Synthesis and anticancer activities of novel 3,5-disubstituted-1,2,4-oxadiazoles

Article abstract of DOI:10.1016/j.bmcl.2009.03.158

A series of 3,5-disubstituted-1,2,4-oxadiazoles were synthesized and evaluated for their in vitro anti-proliferative activities against various cancer cell lines. Formation of 1,2,4-oxadiazole ring was accomplished by the reaction of amidoxime with carbox

Full text of DOI:10.1016/j.bmcl.2009.03.158

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2739–2741
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anticancer activities of novel 3,5-disubstituted-1,2,4-oxadiazoles
a
b
b,
Dalip Kumar a, , Gautam Patel , Emmanuel O. Johnson , Kavita Shah
*
*
a Chemistry Group, Birla Institute of Technology and Science, Pilani 333 031, India
b Department of Chemistry and Purdue Cancer Center, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3,5-disubstituted-1,2,4-oxadiazoles were synthesized and evaluated for their in vitro anti-pro-
liferative activities against various cancer cell lines. Formation of 1,2,4-oxadiazole ring was accomplished
by the reaction of amidoxime with carboxylic acids. The in vitro cytotoxic effects of 3,5-disubstituted-
1,2,4-oxadiazoles have been demonstrated across a wide array of tumor cell types and a few compounds
exhibited specificity towards pancreatic (3f, 3h, 3j, and 3k) and prostate (3n) cancer cells. Among the pre-
pared 3,5-disubstituted-1,2,4-oxadiazoles, compound 3n is the most selective (>450-fold) and compound
3p is the most cytotoxic (10 nM) against prostate cancer cell lines.
Received 11 February 2009
Revised 17 March 2009
Accepted 25 March 2009
Available online 5 April 2009
Keywords:
1,2,4-Oxadiazole
Anticancer agent
Selective cytotoxicity
Ó 2009 Elsevier Ltd. All rights reserved.
Recently, there has been wide interest in compounds containing
the 1,2,4-oxadiazole scaffold because of their unique chemical
structure and broad spectrum of biological properties including
tyrosine kinase inhibition,1 muscarinic agonism,2 histamine H3
antagonism,3 anti-inflammation,4 antitumor,5 and monoamine oxi-
dase inhibition.6 The 1,2,4-oxadiazoles are also widely used as het-
erocyclic amide or ester bioisosters7 and in the design of
dipeptidomimetics as peptide building blocks.8,9
Cancer is a fatal disease that has posed serious threat to human
health. Many classes of anticancer drugs have been developed.
However, most drugs cause undesirable side effects due to lack
of tumor specificity and multidrug resistance. Several factors aid
in cancer cell survival. These include variation in metabolism,
absorption and delivery of drug to target tissues, and tumor loca-
tion. For example, chemotherapy agents such as paclitaxel, vincris-
tine, and doxorubicin have lost their efficacy due to the
development of drug resistance from prolonged exposure. Com-
pounds such as epothilone and discodermolide exhibit activities
against multi-drug resistant cancer cells and have been evaluated
in clinical trials. Recently, ixabepilone, an epothilone derivative
was approved for breast cancer in the US. Several other epothilone
derivatives are in advanced clinical trials. Discodermolide, how-
ever, proved toxic and has been dropped.10 Therefore, the develop-
ment of novel and effective anticancer agents remains a major
challenge. In this letter, we report the synthesis of novel 3,5-disub-
stituted-1,2,4-oxadiazoles and measure their anticancer activities
in various cancer cell lines.
The 3,5-disubstituted-1,2,4-oxadiazoles
3 were synthesized
according to Scheme 1. The reaction of appropriate benzonitrile 1
with hydroxylamine hydrochloride under refluxing condition
afforded amidoxime 211a which was coupled with the correspond-
ing carboxylic acid using DCC in DMF to give 3,5-disubstituted-
1,2,4-oxadiazoles 3 in moderate yield.11b All synthesized com-
pounds were characterized by NMR and mass spectroscopy.12
A library of 14 compounds was tested in vitro for their antican-
cer potential in various human cancer cell lines: prostate (PC3,
DU145 and LnCaP), breast (MCF7 and MDA231), colon (HCT116),
and pancreas (PaCa2).13
All compounds decreased cell viability as determined by color-
imetric MTT assay, with IC50 values ranging from nanomolar to
greater than 1 mM (Table 1). More importantly, a few compounds
were highly potent and exhibited specificity towards one cell type
relative to others.
The activities of various 3,5-disubstituted-1,2,4-oxadiazoles re-
vealed3f, 3g, 3n, and 3p as potent cytotoxic agents against various
cancer cell lines (IC50 ranging from 10 nM to 860 nM). All of these
compounds possess common 1,2,4-oxadiazole nucleus in which
substitution at C-3 and C-5 positions play important roles in po-
tency and selectivity of 3,5-disubstituted-1,2,4-oxadiazoles 3.
Compounds 3a and 3b, with C-3 phenyl and C-5 4-fluorophenyl/
pyridin-4-yl groups, displayed weak inhibitory activity against
tested cancer cell lines.
Introduction of 30-cyclopentyloxy and 40-methoxy moieties at
C-3 aryl ring enhances the activity (3e, 3h in comparison to 3a,
3b, respectively). However, a larger group like benzyloxy at para
position of C-3 aryl ring (3c) is detrimental to the activity. Replace-
* Corresponding authors. Tel.: +91 1596 245073 279; fax: +91 1596 244183
(D.K.); tel.: +1 765 496 9470 (K.S.).
E-mail addresses: dalipk@bits-pilani.ac.in (D. Kumar), shah23@purdue.edu (K.
Shah).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.158

2740
D. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2739–2741
OH
O
O
N
N
a
N
R3
R1
R2
CN
R1
R2
R1
R2
a
b
N
NH2
R3
R1
R2
H or OH
Decreases potency
N
1
2
3
c
Provides potency
and specificity
H or OBn
Decreases potency
3k
3a: R1 = R2 = H, R3 = 4-FC6H4
3b: R1 = R2 = H, R3 = pyridin-4-yl
Piperidin-4-yl
(Highly potent and
b
C6H5(Not Potent)
3c: R1 = OCH3, R2 = OBn, R3 = pyridin-4-yl
3d: R1 = cyclopentyloxy, R2 = OCH3, R3 = C6H5
4-FC6H4
(MCF7 and PaCa2)
specific for several cancer
cell lines)
3e: R1 = cyclopentyloxy, R2 = OCH3, R3 = 4-FC6H4
3f: R1 = cyclopentyloxy, R2 = OCH3, R3 = 4-ClC6H4
3g: R1 = cyclopentyloxy, R2 = OCH3, R3 = 4-HOC6H4
3h: R1 = cyclopentyloxy, R2 = OCH3, R3 = pyridin-4-yl
3i: R1 = cyclopentyloxy, R2 = OCH3, R3 = pyridin-3-yl
3j: R1 = cyclopentyloxy, R2 = OCH3, R3 = (indol-3-yl)methyl
3k: R1 = cyclopentyloxy, R2 = OCH3, R3 = CH2COCH3
3l: R1 = OH, R2 = OCH3, R3 = 4-FC6H4
3d
3p
Pyrrolidin-2-yl
3e
(Highly potent and
specific for LnCaP)
3n
4-ClC6H4
(Highly potent and
specific for PaCa2)
3f
R3
3k
CH2COCH3
(PaCa2)
4-HOC6H4
3g
3j
(Highly potent for
Several cancer cell
lines)
3h
3i
3m: R1 = cyclopentyloxy, R2 = OCH3, R3 = N-Boc-pyrrolidin-2-yl
3n: R1 = cyclopentyloxy, R2 = OCH3, R3 = pyrrolidin-2-yl
3o: R1 = cyclopentyloxy, R2 = OCH3, R3 = N-Boc-piperidin-4-yl
3p: R1 = cyclopentyloxy, R2 = OCH3, R3 = piperidin-4-yl
(Indol-3-yl)methyl
(PaCa2)
d
d
Pyridin-4-yl
(PaCa2)
Pyridin-3-yl
(PaCa2)
Figure 1. SAR of 1,2,4-oxadiazoles (a) Substitution at R3 provides both specificity
and potency. (b) In this panel, R1 = OC5H9 and R2 = OCH3. Phenyl or Pyrindin-3-yl
substitution at R3 position decreases potency. CH2COCH3, pyrindin-4-yl or (Indol-3-
Scheme 1. Synthesis of 1,2,4-oxadiazoles. Reagents and conditions: (a) EtOH, H2O,
NH2OH.HCl, NaOH, reflux, 18 h; (b) DMF, DCC, aromatic/heteroaromatic/aliphatic
carboxylic acid, 0 °C to 30 °C, 3 h then 110 °C 12 h; (c) ethylacetoacetate, reflux; (d)
trifluoroacetic acid, DCM.
yl)methyl at R3 position confer specificity for PaCa2 cells (IC50 3.0–9.56
lM).
Substituents at R3 shown in bold confer high potency. While compounds containing
4-HOC6H5 and piperidin-4-yl substituents at R3 position are highly cytotoxic in
multiple cancer cell lines, both 4-ClC6H5 and pyrrolidin-2-yl are highly potent and
specific for PaCa2 and LnCaP cells, respectively.
ment of 4-fluorophenyl in 3a with 4-chlorophenyl or 4-hydroxy-
phenyl group yielded compounds 3f and 3g, respectively, with sig-
nificant improved activities against different cancer cell lines.
Compounds 3f with 4-chlorophenyl, and 3h with pyridin-4-yl
groups show selectivity against pancreatic cancer cell line with
pancreatic cancer cell line (3.01 lM). Replacement of C-5 phenyl
group of 3d with pyrrolidin-2-yl yielded compound 3n, which
exhibited high potency as well as selectivity against prostate can-
cer cell line (LnCaP, 43 nM). Substitution with piperidin-4-yl group
in 3d at C-5 position led to the compound 3p with significant im-
proved activity against most cancer cell lines and 6-fold selectivity
against LnCaP (10 nM), a prostate cancer cell line.
IC50 values of 0.74 lM and 9.33 lM, respectively. Significant loss
in activity was observed upon changing the position of nitrogen
atom in the C-5 pyridyl ring of compound 3h to obtain the isomer
3i. When C-5 aryl ring is replaced with an aliphatic ketone moiety
as represented by 3k, improved cytotoxicity was observed. Inter-
estingly, compound 3k also exhibited selective cytotoxicity against
The compound 3j with an (indol-3-yl)methyl group at the C-5
position was also selectively cytotoxic towards pancreatic
Table 1
Cytotoxicity profile of 3,5-disubstituted-1,2,4-oxadiazoles against selected human cancer cell lines, IC50 (lM)
1
2
O
N
R3
R1
R2
N
4
3
Compd
R1
R2
R3
PC3
DU145
>103
LnCaP
MCF7
MDA231
HCT 116
PaCa2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3n
3p
H
H
H
H
OBn
4-FC6H4
>103
23.7 0.5
710 0.5
>103
>103
>103
>103
>103
Pyridin-4-yl
Pyridin-4-yl
C6H5
4-FC6H4
4-ClC6H4
4-HOC6H4
Pyridin-4-yl
Pyridin-3-yl
(Indol-3-yl)methyl
CH2COCH3
4-FC6H4
>103
656.6 0.1
>103
>103
> 103
497 0.09
OCH3
OC5H9
OC5H9
OC5H9
OC5H9
OC5H9
OC5H9
OC5H9
OC5H9
OH
>103
>103
>103
>103
>103
>103
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
>103
>103
>103
>103
>103
>103
>103
>103
20.65 0.1
122.5 0.1
0.78 0.08
>103
87.93 0.5
20.8 0.4
0.86 0.05
>103
3.88 0.3
174.2 0.1
0.39 0.3
>103
55.47 0.1
395.5 0.1
0.31 0.05
240.1 0.1
>103
209.7 0.6
6.09 0.08
0.74 0.02
0.48 0.08
9.33 0.06
>103
9.56 0.08
3.01 0.03
350.7 0.09
19.49 0.08
0.54 0.03
>103
>103
162.3 0.08
>103
0.27 0.05
>103
>103
>103
>103
>103
>103
>103
>103
>103
>103
>103
>103
>103
302.1 0.1
28.34 0.4
174.2 0.1
53.3 0.1
184.8 0.3
>103
>103
>103
>103
>103
>103
OC5H9
OC5H9
Pyrrolidin-2-yl
Piperidin-4-yl
>103
242 0.1
0.057 0.07
0.043 0.04
0.010 0.02
>103
212.2 0.3
0.37 0.08
184.8 0.1
1.54 0.03
>103
3.88 0.3
These experiments were conducted in triplicates at three independent times. IC50 values were obtained using a dose response curve by nonlinear regression using a curve
fitting program, GraphPad Prism 5.0. Bold values show IC50 of less than 1 M. OC5H9 = Cyclopentyloxy.
l

D. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2739–2741
2741
(9.56 l
M) cancer cell line. Removal of 30-cyclopentyl group in com-
under nitrogen atmosphere and stirred the reaction mixture at the same
temperature for 1 h. To this reaction mixture appropriate benzamidoxime 2
(0.8 mmol) was added and stirred at 0 °C for 0.5 h. The reaction mixture was
slowly brought to 30 °C, and stirring continued for another 3 h. Gradually
temperature was raised to 110 °C, and further stirred for 10 h. The reaction
mixture was cooled to 25 °C and poured into ice-cold water (25 mL). Upon
addition of ethylacetate (25 mL) and stirring for 10 min, crystals of
dicyclohexylurea separated out and removed by filtration. Separated aqueous
phase was extracted with ethylacetate (2 ꢁ 20 mL) and the combined organic
phase was washed with brine, dried over anhydrous sodium sulfate.
Ethylacetate was distilled off and the residue thus obtained was purified by
flash column chromatography using ethylacetate–hexane (0–25%) as eluent to
afford pure oxadiazole 3.
pound 3e led to the compound 3l having 30-hydroxy-40-methoxy-
phenyl group at C-3 position, which results in complete loss of
activity. Aryl/Heteroaryl substituents with a polar functionality at
C-5 and 30-cyclopentyloxy-40-methoxyphenyl moiety at C-3 posi-
tion of 1,2,4-oxadiazole core is very important for the activity
and selectivity. The observed structure–activity relationship is
illustrated in Figure 1. So far, molecular targets responsible for
the observed cytotoxicity of this new series of 3,5-disubstituted-
1,2,4-oxadiazoles 3 have not been identified, and a reasonable
explanation of the substitutions described above is not yet possi-
ble. Among synthesized 3,5-disubstituted-1,2,4-oxadiazoles, com-
pounds 3f, 3g, 3n, and 3p were most potent and selective in the
in vitro assay.
12. Data for selected compounds 3a: 1H NMR (CDCl3, 400 MHz): dH = 8.26–8.23 (m,
2H), 8.18–8.16 (m, 2H), 7.54–7.50 (m, 3H), 7.27–7.23 (m, 2H). Compound 3b:
1H NMR (CDCl3, 400 MHz): dH = 8.89 (dd, 2H, J = 4.48, 1.64 Hz), 8.19–8.17 (m,
2H), 8.07 (dd, 2H, J = 4.44, 1.68 Hz), 7.56–7.52 (m, 3H). Compound 3c: 1H NMR
(CDCl3, 400 MHz): dH = 8.88 (d, 2H, J = 5.0 Hz), 8.06 (dd, 2H, J = 4.56, 1.56 Hz),
7.72 (dd, 1H, J = 8.36, 2.0 Hz), 7.67 (d, 1H, J = 1.96 Hz), 7.46 (d, 2H, J = 8.76 Hz),
7.41–7.32 (m, 3H), 7.00 (d, 1H, J = 8.4 Hz), 5.25 (s, 2H), 4.00 (s, 3H). Compound
3d: 1H NMR (CDCl3, 400 MHz): dH = 8.22 (dd, 2H, J = 5.20, 0.9 Hz), 7.76 (dd, 1H,
J = 6.24, 1.44 Hz), 7.67 (d, 1H, J = 1.44 Hz), 7.61–7.54 (m, 3H), 6.97 (d, 1H,
J = 6.3 Hz), 4.94–4.91 (m, 1H), 3.92 (s, 3H), 2.17–1.84 (m, 6H), 1.67–1.62 (m,
2H). Calcd m/z for C20H20N2O3: 336.1, found: 337.2 (M+H)+, 359.3 (M+Na).
Compound 3e: 1H NMR (CDCl3, 400 MHz): dH = 8.25–8.21 (m, 2H), 7.74 (dd, 1H,
J = 8.36, 2.04 Hz), 7.66 (d, 1H, J = 1.92 Hz), 7.27–7.22 (m, 2H), 6.97 (d, 1H,
J = 8.4 Hz), 4.93–4.90 (m, 1H), 3.92 (s, 3H), 2.04–1.84 (m, 6H), 1.67–1.62 (m,
2H). Calcd m/z for C20H19FN2O3: 354.1, found: 355.2 (M+H)+, 377.2 (M+Na).
Compound 3f: 1H NMR (CDCl3, 400 MHz): dH = 8.16 (d, 2H, J = 8.64 Hz), 7.74
(dd, 1H, J = 8.40, 1.92 Hz), 7.56 (d, 1H, J = 1.92 Hz), 7.53 (d, 2H, J = 8.64 Hz), 6.97
(d, 1H, J = 8.40 Hz), 4.93–4.90 (m, 1H), 3.92 (s, 3H), 2.06–1.84 (m, 6H), 1.67–
1.60 (m, 2H). Compound 3g: 1H NMR (CDCl3, 400 MHz): dH = 8.11 (d, 2H,
J = 8.80 Hz), 7.74 (dd, 1H, J = 8.28, 1.92 Hz), 7.66 (d, 1H, J = 1.92 Hz), 7.01–6.95
(m, 3H), 6.50 (s, 1H (OH)), 4.93–4.88 (m, 1H), 3.91 (s, 3H), 2.04–1.86 (m, 6H),
1.65–1.56 (m, 2H). Compound 3h: 1H NMR (CDCl3, 400 MHz): dH = 8.88 (dd,
2H, J = 4.48, 1.68 Hz), 8.06 (dd, 2H, J = 4.08, 1.68 Hz), 7.76 (dd, 1H, J = 8.40,
2.04 Hz), 7.66 (d, 1H, J = 1.96 Hz), 6.98 (d, 1H, J = 8.40 Hz), 4.94–4.90 (m, 1H),
3.93 (s, 3H), 2.06–1.85 (m, 6H), 1.68–1.61 (m, 2H). Calculate m/z for
C19H19N3O3: 337.1, found: 338.2 (M + H)+, 360.2 (M+Na). Compound 3i: 1H
NMR (CDCl3, 400 MHz): dH = 9.45 (dd, 1H, J = 2.08, 0.56 Hz), 8.84 (dd, 1H,
J = 4.88, 1.68 Hz), 8.48 (dt, 1H, J = 8.04, 2.0 Hz), 7.76 (dd, 1H, J = 8.4, 2.0 Hz),
7.67 (d, 1H, J = 1.92 Hz), 7.52 (ddd, 1H, J = 8, 4.8, 0.6 Hz), 6.98 (d, 1H,
J = 8.44 Hz), 4.93–4.92 (m, 1H), 3.93 (s, 3H), 2.04–1.85 (m, 6H), 1.71–1.62 (m,
2H), Calcd m/z for C19H19N3O3: 337.1, found: 338.2 (M+H)+, 360.2 (M+Na).
Compound 3j: 1H NMR (CDCl3, 400 MHz): dH = 8.22 (s, 1H), 7.70 (d, 1H,
J = 8.84 Hz), 7.64 (dd, 1H, J = 8.32, 1.92 Hz), 7.57 (d, 1H, J = 1.96 Hz), 7.37 (d, 1H,
J = 8.08 Hz), 7.24–7.13 (m, 3H), 6.91 (d, 1H, J = 8.44 Hz), 4.87–4.84 (m, 1H), 4.43
(s, 2H), 3.88 (s, 3H), 2.04–1.80 (m, 6H), 1.67–1.59 (m, 2H). Calcd m/z for
C23H23N3O3: 389.2, found: 390.3 (M+H)+, 412.3 (M+Na). Compound 3k: 1H
NMR (CDCl3, 400 MHz): dH = 7.65 (dd, 1H, J = 8.40, 2.04 Hz), 7.57 (d, 1H,
J = 1.96 Hz), 6.94 (d, 1H, J = 8.44 Hz), 4.89–4.86 (m, 1H), 4.1 (s, 2H), 3.91 (s, 3H),
2.35 (s, 3H), 2.17–1.80 (m, 6H), 1.64–1.58(m, 2H). Calcd m/z for C17H20N2O4:
316.1, found: 317.3 (M+H)+, 339.3 (M+Na). Compound 3l: 1H NMR (CDCl3,
400 MHz): dH = 8.24–8.20 (m, 2H), 7.73–7.69 (m, 2H), 7.23 (d, 2H, J = 4.6 Hz),
6.96 (d, 1H, J = 8.20 Hz), 5.72 (s, 1H (OH)), 3.97 (s, 3H). Compound 3n: 1H NMR
(CDCl3, 400 MHz): dH = 7.66 (dd, 1H, J = 8.36, 2.0 Hz), 7.57 (d, 1H, J = 1.96 Hz),
6.93 (d, 1H, J = 8.44 Hz), 4.89–4.87 (m, 1H), 4.56–4.52 (m, 1H), 3.90 (s, 3H),
3.24–3.20 (m, 1H), 3.12–3.08 (m, 1H), 2.33–2.29 (m, 1H), 2.17–1.80 (m, 8H),
1.64–1.58(m, 2H). Calcd m/z for C18H23N3O3: 329.2, found: 330.3 (M+H)+, 352.3
(M+Na). Compound 3o: 1H NMR (CDCl3, 200 MHz): dH = 7.63 (dd, 1H, J = 8.00,
2.00 Hz), 7.55 (d, 1H, J = 2.00 Hz), 6.91(d, 1H, J = 8.00 Hz), 4.89–4.78 (m, 1H),
4.16–4.09 (m, 2H), 3.89 (s, 3H), 3.19–2.87 (m, 2H), 2.14–1.85 (m, 10H), 1.70–
1.55 (m, 3H), 1.46 (s, 9H). Compound 3p: 1H NMR (CDCl3, 200 MHz): dH = 7.63
(dd, 1H, J = 8.00, 2.00 Hz), 7.54 (d, 1H, J = 2.00 Hz), 6.92 (d, 1H, J = 8.00 Hz),
4.86–4.78 (m, 1H), 3.88 (s, 3H), 3.40–2.87 (m, 9H), 2.16–1.61 (m, 8H), Calcd m/z
for C19H25N3O3: 343.2, found: 344.2 (M+H)+.
In summary, our SAR study shows that the 3,5-disubstituted-
1,2,4-oxadiazoles decrease cell viability in various cancer cell lines
with IC50 values ranging from 10 nM to >1 mM. While 3n was
highly specific and potent for LnCaP cells, a few others (3f, 3h, 3j,
and 3k) exhibited specificity towards pancreatic cell line. The sub-
stituents at C-3 and C-5 positions of 1,2,4-oxadiazole ring were
shown to be vital for potency, suggesting specific interactions of
these groups with biological targets. Extensive exploration of
structure–activity relationship of this novel 1,2,4-oxadiazole scaf-
fold and its biological target studies are underway.
Acknowledgment
The authors wish to thank the University Grants Commission,
New Delhi (Project F. No. 32-216/2006) for financial support.
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11. (a) Synthesis of amidoxime 2: To
a mixture of appropriate benzonitrile
(10 mmol) and hydroxyl-amine hydrochloride (20 mmol) in 50 mL of ethanol
was added dropwise aqueous solution of sodium hydroxide (20 mmol, 10 mL)
while maintaining temperature at 0 °C. The resulting mixture was allowed to
reflux with stiring for 18 h. Ethanol was distilled off under reduced pressure
and the crude product was taken into 50 mL of water. The pH (ꢀ2) of the
solution was adjusted with 1N HCl and the aqueous phase was washed with
ethylacetate (2 ꢁ 25 mL). Upon cooling (0 °C) and neutralization with sodium
carbonate gave off white precipitate which was filtered, washed and air dried
at 60 °C to afford pure amidoxime 2.; b Synthesis of 3,5-disubstituted-1,2,4-
oxadiazole 3: A solution of appropriate carboxylic acid (0.8 mmol) in dry DMF
(1 mL) was cooled to 0 °C and added dicyclohexylcarbodiimide (1.2 mmol)
13. Cells were cultured in RPMI-1640 media supplemented with 10% heat-
inactivated foetal bovine serum and 1% penicillin/streptomycin. For MTT
assay, cells were seeded in 96 well plates at a density of 4.0 ꢁ 103 cells per
well for 12 h. Cells were incubated with various concentrations of the
compounds ranging from 10 nM–1 mM. After 24 h, MTT (3-(4,5-
dimethyldiazol-2-yl)-2,5-diphenyltetrazoliumbromide) was added to the
final concentration of 0.5 mg/ml and incubated for 30 min. The cells were
washed twice with PBS and lysed in dimethylsulfoxide, and the absorbance
was measured at 570 nm.