Pyrazole-oxadiazole conjugates: Synthesis, antiproliferative activity and inhibition of tubulin polymerization

Article abstract of DOI:10.1039/c4ob01152j

A number of pyrazole-oxadiazole conjugates were synthesized and evaluated for their ability to function as antiproliferative agents on various human cancer cell lines. These conjugates are comprised of pyrazole and oxadiazole scaffolds closely attached to each other without any spacer as two structural classes. The Type I class has a trimethoxy substituent and the type II class has a 3,4-(methylenedioxy) substituent on their A rings. Among these conjugates, 11a, 11d and 11f manifest potent cytotoxicity with IC₅₀values ranging from 1.5 μM to 11.2 μM and inhibit tubulin polymerization with IC₅₀values of 1.3 μM, 3.9 μM and 2.4 μM respectively. The cell cycle assay showed that treatment with these conjugates results in accumulation of cells in the G2/M phase and disrupts the microtubule network. Elucidation of zebrafish embryos revealed that the conjugates cause developmental defects. Molecular docking simulations determined the binding modes of these potent conjugates at the colchicine site of tubulin.

Full text of DOI:10.1039/c4ob01152j

Organic &
Biomolecular Chemistry
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Pyrazole–oxadiazole conjugates: synthesis,
antiproliferative activity and inhibition of tubulin
Cite this: Org. Biomol. Chem., 2014,
2, 7993
1
polymerization†
a,d
a
b
a
Ahmed Kamal,* Anver Basha Shaik, Sowjanya Polepalli, Vangala Santosh Reddy,
a
a
c
G. Bharath Kumar, Soma Gupta, K. V. S. Rama Krishna,
e
e
b
Ananthamurthy Nagabhushana, Rakesh K. Mishra and Nishant Jain
A number of pyrazole–oxadiazole conjugates were synthesized and evaluated for their ability to function
as antiproliferative agents on various human cancer cell lines. These conjugates are comprised of pyrazole
and oxadiazole scaffolds closely attached to each other without any spacer as two structural classes. The
Type I class has a trimethoxy substituent and the type II class has a 3,4-(methylenedioxy) substituent on
their A rings. Among these conjugates, 11a, 11d and 11f manifest potent cytotoxicity with IC50 values
ranging from 1.5 μM to 11.2 μM and inhibit tubulin polymerization with IC50 values of 1.3 μM, 3.9 μM and
2.4 μM respectively. The cell cycle assay showed that treatment with these conjugates results in accumu-
lation of cells in the G2/M phase and disrupts the microtubule network. Elucidation of zebrafish embryos
revealed that the conjugates cause developmental defects. Molecular docking simulations determined
the binding modes of these potent conjugates at the colchicine site of tubulin.
Received 5th June 2014,
Accepted 8th August 2014
DOI: 10.1039/c4ob01152j
www.rsc.org/obc
of cell division, often at the metaphase. As a result, various
efforts aimed at blocking mitosis like inhibition of tubulin
Introduction
In eukaryotic cells, the microtubules are hollow tubes of polymerization by tubulin targeting agents have emerged as an
8,9
α- and β-tubulin heterodimers. The microtubule system is an attractive approach to cancer therapy.
important constituent of the cytoskeleton and functions in Microtubules possess three ligand-binding sites: vinca, col-
many fundamental cellular processes, including chromo- chicine and taxol domains. Combretastatin A-4 (1) is one of
some segregation during cell division, intracellular transport, the naturally occurring tubulin targeting agents that inhibit
development and maintenance of the cell shape, cell motility tubulin polymerization through the colchicine domain of the
1–4
10
and distribution of molecules on cell membranes.
In microtubule (Fig. 1).
Among the common antitubulin
addition, microtubules are involved in a host of cell signaling
pathways which are responsible for cellular apoptosis. The
microtubule dynamics are regulated by various important pro-
5
,6
teins such as kinesin and dynein. Many experiments have
identified that microtubule dependence forces the underlying
7
chromosomal translocation and spindle formation. Hence
altered/aberrant microtubule dynamics result in the obstruction
a
Medicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500 007, India. E-mail: ahmedkamal@iict.res.in;
Fax: (+)91-40-27193189; Tel: (+)91-40-27193157
b
Centre for Chemical Biology CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India
c
Nuclear Magnetic Resonance Centre, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India
d
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education
and Research (NIPER), Hyderabad 500 037, India
e
CSIR – Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
Electronic supplementary information (ESI) available: H NMR and C NMR
Fig. 1 Chemical structures of microtubule targeting agents: CA-4 (1),
azapodophyllotoxin (2), nocodazole (3), anilinonicotinyl–oxadiazole
conjugate (4) and pyrazole–oxadiazole conjugates (5, 6).
1
13
†
spectra of the final compounds are provided. See DOI: 10.1039/c4ob01152j
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agents, nocodazole (3) shows a preferential interference in the
destabilization of microtubule polymerization (Fig. 1). Never-
theless, due to the incredible structural complexity of the
Results and discussion
Chemistry
1
1
microtubule, inhibitors of tubulin exhibit tremendous diver- The synthesis of pyrazole–oxadiazole conjugates 11a–i and
sity. This feature provides many possibilities for designing new 12a–i was successfully carried out by performing the reaction
molecular scaffolds and optimization to circumvent undesir- between 3-substituted phenyl-1H-pyrazole-5-carbohydrazides
able side effects like reduced bioavailability, toxicity and multi- (10a–i) and 3,4,5-trimethoxy cinnamic acid/3,4-(methylene-
drug-resistance.
dioxy)cinnamic acid in the presence of POCl as shown in
3
2
5b
Pyrazoles are well known representative compounds of Scheme 1.
The crucial intermediates 3-substituted phenyl-
potential importance for the design and development of anti- 1H-pyrazole-5-carbohydrazides (10a–i) were prepared con-
cancer agents that are considered as building blocks in syn- veniently in three sequential steps. Initially, different substi-
1
2,13
thetic chemistry.
Moreover, some of the polysubstituted tuted acetophenones (7a–i) upon treatment with diethyl
pyrazole derivatives showed a broad spectrum of antitumor oxalate in the presence of sodium ethoxide afforded the corres-
1
4
25c
potential towards most of the tumor cell lines. Recently, ponding phenyl diketo esters (8a–i).
Magedov and coworkers performed biochemical studies on diketo esters underwent dehydrative cyclization with NH
various dihydropyridopyrazoles (2) that inhibit tubulin NH ·2HCl in refluxing ethanol to provide ethyl 3-substituted
Subsequently, these
2
–
2
1
5
25a
polymerization to disrupt mitotic spindles (Fig. 1). Some tri- phenyl-1H-pyrazole-5-carboxylates (9a–i) in good yields.
and tetra-substituted pyrazole derivatives exhibit impressive the next step, these carboxylates (9a–i) were reacted with NH
activity against Src, B-Raf wt, B-Raf V600E, EGFRs and NH ·H O in ethanol to furnish 3-phenyl-1H-pyrazole-5-carbo-
VEGFR-2, thereby making them promising lead molecules for hydrazide (10a–i). Employing the same synthetic procedures
In
2
–
2
2
1
6
the development of chemotherapeutics. Along with this, a described above, the conjugates 17a–b were synthesized start-
number of oxadiazoles are known to display a wide range of ing with acetone (13) instead of substituted acetophenones as
biological activities including anticancer properties.
1
7,18
shown in the reaction sequence reported in Scheme 2. The
Recently, some 2,5-diaryl-1,3,4-oxadiazoline analogs of com- desired target conjugates were characterized by IR, NMR and
bretastatin A-4 (CA-4) were reported as potent microtubule dis- mass spectral data.
rupting agents and circumvent drug resistance mediated by
1
9
Biology
P-glycoprotein and β-III tubulin expression. In addition,
some benzimidazole linked oxadiazole conjugates were
Antiproliferative activity. Initially, the pyrazole–oxadiazole
2
0
reported to express significant anticancer effects.
More conjugates were evaluated for their in vitro antiproliferative
recently, few chemical entities that possess an oxadiazole activity against a panel of four cancer cell lines such as HeLa,
group attached to hetero aromatic rings with an adjacent sub- A549, MCF-7 and IMR32 employing the sulphorhodamine B
stituted amino functionality were documented as lead micro- assay with nocodazole as a reference standard. The IC50 values
2
1
tubule disrupting agents. In this connection, we have also determined are summarized in Table 2. The majority of the
noticed a report describing significant growth inhibitory activi- conjugates significantly inhibit growth of the cells in a dose-
ties of pyrazole incorporated oxadiazoles as potential anti- dependent manner. The conjugates 11a–i with trimethoxy sub-
2
2
microbial agents.
Therefore, it was observed that the stituents on the A ring demonstrated better antiproliferative
combination of two heterocyclic moieties as a single molecular activity than 12a–i that possess a 3,4-(methylenedioxy) group
2
3,24
scaffold is known to enhance the anticancer activity.
on the same A ring (Fig. 2). In detail, the conjugates with
Previously we showed that incorporation of an oxadiazole electronegative substituents i.e. –Cl and –F on the D-ring
ring system to a 2-anilinonicotinyl linked sulfonyl hydrazide expressed a superior growth inhibition effect than electro-
scaffold (4) resulted in promising antitumor activity that sig- positive (–OCH ) groups on the same D-ring. Interestingly, the
3
2
5a
nificantly inhibited tubulin polymerization (Fig. 1).
There- type I class of conjugates 11a, 11d and 11f exhibit pronounced
fore, in our present endeavor to obtain potent anticancer cytotoxicity towards all the cell lines with IC50 values ranging
agents through the combination of two pharmacophores, we from 1.5 μM to 11.2 μM. Specifically, the type I lead conjugate
designed and synthesized a new series of pyrazole–oxadiazole 11a with a monochloro substitution at the C4 position on the
conjugates that comprise a scaffold of four rings (A, B, C and D-ring manifested significant inhibitory activity with an IC50
D). Consequently, for comprehensive analysis of structural value of 1.5 μM in A549 cells. In comparison, 11d is devoid of
activity relationships (SAR) by modifying the A-ring, we syn- any substitution and 11f with a dichloro at C3 and C4 (D-ring)
thesized two types of compounds. The type I class has a tri- showed an IC50 of 3.1 μM and 2.3 μM in A549 cells. In contrast,
methoxy substituent on the A ring, whereas the type II class 11c with difluoro substitution demonstrated moderate effects
has 3,4-(methylenedioxy) as a substituent on the A ring with an IC50 value of 14 μM in A549 cells. Moreover, conjugate
(Table 1). These pyrazole–oxadiazole conjugates were evaluated 11a exhibited profound potency with an IC50 value of 1.8 μM
for their anticancer activity and ability to inhibit tubulin in MCF-7, 2.3 μM in HeLa and 3.2 μM in IMR32 cell lines.
assembly. Out of the twenty conjugates three conjugates 11a, However, the type II class of compounds of 12a–i displayed
1
1d and 11f showed pronounced anticancer activities towards moderate activities. Among these conjugates, substitution with
most of the cell lines.
monochloro- (12a) and dichloro-(12f) on the D-ring resulted in
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Table 1 Structures of pyrazole–oxadiazole conjugates 11a–i, 12a–i and 17a–b
1
2
3
Structure
Compd
R
R
R
Mol. for
Mol. wt
1
1
1
1
1
1
1
1
1
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
H
H
H
H
H
H
OCH
H
Cl
OCH
F
H
CH
Cl
–OCH
OCH
OCH
H
H
F
H
CH
Cl
C
22
C
23
C
22
C
22
C
24
C
22
C
23
C
25
C
24
H
H
H
H
H
H
H
H
H
19ClN
4
O
5
O
4
4
438.8
434.4
440.4
404.4
432.4
473.3
448.4
494.5
464.4
3
22
18
20
24
N
4
O
F N
2 4
N
N
4
O
4
O
4
3
3
4
18Cl
2
N
4
O
4
2
O–
20
N
26
N
24
N
4
4
4
O
O
O
6
7
6
3
3
OCH
OCH
3
3
3
1
1
1
1
1
1
1
1
1
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
H
H
H
H
H
H
OCH
H
Cl
OCH
F
H
CH
Cl
–OCH
OCH
OCH
H
H
F
H
CH
Cl
C
20
C
21
C
20
C
20
C
22
C
20
C
21
C
23
C
22
H
H
H
H
H
H
H
H
H
13ClN
4
O
3
392.8
388.3
394.3
358.3
386.4
427.2
402.3
448.4
418.4
3
16
12
14
18
4 4
N O
F
2 4
N O
O
O
3
N
N
4
3
3
3
4
3
12Cl
2
N
4
O
3
2
O–
14
N
20
N
18
N
4
4
4
O
O
O
5
6
5
3
3
OCH
OCH
3
3
3
17a
—
—
—
C
17
H N O
18 4 4
342.3
17b
—
—
—
C
15
H N O
12 4 3
296.2
an increased cytotoxicity against most of the cell lines. Further- all the conjugates at 5 μM concentration for a duration of 24 h
more, conjugate 12d devoid of any substitutions manifested and performed dot-blot analysis for cyclin-B1. Interestingly,
an activity of 10 μM (IC50) towards A549 cells. Notably, the 11a and 11f exhibited a marked increase in cyclin-B1 protein
presence of bulky groups on the D-ring diminished the activi- levels similar to colchicine and CA-4 treated cells. In addition,
ties of the compounds in the type II class. Similarly, conju- 11d, 11g, and 11h showed a slight increase in cyclin-B1
gates of the type I class, such as 11h harboring trimethoxy protein. Therefore, these results support the suggestion that
substitution on the D-ring, abrogated the potency of the conju- congeners, 11a, 11f, of pyrazole–oxadiazole conjugates mani-
gate. Based on these observations, removal of the D-ring in fest anti-proliferative effects through cell cycle arrest at mitosis
1
7a and 17b substantially reduced the potency, suggesting that (Fig. 3).
the D-ring is essential for the optimal activity of these conju- Effect on tubulin polymerization. Since conjugates of the
gates. Therefore, these results suggest that the trimethoxy sub- type I class of compounds harbor trimethoxy phenyl, oxadia-
stituent of the A-ring conjugated to chloro or un-substituted at zole and pyrazole moieties, we presumed that these conjugates
the C-4 position of the D-ring is the most potent agent can prevent tubulin assembly. Based on our SAR analysis and
between the two structural classes of these conjugates.
dot-blot results we selected 11a and 11f as potent congeners.
Dot-blot analysis for cyclin-B1. Previously our studies Intriguingly, 11d treated cells showed a modest increase in
showed that 2-anilino-nicotinyl 1,3,4-oxadiazoles, with a tri- cyclin B1 protein levels. In contrast, 11d exhibited potent cyto-
methoxy phenyl group, an oxadiazole and pyridine moiety, toxicity compared to the other conjugates in the series. Conse-
function as potent anti-tubulin agents. Therefore, we hypo- quently, we elucidated the ability of 11a, 11d and 11f to inhibit
thesize that these conjugates induce cytotoxicity through cell tubulin assembly. Thus, we incubated varying concentrations
cycle arrest at the G2/M phase. Accumulation of cyclin-B1 is an of the lead conjugates with tubulin to determine the IC50
2
6
indicator of G2/M arrest. Hence, we treated A549 cells with values for inhibition of tubulin polymerization. As expected,
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a
Table 2 In vitro cytotoxic effect (IC50 µM) of pyrazole–oxadiazole
conjugates
b
c
d
e
Entry
Compd
A549
HeLa
MCF-7
IMR32
1
2
3
4
5
6
7
8
9
11a
11b
11c
11d
11e
11f
11g
11h
11i
12a
12b
12c
12d
12e
12f
12g
12h
12i
1.5 ± 0.1
20 ± 1.2
14 ± 2.1
3.1 ± 0.5
13 ± 1.4
2.3 ± 0.2
20 ± 1.6
12 ± 1.3
21 ± 1.5
4.1 ± 1.2
33 ± 1.5
22 ± 1.1
10 ± 1.7
27 ± 1.2
13 ± 0.6
16 ± 1.5
12 ± 1.1
24 ± 1.6
19 ± 1.0
23 ± 1.5
0.9 ± 0.2
2.3 ± 0.5
4.6 ± 0.4
17 ± 1.5
9.5 ± 1.0
16.2 ± 1.0
2.8 ± 0.5
32.3 ± 2.5
10 ± 1.2
1.8 ± 0.3
14.8 ± 1.4
21.8 ± 0.8
3.9 ± 0.6
21 ± 1.9
3.9 ± 0.7
11.2 ± 1.0
9.0 ± 1.0
19 ± 2.1
3.2 ± 1.2
16.2 ± 2.1
30 ± 1.6
11.0 ± 1.8
13 ± 1.1
8.9 ± 1.7
13 ± 1.8
23 ± 1.7
14.1 ± 1.6
19.2 ± 0.9
15.5 ± 1.8
1.25 ± 0.9
3.2 ± 0.6
2.5 ± 0.5
14.8 ± 1.4
11.2 ± 0.7
17.9 ± 1.3
4.1 ± 0.6
24.3 ± 1.4
4.6 ± 1.2
16.7 ± 1.8
8.9 ± 0.9
20.9 ± 1.8
19.5 ± 0.1
7.6 ± 1.2
16.3 ± 1.0
14.5 ± 1.2
17.9 ± 1.4
21.4 ± 1.2
19.1 ± 0.6
19 ± 1.0
19.4 ± 1.7
6.1 ± 1.1
10 ± 1.2
10
11
12
13
14
15
16
17
18
19
20
21
43.6 ± 1.2
12.0 ± 1.6
33.8 ± 1.9
15.0 ± 1.0
18.8 ± 1.4
18.2 ± 1.2
12.0 ± 1.2
9.3 ± 1.8
18.7 ± 1.0
1.2 ± 0.7
17a
17b
Noc
12.9 ± 1.0
1.3 ± 0.7
a
Concentration required to inhibition 50% cell growth and the values
represent mean ± S.D. from three different experiments performed in
b
c
triplicates. A549: lung adenocarcinoma epithelial cell line. HeLa:
d
cervix cancer cell line. MCF-7: breast adenocarcinoma cell line.
IMR32: neuroblastoma cell line.
Scheme 1 Synthesis of pyrazole–oxadiazole conjugates (11a–i and
e
1
2a–i). Reagents and conditions: (1) NaOEt–ethanol, diethyl oxalate,
°C–rt, 4 h; (2) N ·2HCl–ethanol, reflux, 3–4 h; (3) N ·H O–
ethanol, reflux, 3–4 h; (4) 3,4,5-trimethoxy cinnamic acid, POCl , 110 °C,
h; (5) 3,4-(methylenedioxy)cinnamic acid, POCl , 110 °C, 5 h.
0
2
H
4
2
H
4
2
3
6
3
Fig. 2 SAR of type I pyrazole–oxadiazole conjugates.
also inhibited tubulin assembly at an IC50 value of 3.9 µM and
2.41 µM respectively (Table 3 and Fig. 4).
Effect on cell cycle arrest. Anti-microtubule agents are
known to induce cell cycle arrest in the G2/M phase of the cell
cycle. Flow cytometry is routinely employed to determine the
population of cells in each phase of the cell cycle by measuring
the DNA content of individual cells.
Scheme 2 Synthesis of pyrazole–oxadiazole conjugates (17a–b).
Reagents and conditions: (1) NaOEt–ethanol, diethyl oxalate, 0 °C–rt,
Since conjugates 11a, 11d and 11f bind tubulin and inhibit
4
3
(
h; (2) N
–4 h; (4) 3,4,5-trimethoxy cinnamic acid, POCl
methylenedioxy)cinnamic acid, POCl , 110 °C, 5 h.
2
H
4
·2HCl–ethanol, reflux, 3–4 h; (3) N
2
H
4
·H
2
O–ethanol, reflux, cell growth, we analyzed for their ability to arrest cells at the
3
, 110 °C, 6 h; (5) 3,4-
G2/M phase. Therefore, A549 cells were treated with 5 μM con-
centration of representative compounds for 24 h. Sub-
sequently, harvested cells were subjected to flow cytometry
analysis. The lead conjugate 11a showed an increase of cells in
3
1
1a, which potentially inhibited cell growth, also significantly the G2/M phase to 77.3%. In comparison, cells accumulated to
down-regulated tubulin polymerization with an IC50 value of 75.02% and 75.07% on 11d and 11f treatments in the G2/M
.3 µM when compared to 11d and 11f. However, 11d and 11f phase. Moreover, the cell number significantly decreased in
1
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Fig. 3 Dot-blot analysis of cyclin-B1: A549 cells were treated with 5 μM
concentrations of pyrazole–oxadiazole conjugates for 24 h. Later, cells
were harvested and whole cell lysates were blotted on nitrocellulose
membranes. The blotted proteins were probed with antibodies against
cyclin B1. Tubulin was employed as the loading control.
Fig. 5 Anti-mitotic effects of 11a, 11d and 11f by FACS analysis. (A)
Induction of cell cycle arrest at G2/M by compounds 11a, 11d and 11f.
A549 cells were harvested after treatment at 5 µM for 24 h. Untreated
cells and DMSO treated cells served as controls. The percentage of cells
in each phase of the cell cycle was quantified by flow cytometry. (B) %
of cells arrested in different stages of the cell cycle by compounds 11a,
Table 3 Antitubulin activity of pyrazole–oxadiazole conjugates 11a,
a
11d and 11f, with values expressed in IC50 (μM)
Inhibition of tubulin
polymerization (µM)
Compound
1
1d, 11f and control. (C) Graphical representation of the distribution of
1
1
1
1a
1d
1f
1.3 ± 0.1
3.9 ± 0.05
2.41 ± 0.32
0.91 ± 0.01
the cell cycle.
CA4
resulted in a clear G2/M phase arrest with a concomitant
decrease of cells in other phases of the cell cycle and induction
of cyclin-B1 protein.
a
Effect of conjugates on tubulin polymerization. IC50 values for 11a,
1d and 11f were determined from the tubulin polymerization assays.
1
2
7
Effect on the microtubule network. Spindle poisons exert
their action through the disruption of chromosome separation
during mitosis. The pyrazole–oxadiazole conjugates signifi-
cantly decreased cell growth, inhibited tubulin assembly and
stalled cells in the G2/M phase of the cell cycle. Therefore, we
evaluated their ability to disrupt the microtubule network in
cells. Consequently, A549 cells were treated with 11a, 11d and
11f at 5 μM concentration for 24 h. Immunofluorescence ana-
lysis reveals that cells exhibited a round morphology typical of
mitotic arrest. Moreover, the chromatin was condensed in the
nuclei, suggesting a metaphase arrest (Fig. 6).
Microtubules exhibit dynamic equilibrium with free tubulin
monomers in the cells. Pharmacological agents exploit this
Fig. 4 Effect of lead conjugates 11a, 11d and 11f on tubulin polymeriz- property of microtubules to exert their anticancer effects.28
ation: tubulin polymerization was monitored by the increase in fluo-
rescence at 360 nm (excitation) and 420 nm (emission) for 1 h at 37 °C.
cidated their effect on cytosolic tubulin. Thus, A549 cells were
All the compounds were included at a final concentration of 5 µM. Com-
Since these conjugates disrupt tubulin polymerization, we elu-
treated with 5 μM concentration of 11a, 11d and 11f for 24 h.
bretastatin A-4 (CA-4) and Paclitaxel were used as positive controls.
CA-4 and colchicines were also treated in the same experiment
as the standard reference. Subsequently, these soluble and
the S phase. In contrast, vehicle or DMSO treated cells showed polymerized fractions were collected and subjected to western
a majority of the population in the G1 phase (67.7%) and 11a, blot analysis. Immunoblot results show that the cells exposed
1
1d and 11f treated cells resulted in a significant increase of to these conjugates 11a, 11d, 11f contain more tubulin in the
cyclin-B1 protein, compared to DMSO treated cells (Fig. 5). soluble fraction of cells when compared to the insoluble frac-
Thus treatment with the pyrazole–oxadiazole conjugates tion. Hence, increased tubulin in the soluble fraction of cells
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Fig. 8 Effect of microtubule inhibitors on the development of zebrafish
embryos: zebrafish embryos were treated with DMSO, nocodazole, 11a,
1
1d and 11f at 5 hpf. The embryos were imaged at 30 hpf to observe
morphological changes.
31–33
investigation.
These observations suggest that 11a acts as an
anti-mitotic compound in zebrafish embryos and could be con-
sidered as a potential chemotherapeutic lead (Fig. 8).
Molecular modeling. To explore possible binding modes of
compounds 11a, 11d and 11f to tubulin, we performed mole-
cular docking simulations on β-tubulin with tubulin cocrystal
Fig. 6 Effect of 11a, 11d and 11f on microtubules and nuclear conden- DAMA-colchicine (PDB 3E22). The computational experiments
sation. A549 cells were independently treated with 11a, 11d and 11f at
were performed by employing the Glide 5.8 module in Maestro
5
µM for 24 h. Following treatment, cells were fixed and stained for
9.3 (Schrodinger suite 2012). Molecular docking analysis
tubulin using DAPI as a counter stain. Photographs were taken using an
Olympus confocal fluorescence microscope equipped with FITC and
revealed that the three promising conjugates occupied the
DAPI filter settings. Cells stained for tubulin and DAPI from the same same position corresponding to colchicine (colchicine binding
fields of view are given.
site of tubulin). Moreover, these conjugates are buried at the
interface of α,β-tubulin heterodimers and the major portion is
occupied in the β-subunit (showing maximum interactions)
and there are fewer interactions with residues of the α-subunit
treated with pyrazole–oxadiazole conjugates corroborated well
with the inhibition of tubulin polymerization and arrest of
cells in the G2/M phase (Fig. 7).
3
3,34
(
Fig. 9).
The type I conjugates functionalized with a tri-
methoxy phenyl ring (A-ring) are important basic units for
tubulin binding. The core structure comprising of A, B, C and
D rings is sandwiched between the two subunits and is sur-
rounded by a number of amino acid residues such as N101,
E183, Y202, V181, S178, G143, L248, L242, L255, K254, K352,
M259, A180, A316, C241 and N249.
The docking experiments performed with individual mole-
cules to explore the molecular interactions are depicted in
Fig. 9 (11a, 11d and 11f). Specifically, the docking simulations
of 11a with the trimethoxyphenyl A-ring and C4 chlorophenyl
D-ring have shown important hydrogen bonding interactions
with some representative amino acids. The O atom of the
C4 methoxy group formed a strong hydrogen bond with HN of
K254 (O⋯HN, distance 1.9 Å). In comparison, the next
C4 methoxy O of the same ring formed a strong hydrogen
bond with HN of E181 (O⋯HN, distance 2.0 Å) and the same
methoxy O showed van der Waals interactions with N101
Effect on zebrafish development. Zebrafish (Danio rerio) has
emerged as a powerful model system for chemical genetics. In
order to validate these observations, we investigated the effect
of lead conjugates on the development of zebrafish embryos.
Therefore, embryos treated with 11a showed a remarkable
delay in development as compared to DMSO treated control
embryos. While the control embryos were progressing through
the prim6 stage, 11a treated embryos were arrested at the 18
32
somite stage. Embryos treated with nocodazole also exhibited
severe developmental anomalies. However, embryos treated with
the conjugates 11d and 11f showed minimal phenotypic abnorm-
alities. The inability of 11d and 11f to affect development,
despite their structural similarity, suggests possible differences
in the ADME (absorption, distribution, metabolism, excretion)
properties of the ring substitutes in vivo and needs further
(
O⋯HN, distance 3.1 Å). In addition, a strong hydrogen
bonding was noticed between the N atom of the pyrazole ring
and HS of the important amino acid C241 (N⋯HS, distance
2.8 Å). Most of the residues like S178, G143, L242, K254, K352,
M259, A180, N258 and N249 of both the subunits have shown
hydrophobic interactions with 11a. Interestingly, B and C rings
possessing heteroatoms (oxadiazole–pyrazole) exhibited hydro-
philic interactions with L248 and L255. Furthermore, the
D-ring with a C4 chlorophenyl group forms a π–π interaction
with Y202. The conjugates 11d and 11f also show a similar
type of interaction with the amino acid residues to 11a,
Fig. 7 Distribution of tubulin in polymerized vs. soluble fractions as
analyzed by immunoblotting in 11a, 11d and 11f treated A549 cells:
A549 cells were treated with 5 μM of 11a, 11d and 11f for 24 h. CA4 and
colchicine were used as positive controls. Tubulin was detected by
immunoblot analysis.
7998 | Org. Biomol. Chem., 2014, 12, 7993–8007
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however with varying bonding distance. 11d without any sub-
stitution on the D-ring shows slight deviation in the bonding
interaction and the distances are K254 (O⋯HN, distance
2.0 Å), E181 (O⋯HN, distance 2.1 Å), N101 (O⋯HN, distance
3.3 Å) and C241 (N⋯HS, distance 3.2 Å). The docking pose of
11f with the C3,C4-dichloro phenyl D-ring represents better
simulations when compared to 11d particularly in the case of
given four bonding interactions and these are K254 (O⋯HN,
distance 1.7 Å), E181 (O⋯HN, distance 2.1 Å), N101 (O⋯HN,
distance 3.5 Å) and C241 (N⋯HS, distance 3.8 Å) (Fig. 9). Super-
imposition of 11a, 11d and 11f further confirmed that mode of
binding to tubulin was similar for these conjugates. The pres-
ence of a bulky group in the D-ring, such as monomethoxy-11b,
dimethoxy-11i and trimethoxy-11h groups, does not allow suc-
cessful docking possibly due to steric hindrance. Thus our
results suggest that the pyrazole–oxadiazole conjugates occupy
the colchicine binding pocket of tubulin and exhibit strong
3
5
hydrogen bonding with the surrounding amino acids.
Conclusions
In conclusion, we synthesized a new class of pyrazole–oxadi-
azole conjugates comprised of a four ring scaffold. In order to
present a comprehensive relationship between structure and
activity, we prepared compounds of two structural classes.
The conjugates were functionalized with four substituents at
the A and D rings. Most of the conjugates exhibited poten-
tial antiproliferative activity on different cancer cell lines. The
presence of a monochloro or dichloro group in the D ring was
essential for high antitubulin activity. The compounds 11a,
11d and 11f induce accumulation of cells in the G2/M phase,
activate cyclin-B1 protein and prevent chromosomal separ-
ation. Increased levels of tubulin in the soluble fraction of
cells remarkably corroborated with inhibition of tubulin
polymerization of these compounds. Furthermore, zebrafish
embryos treated with the lead conjugate 11a exhibit severe
developmental defects. Molecular docking studies revealed
that these compounds successfully dock in the colchicine
binding site of tubulin. Therefore, these results suggest that
pyrazole–oxadiazole conjugates are potential inhibitors of
tubulin polymerization. Finally, these conjugates are amenable
to further structural modifications and could serve as useful
pharmacophore templates for the generation of molecules as
potential anticancer agents.
Experimental section
Chemistry
Fig. 9 Docking simulations of lead conjugates in the colchicine binding
site of tubulin: 11a (light blue color), 11d (grey color) and 11f (orange
color) shown in stick model. The O atom of the trimethoxy phenyl ring
formed a strong hydrogen bond with K254, N101 and E183. The pyrazole All the chemicals and reagents were obtained from Aldrich
N formed a hydrogen bond with C241. Red dotted lines indicate the
(
Sigma-Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar,
hydrogen bonds and the distance was measured indicated in red
numerically. The superimposition docking pose of representative conju-
gates 11a, 11d and 11f in the colchicine binding pocket. Stick represen-
tation of selected amino acids (pink color) of tubulin surrounded by
compound 11a, 11d and 11f.
Johnson Matthey Company, Ward Hill, MA, USA), or Spectro-
chem Pvt. Ltd (Mumbai, India) and were used without further
purification. The completion of reactions was monitored by
TLC performed on silica gel glass plates containing 60 GF-254,
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and visualization was achieved by a UV light or iodine indi- 7.02 (s, 1H, ArH), 7.22–7.43 (m, 2H, ArH), 7.72 (d, 1H, J = 6.7 Hz,
cator. Column chromatography was performed with Merck ArH), 9.31 (brs, 1H, –NH) ppm; MS (ESI) m/z 253 [M + H].
6
0–120 mesh silica gel. NMR spectra were recorded on Bruker
Ethyl 3-phenyl-1H-pyrazole-5-carboxylate 9d. Pale yellow
= 0.3 (25% ethyl acetate–hexane);
UXNMR/XWIN-NMR (300 MHz) or Inova Varian-VXR-unity colored solid (yield 85.0%): R
f
1
(400, 500 MHz) instruments. Chemical shifts (δ) are reported
H NMR (500 MHz, CDCl ); δ 1.35–1.40 (t, 3H, J = 6.9 Hz,
3
1
in ppm downfield from an internal TMS standard. ESI spectra –CH
3
), 4.32–4.41 (q, 2H, J
1
= 6.9 Hz, J
2
2
= 7.9 Hz, –CH ), 7.04 (s,
were recorded on a Micro mass Quattro LC using ESI+ software 1H, ArH), 7.36 (d, 2H, J = 7.9 Hz, ArH), 7.36 (d, 3H, J = 7.9 Hz,
with a capillary voltage 3.98 kV and an ESI mode positive ion ArH), 9.31 (brs, 1H, –NH) ppm; MS (ESI) m/z 217 [M + H].
trap detector. High-resolution mass spectra (HRMS) were
Ethyl 3-(3,4-dichlorophenyl)-1H-pyrazole-5-carboxylate 9e.
= 0.3 (20% ethyl acetate–
Melting points were determined with electrothermal melting hexane); H NMR (300 MHz, CDCl ); δ 1.39–1.45 (t, 3H, J =
recorded on a QSTAR XL Hybrid MS–MS mass spectrometer. Yellow colored solid (yield 75.0%): R
f
1
3
point apparatus, and are uncorrected.
3 2
6.5 Hz, –CH ), 4.33–4.46 (q, 2H, J = 7.4 Hz, –CH ), 7.16 (s, 1H,
Preparation of ethyl 2,4-dioxo-4-(substituted phenyl)butano- ArH), 7.49–7.63 (m, 2H, ArH), 7.90–7.94 (m, 1H, ArH), 8.20
ates 8a–i. Initially sodium ethanolate was prepared in situ and (brs, 1H, –NH) ppm; MS (ESI) m/z 285 [M + H].
diethyl oxalate (1.0 mol) was added slowly at 0 °C. The stirring
Ethyl 3-(3,4-dimethylphenyl)-1H-pyrazole-5-carboxylate 9f.
= 0.3 (25% ethyl acetate–
different acetophenones 7(a–i) (1.0 mol) slowly in small por- hexane); H NMR (200 MHz, CDCl ); δ 1.22–1.32 (t, 3H, J =
was continued for 15 minutes followed by the addition of Yellow colored solid (yield 80.0%): R
f
1
3
tions, maintaining the temperature at 0 °C. After completion 5.8 Hz, –CH
3 3
), 2.25 (s, 6H, –CH ), 4.17–4.30 (q, 2H, J = 5.8 Hz,
of addition, the stirring was continued for 4 h at room temp- –CH ), 6.93 (s, 1H, ArH), 7.07 (d, 1H, J = 8.1 Hz, ArH), 7.39
2
erature. The progress of the reaction was monitored by TLC (d, 1H, J = 6.9 Hz, ArH), 7.47 (s, 1H, ArH), 8.20 (brs, 1H, –NH)
using ethyl acetate and hexane as the mobile phase. After com- ppm; MS (ESI) m/z 245 [M + H].
pletion of reaction, the reaction mixture was neutralized using
Ethyl 3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carboxylate
dilute H SO solution and further extracted with ethyl acetate 9g. Pale yellow colored solid (yield 85.0%): R = 0.3 (40% ethyl
2
4
f
1
followed by evaporation of the solvent under reduced pressure acetate–hexane); H NMR (400 MHz, CDCl
to obtain solid products 8(a–i) (yield 80–90%). These were J = 7.5 Hz, –CH
taken as such for the next step without purification. 2H, OCH O), 6.84 (d, 1H, J = 8.3 Hz, ArH), 6.99 (s, 1H, ArH),
Preparation of ethyl 3-substituted phenyl-1H-pyrazole-5-car- 7.21–7.26 (m, 2H, ArH) ppm; MS (ESI) m/z 261 [M + H].
boxylates 9a–i. To each of the ethyl 2,4-dioxo-4-(substituted Ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate
phenyl)butanoates 8(a–i) (1.0 mol) produced in the previous 9h. Yellow colored solid (yield 80.0%): R = 0.3 (40% ethyl
step was added hydrazine dihydrochloride (N
1.5 mol) in ethanol and refluxed for 3–4 h. After completion J = 7.4 Hz, –CH
of the reaction, the ethanol was removed under reduced 4.33–4.45 (q, 2H, J = 7.4 Hz, –CH ), 7.00 (s, 2H, ArH), 7.04 (s,
3
); δ 1.35–1.42 (t, 3H,
), 4.35–4.45 (q, 2H, J = 6.7 Hz, –CH ), 6.0 (s,
2
3
2
f
1
2
H
4
·2HCl) acetate–hexane); H NMR (300 MHz, CDCl
3
); δ 1.34–1.39 (t, 3H,
), 3.87 (s, 3H, –OCH ), 3.88 (s, 6H, –OCH
(
3
3
3
),
1
2
pressure and then 150–200 ml of water was added to the 1H, ArH) ppm; MS (ESI) m/z 307 [M + H].
residue followed by extraction with ethyl acetate (50 ml × 4).
Ethyl 3-(3,4-dimethoxyphenyl)-1H-pyrazole-5-carboxylate 9i.
The organic layer was dried on anhydrous Na SO4 and the Pale yellow colored solid (yield 80.0%): R = 0.3 (30% ethyl
2
f
1
solvent was removed to obtain a crude compound that was acetate–hexane); H NMR (300 MHz, CDCl
further purified by column chromatography using an ethyl J = 7.1 Hz, –CH ), 3.85 (s, 3H, –OCH
acetate and hexane solvent system (3 : 7). The pure compounds 4.19–4.32 (q, 2H, J = 7.1 Hz, –CH ), 6.88 (d, 1H, J = 7.1 Hz,
3
); δ 1.23–1.31 (t, 3H,
), 3.90 (s, 3H, –OCH ),
3 3
3
2
1
9
(a–i) were eluted with 30–40% of ethyl acetate with good ArH), 6.94 (s, 1H, ArH), 7.21–7.28 (m, 1H, ArH), 7.29–7.34 (m,
yields.
Ethyl 3-(4-chlorophenyl)-1H-pyrazole-5-carboxylate 9a. Pale
1H, ArH), 9.67 (brs, 1H, –NH) ppm; MS (ESI) m/z 277 [M + H].
Preparation of 3-substituted phenyl-1H-pyrazole-5-carbohy-
yellow colored solid (yield 80.0%): R
f
= 0.3 (25% ethyl acetate– drazides 10a–i. To the ethyl 3-substituted phenyl-1H-pyrazole-
1
hexane); H NMR (300 MHz, CDCl
3
); δ 1.34–1.42 (t, 3H, J
1
=
5-carboxylates 9(a–i) obtained in the above step was added
6
7
7
.9 Hz, J = 7.9 Hz, –CH ), 4.33–4.41 (q, 2H, J = 6.9 Hz, J = hydrazine hydrate (4.0 mol) in ethanol at room temperature
2
3
1
2
.9 Hz, CH
2
), 7.04 (s, 1H, ArH), 7.36 (d, 2H, J = 7.9 Hz, ArH), and heated to reflux for 3–4 h. The progress of the reaction
was monitored by TLC using ethyl acetate and hexane (1 : 1) as
Ethyl 3-(4-methoxyphenyl)-1H-pyrazole-5-carboxylate 9b. the solvent system. The reaction mixture was allowed to attain
Pale yellow colored solid (yield 80.0%): R = 0.3 (30% ethyl room temperature and then the solvent was distilled off under
); δ 1.22–1.36 (t, 3H, reduced pressure. Then the residue was dissolved in water and
J = 7.5 Hz, –CH ), 3.81 (s, 3H, –OCH ), 4.20–4.35 (q, 2H, J = extracted twice with ethyl acetate, dried over anhydrous Na SO
.70 (d, 2H, J = 7.9 Hz, ArH) ppm; MS (ESI) m/z 251 [M + H].
f
1
acetate–hexane); H NMR (300 MHz, CDCl
3
1
3
3
1
2
4
4
.5 Hz, –CH
ArH), 8.33 (brs, 1H, –NH) ppm; MS (ESI) m/z 248 [M + H].
Ethyl 3-(3,4-difluorophenyl)-1H-pyrazole-5-carboxylate 9c. carbohydrazides obtained by this method were pure and were
Yellow colored solid (yield 75.0%): R = 0.3 (20% ethyl acetate– taken as such for the next step without further purification.
); δ 1.22–1.32 (t, 3H, J Preparation of (E)-2-(3-substituted phenyl-1H-pyrazol-5-yl)-5-
.1 Hz, J = 7.5 Hz, –CH ), 4.20–4.33 (q, 2H, J = 7.1 Hz, –CH ), (3,4,5-trimethoxystyryl)-1,3,4-oxadiazole 11a–i. To each of the
2
), 6.88–6.90 (m, 3H, ArH), 7.60 (d, 2H, J = 9.0 Hz and the solvent was removed under reduced pressure to afford
the corresponding compound 10(a–i) as a crystalline solid. The
f
1
hexane); H NMR (200 MHz, CDCl
7
3
1
=
2
3
2
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3
-substituted phenyl-1H-pyrazole-5-carbohydrazides 10(a–i) –OCH
3
), 6.91 (s, 2H, ArH), 7.09–7.42 (m, 1H, ArH), 7.21 (d, 1H,
(
1.0 mol) produced in the above step was added (E)-3-(3,4,5-tri- J = 16.0 Hz, -trans H), 7.52–7.73 (m, 1H, ArH), 7.62 (d, 1H, J =
methoxyphenyl)acrylic acid (1.0 mol) in POCl and stirred at a 16.0 Hz, -trans H), 7.75–7.88 (m, 1H, ArH), 7.96 (s, 1H, ArH);
temperature of 110 °C for 6 h. The completion of the reaction
was monitored by TLC in the ethyl acetate and hexane solvent 107.2, 107.4, 112.7, 112.8, 116.2, 128.3, 137.0, 137.2, 151.2,
3
1
3
6
C NMR (75 MHz, DMSO-d ): 54.1, 58.1, 101.6, 103.3, 103.4,
−
1
system and then ice cold water was added to the reaction 161.4, 161.7 δ ppm; IR (KBr) (νmax/cm ): ν = 3112, 3049, 2942,
−
1
mixture followed by extraction with ethyl acetate (50 ml × 4). 1648, 1612, 1583, 1468, 1419, 1335, 1236, 1156, 1040 cm ; MS
The organic layer so obtained was washed with saturated (ESI) m/z 441 [M + H]; HR-MS (ESI) m/z for C H N O F cal-
2
2
19 4 4 2
NaHCO
3
solution and dried on anhydrous Na
2
SO
4
. Ethyl culated m/z: 441.13689, found m/z: 441.13681.
(E)-2-(3-Phenyl-1H-pyrazol-5-yl)-5-(3,4,5-trimethoxystyryl)-
acetate was distilled off under vacuum to produce a crude com-
pound and this was purified by column chromatography using 1,3,4-oxadiazole 11d. This compound was prepared using the
ethyl acetate and hexane resulting in pure compounds (E)-2- procedure described above by the addition of 3-phenyl-1H-pyr-
(
3-substituted
styryl)-1,3,4-oxadiazole 11(a–i) in good yields (60–70%).
E)-2-(3-(4-Chlorophenyl)-1H-pyrazol-5-yl)-5-(3,4,5-trimethoxy- compound was obtained as a pale yellow colored solid. Yield:
phenyl-1H-pyrazol-5-yl)-5-(3,4,5-trimethoxy- azole-5-carbohydrazide 10d (202 mg, 1.0 mmol) and (E)-3-
(3,4,5-trimethoxyphenyl)acrylic acid (238 mg, 1.0 mmol). The
(
1
styryl)-1,3,4-oxadiazole 11a. This compound was prepared 270 mg (66.8%); mp: 205–207 °C; H NMR (300 MHz, DMSO-
using the procedure described above by the addition of 3-(4- d ); δ 3.86 (s, 3H, –OCH ), 3.93 (s, 6H, –OCH ), 6.89 (s, 2H,
6
3
3
chlorophenyl)-1H-pyrazole-5-carbohydrazide 10a (236 mg, ArH), 7.10 (d, 1H, J = 16.2 Hz, -trans H), 7.20 (s, 1H, ArH),
.0 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid 7.40 (d, 1H, J = 7.1 Hz, ArH), 7.43–7.52 (t, 1H, J = 7.5 Hz, ArH),
238 mg, 1.0 mmol). The compound was obtained as a yellow 7.63 (d, 1H, J = 16.4 Hz, -trans H), 7.67–7.71 (m, 1H, ArH),
1
(
1
13
colored solid. Yield: 300 mg (68.4%); mp: 225–227 °C; H NMR 7.80 (d, 2H, J = 7.1 Hz, ArH); C NMR (75 MHz, DMSO-d
6
):
(
–
300 MHz, DMSO-d
6
); δ 3.79 (s, 3H, –OCH
3
), 3.90 (s, 6H, δ 55.7, 59.9, 102.4, 104.8, 108.8, 125.2, 128.2, 128.6, 130.0,
−
1
OCH ), 6.94 (s, 2H, ArH), 7.18 (d, 1H, J = 18.3 Hz, -trans H), 138.6, 138.9, 152.91, 158.8, 163.3 ppm; IR (KBr) (νmax/cm ):
3
7
7
.45 (d, 2H, J = 8.4 Hz, ArH), 7.59 (d, 1H, J = 16.2 Hz, -trans H), ν = 3427, 3150, 3110, 3013, 2851, 1644, 1603, 1521, 1447,
.79 (d, 2H, J = 8.4 Hz, ArH), 7.90 (s, 1H, ArH); C NMR 1233, 1035 cm ; MS (ESI) m/z 405 [M + H]; HR-MS (ESI)
1
3
−1
(
75 MHz, DMSO-d ): δ 54.1, 58.2, 101.3, 103.4, 107.2, 125.2, m/z for C H N O calculated m/z: 405.15573, found m/z:
6
22 20 4 4
1
27.1, 128.4, 131.5, 137.2, 151.2, 161.8 ppm; IR (KBr) (νmax
/
405.15483.
(E)-2-(3-(3,4-Dimethylphenyl)-1H-pyrazol-5-yl)-5-(3,4,5-tri-
251, 1176, 1125, 1029, 1003 cm ; MS (ESI) 439 [M + H] m/z methoxystyryl)-1,3,4-oxadiazole 11e. This compound was
−
1
cm ): ν = 3428, 2925, 2846, 1611, 1582, 1503, 1456, 1311,
1
−
1
[
4
M + H]; HR-MS (ESI) m/z for C22
39.11676, found m/z: 439.11597.
H
20
N
4
O
4
Cl calculated m/z: prepared using the procedure described above by the addition
of 3-(3,4-dimethylphenyl)-1H-pyrazole-5-carbohydrazide 11e
(E)-2-(3-(4-Methoxyphenyl)-1H-pyrazol-5-yl)-5-(3,4,5-trimethoxy- (230 mg, 1.0 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)acrylic
styryl)-1,3,4-oxadiazole 11b. This compound was prepared acid (238 mg, 1.0 mmol). The compound was obtained as a
using the procedure described above by the addition of 3-(4- pale yellow colored solid. Yield: 280 mg (64.8%); mp:
1
methoxyphenyl)-1H-pyrazole-5-carbohydrazide 10b (232 mg, 222–224 °C; H NMR (300 MHz, DMSO-d ); δ 2.29 (s, 6H,
6
1
.0 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid –CH
3
), 2.33 (s, 6H, –CH
), 6.90 (s, 3H, ArH), 6.96 (d, 2H, J = 5.4 Hz, ArH), 7.08 (d,
yellow colored solid. Yield: 298 mg (68%); mp: 189–192 °C; H 1H, J = 16.2 Hz, -trans H), 7.12–7.26 (m, 1H, ArH), 7.45 (d, 1H,
3 3
), 3.80 (s, 3H, –OCH ), 3.92 (s, 6H,
(238 mg, 1.0 mmol). The compound was obtained as a pale –OCH
3
1
1
3
NMR (300 MHz, DMSO-d
OCH ), 3.94 (s, 6H, –OCH
ArH), 7.01–7.05 (m, 2H, ArH), 7.11 (s, 1H, ArH), 7.71 (d, 1H, J = 109.0, 109.1, 122.6, 126.3, 129.7, 130.0, 138.0, 138.6, 152.8,
6
); δ 3.56 (s, 3H, –OCH
3
), 3.88 (s, 3H, J = 16.2 Hz, -trans H), 7.48–7.67 (m, 2H, ArH); C NMR
–
3
3
), 6.49 (s, 1H, ArH), 6.96 (s, 1H, (75 MHz, DMSO-d
6
): δ 19.1, 19.3, 55.7, 59.9, 101.8, 104.8,
−
1
1
1
9
1
2
1
5.8 Hz, -trans H), 7.95–7.99 (m, 2H, ArH), 8.42 (d, 1H, J = 163.0 ppm; IR (KBr) (νmax/cm ): ν = 3143, 3000, 2938, 2835,
1
3
5.8 Hz, -trans H); C NMR (75 MHz, DMSO-d
6
): δ 55.3, 55.8, 1647, 1583, 1528, 1504, 1451, 1419, 1333, 1244, 1186, 1154,
7.6, 104.9, 114.4, 120.9, 122.4, 122.9, 133.3, 128.3, 132.8, 1125, 1038 cm− ; MS (ESI) m/z 434 [M + H]; HR-MS (ESI) m/z
55.8, 161.2 ppm; IR (KBr) (νmax/cm ): ν = 3208, 3160, 2932, for
1
−1
C
24
H
25
N
4
O
4
calculated m/z: 433.18703, found m/z:
841, 1625, 1582, 1530, 1509, 1418, 1362, 1241, 1133, 1091, 433.18632.
052 cm− ; MS (ESI) m/z 435 [M + H]; HR-MS (ESI) m/z for
1
(E)-2-(3-(3,4-Dichlorophenyl)-1H-pyrazol-5-yl)-5-(3,4,5-tri-
methoxystyryl)-1,3,4-oxadiazole 11f. This compound was
23 23 4 5
C H N O
calculated m/z: 435.16630, found m/z: 435.16665.
(
E)-2-(3-(3,4-Difluorophenyl)-1H-pyrazol-5-yl)-5-(3,4,5-tri- prepared using the procedure described above by the addition
methoxystyryl)-1,3,4-oxadiazole 11c. This compound was of 3-(3,4-dichlorophenyl)-1H-pyrazole-5-carbohydrazide 10f
prepared using the procedure described above by the addition (271 mg, 1.0 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)acrylic
of 3-(3,4-difluorophenyl)-1H-pyrazole-5-carbohydrazide 10c acid (238 mg, 1.0 mmol). The compound was obtained as a
(
238 mg, 1.0 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)acrylic brown colored solid. Yield: 315 mg (66.5%); mp: 158–160 °C;
1
acid (238 mg, 1.0 mmol). The compound was obtained as a
brown colored solid. Yield: 295 mg (67%); mp: 150–152 °C; H 6H, –OCH ), 6.76 (s, 2H, ArH), 6.80 (s, 1H, ArH), 6.93 (d, 1H,
H NMR (400 MHz, DMSO-d
6 3
); δ 3.89 (s, 3H, –OCH ), 3.90 (s,
1
3
NMR (300 MHz, DMSO-d ); δ 3.81 (s, 3H, –OCH ), 3.92 (s, 6H, J = 15.8 Hz, -trans H), 6.98 (d, 1H, J = 7.1 Hz, ArH), 7.26 (s, 1H,
6
3
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ArH), 7.40 (d, 1H, J = 15.8 Hz, -trans H), 7.52 (d, 1H, J = 8.2 Hz,
Preparation of (E)-2-(2-(benzo[d][1,3]dioxol-5-yl)vinyl)-5-(3-
1
3
ArH); C NMR (75 MHz, DMSO-d ): δ 54.1, 58.2, 103.2, 107.5, substituted phenyl-1H-pyrazol-5-yl)-1,3,4-oxadiazole 12a–i. To
6
1
1
1
1
28.4, 136.3, 137.0, 137.1, 137.3, 151.2, 160.9, 161.4, each of the 3-substituted phenyl-1H-pyrazole-5-carbohydra-
62.1 ppm; IR (KBr) (νmax/cm ): ν = 3049, 3000, 2939, 2835, zides 10(b–i) (1.0 mol) produced in the above step was added
−
1
647, 1583, 1529, 1503, 1466, 1419, 1334, 1245, 1186, 1125, (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic acid (1.0 mol) in POCl3
045 cm− ; MS (ESI) m/z 511 [M + K].
1
and stirred at a temperature of 110 °C for 5 h. The completion
(E)-2-(3-(Benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-5-(3,4,5-tri- of the reaction was monitored by TLC in an ethyl acetate and
methoxystyryl)-1,3,4-oxadiazole 11g. This compound was pre- hexane solvent system and then ice cold water was added to
pared using the procedure described above by the addition of the reaction mixture followed by extraction with ethyl acetate
3
-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbohydrazide 10g (50 ml × 4). The organic layer so obtained was washed with
(
246 mg, 1.0 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)acrylic saturated NaHCO solution and dried on anhydrous Na SO .
3
2
4
acid (238 mg, 1.0 mmol). The compound was obtained as a Ethyl acetate was distilled off under vacuum to produce a
brown red colored solid. Yield: 294 mg (65.6%); mp: crude compound and this was further purified by column
1
1
99–202 °C; H NMR (300 MHz, DMSO-d ); δ 3.84 (s, 3H, chromatography using ethyl acetate and hexane to afford pure
6
–
1
2
3 3 2
OCH ), 3.94 (s, 6H, –OCH ), 6.05 (s, 2H, –OCH O–), 6.86 (d, compounds of each (E)-2-(2-(benzo[d][1,3]dioxol-5-yl)vinyl)-5-
H, J = 7.9 Hz, ArH), 6.94 (d, 1H, J = 16.8 Hz, -trans H), 7.09 (s, (3-substituted phenyl-1H-pyrazol-5-yl)-1,3,4-oxadiazole 12(a–i)
H, ArH), 7.16 (d, 2H, J = 8.9 Hz, ArH), 7.60 (d, 1H, J = 15.8 Hz, in good yields (65–75%).
1
3
-
d
1
trans H), 7.67–7.69 (m, 1H, ArH); C NMR (75 MHz, DMSO-
): δ 55.8, 60.0, 101.2, 102.0, 104.8, 105.7, 108.5, 109.2, 119.3, 1H-pyrazol-5-yl)-1,3,4-oxadiazole 12a. This compound was pre-
30.1, 138.1, 138.8, 147.5, 147.8, 153.0, 162.6 ppm; IR (KBr) pared using the procedure described above by the addition of
(E)-2-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-5-(3-(4-chlorophenyl)-
6
−1
(
νmax/cm ): ν = 3050, 2983, 1645, 1583, 1529, 1504, 1461, 3-(4-chlorophenyl)-1H-pyrazole-5-carbohydrazide 10a (236 mg,
−
1
1
+
4
418, 1334, 1240, 1188, 1125, 1035 cm ; MS (ESI) m/z 449 [M 1.0 mmol) and (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic acid
H]; HR-MS (ESI) m/z for C H N O calculated m/z: (192 mg, 1.0 mmol). The compound was obtained as a brown
2
3
21 4 6
1
49.14556, found m/z: 449.14569.
colored solid. Yield: 263 mg (67%); mp: 164–166 °C; H NMR
); δ 6.04 (s, 2H, –OCH O–), 6.81 (d, 1H, J =
(
E)-2-(3-(3,4,5-Trimethoxyphenyl)-1H-pyrazol-5-yl)-5-(3,4,5-tri- (300 MHz, DMSO-d
6
2
methoxystyryl)-1,3,4-oxadiazole 11h. This compound was pre- 16.4 Hz, -trans H), 6.84 (d, 2H, J = 7.7 Hz, ArH), 7.0 (d, 1H, J =
pared using the procedure described above by the addition of 8.1 Hz, ArH), 7.09–7.21 (m, 2H, ArH), 7.41 (d, 1H, J = 16.4 Hz,
1
3
3
-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carbohydrazide 11h -trans H), 7.46 (s, 2H, ArH), 7.64–7.84 (m, 1H, ArH); C NMR
(
292 mg, 1.0 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)acrylic (75 MHz, DMSO-d ): δ 101.4, 106.0, 108.0, 108.3, 124.0, 129.0,
6
−
1
acid (238 mg, 1.0 mmol). The compound was obtained as a 137.8, 147.9, 148.6, 162.8, 163.9 ppm; IR (KBr) (νmax/cm ): ν =
yellow colored crystal. Yield: 345 mg (69.8%); mp: 184–185 °C; 2924, 2854, 1743, 1638, 1603, 1579, 1530, 1496, 1445, 1355,
1
−1
H NMR (400 MHz, DMSO-d ); δ 3.70 (s, 9H, –OCH ), 3.85 (s, 1316, 1255, 1255, 1227, 1097, 1033, 965 cm ; MS (ESI) m/z 393
6
3
9
H, –OCH
3
), 7.08–7.25 (m, 4H, ArH), 7.37–7.50 (m, 2H, J = [M + H].
(E)-2-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-5-(3-(4-methoxyphenyl)-
1
6.9 Hz, trans H, ArH), 7.63 (d, 1H, J = 16.3 Hz, trans H);
1
3
C NMR (75 MHz, DMSO-d ): δ 56.0, 60.0, 103.0, 105.4, 109.2, 1H-pyrazol-5-yl)-1,3,4-oxadiazole 12b. This compound was
6
1
24.0, 130.2, 137.7, 138.9, 153.1, 153.3, 159.1, 163.6 ppm; prepared using the procedure described above by the addition
−
1
IR (KBr) (νmax/cm ): ν = 3115, 2947, 2832, 1683, 1612, 1587, of 3-(4-methoxyphenyl)-1H-pyrazole-5-carbohydrazide 10b
−
1
1
545, 1463, 1427, 1377, 1346, 1237, 1132, 1070 cm ; MS (ESI) (232 mg, 1.0 mmol) and (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic
m/z 495 [M + H]; HR-MS (ESI) m/z for C25
m/z: 495.18944, found m/z: 495.18350.
H
27
N
4
O
7
calculated acid (192 mg, 1.0 mmol). The compound was obtained as a
yellow colored solid. Yield: 235 mg (65.6%); mp: 186–189 °C;
1
(
E)-2-(3-(3,4-Dimethoxyphenyl)-1H-pyrazol-5-yl)-5-(3,4,5-tri-
H NMR (500 MHz, DMSO-d ); δ 3.81 (s, 3H, –OCH ), 6.09 (s,
6 3
2
methoxystyryl)-1,3,4-oxadiazole 11i. This compound was pre- 2H, –OCH O–), 6.98 (d, 1H, J = 7.9 Hz, ArH), 7.02 (d, 2H, J =
pared using the procedure described above by the addition 7.7 Hz, ArH), 7.16–7.40 (m, 3H, J = 15.2 Hz, -trans H, ArH), 7.53
of 3-(3,4-dimethoxyphenyl)-1H-pyrazole-5-carbohydrazide 10i (s, 1H, ArH), 7.60 (d, 1H, J = 16.4 Hz, -trans H), 7.81 (d, 2H, J =
1
3
(
6
262 mg, 1.0 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)acrylic 7.7 Hz, ArH); C NMR (75 MHz, DMSO-d ): δ 55.2, 101.4,
acid (238 mg, 1.0 mmol). The compound was obtained as a 101.8, 106.1, 106.2, 107.8, 108.4, 114.4, 120.7, 124.2, 126.8,
brown colored solid. Yield: 315 mg (67.8%); mp: 158–160 °C; 129.1, 137.5, 138.5, 143.9, 148.0, 148.8, 159.2, 159.5,
1
−1
H NMR (300 MHz, DMSO-d
6
); δ 3.89 (s, 3H, –OCH
), 3.93 (s, 3H, –OCH ), 6.76 (s, 1H, ArH), 6.80 (d, 1501, 1447, 1257, 1360, 1308, 1177, 1119, 1032, 965 cm ; MS
H, J = 6.2 Hz, ArH), 6.92 (d, 1H, J = 16.3 Hz, -trans H), 6.98 (d, (ESI) m/z 389 [M + H]; HR-MS (ESI) m/z for C H N O calcu-
3
), 3.91 (s, 163.6 ppm; IR (KBr) (νmax/cm ): ν = 3195, 2922, 2852, 1608,
−
1
9
2
1
H, –OCH
3
3
21
17 4 4
H, J = 16.3 Hz, -trans H), 7.04–7.08 (m, 1H, ArH), 7.19–7.25 lated m/z: 389.12443, found m/z: 389.12518.
1
3
(m, 1H ArH), 7.39–7.45 (m, 1H ArH); C NMR (75 MHz,
(E)-2-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-5-(3-(3,4-difluorophenyl)-
DMSO-d ): δ 55.9, 60.0, 105.2, 109.3, 130.2, 138.1, 138.8, 153.0, 1H-pyrazol-5-yl)-1,3,4-oxadiazole 12c. This compound was
6
−1
1
1
1
62.9, 163.9 ppm; IR (KBr) (νmax/cm ): ν = 3150, 2883, 1660, prepared using the procedure described above by the addition
645, 1553, 1529, 1500, 1461, 1418, 1287, 1240, 1188, 1125, of 3-(3,4-difluorophenyl)-1H-pyrazole-5-carbohydrazide 10c
033 cm− ; MS (ESI) m/z 465 [M + H].
1
(238 mg, 1.0 mmol) and (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic
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acid (192 mg, 1.0 mmol). The compound was obtained as a (d, 1H, J = 8.0 Hz, ArH), 7.05 (s, 2H, ArH), 7.26 (s, 3H, ArH),
1
3
yellow colored solid; yield: 250 mg (63.4%); mp: 210–212 °C; 7.40 (d, 1H, J = 16.0 Hz, -trans H); C NMR (75 MHz, DMSO-
1
H NMR (300 MHz, DMSO-d
s, 1H, ArH), 6.99 (d, 1H, J = 16.6 Hz, -trans H), 7.09 (s, 1H, 147.9, 162.8, 163.9 ppm; IR (KBr) (νmax/cm ): ν = 3421,
ArH), 7.23 (s, 1H, ArH), 7.42–7.68 (m, 1H, ArH), 7.59 (d, 1H, J = 2916, 1652, 1635, 1604, 1559, 1499, 1447, 1318, 1256, 1228,
6
); δ 6.04 (s, 2H, –OCH
2
O–), 6.88
6
d ): δ 101.4, 106.0, 108.0, 108.3, 124.0, 129.0, 137.8, 148.6,
−
1
(
6.4 Hz, -trans H), 7.72–7.84 (m, 1H, ArH), 7.88 (s, 2H, ArH), 1102, 1036 cm− ; MS (ESI) m/z 427 [M + H]; HR-MS (ESI) m/z
1
1
1
1
1
1
3
3.93 (brs, 1H, –NH); C NMR (75 MHz, DMSO-d
03.6, 106.1, 107.7, 108.4, 114.5, 117.3, 118.4, 122.3, 123.4, 427.03582.
24.3, 129.0, 138.7, 139.7, 148.0, 148.9, 163.7 ppm; IR (KBr) (Z)-5-Methoxy-3-((3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)-
max/cm ): ν = 3116, 3055, 2921, 2853, 1637, 1609, 1503, methylene)indolin-2-one 12g. This compound was prepared
6 2 4 3
): δ 101.4, for C20H13Cl N O calculated m/z: 427.03592, found m/z:
−1
(
ν
−
1
1
494, 1491, 1446, 1362, 1246, 1036 cm ; MS (ESI) m/z 395 using the procedure described above by the addition of
[
M + H]; HR-MS (ESI) m/z for C20
H
13
F
2
N
4
O
3
calculated 3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbohydrazide 10g
(246 mg, 1.0 mmol) and (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic
m/z:395.09502, found m/z: 395.09491.
(
E)-2-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-5-(3-phenyl-1H-pyrazol- acid (192 mg, 1.0 mmol). The compound was obtained as a
1
5
-yl)-1,3,4-oxadiazole 12d. This compound was prepared using saffron colored solid; yield: 270 mg (67%); mp: 196–198 °C; H
the procedure described above by the addition of 3-phenyl-1H- NMR (300 MHz, DMSO-d ); δ 6.01 (s, 4H, OCH O), 6.53 (d, 1H,
pyrazole-5-carbohydrazide 10d (202 mg, 1.0 mmol) and (E)-3- J = 15.5 Hz, -trans H), 6.81 (d, 2H, J = 7.7 Hz, ArH), 7.0 (d, 1H,
benzo[d][1,3]dioxol-5-yl)acrylic acid (192 mg, 1.0 mmol). The J = 7.7 Hz, ArH), 7.05 (s, 1H, ArH), 7.45–7.63 (m, 4H, J = 15.5
compound was obtained as a saffron colored solid. Yield: Hz, -trans H, ArH); C NMR (75 MHz, DMSO-d
35 mg (65.5%); mp: 220–222 °C; H NMR (400 MHz, DMSO- 106.2, 108.5, 117.4, 123.5, 128.9, 139.8, 147.9, 148.6, 163.8,
6
2
(
1
3
6
): δ 101.4,
1
2
d
(
−
1
6 2
); δ 6.04 (s, 2H, –OCH O–), 6.87 (d, 1H, J = 8.1 Hz, ArH), 6.92 167.7 ppm; IR (KBr) (νmax/cm ): ν = 3198, 3020, 1637, 1601,
d, 1H, J = 16.2 Hz, -trans H), 7.05–7.11 (m, 2H, ArH), 7.20 (s, 1483, 1443, 1365, 1251, 1101, 1037 cm ; MS (ESI) m/z 403
−
1
1
H, ArH), 7.35–7.48 (m, 4H, ArH), 7.61 (d, 1H, J = 16.4 Hz, [M + H].
1
3
-
trans H), 7.77 (d, 1H, J = 7.1 Hz, ArH); C NMR (75 MHz,
DMSO-d ): δ 101.5, 102.8, 106.2, 107.7, 108.4, 124.3, 125.4, phenyl)-1H-pyrazol-5-yl)-1,3,4-oxadiazole 12h. This compound
28.7, 129.0, 129.1, 138.6, 148.0, 148.8, 163.7 ppm; IR (KBr) was prepared using the procedure described above by the
(E)-2-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-5-(3-(3,4,5-trimethoxy-
6
1
−1
(
νmax/cm ): ν = 3448, 3120, 3069, 2995, 2956, 2828, 1677, addition of 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carbohy-
−
1
1
610, 1516, 1337, 1247, 1207, 1167 cm ; MS (ESI) m/z 359 drazide 11h (292 mg, 1.0 mmol) and (E)-3-(benzo[d][1,3]-
[M + H]; HR-MS (ESI) m/z for C H N O calculated m/z: dioxol-5-yl)acrylic acid (192 mg, 1.0 mmol). The compound
20 15 4 3
3
59.11387, found m/z: 359.11377.
E)-2-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-5-(3-(3,4-dimethylphe- (66.9%); mp: 228–230 °C; H NMR (300 MHz, DMSO-d
nyl)-1H-pyrazol-5-yl)-1,3,4-oxadiazole 12e. This compound was (s, 3H, –OCH ), 3.94 (s, 6H, –OCH ), 6.05 (s, 2H, –OCH O–),
was obtained as a light yellow colored solid. Yield: 300 mg
1
(
6
); δ 3.84
3
3
2
prepared using the procedure described above by the addition 6.86 (d, 1H, J = 7.9 Hz, ArH), 6.94 (d, 1H, J = 16.8 Hz, -trans H),
of 3-(3,4-dimethylphenyl)-1H-pyrazole-5-carbohydrazide 10e 7.09 (s, 2H, ArH), 7.16 (d, 1H, J = 8.9 Hz, ArH), 7.60 (d, 1H, J =
1
3
(
230 mg, 1.0 mmol) and (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic 15.8 Hz, -trans H), 7.67–7.69 (m, 1H, ArH); C NMR (75 MHz,
acid (192 mg, 1.0 mmol). The compound was obtained as a DMSO-d ): δ 54.3, 58.5, 99.8, 101.0, 101.3, 104.3, 106.0, 106.7,
pale yellow colored solid. Yield: 265 mg (68.6%); mp: 122.2, 127.4, 136.2, 136.8, 146.5, 147.3, 151.6, 161.9 ppm;
6
1
−1
1
–
87–189 °C; H NMR (400 MHz, DMSO-d ); δ 2.28 (s, 3H, IR (KBr) (νmax/cm ): ν = 3136, 3103, 2924, 1638, 1589, 1527,
6
−
1
CH
3
), 2.32 (s, 3H, –CH
3
), 6.08 (s, 2H, –OCH
2
O–), 6.91 (s, 1H, 1503, 1490, 1468, 1253, 1123, 1036 cm ; MS (ESI) m/z 449
calculated m/z:
ArH), 7.07–7.32 (m, 4H, J = 16.0 Hz, -trans H, ArH), 7.40 (s, 1H, [M + H]; HR-MS (ESI) m/z for C23
21 4 6
H N O
1
3
ArH), 7.58 (d, 3H, J = 16.0 Hz, -trans H, ArH); C NMR 449.14556, found m/z: 449.14424.
75 MHz, DMSO-d ): δ 19.0, 19.2, 101.1, 101.8, 105.7, 108.0, (E)-2-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-5-(3-(3,4-dimethoxy-
22.5, 123.5, 126.2, 128.7, 129.6, 136.5, 136.6, 138.2, 147.9, phenyl)-1H-pyrazol-5-yl)-1,3,4-oxadiazole 12i. This compound
48.7, 163.3 ppm; IR (KBr) (νmax/cm ): ν = 3170, 3068, 2918, was prepared using the procedure described above by the
851, 1663, 1522, 1498, 1485, 1418, 1360, 1253, 1169, addition of 3-(3,4-dimethoxyphenyl)-1H-pyrazole-5-carbohydra-
031 cm− ; MS (ESI) m/z 387 [M + H]; HR-MS (ESI) m/z for zide 10i (262 mg, 1.0 mmol) and (E)-3-(benzo[d][1,3]dioxol-5-
(
6
1
1
2
1
−1
1
C H N O calculated m/z: 387.14517, found m/z: 387.14565.
yl)acrylic acid (192 mg, 1.0 mmol). The compound was
2
2
19 4 3
(
E)-2-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-5-(3-(3,4-dichlorophe- obtained as a light yellow colored solid. Yield: 265 mg (63.3%);
1
nyl)-1H-pyrazol-5-yl)-1,3,4-oxadiazole 12f. This compound was mp: 172–174 °C; H NMR (500 MHz, DMSO-d
6
); δ 3.78 (s, 3H,
prepared using the procedure described above by the addition –OCH ), 3.85 (s, 3H, –OCH ), 6.09 (s, 2H, –OCH O–), 6.99 (s,
3
3
2
of 3-(3,4-dichlorophenyl)-1H-pyrazole-5-carbohydrazide 10f 1H, ArH), 7.02 (s, 1H, ArH), 7.13–7.31 (m, 2H, ArH), 7.36 (s,
271 mg, 1.0 mmol) and (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic 1H, ArH), 7.45 (d, 2H, J = 16.7 Hz, -trans H), 7.36 (s, 1H, ArH),
(
1
3
acid (192 mg, 1.0 mmol). The compound was obtained as a 7.45 (d, 1H, J = 16.7 Hz, -trans H); C NMR (75 MHz, DMSO-
yellow colored solid. Yield: 285 mg (66.7%); mp: 186–188 °C; ): δ 55.6, 59.7, 101.5, 102.2, 106.2, 107.8, 108.5, 109.2, 112.0,
O–), 117.9, 121.2, 124.3, 129.1, 138.6, 149.0, 149.1, 159.0,
d
6
1
H NMR (400 MHz, DMSO-d
6 2
); δ 6.01 (s, 2H, –OCH
−
1
6
.82–6.86 (m, 1H, ArH), 6.82 (d, 1H, J = 16.0 Hz, -trans H), 6.99 163.6 ppm; IR (KBr) (νmax/cm ): ν = 3117, 2917, 1684, 1620,
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1
1
549, 1515, 1461, 1416, 1337, 1244, 1187, 1127, 1079, purified by column chromatography using ethyl acetate and
039 cm− ; MS (ESI) m/z 419 [M + H]; HR-MS (ESI) m/z for hexane to yield a yellow colored pure compound (E)-2-(3-
1
C
22
H
19
N
4
O
5
calculated m/z: 419.13500, found m/z: 419.13453.
methyl-1H-pyrazol-5-yl)-5-(3,4,5-trimethoxystyryl)-1,3,4-oxadia-
1
Preparation of ethyl 2,4-dioxopentanoate 14. Initially zole (17a) in good yield: 240 mg (70%); mp: 15–152 °C;
H
sodium ethanolate was prepared in situ and diethyl oxalate NMR (400 MHz, DMSO-d ); δ 2.57 (s, 3H, –CH ), 3.85 (s, 3H,
6
3
(1.0 mol) was added slowly at 0 °C. The stirring was continued –OCH
3
), 3.89 (s, 6H, –OCH
3
), 6.71 (s, 2H, ArH), 6.76 (s, 1H,
for 15 minutes followed by addition of acetone (13) (1.0 mol) ArH), 6.86 (d, 1H, J = 16.8 Hz, -trans H), 7.36 (d, 1H, J = 15.8
1
3
dropwise maintaining the temperature at 0 °C. After com- Hz, -trans H); C NMR (75 MHz, DMSO-d ): δ 8.9, 54.2, 58.4,
6
pletion of addition the stirring was continued for 4 h at room 103.2, 107.6, 128.5, 136.5, 137.1, 137.3, 137.5, 151.4, 160.9,
−1
temperature. The reaction mixture was neutralized by the 161.6, 162.3 ppm; IR (KBr) (νmax/cm ): ν = 3503, 3049, 2999,
−
1
addition of dilute H SO solution and further extracted with 2940, 2834, 1641, 1583, 1503, 1466, 1334,1245, 1125 cm ; MS
2
4
ethyl acetate to obtain product 14 (yield 80–90%) which was (ESI) m/z 343 [M + H].
taken as such for the next step without purification.
(E)-2-(2-(Benzo[d][1,3]dioxol-5-yl)vinyl)-5-(3-methyl-1H-pyrazol-
Preparation of ethyl 3-methyl-1H-pyrazole-5-carboxylate 5-yl)-1,3,4-oxadiazole 17b. To the 3-methyl-1H-pyrazole-5-carbo-
1
5. The produced ethyl 2,4-dioxopentanoate (14) (1.0 mol) was hydrazide 16 (154 mg, 1.0 mol) produced in the above step was
added to hydrazine dihydrochloride (N ·2HCl) (1.5 mol) in added (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic acid (192 mg,
2
H
4
ethanol and refluxed for 3–4 h. The progress of the reaction 1.0 mmol) in POCl and stirred at a temperature of 110 °C for
3
was monitored by TLC using an ethyl acetate–hexane solvent 5 h. The completion of the reaction was monitored by TLC in
system. The ethanol was distilled under vacuum and then an ethyl acetate and hexane solvent system and then ice cold
water was added to the residue followed by extraction with water was added to the reaction mixture followed by extraction
ethyl acetate (50 ml × 4). The organic layer was dried on anhy- with ethyl acetate (50 ml × 4). The organic layer so obtained
2 4 3
drous Na SO and the solvent was evaporated to obtain a was washed with saturated NaHCO solution and dried on
crude compound that was further purified by column chrom- anhydrous Na SO . Ethyl acetate was distilled off under
2
4
atography using an ethyl acetate and hexane solvent system. vacuum to produce a crude compound and this was further
The pure compound (15) was eluted with 25–30% of ethyl purified by column chromatography using ethyl acetate and
acetate with good yields. Yellow colored solid (yield 80.0%): hexane to afford a pale yellow colored pure compound (E)-
1
R
f
= 0.3 (30% ethyl acetate–hexane); H NMR (300 MHz, 2-(2-(benzo[d][1,3]dioxol-5-yl)vinyl)-5-(3-methyl-1H-pyrazol-5-yl)-
3 3
CDCl ); δ 1.32–1.41 (t, 3H, J = 6.7 Hz, –CH ), 2.36 (s, 3H, 1,3,4-oxadiazole (17b) in good yield: 200 mg (67.5%); mp:
1
–
CH ), 4.32–4.44 (q, 2H, J = 6.7 Hz, J = 7.5 Hz, –CH ), 7.04 (s, 210–212 °C; H NMR (400 MHz, DMSO-d ); δ 2.55 (s, 3H,
2
1
1
2
6
1
H, ArH) ppm; MS (ESI) m/z 155 [M + H].
Preparation of
6. To the ethyl 3-methyl-1H-pyrazole-5-carboxylate (15) Hz, -trans H), 7.24 (s, 1H, ArH), 7.35 (d, 1H, J = 16.0 Hz, -trans
–CH
3
), 5.99 (s, 2H, –OCH
2
O), 6.74 (d, 1H, J = 8.0 Hz, ArH), 6.79
3-methyl-1H-pyrazole-5-carbohydrazide (s, 1H, ArH), 6.95 (d, 1H, J = 8.0 Hz, ArH), 7.04 (d, 1H, J = 16.0
1
1
3
obtained in the above step was added hydrazine hydrate H); C NMR (75 MHz, DMSO-d
6
): δ 8.7, 99.5, 104.1, 106.4,
(
4.0 mol) in ethanol at room temperature and then heated to 106.6, 122.0, 127.2, 135.9, 146.1, 146.8, 160.8, 162.1 ppm;
−1
reflux for 3–4 h. The completion of the reaction was monitored IR (KBr) (νmax/cm ): ν = 3448, 3120, 3069, 2995, 2956, 2828,
by TLC using an ethyl acetate and hexane (1 : 1) system. The 1677, 1610, 1516, 1337, 1247, 1207, 1167 cm ; MS (ESI) m/z
reaction mixture was allowed to attain room temperature and 297 [M + H].
−1
then the solvent was distilled off under reduced pressure. The
residue was dissolved in water and extracted twice with ethyl
Biology
acetate, dried over anhydrous Na
2
SO
4
and the solvent was
Cell cultures, maintenance and antiproliferative evaluation.
removed under reduced pressure to afford the compound (16) All the cell lines used in this study were purchased from the
as a crystalline solid. The carbohydrazide obtained by this American Type Culture Collection (ATCC, United States). A549,
method was pure and was taken as such for the next step MCF-7, HeLa and IMR32 were grown in Dulbecco’s modified
without further purification.
E)-2-(3-Methyl-1H-pyrazol-5-yl)-5-(3,4,5-trimethoxystyryl)- atmosphere of 5% CO
,3,4-oxadiazole 17a. To the 3-methyl-1H-pyrazole-5-carbohy- sub-confluent from T25 flasks/60 mm dishes and seeded in
Eagle’s medium (containing 10% FBS under a humidified
(
2
at 37 °C). Cells were trypsinized when
1
drazide 16 (154 mg, 1.0 mol) produced in the above step was 96-well plates. The pyrazole–oxadiazole conjugates were evalu-
added (E)-3-(3,4,5-trimethoxyphenyl)acrylic acid (238 mg, ated for their in vitro antiproliferative activity in four different
1
.0 mol) in POCl
3
and stirred at a temperature of 110 °C for human cancer cell lines. A protocol of 48 h continuous drug
6
h. The completion of the reaction was monitored by TLC in exposure was used and a SRB cell proliferation assay was
an ethyl acetate and hexane solvent system and then ice cold employed to estimate cell viability or growth. The cell lines
water was added to the reaction mixture followed by extraction were grown in their respective media containing 10% fetal
with ethyl acetate (50 ml × 4). The organic layer so obtained bovine serum and were seeded in 96-well microtiter plates in
was washed with saturated NaHCO
anhydrous Na SO . Ethyl acetate was distilled off under time of individual cell lines. The microtiter plates were incu-
vacuum to produce a crude compound and this was further bated at 37 °C, 5% CO , 95% air, and 100% relative humidity
3
solution and dried on 200 μL aliquots at plating densities depending on the doubling
2
4
2
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for 24 h prior to addition of experimental drugs. Aliquots of length was 360 nm) was measured for 1 h at 1 min intervals in
μL of the test compounds were added to the wells already a multimode plate reader (Tecan M200). To determine the IC50
2
containing 198 μL of cells, resulting in the required final drug values of the compounds against tubulin polymerization, the
concentrations. For each compound, four concentrations (0.1, compounds were pre-incubated with tubulin at varying con-
1
, 10 and 100 μM) were evaluated, and each was done in tripli- centrations (0.001, 0.01, 0.1, 1, 10 and 100 μM). Assays were
cate wells. Plates were incubated further for 48 h, and the performed under similar conditions to those employed for
2
7
assay was terminated by the addition of 100 μL of 10% (wt/vol) polymerization assays as described above.
cold trichloroacetic acid and incubated at 4 °C for 1 h. The Analysis of the cell cycle. A549 cells in 60 mm dishes were
supernatant was discarded. The plate was washed four times incubated for 24 h in the presence or absence of test com-
with tap water and was allowed to air dry. The cells were then pounds 11a, 11d and 11f at 5 μM concentration. Cells were har-
stained with 0.057% SRB dissolved in 1% acetic acid for vested with Trypsin-EDTA, fixed with ice-cold 70% ethanol at
3
0 min at room temperature. Unbound SRB was washed away 4 °C for 30 min, ethanol was removed by centrifugation and
with four washes of 1% acetic acid. The plate was again cells were stained with 1 ml of DNA staining solution [0.2 mg
allowed to air dry and the bound SRB stain, representing sur- of propidium iodide (PI), and 2 mg RNase A] for 30 min as
viving cells, was dissolved in 50 μL of Tris base (10 mM). The described earlier. The DNA contents of 20 000 events were
optical density was determined at 510 nm using a microplate measured using a flow cytometer (BD FACSCanto II). Histo-
2
9
reader (Enspire, Perkin Elmer, USA). Percent growth was calcu- grams were analyzed using a FCS express 4 plus.
lated on a plate by plate basis for test wells relative to control Immunohistochemistry of tubulin and analysis of nuclear
wells. The above determination was repeated thrice. The morphology. A549 cells were seeded on glass cover slips, and
growth inhibitory effects of the compounds were analyzed by incubated for 24 h in the presence or absence of test com-
generating dose response curves as a plot of the percentage of pounds 11a, 11d and 11f at a concentration of 5 μM. Cells
surviving cells versus compound concentration. The sensitivity grown on cover slips were fixed in 3.5% formaldehyde in phos-
of the cancer cells to the test compound was expressed in phate-buffered saline (PBS) pH 7.4 for 10 minutes at room
terms of IC50, a value defined as the concentration of the com- temperature. Cells were permeabilized for 6 minutes in PBS
pound that produced 50% reduction as compared to the containing 0.5% Triton X-100 (Sigma) and 0.05% Tween-20
control absorbance. IC50 values are indicated as means ± SD of (Sigma). The permeabilized cells were blocked with 2% BSA
2
5c
three independent experiments.
(Sigma) in PBS for 1 h. Later, the cells were incubated with a
Dot-blot assay. A549 cells were treated with 5 μM concen- primary antibody for tubulin from Sigma at 1 : 200 dilution in
tration of 11a–i, 12a–i and 17a–b for 24 h. Subsequently, cells a blocking solution for 4 h at room temperature. Subsequently
were harvested and proteins were quantified using Amido the antibodies were removed and the cells were washed thrice
Black followed by densitometry analysis. Equal amounts of with PBS. Cells were then incubated with the FITC labeled
proteins were blotted on a nitrocellulose membrane using Bio- anti-mouse secondary antibody (1 : 500) for 1 h at room temp-
Dot SF microfiltration apparatus (Bio-Rad). Briefly, the nitro- erature. Cells were washed thrice with PBS and mounted in a
cellulose membrane and 3 filter papers (Whatmann 3) were medium containing DAPI. Images were captured using the
soaked in 1× TBS solution for 10 min. Later, the filter papers Olympus confocal microscope FLOW VIEW FV 1000 series and
and membrane were arranged in the apparatus and connected analyzed with the FV10ASW 1.7 series software.
to a vacuum pump (Millipore). The membranes were rehy-
Western blot analysis of soluble versus polymerized tubulin.
5
drated using 100 μl of 1× TBS by vacuum filtration. Sub- Cells were seeded in 12-well plates at 1 × 10 cells per well in
sequently, 50 μl of samples were blotted on the membranes complete growth medium. Following treatment of cells with
and washed with 200 μl of 1× TBS by application of vacuum. the respective compounds (11a, 11d and 11f) for a duration of
The blot was blocked with 5% blotto for 1 h at room tempera- 24 h, cells were washed with PBS and subsequently soluble
ture. Immunoblot analysis was performed as described pre- and insoluble tubulin fractions were collected. To collect the
2
5c
viously using the UVP, biospectrum 810 imaging system.
Tubulin polymerization assay. An in vitro assay for monitor- of pre-warmed lysis buffer [80 mM Pipes-KOH (pH 6.8), 1 mM
ing the time-dependent polymerization of tubulin to microtu- MgCl , 1 mM EGTA, 0.2% Triton X-100, 10% glycerol, 0.1%
soluble tubulin fractions, cells were permeabilized with 200 μL
2
bules was performed employing a fluorescence-based tubulin protease inhibitor cocktail (Sigma-Aldrich)] and incubated for
polymerization assay kit (BK011, Cytoskeleton, Inc.) according 3 min at 30 °C. Lysis buffer was gently removed, and mixed
to the manufacturer’s protocol. The reaction mixture in a final with 100 µL of 3 × Laemmli’s sample buffer (180 mM Tris-Cl
volume of 10 µl in PEM buffer (80 mM PIPES, 0.5 mM EGTA, pH 6.8, 6% SDS, 15% glycerol, 7.5% β-mercaptoethanol and
−
1
2
mM MgCl , pH 6.9) in 384 well plates contained 2 mg ml
0.01% bromophenol blue). Samples were immediately heated
2
bovine brain tubulin, 10 µM fluorescent reporter, and 1 mM to 95 °C for 3 min. To collect the insoluble tubulin fraction,
GTP in the presence or absence of test compounds at 37 °C. 300 μL of 1 × Laemmli’s sample buffer was added to the
Tubulin polymerization was followed by monitoring the fluo- remaining cells in each well, and the samples were heated to
rescence enhancement due to the incorporation of a fluo- 95 °C for 3 min. Equal volumes of samples were run on an
rescence reporter into microtubules as polymerization SDS-10% polyacrylamide gel and were transferred to a nitrocel-
proceeds. Fluorescence emission at 420 nm (excitation wave- lulose membrane employing semidry transfer at 50 mA for 1 h.
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th
Blots were probed with mouse anti-human α-tubulin, diluted edge CSIR for financial support under the 12 Five Year plan
to 1 : 2000 ml (Sigma) and stained with the rabbit anti-mouse project “Affordable Cancer Therapeutics (ACT)” (CSC0301) and
secondary antibody coupled with horseradish peroxidase “Small Molecules in Lead Exploration (SMiLE)” (CSC0111). We
diluted to 1 : 5000 ml (Sigma). Bands were visualized using an thank Dr K. Ravinder for assistance with zebrafish assays.
enhanced chemiluminescence protocol (Pierce) and radio-
graphic film (Kodak).
Zebrafish screening. Wild type zebrafish (Danio rerio) were
raised and maintained at 28.5 °C with 14 h : 10 h light : dark
Notes and references
cycles. 2–3 pairs of zebrafish were synchronously mated and
embryos were pooled. The embryos were dechlorinated and
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MgCl
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3
0
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Acknowledgements
1
A.B.S., G.B.K. and V.S.R. acknowledge CSIR-UGC, New Delhi
for the award of a senior research fellowship. We also acknowl-
8006 | Org. Biomol. Chem., 2014, 12, 7993–8007
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Paper
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