Synthesis and In Vitro Anticancer Activity of Novel 1,3,4-Oxadiazole-Linked 1,2,3-Triazole/Isoxazole Hybrids

Article abstract of DOI:10.1002/jhet.3110

A series of new 1,3,4-oxadiazole-linked 1,2,3-triazole/isoxazole derivatives were designed and synthesized. All the synthesized compounds were screened for in vitro anticancer activity against four human cancer cells: HeLa (cervical), MDA-MB-231 (breast), DU-145 (prostate), and HEPG2 (liver). Among 17 compounds tested, 7a, 7c, and 7d showed potent activity toward four cell lines.

Full text of DOI:10.1002/jhet.3110

Month 2018
Synthesis and In Vitro Anticancer Activity of Novel 1,3,4-Oxadiazole-
Linked 1,2,3-Triazole/Isoxazole Hybrids
B. Madhavilatha,a† Debanjan Bhattacharjee,b† Gowravaram Sabitha,a,c
B. V. Subba Reddy,c,d J. S. Yadav,
c,d
*
*
Nishant Jain,b and B. Jagan Mohan Reddye
aNatural Products Chemistry Division, CSIR Indian Institute of Chemical Technology, Hyderabad 500007, India
bCenter for Chemical Biology, CSIR Indian Institute of Chemical Technology, Hyderabad 500007, India
cAcademy of Scientific and Innovative Research (AcSIR), 2 Rafi Marg, New Delhi 110 001, India
dCenter for Semiochemicals, CSIR Indian Institute of Chemical Technology, Hyderabad 500007, India
eDepartment of Chemistry, Adikavi Nannaya University, Rajahmundry 533296
*
E-mail: gowravaramsr@yahoo.com; sabitha@iict.res.in
†B. M. and D. B. contributed equally to this work and should be treated as first authors.
Received October 3, 2017
DOI 10.1002/jhet.3110
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).
A series of new 1,3,4-oxadiazole-linked 1,2,3-triazole/isoxazole derivatives were designed and synthe-
sized. All the synthesized compounds were screened for in vitro anticancer activity against four human can-
cer cells: HeLa (cervical), MDA-MB-231 (breast), DU-145 (prostate), and HEPG2 (liver). Among 17
compounds tested, 7a, 7c, and 7d showed potent activity toward four cell lines.
J. Heterocyclic Chem., 00, 00 (2018).
INTRODUCTION
Individually, 1,3,4-oxadiazole and triazole/isoxazole
moieties are of significant biological interest owing to
their importance in drugs and pharmaceuticals (Fig. 1).
To assess their combined biological impact, we designed
and synthesized a new series of hybrid molecules
containing 1,3,4-oxadiazole-linked triazole/isoxazole
units in one core and subjected them to in vitro
anticancer evaluation.
Cancer is a life-threatening disease and is considered as
the second most common cause of death worldwide after
cardiovascular diseases. Despite significant progress that
has been achieved in anticancer therapy, the development
of new anticancer drugs represents a major challenge to
medicinal chemist researchers. 1,3,4-Oxadiazoles are a
very important class of heterocyclic compounds, and
much attention has been paid to the chemistry and
biological activities of this nucleus. Oxadiazoles are
known to exhibit a wide range of biological activities,
such as anticancer [1–5], antibacterial [6,7], antifungal
[8,9], antidiabetic [10], antitubercular [11], and anti-
inflammatory [12–14]. In addition, the triazole scaffold is
an attractive moiety not only from a synthetic point of
view but also in the context of biological and drug
discovery applications [15–22] owing to its easy formation
by click chemistry. On the other hand, isoxazoles are core
components of many natural products [23–25], and their
wide range of biological activities [26–30] makes them
important anchors in the field of medicinal chemistry.
RESULTS AND DISCUSSION
Chemistry.
As illustrated in Scheme 1, aryl acid
hydrazides 3a–3d were synthesized by the reaction of
85% hydrazine hydrate with esters 2a–2d, which derived
from substituted benzoic acids 1a–1d, in methanol under
reflux condition. Treatment of compounds 3a–3d with
ethyl chloroformate in toluene in the presence of
potassium carbonate under reflux condition for 5 h gave
ethyl 2-benzoylhydrazinecarboxylates 4a–4d. The starting
materials, 5-aryl-1,3,4-oxadiazol-2(3H)-ones (5a–5d)
[31], were prepared in good yields by cyclization of
© 2018 Wiley Periodicals, Inc.

Vol 000
B. Madhavilatha, D. Bhattacharjee, G. Sabitha, B. V. S. Reddy, J. S. Yadav, N. Jain,
and B. J. M. Reddy
Figure 1. 1,3,4-Oxadiazole moiety containing pharmaceutical agents. IC50, half maximal inhibitory concentration.
Scheme 1. Synthesis of oxadiazole–triazole/isoxazole hybrids.
compounds 4a–4d under reflux conditions using POCl3.
Our study started with the reaction between 5-aryl-1,3,4-
oxadiazol-2(3H)-ones (5a–5d) and propargyl bromide
in the presence of NaH in tetrahydrofuran/
dimethylformamide (1:1) to obtain the 5-aryl-3-(prop-
2-ynyl)-1,3,4-oxadiazol-2(3H)-ones (6a–6d) [32]. In the
second step, these N-propargylated compounds 6a–6d
were independently reacted with various azides using
“click” chemistry and aldoximes, respectively giving
novel 3-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-aryl-
1,3,4-oxadiazol-2(3H)-one hybrids (7a–7h) and 5-aryl-
3-((3-arylisoxazol-5-yl)methyl)-1,3,4-oxadiazol-2(3H)-one
hybrids (8a–8i) in good yields (Scheme 1).
In this study, 17 derivatives, 7a–7h and 8a–8i, were
synthesized and reported for the first time. All the
1
synthesized compounds were characterized by H-NMR,
13C-NMR, and HRMS (the spectral data are included in
the supporting information) data.
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Month 2018
Novel 1,3,4-Oxadiazole Linked 1,2,3-Triazole/Isoxazole Hybrids
Biology. Antiproliferative activity. Meanwhile, in vitro
antitumor activities of these compounds were evaluated
against HeLa, MDA-MB-231, DU-145, and HEPG2 cells
with the ultimate aim of developing novel potent
antitumor agents.
Nocodazole and paclitaxel were employed as positive
controls (Table 1). In general, antimitotic drugs are used as
frontline therapies; hence, we choose these 2 antimitotic
agents to compare the activities of the congeners.
level of cyclin B1 is an indicator for G2/M arrest. Thus,
HeLa cells were treated with 5 μM of compounds 7a, 7c,
and 7d for 24 h and subjected to an immunoblot for
cyclin B1. We used nocodazole as a positive control and
tubulin as a loading control. Interestingly, 7a, 7c, and 7d
showed a marked increase in cyclin B1 level comparable
to nocodazole-treated cells (Fig. 2).
Immunoblot analysis of soluble versus insoluble
(polymerized) tubulin in HeLa cells. To further examine
whether these compounds disrupt microtubule dynamics,
we checked the levels of soluble versus insoluble
(polymerized) forms of tubulin. HeLa cells were treated
with 5 μM of compounds 7a, 7c, and 7d. Nocodazole
and dimethyl sulfoxide were used as positive and
negative controls, respectively (Fig. 3).
After 24 h of treatment, cells were permeabilized
to collect the soluble fraction, and the remaining cells
were collected as the insoluble fraction and subjected
to immunoblot analysis. This analysis revealed that
these compounds showed potent inhibition of tubulin
On the basis of the GI50 values, we have predicted the
structure–activity relationship of all the synthesized
compounds (7a–7h and 8a–8i), and we assumed that the
compounds containing methyl-substituted aromatic
groups present on the oxadiazole moiety such as 7a, 7c,
and 7d are potent toward the four human anticancer cells.
Halogens present on the aromatic rings attached to the
oxadiazole moiety affect the potentiality downwards.
As the bulkiness of the atoms (or) groups present on the
benzylic group attached to the triazole moiety shows
greater effects on the potentiality toward the cytotoxicity,
it was observed that the smaller the atom (or) group, the
greater was the potentiality in the case of 7a, 7c, and 7d.
The congeners having an isoxazole link to the
oxadiazole moiety showed moderate activity, which
indicates that triazole-ring-linked congeners are more
potent than the isoxazole-linked congeners. All these
structure activity relationship (SAR) assumptions were
deduced based on the preliminary GI50 results.
Immunoblot analysis of cellular cyclin B1 levels. As three
compounds in this series showed significant cytotoxicity,
we tested their effect on cellular cyclin B1. This is one of
the essential regulatory proteins of mitosis, and a high
Figure 2. Western blot analysis of cyclin B1. DMSO, dimethyl sulfoxide;
NOC, nocodazole.
Table 1
Growth inhibition of 50% (GI50)/μM values of compounds triazole and isoxazole series, 7a–7h and 8a–8i.
Compound
HeLa
MDA-MB-231
DU-145
HEPG2
7a
7b
7c
7d
1.28 0.3
3.62 1.1
1.7 0.25
0.82 0.15
1.75 0.2
6.4 0.7
2.14 0.6
1.04 0.2
2.39 0.4
7.21 0.8
1.72 0.04
0.96 0.04
1.84 0.2
4.25 0.6
1.78 0.1
1.42 0.25
7e
7f
7g
7h
8a
8b
8c
8d
8e
8f
8g
8h
4.25 0.8
6.13 0.7
8.23 0.9
6.39 0.5
7.29 0.9
5.7 0.9
8.16 0.3
6.95 0.6
7.82 0.8
10.32 1.1
6.2 0.8
6.24 0.04
6.92 0.02
9.58 1.2
8.11 0.2
10.41 1.2
8.8 0.6
6.26 0.7
8.12 0.08
6.26 0.9
9.13 0.9
8.82 1.2
6.34 0.5
10.66 1.1
0.027 0.002
0.034 0.003
8.82 0.6
9.65 0.8
8.24 0.7
7.25 1.1
6.47 0.8
11.27 1.4
9.43 1.1
5.17 0.2
7.55 0.4
6.72 0.45
7.51 0.4
9.3 1.2
6.8 0.05
8.26 0.5
10.22 0.6
5.16 0.08
9.92 0.6
6.01 0.2
11.23 1.2
8.27 0.4
8.13 1.1
7.34 0,6
6.81 0.8
9.29 0.8
7.92 0.7
8.37 0.9
0.034 0.003
0.021 0.001
8i
9.18 0.8
<0.016 0.004
0.023 0.002
8.45 0.9
0.05 0.002
<0.011 0.002
Paclitaxel
Nocodazole
Potent towards the four human anti-cancer cells are shown in bold font.
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B. Madhavilatha, D. Bhattacharjee, G. Sabitha, B. V. S. Reddy, J. S. Yadav, N. Jain,
and B. J. M. Reddy
precoated silica gel 60 F254 (mesh); spots were visualized
with UV light. Merck silica gel (180–200 mesh) was
used for column chromatography.
General
procedure
for
the
synthesis
of
5-aryl-1,3,4-oxadiazol-2(3H)-ones (5a–5d).
The 5-aryl-
1,3,4-oxadiazol-2(3H)-ones (5a–5d) were synthesized by
a modified literature method as follows: POCl3 (200 mL,
mmol) was taken in a round-bottomed flask, and much
ethyl 2-benzoylhydrazinecarboxylate (4a–4d) (mmol) was
added to it at room temperature. After the addition is
over, it was slowly heated to reflux for 5 h. POCl3 was
distilled, and then the reaction mass was poured into
crushed ice and filtered, and the residue was purified by
recrystallization from methanol to afford the
corresponding products.
Figure 3. Western blot analysis of distribution of tubulin in soluble (S)
versus insoluble (IN) fractions. DMSO, dimethyl sulfoxide; NOC,
nocodazole.
polymerization and possessed increased levels of
insoluble tubulin fraction similar to nocodazole-treated
cells.
General procedure for the synthesis of 5-aryl-3-(prop-2-
ynyl)-1,3,4-oxadiazol-2(3H)-ones
(6a–6d).
To
a
suspension of NaH (12.3 mmol, 2 equiv) in anhydrous
tetrahydrofuran (20 mL) was added a solution of 5-aryl-
1,3,4-oxadiazol-2(3H)-one (5a–5d) (6.17 mmol, 1 equiv)
in anhydrous dimethylformamide (10 mL) in a dropwise
manner at 0°C. After 30 min, propargyl bromide
(9.87 mmol, 1.6 equiv) was added at 0°C and allowed to
stir for 30 min at room temperature. After completion,
followed by TLC, the mixture was quenched with ice
H2O (15 mL) and extracted with ethyl acetate
(2 × 50 mL). The combined organic layer was dried over
anhydrous Na2SO4, concentrated, and purified by silica
gel column chromatography (hexane : ethyl acetate, 9:1)
CONCLUSION
In the present study, a focused library of 1,3,4-
oxadiazole-triazole/isoxazole moieties conjugated through
a nitrogen linkage is synthesized and evaluated for their
anticancer activity against four human cancer cell lines:
HeLa, MDA-MB-231, DU-145, and HEPG2. Among
them, 7a, 7c, and 7d showed a potent anticancer activity
against all the tested cancer cell lines and inhibited tubulin
polymerization. Western blot analysis in the HeLa cell
line showed that these lead compounds induced cyclin B1
protein level, which is a strong indicator of G2/M cell
cycle arrest. These compounds also disrupted microtubule
assembly and increased the amount of insoluble tubulin
fractions of cells like other potent tubulin polymerization
inhibitors such as nocodazole. These results demonstrate
that the triazole and isoxazole series compounds are
potent tubulin polymerization inhibitors, which can serve
as a useful template for the generation of a related new
class of molecules as potential anticancer drugs.
to afford the pure O-propargylated compounds 6a–6d.
5-Phenyl-3-(prop-2-yn-1-yl)-1,3,4-oxadiazol-2(3H)-one
(6a). Colorless solid; yield 98%; mp 60–62°C; IR (KBr):
1
2969, 1782, 1670, 1351, 740 cmꢀ1; H-NMR (400 MHz,
CDCl3): δ 7.89–7.85 (m, 2H), 7.55–7.45 (m, 3H), 4.61
(d, J = 2.6 Hz, 2H), 2.42 (t, J = 2.4 Hz, 1H); 13C-NMR
(125 MHz, CDCl3): δ 153.7, 152.7, 131.8, 128.9 (2C),
125.8 (2C), 123.5, 75.7, 73.8, 35.7; HRMS (ESI) for
C11H9N2O2 [M + H]+, found 201.0668, Calcd 201.0664.
3-(Prop-2-yn-1-yl)-5-(p-tolyl)-1,3,4-oxadiazol-2(3H)-one
(6b). Colorless solid; yield 96%; mp 76–77°C; IR (KBr):
1
2965, 1782, 1608, 1350, 741 cmꢀ1; H-NMR (400 MHz,
EXPERIMENTAL
CDCl3): δ 7.75 (d, J = 8.3 Hz, 2H), 7.28 (d, J = 7.9 Hz,
2H), 4.59 (d, J = 2.6 Hz, 2H), 2.42–2.40 (m, 4H); 13C-
NMR (100 MHz, CDCl3): δ 153.9, 152.8, 142.4, 129.6
(2C), 125.7 (2C), 120.7, 75.8, 73.7, 35.6, 21.6; HRMS
(ESI) for C12H11N2O2 [M + H]+, found 215.0828, Calcd
Chemistry.
Melting points were recorded on an
Electrothermal melting point apparatus (Hyderabad,
India) and are uncorrected. IR spectra were recorded on a
Perkin-Elmer
spectrophotometer (Hyderabad, India) using KBr optics.
1H-NMR
spectra were recorded on Bruker
Fourier
transform
IR
240-C
215.0820.
5-(4-Chlorophenyl)-3-(prop-2-yn-1-yl)-1,3,4-oxadiazol-
2(3H)-one (6c). Colorless solid; yield 98%; mp 62–64°C;
AV (Hyderabad, India) 300 MHz, CDCl3, using
tetramethylsilane as an internal standard. Electron spray
1
IR (KBr): 3092, 2923, 1781, 1384, 837 cmꢀ1; H-NMR
ionization (ESI) and HRMS were recorded on
QSTARXL hybrid MS/MS system (Applied
Biosystems, Hyderabad, India). All the reactions were
monitored by thin-layer chromatography (TLC) on
a
(400 MHz, CDCl3): δ 7.80 (d, J = 8.8 Hz, 2H), 7.46 (d,
J = 8.8 Hz, 2H), 4.60 (d, J = 2.4 Hz, 2H), 2.42 (t,
J = 2.4 Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ 152.9,
152.5, 138.1, 129.4 (2C), 127.1 (2C), 122.0, 75.6, 73.9,
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Month 2018
Novel 1,3,4-Oxadiazole Linked 1,2,3-Triazole/Isoxazole Hybrids
35.8; HRMS (ESI) for C11H8ClN2O2 [M + H]+, found
235.0279, Calcd 234.0274.
125.6 (2C), 122.7, 120.8, 54.2, 41.4, 21.6; HRMS (ESI)
for C19H17N5O2 Na [M + Na]+, found 370.1298, Calcd
5-(4-Bromophenyl)-3-(prop-2-yn-1-yl)-1,3,4-oxadiazol-
2(3H)-one (6d). Colorless solid; yield 96%; mp 70–72°C;
370.1274.
3-((1-(2-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-5-(p-
tolyl)-1,3,4-oxadiazol-2(3H)-one (7d). Colorless solid; yield
IR (KBr): 2969, 2926, 1779, 820 cmꢀ1
;
1H-NMR
(400 MHz, CDCl3): δ 7.73 (d, J = 8.7 Hz, 2H), 7.63 (d,
J = 8.6 Hz, 2H), 4.60 (d, J = 2.6 Hz, 2H), 2.42 (t,
J = 2.6 Hz, 1H); 13C-NMR (100 MHz, CDCl3): δ 153.0,
152.5, 132.3 (2C), 127.2 (2C), 126.5, 122.4, 75.6, 73.9,
35.8; HRMS (ESI) for C11H8BrN2O2 [M + H]+, found
278.9779, Calcd 278.6769.
76%; mp 176–178°C; IR (KBr): 3071, 2985, 1772, 1615,
1
1343, 766 cmꢀ1; H-NMR (400 MHz, CDCl3): δ 7.68 (t,
J = 8.2 Hz, 3H), 7.38–7.27 (m, 2H), 7.24(d, J = 8.1 Hz,
2H) 7.17–7.09 (m, 2H), 5.58 (s, 2H), 5.09 (s, 2H), 2.39
(s, 3H); 13C-NMR (75 MHz, CDCl3): δ 160.5 (d, JC-
= 247.9 Hz), 153.6, 153.2, 142.2, 142.0, 131.0 (d, JC-
F
= 8.1 Hz) 130.6 (d, JC-F = 2.9 Hz), 129.6 (2c), 125.7
(2c), 124.8 (d, JC-F = 2.9 Hz), 122.9, 121.5 (d, JC-
= 14.5 Hz), 120.8, 115.8 (d, JC-F = 20.5 Hz), 47.7 (d,
General procedure for the synthesis of 3-[(1-benzyl-1H-
1,2,3-triazol-4-yl)methyl]-5-aryl-1,3,4-oxadiazol-2(3H)-ones
F
(7a–7h).
To a mixture of (azidomethyl)-benzenes
F
(0.55 mmol) were added propargyl compounds (6a–6d)
(0.5 mmol) in tBuOH : H2O (1:1, 15 mL), CuSO4·5H2O
(0.1 mmol), and sodium ascorbate (0.47 mmol) at room
temperature. The mixture was stirred for 2 h at the same
temperature. The mixture was poured into ice water
(10 mL) with stirring. The solution was extracted with
ethyl acetate (3 × 20 mL), washed with brine, dried over
MgSO4, and concentrated in vacuo. The crude product
was purified by column chromatography (petroleum
ether : ethyl acetate, 5:5) to afford the pyrazolotriazole
JC-F
=
3.7 Hz), 41.4, 21.6; HRMS (ESI) for
C19H17FN5O2 [M + H]+, found 366.1375, Calcd 366.1366.
5-(4-Chlorophenyl)-3-((1-(4-methoxybenzyl)-1H-1,2,3-
triazol-4-yl)methyl)-1,3,4-oxadiazol-2(3H)-one
(7e).
Colorless solid; yield 88%; mp 178–180°C; IR (KBr):
3073, 1780, 1613, 1492, 1095, 836 cmꢀ1 1H-NMR
;
(500 MHz, CDCl3): δ 7.74 (d, J = 8.7 Hz, 2H), 7. 53 (s,
1H), 7.43 (d, J = 8.7 Hz, 2H), 7.27–7.21 (m, 2H), 6.89
(d, J = 8.7 Hz, 2H), 5.46 (s, 2H), 5.07 (s, 2H), 3.81 (s,
3H); 13C-NMR (100 MHz, CDCl3): 160.0, 152.9, 152.6,
141.6, 137.8, 129.7 (2C), 129.3 (2C), 126.9 (2C), 126.1,
122.5, 122.1, 114.5 (2C), 55.3, 53.9, 41.5; HRMS (ESI)
for C19H16ClN5O3Na [M + Na]+, found 420.0866, Calcd
hybrid compounds (7a–7h).
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-phenyl-1,3,4-
oxadiazol-2(3H)-one (7a). Colorless solid; yield 89%; mp
160–162°C; IR (KBr): 3141, 2925, 1773, 1610,
420.0839.
1
830 cmꢀ1; H-NMR (400 MHz, CDCl3): δ 7.83–7.78 (m,
5-(4-Chlorophenyl)-3-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-
2H), 7.57 (s, 1H), 7.52–7.41 (m, 3H), 7.40–7.34 (m, 3H),
7.30–7.25 (m, 2H), 5.52 (s, 2H), 5.09 (s, 2H); 13C-NMR
(100 MHz, CDCl3): δ 152.9, 152.7, 141.7, 134.2, 132.2
(2C), 129.1(2C), 128.9, 128.1(2C), 127.1 (2C), 126.3,
122.7, 122.5, 54.3, 41.5; HRMS (ESI) for
C18H15N5O2Na [M + Na]+, found 356.1148, Calcd
yl)methyl)-1,3,4-oxadiazol-2(3H)-one (7f).
Pale yellow
solid; yield 80%; mp 150–151°C; IR (KBr): 3086, 2922,
1774, 1521, 1345, 729 cmꢀ1 1H-NMR (4500 MHz,
;
CDCl3): δ 8.23 (d, J = 8.6 Hz, 2H), 7. 75 (d, J = 8.6 Hz,
2H), 7.66 (s, 1H), 7.47–7.40 (m, 4H), 5.64 (s, 2H), 5.12
(s, 2H); 13C-NMR (100 MHz, CDCl3): δ 152.9, 152.7,
148.1, 142.3, 141.1, 138.0, 129.3 (2C), 128.6 (2C), 126.9
(2C), 124.3 (2C), 123.0, 121.9, 53.2, 41.5; HRMS (ESI)
for C18H14ClN6O4 [M + H]+, found 413.0787, Calcd
356.1118.
3-((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-5-(p-
tolyl)-1,3,4-oxadiazol-2(3H)-one (7b). Colorless solid; yield
83%; mp 147–149°C; IR (KBr): 3108, 2922, 1776, 1615,
413.0765.
1
1252, 823 cmꢀ1; H-NMR (400 MHz, CDCl3): δ 7.68 (d,
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-(4-
bromophenyl)-1,3,4-oxadiazol-2(3H)-one (7g). Pale yellow
J = 8.2 Hz, 2H), 7.52 (s, 1H), 7.27–7.21 (m, 4H), 6.81
(d, J = 8.6 Hz, 2H), 5.45 (s, 2H), 5.06 (s, 2H), 3.80 (s,
3H), 2.39 (s, 3H); 13C-NMR (125 MHz, CDCl3): δ
159.9, 153.6, 153.2, 142.2, 129.7 (2c), 129.6 (3c), 126.2,
125.6 (2c), 122.5, 120.8, 114 (2c), 55.2, 53.8, 41.4, 21.6;
HRMS (ESI) for C20H20N5O3 [M + H]+, found 378.1575,
Calcd 378.1566.
3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)-5-(p-tolyl)-1,3,4-
oxadiazol-2(3H)-one (7c). Colorless solid; yield 80%; mp
solid; yield 82%; mp 197–198°C; IR (KBr): 3276, 2930,
1776, 1608, 1402, 836 cmꢀ1 1H-NMR (500 MHz,
;
CDCl3): δ 7.69 (d, J = 8.9, 2.1 Hz, 2H), 7. 59 (d, J = 8.8,
2.1 Hz, 2H), 7.56 (s, 1H), 7.40–7.35 (m, 3H), 7.30–7.26
(m, 2H), 5.52 (s, 2H), 5.08 (s, 2H); 13C-NMR (125 MHz,
CDCl3): δ 153.5, 153.2, 141.9, 133.8, 133.2, 131.6,
130.5, 130.4, 128.9 (2c), 128.2, 125.7 (2c), 123.6, 123.5,
123.1, 53.9, 41.5; HRMS (ESI) for C18H15BrN5O2
[M + H]+, found 412.0213, Calcd 412.0209.
167–169°C; IR (KBr): 3132, 2995, 1770, 1609, 1347,
747 cmꢀ1 1H-NMR (400 MHz, CDCl3): δ 7.69 (d,
;
5-(4-Bromophenyl)-3-((1-(2-fluorobenzyl)-1H-1,2,3-triazol-
J = 8.3 Hz, 2H), 7.58 (brs, 1H), 7.40–7.34 (m, 4H),
7.29–7.21 (m, 3H), 5.52 (s, 2H), 5.08 (s, 2H), 2.39 (s,
3H); 13C-NMR (125 MHz, CDCl3): δ 153.6, 153.2,
142.2, 134.2, 129.6 (3C), 129.1 (2C), 128.8, 128.1 (2C),
4-yl)methyl)-1,3,4-oxadiazol-2(3H)-one (7h).
Pale yellow
solid; yield 75%; mp 174–176°C; IR (KBr): 3130, 2985,
1
1765, 1380, 823 cmꢀ1; H-NMR (400 MHz, CDCl3): δ
7.69–7.65 (m, 3H), 7.60–7.57 (m, 2H), 7.40–7.34 (m,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Vol 000
B. Madhavilatha, D. Bhattacharjee, G. Sabitha, B. V. S. Reddy, J. S. Yadav, N. Jain,
and B. J. M. Reddy
1H), 7.31–7.26 (m, 1H), 7.17–7.09 (m, 2H), 5.59 (s, 2H),
5.09 (s, 2H); 13C-NMR (100 MHz, CDCl3): δ 161.7 (d,
JC-F = 247.3 Hz), 152.9, 152.7, 141.7, 132.2 (2c), 131.0
(d, JC-F = 8.1 Hz), 130.6 (d, JC-F = 2.9 Hz), 127.1 (2c),
126.3, 124.8 (d, JC-F = 3.7 Hz), 122.9, 122.5, 121.5 (d,
JC-F = 14.7 Hz), 115.8 (d, JC-F = 20.5 Hz), 47.8(d, JC-
HRMS (ESI) for C19H16N3O4 [M + H]+, found 350.1151,
Calcd 350.1140.
5-(p-Tolyl)-3-((3-(p-tolyl)isoxazol-5-yl)methyl)-1,3,4-
oxadiazol-2(3H)-one (8d). Colorless solid; yield 72%; mp
113–115°C; IR (KBr): 3127, 2824, 1761, 1611, 1345,
824 cmꢀ1 1H-NMR (400 MHz, CDCl3): δ 7.74 (d,
;
J = 8.3 Hz, 2H), 7.50–7.46 (m, 1H), 7.37–7.23 (m, 5H),
6.52 (s, 1H), 5.18 (s, 2H), 2.47 (s, 3H), 2.41 (s, 3H); 13C-
NMR (100 MHz, CDCl3): δ 164.8, 163.3, 154.1, 153.0,
142.5, 136.8, 131.0, 129.7 (2c), 129.6, 129.4, 128.1,
125.9, 125.7 (2c), 120.5, 104.3, 41.3, 21.6, 21.1; HRMS
(ESI) for C20H17N3O3Na [M + Na]+, found 370.1194,
= 4.4 Hz), 41.5; HRMS (ESI) for C18H14BrFN5O2
F
[M + H]+, found 432.0344, Calcd 432.0314.
General procedure for the synthesis of 5-aryl-3-((3-
phenylisoxazol-5-yl)methyl)-1,3,4-oxadiazol-2(3H)-ones (8a–
8i). Propargylated compounds (6a–6d) (0.5 mmol) and
triethylamine (0.5 mmol) were taken in dichloromethane
(5 mL), and the resulting mixture was cooled to 0°C.
Aqueous sodium hypochlorite (11%, 10 mL) of 10 mL
was added over 30 min at 0°C, followed by aryl aldehyde
oximes (1.0 mmol), and the reaction mixture was
maintained at room temperature. After completion of the
reaction monitored by TLC, the reaction mixture was
concentrated under vacuum and extracted with ethyl
acetate. The organic layers were separated and dried over
anhydrous sodium sulfate. The resulting crude product
was washed with n-hexane to obtain pyrazoloisoxazole
Calcd 370.1162.
5-(4-Chlorophenyl)-3-((3-(p-tolyl)isoxazol-5-yl)methyl)-
1,3,4-oxadiazol-2(3H)-one (8e).
White solid; yield 78%;
mp 154–156°C; IR (KBr): 3127, 2966, 1773, 1614, 1392,
834 cmꢀ1 1H-NMR (400 MHz, CDCl3): δ 7.78 (d,
;
J = 8.5 Hz, 2H), 7.49–7.43 (m, 3H), 7.36–7.23 (m, 3H),
6.53 (s, 1H), 5.16 (s, 2H), 2.47 (s, 3H); 13C-NMR
(100 MHz, CDCl3): δ 164.5, 163.3, 153.1, 152.8, 138.2,
136.9, 131.0, 129.6, 129.3 (3c), 128.1, 127.0 (2c), 125.9,
121.8, 104.4, 41.3, 21.1; HRMS (ESI) for
C19H14ClN3O3Na [M + Na]+, found 390.0643, Calcd
hybrid products (8a–8i).
5-Phenyl-3-((3-(p-tolyl)isoxazol-5-yl)methyl)-1,3,4-
390.0621.
3-((3-(4-Bromophenyl)isoxazol-5-yl)methyl)-5-(4-
oxadiazol-2(3H)-one (8a).
White solid; yield 71%; mp
115–117°C; IR (KBr): 3140, 2995, 1785, 1573, 1397,
830 cmꢀ1 1H-NMR (400 MHz, CDCl3): δ 7.87–7.83
chlorophenyl)-1,3,4-oxadiazol-2(3H)-one (8f).
Pale yellow
solid; yield 67%; mp 119–121°C; IR (KBr): 3117, 2956,
;
1
1775, 1602, 1362, 829, 764 cmꢀ1; H-NMR (500 MHz,
(m, 2H), 7.54–7.45 (m, 5H), 7.34–7.24 (m, 2H), 6.53
(s, 1H), 5.17 (s, 2H), 2.47 (s, 3H); 13C NMR
(125 MHz, CDCl3): δ 164.7, 163.3, 153.9, 153.0,
136.9, 131.9, 131.1, 129.6, 129.4, 129.0 (2c), 128.9,
125.9, 125.8 (2c), 123.4, 104.4, 41.3, 21.1; HRMS
(ESI) for C19H15N3O3Na [M + Na]+, found 356.1035,
Calcd 356.1006.
3-((3-(4-Bromophenyl)isoxazol-5-yl)methyl)-5-phenyl-1,3,4-
oxadiazol-2(3H)-one (8b). Colorless solid; yield 80%; mp
CDCl3): δ 7.81–7.76 (m, 2H), 7.67(d, J = 8.5 Hz, 2H),
7.59(d, J = 8.5 Hz, 2H), 7.49–7.42 (m, 2H), 6.64 (s, 1H),
5.15 (s, 2H); 13C-NMR (100 MHz, CDCl3): 165.8, 161.8,
153.2, 152.8, 138.3, 132.2 (2c), 129.4 (2c), 129.3, 128.3
(2c), 127.1 (2c), 124.6, 121.8, 101.8, 41.3; HRMS (ESI)
for C18H12BrClN3O3 [M
Calcd431.9750.
+
H]+, found 431.9762,
5-(4-Bromophenyl)-3-((3-phenylisoxazol-5-yl)methyl)-1,3,4-
oxadiazol-2(3H)-one (8g). Pale yellow solid; yield 82%;
155–157°C; IR (KBr): 3066, 2927, 1794, 1613, 1291,
897 cmꢀ1 1H-NMR (400 MHz, CDCl3): δ 7.85 (d,
;
mp 122–124°C; IR (KBr): 3276, 2930, 1776, 1608, 1402,
1
836 cmꢀ1; H-NMR (400 MHz, CDCl3): 7.83–7.66 (m,
J = 6.9 Hz, 1H), 7.78 (d, J = 7.1 Hz, 1H), 7.67 (d,
J = 8.4 Hz, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.56–7.43
(m, 5H) 6.64 (s, 1H), 5.16 (s, 2H); 13C-NMR
(125 MHz, CDCl3): δ 166.0, 161.8, 154.0, 153.0, 132.2
(2C), 132.0, 129.0 (2C), 128.7, 128.3 (2C), 126.9,
125.8 (2C), 123.3, 101.7, 41.3; HRMS (ESI) for
C18H13BrN3O3 [M + H]+, found 398.0170, Calcd
4H), 7.64–7.57 (m, 3H), 7.46–7.40 (m, 2H), 6.65 (s, 1H),
5.14 (s, 2H); 13C-NMR (125 MHz, CDCl3): δ 165.4,
162.7, 153.1, 152.9, 132.3 (2c), 130.2, 128.9 (2c), 128.4,
127.1 (2c), 126.7 (2C), 126.4, 122.4, 101.9, 41.3; HRMS
(ESI) for C18H13BrN3O3 [M + H]+, found 398.0130,
Calcd 398.0140.
5-(4-Bromophenyl)-3-((3-(p-tolyl)isoxazol-5-yl)methyl)-
1,3,4-oxadiazol-2(3H)-one (8h). Pale yellow solid; yield
399.0140.
3-((3-(4-Methoxyphenyl)isoxazol-5-yl)methyl)-5-phenyl-
1,3,4-oxadiazol-2(3H)-one (8c).
Colorless solid; yield
76%; mp 169–171°C; IR (KBr): 3139, 2984, 1779,
1659, 1291, 820 cmꢀ1 1H-NMR (400 MHz, CDCl3):
;
68%; mp 139–141°C; IR (KBr): 3129, 2956, 1771, 1616,
1
1392, 834, 696 cmꢀ1; H-NMR (500 MHz, CDCl3): δ
δ 7.71 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H),
7.48 (d, J = 7.6 Hz, 1H), 7.37–7.22 (m, 3H), 6.53 (s,
1H), 5.16 (s, 2H), 2.47 (s, 3H); 13C-NMR (125 MHz,
CDCl3): δ 164.4, 163.3, 153.1, 152.7, 136.8, 132.3
(2c), 131.1, 129.6, 129.3, 128.0, 127.1 (2c), 126.6,
125.9, 122.2, 104.4, 41.3, 21.1; HRMS (ESI) for
7.87–7.83 (m, 2H), 7.73(d, J = 8.8 Hz, 2H), 7.53–7.44
(m, 3H), 6.96 (d, J = 8.8 Hz, 2H), 6.59 (s, 1H), 5.13 (s,
2H), 3.85 (s, 3H); 13C-NMR (100 MHz, CDCl3): 165.4,
162.3, 161.1, 153.9, 153.0, 131.9, 128.9 (2c), 128.2 (2c),
125.8 (2c), 123.4, 120.9, 114.3 (2c), 101.6, 55.3, 41.5;
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Novel 1,3,4-Oxadiazole Linked 1,2,3-Triazole/Isoxazole Hybrids
C19H15BrN3O3 [M + H]+, found 412.0299, Calcd
412.0296.
5-(4-Bromophenyl)-3-((3-(4-methoxyphenyl)isoxazol-5-yl)
methyl)-1,3,4-oxadiazol-2(3H)-one (8i). Pale yellow solid;
½ðTi ꢀ TzÞ=ðC ꢀ TzÞꢁꢂ100
for concentrations for which Ti≥Tz and
½ðTi ꢀ TzÞ=Tzꢁꢂ100
for concentrations for which Ti < Tz:
yield 70%; mp 162–164°C; IR (KBr): 3101, 2924, 1761,
1
1510, 1345, 824 cmꢀ1; H-NMR (400 MHz, CDCl3): δ
7.76–7.66 (m, 4H), 7.60 (d, J = 8.8 Hz, 2H), 6.95 (d,
J = 8.8 Hz, 2H), 6.60 (s, 1H), 5.13 (s, 2H), 3.84 (s, 3H);
13C-NMR (100 MHz, CDCl3): δ 165.1, 162.3, 161.1,
153.1, 152.7, 132.3 (2c), 129.9, 128.6, 128.2 (2c), 127.1
(2c), 122.2, 114.3 (2c), 101.7, 55.3, 41.3; HRMS (ESI)
for C19H15BrN3O4 [M + H]+, found 428.0239, Calcd
428.0245.
The dose–response parameter, GI50, was calculated for
each experimental drug. GI50 was calculated from
[(Ti ꢀ Tz)/(C ꢀ Tz)] × 100 = 50, which is the drug
concentration resulting in a 50% reduction in net protein
increase (as measured by SRB assay) in control cells
during the drug treatment.
Western blot analysis of cyclin B1 and soluble versus
insoluble (polymerized) tubulin. HeLa cells were plated
BIOLOGY
in six-well plates at 2 × 105 cells per well in Dulbecco’s
modified Eagle’s medium with 10% fetal bovine serum.
Cells were treated with 5 μM of compounds 7a, 7c, and
7d for 24 h. After treatment, cells were washed twice
with phosphate-buffered saline and lysed with 200 μL of
1× Laemmli sample buffer (180 mM of Tris-HCl pH 6.8,
6% sodium dodecyl sulfate, 15% glycerol, 7.5% b-
mercaptoethanol, and 0.01% bromophenol blue). Then
samples were heated at 98°C for 5 min. An equal amount
of samples was run on a 10% sodium dodecyl sulfate–
polyacrylamide gel and was transferred to a nitrocellulose
membrane using semidry transfer at 60 mA for 70 min.
Then the blot was probed with the primary rabbit anti-
cyclin-B1 antibody at 1:1000 μL (Sigma, Hyderabad,
India) and horseradish-peroxidase-coupled goat antirabbit
secondary antibody at 1:5000 (Sigma) dilution. Mouse
anti-α-tubulin antibody 1:5000 (Sigma) was probed as a
loading control. Bands were visualized by using
radiographic film (Kodak, Hyderabad, India). For soluble
versus insoluble tubulin, HeLa cells were seeded in six-
well plates at 2 × 105 cells per well in a complete growth
medium (Dulbecco’s modified Eagle’s medium) and treated
with 5 μM of compounds 7a, 7c, and 7d for 24 h. After
that, cells were washed with phosphate-buffered saline, and
soluble and insoluble tubulin fractions were collected. For
collection of soluble tubulin fraction, cells were
permeabilized with 300 μL of Triton lysis buffer [80 mM
Pipes-KOH (pH 6.8), 1 mM of MgCl2, 1 mM of egtazic
acid, 0.2% Triton X-100, 10% glycerol, 0.1% protease
inhibitor cocktail (Sigma)] and incubated for 2 min at room
temperature. After incubation, lysis buffer was gently
removed, mixed with 150 μL of 3× Laemmli sample buffer
and stored as soluble fraction. To collect insoluble fraction,
300 μL of 1× Laemmli sample buffer was added to the
remaining cells in each well. The samples were boiled at
98°C for 5 min. Then the protein samples were separated
in a 10% sodium dodecyl sulfate–polyacrylamide gel,
transferred, then probed with mouse anti-α-tubulin diluted
1:5000 μL (Sigma) and with goat antimouse secondary
Materials and methods: cell cultures, maintenance, and
antiproliferative evaluation.
The cell lines MDA-MB-
231, HEPG2, DU-145, and HeLa (breast, liver, prostate,
and cervical, respectively), which were used in this
study, were procured from the American Type Culture
Collection, United States, were grown in Dulbecco’s mod-
ified Eagle’s medium (containing 10% fetal bovine serum)
under a humidified atmosphere of 5% CO2 at 37°C. The
compounds triazole and isoxazole series were evaluated
for their in vitro antiproliferative activity in these four dif-
ferent human cancer cell lines. A protocol of 48 h of con-
tinuous drug exposure was employed, and
a
sulforhodamine B (SRB) cell proliferation assay was per-
formed to estimate cell viability or growth. Cells were
trypsinized when subconfluent from 60-mm dishes and
plated in 96-well plates in 100-μL aliquots at plating den-
sities depending on the doubling time of the respective
cell lines. The 96-well plates were incubated at 37°C,
5% CO2, 95% air, and 100% relative humidity. After
24 h, experimental compounds were added and incubated
for 48 h with different doses (0.01, 0.1, 1, 10, and
100 μM). After 48 h of incubation, cell monolayers were
fixed by the addition of 10% (wt/vol) cold trichloroacetic
acid and incubated at 4°C for 1 h, after which they were
washed with tap water and allowed to air dry. Cells were
then stained with 0.057% SRB dissolved in 1% acetic
acid at room temperature for 30 min. Unbound SRB
was washed two to three times with 1% acetic acid and
again allowed to air dry. The protein-bound dye
representing surviving cells was dissolved in 10 mM Tris
solution for Optical Density (OD) determination at
510 nm using a microplate reader (Enspire, Perkin-
Elmer). With the seven absorbance measurements [time
zero (Tz), growth control (C), and the five dose-treated
cells (Ti)], the percentage of growth was calculated at
each of the drug concentration levels. Percentage growth
inhibition was calculated as follows:
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Vol 000
B. Madhavilatha, D. Bhattacharjee, G. Sabitha, B. V. S. Reddy, J. S. Yadav, N. Jain,
and B. J. M. Reddy
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Acknowledgement. G. S. and B. V. S. R thank CSIR, India, for
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SUPPORTING INFORMATION
[16] Giffin, M. J.; Heaslet, H.; Brik, A.; Lin, Y. C.; Cauvi, G.;
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Additional Supporting Information may be found online
in the supporting information tab for this article.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet