropriate filters. As evidenced in Panels c and d of (Fig. 3), com-
pound 6 and 11 resulted in a significant number of TUNEL-positive
colon cancer cells.
indole moiety, and 5,7-dibromo-N-benzylisatin derivatives inter-
fere with microtubule dynamics. Compounds 5, 6, 11, and 13 were
chosen as representative molecules to further investigate their
ability to alter tubulin polymerization in vitro. To investigate
whether the antiproliferative activities of compounds 5, 6, 11
and 13 derived from an interaction with tubulin, they were evalu-
ated for their inhibition of tubulin polymerization in a cell-free
in vitro assay. Paclitaxel and vinblastine sulfate were used as a
2.2.3. Effects on tubulin polymerization and microtubule
formation
Several tubulin polymerization inhibitors characterized by the
presence of an indole nucleus have been obtained from natural
sources or have been prepared by semi-synthesis. The indole het-
erocyclic nucleus is central to a large number of tubulin polymer-
ization inhibitors.12,14,52 Isatins are oxidized derivatives of an

G. Krishnegowda et al. / Bioorg. Med. Chem. 19 (2011) 6006–6014
6011
strongly suppressed the phosphorylation of Akt. Introduction of a
selenocyanate moiety in the alkyl chain (compounds 5, 9, and
12) showed higher cytotoxicity than thiocyanate and isothiocya-
nate against MCF-7 breast cancer cell lines. Overall compounds
11 and 13 emerged as dual inhibitors of tubulin polymerization
and the Akt signaling pathway and the most promising candidates
for further investigation in vivo as antitumor agents for colon
cancer.
4. Experimental section
4.1. Chemistry
Figure 5. Inhibition of Akt phosphorylation by compounds 6, 11, and 13. HT29 cells
were treated for 24 h with drugs at either 1 or 2 M, or with no drug. Cell lysates
l
were assayed by Western blot analysis for phospho Akt or total Akt. Lower panel
shows actin loading control.
Isatin was purchased from Sigma–Aldrich, 5,7-dibromoisatin
was synthesized using previously reported methods.11 All other
chemicals and solvents were purchased from the major vendors.
Anhydrous solvents were used as received. Reactions were carried
out using dried glassware and under an atmosphere of nitrogen.
Reaction progress was monitored with analytical thin-layer chro-
matography (TLC) on aluminum backed precoated silica gel 60
F254 plates (E. Merck). The N-alkylisatins were highly colored
and would usually be clearly seen on a TLC plate; colorless com-
pounds were detected using UV light and/or iodine vapor. Column
chromatography was carried out using silica gel 60 (230–400
mesh, E. Merck) with the solvent system indicated in the individual
procedures. All solvent ratios are quoted as vol/vol. NMR spectra
were recorded using a Bruker Avance 500 MHz spectrometer.
Chemical shifts (d) were reported in parts per million downfield
from the internal standard. The signals are quoted as s (singlet),
d (doublet), t (triplet), m (multiplet), dd (doublet of doublet). Spec-
tra are referenced to the residual solvent peak of the solvent stated
in the individual procedure. High-resolution mass spectra (HRMS)
were determined on Thermo Electron MAT 95XL magnetic sector
mass spectrometer operating at 70 eV for EI with a source temper-
ature at 180 °C and were referenced with PFK and at 5 kV for ESI
operating with a source temperature at 250 °C and were referenced
with polyethylene amine. Melting points were determined on a
Fischer–Johns melting point apparatus and are uncorrected.
known microtubule stabilizer and destabilizer, respectively. The
results of both paclitaxel and vinblastine were consistent with
the literature reports.53,54 At 10
lM, paclitaxel stabilized microtu-
bules, in comparison to the vehicle control, while vinblastine
strongly inhibited microtubule formation at the same concentra-
tion (Fig. 4). The test compounds 11 and 13 more strongly inhibited
about 71% and 77% respectively the rate of microtubule polymeri-
zation at 10 lM, than vinblastine (66%) (Fig. 4). Analogs 5 (63%)
and 6 (62%) were almost similar to vinblastine as inhibitors of
tubulin assembly. This suggests that both benzyl and thiocya-
nate/isothiocyanate groups in 11 and 13 are playing a role in the
maximum inhibition of tubulin assembly. By comparison, both
compounds 6 and 13 have isothiocyanate functional group, com-
pound 6 was slightly less active than 13 as a microtubule destabi-
lizer, suggesting that, N-benzyl substitution is more important
than N-propyl for microtubule destabilization.
2.2.4. Inhibition of Akt phosphorylation
To determine the effects of the compounds on Akt, Western blot
analysis was performed. Cells were treated for 24 h, and Western
blots were performed on the lysates (Fig. 5). The blots were probed
for phospho-Akt (top panel) and for total Akt (middle panel).
Results show that compound 6 at 1
either expression or phosphorylation of Akt, while at 2
l
M had very little effect on
4.1.1. 5,7-Dibromo-N-(3’-chloropropyl)isatin (3)
lM both
5,7-dibromoisatin 2 (1 g, 3.28 mmol) was taken up in anhy-
drous DMF (30 mL) and cooled on ice with stirring. Solid K2CO3
(544 mg, 3.94 mmol) was added in one portion, and the dark col-
ored suspension was brought to room temperature and stirred
for a further 1 h. 1-Bromo-3-chloropropane (620 mg, 3.94 mmol,
0.387 mL) was added slowly with constant stirring and the reac-
tion mixture was stirred at 80 °C for 4–8 h, until the 5,7-dibromoi-
satin starting material had been consumed (TLC). The reaction
mixture was poured into HCl (0.5 M, 50 mL) and extracted with
ethyl acetate (3 Â 50 mL). The ethyl acetate layer was washed with
brine and dried over MgSO4. The solvent was removed, and the
crude product was purified by silica gel column chromatography
(CH2Cl2 as eluent) to give pure 3 (0.93 g, 74%) as orange red crys-
tals, mp 130–132 °C. 1H NMR (CDCl3, 500 MHz) d 2.24–2.39 (m,
2H, H2’), 3.50(t, 1H, J = 6.5 Hz, H3’), 3.67 (t, 1H, J = 6.5 Hz, H3’),
4.31–4.36 (m, 2H, H1’), 7.72 (d, J = 2 Hz, 1H, isatin ArH), 7.91 (d,
J = 2.5 Hz, 1H, isatin ArH). HRMS (EI) M+ calcd for C11H8-
O2N179Br235Cl1, 378.8605; found, 378.8609.
levels and phosphorylation state of Akt were reduced. For com-
pounds 11 and 13, Akt levels were reduced but the phosphoryla-
tion was virtually eliminated, indicating that the Akt present was
not active. These results indicate that both compounds 11 and 13
are more potent Akt inhibitors than compound 6 and that in addi-
tion to inhibition of activity, the drugs down regulate the expres-
sion of the proteins. Comparison with N-propyl isothiocyanate
(6) and N-benzyl thiocyanate/isothiocyanate (11 and 13), benzyl
group gave more potency to the isothiocyanate/thiocyanate for
the Akt inhibition.
3. Conclusions
From Table 1, it is interesting to note that the N-alkylation with
thiocyanate, isothiocyanate and selenocyanate moieties apparently
play an important role in the activity of these compounds. Com-
pounds with N-propyl or N-benzyl are essential for activity, but
N-butyl does not improve the potency of the compounds. Com-
pounds 5 and 6 inhibited tubulin polymerization to the same ex-
tent as anticancer drug vinblastine sulfate. Compounds 11 and 13
were found to inhibit tubulin polymerization greater than the vin-
blastine. Further western blot analysis suggested that compound 6,
4.1.2. 5,7-Dibromo-N-(3’-thiocyanatopropyl)isatin (4)
Compound 3 (200 mg, 0.52 mmol) was dissolved in anhydrous
acetonitrile (15 mL), and KI (300 mg, 1.81 mmol) and KSCN
(200 mg, 2.05 mmol) were added. The mixture was stirred over-
night until a substantial amount of product was formed. The reac-
tion mixture was diluted with water and extracted with ethyl
at 1
tion of Akt, while at 2
were reduced. Compounds 11 and 13 reduced Akt levels and
lM had very little effect on either expression or phosphoryla-
lM both levels and phosphorylation state

6012
G. Krishnegowda et al. / Bioorg. Med. Chem. 19 (2011) 6006–6014
acetate (3 Â 50 mL). The organic layer was washed with water,
dried over anhydrous MgSO4, and evaporated to dryness on the
rotary evaporator. The crude product was purified by silica gel col-
umn chromatography (CH2Cl2–hexane, 7:3) eluted the product 4 as
a yellow–orange solid (140 mg, 65%), mp 118–120 °C. 1H NMR
(CDCl3, 500 MHz) d 2.36 (m, 2H, H2’), 3.06(t, 1H, J = 7 Hz, H3’),
4.35 (t, 2H, J = 6.5 Hz, H1’), 7.74 (d, J = 2 Hz, 1H, isatin ArH), 7.93
(d, J = 2 Hz, 1H, isatin ArH). 13C NMR (CDCl3, 126 MHz) d 30.1,
31.2, 39.8, 104.7, 111.7, 117.4, 121.4, 127.8, 145.3, 146.1, 158.3,
180.9. HRMS (EI) M+ calcd for C12H8O2N279Br2S1, 401.8668; found,
401.8675.
4.1.6. 5,7-Dibromo-N-(4’-thiocyanatobutyl)isatin (8)
The procedure used for was followed with (200 mg,
4
3.66 mmol) of 7, KI (300 mg, 1.81 mmol) and KSCN (200 mg,
2.06 mmol) to obtain 8. The product was chromatographed on
the silica gel with CH2Cl2–hexane (7:3), yielded 8 as an orange–yel-
low solid (144 mg, 68%), mp 120–122 °C). 1H NMR (CDCl3,
500 MHz) d 1.96 (m, 4H, H2’ and H3’), 3.05(t, 2H, J = 7 Hz, H4’),
4.21 (t, 2H, J = 6.5 Hz, H1’), 7.72 (d, J = 2 Hz, 1H, isatin ArH), 7.91
(d, J = 2 Hz, 1H, isatin ArH). HRMS (EI) M+ calcd for
C
13H10O2N279Br2S, 415.8824; found, 415.8824.
4.1.7. 5,7-Dibromo-N-(4’-selenocyanatobutyl)isatin (9)
The procedure used for was repeated with (200 mg,
4.1.3. 5,7-Dibromo-N-(3’-selenocyanatopropyl)isatin (5)
The procedure used for 4 was repeated with (200 mg, 0.52 mmol)
of 3, KI (300 mg, 1.81 mmol) and KSeCN (200 mg, 1.39 mmol). The
product was a bright orange solid (158 mg, 67%), mp 120–122 °C.
1H NMR (CDCl3, 500 MHz) d 2.45 (m, 2H, H2’), 3.11(t, 1H, J = 7 Hz,
H3’), 4.35 (t, 2H, J = 6.5 Hz, H1’), 7.73 (d, J = 2 Hz, 1H, isatin ArH),
7.92 (d, J = 2 Hz, 1H, isatin ArH). 13C NMR (CDCl3, 126 MHz) d 26.1,
31.0, 40.7, 101.1, 104.7, 117.4, 121.4, 127.8, 145.3, 146.2, 158.5,
180.8. HRMS (EI) M+ calcd for C12H8O2N279Br280Se, 449.8112; found,
449.8113.
5
3.66 mmol) of 7, (300 mg, 1.81 mmol) of KI, (200 mg, 1.39 mmol)
of KSeCN. The product was an orange red solid (158 mg, 67%),
mp 123–125 °C. 1H NMR (CDCl3, 500 MHz) d 1.95–2.06 (m, 4H,
H2’and H3’), 3.12(t, 1H, J = 7 Hz, H4’), 4.21 (t, 2H, J = 7.5 Hz, H1’),
7.71 (d, J = 1.5 Hz, 1H, isatin ArH), 7.90 (d, J = 2 Hz, 1H, isatin
ArH). HRMS (EI) M+ calcd for C13H10O2N279Br280Se, 463.8269;
found, 463.8273.
4.1.8. 5,7-Dibromo-N-(p-bromomethylbenzyl)isatin (10)
The procedure used for 3 was repeated with 5,7-dibromoisatin
(1 g, 3.28 mmol), (544 mg, 3.94 mmol) of K2CO3, anhydrous DMF
(30 mL) and of 1,4-bis(bromomethyl)benzene (1.04 g, 3.94 mmol).
Compound 10 was obtained as an orange red solid (1.11 g, 75%),
mp 140–142 °C). 1H NMR (CDCl3, 500 MHz) d 4.49 (s, 2H, H1’),
5.42 (s, 2H, CH2Br), 7.24 (d, J = 8 Hz, 2H, phenyl ArH), 7.39 (d,
J = 8.5 Hz, 2H, phenyl ArH), 7.75 (d, J = 2 Hz, 1H, isatin ArH) 7.85
(d, J = 2 Hz, 1H, isatin ArH). HRMS (EI) M+ calcd for C16H10O2N79Br3,
484.8256; found, 484.8256.
4.1.4. 5,7-Dibromo-N-(3’-isothiocyanatopropyl)isatin (6)
The procedure used for
3 was repeated with (500 mg,
1.64 mmol) of 2, 15 ml of anhydrous DMF, KI (300 mg, 1.81 mmol),
K2C03 (272 mg, 1.97 mmol) and (470 mg, 1.97 mmol) of tert-butyl
3-bromopropyl carbamate. The crude product was purified by sil-
ica gel column chromatography (hexanes/EtOAc, 80:20) to obtain
5,7-dibromo-N-[3’-(tert-butylcarbamate)propyl]isatin 14 as an or-
ange-yellow solid (470 mg, 62%), mp 94–96 °C. 1H NMR (CDCl3,
500 MHz) d 1.46 (s, 9H, H t-butyl), 1.98 (m, 2H, H2’), 3.22 (q, 2H,
J = 6 Hz, H3’), 4.23 (t, 2H, J = 7 Hz, H1’), 7.70 (d, J = 2 Hz, 1H, isatin
ArH), 7.89 (d, J = 2 Hz, 1H, isatin ArH). HRMS (EI) M+ calcd for
4.1.9. 5,7-Dibromo-N-(p-thiocyanomethylbenzyl)isatin (11)
Compound 11 in the pure form was prepared following the pro-
cedure used for 4 with (200 mg, 0.44 mmol) of 10, (300 mg,
1.81 mmol) of KI, and (200 mg, 2.06 mmol) of KSCN. The product
was a yellow–orange solid (144 mg, 74%), mp 162–164 °C. 1H
NMR (CDCl3, 500 MHz): d 4.16 (s, 2H, H1’), 5.44 (s, 2H, CH2SCN),
7.31 (d, J = 8 Hz, 2H, phenyl ArH), 7.38 (d, J = 8.5 Hz, 2H, phenyl
ArH), 7.76 (d, J = 2 Hz, 1H, isatin ArH) 7.86 (d, J = 2 Hz, 1H, isatin
ArH). 13C NMR (CDCl3, 126 MHz): d 37.8, 44.4, 105.1, 111.7,
117.4, 121.4, 127.6, 129.5, 134.0, 136.6, 145.3, 146.5, 158.2,
181.1. HRMS (EI) M+ calcd for C17H10O2N279Br2S, 463.8824; found,
463.8831.
C
16H18O4N279Br2, 459.9633; found, 459.9613.
To a solution of 5,7-dibromo-N-[3’-(tert-butylcarbamate)pro-
pyl]isatin 14 (400 mg, 0.89 mmol) in methylene chloride (20 mL)
was added trifluroacetic acid (1.5 mL) at 0 °C, and the reaction
was stirred at room temperature for 12 h. The mixture was then
concentrated to give a solid which was washed with diethyl ether,
affording trifluroacetate salt of 5,7-dibromo-N-(3’-aminopro-
pyl)isatin as an orange red solid (460 mg, 96%). The whole salt
was carried to synthesis of isothiocyanate, suspended in CH2Cl2
and (1.3 g, 9.4 mmol) of K2CO3. Thiophosgene (112 mg, 74 ll,
0.96 mmol) was added slowly and stirred 12 h at RT. The product
was extracted with more CH2Cl2 and washed with water, dried
with MgSO4 and chromatographed on silica gel column, (hex-
anes/CH2Cl2, 20:80) obtained 6 as a bright orange red solid
(210 mg, 54%), mp 131–133 °C. 1H NMR (CDCl3, 500 MHz) d 2.18
(m, 2H, H2’), 3.22 (t, 2H, J = 6 Hz, H3’), 4.34 (t, 2H, J = 7 Hz, H1’),
7.72 (d, J = 2 Hz, 1H, isatin ArH), 7.91 (d, J = 2 Hz, 1H, isatin ArH).
13C NMR (CDCl3, 126 MHz) d 29.7, 39.2, 42.7, 104.7, 117.2, 121.4,
127.7, 131.5, 145.2, 146.2, 158.1, 180.9. HRMS (EI) M+ calcd for
4.1.10. 5,7-Dibromo-N-(p-selenocyanomethylbenzyl)isatin (12)
(200 mg 0.44 mmol) of 10, (300 mg, 1.81 mmol) of KI, (200 mg,
1.39 mmol) of KSeCN, 15 mL of acetonitrile were stirred and pro-
cessed according to the procedure used for 5. Purification by col-
umn chromatography using CH2Cl2–hexane (7:3) as an eluent
afforded 12 as a bright red solid (162 mg, 76%), mp 173–175 °C).
1H NMR (CDCl3, 500 MHz): d 4.30 (s, 2H, H1’), 5.43 (s, 2H,
CH2SeCN), 7.29 (d, J = 8 Hz, 2H, phenyl ArH), 7.37 (d, J = 8.5 Hz,
2H, phenyl ArH), 7.76 (d, J = 2 Hz, 1H, isatin ArH) 7.85 (d, J = 2 Hz,
1H, isatin ArH). 13C NMR (CDCl3, 126 MHz): d 32.2, 44.4, 101.6,
105.1, 117.3, 121.4, 127.3, 127.6, 129.5, 135.1, 136.4, 145.3,
146.5, 158.2, 181.1. HRMS (ESI) (M+Na)+ calcd for C17H10O2-
N279Br280SeNa, 534.8166; found 534.8167.
C
12H8O2N279Br2S, 401.8668; found, 401.8675.
4.1.5. 5,7-Dibromo-N-(4’-chlorobutyl)isatin (7)
The procedure used for 3 was repeated with (1 g, 3.28 mmol) of
2, (544 mg, 3.94 mmol) of K2CO3, anhydrous DMF (30 mL) and of 1
bromo-4-chlorobutane (452 lL, 3.94 mmol). The product was
chromatographed on the silica gel with CH2Cl2 as eluent yielded
7 as an orange-red solid (0.98 g, 75%), mp 80–82 °C. 1H NMR
(CDCl3, 500 MHz) d 1.89–1.99 (m, 4H, H2’ and H3’), 3.48(t, 1H,
J = 6 Hz, H4’), 3.61 (t, 1H, J = 6.0 Hz, H4’), 4.19 (t, J = 7 Hz, 2H,
H1’), 7.71 (d, J = 2 Hz, 1H, isatin ArH), 7.90 (d, J = 2.5 Hz, 1H, isatin
ArH). HRMS (EI) M+ calcd for C12H10O2N79Br235Cl, 392.8761; found,
392.8770.
4.1.11. 5,7-Dibromo-N-(p-isothiocyanatomethylbenzyl)isatin
(13)
The experimental procedure used for 3 was employed with
(500 mg, 1.64 mmol) of 2, 15 ml of anhydrous DMF, (100 mg,
0.60 mmol) of KI, (272 mg, 1.97 mmol) of K2CO3, and (590 mg,
1.97 mmol) of tert-butyl(4-bromomethyl-benzyl)carbamate. The
product
5,7-dibromo-N-(tert-butyl-carbamate-methylbenzyl)-

G. Krishnegowda et al. / Bioorg. Med. Chem. 19 (2011) 6006–6014
6013
isatin 15 was obtained as an orange red solid (620 mg, 72%), mp
added to each well of a black 96-well plate containing 100 ll of
125–127 °C. 1H NMR (CDCl3, 500 MHz): d 1.47 (s, 9H, H t-butyl),
5.42 (s, 2H, H1’), 4.32 (d, J = 5 Hz, 2H, CH2NHBOC), 7.22–7.28 (m,
4H, phenyl ArH), 7.74 (d, J = 2 Hz, 1H, isatin ArH) 7.84 (d, J = 2 Hz,
1H, isatin ArH). HRMS (ESI) (M+Na)+ calcd for C21H20O4N279Br2Na,
546.9662; found, 546.9692. The procedure used for 6 was repeated
to convert trifluroacetate salt into isothiocyanate product 13
(277 mg, 52%), mp 167–69 °C. 1H NMR (CDCl3, 500 MHz): d 5.44 (s,
2H, H1’), 4.73 (s, 2H, CH2NCS), 7.31 (d, J = 3 Hz, 4H, phenyl ArH),
7.76 (d, J = 2 Hz, 1H, isatin ArH) 7.86 (d, J = 2 Hz, 1H, isatin ArH).
13C NMR (CDCl3, 126 MHz): d 44.4, 48.3, 105.3, 117.5, 121.4, 127.1,
127.5, 127.8, 133.8, 136.0, 145.5, 146.5, 158.3, 181.3. HRMS (EI) M+
calcd for C17H10O2N279Br2S, 463.8824; found, 463.8831.
blank, control or cells in culture. The plate was gently mixed with
a plate shaker at 300–500 rpm for 30 s. The plate was then incu-
bated at room temperature for 2 h. Caspase 3/7 activity was deter-
mined by measuring the fluorescence of the cleaved substrate
using a microplate reader (excitation 498 nm; emission 521 nm).
4.6. Terminal deoxynucleotide transferase dUTP nick end
labeling (TUNEL) assay
HT-29 cells were plated at 1 Â 105 cells per well in a 6 well
plate, and grown in 10% serum fortified media for 24 h prior to
treatment. Cells were exposed to compounds 6 and 11 at 5 lM
for 24 h. Fragmented DNA of apoptotic cells were stained using
an Apop Tag Red In Situ Apoptosis Detection Kit according to the
manufacturer’s instructions (Chemicon, Temecula, CA), and visual-
ized by fluorescence microscopy using appropriate filters.
4.2. Cell lines and culture conditions
The cell lines used in this study were as follows: HT29 (human
colorectal cancer), MCF-7 (human breast cancer), A549 (human
lung epithelial carcinoma), and UACC903 (human melanoma can-
cer) were obtained from American Type Culture Collection (Manas-
sas, VA). Cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM), supplemented with 10% fetal bovine serum
(FBS) and maintained at 37 °C in 5% CO2. HT29 cells were cultured
in RPMI medium with 10% FBS.
4.7. Tubulin polymerization assay
Compounds 5, 6, 11 and 13 were tested for their anti-mitotic
ability in reference to vinblastine sulfate and paclitaxel positive
controls in a fluorescence-based Tubulin Polymerization Assay.55
Briefly, 50 lL of tubulin reaction mix 2 mg/mL purified bovine
brain tubulin (Cytoskeleton Inc., Denver, CO, USA) in 80 mM PIPES
pH 6.9, 2.0 mM MgCl2, 0.5 mM EGTA, 1.0 mM GTP, and 20% glyc-
erol) was added to duplicate wells of a half area 96-well black plate
4.3. Cellular viability assay
A colorimetric cell proliferation assay was used in each of the
HT29, MCF-7, A549, and UACC903 to assess the effect on cell pro-
containing 5
fate, or compounds 5, 6, 11 and 13 all at a final concentration of
10 M. The rate of polymerization was followed for 1 h at 37 °C,
data points were collected at every 10 s, using an excitation wave-
length of 360 10 nm, and the fluorescence was collected at
440 10 nm.
lL of either vehicle control, paclitaxel, vinblastine sul-
liferation of
a
series of 5,7-dibromoisatin derivatives. Cells
l
(5 Â 103) were seeded into each well of a 96-well plate in 100
l
l
of tissue culture medium. After 24 h incubation to allow cells to ad-
here, cells were treated with compounds 2–13 were dissolved in
DMSO and diluted in DMEM at concentrations of 0.5–50 lM for
48 h. Cell viability was then determined by the colorimetric MTS
assay using Cell Titer 96 AQueous One Solution Cell Proliferation
Assay according to the manufacturer’s instruction (Promega, Mad-
ison, WI, USA). Viability was determined by measuring absorbance
at 490 nm using a micro plate reader and normalizing to the viabil-
ity observed under control conditions.
4.8. Western analysis
Antibodies used were: Pan-Akt rabbit polyclonal, phospho-Akt
rabbit polyclonal, (Cell Signaling, Danvers, MA), and b-actin mouse
monoclonal (Sigma, Saint Louis, MO). HT29 cells were treated with
compounds 6, 11 and 13 at different concentrations for 24 h.
Plates were washed with cold PBS and the cells were lysed into ly-
sis buffer (50 mM HEPES, 100 mM NaCl, 10 mM EDTA, 0.5% NP40,
10% glycerol, 0.0001% Tween 20, supplemented with 0.1 mM
4.4. Determination of apoptosis by flow cytometry
Apoptosis was measured by staining cells with PE Annexin V
and 7-AAD. Cells were analyzed and quantified by flow cytome-
try (BD FACScan). Human colon cancer cells HT29 were treated
PMSF, 0.1 mM NaVO4, 0.5 mM NaF, 5 lg/ml leupeptin, 0.1 mM
DTT). The proteins were quantified according to the Bradford As-
say and loaded equally onto 10% polyacrylamide gels. Proteins
were electrophoresed at 150 v and transferred to nitrocellulose
membranes using a semi-dry blotter (BioRad, Hercules, CA). Mem-
branes were blocked with 5% non-fat dry milk for 2 h and incu-
bated with primary antibody overnight at 4 °C. The blots were
washed 3X in TBS-Tween and incubated for 1 h in appropriate
HRP-conjugated secondary antibodies (GE Healthcare, Little Chal-
font, Buckinghamshire, UK). Blots were washed and developed
using the SuperSignal West Pico Chemiluminescent Substrate
(Thermo Scientific, Rockford, IL, USA). The blots were exposed to
autoradiography film.
with or without 5,7-dibromoisatin derivatives (1.25–5.0 lM) for
24 h at 37 °C. At the end of the incubation, cells were harvested
by low speed centrifugation. Cells were washed twice with cold
PBS and then resuspended in binding buffer at a concentration of
1 Â 106 cells/ml and stained with 7-AAD and PE Annexin V. Cells
were incubated for 15 min at RT in the dark according to the
manufacturer’s instructions (Promega, Madison, Wisconsin,
USA). 7-AAD and Annexin V negative were identified as live
cells, 7-AAD positive were identified as dead cells. Cells which
were Annexin V positive and 7-AAD negative were identified
as early apoptotic. Cells which were Annexin V positive and 7-
AAD positive were identified as cells in late apoptosis or
necrotic.
Acknowledgment
This study was supported in part by the National Institutes of
Health National Cancer Institute Grant R03-CA143999 (A. K. S.),
and by Penn State Hershey Cancer Institute. The authors thank
the Flow Cytometry and Solution Phase NMR Facility (Dr. Jyh-Ming
Lin) at Core Research Facilities of the Pennsylvania State Univer-
sity, College of Medicine, Hershey, PA, for recording of NMR
spectra.
4.5. Caspase 3/7 activity assay
HT-29 cells were plated at 1 Â 104 cells per well in a black 96
well plate, and grown for 24 h prior to treatment. Cells were ex-
posed to DMSO, or to compounds 5, 6, 11 and 13 at 1.25–5
lM con-
centrations for 24 h. 100
l
l of Apo-ONEÒ Caspase-3/7 Reagent was

6014
G. Krishnegowda et al. / Bioorg. Med. Chem. 19 (2011) 6006–6014
27. Maheo, K.; Morel, F.; Langouet, S.; Kramer, H.; Le Ferrec, E.; Ketterer, B.;
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