Design, synthesis and pro-apoptotic antitumour properties of indole-based 3,5-disubstituted oxadiazoles

Article abstract of DOI:10.1016/j.ejmech.2010.07.012

A series of new indole-based 3,5-disubstituted 1,2,4-oxadiazoles has been designed and synthesised as potential pro-apoptotic antitumour agents, via the base-catalysed condensation reaction between substituted amidoximes and indole esters. Evaluation of a

Full text of DOI:10.1016/j.ejmech.2010.07.012

European Journal of Medicinal Chemistry 45 (2010) 4523e4530
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and pro-apoptotic antitumour properties of indole-based
3,5-disubstituted oxadiazoles
,
a,
Noha I. Ziedan a, Fabio Stefanelli b, Stefano Fogli b , Andrew D. Westwell
*
**
a Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB, UK
b Department of Psychiatry, Pharmacology and Biotechnology, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new indole-based 3,5-disubstituted 1,2,4-oxadiazoles has been designed and synthesised as
potential pro-apoptotic antitumour agents, via the base-catalysed condensation reaction between
substituted amidoximes and indole esters. Evaluation of antiproliferative activity against the human
cancer cell lines COLO 320 (colorectal) and MIA PACA-2 (pancreatic) revealed IC50 values in the low
micromolar range. Selected compounds were able to trigger apoptosis in sensitive cell lines, for example
via activation of caspase-3/7, demonstrating that indole-based oxadiazoles possess in vitro antitumour
and pro-apoptotic activity.
Received 11 March 2010
Received in revised form
2 July 2010
Accepted 5 July 2010
Available online 21 July 2010
Keywords:
3,5-Disubstituted oxadiazoles
Indoles
Ó 2010 Elsevier Masson SAS. All rights reserved.
Pro-apoptotic antitumour agents
Anticancer activity
Caspase induction
1. Introduction
promising pre-clinical drug candidates into registered drugs in this
category has not yet been realised [4].
The fundamental roles of defective apoptotic pathways in
a range of disease states are well characterised. Insufficient
apoptosis is a characteristic feature of proliferative diseases such as
cancer or rheumatoid arthritis, whereas too much apoptosis is
known to contribute to the development of degenerative disease
such as Alzheimer’s disease and multiple sclerosis [1]. Apoptotic
pathways are particularly well characterised in the cancer field,
where resistance to apoptotic stimuli is known as one of the six
hallmarks of cancer articulated by Hanahan and Weinberg [2].
Mitochondria play a prominent and central role in the execution
of the cell death process via apoptosis, with mitochondrial
membrane permeabilisation and subsequent release of apopto-
genic factors usually marking the point-of-no-return for a cell
destined to undergo apoptotic cell death. The discovery of small
molecules able to selectively induce apoptosis in cancer cells via
a variety of mechanisms including those centred on the mito-
chondrial membrane represents an enticing prospect for future
cancer therapeutics [3]. A number of articles published in recent
years suggest that the goal of selective pro-apoptotic therapy for
cancer treatment might be attainable, although translation of
Although cell death through apoptosis is an indirect conse-
quence of many, if not most, cancer therapeutic agents, selective
pro-apoptotic antitumour agents such as those directly targeting
cancer cell mitochondria would be expected to have a number of
beneficial properties compared to existing cytotoxic and molecu-
larly targeted drugs. For example, tumour cells eliminated by pro-
apoptotic agents by definition will not contribute to tumour
relapse, as may be the case for cytostatic agents. In addition,
selective pro-apoptotic agents would likely bypass the severe
toxicological side effects associated with cytotoxic cancer chemo-
therapy, since genotoxic effects would be mediated through
processes such as production of Reactive Oxygen Species (ROS) and
oxidative DNA damage rather than direct DNA interaction. Finally,
apoptosis is a particularly efficient mode of cell death (compared to
necrosis for example), and would not lead to local inflammation
and damage to surrounding tissue [3]. A potential disadvantage of
pro-apoptotic agents is their likely diverse mode of action, making
optimisation through structureeactivity relationships studies
difficult; for this reason targeted therapies are often preferred in
drug development.
Targeting the physiological process of apoptosis as a therapeutic
strategy relies on the assumption that cancer cells, particularly
cancer cell mitochondria, have fundamentally different properties
compared to their normal tissue counterparts. The emerging field of
* Corresponding author. Tel.: þ39 050 2219511; fax: þ39 050 2219609.
** Corresponding author. Tel.: þ44 2920 875800; fax: þ44 2920 874149.
E-mail addresses: s.fogli@farm.unipi.it (S. Fogli), westwella@cf.ac.uk (A.D. Westwell).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.012

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N.I. Ziedan et al. / European Journal of Medicinal Chemistry 45 (2010) 4523e4530
tumour bioenergetics provides an important clue as to how cancer
cells differ from their normal counterparts at the level of cell
mitochondria. Alongside their role as the gateway to apoptosis,
mitochondria perform a vital role as the “energy factory” of the cell
through production of ATP orchestrated under normal conditions
through the cycle of oxidative phosphorylation. It is well established
that cancer cells exhibit a unique metabolic profile whereby aerobic
glycolysis is the main source of energy compared to oxidative
phosphorylation in normal cells, a phenomenon first observed
back in the 1930’s and now known as the “Warburg effect” [5].
In an intriguing study, Bonnet et al. demonstrated that a simple
commercially available chemical, the sodium salt of dichloroacetate
(DCA), selectively induced apoptosis, reduced proliferation and
inhibited growth of cancer cells by reversing this metabolic
remodeled process [6] through the inhibition of mitochondrial
pyruvate dehydrogenase kinase (PDK2). In vivo studies demon-
strated that using clinically relevant DCA doses in the drinking
water of rats effectively led to the inhibition and prevention of
tumour growth with no observed toxicity [6], raising hopes for
a potent orally active and cost-effective antitumour agent.
2-yl ring in the oxadiazole 5-position. Indoles are an important
heterocyclic component of a number of antitumour agents; for
example, the use of indole derivatives as inhibitors of tubulin
polymerisation has been reviewed [15]. The present work builds on
our research interest and experience in the discovery of novel pro-
apoptotic antitumour agents [3,16,17].
2. Chemistry results and discussion
Amidoximes 5aed were readily accessible in high isolated
yields (76e99%) via the reaction between benzonitriles and
hydroxylamine hydrochloride under basic conditions, according
to the method of Quan and Kurth [18]. Indole-based starting
materials (6aee) for oxadiazole synthesis were readily available
either commercially (ethyl-5-chloroindole-2-carboxylate 6e) or
via simple esterification of commercially available materials
(in the case of methylindole-2-carboxylate 6a and methyl-
5-methoxyindole-2-carboxylate 6b). Both ethyl-5-fluoroindole-2-
carboxylate 6c and ethyl-5-nitroindole-2-carboxylate 6d were
prepared from the corresponding anilines via the Fischer indole
synthesis in 60% and 52% yield respectively [19].
The key step in the synthesis of target structures 7aeu was the
base-catalysed condensation reaction between substituted ami-
doximes (5aed) and indole esters (6aee), using sodium ethoxide in
refluxing ethanol (Scheme 1) to give concise access to oxadiazole
products in low to moderate yields (11e47%) [20]. Oxadiazole 7g
containing a phenol group at the indole 5-position was obtained via
demethylation of 7f using boron tribromide in dichloromethane in
85% yield, and the two N-methylindole products (7p and 7r) were
accessible by sodium hydride-promoted N-methylation using
iodomethane in DMF (83% and 80% yield respectively).
3-Amino-5-(indol-2-yl)-1,2,4-oxadiazole (10) was obtained in
two steps from indole-2-carboxylic acid chloride 8 (Scheme 2) [21].
Base-promoted (NaH) reaction between the acyl chloride 8 and
cyanamide in DMF gave the intermediate N-cyano-1H-indole-
2-carboxamide 9 in 60% yield. Compound 9 was then used without
further purification in reaction with hydroxylamine hydrochloride
in pyridine/ethanol (1:2) to afford the desired product 3-amino-
5-(indol-2-yl)-1,2,4-oxadiazole (10) in low yield (8%) following
purification using column chromatography.
More relevant to the present study was the identification of
3,5-diaryl-1,2,4-oxadiazoles such as compound 1 (Fig. 1) as novel
inducers of apoptosis using a high-throughput caspase- and cell-
based screening assay [7]. The pro-apoptotic activity of 1 was
characterised in human SKBr3 cancer cells, where a reduction in
pro-caspase levels and appearance of active caspases-3 and -7 were
observed. Induction of apoptosis selectively in human cancer cells
(such as breast T47D and ZR75-1, and colorectal DLD-1 and HT29)
was found, with EC50 values in the range 0.44e3.34
mM. Photo-
affinity-labelling studies led to the identification of the mannose-6-
phosphate receptor trafficking protein TIP47 as a molecular target
of the diaryl-oxadizoles [8]. Over-expression of TIP47 in several
tumour types is known [9], illustrating the utility of this chemical
genetics approach to cancer drug discovery. Further recent devel-
opment of the 3,5-diaryl-1,2,4-oxadiazoles as apoptosis-inducing
anticancer agents has led to the identification of more water soluble
analogues with good in vivo efficacy in cancer xenograft models
[10]. Other notable small molecules of current interest known to
exert pro-apoptotic effects selectively in cancer cells include the
anti-apoptotic Bcl-2-targeting Flex-Hets such as 2 [11], the mito-
chondria targeting jasmonates such as methyl jasmonate 3 [12],
and the 4-anilinoquinazolines such as 4 [13] (Fig. 1).
3. Biological results and discussion
In view of the utility of the oxadiazole scaffold for the discovery
of novel pro-apoptotic antitumour agents [14], we chose to focus on
the synthesis and evaluation of new disubstituted oxadiazoles in
our own work, in order to design new antitumour agents and
further understand structureeactivity relationships within this
series. In this study we report our antitumour and pro-apoptotic
data on a new series of 3,5-diaryloxadiazoles containing an indol-
3.1. Structureeactivity relationship (SAR) analysis e in vitro
inhibition of cellular proliferation
The antitumour effect in vitro of compounds 7aeu and 10 was
assessed against a panel of cancer cell lines derived from different
human tissues including colon (COLO 320), pancreas (MiaPaCa-2),
breast (MCF-7) and peripheral blood of a T cell leukemia patient
(Jurkat). Antiproliferative data was compared with the previously
reported pro-apoptotic oxadiazole 1 as control [7]. The screening
was performed by using a cell proliferation assay based on the
mitochondrial dehydrogenase-mediated cleavage of the tetrazo-
lium salt WST-1 to formazan, following 72-h drug exposure.
Tetrazolium-based assays (such as MTT, MTS or WST-1) represent
one of the most commonly used methods for anticancer drug
screening; of relevance here is their application in the assessment
of antiproliferative activity of related pro-apoptotic agents [11,12].
Overall, the COLO 320 (colon) and MIA PaCa-2 (pancreatic) cell lines
were most responsive to indolyl-oxadiazoles and exposure of the
cells to compounds resulted in dose-dependent decrease in cell
viability with mean 50% inhibition (IC50) in the micromolar
concentration range (Table 1). The most active compounds in the
Cl
H
N
H
N
N
N
S
S
CF3
S
NO2
O
2 (SHetA2)
1 (diaryl oxadiazole)
OMe
O
N
N
COOMe
N
Me
3 (methyl jasmonate)
4 (4-anilinoquinazoline)
Fig. 1. Representative selective pro-apoptotic antitumor agents.
COLO 320 screen (7n, IC50 7.7 mM; 7u, IC50 9.6 mM) were found to be

N.I. Ziedan et al. / European Journal of Medicinal Chemistry 45 (2010) 4523e4530
4525
R2
R2
O
N
OH
O
N
(i)
N
R1
+
6a-u
R4O
N
R3
N
R3
R3
R1
R1
NH2
R2
R1
R2
R3
R2 R3 R4
5a (R1=Me)
5b (R1=Ph)
7l
4-Cl-Ph
OMe
F
NO2
Cl
Cl Me
H
H
H
H
H
H
OMe
F
Cl
H
OMe
OH
F
NO2
Cl
H
7a
7b
7c
7d
Me
Me
Me
Me
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
Me
Me
Et
6a
H
7m 4-Cl-Ph
7n 4-Cl-Ph
7o 4-Cl-Ph
7p 4-Cl-Ph
7q 4-MeO-Ph
5c (R1=4-ClPh)
H
H
H
H
6b OMe
5d (R1=4-MeOPh)
6c
F
(iii)
(iii)
6d NO2
Et
Et
7e
6e
Cl
H
Me
H
H
H
7f
7g
7h
7i
H
H
H
H
H
H
(ii)
7r 4-MeO-Ph
H
7s
4-MeO-Ph OMe
4-MeO-Ph
7u 4-MeO-Ph Cl
7t
F
7j
7k
4-Cl-Ph
H
aReagents and conditions: (i) NaOEt, EtOH, reflux; (ii) BBr3, CH2Cl2; (iii) NaH, MeI, DMF.
Scheme 1. Reagents and conditions: (i) NaOEt, EtOH, reflux; (ii) BBr3, CH2Cl2; (iii) NaH, MeI, DMF.
less active than the previously reported lead compound 1 (IC50
1.2 M), however a number of active compounds from the present
series with low micromolar IC50 values compared favourably with
compounds 7p and 7r, where R3 ¼ methyl. Compounds 7p and 7r
were amongst the least active in the series, implying (albeit in this
limited study) that methyl substitution at the indole nitrogen
decreases activity.
m
compound 1 in the MiaPaCa-2, MCF-7 and Jurkat cancer cell lines
where compound 1 was found to be inactive (IC50 > 50 mM).
In general, structureeactivity relationships for cell growth
inhibition are difficult to determine within the indolyl-oxadiazole
series, and analyses of activity need to be examined individually on
each cell line to give meaningful SAR insights. The most active
compounds overall in the most responsive COLO 320 and MIA
PaCa-2 cell lines were found to be compounds 7n and 7u.
3.4. In vitro assessment of cardiac cell viability
Potential side effects of test compounds could arise by affecting
mitochondria in non-cancerous tissues, particularly those charac-
terised by high abundance of mitochondria (e.g. cardiomyocytes).
Therefore, the effect of test compounds was evaluated over 72 h
against the H9c2 cardiac muscle cell line at the IC50 values deter-
mined on COLO320 cells (i.e. the most responsive cells to test
compounds). The data presented in Fig. 2 suggests that indolyl-
oxadiazole derivatives may exhibit some selectivity for cancer cells
since, for all compounds except 7o, cardiac cell viability was
significantly higher than 50% (P < 0.05). Similarly, compound 1 was
also found to exhibit no substantial effects on cardiac cell viability
at the COLO 320 cell IC50 concentration. These results appear to be
in line with evidence demonstrating that mitochondriotoxic effects
at the tumour site may occur in the presence of minimal side effects
and a complete lack of cardiotoxicity [22], probably due to differ-
ences in the mitochondrial contribution to the cancer cell physi-
ology compared to their normal tissue counterparts.
3.2. Effect of R1 (oxadiazole 3-substituent)
In the COLO 320 cell line, the methyl R1 substituent (compounds
7aed) gave some of the most potent activity in the series (IC50 of
10e20
m
M). SAR in the R1 ¼ (substituted)phenyl series was more
variable and difficult to determine, where compounds 7n and 7u
(R1 ¼ 4-Cl-Ph and 4-MeO-Ph respectively) were the most active
compounds in this series. Similarly SAR’s for different R1 substitu-
ents in the MIA PaCa-2 cell line were more difficult to determine.
3.3. Effect of R2 and R3 (indole substituents)
The data clearly demonstrates that both electron-withdrawing
groups (e.g. R2 ¼ NO2, compound 7n) and electron-donating groups
(e.g. R2 ¼ OMe, compounds 7l, 7s) are tolerated on the indole
5-position with respect to potency in COLO 320 and MIA PaCa-2 cell
lines. The specific effect of different R2 substituents is difficult to
determine, with related compounds with the same R2 group giving
quite different results (e.g. compound 7u is the most active of the
series in COLO 320 and MIA PaCa-2, whereas the close analogue 7o
is amongst the least active). For most compounds in the series
the indole nitrogen was unsubstituted, with the exception of
3.5. In vitro apoptosis induction
Since the cytotoxicity assay did not indicate a specific cellular
death mechanism, we investigated the ability of selected
compounds to induce internucleosomal degradation of genomic
DNA (a hallmark of apoptosis) against sensitive cancer cell lines.
Overall, the exposure of COLO 320 cells to compounds resulted
in apoptotic cell death; in particular, the active compounds, 7g and
7h, displayed similar abilities to induce DNA degradation and
Scheme 2. Reagents and conditions: (i) H2NeCN, NaH, DMF; (ii) NH2OH$HCl, pyridine/EtOH.

4526
N.I. Ziedan et al. / European Journal of Medicinal Chemistry 45 (2010) 4523e4530
Table 1
Table 2
Activity of new substituted indolyl-oxadiazoles 6aeu and 9 against human cancer
Induction of internucleosomal DNA fragmentation (enrichment factor) after treat-
cell lines.a
ment with new substituted indolyl-oxadiazoles in the sensitive cell lines.a
IC50 values (
Compd.
m
M)b in cell linesc
COLO 320
Compd.
Cell line
MIA PaCa-2
MCF-7
Jurkat
COLO 320
MIA PaCa-2
1
1.2 ꢀ 0.13
11.8 ꢀ 0.8
10.4 ꢀ 1.0
18.1 ꢀ 1.2
19.5 ꢀ 1.0
12.0 ꢀ 0.9
18.7 ꢀ 0.7
15.7 ꢀ 0.5
10.8 ꢀ 1.2
12.5 ꢀ 1.0
16.5 ꢀ 1.1
13.9 ꢀ 1.3
12.8 ꢀ 1.2
>50
7.7 ꢀ 1.0
25.0 ꢀ 0.8
16.9 ꢀ 0.9
27.7 ꢀ 0.9
>50
28.3 ꢀ 1.2
14.8 ꢀ 1.0
9.6 ꢀ 1.0
27.7 ꢀ 1.1
>50
>50
>50
>50
>50
19.9 ꢀ 1.0
22.9 ꢀ 1.2
21.6 ꢀ 1.3
>50
7c
7e
7g
7h
7j
7k
7l
7n
7s
7t
1.0 ꢀ 0.2
2.5 ꢀ 0.1
4.3 ꢀ 1.8
5.3 ꢀ 0.6
2.1 ꢀ 0.6
3.0 ꢀ 0.7
1.8 ꢀ 0.3
1.8 ꢀ 0.2
1.2 ꢀ 0.3
1.4 ꢀ 0.4
1.2 ꢀ 0.2
7.3 ꢀ 0.8
1.4 ꢀ 0.1
7.3 ꢀ 0.8
6.9 ꢀ 0.3
1.9 ꢀ 0.1
1.0 ꢀ 0.1
1.2 ꢀ 0.2
2.7 ꢀ 0.9
1.9 ꢀ 0.1
2.2 ꢀ 0.3
1.4 ꢀ 0.1
7a
7b
7c
7d
7e
7f
7g
7h
7i
30.5 ꢀ 1.0
>50
16.7 ꢀ 1.0
12.4 ꢀ 1.0
34.4 ꢀ 1.4
45.7 ꢀ 2.2
>50
26.0 ꢀ 1.7
35.0 ꢀ 2.1
>50
19.4 ꢀ 1.0
>50
22.0 ꢀ 1.0
32.6 ꢀ 1.1
13.0 ꢀ 1.2
33.6 ꢀ 1.1
>50
23.1 ꢀ 1.3
19.2 ꢀ 1.2
22.7 ꢀ 1.0
>50
>50
26.9 ꢀ 1.5
33.8 ꢀ 1.2
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
7j
7k
7l
29.1 ꢀ 1.9
7u
>50
a
Determined by Cell Death Detection ELISA assay (100
m
M for 24 h drug expo-
>50
>50
>50
>50
>50
>50
>50
>50
sure); see Biological Experimental for details. Compounds were tested in duplicate,
data expressed as mean ꢀ SEM.
7m
7n
7o
7p
7q
7r
7s
7t
17.6 ꢀ 1.1
>50
>50
>50
anticancer action. Otherwise, while the compound 7c was inactive
in the COLO 320 cell line, it was able to consistently induce
apoptosis in the MIA PaCa-2 cells (Table 2). Treatment of MIA PaCa-
2 cells with compounds 7j, 7n, 7s, and 7t was associated with an
increase in the extent of DNA fragmentation from 1.9 to 2.7-fold,
while the effect of compounds 7e, 7k, 7l and 7u was negligible
(Table 2).
An intriguing aspect of this work is that some of test compounds
that were found to be very active in the cytotoxicity assay (such as
7u), did not induce apoptosis in MIA PaCa-2 cells. To clarify this
apparent discrepancy, we evaluated whether the compound 7u
(taken as a paradigmatic example) was able to promote necrosis
23.1 ꢀ 1.0
5.6 ꢀ 1.2
10.3 ꢀ 1.0
7.8 ꢀ 1.0
18.7 ꢀ 1.1
>50
>50
>50
7u
10
a
Determined by WST-1 assay (72 h drug exposure), see Biological Experimental
for details.
b
Compounds tested in triplicate, data expressed as mean values of independent
experiments ꢀ standard error of the mean (SEM).
c
Cancer cell line origin: COLO 320 (colon), MIA PaCa-2 (pancreas), MCF-7 (breast;
ER þve), Jurkat (leukemia). When the IC50 was not reached at 50
mM, compounds
were considered to be inactive.
against MIA PaCa-2 cells. As a matter of fact, when given at 100 mM
for 24 h, compound 7u induced plasma membrane ruptures with
the consequent accumulation of oligonucleosomes in the super-
natant (necrosis), but not in the cytoplasmic fraction (apoptosis) of
MIA PaCa-2 cells (Fig. 3).
formation of oligonucleosomal fragments (4.3-fold and 5.3-fold
respectively over control), whereas the amount of apoptotic DNA
generated upon exposure to compounds 7e, 7j, 7k, 7l and 7n to
COLO 320 cells was from 1.8-fold to 3.0-fold higher than that of
untreated control. On the contrary, the production of cytoplasmic
oligonucleosomes induced by compounds 7c, 7s, 7t and 7u was
negligible (Table 2). Notably, as observed for COLO 320 cells,
treatment of MIA PaCa-2 cells with compounds 7g and 7h signifi-
cantly increased the amount of cytoplasmic histone-associated
DNA fragments (7.3-fold and 6.9-fold respectively over control)
suggesting that apoptosis may be a major contributor to their
Fig. 2. H9c2 cardiac cell viability following 72 h exposure to test compound concen-
trations equal to their IC50 mean values obtained in the most responsive human cancer
cell line (COLO 320). Data are the mean of three experiments and are reported as
mean ꢀ standard error of the mean (SEM).
Fig. 3. Enrichment of nucleosomes in the cytoplasm (white bar) or in the supernatant
(black bar) of MIA PaCa-2 cells treated with the compound 7u at 100 mM for 24 h.

N.I. Ziedan et al. / European Journal of Medicinal Chemistry 45 (2010) 4523e4530
4527
To explore in more detail the underlying mechanism of
apoptosis induced by the indolyl-oxadiazole compounds, we
assessed whether caspase-3 and/or caspase-7, two enzymes
involved in the effector phase of apoptosis, were a target of the
new indolyl-substituted oxadiazole compounds. Caspase activation
(2.1- to 6.4-fold induction) was consistently observed after COLO
320 cells were incubated with compounds 7e, 7g, 7h, 7l, 7n, 7t and
7u (Table 3). Of note, for most test compounds, such an effect was
associated with DNA fragmentation (see Table 2) suggesting that, at
least in this specific cell type, caspase activation may contribute to
their pro-apoptotic action. Results in Table 3 show that caspase-3/7
activity was marginally altered in MIA PaCa-2 cells after treatment
with the pro-apoptotic compounds 7c, 7e, 7g, 7h, 7k and 7u;
suggesting that apoptosis may also occur through a caspase-inde-
pendent pathway, a mechanism already observed in human
pancreatic adenocarcinoma cell lines [23]. Therefore, in the
pancreatic cancer cell line, MIA PaCa-2, the analysis of caspase-3/7
activity was not able to entirely predict the occurrence of apoptosis;
accordingly, the compound 7l significantly induced caspase activity
without DNA fragmentation (see Table 2). This notion seems to be
in line with data showing that, in the absence of apoptotic stimuli,
tumour cells with high levels of the anti-apoptotic proteins, sur-
vivin and XIAP, such as MIA PaCa-2, displayed a low sensitivity to
apoptosis induction by overexpression of caspase genes [24].
However, when the activity of caspase-3/7 was markedly increased
in MIA PaCa-2 cells (see 7n and 7t; Table 3), such an effect was
associated with a modest but significant induction of apoptotic cell
death (Table 2).
a phenyl substituent at the R1 position (7g and 7h) were able to
induce apoptotic cell death in both COLO 320 and MIA PaCa-2 cell
lines, whereas the structurally-related compounds 7e and 7k
induced apoptosis only in COLO 320 cells.
Some of the test compounds were found to be active in
promoting apoptosis via activation of caspase-3/7, most notably
compounds 7l (R1 ¼ 4-Cl-Ph; R2 ¼ OMe), 7n (R1 ¼ 4-Cl-Ph;
R2 ¼ NO2) and 7t (R1 ¼ 4-MeO-Ph; R2 ¼ F) or through a caspase-
independent pathway, depending on the cell type and compound
examined. For example, cell necrosis was demonstrated for the
compound 7u (R1 ¼ 4-MeO-Ph; R2 ¼ Cl) and this type of cell death
may be also taken into account to interpret the cytotoxic effects of
other compounds. These results are relevant considering that
development of resistant mechanisms for apoptosis confers high
survival ability and low drug sensitivity of tumour cells.
5. Experimental
5.1. Chemistry
Melting points were measured on a Griffin apparatus and are
uncorrected. NMR spectra were recorded on a Bruker AVANCE
500 MHz instrument; coupling constants (J values) are in Hz. Merck
silica gel 60 (40e60 mM) was used for column chromatography. All
commercially available starting materials were used without
further purification, and purchase/synthesis of the required ami-
doxime (5aed) and indolyl ester (6aee) starting materials was
carried out according to well established literature methods as
described in the chemistry results section. Compound 1 was syn-
thesised as previously described [7]. The purity of tested
compounds was found to be ꢁ95% as determined by combustion
analysis (% C, H, N values with ꢀ0.4% of theoretical; carried out in
duplicate by Medac Ltd, U.K., www.medacltd.com) in the case of
new compounds 7a-7u, and accurate mass spectrometry (EPSRC
National Mass Spectrometry Centre, Swansea, U.K.) in the case of
the 3-aminooxadiazole 10.
4. Conclusions
Antitumour data from the present study demonstrates in vitro
anticancer effects of new indole-based 3,5-substituted oxadiazoles,
particularly against cancer cell lines derived from colon (COLO 320)
and pancreatic (MiaPaCa-2) tissues. The new compounds were
found to be both synthetically accessible in one key chemical step,
and amenable to generation of a small library of compounds for
structureeactivity analysis. A number of compounds were found to
have IC50 values in the low micromolar range (notably 7n and 7u),
and corresponding tests on the H9c2 cardiac muscle cell line
(characterised by a high abundance of mitochondria) revealed
selectivity for cancer cell lines in most cases.
5.1.1. General method for the synthesis of 3-substituted-5-(5-
substituted-1H-indol-2-yl)-1,2,4-oxadiazoles (7aef, 7heo, 7q and
7seu)
To a stirred solution of sodium ethoxide in ethanol (2.6 mL of
21% solution in ethanol, 8 mmol) was added amidoxime (5aed,
4 mmol). The mixture was stirred at room temperature for 30 min,
then indole-2-carboxylate (6aee, 4 mmol) was added and the
mixture was heated under reflux for 2e5 h. The reaction mixture
was left to cool to room temperature, then H2O was added, and the
precipitate formed was collected by filtration and recrystallised
from ethanol to give pure indolyl-oxadiazole product in 8e48%
yield.
Through structureeactivity relationship studies of indole-based
3,5-disubstituted oxadiazoles, we found in particular that certain
compounds characterised by an unsubstituted indole nitrogen and
Table 3
Induction of caspase-3/7 activity (fold change over control values) after treatment
with new substituted indolyl-oxadiazoles in the sensitive cell lines.a
5.1.1.1. 5-(Indol-2-yl)-3-methyl-1,2,4-oxadiazole (7a). Yield 11%;
Compd.
Cell line
mp 121e122 ꢂC. 1H NMR (CDCl3)
d 2.47 (3H, s, CH3), 7.12 (1H, td,
COLO 320
MIA PaCa-2
J ¼ 7.5, 0.9 Hz, Ar-H), 7.28 (1H, td, J ¼ 7.7, 1.1 Hz, Ar-H), 7.31
(1H, dd, J ¼ 7.1, 0.9 Hz, Ar-H), 7.38 (1H, dd, J ¼ 8.3, 0.9 Hz, Ar-H), 7.65
(1H, dd, J ¼ 8.15, 0.80 Hz, Ar-H), 8.98 (1H, s, NH). 13C NMR (CDCl3)
7c
7e
7g
7h
7j
7k
7l
7n
7s
7t
1.5 ꢀ 0.2
3.1 ꢀ 0.1
3.6 ꢀ 0.1
4.4 ꢀ 0.6
1.3 ꢀ 0.1
0.3 ꢀ 0.1
4.5 ꢀ 0.1
6.4 ꢀ 0.2
1.1 ꢀ 0.2
2.1 ꢀ 0.3
4.2 ꢀ 0.4
1.3 ꢀ 0.2
1.2 ꢀ 0.3
0.7 ꢀ 0.1
1.2 ꢀ 0.3
1.9 ꢀ 0.4
1.2 ꢀ 0.1
10.1 ꢀ 1.7
23.9 ꢀ 6.4
2.0 ꢀ 0.4
5.0 ꢀ 1.6
1.4 ꢀ 0.6
d
11.9 (CH3), 108.19, 111.8, 121.3, 122.4, 125.7 (C3, C4, C5, C6, C7 of
indole), 121.5, 127.8, 137.4 (C2, C3a, C7a of indole), 167.4, 169.8 (C3,
C5 of oxadiazole). Anal. calcd for C11H9N3O: C, 66.32; H, 4.55;
N, 21.09. Found: C, 66.13; H, 4.54; N, 21.09.
5.1.1.2. 5-(5-Methoxyindol-2-yl)-3-methyl-1,2,4-oxadiazole(7b). Yield
13%; mp 127e130 ꢂC. 1H NMR (CDCl3)
d 2.48 (3H, s, CH3), 3.90 (3H,
7u
s, OCH3), 7.02 (1H, dd, J ¼ 8.8, 2.5 Hz, Ar-H), 7.10 (1H, s, Ar-H), 7.25
a
Determined by Apo-ONE Homogeneous Caspase-3/7 assay (100 mM for 24 h
(1H, d, J ¼ 2.9 Hz, Ar-H), 7.32 (1H, d, J ¼ 8.8 Hz, Ar-H), 9.05 (1H, s, NH).
drug exposure); see Biological Experimental for details. Compounds were tested in
duplicate, data expressed as mean ꢀ SEM.
13C NMR (CDCl3)
d 11.6 (CH3), 55.5 (OCH3), 102.4, 107.7, 112.7, 117.3

4528
N.I. Ziedan et al. / European Journal of Medicinal Chemistry 45 (2010) 4523e4530
(C3, C4, C6, C7 of indole), 121.9, 128.3, 132.8, 155.1 (C2, C3a, C5, C7a of
indole),167.4,169.7 (C3, C5 of oxadiazole). Anal. calcd for C12H11N3O2:
C, 62.87; H, 4.84; N, 18.33. Found: C, 62.67; H, 4.87; N, 18.17.
5.1.1.9. 5-(5-Chloroindol-2-yl)-3-phenyl-1,2,4-oxadiazole (7j). Yield
45%. mp 184e185 ꢂC. 1H NMR (CDCl3)
d
7.32 (1H, dd, J ¼ 8.8, 2.1 Hz,
Ar-H), 7.38 (1H, dd, J ¼ 2.1, 0.9 Hz, Ar-H), 7.41 (1H, d, J ¼ 8.8 Hz,
Ar-H), 7.50e7.56 (3H, m, Ar-H), 7.72 (1H, d, J ¼ 2.0 Hz, Ar-H), 8.15
5.1.1.3. 5-(5-Fluoroindol-2-yl)-3-methyl-1,2,4-oxadiazole (7c). Yield
(2H, m, Ar-H), 9.15 (1H, s, NH). 13C NMR (CDCl3)
d 107.5, 112.9, 121.6,
15%; mp 148e149 ꢂC. 1H NMR (DMSO-d6)
d
2.45 (3H, s, CH3),
7.15e7.18 (1H, m, Ar-H), 7.34 (1H, s, Ar-H), 7.46e7.50 (2H, m, Ar-
H), 12.5 (1H, s, NH). 13C NMR (DMSO-d6)
11.1 (CH3) 105.9, 106.8,
126.3 (C3, C4, C6, C7 of indole), 127.6, 129.0, 131.4 (C2, C3, C4, C5, C6
of phenyl), 122.8, 126.5, 127.1, 128.8, 135.7 (C2, C3a, C5, C7a of índole
and C1 of phenyl), 168.7, 169.5 (C3, C5 of oxadiazole). Anal. calcd for
C16H10ClN3O: C, 64.98; H, 3.41; N, 14.21. Found: C, 65.05; H, 3.27;
N, 14.13.
d
113.7, 113.9 (C3, C4, C6, C7 of indole), 122.8, 127.2, 134.8, 156.5, 158.4
(C2, C3a, C5, C7a of indole), 167.5, 169.2 (C3, C5 of oxadiazole).
Anal. calcd for C11H8FN3O: C, 60.83; H, 3.71; N 19.35. Found:
C, 60.79; H, 3.61; N, 18.87.
5.1.1.10. 3-(4-Chlorophenyl)-5-(indol-2-yl)-1,2,4-oxadiazole (7k). Yield
41%. mp 188e189 ꢂC. 1H NMR (DMSO-d6)
d
7.15 (1H, t, J ¼ 7.5 Hz,
5.1.1.4. 5-(5-Chloroindol-2-yl)-3-methyl-1,2,4-oxadiazole (7d). Yield
Ar-H), 7.33 (1H, t, J ¼ 7.7 Hz, Ar-H), 7.34 (1H, d, J ¼ 8.3 Hz, Ar-H),
7.49 (1H, s, Ar-H), 7.70 (1H, dd, J ¼ 6.8, 1.9 Hz, Ar-H), 7.75 (1H, d,
J ¼ 8.1 Hz, Ar-H), 8.12 (2H, dd, J ¼ 6.8, 1.9 Hz, Ar-H), 12.50 (1H, s,
14%; mp 174e175 ꢂC. 1H NMR (CDCl3)
d
2.50 (3H, s, CH3),
7.28e7.31 (2H, m, Ar-H), 7.37 (1H, d, J ¼ 8.8 Hz, Ar-H), 7.69 (1H, s,
Ar-H), 9.13 (1H, s, NH). 13C NMR (CDCl3)
11.6 (CH3), 107.4, 112.9,
d
NH). 13C NMR (DMSO-d6)
d 107.7, 112.6, 120.7, 121.9, 125.2 (C3, C4,
121.6, 126.2 (C3, C4, C6, C7 of indole), 121.7, 127.1, 128.8, 135.6 (C2,
C3a, C5, C7a of indole), 167.5, 169.8 (C3, C5 of oxadiazole). Anal.
calcd for C11H8ClN3O: C, 56.54; H, 3.45; N, 17.98. Found: C, 56.28;
H, 3.34; N, 17.72.
C5, C6, C7 of indole), 128.9, 129.7 (C2, C3, C5, C6 of phenyl), 120.9,
127.2, 138.4 (C2, C3a, C7a of indole), 125.0 (C1 of phenyl), 136.4 (C4
of phenyl), 167.3, 170.4 (C3, C5 of oxadiazole). Anal. calcd for
C16H10ClN3O: C, 64.98; H, 3.41; N, 14.21. Found: C, 64.85; H, 3.41;
N, 13.79.
5.1.1.5. 5-(Indol-2-yl)-3-phenyl-1,2,4-oxadiazole (7e). Yield 42%. mp
168e169 ꢂC. 1H NMR (DMSO-d6)
d
7.15 (1H, td, J ¼ 7.6, 0.9 Hz, Ar-H),
5.1.1.11. 3-(4-Chlorophenyl)-5-(5-methoxyindol-2-yl)-1,2,4-oxadiazole
7.33 (1H, td, J ¼ 7.6, 1.0 Hz, Ar-H), 7.49 (1H, d, J ¼ 1.1 Hz, Ar-H), 7.55
(1H, dd, J ¼ 8.3, 0.9 Hz, Ar-H), 7.61e7.67 (3H, m, Ar-H), 7.74 (1H, d,
J ¼ 8.0 Hz, Ar-H), 8.11e8.13 (2H, m, Ar-H),12.49 (1H, s, NH). 13C NMR
(7l). Yield 35%; mp 184e185 ꢂC. 1H NMR (DMSO-d6)
d 3.83 (3H, s,
OCH3), 7.00 (1H, dd, J ¼ 8.9, 2.5 Hz, Ar-H), 7.18 (1H, d, J ¼ 2.3 Hz, Ar-
H), 7.38 (1H, s, Ar-H), 7.43 (1H, d, J ¼ 8.9 Hz, Ar-H), 7.70 (2H, dd,
J ¼ 6.7, 1.8 Hz, Ar-H), 8.11 (2H, dd, J ¼ 6.7, 1.8 Hz, Ar-H), 12.40 (1H, s,
(DMSO-d6)
d
107.5, 112.6, 120.7, 122.0, 125.9 (C3, C4, C5, C6, C07 of
indole), 127.2, 129.3, 131.7 (C2, C3, C4, C5, C6 of phenyl), 121.0, 127.1,
138.2 (C2, C3a, C7a of indole), 126.1 (C1 of phenyl), 168.1, 170.2 (C3,
C5 of oxadiazole). Anal. calcd for C16H11N3O: C, 73.55; H, 4.24; N,
16.08. Found: C, 73.38; H, 4.07; N, 15.98.
NH). 13C NMR (DMSO-d6)
d 55.2 (OCH3), 101.8, 107.2, 113.5, 116.8 (C3,
C4, C6, C7 of indole), 128.9, 129.3 (C2, C3, C5, C6 of phenyl), 121.0,
127.5, 133.6, 154.3 (C2, C3a, C5, C7a of indole), 125.0 (C1 of phenyl),
136.3 (C4 of phenyl), 167.3, 170.3 (C3, C5 of oxadiazole). Anal. calcd
for C17H12ClN3O2: C, 62.68; H, 3.71; N,12.90. Found: C, 62.40; H, 3.58;
N, 12.64.
5.1.1.6. 5-(5-Methoxyindol-2-yl)-3-phenyl-1,2,4-oxadiazole (7f). Yield
37%; mp 147e149 ꢂC. 1H NMR (CDCl3)
d 3.89 (3H, s, OCH3), 7.03
(1H, dd, J ¼ 9.1, 2.5 Hz, Ar-H), 7.12 (1H, d, J ¼ 2.4 Hz, Ar-H), 7.25
5.1.1.12. 3-(4-Chlorophenyl)-5-(5-fluoroindol-2-yl)-1,2,4-oxadiazole
(1H, s, Ar-H), 7.35 (1H, m, Ar-H), 7.48e7.54 (3H, m, Ar-H), 8.15 (2H,
(7m). Yield 15%; mp 225e226 ꢂC. 1H NMR (DMSO-d6)
d 7.20
m, Ar-H), 9.02 (1H, s, NH). 13C NMR (CDCl3)
d
55.7 (OCH3), 102.5,
(1H, td, J ¼ 9.3, 2.6 Hz, Ar-H), 7.47 (1H, s, Ar-H), 7.51 (1H, dd, J ¼ 9.0,
2.5 Hz, Ar-H), 7.55 (1H, q, J ¼ 4.6 Hz, Ar-H), 7.71 (2H, dd, J ¼ 6.9,
1.9 Hz, Ar-H), 8.12 (2H, dd, J ¼ 6.7, 1.9 Hz, Ar-H), 12.62 (1H, s, NH).
107.8, 112.7, 117.4 (C3, C4, C6, C7 of indole), 127.5, 128.9, 131.3
(C2, C3, C4, C5, C6 of phenyl), 121.5, 128.4, 132.8, 155.1 (C2, C3a, C5,
C7a of indole), 126.7 (C1 of phenyl), 168.6, 169.9 (C3, C5 of oxa-
diazole). Anal. calcd for C17H13N3O2: C, 70.09; H, 4.05; N, 14.42.
Found: C, 69.90; N, 4.37; N, 14.37.
13C NMR (CDCl3)
d 105.0, 107.5, 113.7, 114.8 (C3, C4, C6, C7 of
indole), 127.2, 128.9 (C2, C3, C5, C6 of phenyl), 122.5, 127.2, 135.0,
156.5, 158.4 (C2, C3a, C5, C7a of indole), 124.9 (C1 of phenyl), 136.4
(C4 of phenyl), 167.3, 170.1 (C3, C5 of oxadiazole). Anal. calcd
for C16H9FClN3O: C, 61.26; H, 2.89; N, 13.39. Found: C, 61.01;
H, 2.80; N, 13.17.
5.1.1.7. 5-(5-Fluoroindol-2-yl)-3-phenyl-1,2,4-oxadiazole (7h). Yield
36%; mp 169e170 ꢂC. 1H NMR (DMSO-d6)
d
7.20 (1H, td, J ¼ 9.3,
2.6 Hz, Ar-H), 7.45 (1H, s, Ar-H), 7.50 (1H, dd, J ¼ 9.7, 2.5 Hz, Ar-H),
7.55 (1H, dd, J ¼ 9.0, 4.6 Hz, Ar-H), 7.60e7.66 (3H, m, Ar-H), 8.11
5.1.1.13. 3-(4-Chlorophenyl)-5-(5-nitroindol-2-yl)-1,2,4-oxadiazole
(2H, m, Ar-H), 12.60 (1H, s, NH). 13C NMR (CDCl3)
d
106.8, 108.1,
(7n). Yield 27%; mp 257e259 ꢂC. 1H NMR (CDCl3)
d 7.51
112.8, 114.8 (C3, C4, C6, C7 of indole), 127.6, 129.0, 131.5 (C2, C3,
C4, C5, C6 of phenyl), 122.6, 127.3, 131.9, 156.5, 158.4 (C2, C3a, C5,
C7a of indole), 126.0 (C1 of phenyl), 168.2, 169.9 (C3, C5 of oxa-
diazole). Anal. calcd for C16H10FN3O: C, 68.81; H, 3.61; N, 15.05.
Found: C, 68.73; H, 3.52; N, 14.79.
(2H, dd, J ¼ 8.4, 1.8 Hz, Ar-H), 7.58 (1H, d, J ¼ 9.1 Hz, Ar-H), 7.62 (1H,
s, Ar-H), 8.10 (2H, dd, J ¼ 8.1, 1.8 Hz, Ar-H), 8.28 (1H, dt, J ¼ 9.1,
2.0 Hz, Ar-H), 8.74 (1H, s, Ar-H), 9.48 (1H, s, NH). 13C NMR (CDCl3)
d
109.9, 113.3, 119.4, 119.8 (C3, C4, C6, C7 of indole), 129.0, 129.5 (C2,
C3, C5, C6 of phenyl), 124.8, 136.5 (C1, C4 of phenyl), 126.4, 141.0,
141.8 (C2, C3a, C5, C7a of ndole),167.5,169.6 (C3, C5 of oxadiazole).
n
5.1.1.8. 5-(5-Nitroindol-2-yl)-3-phenyl-1,2,4-oxadiazole (7i). Yield
Anal. calcd for C16H9ClN4O3.H2O: C, 53.57; H, 3.09; N, 15.62. Found:
C, 53.14; H, 3.40; N, 15.15.
39%; mp 240e241 ꢂC. 1H NMR (DMSO-d6)
d 7.67e7.73 (3H, m, Ar-H),
7.75 (1H, d, J ¼ 9.1 Hz, Ar-H), 7.82 (1H, s, Ar-H), 8.17 (2H, m, Ar-H),
8.28 (1H, dd, J ¼ 9.1, 2.4 Hz, Ar-H), 8.84 (1H, d, J ¼ 2.3 Hz, Ar-H), 13.3
5.1.1.14. 5-(5-Chloroindol-2-yl)-3-(4-chlorophenyl)-1,2,4-oxadiazole
(1H, s, NH). 13C NMR (CDCl3)
d
109.7, 112.1, 119.6, 120.9 (C3, C4, C6,
(7o). Yield 47%; mp 212e214 ꢂC. 1H NMR (DMSO-d6)
d 7.33 (1H, dd,
C7 of indole), 127.6, 129.0, 133.4 (C2, C3, C4, C5, C6 of phenyl), 124.6,
125.9, 140.8, 141.8 84 (C2, C3a, C5, C7a of índole), 126.43 (C1 of
phenyl), 168.3, 169.5 (C3, C5 of oxadiazole). Anal. calcd for
C16H10N4O3.0.67H2O: C, 60.30; H, 3.69; N, 17.58. Found: C, 60.09; H,
3.58; N, 17.93.
J ¼ 8.8, 2.1 Hz, Ar-H), 7.46 (1H, s, Ar-H), 7.54 (1H, d, J ¼ 8.8 Hz, Ar-H),
7.70 (2H, dd, J ¼ 6.7, 2.0 Hz, Ar-H), 7.81 (1H, d, J ¼ 1.9 Hz, Ar-H), 8.15
(2H, dd, J ¼ 6.7, 2.0 Hz, Ar-H), 12.70 (1H, s, NH). 13C NMR (DMSO-d6)
d
107.1, 114.2, 121.0, 125.2 (C3, C4, C6, C7 of indole), 128.9, 129.5 (C2,
C3, C5, C6 of phenyl), 122.6, 125.2, 128.1, 136.4 (C2, C3a, C5, C7a of

N.I. Ziedan et al. / European Journal of Medicinal Chemistry 45 (2010) 4523e4530
4529
indole), 124.9 (C1 of phenyl), 136.6 (C4 of phenyl), 167.3, 170.4 (C3,
C5 of oxadiazole). Anal. calcd for C16H9Cl2N3O: C, 58.20; H, 2.75;
N, 12.73. Found: C, 58.27; H, 2.59; N, 12.63.
Ar-H), 7.60 (3H, m, Ar-H), 8.11 (2H, d, J ¼ 7.8 Hz, Ar-H), 9.03
(1H, s, OH), 12.50 (1H, s, NH). 13C NMR (DMSO-d6)
104.3, 106.5,
d
113.1, 116.6 (C3, C4, C6, C7 of indole), 127.1, 129.2, 131.6 (C2, C3, C4,
C5, C6 of phenyl), 121.5, 128.4, 132.8, 155.1 (C2, C3a, C5, C7a of
indole), 126.7 (C1 of phenyl), 168.6, 169.9 (C3, C5 of oxadiazole).
Anal. calcd for C16H11N3O2.H2O: C, 65.08; H, 4.44; N, 14.23. Found:
C, 64.92; H, 4.36; N, 14.40.
5.1.1.15. 5-(Indol-2-yl)-3-(4-methoxyphenyl)-1,2,4-oxadiazole (7q).
Yield 39%; mp 157e159 ꢂC. 1H NMR (CDCl3)
d 3.90 (3H, s, OCH3), 7.02
(2H, m, Ar-H), 7.20 (1H, td, J ¼ 7.6, 0.9 Hz, Ar-H), 7.37 (1H, td, J ¼ 7.7,
1.1 Hz, Ar-H), 7.44 (1H, dd, J ¼ 2.1, 0.9 Hz, Ar-H) 7.49 (1H, dd, J ¼ 8.3,
0.9 Hz, Ar-H), 7.74 (1H, dd, J ¼ 8.1, 0.8 Hz, Ar-H), 8.12 (2H, m, Ar-H),
5.1.3. Synthesis of N-methylated indolyl-oxadiazoles (7p and 7r)
Sodium hydride (24.3 mg, 1 mmol) was added to a stirred
solution of the 1H-indolyl-oxadiazole precursor 7o or 7q
(1 mmol) in dry DMF (5 mL) under N2 atmosphere at 5 ꢂC. The
reaction mixture was stirred at 5 ꢂC for 30 min, then iodo-
methane (0.17 g, 1.2 mmol) was added and the mixture stirred at
ambient temperature for 8 h. Water (50 mL) was added and the
mixture extracted using ethyl acetate (3 ꢃ 50 mL). The organic
extracts were dried (MgSO4) and concentrated in vacuo. Recrys-
tallisation of the residue from ethanol afforded the required
product 7p or 7r.
9.09 (1H, s, NH). 13C NMR (CDCl3)
d 55.4 (OCH3), 108.1, 111.8, 121.3,
122.4, 125.7 (C3, C4, C5, C6, C7 of indole), 114.3, 129.2 (C2, C3, C5, C6
of phenyl), 121.7, 127.9, 137.4 (C2, C3a, C7a of índole), 119.1, 162.1 (C1,
C4 of phenyl), 168.3, 169.7 (C3, C5 of oxadiazole). Anal. calcd for
C17H13N3O2: C, 70.09; H, 4.05; N, 14.42. Found: C, 69.81; H, 4.39; N.
14.30.
5.1.1.16. 5-(5-Methoxyindol-2-yl)-3-(4-methoxyphenyl)-1,2,4-oxadia-
zole (7s). Yield 27%; mp 174e176 ꢂC. 1H NMR (DMSO-d6)
d 3.90 (3H,
s, OCH3), 3.95 (3H, s, OCH3), 7.03 (1H, dd, J ¼ 9.0, 2.5 Hz, Ar-H), 7.22
(1H, d, J ¼ 2.4 Hz, Ar-H), 7.41 (1H, s, Ar-H), 7.48 (1H, d, J ¼ 8.9 Hz, Ar-
H), 7.24 (2H, d, J ¼ 8.9 Hz, Ar-H), 8.10 (2H, d, J ¼ 8.9 Hz, Ar-H), 12.35
5.1.3.1. 3-(4-Chlorophenyl)-5-(5-chloro-1-methylindol-2-yl)-1,2,4-oxa-
(1H, s, NH). 13C NMR (DMSO-d6)
d
55.3 (OCH3), 55.4 (OCH3), 101.9,
diazole (7p). Yield 83%; mp 182e184 ꢂC.1H NMR (CDCl3)
d 4.25 (3H, s,
106.9,113.4,116.6 (C3, C4, C6, C7 of indole),114.7,128.8 (C2, C3, C5, C6
of phenyl), 121.3, 127.6, 133.5, 154.3 (C2, C3a, C5, C7a of indole), 118.4
(C1 of phenyl), 161.8 (C4 of phenyl), 167.4, 169.3 (C3, C5 of oxadia-
zole). Anal. calcd for C18H15N3O3: C, 67.28; H, 4.71; N,13.08. Found: C,
67.14; H, 4.66; N, 12.93.
NCH3), 7.36 (1H, dd, J ¼ 8.9,1.9 Hz, Ar-H), 7.40 (1H, d, J ¼ 8.9 Hz, Ar-H),
7.45 (1H, s, Ar-H), 7.52 (2H, d, J ¼ 6.7 Hz, Ar-H), 7.72 (1H, d, J ¼ 1.8 Hz,
Ar-H), 8.15 (2H, d, J ¼ 6.7 Hz, Ar-H). 13C NMR (CDCl3)
d32.3(NCH3),
108.5,111.5,121.4,126.9 (C3, C4, C6, C7 of indole), 128.9, 129.2 (C2, C3,
C5, C6 of phenyl),122.6,125.2,128.1,136.4 (C2, C3a, C5, C7a of indole),
124.9 (C1 of phenyl), 136.6 (C4 of phenyl), 167.8, 169.2 (C3, C5 of
oxadiazole). Anal. calcd for C17H11Cl2N3O: C, 59.32; H, 3.22; N, 12.21.
Found: C, 59.12; H, 2.97; N, 11.91.
5.1.1.17. 5-(5-Fluoroindol-2-yl)-3-(4-methoxyphenyl)-1,2,4-oxadiazole
(7t). Yield 20%; mp 190e191 ꢂC.1H NMR (CDCl3)
d 3.90 (3H, s, OCH3),
7.02 (2H, dd, J ¼ 6.9, 2.1 Hz, Ar-H), 7.13 (1H, td, J ¼ 9.0, 2.5 Hz, Ar-H),
7.36 (1H, d, J ¼ 2.5 Hz, Ar-H), 7.39 (1H, m, Ar-H), 8.42 (1H, dd, J ¼ 9.0,
4.3 Hz, Ar-H), 8.09 (2H, dd, J ¼ 6.9, 2.1 Hz, Ar-H), 9.1 (1H, s, NH). 13C
5.1.3.2. 3-(4-Methoxyphenyl)-5-(1-methylindol-2-yl)-1,2,4-oxadiazole
(7r). Yield 80%; mp 128e129 ꢂC. 1H NMR (CDCl3)
d 3.90
NMR (CDCl3)
d
55.4 (OCH3), 106.7, 107.9, 112.6,114.8 (C3, C4, C6, C7 of
(3H, s, OCH3), 4.30 (3H, s, NCH3), 7.02 (2H, dd, J ¼ 6.9, 2.0 Hz, Ar-H),
7.19 (1H, td, J ¼ 7.5, 0.9 Hz, Ar-H), 7.38 (1H, td, J ¼ 7.5, 1.0 Hz,
Ar-H), 7.44 (1H, d, J ¼ 8.4 Hz, Ar-H) 7.49 (1H, s, Ar-H), 7.72
(1H, d, J ¼ 8.1 Hz, Ar-H), 8.12 (2H, dd, J ¼ 6.9, 2.0 Hz, Ar-H). 13C
indole), 114.4, 129.2 (C2, C3, C5, C6 of phenyl), 118.3, 161.8 (C1, C4 of
phenyl), 122.8, 127.3, 134.9, 156.5 (C2, C3a, C5, C7a of índole), 167.8,
169.6 (C3, C5 of oxadiazole). Anal. calcd for C17H12FN3O2: C, 66.02; H,
3.91; N, 13.59. Found: C, 65.95; H, 3.84; N, 13.42.
NMR (CDCl3)
d 32.0 (NCH3), 55.2 (OCH3), 109.0, 110.3, 120.9, 122.4,
125.1 (C3, C4, C5, C6, C7 of indole), 114.3, 129.2 (C2, C3, C5, C6 of
phenyl), 122.4, 127.0, 139.8 (C2, C3a, C7a of índole), 118.9 (C1 of
phenyl), 162.0 (C4 of phenyl), 168.2, 170.0 (C3, C5 of oxadiazole).
Anal. calcd for C18H15N3O2: C, 70.81; H, 4.95; N, 13.76. Found:
C, 70.76; H, 5.19; N, 13.34.
5.1.1.18. 5-(5-Chloroindol-2-yl)-3-(4-methoxyphenyl)-1,2,4-oxadia-
zole (7u). Yield 46%; mp 221e222 ꢂC. 1H NMR (DMSO-d6)
d 3.90
(3H, s, OCH3), 7.03 (2H, dd, J ¼ 6.5, 2.1 Hz, Ar-H), 7.31 (1H, dd, J ¼ 8.8,
2.0 Hz, Ar-H), 7.37 (1H, d, J ¼ 2.1 Hz, Ar-H), 7.42 (1H, d, J ¼ 8.8 Hz,
Ar-H), 7.72 (1H, d, J ¼ 2.0 Hz, Ar-H), 8.08 (2H, dd, J ¼ 6.9, 2.1 Hz,
Ar-H), 12.65 (1H, s, NH). 13C NMR (DMSO-d6)
d
55.4 (OCH3), 107.4,
5.1.4. Synthesis of 1H-indole-2-carbonyl cyanamide (9)
112.9, 121.6, 126.2 (C3, C4, C6, C7 of indole), 114.4, 129.2 (C2, C3, C5,
C6 of phenyl), 123.0, 127.1, 128.8, 135.7 (C2, C3a, C5, C7a of indole),
119.0 (C1 of phenyl), 162.2 (C4 of phenyl), 168.4, 169.3 (C3, C5 of
oxadiazole). Anal. calcd for C17H12ClN3O2: C, 62.68; H, 3.71;
N, 12.90. Found: C, 62.40; H, 3.58; N, 12.64.
Sodium hydride (0.49 g, 20 mmol) was added slowly to a solu-
tion of cyanamide (0.84 g, 20 mmol) in anhydrous DMF (20 mL)
under nitrogen atmosphere at 0e5 ꢂC. The mixture was stirred for
30 min at 5 ꢂC, then a solution of índole-2-carbonyl chloride (1.80 g,
10 mmol) in anhydrous DMF (10 mL) was added, followed by stir-
ring at ambient temperature for 1 h. Diethyl ether (200 mL) was
added and the resulting precipitate collected by vacuum filtration.
The filtrate was extracted with water (100 mL), and the aqueous
extract acidified to pH 2 using HCl (aq) and extracted using ethyl
acetate (3 ꢃ 100 mL). The combined organic layers were dried
(MgSO4) and concentrated in vácuo to afford the required product
in 60% yield, which was used in the next step without further
5.1.2. Synthesis of 5-(5-hydroxyindol-2-yl)-3-phenyl-1,2,4-
oxadiazole (7g)
An ice-cold solution of 5-(methoxyindol-2-yl)-3-phenyl-
1,2,4-oxadiazole 7f (0.766 g, 2.63 mmol) was slowly added to
a solution of boron tribromide (5.4 mL of 1 M in CH2Cl2, 5.4 mmol),
followed by stirring at ambient temperature overnight. The
resulting mixture was then poured onto ice (250 g) and the aqueous
phase extracted with ethyl acetate (3 ꢃ 100 mL). The combined
organic extracts were washed with water and brine (100 mL), dried
(Na2SO4) and concentrated in vacuo. The resulting solid was
washed with excess diethyl ether and dried under vacuum to give
purification. 1H NMR (DMSO-d6)
d
7.10 (1H, td, J ¼ 7.5, 0.9 Hz, Ar-H),
7.29 (1H, td, J ¼ 7.6, 1.0 Hz, Ar-H), 7.35 (1H, d, J ¼ 1.3 Hz, Ar-H), 7.46
(1H, d, J ¼ 8.4 Hz, Ar-H), 7.71 (1H, d, J ¼ 8.1 Hz, Ar-H), 12.0
(1H, s, NH).
the required product 7g as
a
d
yellow solid. Yield 85%; mp
6.82 (1H, t, J ¼ 7.6 Hz, Ar-H), 7.01
5.1.5. Synthesis of 3-amino-5-(indol-2-yl)-1,2,4-oxadiazole (10)
A solution of indole-2-carbonyl cyanamide (10 mmol) in ethanol
(20 mL) was added portionwise to a solution of hydroxylamine
205e207 ꢂC. 1H NMR (DMSO-d6)
(1H, t, J ¼ 8.1 Hz, Ar-H), 7.30 (1H, s, Ar-H), 7.43 (1H, d, J ¼ 7.9 Hz,

4530
N.I. Ziedan et al. / European Journal of Medicinal Chemistry 45 (2010) 4523e4530
hydrochloride (10 mmol) in pyridine (3 mL) with cooling (ice bath)
for 15 min. The mixture was stirred at ambient temperature for 1 h,
followed by standing for a further 16 h. 2 N NaOH (20 mL) was
added to the reaction mixture to afford a precipitate, which was
collected, washed with H2O (50 mL), and dried in vacuo. Purification
by column chromatography (ethyl acetate:hexane) afforded the
required product. Yield 8%; mp 196e200 ꢂC. 1H NMR (DMSO-d6)
cytoplasmic (apoptosis) or in the supernatant fraction (necrosis) of
cell lysates.
5.2.3. Caspase activity assay
Enzyme activity was assessed by the Apo-ONEÔ Homogeneous
Caspase-3/7 assay (Promega, Madison, WI, USA). Cells were seeded
at 7 ꢃ 103/well and treated with compounds at 100
mM for 24 h.
d
6.40 (2H, s, NH2), 7.10 (1H, t, J ¼ 7.5 Hz, Ar-H), 7.24 (1H, s, Ar-H),
Subsequently, the caspase-3/7 assay substrate was added and the
fluorescence was measured by spectrofluorimeter at excitation and
emission wavelengths of 485 and 530 nm, respectively. Values were
expressed as ratio between fluorescent signals generated in cells
treated with compounds and those produced in untreated cells
(vehicle alone).
7.27 (1H, t, J ¼ 7.7 Hz, Ar-H), 7.49 (1H, d, J ¼ 8.3 Hz, Ar-H), 7.68
(1H, d, J ¼ 7.5 Hz, Ar-H), 12.15 (1H, s, NH). 13C NMR (DMSO-d6)
d
106.1, 112.4, 120.4, 121.7, 124.5 (C3, C4, C5, C6, C7 of indole), 122.4,
127.0, 139.8 (C2, C3a, C7a of indole), 168.8, 170.2 (C3, C5 of oxa-
diazole). Acc. Mass (C10H8N4O), found 200.0691, theoretical
200.0693.
Acknowledgements
5.2. Biology
We thank the Egyptian Ministryof Higher Education for the award
of a PhD studentship (to NIZ). Support of the EPSRC National Mass
Spectrometry Service Centre, Swansea, U.K. is also acknowledged.
5.2.1. Cell viability assay
The human cancer cell lines COLO 320 (colon), MIA PaCa-2
(pancreas), MCF-7 (breast; ER þve) and Jurkat (leukemia)
(American Type Culture Collection, Manassas, VA), were cultured in
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