Synthesis and evaluation of indole-containing 3,5-diarylisoxazoles as potential pro-apoptotic antitumour agents

Article abstract of DOI:10.1016/j.ejmech.2012.08.009

A series of novel indole-containing diarylisoxazoles has been synthesised, based on our previous work on the synthesis and pro-apoptotic antitumour activity of indole-based diaryl 1,2,4-oxadiazoles. Concise synthetic routes to both 3-(indol-2-yl)-5-phenylisoxazoles and 5-(indol-2-yl)-3-phenylisoxazoles have been developed with full regiochemical control, bearing substituents on the indole ring, indole nitrogen, and/or phenyl group. Additionally a series of the related 5-(1H-indol-5-yl)-3-phenylisoxazoles has been prepared. In vitro evaluation in human cancer cell lines Colo320 (colon) and Calu-3 (lung) revealed preferential antiproliferative activity within the 5-(indol-5-yl)-3- phenylisoxazole series (low micromolar IC₅₀). Further analysis revealed the ability of the indol-5-yl series to induce expression of effector caspases-3 and -7, and retention of viability of the human bronchial smooth muscle cell (BSMC) control cell population (particularly for compounds 18c and 18e).

Full text of DOI:10.1016/j.ejmech.2012.08.009

European Journal of Medicinal Chemistry 56 (2012) 263e270
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of indole-containing 3,5-diarylisoxazoles as potential
pro-apoptotic antitumour agents
,
a,
Siti Farah Md Tohid a, Noha I. Ziedan a, Fabio Stefanelli b, Stefano Fogli b , Andrew D. Westwell
*
**
a School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff, CF10 3NB, Wales, 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 novel indole-containing diarylisoxazoles has been synthesised, based on our previous work on
the synthesis and pro-apoptotic antitumour activity of indole-based diaryl 1,2,4-oxadiazoles. Concise
synthetic routes to both 3-(indol-2-yl)-5-phenylisoxazoles and 5-(indol-2-yl)-3-phenylisoxazoles have
been developed with full regiochemical control, bearing substituents on the indole ring, indole nitrogen,
and/or phenyl group. Additionally a series of the related 5-(1H-indol-5-yl)-3-phenylisoxazoles has been
prepared. In vitro evaluation in human cancer cell lines Colo320 (colon) and Calu-3 (lung) revealed
preferential antiproliferative activity within the 5-(indol-5-yl)-3-phenylisoxazole series (low micromolar
IC50). Further analysis revealed the ability of the indol-5-yl series to induce expression of effector
caspases-3 and -7, and retention of viability of the human bronchial smooth muscle cell (BSMC) control
cell population (particularly for compounds 18c and 18e).
Received 9 May 2012
Received in revised form
3 August 2012
Accepted 4 August 2012
Available online 14 August 2012
Keywords:
3,5-Disubstituted isoxazoles
Indoles
Pro-apoptotic antitumour agents
Anticancer activity
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
activation. In this paper we extend our studies of new indole-
substituted azoles to the synthesis and in vitro antitumour evalu-
A number of promising new antitumour agents based on a 3,5-
diaryl-substituted azole scaffold have been reported. Notable
among these are the pro-apoptotic and antitumour 3,5-diaryl-
1,2,4-oxadiazoles (e.g. 1, Fig. 1), identified using a high-throughput
caspase- and cell-based screening assay [1,2]. Extension of this
work to identify more water-soluble derivatives (2aeb, Fig. 1) with
in vivo efficacy in cancer xenograft models has been reported [3].
Our own recent work has led to the identification of a series of pro-
apoptotic indole-based 3,5-diaryl-1,2,4-oxadiazole antitumour
agents (e.g. 3aeb, Fig. 1) [4,5]. In addition, the 3,5-diaryl isoxazole-
based compound 4 has also been reported to possess affinity for the
retinoic acid receptor (RAR) and selective apoptosis-inducing
activity [6].
Indole-based compounds are amongst the most commonly
occurring heterocycles in cancer drug discovery and development
[7]. Our indole-based 1,2,4-oxadiazoles such as 3aeb were found to
possess antiproliferative activity in the low micromolar IC50 range
(COLO320 and MIA PaCa-2 human cancer cell lines) with the ability
to trigger apoptosis in sensitive cell lines through caspase
ation of indole-containing 3,5-diarylisoxazoles, related in structure
to previously described 1,2,4-oxadiazoles.
2. Chemistry results and discussion
2.1. Synthesis of 3-(indol-2-yl)-5-phenylisoxazoles
The synthesis of 3-(indol-2-yl)-5-phenylisoxazoles was ach-
ieved in two chemical steps from indole-2-carbonyl chlorides 5aec
(Scheme 1). Compounds 5aec were subjected to palladium-
catalysed coupling reaction with substituted phenylacetylenes
according to previously reported methodology [8] to give the
intermediate alkynyl ketones 6aee. Condensation/cyclisation of
the intermediate alkynyl ketones with hydroxylamine hydrochlo-
ride in refluxing pyridine:EtOH (1:3) then gave rise to the product
3-(indol-2-yl)-5-phenylisoxazoles 7aee as single regioisomers in
low to moderate yield (20e35%).
In the case of products 7aee, a single regioisomeric product was
obtained from the initial condensation reaction between the
nucleophilic nitrogen of hydroxylamine and the ketone group of
the alkynyl ketone (6). However cyclisation of further alkynyl
ketones 6f and 6g gave rise to a regioisomeric mixture of isoxazole
products (7f/8f and 7g/8g respectively) that could be separated by
column chromatography (using ethyl acetate/hexane as eluant).
* Corresponding author. Tel.: þ39 050 2219511; fax: þ39 050 2219609.
** Corresponding author. Tel.: þ44 2920 875800; fax: þ44 2920 874149.
E-mail address: stefano.fogli@farm.unipi.it (S. Fogli).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.009

264
S.F. Md Tohid et al. / European Journal of Medicinal Chemistry 56 (2012) 263e270
oxopropylphosphonate) according to the method of Roth et al.
[13]. The partner aldoxime (10aeb) was readily accessible from the
corresponding substituted benzaldehyde using hydroxylamine as
reagent under basic conditions. Dipolar cycloaddition between
2-ethynyl-1-methylindole (9) and aldoxime (10a or 10b) promoted
by sodium hypochlorite solution (13%) and triethylamine as base
according to the method of Lee [14] gave the desired 5-(1-
methylindol-2-yl)-3-phenylisoxazole products 8c and 8h with full
regiochemical control in moderate yield following column chro-
matography (Scheme 3).
2.3. Alternative regiospecific synthesis of 3-(indol-2-yl)-5-
phenylisoxazoles
Fig. 1. Examples of previously reported antitumour diaryl-oxadiazoles and -isoxazoles.
A related synthetic procedure to that outlined in Scheme 3 was
used to secure an alternative regiospecific route to 3-(indol-2-yl)-5-
phenylisoxazoles, albeit containing a chloro group in the 3-position
of the indole ring, arising from the use of sodium hypochlorite to
promote isoxazole formation. In this procedure,1-methyl-indole-2-
carboxaldehyde (11) was first converted to the corresponding
oxime (12) using hydroxylamine hydrochloride. Reaction of oxime
(12) with commercially available substituted phenylacetylenes
(13aeb) promoted by sodium hypochlorite and triethylamine [14]
then gives rise to the required 3-(1-methyl-indol-2-yl)-5-
phenylisoxazoles (14aeb) as shown in Scheme 4.
The reasons for the regiochemical outcome of this isoxazole-
forming reaction are not well understood and are dependent on
both substrates and reaction conditions. The use of pyridine as base
in ethanol for this type of reaction has been reported to be both
regiospecific [9] and to result in a mixture of isoxazole isomers in
some cases [10]. Determination of regiochemistry for the isoxazole
product can be achieved either by careful analysis of the 13C NMR
spectrum (chemical shift of the C-4 isoxazole peak) or by analysis
of the mass spectrometry fragmentation pattern according to re-
ported methodology [11]. For example, in the case of 5-(3-
fluorophenyl)-3-(5-methoxy-1H-indolyl-2-yl)isoxazole 7g the ex-
pected mass spectrometry (electron impact) fragmentation via the
acylaziridine will give rise to an acylium ion with m/z 123, whereas
the regioisomeric 3-(3-fluorophenyl)-5-(1H-indolyl-2-yl)isoxazole
8g will give an acylium ion at m/z 174 (Fig. 2). Assignment of acy-
lium ion EI mass spectrometry fragmentation peaks alongside 13C
NMR analysis secured structural verification of regioisomers 7f/8f
and 7g/8g, and the synthetic route to these diarylisoxazole
regioisomers is shown in Scheme 2.
2.4. Synthesis of 5-(1H-indol-5-yl)-3-phenylisoxazoles
The synthesis of 5-(indol-5-yl)-3-phenylisoxazoles was under-
taken to provide comparative data with the alternative regioiso-
meric 5-(indol-2-yl)-3-phenylisoxazoles (8c, 8feh) described
above. The preparative route to these 5-indolyl isoxazoles is shown
in Scheme 5. Commercially available 5-iodoindole (15) was con-
verted to 5-ethynylindole (16) in two synthetic steps involving
palladium-catalysed coupling with (trimethylsilyl)acetylene fol-
lowed by removal of the trimethylsilyl group using tetrabuty-
lammonium fluoride. 5-Ethynylindole was then reacted with the
benzaldehyde oximes (17aee, prepared from the corresponding
benzaldehyde using hydroxylamine) under the same reaction
conditions as outlined in Schemes 3 and 4 to give the 5-(1H-indol-
5-yl)-3-phenylisoxazole products 18aee as single regioisomers.
2.2. Alternative regiospecific synthesis of 5-(1-methylindol-2-yl)-3-
phenylisoxazoles
Since the cyclisation route between alkynyl ketones and
hydroxylamine outlined in Scheme 2 (unpredictably) gave regioi-
someric diarylisoxazole mixtures that required careful and labo-
rious separation by column chromatography, we devised an
alternative regiospecific route to new 5-(1-methylindol-2-yl)-3-
phenylisoxazoles based on literature procedures. This alternative
regiospecific synthetic route is based on the 1,3-dipolar cycload-
dition between a terminal alkyne (attached to the indole ring) and
an aryl aldoxime [12]. The required 2-ethynyl-1-methylindole (9)
was synthesised from the corresponding aldehyde using the pre-
3. Biological results and discussion
New compounds have been evaluated for in vitro antitumour
activity in the human cancer cell lines, Colo320 (colon) and Calu-3
(lung), using the previously described WST-1 assay [4]. Potency
(50% inhibitory concentration, IC50) following test compound
treatment for 72 h was determined, and the results are presented in
Table 1, as mean values of experiments carried out in triplicate.
prepared
BestmanneOhira
reagent
(dimethyl-1-diazo-2-
Scheme 1. Regiospecific synthesis of 3-(indol-2-yl)-5-phenylisoxazoles 7aee.

S.F. Md Tohid et al. / European Journal of Medicinal Chemistry 56 (2012) 263e270
265
Fig. 2. EI mass spectrometric degradation of diarylisoxazole regioisomers to illustrate structural assignment.
Stock solutions of test compound were made in DMSO, followed
by serial dilution in buffer solution to maintain total DMSO
concentration below 0.2%. The clinically used anticancer agent
5-fluorouracil was used as a positive control.
enzymes involved in the effector phase of apoptosis, were a target
of the new indolyl-substituted oxadiazole compounds. Overall,
caspase activation (2- to 4-fold induction) was consistently
observed after Colo320 and CaLu-3 cells were incubated with
Overall, exposure of the cells to a number of compounds from
the present series resulted in dose-dependent decrease in cell
viability with IC50’s in the low micromolar concentration range.
compounds 18a, 18c, 18d, and 18e (10 mM and 20 mM respectively),
suggesting that caspase activation may contribute to their anti-
cancer activity (Table 2).
Those with IC50 mean values >100
m
M were considered to be
Potential side effects of test compounds could arise by affecting
cell viability in non-cancerous tissues. Therefore, the effect of the
most active compounds was evaluated over 72 h against human
bronchial smooth muscle cells (BSMC), as an in vitro model of
inactive. The most active compounds were the 5-(1H-indol-5-yl)-3-
phenylisoxazoles 18a (R ¼ 4-NO2), 18c (R ¼ 4-OCH3), 18d (R ¼ 4-F),
and 18e (R ¼ 3,4,5-tri-OCH3) with growth inhibitory activity similar
to that observed for 5-fluorouracil in the Colo320 cells (Table 1).
The cell line data indicated a clear preference for activity in the 5-
substituted indole series, with the exception of compound 18b
(containing an ester substituent at the phenyl 4-position), which
was found to be inactive. It is noteworthy that activity (low
micromolar IC50) within the indol-5-yl series appeared to be
tolerant of both electron-withdrawing (e.g. nitro) and electron-
donating (e.g. methoxy) substituents. Of the 2-substituted indole
series, only the 3-(indol-2-yl)-5-phenylisoxazaoles (7f and 14a;
normal cells. Compounds were tested at 20 mM, that is, a concen-
tration similar to the IC50 values determined on cancer cells. The
data presented in Fig. 3 suggests that active 5-substituted indole
compounds may exhibit some selectivity for cancer cells since
smooth muscle cell viability after 72 h of exposure was comparable
to that of control (P > 0.05). Although preliminary in nature, these
results suggest that cytotoxic effects in cancer cells could occur in
the presence of minimal side effects in healthy tissues. Such an
in vitro safety profile appeared to be related to the nature of the
phenyl 4-position, since the strongly electron-withdrawing 4-
nitrophenyl-substituted compound (18a) was found to exhibit
substantial effects on human BSMC viability (Fig. 3). The 4-fluoro-
substituted analogue (18d) also reduced viability in the BSMC cell
line to a lesser extent, whereas the methoxy-substituted analogues
18c and 18e had little effect on BSMC viability.
containing
a methoxyphenyl substituent) showed moderate
growth inhibitory activity, particularly in the Calu-3 cell line.
Remaining compounds of the 2-substituted indole series were
found to be inactive in our test assays.
To explore in more detail the underlying mechanism of selected
compounds, we assessed whether caspase-3 and/or caspase-7, two
Scheme 2. Cyclisation of alkynyl ketones 6feg to give diarylisoxazole regioisomeric mixtures 7f/8f and 7g/8g.
Scheme 3. Regiospecific synthesis of 5-(indol-2-yl)-3-phenylisoxazoles 8c and 8h via dipolar cycloaddition between 2-ethynyl-1-methylindole and aldoximes.

266
S.F. Md Tohid et al. / European Journal of Medicinal Chemistry 56 (2012) 263e270
Table 1
Potency (IC50) for isoxazole products in human cancer cell lines.
Cpd
Potency (IC50
Colo320
, m
M)a
Calu-3
5-FU
7a
12.5 ꢁ 1.1
>100
NDb
>100
7b
>100
>100
7c
>100
>100
7d
>100
>100
7e
>100
>100
7f
7g
69.5 ꢁ 0.8
17.3 ꢁ 0.8
>100
>100
8c
>100
>100
8f
>100
>100
8g
8h
>100
>100
97.9 ꢁ 0.9
>100
14a
14b
18a
18b
18c
18d
18e
78.1 ꢁ 0.9
60.9 ꢁ 0.9
>100
>100
Scheme 4. Regiospecific synthesis of 3-(indol-2-yl)-5-phenylisoxazoles 14aeb via 1,3-
dipolar cycloaddition between indolyl-oxime and phenylacetylenes.
14.0 ꢁ 0.9
>100
13.5 ꢁ 0.9
13.9 ꢁ 0.9
9.0 ꢁ 0.9
25.8 ꢁ 0.8
>100
22.5 ꢁ 0.8
16.8 ꢁ 0.8
13.3 ꢁ 0.8
4. Conclusions
a
Results are expressed as mean values ꢁ SEM (n ¼ 3) after 72 h treatment.
ND ¼ not determined.
b
The concise synthesis of new series of diarylisoxazoles con-
taining an indole group has been accomplished. Synthetic routes
have been developed to access new compounds where the indol-2-
yl and phenyl groups are attached interchangeably to the 3- and 5-
positions of the isoxazole core, with full regiochemical control in
most cases. Additionally the point of attachment of the indole
function to the isoxazole ring has been varied through preparation
of the corresponding 5-(1H-indol-5-yl)-3-phenylisoxazole prod-
ucts. Assessment of the in vitro growth inhibitory activity of the
new isoxazoles against the Colo320 (colon) and Calu-3 (lung)
human cancer cell lines has been carried out using the WST-1
assay. The indol-5-yl substituted compounds 18a and 18cee
were the most active compounds in this assay, with IC50 values
in the low micromolar range. The 5-substituted indole compounds
were furthermore able to induce expression (2e4 fold over
controls) of the apoptotic effector enzymes caspase-3 and caspase-
7. Further studies on human bronchial smooth muscle cells
(BSMC), indicated that the 5-(1H-indol-5-yl)-3-(methoxyphenyl)
isoxazoles 18c and 18e had little or no effect on cell viability in this
normal cell line control, suggestive of selective pro-apoptotic
antitumour effects.
coupling constants (J values) are in Hz. Mass spectrometry (ESI) was
performed on a Bruker microTof instrument, with EI/CI modes of
ionisation carried out within the School of Chemistry, Cardiff
University, as a service. Merck silica gel 60 (40e60 mM) was used for
column chromatography. All commercially available starting
materials were used without further purification. Synthesis of
the required indole 2-carbonyl chlorides (5aec) from commercially
available indole 2-carboxylic acids was achieved by treatment
with oxalyl chloride in dichloromethane according to published
methods [15]. 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) and accurate mass spec-
trometry (EPSRC National Mass Spectrometry Centre, Swansea,
U.K.).
5.1.1. General method for the synthesis of 1-(1H-indol-2-yl)-3-
(phenyl)prop-2-yn-1-ones and 1-(1-methyl-1H-indol-2-yl)-3-
(phenyl)prop-2-yn-1-ones (6aeg)
To a solution of indole-2-carbonylchloride (5aec, 1.55 mmol)
and substituted phenylacetylene (1.1 mmol) in anhydrous THF
(10 mL) was added triethylamine (0.15 mL, 1.1 mmol), CuI (8.9 mg,
0.047 mmol, 3 mol%) and PdCl2(PPh3)2 (98 mg, 0.14 mmol, 0.9 mol
%) with stirring at room temperature under nitrogen atmosphere.
The reaction was completed in 30 min to 1 h by TLC analysis. Water
(50 mL) was added and the product extracted with ethyl acetate
(3 ꢂ 50 mL), and washed with water (50 mL) to remove the
amine hydrochloride salt. The organic layer was dried over
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; chemical shift (
ppm relative to tetramethylsilane (as zero ppm reference), and
d values) were measured in
Scheme 5. Regiospecific synthesis of 5-(indol-5-yl)-3-phenylisoxazoles 18aee via dipolar cycloaddition between 5-ethynylindole and benzaldehyde oximes.

S.F. Md Tohid et al. / European Journal of Medicinal Chemistry 56 (2012) 263e270
267
Table 2
5.1.1.4. 1-(1-Methylindol-2-yl)-3-(3-fluorophenyl)prop-2-yn-1-one
(6d). Yield 64%; mp 94 ꢃC. 1H NMR (CDCl3)
d 4.12 (3H, s, CH3),
Induction of caspase-3/7 activity (fold change over control values) after treatment
with new substituted indolyl-oxadiazoles in the sensitive cell lines.
7.19e7.23 (2H, m, H-20, H-60), 7.37 (1H, dt, J ¼ 9.5, 2.5 Hz, H-40),
7.39 (1H, m, H-50), 7.42 (1H, t, J ¼ 8.0 Hz, H-5), 7.45 (1H, t,
J ¼ 8.0 Hz, H-6), 7.50 (1H, dd, J ¼ 8.0, 2.0 Hz, H-4), 7.65 (1H, s, H3),
Compound
Cell linea
Colo320
Calu-3
7.76 (1H, dd, J ¼ 2.0, 8.0 Hz, H-7). 13C NMR (CDCl3)
d 31.62 (CH3),
18a
18c
18d
18e
2.84 ꢁ 0.06
2.44 ꢁ 0.13
1.94 ꢁ 0.21
3.39 ꢁ 0.24
3.76 ꢁ 0.46
2.18 ꢁ 0.10
1.81 ꢁ 0.19
2.38 ꢁ 0.18
87.72 (alkyne), 88.28 (alkyne), 110.60, 116.68, 121.16, 123.20,
127.08, 117.85 (d, J ¼ 20.2 Hz), 119.45 (d, J ¼ 22.7 Hz),128.82,130.41
(d,
J
¼
8.8 Hz), 122.30, 125.99, 135.93, 141.13, 161.35 (d,
a
Results are expressed as fold change over control, mean values ꢁ SEM (n ¼ 3).
J ¼ 246.5 Hz, C-3), 169.20 (C]O).
5.1.1.5. 1-(5-Methoxy-1H-indol-2-yl)-3-(3-methoxyphenyl)prop-2-yn-
1-one (6e). Yield 23%; mp 147 ꢃC. 1H NMR (CDCl3)
d 3.88 (3H, s,
MgSO4, filtered and concentrated in vacuo. The resulting crude
compound was purified by column chromatography (hexane:ethyl
acetate ¼ 4:1) to give intermediate propanone product (6aeg) in
23e95% yield.
OCH3), 3.89 (3H, s, OCH3), 7.06 (1H, d, J ¼ 2.0 Hz, H-20), 7.08 (1H, d,
J ¼ 2.5 Hz, H-3), 7.11 (1H, dd, J ¼ 8.0, 2.0 Hz, H-40), 7.15 (1H, d,
J ¼ 2.5 Hz, H-4), 7.23 (1H, t, J ¼ 8.0 Hz, H-50), 7.39 (1H, d, J ¼ 8.0 Hz, H-
6), 7.41 (1H, d, J ¼ 8.0 Hz, H-7), 7.45 (1H, dd, J ¼ 8.0, 2.0 Hz, H-60), 8.97
(1H, s, NH). 13C NMR (CDCl3)
d 55.45 (OCH3), 55.67 (OCH3), 86.52
5.1.1.1. 1-(1H-Indol-2-yl)-3-(4-methoxyphenyl)prop-2-yn-1-one (6a).
Yield 88%; mp 158e160 ꢃC. 1H NMR (CDCl3)
d 3.75 (3H, s, OCH3), 6.90
(alkyne), 91.61 (alkyne), 102.82, 113.03, 113.26, 117.51, 118.96, 119.35,
127.57, 129.81, 121.15, 127.89, 133.73, 137.12, 155.04, 159.50, 168.50
(C]O).
(2H, d, J ¼ 8.0 Hz, C-30, C-50), 7.15 (1H, dt, J ¼ 8.2, 2.1 Hz, AreH), 7.35
(1H, t, J ¼ 7.6 Hz, AreH), 7.45 (1H, d, J ¼ 3.5 Hz, AreH), 7.37 (1H, dd,
J ¼ 7.2, 1.9 Hz, AreH), 7.75 (1H, d, J ¼ 7.8 Hz, AreH), 7.65 (2H, d,
J ¼ 8.0 Hz, C-20, C-60), 9.15 (1H, s, NH).
5.1.1.6. 1-(1-Methylindol-2-yl)-3-(3-methoxyphenyl)prop-2-yn-1-one
(6f). Yield 95%; mp 75 ꢃC. 1H NMR (CDCl3)
d 3.87 (3H, s, OCH3),
4.13 (3H, s, CH3), 7.06 (1H, dt, J ¼ 8.0, 1.5 Hz, H-40), 7.21 (1H, dt,
J ¼ 8.0, 2.0 Hz, H-50), 7.23 (1H, s, H-20), 7.33 (1H, dt, J ¼ 8.0, 2.0 Hz,
H-5), 7.36 (1H, d, J ¼ 8.0 Hz, H-4), 7.40 (1H, dd, J ¼ 8.0, 2.0 Hz, H-
60), 7.46 (1H, td, J ¼ 8.0, 2.0 Hz, H-6), 7.68 (1H, s, H-3), 7.76 (1H, d,
5.1.1.2. 1-(1H-Indol-2-yl)-3-(3-methoxyphenyl)prop-2-yn-1-one (6b).
Yield 62%; mp 124e127 ꢃC. 1H NMR (CDCl3)
d 3.90 (3H, s, OCH3), 6.97
(1H, dd, J ¼ 8.1, 2.6 Hz, AreH), 7.10 (1H, t, J ¼ 7.1 Hz, AreH); 7.13 (1H,
m, AreH), 7.18 (1H, s, H-3), 7.25e7.35 (3H, m, AreH), 7.44 (1H, d,
J ¼ 1.2 Hz, AreH), 7.68 (1H, d, J ¼ 8.1 Hz, AreH), 9.15 (1H, s, NH). 13C
J ¼ 8.0 Hz, H-7). 13C NMR (CDCl3)
d 32.07 (CH3), 55.44 (OCH3),
NMR (CDCl3)
d 55.46 (OCH3), 86.40 (alkyne), 91.74 (alkyne), 112.32,
87.71 (alkyne), 89.59 (alkyne), 110.45, 117.27, 117.51, 117.59, 121.20,
123.39, 125.46, 126.49, 127.73, 121.36, 126.01, 136.11, 141.05, 159.50,
169.59 (C]O).
113.56, 121.36, 123.16, 126.29, 117.57, 117.59, 125.60, 129.82, 127.53,
136.70, 138.06, 121.06, 159.51, 168.78 (C]O).
5.1.1.3. 1-(1-Methylindol-2-yl)-3-(4-methoxyphenyl)prop-2-yn-1-one
5.1.1.7. 1-(5-Methoxy-1H-indol-2-yl)-3-(3-fluorophenyl)prop-2-yn-
(6c). Yield 64%; mp 112 ꢃC. 1H NMR (CDCl3)
d 3.88 (3H, s, OCH3), 4.15
1-one (6g). Yield 33%; mp 142 ꢃC. 1H NMR (CDCl3)
d 3.89 (3H, s,
(3H, s, CH3), 7.97 (2H, d, J ¼ 9.0 Hz, H-30, H-50), 7.20 (1H, dt, J ¼ 8.0,
2.0 Hz, H-5), 7.40 (dt, J ¼ 8.0, 2.5 Hz, H-6), 7.46 (1H, d, J ¼ 9.0 Hz, H-4),
7.66 (1H, s, H-3), 7.67 (2H, d, J ¼ 9.0 Hz, H-20, H-60), 7.77 (1H, d,
OCH3), 7.11 (1H, dd, J ¼ 9.0, 2.5 Hz, H-60), 7.14 (1H, d, J ¼ 2.5 Hz, H-3),
7.23 (1H, td, J ¼ 8.0, 2.5 Hz, AreH), 7.37 (1H, d, J ¼ 8.5 Hz, H-6), 7.41
(1H, d, J ¼ 8.5 Hz, H-7), 7.45 (2H, m, AreH), 7.51 (1H, d, J ¼ 7.5 Hz,
J ¼ 7.5 Hz, H-7). 13C NMR (CDCl3)
d 32.08 (CH3), 55.45 (OCH3), 87.81
(alkyne), 90.75 (alkyne), 110.41, 114.40, 115.97, 120.97, 123.29, 126.67,
AreH), 9.17 (1H, s, NH). 13C NMR (CDCl3)
d 55.67 (OCH3), 87.07
(alkyne), 89.63 (alkyne), 102.83, 113.20, 113.32, 130.40, 118.08 (d,
J ¼ 21.4 Hz), 119.56 (d, J ¼ 22.7 Hz), 122.0 (d, J ¼ 10.0 Hz), 127.89,
128.89 (d, J ¼ 2.5 Hz), 130.46, 133.75, 136.96, 155.11, 161.35 (d,
J ¼ 249.5 Hz, C-30), 168.18 (C]O).
112.23, 126.01, 134.90, 136.28, 140.93, 161.52, 169.87 (C]O).
5.1.2. General method for the synthesis of 3-(1H-indol-2-yl)-5-
phenylisoxazole and 3-(1-methyl-1H-indol-2-yl)-5-phenylisoxazole
(7aeg)
To a stirred solution of indole-substituted prop-2-yn-1-one
(6aeg, 1.4 mmol) in ethanol (15 mL) and pyridine (5 mL) was
added hydroxylamine hydrochloride (0.19 g, 2.8 mmol). The
mixture was heated under reflux for 5 h and monitored by TLC. The
reaction mixture was cooled and the solvent was evaporated to
dryness. Water (10 mL) was added to the residue and the crude
product extracted with ethyl acetate (3 ꢂ 10 mL). The organic layer
was dried (MgSO4), filtered and evaporated in vacuo. The crude
compound was purified by column chromatography (hexane:ethyl
acetate ¼ 3:1).
5.1.2.1. 3-(1H-Indolyl-2-yl)-5-(4-methoxyphenyl)isoxazole (7a). Yield
20%; mp 194e195 ꢃC. 1H NMR (CDCl3)
d 3.81 (3H, s, OCH3), 6.70 (1H,
s, isoxazole H-4), 6.93 (3H, m, AreH), 7.22 (1H, t, J ¼ 7.0 Hz, AreH),
7.35 (1H, t, J ¼ 7.5 Hz, AreH), 7.38 (1H, d, J ¼ 8.0 Hz, AreH), 7.61
(1H, d, J ¼ 8.0 Hz, ArH), 7.73 (2H, d, J ¼ 9.0 Hz, H-20, H-60), 8.62 (1H, s,
Fig. 3. Effects of selected compounds on human bronchial smooth muscle cell (BSMC)
viability. Cells were treated with compounds at 20 mM for 72 h. Results are mean S.E.M.
NH). 13C NMR (CDCl3)
d 55.4 (OCH3), 97.20 (C-4 isoxazole), 103.46,
(n ¼ 3).
111.44, 120.94, 121.52, 124.20, 114.40, 128.30. Anal. calcd. for

268
S.F. Md Tohid et al. / European Journal of Medicinal Chemistry 56 (2012) 263e270
C18H14N2O2: C, 74.47; H, 4.86; N, 9.64. Found: C, 74.36; H, 4.74; N,
9.49.
J ¼ 9.5, 2.5 Hz, AreH), 7.14 (1H, d, J ¼ 2.0 Hz, H-3), 7.20 (1H, m,
AreH), 7.38 (1H, d, J ¼ 9.0 Hz, AreH), 7.51 (1H, dd, J ¼ 5.0, 9.5 Hz,
AreH), 7.59 (1H, d, J ¼ 9.5 Hz, AreH), 7.67 (1H, d, J ¼ 8.0 Hz, H-7),
5.1.2.2. 3-(1H-Indolyl-2-yl)-5-(3-methoxyphenyl)isoxazole (7b).
8.94 (1H, s, NH). 13C NMR (CDCl3)
d 55.82 (OCH3), 98.19 (C-4 iso-
Yield 33%; mp 120e122 ꢃC.1H NMR (CDCl3)
d
3.81 (3H, s, OCH3), 6.73
xazole), 102.44, 104.12, 112.19, 114.83, 113.05 (d, J ¼ 23.4 Hz), 117.30
(d, J ¼ 21.8 Hz), 121.64, 131.80 (d, J ¼ 7.8 Hz, C-50), 126.77, 129.84,
131.23, 134.77, 151.32, 155.17, 164.23 (d, J ¼ 246.2 Hz, C-30), 166.13.
MS (EIþ) m/z 308.10 (Mþ). HRMS (ESI) found for C18H13FN2O2 m/
z ¼ 309.1034 [M þ H]þ; calcd.: 308.0961.
(1H, s, isoxazole H-4), 6.95 (2H, m, AreH), 7.1 (1H, td, J ¼ 7.5, 0.85 Hz,
AreH), 7.18 (1H, s, H-3), 7.22 (1H, td, J ¼ 7.6,1.1 Hz, AreH), 7.32e7.38
(3H, m, AreH), 7.61 (1H, d, J ¼ 7.9 Hz, AreH), 8.7 (1H, s, NH). 13C NMR
(CDCl3)
d 55.44 (OCH3), 97.52 (C4 isoxazole), 103.63, 111.48, 120.97,
121.55, 124.27, 111.88, 116.32, 119.42, 130.05, 128.15, 136.83, 138.06,
139.06,163.01,160.04,163.77. Anal. calcd. for C18H14N2O2: C, 74.47; H,
4.86; N, 9.64. Found: C, 74.28; H, 4.74; N, 9.51.
5.1.2.8. 5-(1-Methylindol-2-yl)-3-(3-methoxyphenyl)isoxazole (8f)
e alternative regioisomer. Yield 4%; mp 125 ꢃC. 1H NMR (CDCl3)
d
3.92 (3H, s, OCH3), 4.18 (3H, s, CH3), 6.86 (1H, s, isoxazole H-4),
5.1.2.3. 3-(1-Methylindolyl-2-yl)-5-(4-methoxyphenyl)isoxazole (7c).
6.98 (1H, s, AreH), 7.04 (1H, dq, J ¼ 1.5, 8.0 Hz, AreH), 7.19 (1H, td,
J ¼ 8.0, 2.0 Hz, AreH), 7.34 (1H, td, J ¼ 8.0, 2.0 Hz, AreH), 7.42 (1H,
d, J ¼ 8.0 Hz, AreH), 7.43 (1H, dd, J ¼ 8.0, 2.0 Hz, AreH), 7.46 (1H, s,
AreH), 7.47 (1H, td, J ¼ 2.0, 8.0 Hz, AreH), 7.71 (1H, d, J ¼ 8.0 Hz,
Yield 56%; mp 168 ꢃC. 1H NMR (CDCl3)
d 3.90 (3H, s, OCH3), 4.07 (3H,
s, CH3), 6.79 (1H, s, isoxazole H-4), 7.03 (2H, d, J ¼ 9.0 Hz, H-30, H-50),
7.05 (1H, s, H-3), 7.20 (1H, t, J ¼ 8.0 Hz, AreH), 7.36 (1H, t, J ¼ 8.0 Hz,
AreH), 7.43 (1H, d, J ¼ 9.0 Hz, AreH), 7.69 (1H, d, J ¼ 7.5 Hz, AreH),
AreH). 13C NMR (CDCl3)
d 32.44 (CH3), 55.45 (OCH3), 99.70 (iso-
7.86 (2H, d, J ¼ 9.0 Hz, H-20, H-60). 13C NMR (CDCl3)
d
31.83 (CH3),
xazole C-4), 104.94, 109.93, 111.16, 120.19, 121.22, 116.34, 118.42,
123.29, 130.19, 127.32, 128.33, 128.73, 139.21, 157.42, 160.05,
169.18. MS (EIþ) m/z 304.13 (Mþ). HRMS (ESI) found for
C19H16N2O2 m/z ¼ 305.1281 [M þ H]þ; calcd.: 304.1212.
55.39 (OCH3), 99.55 (isoxazole C-4), 109.82, 104.69, 114.41, 120.51,
121.52, 123.65, 121.18, 127.25, 128.28, 127.30, 138.88, 161.16, 162.27,
164.05. MS (EIþ) m/z 304.13 (Mþ). Anal. calcd. for C19H16N2O2; C,
74.98; H, 5.30; N, 9.20; found C, 74.84; H, 5.29; N, 9.20.
5.1.2.9. 5-(5-Methoxy-1H-indol-2-yl)-3-(3-fluorophenyl)isoxazole (8g) e
alternative regioisomer. Yield 5%; mp 117e118 ꢃC. 1H NMR (CDCl3)
d 3.90 (3H, s, OCH3), 6.79 (1H, s, isoxazole H-4), 6.99 (1H, d,
5.1.2.4. 3-(1-Methylindolyl-2-yl)-5-(3-fluorophenyl)isoxazole (7d).
Yield 18%; mp 124 ꢃC. 1H NMR (CDCl3)
d
4.07 (3H, s, CH3), 6.83 (1H,
s, isoxazole H-4), 7.08 (1H, d, J ¼ 2.0 Hz, ArH), 7.19e7.23 (2H, m,
AreH), 7.37 (1H, td J ¼ 9.5, 2.5 Hz, AreH), 7.43 (1H, d, J ¼ 8.0 Hz,
AreH), 7.49 (1H, dd, J ¼ 9.5, 5.0 Hz, AreH), 7.64 (1H, dd, J ¼ 9.5,
2.5 Hz, AreH), 7.68 (1H, d, J ¼ 8.0 Hz, AreH), 7.72 (1H, t, J ¼ 8.0 Hz,
J ¼ 2.0 Hz, AreH), 7.00 (1H, dd, J ¼ 9.0, 2.5 Hz, AreH), 7.13 (1H, d,
J ¼ 2.0 Hz, AreH), 7.20 (1H, m, AreH), 7.36 (1H, d, J ¼ 9.0 Hz, AreH),
7.49 (1H, dd, J ¼ 9.0, 5.0 Hz, AreH), 7.60 (1H, d, J ¼ 9.5 Hz, AreH),
7.67 (1H, d, J ¼ 8.0 Hz, AreH), 8.67 (1H, s, NH). 13C NMR (CDCl3)
AreH). 13C NMR (CDCl3)
d
31.82 (CH3), 99.54 (isoxazole C-4), 105.01,
d 55.77 (OCH3), 97.07 (isoxazole C-4), 102.37, 103.51, 112.19, 115.37,
109.87, 120.62, 121.60, 122.61, 113.80 (d, J ¼ 22.7 Hz), 117.02 (d,
J ¼ 21.4 Hz), 123.86, 130.62 (d, J ¼ 8.8 Hz), 127.20, 130.89, 138.95,
161.73, 162.08 (d, J ¼ 248.2 Hz, C-30), 164.66. MS (EIþ) m/z 292.11
(Mþ). Anal. calcd. for C18H13N2OF; C, 73.96; H, 4.48; N, 9.58; found
C, 73.93; H, 4.40; N, 9.58.
114.01 (d, J ¼ 23.9 Hz), 117.21 (d, J ¼ 21.4 Hz), 122.61, 130.60 (d,
J ¼ 8.8 Hz), 125.34, 128.60, 130.67, 132.12, 150.90, 154.96, 162.08 (d,
J ¼ 245.7 Hz, C-30), 164.13. MS (EIþ) m/z 308.10 (Mþ). HRMS (ESI)
found for C18H13FN2O2 m/z ¼ 309.1031 [M þ H]þ; calcd.: 308.0961.
5.1.3. Generalmethodfortheregiospecific synthesisof5-(1-methylin-
dol-2-yl)-3-phenylisoxazoles (8c and 8h)
5.1.2.5. 3-(5-Methoxy-1H-indolyl-2-yl)-5-(3-methoxyphenyl)isoxazole
(7e). Yield 25%; mp 202 ꢃC. 1H NMR (CDCl3)
d
3.90 (3H, s, OCH3), 3.91
To a solution of 2-ethynyl-1-methylindole 9 (0.22 g, 1.4 mmol)
(3H, s, OCH3), 6.80 (s, 1H, isoxazole H-4), 6.97 (1H, d, J ¼ 2.0 Hz,
AreH), 6.99 (1H, d, J ¼ 2.5 Hz, AreH), 7.05 (1H, dd, J ¼ 2.0, 8.0 Hz,
AreH), 7.12 (1H, d, J ¼ 2.5 Hz, AreH), 7.35 (1H, d, J ¼ 9.0 Hz, AreH),
7.40 (1H, d, J ¼ 8.0 Hz, AreH), 7.42 (1H, d, J ¼ 8.0 Hz, AreH), 7.46 (1H,
and triethylamine (19 mL, 0.14 mmol) in dichloromethane (10 mL)
was added sodium hypochlorite (1.40 mL of 13% aqueous solution,
2.6 mmol). The mixture was cooled to 0 ꢃC, and a solution of
benzaldehyde oxime 10aeb (1.4 mmol) in dichloromethane
(10 mL) was added dropwise. The reaction was stirred at room
temperature for 24 h. After reaction completion as indicated by TLC,
water (15 mL) was added, the layers separated and the aqueous
layer was further extracted with dichloromethane (15 mL). The
organic layer was dried (MgSO4), concentrated in vacuo and the
residue was purified by column chromatography using hexane/
ethyl acetate (5:1) as eluent.
dd, J ¼ 8.0, 2.0 Hz, AreH), 8.76 (1H, s, NH). 13C NMR (CDCl3)
d 55.43
(OCH3), 55.78 (OCH3), 97.32 (C-4 isoxazole), 102.35, 103.31, 111.87,
112.32, 115.18, 116.29, 119.41, 130.04, 125.57, 128.62, 131.88, 132.11,
154.90, 160.03, 162.97, 163.80. MS (EIþ) m/z 320.12 (Mþ). HRMS (ESI)
found for C19H16N2O3 m/z ¼ 321.1236 [M þ H]þ; calcd.: 320.1161.
5.1.2.6. 3-(1-Methylindolyl-2-yl)-5-(3-methoxyphenyl)isoxazole (7f).
Yield 20%; mp 73e74 ꢃC. 1H NMR (CDCl3)
d 3.92 (3H, s, OCH3), 4.04
(3H, s, CH3), 6.81 (1H, s, isoxazole H-4), 7.05 (1H, dq, J ¼ 8.0, 1.5 Hz,
AreH), 7.07 (1H, s, AreH), 7.21 (1H, td, J ¼ 8.0, 2.0 Hz, AreH), 7.35
(1H, td, J ¼ 8.0, 2.0 Hz, AreH), 7.42 (1H, d, J ¼ 8.0 Hz, AreH), 7.43 (1H,
dd, J ¼ 8.0, 2.0 Hz, AreH), 7.45 (1H, td, J ¼ 2.0, 8.0 Hz, AreH), 7.49 (1H,
5.1.3.1. 5-(1-Methylindol-2-yl)-3-(4-methoxyphenyl)isoxazole (8c).
Yield 23%; mp 169 ꢃC. 1H NMR (CDCl3)
d 3.91 (3H, s, OCH3), 4.06 (3H,
s, CH3), 6.79 (1H, s, isoxazole H-4), 7.03 (2H, dd, J ¼ 8.5, 1.5 Hz, H30,
H50), 7.20 (1H, t, J ¼ 8.0 Hz, AreH), 7.35 (1H, t, J ¼ 8.0, 0.5 Hz, AreH),
7.42 (1H, d, J ¼ 8.5 Hz, AreH), 7.70 (1H, d, J ¼ 8.0 Hz, AreH), 7.86 (2H,
s, AreH), 7.72 (1H, d, J ¼ 8.0 Hz, AreH). 13C NMR (CDCl3)
d 31.82
(CH3), 55.44 (OCH3), 99.81 (isoxazole C-4), 104.83, 109.85, 111.88,
120.56, 121.55, 116.26, 119.14, 123.74, 130.05, 127.15, 127.24, 130.06,
138.91, 160.06, 162.60, 164.30. MS (EIþ) m/z 304.12 (Mþ). Anal. calcd.
for C19H16N2O2; C, 74.98; H, 5.30; N, 9.20; found C, 74.91; H, 5.22; N,
9.23.
d, J ¼ 8.5 Hz, H20, H60). 13C NMR (CDCl3)
d 31.84 (CH3), 55.39 (OCH3),
99.56 (isoxazole C-4), 104.96, 109.82, 114.41, 120.51, 121.52, 121.33,
124.30, 123.65, 128.28, 127.30, 138.88, 161.16, 162.27, 164.05. MS
(EIþ) m/z 304.13 (Mþ). Anal. calcd. for C19H16N2O2; C, 74.98; H, 5.30;
N, 9.20; found C, 74.55; H, 5.27; N, 9.19.
5.1.2.7. 3-(5-Methoxy-1H-indolyl-2-yl)-5-(3-fluorophenyl)isoxazole
5.1.3.2. 5-(1-Methylindol-2-yl)-3-(4-fluorophenyl)isoxazole (8h). Yield
(7g). Yield 2%; mp 214 ꢃC. 1H NMR (CDCl3)
d
3.90 (3H, s, OCH3), 6.88
29%; mp 145 ꢃC. 1H NMR (CDCl3)
d
4.06 (3H, s, CH3), 6.80 (1H, s,
(1H, s, isoxazole H-4), 6.91 (1H, d, J ¼ 2.5 Hz, AreH), 6.99 (1H, dd,
isoxazole H-4), 7.07 (1H, s, H-3), 7.21 (1H, t, J ¼ 8.0 Hz, AreH), 7.22

S.F. Md Tohid et al. / European Journal of Medicinal Chemistry 56 (2012) 263e270
269
(2H, dd, J ¼ 9.0, 2.0 Hz, H20, H60), 7.36 (1H, dt, J ¼ 8.0, 1.0 Hz, AreH),
7.44 (1H, d, J ¼ 8.5 Hz, AreH), 7.72 (1H, d, J ¼ 8.0 Hz, AreH), 7.90
J ¼ 8.5 Hz, AreH), 7.62 (1H, s, AreH), 10.69 (1H, s, NH). 13C NMR
(CDCl3) d 74.02 (acetylene), 85.26 (acetylene), 101.25, 110.97, 112.58,
(2H, q, J ¼ 9.0, 2.0 Hz, H30, H50). 13C NMR (CDCl3)
d
31.84 (CH3), 99.52
123.92, 124.38, 125.81, 127.81, 136.23.
(isoxazole C-4), 104.92, 109.85, 120.60, 121.57, 123.81, 116.22 (d,
J ¼ 22.7 Hz, C-30, C-50), 125.08, 127.03, 128.85 (d, J ¼ 8.0 Hz, C-20, C-
60), 127.21, 138.93, 161.75, 162.94 (d, J ¼ 250.7 Hz, C-40), 164.50. MS
(EIþ) m/z 292.11 (Mþ). Anal. calcd. for C18H13FN2O; C, 73.96; H, 4.48;
N, 9.58; found C, 73.93; H, 4.50; N, 9.59.
5.1.5.2. General method for synthesis of 5-(1H-indole-5-yl)-3-
phenylisoxazoles (18aee). To a solution of 5-ethynyl-1H-indole
(16, 1.4 mmol) and Et3N (0.14 mmol) in CH2Cl2 (20 mL) was
added sodium hypochlorite (1.40 mL of 13% aqueous solution,
2.6 mmol) under N2. After cooling to 0 ꢃC, a solution of oxime (17,
1.4 mmol) in CH2Cl2 was added dropwise over a period of 1 h.
The reaction was then stirred at room temperature for a further
24 h. After reaction completion as indicated by TLC, H2O (20 mL)
was added and the mixture was extracted with CH2Cl2
(3 ꢂ 20 mL). The organic layer was dried over MgSO4, filtered and
evaporated under reduced pressure, and the residue was purified
by column chromatography using EtOAc/hexane (1:2) as eluent.
5.1.5.2.1. 5-(1H-Indol-5-yl)-3-(4-nitrophenyl)isoxazole
5.1.4. Synthesis of 3-(3-chloro-1-methylindol-2-yl)-5-phenylisoxazoles
(14aeb)
To
a solution of terminal alkyne (1.4 mmol) and Et3N
(0.14 mmol) in CH2Cl2 (10 mL) under N2 atmosphere was added
sodium hypochlorite (1.40 mL of 13% aqueous solution, 2.6 mmol).
The reaction mixture was cooled to 0 ꢃC, then a solution of oxime
(1.4 mmol) in CH2Cl2 (10 mL) was added dropwise over a period of
1 h. The reaction was stirred at room temperature for 24 h. After
reaction completion as indicated by TLC, H2O (20 mL) was added
and the mixture was extracted with CH2Cl2 (3 ꢂ 20 mL). The
organic layer was dried over MgSO4, evaporated under reduced
pressure and the residue was purified by column chromatography
using EtOAc/hexane (1:2) as eluent.
(18a). Yield 50%; mp 252e253 ꢃC. 1H NMR (DMSO-d6)
d 6.60 (1H, s,
AreH), 7.48 (1H, t, J ¼ 2.5 Hz, AreH), 7.58 (1H, d, J ¼ 8.5 Hz, AreH),
7.61 (1H, s, AreH), 7.66 (1H, dd, J ¼ 8.5, 1.5 Hz, AreH), 8.17 (1H, s,
AreH), 8.21 (2H, d, J ¼ 9.0 Hz, AreH), 8.41 (2H, d, J ¼ 9.0 Hz, AreH),
11.44 (1H, s, NH). 13C NMR (DMSO-d6)
d 96.83 (C4 isoxazole), 102.16,
112.31, 118.88, 123.63, 124.33, 127.65, 127.80, 129.15, 130.63, 136.13,
141.56, 148.78, 154.67, 167.95. Anal. calcd. for C17H11N3O3; C, 66.88;
H, 3.63; N, 13.76; found C, 67.20; H, 3.54; N, 13.71.
5.1.4.1. 3-(3-Chloro-1-methylindol-2-yl)-5-(4-methoxyphenyl)iso-
xazole (14a). Yield 67%; mp 150e152 ꢃC. 1H NMR (DMSO-d6)
d 3.86
(3H, s, OCH3), 3.97 (1H, s, NCH3), 7.13 (2H, dt, J ¼ 9.0, 2.0 Hz, AreH),
7.24 (1H, td, J ¼ 7.5, 0.5 Hz, AreH), 7.40 (1H, td, J ¼ 7.5,1.5 Hz, AreH),
7.47 (1H, s, isoxazole H-4), 7.63 (1H, d, J ¼ 8.0 Hz, AreH), 7.68 (1H, d,
J ¼ 8.0 Hz, AreH), 8.00 (1H, dt, J ¼ 9.0, 2.5 Hz, AreH). 13C NMR
5.1.5.2.2. Methyl4-(5-(1H-indol-5-yl)isoxazol-3-yl)benzoate(18b).
Yield 57%; mp 209e210 ꢃC. 1H NMR (DMSO-d6)
d 3.90 (3H, s, OCH3)
6.59 (1H, s, AreH), 7.48 (1H, s, AreH), 7.35 (1H, s, AreH), 7.57 (1H, d,
J ¼ 8.5 Hz, AreH), 7.66 (1H, d, J ¼ 8.45, AreH), 8.08 (2H, d,
J ¼ 8.3 Hz, AreH), 8.12 (2H, d, J ¼ 8.3 Hz, AreH), 8.16 (1H, s, AreH),
(DMSO-d6)
d 32.31 (NCH3), 55.42 (OCH3), 99.67 (C4 isoxazole),
105.33, 111.05, 114.73, 118.00, 119.05, 120.88, 124.06, 124.38, 127.59,
136.63, 154.39, 161.12, 169.53. Anal. calcd. for C18H13ClN2O2; C,
67.36; H, 4.46; N, 8.26; found C, 67.37; H, 4.38; N, 8.25.
11.43 (1H, s, NH). 13C NMR (DMSO-d6)
d 52.27 (OCH3), 96.06 (C4
isoxazole),103.68,111.64,119.06,120.18,125.59,126.77,128.15, 129.19,
130.16, 130.97, 135.89, 140.78, 154.67, 167.95, 168.78 (C]O). Anal.
calcd. for C19H14N2O3; C, 71.69; H, 4.43; N, 8.80; found C, 72.06; H,
4.27; N, 8.82.
5.1.4.2. 3-(3-Chloro-1-methylindol-2-yl)-5-(4-trifluoromethylphenyl)
isoxazole (14b). Yield 70%; mp 148e149 ꢃC. 1H NMR (DMSO-d6)
5.1.5.2.3. 5-(1H-Indol-5-yl)-3-(4-methoxyphenyl)isoxazole (18c).
d
4.08 (1H, s, NCH3), 7.24 (1H, m, AreH), 7.39 (3H, m, AreH), 7.70 (1H,
Yield 17%; mp 151e152 ꢃC. 1H NMR (CDCl3)
d 3.90 (3H, s, OCH3), 6.67
d, J ¼ 8.0 Hz, AreH), 7.78 (2H, d, J ¼ 8.0 Hz, AreH), 8.04 (2H, d,
(1H, s, AreH), 6.76 (1H, s, AreH), 7.02 (2H, d, J ¼ 8.0 Hz, H-30, H-50),
7.32 (1H, s, AreH), 7.50 (1H, d, J ¼ 8.0 Hz, AreH), 7.70 (1H, d,
J ¼ 8.0 Hz, AreH), 7.85 (2H, d, J ¼ 8.0 Hz, H-20, H-60), 8.19 (1H, s,
J ¼ 8.5 Hz, AreH). 13C NMR (DMSO-d6)
d 32.83 (NCH3), 101.80 (C4
isoxazole), 104.87, 110.14, 118.94, 119.15, 120.20, 120.81, 124.22,
124.72, 126.13, 126.25, 131.34, 136.12, 140.52, 155.45, 168.75. Anal.
calcd. for C18H10F3N2O; C, 60.57; H, 3.21; N, 7.43; found C, 61.07; H,
3.25; N, 7.31.
AreH), 8.33 (1H, s, NH). 13C NMR (CDCl3)
d 55.74 (OCH3), 96.13 (C4
oxazole), 103.64, 112.06, 114.68, 119.01, 120.05, 120.32, 121.37,
126.19, 128.47, 129.70, 137.13, 161.43, 162.86, 163.09. MS (EIþ) 290.11
(Mþ). HRMS (ESI) found for C18H14N2O2 m/z ¼ 291.1122 [M þ H]þ;
calcd.: 290.1055.
5.1.5. Synthesis of 5-(1H-indol-5-yl)-3-phenylisoxazoles
5.1.5.1. Synthesis of 5-ethynyl-1H-indole (16). A mixture of 5-iodo-
indole (15, 0.20 g, 0.82 mmol), triethylsilylacetylene (1.28 mmol),
Pd(PPh3)4 (0.024 mmol), CuI (0.042 mmol), and triethylamine
(0.23 mL) in acetonitrile (10 mL) was heated under reflux for 4 h.
When TLC indicated complete consumption of the starting material,
H2O was added and the mixture was extracted with ethyl acetate, the
organic layer was dried over MgSO4 and evaporated to dryness. The
residue containing 5-(2-triethylsilylethynyl)indole was used for the
next step without further purification.
The residue was dissolved in THF, 1 mmol of 1 M tetrabuty-
lammonium fluoride (TBAF, aqueous) was added, and the mixture
was stirred at room temperature for 24 h. Water (15 mL) was added,
and the mixture was extracted with ethyl acetate (3 ꢂ 15 mL). The
organic layer was dried over MgSO4, filtered and evaporated under
reduced pressure. The residue was purified by column chroma-
tography using EtOAc/petroleum ether (1:2) as eluent. The product
5-ethynyl-1H-indole (16) was obtained in good purity, with an
overall yield for the two reactions of 76%, M.p. 59e60 ꢃC. 1H NMR
5.1.5.2.4. 5-(1H-Indol-5-yl)-3-(4-fluorophenyl)isoxazole (18d).
Yield 36%; mp 162e163 ꢃC. 1H NMR (CDCl3)
d 6.69 (1H, s, AreH),
6.77 (1H, s, AreH), 7.19 (2H, dd, J ¼ 9.5, 2.5 Hz, AreH), 7.32 (1H, s,
AreH), 7.51 (1H, d, J ¼ 8.0 Hz, AreH), 7.70 (1H, d, J ¼ 8.0 Hz, AreH),
7.90 (2H, dd, J ¼ 8.5, 2.5 Hz, AreH), 8.19 (1H, s, AreH), 8.34 (1H, s,
NH). 13C NMR (CDCl3)
d 95.86 (C4 isoxazole), 103.63, 111.63, 115.89
(d, J ¼ 25.1 Hz, C-30, C-50), 119.00, 120.16, 120.28, 125.08, 125.59,
128.06, 128.72 (d, J ¼ 10.2 Hz, C-20, C-60), 136.75, 162.00, 162.90 (d,
J ¼ 248.5 Hz, C-40), 169.03. MS (EIþ) 278.09 (Mþ). HRMS (ESI) found
for C17H11FN2O m/z ¼ 279.0932 [M þ H]þ; calcd.: 278.0855.
5.1.5.2.5. 5-(1H-Indol-5-yl)-3-(3,4,5-trimethoxyphenyl)isoxazole
(18e). Yield 29%; mp 57e58 ꢃC. 1H NMR (CDCl3)
d 3.94 (3H, s,
OCH3), 3.95 (6H, s, 2ꢂ OCH3), 6.66 (1H, s, AreH), 6.78 (1H, s, AreH),
7.14 (2H, s, H-20, H-60), 7.30 (1H, s, AreH), 7.50 (1H, d, J ¼ 8.0 Hz,
AreH), 7.70 (1H, d, J ¼ 8.0 Hz, AreH), 8.19 (1H, s, AreH), 8.77 (1H, s,
NH). 13C NMR (CDCl3)
d
56.31 (2ꢂ OCH3), 60.93 (OCH3), 96.01 (C4
isoxazole), 103.47, 104.16, 111.67, 118.94, 119.53, 120.07, 124.98,
125.71, 128.07, 136.80, 139.54, 153.61, 162.85, 172.17. MS (EIþ) 350.13
(Mþ). HRMS (ESI) found for C20H18N2O4 m/z ¼ 351.1343 [M þ H]þ;
calcd.: 350.1267.
(CDCl3)
d 3.06 (1H, s, acetylene), 6.33 (1H, s, AreH), 7.10 (1H, d,
J ¼ 1.5 Hz,1H, AreH), 7.12 (1H, dd, J ¼ 3.5, 2.0 Hz, AreH), 7.25 (1H, d,

270
S.F. Md Tohid et al. / European Journal of Medicinal Chemistry 56 (2012) 263e270
5.2. Biology
Education and Universiti Putra Malaysia for the award of a PhD
studentship (to SFMT). Support of the EPSRC National Mass Spec-
trometry Service (Swansea, U.K.) is also acknowledged.
5.2.1. Cell viability assay
The human cancer cell lines Colo320 (colon) and Calu-3 (lung)
(American Type Culture Collection, Manassas, VA), were cultured
in DMEM medium supplemented with L-glutamine (2 mM), 10%
Appendix A. Supplementary data
foetal bovine serum, 2.5% horse serum, 50 IU/mL penicillin and
50 mg/mL streptomycin (SigmaeAldrich, Milano, Italy) at 37 ꢃC in
an atmosphere of 5% CO2. Cell viability was measured using
a method based on the cleavage of the tetrazolium salt WST-1 to
formazan by mitochondrial dehydrogenase activity (cell prolif-
eration reagent WST-1; Roche, Mannheim, Germany). Cells
(2 ꢂ 103/well) were seeded into 96-well microtitre plates and
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.08.
009.
References
[1] N.I. Ziedan, H. Kadri, A.D. Westwell, The development of pro-apoptotic cancer
therapeutics, Mini. Rev. Med. Chem. 8 (2008) 711e718.
received compounds from 0.1 to 100 mM for 72 h. Following drug
[2] H.-Z. Zhang, S. Kasibhatla, J. Kuemmerle, W. Kemnitzer, K. Ollis-Mason, L. Qiu,
C. Crogan-Grundy, B. Tseng, J. Drewe, S.X. Cai, Discovery and structure-
activity relationship of 3-aryl-5-aryl-1,2,4-oxadiazoles as a new series of
apoptosis inducers and potential anticancer agents, J. Med. Chem. 48 (2005)
5215e5223.
exposure, WST-1 was added and the absorbance was measured at
450 nm using a microplate reader. Inhibition of proliferation was
assessed as the percentage reduction of absorbance of treated
cells versus control cultures. Potency (the concentration of
compounds that decreased cell viability by 50%, IC50) and efficacy
(maximum cell growth inhibition, Emax) was calculated by non-
linear least squares curve fitting (GraphPad Software, San Diego,
CA, USA). DMSO concentration in the culture medium never
exceeded 0.2%.
We also performed additional in vitro experiments on human
bronchial smooth muscle cells (Lonza, Walkersville, MD, USA) as
a potential measure of side effects of the compounds in vitro. Cells
were routinely grown in DMEM containing 10% FBS and exposed to
test compound concentrations equal to their IC50 mean values in
the relatively less responsive human cancer cell line (Calu-3). All
other experimental conditions, such as cell number and time of
exposure, were identical to those used in the anticancer screening
experiments described above.
[3] W. Kemnitzer, J. Kuemmerle, H.-Z. Zhang, S. Kasibhatla, B. Tseng, J. Drewe,
S.X. Cai, Discovery of 3-aryl-5-aryl-1,2,4-oxadiazoles as
a new series of
apoptosis inducers. 2. Identification of more aqueous soluble analogs as
potential anticancer agents, Bioorg. Med. Chem. Lett. 19 (2009) 4410e4415.
[4] N.I. Ziedan, F. Stefanelli, S. Fogli, A.D. Westwell, Design, synthesis and pro-
apoptotic antitumour properties of indole-based 3,5-disubstituted oxadia-
zoles, Eur. J. Med. Chem. 45 (2010) 4523e4530.
[5] N.I. Ziedan, A.D. Westwell, S. Fogli, Design and synthesis of novel indole
derivatives as selective apoptosis-inducers, Eur. J. Cancer Suppl. 6 (2008)
45e46.
[6] D. Simoni, M. Roberti, F.P. Invidiata, R. Rondanin, R. Baruchello, C. Malagutti,
A. Mazzali, M. Rossi, S. Grimaudo, F. Capone, L. Dusonchet, M. Meli,
M.V. Raimondi, M. Landino, N. D’Alessandro, M. Tolomeo, D. Arindam, S. Lu,
D.M. Benbrook, Heterocycle-containing retinoids. Discovery of a novel iso-
xazole arotinoid possessing potent apoptotic activity in multidrug and drug-
induced apoptosis-resistant cells, J. Med. Chem. 44 (2001) 2308e2318.
[7] A. Brancale, R. Silvestri, Indole, a core nucleus for potent inhibitors of tubulin
polymerization, Med. Res. Rev. 27 (2007) 209e238.
[8] R.J. Cox, D.J. Ritson, T.A. Dane, J. Berge, J.P.H. Charmant, A. Kantacha, Room
temperature palladium catalysed coupling of acyl chlorides with terminal
alkynes, Chem. Commun. (2005) 1037e1039.
[9] K.M. Johnston, R.G. Shotter, Conversion of some alpha-acetylenic ketones and
related alpha, beta-dibromoketones into 3,5-diarylisoxazoles, J. Chem. Soc. C
(1968) 1774.
[10] R.M. Adlington, J.E. Baldwin, D. Catterick, G.J. Pritchard, L.T. Tang, The
synthesis of novel heterocyclic substituted alpha-amino acids; further
exploitation of alpha-amino acid alkynyl ketones as reactive substrates,
J. Chem. Soc. Perkin Trans. 1 (2000) 2311e2316.
[11] C.E. Stephens, R.K. Arafa, 3,5-Diarylisoxazoles: individualized three-step
synthesis and isomer determination using C-13 NMR or mass spectroscopy,
J. Chem. Educ. 83 (2006) 1336e1340.
5.2.2. Caspase activity assay
Enzyme activity was assessed by the Apo-ONE Homogeneous
Caspase-3/7 assay (Promega, Madison, WI, USA). Cells were seeded
at 7 ꢂ 103/well and treated with compounds at 10
mM (Colo320)
and 20 mM (Calu-3) for 4 h. Subsequently, the caspase-3/7 assay
substrate was added and the fluorescence was measured by spec-
trofluorimeter 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).
[12] A.A. Vieira, F.R. Bryk, G. Conte, A.J. Bortoluzzi, H. Gallardo, 1,3-Dipolar cyclo-
addition reaction applied to synthesis of new unsymmetric liquid crystal
compounds-based isoxazole, Tetrahedron Lett. 50 (2009) 905e908.
[13] G.J. Roth, B. Liepold, S.G. Muller, H.J. Bestmann, Further improvements of the
synthesis of alkynes from aldehydes, Synthesis (2004) 59e62.
[14] G.A. Lee, A simplified synthesis of unsaturated nitrogen-heterocycles using
nitrile betaines, Synthesis (1982) 508e509.
Acknowledgements
We thank the Egyptian Ministry of Higher Education for the
award of a PhD studentship (to NIZ); and the Ministry of Higher
[15] R. Pellegata, A. Italia, M. Villa, A facile synthesis of primary carboxamides,
Synthesis (1985) 517e519.