Preliminary biological evaluation and mechanism of action studies of selected 2-arylindoles against glioblastoma

Article abstract of DOI:10.1016/j.bmc.2013.01.032

A series of related 2-arylindoles have been evaluated for their anticancer activity against a range of glioblastoma cell lines using a number of different cell-based assays to determine cell viability after treatment with the compounds. The best indoles,

Full text of DOI:10.1016/j.bmc.2013.01.032

Bioorganic & Medicinal Chemistry 21 (2013) 1918–1924
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Preliminary biological evaluation and mechanism of action studies
of selected 2-arylindoles against glioblastoma
a
a
a
b
a
Saurabh Prabhu , Zaheer Akbar , Frederick Harris , Katherine Karakoula , Robert Lea ,
b
b
a,
⇑
Farzana Rowther , Tracy Warr , Timothy Snape
a
School of Pharmacy and Biomedical Sciences, Maudland Building, University of Central Lancashire, Preston PR1 2HE, UK
Brain Tumour UK Neuro-Oncology Research Centre, University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1LY, UK
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of related 2-arylindoles have been evaluated for their anticancer activity against a range of glio-
blastoma cell lines using a number of different cell-based assays to determine cell viability after treat-
ment with the compounds. The best indoles, which showed comparable activity to cisplatin against a
U87MG cell line in the MTS assay, were taken forward and initial studies suggest that their mechanism
of action is consistent with the generation of reactive oxygen species followed by autophagic cell death.
Furthermore, activity was also observed in glioblastoma short-term cell cultures for the best lead com-
pound and in some cases gave low micromolar IC50s.
Received 25 October 2012
Revised 9 January 2013
Accepted 14 January 2013
Available online 4 February 2013
Keywords:
2
-Arylindoles
Ó 2013 Elsevier Ltd. All rights reserved.
Glioblastoma
Reactive oxygen species (ROS)
Autophagy
Cancer
1
1
. Introduction
Indole-3-carbinol (I3C (1), Fig. 1) has received a great deal of
parent indole (5, R = CHO, R = H) inhibits cell growth, but that 3-
6
formylindole, which lacks the 2-phenyl group, did not.
attention over the past circa 30 years for the chemoprevention
and treatment of a number of cancers, including: breast, prostate,
keratinocyte, endometrial and non-small cell lung adenocarcinoma
2
. Results and discussion
We are currently interested in developing new and improved
1
–3
cells,
but not, to our knowledge, for the treatment of glioma.
chemotherapeutic agents for the treatment of glioma and also have
an interest in the development of new methods for the synthesis of
Similarly, it has been demonstrated that other indoles, so-called
4
1
4
privileged structures, such as 2-arylindoles (Fig. 1), possess bio-
substituted 2-arylindoles, therefore a study was embarked upon
in order to examine what effects indoles would have on various gli-
oma cell lines to see if new lead compounds could be identified.
Both I3C (1) and 2-arylindoles (such as 2) have established
activity against cancer cells and thus the initial investigation began
by ascertaining what kind of activity these two compounds have
against the established glioma cell lines 1321N1 and U87MG. The
results (Table 1, entries 1 and 2) reveal that whilst I3C (1) has mod-
est activity [IC50 = 309 ± 2 lM (1321N1) and 526 ± 2 lM (U87MG)],
the 2-arylindole (2) does not reach its IC50 over the same time
frame (48 h) and only reaches a maximum growth inhibition of
7
5
logical activity in a number of therapeutic areas, including cancer.
Indole-3-carbinol, a natural product obtained from cruciferous
vegetables, has been identified as a potential treatment/preventa-
tive for breast cancer, where it has been shown to act through var-
ious mechanisms, such as: affecting DNA repair and nuclear
regulatory factors, induction of cell cycle arrest and induction of
apoptosis, effects which have been observed in both mice and hu-
2
man volunteers. Additionally, a substantial amount of research
has been undertaken studying structure activity relationships of
more heavily substituted indoles for the treatment of breast
6
–13
cancer,
where it has been established that the 2-phenyl group
% (U87MG) at 400 lM, however, 2 is not able to inhibit the
appears to be important for biological activity in certain cases.
For example, in studies to identify new 3-formyl-2-phenylindoles
as inhibitors of tubulin polymerization, it was found that the
growth of the 1321N1 cell line.
Based on these results, whilst bearing in mind the observed
importance of both I3C and the 2-phenyl group in indoles,6 it
was of interest to know that if making hybrid structures of 1 and
2, thus generating 3 and 4 (Figs. 1 and 2), would result in improved
activity against these cell lines when compared to 1 and 2. In the
event, indoles 3 and 4 were prepared as previously reported
⇑
Corresponding author. Tel.: +44 (0)1772 895805.
E-mail address: tjsnape@uclan.ac.uk (T. Snape).
1
5
14
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.032

S. Prabhu et al. / Bioorg. Med. Chem. 21 (2013) 1918–1924
1919
OH
N
H
N
H
Indole-3-carbinol
I3C, 1)
2-Phenyl-1H-indole, 2
(
(a 2-arylindole)
OH
HO
R
R1
N
H
3
N
H
N
H
4
Generic structure, 5
Possible hybrid structures
Figure 1. Chemical structures of indole-3-carbinol (I3C, 1), a 2-arylindole (2) and their structural hybrids (3 and 4), and their generic structure, 5.
Table 1
OH
OH
A table to show the IC50 values of the indoles against two different cell lines using the
MTS assay
Entry
Compounds
MTS assay IC50 valuesa
N
H
N
H
N
H
1
321N1 (lM)
U87MG (lM)
1
, Sigma-Aldrich
2, Sigma-Aldrich
3
1
2
3
4
5
6
7
1 (I3C)
309 ± 2
—
111 ± 1
445 ± 4
—
526 ± 2
—
176 ± 1
379 ± 4
—
b
2
3
4
6
7
b
b
O
HO
MeO
—
11 ± 2
—
Cisplatin
310 ± 4
N
H
N
H
N
The values shown are the average and standard deviations of four repeats.
H
a
IC50 values reached after 48 h.
b
4
6, Sigma-Aldrich
7
IC50 value not reached.
Figure 2. The compounds screened. Compounds 1, 2, and 6 were purchased from
Sigma–Aldrich, UK, whilst 3,15 4, and 7 were prepared as referenced here.
14
16
and both were subjected to the same assay as indoles 1 and 2; the
activities (Table 1, entries 3 and 4) demonstrate that both hybrid
compounds were more active than 2, in both cell lines, and both
were more active than 1 in the malignant U87MG glioma cell line.
morethat, since a potentanticanceragent such as cisplatinalso gives
high IC50 values with these cell lines, it is promising that activity is
observed at all with these simple indoles, and thus they are worthy
of further investigation. Evidently, the 2-Ph group is having a large
influence on the activity of these indoles (compare entry 1 to entry
3 in Table 1), as has been reported against breast cancer cell lines.6
Having discovered potential new lead cytotoxic hybrid indoles
against these glioma cell lines, the mechanism of action was inves-
tigated of the more active indoles (3 and 4) in the hope that the
biological target(s) identified could be used to help with future
rounds of lead development by incorporating in silico modelling.
Preliminary insights into their mechanism of action were
gleaned from the structure of the active compounds. It was insight-
ful that compounds 1, 3 and 4 were active, yet analogues such as 2,
6 and 7 were not; the hydroxyl group was evidently having an ef-
fect in addition to the 2-phenyl ring. It was assumed that such a
difference in activity could not be due merely to differences in
hydrogen-bonding, and so attention was turned to the hydroxy
or phenoxy groups in these compounds to investigate if they were
responsible for the observed cytotoxic effects, as has been ob-
served for other hydroxy- and phenoxy-containing com-
1
6
Indoles 6 and 7 (Fig. 2) were also prepared or purchased, and
evaluated in the same assay (Table 1, entries 5 and 6) as a test to
determine the influence of the hydroxyl group in these com-
pounds. Interestingly, these two indoles (6 and 7) which lack the
hydroxyl group are both inactive, which in addition to the result
with indole 2 (Table 1, entry 2), which also lacks this group, high-
lights the hydroxyl’s apparent importance for activity against these
cell lines. All indoles assessed (Fig. 2 and Table 1) were screened
alongside the known anticancer agent—cisplatin—to gain a rudi-
mentary measure of their therapeutic efficacy. Cisplatin was cho-
sen because, in previous studies, it was shown to possess the
best cytotoxic effects against these cell lines when compared to
other known anticancer agents (temozolomide, etoposide and
1
7
carmustine).
Only 1, 3, 4 and cisplatin were active against the cell lines, with
cisplatin showing an order of magnitude better activity than the in-
doles in the 1321N1 cell line. However, it is of note that, against the
U87MG cell line, compound 4 has an IC50 value of a similar order of
magnitude to the established anticancer drug cisplatin, whilst in-
dole 3 possesses better activity than cisplatin. A result which adds
weight to the potential of these indoles as lead compounds, how-
ever, it is not suggested that indoles 3 and 4 have a mechanism of ac-
tion similar to the DNA-alkylation behavior of the platinum drug,
1
8–24
pounds.
The hypothesis was that the active indoles were acting through
a radical-based mechanism, which was most likely related to the
1
9
generation of reactive oxygen species (ROS).
As such, the

1
920
S. Prabhu et al. / Bioorg. Med. Chem. 21 (2013) 1918–1924
Image-iT™ LIVE Green Reactive Oxygen Species Detection Kit from
Invitrogen Ltd, UK, was chosen, which is able to detect ROS in live
cells, to assess the selected indoles. Oxidatively stressed cells,
when labeled with the carboxy-H DCFDA dye provided, should
2
generate fluorescence which is detectable using confocal micros-
copy and flow cytometry.
In the event, to confirm and quantify the amount of fluores-
cence produced the dye was used to detect the presence of ROS
in glioma cell lines 1321N1 and U87MG in the presence of indoles
group in their structures being able to form radical species in vitro
upon reaction with redox enzymes. Indole 7 was used as a compar-
ison (which has the hydroxyl group masked as a methoxy group)
and it was found that ROS was not induced in this case, as would
be expected if the free hydroxyl group was important for ROS gen-
eration. Nor was it induced by indole 2, which lacks any oxygena-
tion on the 2-phenyl group.
The positive control used in this experiment was tert-butyl
hydroperoxide (TBHP) (100 lM) which is a common inducer of
2
, 3, 4 and 7 (500
l
M). The results (Fig. 3) suggest that the cells
ROS and was suggested and provided by Invitrogen Ltd. Impor-
tantly, the indoles inducing ROS (3 and 4) appeared to induce it
to a greater level than the positive control (TBHP), suggesting that
these compounds are powerful inducers of reactive oxygen species
and that this presumably is intrinsic in their ability to affect the
viability and hence proliferation of the cell lines tested. In particu-
lar, the results suggest that the fluorescence generated in the
were under oxidative stress when in the presence of indoles 3
and 4 (Fig. 3D and E), as identified by the shift of the fluorescence
signal in these cases compared to the control signal (Fig. 3A), but
that oxidative stress was not observed with 2 and 7 (Fig. 3C and
F) in which no shift was observed compared to the control signal.
Presumably, only 3 and 4 generated ROS as a result of the hydroxyl
Figure 3. (Left) 1321N1 cells were treated with analogues of indole 4 for 1 h.25 (A) Cells + dye. (B) Cells treated with positive control [100
h. (C) Cells treated with indole 2 (500 M) for 1 h. (D) Cells treated with indole 3 (500 M) for 1 h. (E) Cells treated with indole 4 (500
indole 7 (500 M) for 1 h. (Right) U87MG cells were treated with analogues of indole 4 for 1 h. (A) Cells + dye. (B) Cells treated with positive control [100
hydroperoxide (TBHP)] for 1 h. (C) Cells treated with indole 2 (500 M) for 1 h. (D) Cells treated with indole 3 (500 M) for 1 h. (E) Cells treated with indole 4 (500
F) Cells treated with indole 7 (500 M) for 1 h. Carboxy-H DCFDA staining intensity was quantified by flow cytometry. Representative data from one of three experiments
yielding similar results is shown.
l
l
M tert-butyl hydroperoxide] for
M) for 1 h. (F) Cells treated with
M tert-butyl
M) for 1 h.
1
l
l
l
l
l
l
l
(
l
2

S. Prabhu et al. / Bioorg. Med. Chem. 21 (2013) 1918–1924
1921
1
321N1 and U87MG cell lines treated with indole 4 were signifi-
induces ROS generation which mediates autophagic cell death in
2
5
cantly higher when compared to the controls (Fig. 3), suggesting
that the lack of cell viability observed is mediated through the for-
mation of excessive ROS placing the cells under oxidative stress. As
a control experiment, the fluorescence of indole 4 itself plus the
cells was measured, and was found to be comparable to the cells
alone (data not shown), confirming that it is not the indole itself
that is generating false positive results, but that the results shown
in Figure 3 are due to the formation of ROS in the presence of 3 and
U87MG cells. The phenolic compound, resveratrol, has also been
shown to induce autophagy in human U251MG glioma cells.41
Based on such precedent it was important to see if autophagy
was being induced in the 1321N1 and U87MG cells upon treatment
with the most active, stable compound, indole 4. The process of
autophagy is in-part characterized by the formation of acidic vesic-
ular organelles (AVOs) which can be detected and measured by
staining with acridine orange. Acridine orange moves freely across
biological membranes and accumulates in acidic compartments
upon protonation where it can be observed and quantified by fluo-
rescence at a distinct wavelength. Therefore, in view of the finding
that AVOs accumulate in cells undergoing autophagy, an investiga-
tion into whether indole 4 induced AVO formation by treating the
cells with indole 4 was carried out, staining them with acridine or-
ange and analyzing the results by flow cytometry.
4.
Ascorbic acid is a naturally occurring organic compound with
antioxidant properties and is a scavenger of hydroxyl radicals,
reacting with reactive oxygen species, such as the hydroxyl radical,
2
6
and neutralizing it. Recent studies have found that ascorbic acid
2
7
inhibits ROS production by neutralizing the radicals formed, and
therefore ascorbic acid was chosen here to validate the ROS pro-
duction induced by indole 4 (500
cell lines following the assumption that reduced amounts of ROS
production would be seen after the co-treatment of the cells with
l
M) in the 1321N1 and U87MG
The results (Fig. 5) indicated the formation of AVOs had oc-
curred, as concluded by the increased amount of red fluorescence
in the cell lines studied.
ascorbic acid (500
concentrate on the phenol derivative 4, since in our hands, carbinol
l
M) and indole 4. At this stage it was decided to
The results were confirmed on both cell lines by repeating the
experiments in the presence of an autophagy inhibitor, 3-methyl-
4
2
3
had proved to be slightly unstable under acidic conditions, as is
adenine (3-MA, 5 mM). The results (Fig. 5) indicate that there is
a reduction in the amount of fluorescence produced in the experi-
ments with 3-MA, indicating that autophagy is inhibited, and that
indole 4 induces AVO formation as a result of the cells being in an
autophagic process, which may be mediated by reactive oxygen
species generation in these particular cell lines. This potentially re-
veals a new class of compound which could possibly be optimized
further for glioma treatment through analogue preparation.
Based on these preliminary results it was decided to ascertain if
the effects were general against other glioblastoma cells and thus
set out to screen indole 4 against the short term cell cultures
IN1472, IN1265, IN1760 and IN859, and another established cell
line—U251MG. The IC50 values obtained were better for U251MG,
IN1472 and IN1265 (Table 2) compared to 1321N1 and U87MG,
possibly suggesting that indole 4 is even more potent against these
cells. In contrast however, it was completely inactive against the
highly resistant cell culture IN1760, a cell culture which has proved
to be highly resistant to a number of commercially available anti-
1–3
well documented for similar structures,
and thus did not justify
further investigation here since its stability would be an issue in
the presence of an additional acid (ascorbic acid). Moreover, it is
possible that the addition of the 2-aryl ring in 3 may cause further
instability through increasing the conjugation of the vinylogous
hemiaminal of I3C (1), and thus increasing the stability of the acid
3
degradation products, Figure 4. Nevertheless related indole-3-car-
binol derivatives have recently demonstrated activity against pros-
2
8
29
tate
and breast
cancers where the structures have been
modified on nitrogen to reduce its inherent acid-lability.
The results of the ascorbic acid treatment reveals that the level
of fluorescence produced by both the cell lines treated with a com-
bination of indole 4 and ascorbic acid was shifted back towards the
levels shown by the vehicle control cells alone (data not shown),
thus further validating that indole 4 expresses its cytotoxic behav-
iour through ROS generation, but that this cannot happen in the
presence appropriate levels of radical scavengers.30 This was also
supported by the MTS assay on both cell lines, which showed that
cell viability was unaffected by indole 4 when in the presence of
ascorbic acid.
4
3
cancer drugs (CCNU, vincristine and doxorubicin). Furthermore,
the IC50 value against the short-term cell cultures (except
IN1760) are comparable, if not better, than that for U87MG sug-
gesting that indole 4 possesses the ability to kill short-term cell
cultures as well as established cancer cell lines.
To further understand the effects that such ROS generation was
having on the cells the examination of whether indole 4 was
responsible for cell death through the induction of autophagy, as
In conclusion, these combined results suggest that indole 4 (and
possibly indole 3) possess promising anticancer activity against a
range of glioblastoma cells and that further development and ana-
logue preparation is warranted in order to improve activity further.
has been observed for other small molecules,2
investigated.
5,31–36
was
Oxidative stress is known to induce autophagy,31,33,35–37 and
cell death associated with autophagy in tumour cell lines treated
3
8
with chemotherapeutic agents has been proposed recently. Such
research has also shown that cell death is associated with
3
. Materials and methods
25
39
autophagy in malignant gliomas,
mammary carcinomas,40 and that hydrogen peroxide (H
-methoxyestradiol (2-ME) induce oxidative stress, causing auto-
ovarian carcinomas
and
3.1. MTS assay
2
O
2
) and
2
The 1321N1 cells (obtained from ECACC, UK) were maintained
in Dulbeccos’s Modified Eagle’s Medium (DMEM) supplemented
phagic cell death, in the transformed cell line HEK293 and cancer
cell lines U87MG and HeLa cells.37 Furthermore, b-lapachone
OH
OH
+
H+
+H+
N
H
N
H
N
H
N
H
Increased conjugation
and stability
1
3
Figure 4. Figure depicting the acid instability of I3C (1) and the analogous structure for the presumably more stable acid-degradation product from 3.3

1
922
S. Prabhu et al. / Bioorg. Med. Chem. 21 (2013) 1918–1924
Figure 5. Acidic vesicular organelle (AVO) formation in 1321N1 cells (Left) and U87MG cells (Right). (A) Cells + acridine orange. (B) Cells treated with indole 4 (500
lM) for
1
h + acridine orange. (C) Cells treated with indole 4 (500 M) for 1 h, acridine orange + 3-MA (5 mM for 1 h). Staining intensity was quantified by flow cytometry.
l
Representative data from one of two experiments yielding similar results is shown.
Table 2
A table to show the IC50 values of indole 4 against different cells using the MTS and MTT assays.
Compound
MTS assay IC50 valuesa
321N1 ( M)
445 ± 4
MTT assay IC50 valuesb
IN1472 ( M) IN1265 (
48.0 ± 0.2 90.0 ± 0.5
1
l
U87MG (
l
M)
IN859c
400 ± 1
(lM)
l
l
M)
U251MG (lM)
IN1760 (lM)
4
379 ± 4
92.0 ± 0.6
>1000
The values shown are the average and standard deviations of at least two repeats.
a
IC50 values reached after 48 h.
IC50 values reached after 72 h.
IC50 value reached after 2 h.
b
c
with 10% (v/v) foetal bovine serum (FBS) and 2 mM
L
-glutamine,
2
fied incubator supplied with 5% CO for 60 min. At the end of
while the U87MG cell line (obtained from ECACC, UK) was main-
tained in Essential Minimum Eagle Medium (EMEM) supple-
60 min, the absorbance was recorded at 490 nm using a Tecan
GENios ProÒ microplate reader. The concentration of compound
that inhibits 50% of cell growth (IC50) compared to untreated vehi-
cle controls containing 60.5% absolute ethanol was calculated by
nonlinear regression analysis. The untreated vehicle controls con-
tained 60.5% absolute ethanol or DMSO, as indole 4 was dissolved
in absolute ethanol while indole 2 was dissolved in DMSO.
mented with 10% (v/v) FBS, 2 mM
pyruvate and 1% (v/v) non-essential amino acids (NEAA) (Lonza,
UK) in a 37 °C humidified incubator supplied with 5% CO . The
321N1 and the U87MG cells were trypsinized and cultured over-
L-glutamine, 1 mM sodium
2
1
night at a cell density of 3500 and 4000 cells/200 ll respectively in
9
6-well microtitre plates (Sarstedt, UK). The following day when
the cells were 50–60% confluent, each concentration of the com-
mercial drugs and novel compounds (see text) were added in qua-
3.2. MTT assay
druplicates and left for incubation for 48 h. After 48 h, 20
pre-warmed MTS reagent (Promega, UK) was added to each of the
wells and the 96-well plates were incubated at 37 °C in a humidi-
l
l of the
Three short-term glioblastoma multiforme (GBM) cell cultures
(IN1472, IN1265 and IN1760) derived in our laboratory from tu-
mour biopsies, as described by Lewandowicz et al., and the
4
4

S. Prabhu et al. / Bioorg. Med. Chem. 21 (2013) 1918–1924
1923
established GBM cell line U251MG (obtained from ECACC, UK)
were used in this study. Cells were maintained in Hams F10 nutri-
ent mix (Invitrogen Ltd., UK) with 10% foetal calf serum in a 37 °C
(BD Biosciences). The untreated vehicle controls contained 60.5%
absolute ethanol as indole 4 was dissolved in absolute ethanol,
while 3-MA was directly soluble in the cell culture medium.
2
non-CO incubator. The anti-proliferative effects of the compounds
were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltet-
3
3
.5. Synthesis and characterization of indoles
4
5
razolium bromide (MTT) assay as previously described. Cells
3
were seeded at a density of 10 cells/well into 96-well plates and
.5.1. General synthetic procedures
Commercially available reagents were used as received without
incubated in growth media for 48 h prior to treatment with indole
4
at concentrations between 0.01 and 1000 lM. The compound
purification. Analytical thin layer chromatography (TLC) were per-
formed with plastic-backed TLC plates coated with silica G/UV254
in a variety of solvents. The plates were visualised by UV light
254 nm). Flash column chromatography was conducted with
Davisil silica 60 Å (40–63 m) under bellows pressure. Low resolu-
tion mass spectra were recorded on a Thermo Finnigan LCQ Advan-
was renewed at 24 h intervals and each dose was tested in quadru-
plicate. Cells were then allowed a 24-h recovery period at 37 °C in
compound-free media before media was aspirated and 100 ll of
MTT solution (1 mg/mL in Ham’s F10 medium without FCS) was
,
(
l
added to each well. Four hours later, formazan crystals were dis-
solved in DMSO (100
l
l/well) and the plates were agitated for 2–
1
13
tage MAX using chemical ionisation (CI). H and C NMR spectra
were recorded on a Bruker Avance DPX 250 (250 MHz) or a Bruker
3
min before the optical density at 570 nm was determined on a
plate reader (Fluoroskan Ascent, Thermo Labsystems, Cheshire,
UK). The concentration of compound that inhibits 50% of cell
growth (IC50) compared to untreated controls was calculated by
nonlinear regression analysis.
4
00 (400 MHz) spectrometer. All chemical shifts (d) are quoted in
parts per million (ppm) relative to a calibration reference of the
residual protic solvent; CHCl (d 7.26, s) or DMSO (d 2.53, m)
was used as the internal standard in H NMR spectra, and
NMR shifts were referenced using CDCl (d 77.0, t) or DMSO (d
3
H
H
1
13
C
3
C
C
3
.3. Flow cytometry
4
0.5, sept) with broad band decoupling.
The 1321N1 cells were maintained in Dulbeccos’s Modified Ea-
0
16
3
.5.2. Synthesis of 2-(2 -methoxyphenyl)-1H-indole, 7
2
gle’s Medium (DMEM) supplemented with 10% (v/v) foetal bovine
serum (FBS) and 2 mM -glutamine, while the U87MG cell line was
maintained in Essential Minimum Eagle Medium (EMEM) supple-
mented with 10% (v/v) FBS, 2 mM -glutamine, 1 mM sodium pyru-
vate and 1% (v/v) non-essential amino acids (NEAA) (Lonza, UK) in
a 37 °C humidified incubator supplied with 5% CO . The 1321N1
0
-Methoxyacetophenone (1.38 mL, 10 mmol) was mixed with
L
phenylhydrazine (0.99 mL, 10 mmol) in ethanol (5 mL) and four
drops of glacial acetic acid added. The pale yellow solution was
heated to 80 °C with stirring for 1 h which produced a red/brown
solution. The solvent was evaporated to yield the phenylhydrazone
intermediate as red/brown oil. To this oil was added polyphospho-
ric acid (20 g) and the reaction heated to 120 °C with stirring for
h. After completion of the reaction (TLC) the reaction mixture
was poured onto crushed ice, followed by the addition of NaOH un-
til a neutral pH was reached. The product was extracted with DCM
L
2
and the U87MG cells were trypsinized and cultured overnight at
4
a cell density of 10 cells/well/mL and were seeded in six well
2
plates (BD Biosciences, UK) and were allowed to attach overnight;
2
mL of this cell suspension was added into each well resulting in a
final concentration of 20,000 cells/well. Cells were labelled with
sufficient amount of 25 M carboxy-H DCFDA dye and were incu-
bated for 30 min at 37 °C, protected from light. Cells were then
treated with 500 M of the test compounds and positive control
tert-butyl hydroperoxide (TBHP, 100 M)] and negative control
indole 2, 500 M) for 60 min. Cells were gently washed with
(
(
3 ꢀ 50 mL) and the combined extracts washed with water
l
2
50 mL), brine (50 mL), dried (MgSO ), filtered and the solvent
4
evaporated. The product was purified using flash chromatography
SiO 50% toluene:50% petroleum ether) to yield the title
compound as an off-white solid (532 mg, 24%). R 0.64 (100%
f
l
(
2
;
[
(
l
l
toluene).
Hanks balanced salt solution (HBSS) warm buffer three times and
then trypsinized, centrifuged and resuspended in 300 l PBS. Cells
were filtered through cell strainer caps (35 m mesh) to obtain a
1H NMR (250 MHz, CDCl
3
H
) d 4.03 (s, 3H), 6.93 (br s, 1H). 7.03–
l
7
1
(
1
.34 (m, 5H), 7.46 (d, J = 8.0 Hz, 1H), 7.67 (dd, J = 0.5 and 7.5 Hz,
l
13
H), 7.87 (dd, J = 1.5 and 8.0 Hz, 1H), 9.70 (br s, 1H); C NMR
single cell suspension and analyzed using a FACSAria flow cytom-
eter using FITC fluorochrome at an excitation/emission of 488/
19 nm (BD Biosciences). This same procedure and time point
3 C
62.5 MHz, CDCl ) d 55.8, 99.5, 110.9, 111.8, 119.8, 120.2, 120.5,
21.5, 121.8, 128.0, 128.3, 128.6, 135.9, 136.1, 155.7;
mmax (film,
5
ꢁ1
+
cm ) 3443, 1577, 1463, 1435, 1308, 1232; m/z (CI) 224 ([M+H] ,
was used for ascorbic acid (500 M) studies. The untreated vehicle
l
1
00%).
controls contained 60.5% absolute ethanol and DMSO as indole 4
was dissolved in absolute ethanol while TBHP and indole 2 were
dissolved in DMSO.
.5.3. Synthesis of (2-phenyl-1H-indol-3-yl)methanol, 315
3
2
-Phenylindole-3-carboxaldehyde (300 mg, 1.36 mmol) and
NaBH (103 mg, 2.72 mmol) were stirred at reflux in ethanol
(10 mL) for 1 min followed by stirring at room temperature for
h. 1% NaOH (10 mL) was added to the reaction mixture and the
product was extracted with Et
O (3 ꢀ 10 mL). The combined ex-
tracts were dried (MgSO ), filtered and the solvent evaporated to
yield crude product which was re-crystallised in
4
3
.4. Acridine orange assay
2
The same procedure was used to prepare the cells as in the flow
2
cytometry experiments above with the carboxy-H
to the point of trypsinization and attaching overnight. After 24 h
of incubation, cells were treated with 500 M of indole 4 or indole
(500 M) + 3-methyladenine (5 mM) for 60 min. After 60 min
incubation with the compounds, the cells were stained with
M acridine orange and incubated for 15 min at 37 °C, protected
from light. Cells were gently washed with PBS, trypsinised, centri-
fuged and were resuspended in 300 l PBS. Cells were filtered
through cell strainer caps (35 m mesh) to obtain a single cell sus-
2
DCFDA dye, up
4
a
l
PhMe:EtOAc:petroleum ether, yielding the title compound as a
4
l
white solid 133 mg, 44%).
1
6 H
H NMR (250 MHz, DMSO-d ) d 4.81 (d, J = 5.0 Hz, 2H), 5.10 (t,
J = 5.0 Hz, 1H), 7.14–7.28 (m, 2H), 7.48–7.53 (m, 2H), 7.64 (t,
J = 7.5 Hz, 2H), 7.80 (d, J = 7.5 Hz, 1H), 7.92 (d, J = 7.5 Hz, 2H),
), d 54.8, 112.1,
6 C
113.3, 119.9, 122.6. 128.5, 128.9, 129.67, 129.71, 133.4, 136.8,
3
l
1
3
l
11.47 (s, 1H); C NMR (62.5 MHz, DMSO-d
l
ꢁ1
pension and analysed using a FACSAria flow cytometer using a PE-
Texas Red fluorochrome at an excitation/emission of 488/615 nm
136.9;
mmax (film, cm ) 3488, 3183 (br s), 1638, 1491, 1452,
+
1392; m/z (CI) 206 ([MꢁOH] , 100%).

1
924
S. Prabhu et al. / Bioorg. Med. Chem. 21 (2013) 1918–1924
0
14
3
.5.4. Synthesis of 2-(2 -hydroxyphenyl)-1H-indole, 4
1
14. Snape, T. J. Synlett 2008, 2689.
15. Leete, E. J. Am. Chem. Soc. 1959, 81, 6023.
-(2-Hydroxyphenyl)-2-(2-nitrophenyl)ethanone,14 (105 mg,
.41 mmol) was dissolved in methanol (4.1 mL). Pd/C (10 mg,
0 wt %) was added and the flask evacuated and backfilled with
hydrogen (three cycles). The reaction was then stirred under an
atmosphere of hydrogen for 18 h. The reaction was filtered through
CeliteÒ and eluted with methanol (10 mL) and the solvent removed
in vacuo. The crude product was purified by column chromatogra-
phy on silica gel (10% EtOAc in petroleum ether) to give the title
16. So, C. M.; Lau, C. P.; Kwong, F. Y. Org. Lett. 2007, 9, 2795.
0
1
17. Prabhu, S.; Harris, F.; Lea, R.; Snape, T. J. Neuro Oncol. 2011, 13, 12.
18. Arnao, M. B.; SanchezBravo, J.; Acosta, M. Biochem. Mol. Biol. Int. 1996, 39, 1125.
1
2
2
9. Han, Y.-M.; Shin, D.-S.; Lee, Y.-J.; Ismail, I. A.; Hong, S.-H.; Han, D. C.; Kwon, B.-
M. Bioorg. Med. Chem. Lett. 2011, 21, 747.
0. Iwasaki, Y.; Hirasawa, T.; Maruyama, Y.; Ishii, Y.; Ito, R.; Saito, K.; Umemura, T.;
Nishikawa, A.; Nakazawa, H. Toxicol. In Vitro 2011, 25, 1320.
1. Kagan, V. E.; Kuzmenko, A. I.; Shvedova, A. A.; Kisin, E. R.; Li, R.; Martin, I.;
Quinn, P. J.; Tyurin, V. A.; Tyurina, Y. Y.; Yalowich, J. C. Biochim. Biophys. Acta
2
003, 1620, 72.
22. Siraki, A. G.; O’Brien, P. J. Toxicol. In Vitro 2002, 177, 81.
3. Steenken, S.; Neta, P. J. Phys. Chem. 1982, 86, 3661.
24. Fujisawa, S.; Atsumi, T.; Murakami, Y.; Kadoma, Y. Arch. Immunol. Ther. Exp.
005, 53, 28.
25. Parka, E. J.; Choi, K. S.; Kwon, T. K. Chem. Biol. Interact. 2011, 189, 37.
compound as a pale yellow solid (62 mg, 72%). R
f
0.39 (30% ethyl
acetate in petroleum ether).Mp (EtOAc:petroleum ether) 170–
2
ꢁ1
1
72 °C;
s), 7.70 (1H, dd, J = 1.6 and 7.8 Hz, Ar), 7.66 (1H, d, J = 7.8 Hz, Ar),
.42 (1H, d, J = 8.1 Hz, Ar), 7.26–7.11 (3H, m, Ar), 7.04 (1H, td,
J = 1.1 and 7.6 Hz, Ar), 6.91 (1H, dd, J = 0.9 and 8.1 Hz, Ar), 6.87
1H, m, Ar), 6.0–5.0 (1H, br s); d (75 MHz; CDCl ) 152.0, 136.4,
34.8, 128.9, 128.4, 128.3, 122.2, 121.5, 120.4, 120.1, 119.1,
H 3
mmax/cm 3500, 3425; d (300 MHz; CDCl ) 9.22 (1H, br
2
2
2
6. Biswas, S.; Bhattacharyya, J.; Dutta, A. G. Mol. Cell. Biochem. 2005, 276, 205.
7. Peng, Y.; Kwok, K. H. H.; Yang, P. H.; Ng, S. S. M.; Liu, J.; Wong, O. G.; He, M. L.;
Kung, H. F.; Lin, M. C. M. Neuropharmacology 2005, 48, 426.
7
(
C
3
28. Weng, J.-R.; Tsai, C.-H.; Kulp, S. K.; Wang, D.; Lin, C.-H.; Yang, H.-C.; Ma, Y.;
Sargeant, A.; Chiu, C.-F.; Tsai, M.-H.; Chen, C.-S. Cancer Res. 2007, 67, 7815.
29. Weng, J.-R.; Tsai, C.-H.; Omar, H. A.; Sargeant, A. M.; Wang, D.; Kulp, S. K.;
Shapiro, C. L.; Chen, C.-S. Carcinogenesis 2009, 30, 1702.
1
1
2
+
16.6, 111.0, 100.2; m/z (ES+) 210 ([M+H] , 100%); found
+
10.0920, C14H12NO (M+H ) requires 210.0919.
3
3
0. Tsai, C.-W.; Lin, C.-Y.; Lin, H.-H.; Chen, J.-H. Neurochem. Res. 2011, 36, 2442.
1. Duan, W.-J.; Li, Q.-S.; Xia, M.-Y.; Tashiro, S.-I.; Onodera, S.; Ikejima, T. J. Asian
Nat. Prod. Res. 2011, 13, 27.
Acknowledgement
3
2. Gonzalez-Polo, R. A.; Niso-Santano, M.; Ortiz-Ortiz, M. A.; Gomez-Martin, A.;
Moran, J. M.; Garcia-Rubio, L.; Francisco-Morcillo, J.; Zaragoza, C.; Soler, G.;
Fuentes, J. M. Autophagy 2007, 3, 366.
The authors would like to thank Brain Tumour North West
3
3
3. Li, J.; Wang, X.-L.; Fang, Y.-C.; Wang, C.-Y. J. Asian Nat. Prod. Res. 2010, 12, 992.
4. Wu, J.-Y.; Tsai, K.-W.; Shee, J.-J.; Li, Y.-Z.; Chen, C.-H.; Chuang, J.-J.; Liu, Y.-W.
Acta Pharmacol. Sin. 2010, 31, 81.
(
BTNW) for helpful advice.
References and notes
35. Xiao, D.; Powolny, A. A.; Antosiewicz, J.; Hahm, E.-R.; Bommareddy, A.; Zeng,
Y.; Desai, D.; Amin, S.; Herman-Antosiewicz, A.; Singh, S. V. Pharm. Res. 2009,
2
6, 1729.
1
2
3
4
5
6
.
.
.
.
.
.
Bradlow, H. L. In Vivo 2008, 22, 441.
Rogan, E. G. In Vivo 2006, 20, 221.
Weng, J.-R.; Tsai, C.-H.; Kulp, S. K.; Chen, C.-S. Cancer Lett. 2008, 262, 153.
Horton, D. A.; Bourne, G. T.; Smythe, M. L. Chem. Rev. 2003, 103, 893.
Lal, S.; Snape, T. J. Curr. Med. Chem. 2012, 19(28), 4828–4837.
Gastpar, R.; Goldbrunner, M.; Marko, D.; von Angerer, E. J. Med. Chem. 1998, 41,
3
3
6. Zhang, Y.-H.; Wu, Y.-L.; Tashiro, S.-I.; Onodera, S.; Ikejima, T. Acta Pharmacol.
Sin. 2011, 32, 1266.
7. Chen, Y.; McMillan-Ward, E.; Kong, J.; Israels, S. J.; Gibson, S. B. Cell Death Differ.
2
008, 15, 171.
3
3
8. Levine, B.; Yuan, J. Y. J. Clin. Invest. 2005, 115, 2679.
9. Opipari, A. W.; Tan, L. J.; Boitano, A. E.; Sorenson, D. R.; Aurora, A.; Liu, J. R.
Cancer Res. 2004, 64, 696.
4
965.
7.
8.
9.
Kaufmann, D.; Pojarova, M.; Vogel, S.; Liebl, R.; Gastpar, R.; Gross, D.; Nishino,
T.; Pfaller, T.; von Angerer, E. Bioorg. Med. Chem. 2007, 15, 5122.
Pojarova, M.; Kaufmann, D.; Gastpar, R.; Nishino, T.; Reszka, P.; Bednarski, P. J.;
von Angerer, E. Bioorg. Med. Chem. 2007, 15, 7368.
Vogel, S.; Kaufmann, D.; Pojarova, M.; Mueller, C.; Pfaller, T.; Kuehne, S.;
Bednarski, P. J.; von Angerer, E. Bioorg. Med. Chem. 2008, 16, 6436.
4
0. Bursch, W.; Ellinger, A.; Kienzl, H.; Torok, L.; Pandey, S.; Sikorska, M.; Walker,
R.; Hermann, R. S. Carcinogenesis 1996, 17, 1595.
4
4
1. Li, J.; Qin, Z. H.; Liang, Z. Q. BMC Cancer 2009, 9.
2. McFarland, A. J.; Anoopkumar-Dukie, S.; Perkins, A. V.; Davey, A. K.; Grant, G. D.
Arch. Toxicol. 2012, 86, 275.
3. Personal communicaion with Dr Tracy Warr, University of Wolverhampton,
UK.
4. Lewandowicz, G. M.; Harding, B.; Harkness, W.; Hayward, R.; Thomas, D. G. T.;
Darling, J. L. Eur. J. Cancer 1955, 2000, 36.
5. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
4
4
4
10. Vonangerer, E.; Knebel, N.; Kager, M.; Ganss, B. J. Med. Chem. 1990, 33, 2635.
11. Vonangerer, E.; Prekajac, J.; Strohmeier, J. J. Med. Chem. 1984, 27, 1439.
12. Liao, S. Y.; Qian, L.; Miao, T. F.; Lu, H. L.; Zheng, K. C. Eur. J. Med. Chem. 2009, 44,
2
822.
1
3. Greenberger, L. M.; Annable, T.; Collins, K. I.; Komm, B. S.; Lyttle, C. R.; Miller, C.
P.; Satyaswaroop, P. G.; Zhang, Y. X.; Frost, P. Clin. Cancer Res. 2001, 7, 3166.