Design and synthesis of pyrazole/isoxazole linked arylcinnamides as tubulin polymerization inhibitors and potential antiproliferative agents

Article abstract of DOI:10.1039/c5ob01257k

As pyrazole and isoxazole based derivatives are well-known for displaying a considerable biological profile, an attempt has been made to unravel their cytotoxic potential. In this context, a number of pyrazole/isoxazole linked arylcinnamide conjugates (15

Full text of DOI:10.1039/c5ob01257k

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Design and synthesis of pyrazole/isoxazole linked
arylcinnamides as tubulin polymerization
Cite this: DOI: 10.1039/c5ob01257k
inhibitors and potential antiproliferative agents†
Ahmed Kamal,*a,b Anver Basha Shaik,a Bala Bhaskara Rao,c Irfan Khan,a
G. Bharath Kumara and Nishant Jainc
As pyrazole and isoxazole based derivatives are well-known for displaying a considerable biological
profile, an attempt has been made to unravel their cytotoxic potential. In this context, a number of pyr-
azole/isoxazole linked arylcinnamide conjugates (15a–o and 21a–n) have been synthesized by employing
a straight forward route. The basic structure comprised three ring scaffolds (A, B and C): methoxyphenyl
rings as A and C rings and a five membered heterocyclic ring (pyrazole or isoxazole) as the B-ring. To
achieve clear understanding, these derivatives are categorized as pyrazole-phenylcinnamides (PP) and
isoxazole-phenylcinnamides (IP). These compounds have been evaluated for their ability to inhibit the
growth of various human cancer cell lines such as HeLa, DU-145, A549 and MDA-MB231 and most of
them exhibit considerable cytotoxic effects. Some of them like 15a, 15b, 15e, 15i and 15l exhibit promis-
ing cytotoxicity in HeLa cells (IC50 = 0.4, 1.8, 1.2, 2.7 and 1.7 μM). Amongst them 15a, 15b and 15e were
taken up for detailed biological studies, they were found to arrest the cells in the G2/M phase of the cell
cycle. Moreover, they were investigated for their effect on the microtubular cytoskeletal system by using a
tubulin polymerization assay, immunofluroscence and molecular docking studies; interestingly they
demonstrate a significant inhibition of tubulin polymerization.
Received 19th June 2015,
Accepted 14th August 2015
DOI: 10.1039/c5ob01257k
www.rsc.org/obc
tubulin polymerization, thereby inducing mitotic arrest. More-
over, various categories of drugs that are used for inhibiting
Introduction
Microtubules are important protein biopolymers formed microtubule dynamics, include taxanes, vinca alkaloids and
through polymerization of α,β-tubulin heterodimers. The func- colchicines.3 Taxanes accelerate the polymerization of tubulin
tion of tubulin polymerization is reversible and the dynamic by stabilizing the assembled microtubules whereas the latter
assembly and disassembly of microtubules occur in a number candidates depolymerise the tubulin by destabilising the
of cellular activities, including cell division, migration and microtubule.
morphological alteration.1,2 In addition, microtubules are
Based on the literature it is well established that the micro-
involved in a host of cell signaling pathways related to apopto- tubules possess three important ligand binding sites namely
sis. Consequently, microtubule network has become an impor- vinca domain, colchicine domain and taxan sites.4–6 These
tant drug target in the design of newer antimitotic cytotoxic sites specifically recognized in the α/β-tubulin heterodimers
agents. Drugs that inhibit microtubule polymerization are and the agents that interact with them are the foremost focus
effective in the treatment of lung, breast, ovarian, and other of the current researchers.4–6 Some of the most important
cancers. These drugs disrupt the dynamic equilibrium of tubulin-binding agents that are employed as positive controls
for the tubulin depolimerisation assay are colchicine (1),
combretastatin A-4 (CA-4) (2) and nocodazole (3) (Fig. 1).7–11
CA-4 is a natural cis-stilbene that has a strong inhibitory
effect on tubulin polymerization by binding at the colchicine
binding site.12–14
Pyrazole and isoxazole molecular scaffolds have gained
much attention for research in medicinal chemistry and many
investigations on their synthesis as well as biological activities
have been reported.15 Recently analogs of combretastatin A-4
(CA-4) and 3,4-diarylpyrazoles were concisely synthesized,
aMedicinal Chemistry and Pharmacology, CSIR – Indian Institute of Chemical
Technology, Hyderabad 500007, India. E-mail: ahmedkamal@iict.res.in
bCatalytic Chemistry Research Chair, Chemistry Department, College of Science,
King Saud University, Riyadh 11451, Saudi Arabia
cCentre for Chemical Biology, CSIR – Indian Institute of Chemical Technology,
Hyderabad 500007, India
†Electronic supplementary information (ESI) available: 1H NMR and 13C NMR
spectra of the conjugates and experimental procedures for biological activities.
See DOI: 10.1039/c5ob01257k
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phenyl rings anchored on the A and C rings, while a five mem-
bered pyrazole or isoxazole as the B ring (Fig. 1).
Results and discussion
Chemistry
The synthesis of pyrazole linked phenylcinnamide conjugates
15a–o described in this work is outlined in Scheme 1. The syn-
thesis was initiated with the sodium ethoxide-mediated con-
densation of different acetophenones 7a–d with diethyl
oxalate. The diketoesters 8a–d thus obtained were converted to
their corresponding pyrazole esters 9a–d upon treatment with
hydrazine in ethanol. In the next step, lithium aluminium
hydride reduction of these esters afforded the corresponding
primary alcohols 10a–d. These were oxidized to the pyrazole
carbaldehydes (11a–d) by using IBX in DMSO.24 These pyrazole
carbaldehydes upon reaction with (carbethoxymethylene)tri-
phenylphosphine, Ph3PCHCO2C2H5 (C2-Wittig salt) in toluene
afforded α,β-unsaturated esters 12a–d that subsequently under-
went a base hydrolysis to give α,β-unsaturated carboxylic acids
13a–d.26,27 Finally, these carboxylic acids conveniently coupled
with substituted arylamines 14a–d in the presence of EDC/
Hobt produce the desired pyrazole linked phenylcinnamides
Fig. 1 Structures of some cytotoxic agents: colchicine (1), combretas-
tatin-A4 (2), nocodazole (3), phenylcinnamides (4), thiadiazole ring
based cinnamide (5), acrylylpiperazine (6), most active compounds (15a,
15b and 15e), pyrazole/isoxazole linked arylcinnamide conjugates (15a–
o and 21a–m).
among which, 3-methoxy-6-[4-(3,4,5-trimethoxyphenyl)-1H- (PP).19 By employing the same methodology isoxazole linked
pyrazol-3-yl]benzene-1,2-diol exhibits low nanomolar potency
towards the cytotoxic and anti-tubulin activities.16,17 Hergenr-
other and coworkers have synthesized some phenylcinnamides
(4) that cause cytotoxicity in various human cancer cell lines
apart from the accumulation of cells at the G2/M-phase of the
cell cycle. Further investigations on cinnamide derivatives
suggested that they exert their potential cytotoxic effects
through the disruption of the microtubule network.18a,b More-
over, some 1,3,4-thiadiazole ring based cinnamide derivatives
(5) showed a higher growth inhibition effect against MCF-7
and A549 cell lines apart from potent tubulin polymerization
inhibitory activity.19 In addition, a series of (E)-3-(3,4-dihydroxy-
phenyl)acrylylpiperazine conjugates (6) displayed potential
cytotoxic activity in a panel of cancer cell lines that showed sig-
nificant inhibition of tubulin polymerization.20
Based on the recent reports it is acknowledged that the
derivatives of pyrazole, isoxazole and phenylcinnamides play a
more significant role towards the development of potential
cytotoxic agents.21–23
Previously we have reported that a series of pyrazole based
conjugates have shown profound cytotoxic effects by arresting
the cells in the G2/M phase.24–26 In view of the attractive bio-
logical activities exhibited by them, considerable interest in
the development of newer cytotoxic agents has been aroused.
The present work illustrates the design and synthesis of some
pyrazole/isoxazole linked arylcinnamide conjugates and evalu-
ates their ability to inhibit the growth of a panel of four
human cancer cell lines. The cellular effects shown by these
compounds have been discussed in the following sections. All
the desired compounds comprised three ring molecular
scaffolds (A, B and C): different methoxy and fluro substituted
Scheme 1 Synthesis of pyrazole linked arylcinnamide conjugates.
Reagents and conditions: (i) NaOEt, EtOH, 4 h, 0 °C–rt, (85–90%); (ii)
NH2–NH2·2HCl, EtOH, 3–4 h, reflux, (70–80%); (iii) LiAlH4, THF, 1–2 h,
0
°C–rt, (75–80%); (iv) IBX, dry DMSO, 1 h, rt (80–85%); (v)
Ph3PCHCO2C2H5, toluene, 4 h, rt, (70–75%); (vi) LiOH·H2O, THF : MeOH :
H2O (3 : 1 : 1), 3–4 h, rt; (80–85%) (vii) EDC/Hobt, dry CH2Cl2 + DMF,
8 h, 0 °C–rt (60–80%).
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phenylcinnamide conjugates 21a–n were synthesized starting Biology
from isoxazole esters (16a–d) as shown in Scheme 2. The latter
esters were obtained when diketoesters 8a–d were treated with
Cytotoxic activity. To investigate the cytotoxic potential of
hydroxylamine hydrochloride in refluxing ethanol.25 Sub- these phenylcinnamides we evaluated the antiproliferative
sequently these esters underwent a sequential reduction and activity on different cancer cell lines like HeLa (cervical
oxidation to afford isoxazole carbaldehydes 18a–d that reacted cancer), DU-145 (prostate cancer), A549 (lung adeno-
with Ph3PCHCO2C2H5 (C2-Wittig reagent) in toluene to yield carcinoma) and MDA-MB231 (breast carcinoma). The results
α,β-unsaturated esters 19a–d. Selective oxidation of pyrazole or are summarized in Table 1 and nocodazole was employed as a
isoxazole alcohols is very critical as used IBX in DMSO. Due to standard. Both the types of compounds (PP and IP) bearing
miscibility of DMSO in ethyl acetate, crushed ice was added to the cinnamoyl moiety exhibited significant antiproliferative
the reaction mixture after extraction with ethyl acetate to activity. Some of the conjugates of PP possessing pyrazole as
obtain the DMSO free product. These unsaturated esters the B-ring displayed profound cytotoxicity that is comparable
underwent a base hydrolysis to give the corresponding carb- with the reference standard. Based on the obtained results
oxylic acids 20a–d that were coupled with different anilines to most of the conjugates (15a, 15b, 15e, 15i and 15l) possessing
afford the desired isoxazole linked phenylcinnamides (IP). pyrazole as the B-ring exhibited promising cytotoxicity in HeLa
Some sort of difficulty was observed when the crude com- cells (IC50 range 0.4–2.7 μM) and the results were comparable
pounds of pyrazoles and isoxazole aryl cinnamides were with positive control, nocodazole. Whereas some of the conju-
washed with NaHCO3, water and dil. HCl. From the starting gates 21a, 21b and 21e that contain isoxazole as the B-ring also
point of synthesis we observed that all the reaction products of showed moderate cytotoxicity, however, reduced activity was
pyrazoles were more polar when compared to isoxazole reac- observed when compared to pyrazole containing conjugates.
tion products. The pyrazole conjugates eluted very slowly from All the compounds having electron donating methoxy/methyl-
the column as they are highly polar when compared to isox- enedioxy groups on their A-ring and electron donating
azole conjugates. All the synthesized compounds were methoxy as well as electron withdrawing groups like fluoro on
characterized by standard spectroscopic analysis such as IR, the C-ring have been examined. Among these compounds 15a,
1H NMR, 13C NMR, and HRMS spectra.
15e, 15i and 15l bearing trimethoxy substituents on the C-ring
have shown significant cytotoxicity with the highest inhibitory
effect against HeLa cells, whereas 21a, 21e, 21i and 21l inhibit
the growth of cells moderately. The lead compounds 15a and
15e with trimethoxy and dimethoxy groups on the A-ring and a
similar substitution on the C-ring showed excellent potency in
HeLa cells with IC50 values of 0.4 μM and 1.2 μM respectively.
However, the presence of monomethoxy and 3,4-(methyl-
enedioxy) groups on the A-ring diminished the cytotoxic effect
(IC50 = 2.7 μM for 15i and IC50 = 1.7 μM for 15l in HeLa cells).
In addition, the representative compounds inhibit the growth
of MDA-MB231 cells significantly (IC50 = 1.0 μM, 1.6 μM,
3.8 μM and 2.3 μM). Further promising activity was also
observed in dimethoxy substituted derivatives, C-ring like 15b
(1.8 μM), 15f (5.5 μM), 15j (13.5 μM) and 15m (12.2 μM),
whereas 21b, 21f, 21j and 21m showed moderate effects.
However, monomethoxy substituted derivatives showed dele-
terious effects on cytotoxicity. Some of the compounds like
15d, 15h, 15k, 15o, 21d, 21h, 21k and 21n that possess
difluoro substituents on the C-ring showed minimum cytotoxi-
city. Finally, it is observed that the presence of more methoxy
groups on the C-ring increases the cytotoxicity, indicating that
electron donating groups are important for the activity com-
pared to electron withdrawing groups (OMe > F2), however
change of substitutions on the A-ring evidently plays a critical
role towards the activity. Based on the structure–activity
Scheme 2 Synthesis of isoxazole linked arylcinnamide conjugates. relationship, the optimal order of the substitution effect on
Reagents and conditions: (i) NaOEt, EtOH, 4 h, 0 °C–rt (85–90%); (ii)
NH2–OH·HCl, EtOH, 3 h, reflux, (75–80%); (iii) LiAlH4, THF, 1–2 h, 0 °C–
rt (70–80%); (iv) IBX, dry DMSO, 1 h, rt (80–85%); (v) Ph3PCHCO2C2H5,
toluene, 4 h, rt (70–75%); (vi) LiOH·H2O, THF : MeOH : H2O (3 : 1 : 1)
the A-ring is trimethoxy > dimethoxy ≥ 3,4-(methylenedioxy) >
monomethoxy (Fig. 2).24,28,29
Effect on cell morphology. Since these compounds (15a,
15b and 15e) demonstrated potent cytotoxicity it was neccesary
to understand their effects on the cell morphology. Phase
3–4 h, rt (80–85%); (vii) EDC/Hobt, dry CH2Cl2 + DMF, 8 h, 0 °C–rt
(65–78%).
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Table 1 Structures and IC50 (μM) values of the synthesized compounds on HeLa,b DU-145,c MDA-MB231d and A549e cells determined by the MTT
assay
IC50 valuesa (μM)
Com
X
R1
R2
R3
R4
R5
R6
HeLab
DU-145c
MDA-MB231d
A549e
15a
15b
15c
15d
15e
15f
15g
15h
15i
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
O
O
O
O
O
O
O
O
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
H
H
H
OMe
OMe
H
OMe
OMe
OCH3
F
OMe
OMe
OMe
F
OMe
OMe
F
OCH3
OCH3
OCH3
F
OMe
OMe
OMe
F
OMe
OMe
OMe
F
OMe
OMe
F
OMe
OMe
F
OMe
H
H
H
OMe
H
H
H
OMe
H
H
OCH3
H
H
H
OMe
H
H
H
OMe
H
H
0.4 0.06
1.8 0.13
3.5 0.25
13.7 1.1
1.2 0.12
5.5 0.18
10.4 0.03
14.2 0.6
2.7 0.33
13.5 0.5
16.1 1.3
1.7 0.18
12.2 0.9
13.4 1.1
29.7 0.7
2.9 0.16
3.7 0.12
12.3 0.1
17.3 1.5
3.3 0.2
13.8 1.0
20.1 0.6
31.9 0.7
5.4 0.1
20.1 0.6
34.9 0.7
4.2 0.1
20.1 0.6
19.8 0.7
0.5 0.02
1.6 0.13
2.4 0.15
4.8 0.9
12.3 0.5
1.8 0.19
11 0.2
22.7 1.7
23.5 1.5
6.8 0.4
18.8 0.9
18.9 0.2
1.9 0.14
12.1 0.1
3.1 0.70
19.2 1.12
3.2 0.10
2.8 0.38
14.2 + 0.3
21.8 0.3
4.4 0.5
15.8 0.9
22.3 0.4
28.0 1.9
6.1 0.6
39.8 1.1
25.6 1.3
6.2 0.21
19.9 1.6
31.2 1.7
1.1 0.03
1.0 0.03
2.6 0.11
5.6 0.23
19.2 1.1
1.6 0.17
13.2 1.2
36.5 1.5
14.2 1.1
3.8 0.6
15.6 0.8
15.4 1.0
2.3 0.28
17.8 1.0
17.2 1.7
23.1 0.6
1.8 0.11
2.5 0.3
22.1 1.5
27.8 0.9
2.3 0.4
18.0 1.9
19 0.6
32.7 1.7
3.8 0.05
15.8 0.8
28.9 0.9
4.9 0.12
11.2 0.9
22.1 1.0
0.7 + 0.03
1.7 0.05
2.2 0.12
6.4 0.18
15.7 1.0
2.2 0.10
10.7 0.3
13.3 0.5
22.1 1.0
7.0 0.8
15.2 1.2
23.4 1.1
2.9 0.1
20.3 1.2
9.7 0.12
18.7 1.1
4.3 0.5
F
OMe
OMe
H
F
OMe
OMe
F
OCH3
OCH3
H
15j
15k
15l
H
H
–OCH2O–
–OCH2O–
–OCH2O–
–OCH2O–
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
15m
15n
15o
21a
21b
21c
21d
21e
21f
21g
21h
21i
21j
21k
21l
F
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
5.9 0.2
23.8 1.1
25.6 1.2
4.2 0.6
10.7 0.7
13.9 0.5
22.1 1.0
10.0 0.6
10.2 0.3
13.4 0.1
3.9 0.7
F
OMe
OMe
H
H
H
H
H
H
H
H
—
F
H
OMe
H
H
OMe
H
OMe
OMe
F
OMe
OMe
F
O
O
O
O
O
—
H
H
–OCH2O–
–OCH2O–
–OCH2O–
—
21m
21n
Noc
20.9 1.6
29.7 1.2
0.9 + 0.04
H
—
—
—
a Each data represent mean + S.D. from three different experiments performed in triplicate. b HeLa: human cervix cancer cell line. c DU-145:
human prostate cancer cell line. d MDA-MB231: human breast carcinoma cell line. e A549: human lung adenocarcinoma epithelial cell.
contrast images were observed to elucidate the morphology of
cells treated with these compounds. Interestingly, HeLa cells
treated by these compounds at 2 μM concentration manifested
a rounded morphology similar to mitotic arrested cells. These
results suggest that the possible mode of action for them is
through the inhibition of mitosis (Fig. 3).
Effect on cell cycle arrest. Since the compound treated cells
showed a predominant mitotic phenotype, we examined
whether the cytoxicity induced by these derivatives was due to
cell cycle arrest. Thus we performed flow cytometry analysis for
the compounds that exhibited potent cytotoxicity HeLa cells
were treated with 2 μM concentration of 15a, 15b and 15e for
Fig. 2 SAR of pyrazole/isoxazole linked arylcinnamides. The com-
pounds with a trimethoxy aryl group as the A-ring were comparatively
24 h. Cells treated with 15a at 2 μM showed 75.53% arrest of
more potent in the series. C-ring with more electropositive units
cells in the G2/M phase, whereas 15b and 15e resulted in an
enhances the cytotoxicity.
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Fig. 4 Anti-mitotic effects of 15a, 15b and 15e by FACS analysis:
induced G2/M cell cycle arrest by compounds 15a, 15b and 15e. HeLa
cells were harvested after treatment at 2 µM for 24 h. Untreated cells
and DMSO treated cells taken as controls. The percentage of cells in
each phase of the cell cycle was measured quantitatively by flow
cytometry.
Fig. 3 Morphological representation of HeLa cells in the presence of
the most active compounds: HeLa cells were treated with 2 μM of 15a,
15b and 15e for 24 h. There is an abnormal cellular morphology noticed
in compound treated cells indicating antiproliferative effects. DMSO was
employed as negative control and such morphological abnormality was
not observed.
Table 2 Tubulin polymerization inhibitory effect of compounds 15a,
15b and 15e
increase of mitotic cell arrest by 49.22% and 65.98% respecti-
vely (Fig. 4).
Effect on tubulin polymerization. These pyrazole/isoxazole
linked arylcinnamide conjugates were examined for their
ability to inhibit tubulin polymerization as they exhibit poten-
tial cytotoxicity. Therefore, we incubated tubulin with varying
concentrations of these conjugates to determine IC50 values
for tubulin depolymerization activity and results are shown in
Table 2. All the examined conjugates inhibit the polymeriz-
ation of tubulin. Conjugate 15a induces tubulin depolymeriza-
tion significantly (IC50 value of 1.5 µM), whereas 15b and 15e
inhibit tubulin assembly with IC50 values of 3.2 µM and
2.6 µM respectively (Table 2).
Compound
IC50 in µM
15a
15b
15e
Nocodazole
1.5 0.11
3.2 0.23
2.6 0.15
0.87 0.02
Effect of congeners on tubulin polymerization. IC50 values for 15a, 15b
and 15e were determined from the tubulin polymerization assay.
Nocodazole was employed as positive control.
Effect on the microtubule network. The presence of anoma- their effect on cellular tubulin. To elucidate this, HeLa cells
lous spindle fibres due to disrupted microtubule dynamics is a were treated with these compounds at 2 μM concentration for
hallmark of cells treated with antimitotic agents.30 In order to 24 h.
ascertain whether the cell cycle arrest is due to spindle
Subsequently, cells were permeabilized to collect the
abnormality, HeLa cells were treated with 2 μM concentrations soluble fraction and the remaining cells were collected as the
of these conjugates (15a, 15b and 15e) and stained with the polymerized fraction. Immunoblot analysis revealed that the
tubulin antibody, whereas the control treated cells exhibit an
cells exposed to pyrazole/isoxazole linked arylcinnamide conju-
organized network of microtubules. In contrast cells exposed gates restrained more tubulin in the soluble fraction. There-
to 15a and 15b showed multipolar spindle fibres, and 15e fore, increased tubulin in soluble fraction of cells treated with
treated cells exhibited a metaphase arrest (Fig. 5).
potent conjugates corroborated with the inhibition of tubulin
Effect on cellular tubulin polymerization and cyclin- assembly and arrest of cells in the G2/M phase of the cell cycle
B1. These observations suggest that the aberrant spindle (Fig. 6).
dynamics in cells treated with 15a, 15b and 15e result in cell
To further authenticate our observations whether the pyr-
cycle arrest. Tubulin assembly assays reveal that the congeners azole/isoxazole arylcinnamides function as potent tubulin
inhibit microtubule polymerization, consequently we analyzed polymerization inhibitors, HeLa cells were treated with the
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Fig. 7 Western blot analysis for cyclin-B1: HeLa cells were treated with
2 μM concentration of compounds 15a and 15e for 24 h. Subsequently,
whole lysates were prepared and analyzed for cyclin-B1. Results illus-
trate an increased level of cyclin B1 in the treatments. The potent com-
pound 15a shows a pronounced expression of cyclin B1 levels. Tubulin
was employed as loading control. Nocodazole (Noc) and taxol (Tax)
were used as positive controls.
Fig. 5 Effect on microtubules and nuclear condensation: HeLa cells
were independently treated with 15a, 15b and 15e at 2 μM concentration
for 24 h. Following the termination of the experiment, cells were fixed
and stained for tubulin. DAPI was used as counter stain. The merged
images of cells stained for tubulin and DAPI are represented.
Fig. 8 RT-PCR analysis for cyclin B1 of the treated compounds 15a and
15e: HeLa cells treated with 2 μM concentration of compounds 15a and
15e for 24 h. Later, whole cell lysates were prepared and RNA was iso-
lated. Semi-quantitative RT-PCR analysis was performed for cyclin-B1.
GAPDH served as internal control.
site of the tubulin (PDB code: 3E22) by using the Autodock4
program.31,32 Based on the previous reports it is well estab-
lished that the colchicine site is commonly positioned at the
interface of α and β protein heterodimers.33 The conjugates
upon docking are specifically surrounded by significant amino
acid residues such as αSer-α178, αTyr-224, αVal-405, αTrp-407,
αThr-223, and αAsn349 of the α subunit, βLeu-255, βLeu252,
βCys-241, βGln-434 and βPhe-268, βThr-353, βPhe-343, βTyr-
202, βAsn-258 and βAla-256 of the β subunit. The docking pose
observed for 15a showed a very similar binding to the cocrys-
tallized DAMA-colchicine34 with the trimethoxyphenyl ring
(A-ring) in very close contact with βCys-241 (electrostatic inter-
Fig. 6 Distribution of tubulin in soluble vs. insoluble fractions: HeLa
cells were treated with 2 µM of 15a and 15e for 24 h. This image shows
soluble and insoluble (polymerized) fractions of tubulin for 15a and 15e.
A more soluble fraction is observed in 15a treated cells when compared
to 15e. Nocodazole and Taxol were employed as reference standards.
The amount of tubulin was detected by western blot analysis.
representative analogues 15a and 15e to demonstrate the elev- actions). However, the C-ring positioned in the binding pocket
ated amount of cyclin B1 protein, a well recognized G2/M of the α subunit resulting the rest pyrazole bridge (B-ring) at
marker (Fig. 7). In addition, we further substantiated our the border of the two subunits. Additionally, the O atom of the
results by performing semi-quantitative RT-PCR analysis for unsaturated carbonyl structural unit forms a weak hydrogen
cyclin B1 mRNA levels in compounds 15a and 15e treated bond with OH of αSer-178 (O⋯HO). Moreover, a weak hydro-
HeLa cells. Notably, treatments with the congeners showed a gen bond formed between the O atom of the trimethoxy group
profound increase in cyclin B1 mRNA levels compared to positioned at C3 of the C-ring, and NH of αThr-223 (O⋯HN).
control. Thus our results support the suggestion that the pyr- Significantly a strong hydrogen bond was observed between
azole/isoxazole arylcinnamides function as potent tubulin HN of the acrylamide linker and carbonyl O of βThr-353 of the
inhibitors that cause an accumulation of cells at the G2/M β subunit (O⋯HN distance = 1.7 Å). Therefore, 15a with tri-
phase of the cell cycle (Fig. 8).
methoxy benzene groups on both the A and C-rings occupies a
Molecular modeling studies. Molecular docking studies similar position where colchicine binds to tubulin with little
were also performed for the selected compounds like 15a and space orientation. However, superimposition poses explicate
15b to authenticate the obtained experimental results. These the important basic unit, a trimethoxy phenyl ring of both col-
compounds were succesfully docked in the colchicine binding chicine and 15a occupy the exact location in the β subunit.
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Fig. 9 Molecular modeling poses of the lead conjugates 15a, 15b and colchicine with 15a: the panel of images represents the docking poses of the
proposed ligands at the interface of α,β-tubulin. All the ligands are visualized in stick models (green color). The grey and salmon color ribbons depict
α- and β-tubulin subunits respectively. The interacting residues that are shown in magenta stick model and potential inter molecular hydrogen
bonding interactions were indicated with black dots. The poses (15a+col) represent 15a along with colchicine occupy the same site in the tubulin.
Images were generated with the PyMol programme.
The next tubulin polymerization inhibitor 15b with
a
experimental results; in particular, methoxy groups placed in a
dimethoxy phenyl unit as the C-ring and the trimethoxy group tight hydrophobic region at the interface of α and β hetero-
on the A-ring is sandwiched between the two subunits dimers (Fig. 9).
showing strong hydrogen bonding with αTyr-224, and αTyr-
202. In addition, a weak hydrogen bond is noted between the
O atom of the dimethoxy group at the C3 position of the
C-ring, and SH of βCys-241 (O⋯HS). Moreover, both the
Conclusions
ligands exhibit few hydrophobic interactions with the residues In summary, we synthesized a new class of pyrazole/isoxazole
like αVal-405, αTrp-407, αAsn349, βLeu-255, βLeu252, βGln- linked arylcinnamide conjugates and investigated their anti-
434, andβPhe-268, βPhe-343, βAsn-258 and βAla-256. Although proliferative activity against a panel of five cancer cell lines.
the trimethoxy benzene group (A-ring) is identified as the sig- These conjugates comprised a three ring molecular scaffold;
nature for binding to tubulin, we noted that dimethoxy deriva- A, B and C rings and these rings were decorated with various
tives of 15b inhibit the tubulin polymerization to a less extent substitutions to present a comprehensive structure–activity
than 15a. These results are in accordance with the observed relationship. Most of the compounds displayed significant
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cytotoxic activity wherein the presence of a trimethoxy phenyl
(E)-Ethyl 3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)acryl-
group on the A-ring as well as C-rings was necessary for the ate 12a. This compound was prepared by the addition
tubulin inhibitory activity. Some of the promising compounds of 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11a
like 15a, 15b and 15e inhibited the growth of cells in the G2/M (262 mg 1.0 mmol) and Ph3PCHCO2C2H5 (248 mg, 1.0 mmol).
phase and blocked the chromosomal translocation as well. Yellow solid (242 mg, yield 73%): Rf = 0.4 (30% ethyl acetate/
1
The presence of increased levels of the cyclin B1 protein and hexane); H NMR (300 MHz, CDCl3); δ 1.33 (t, 3H, J = 7.1 Hz,
tubulin in the soluble fraction of cells corroborate well with –CH3), 3.92 (s, 3H, –OCH3), 3.93 (s, 6H, –OCH3), 4.15–4.33
the tubulin polymerization inhibition. A significant upregula- (q, 2H, J1 = 7.0 Hz, J2 = 7.1 Hz, –CH2), 6.42 (d, 1H, transH
tion of cyclin B1 mRNA by 15a and 15e indicates the activation J = 16.2 Hz), 6.72 (s, 1H, ArH), 6.92 (d, 1H, J = 8.6 Hz, ArH),
of a cell cycle dependent apoptotic pathway as the mechanism 6.42 (d, 1H, transH J = 16.2 Hz), 7.17–7.23 (m, 1H, ArH)
of their cytotoxic effects. Molecular modeling analysis demon- ppm; 13C NMR (75 MHz, CDCl3): 14.2, 56.0, 60.8, 101.9, 102.9,
strated that the compounds showed significant interactions to 119.1, 126.5, 128.4, 128.5, 131.6, 131.9, 132.0, 132.4, 134.1,
the colchicine binding site of tubulin. Based on the results, 138.0, 153.4, 166.8 ppm; MS (ESI) m/z 333 [M + H]; HR-MS
the design and synthesis of the compounds containing a pyr- (ESI) m/z for C17H21O5N2 calculated m/z: 333.1405, found m/z:
azole/isoxazole moiety are effective tubulin polymerization 333.1408.
inhibitors, which can be further amenable for the generation
of different conjugates as potential anticancer drugs.
(E)-Ethyl 3-(3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)acrylate
12b. This compound was prepared by the addition of 3-(3,4-
dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 11b (232 mg
1.0 mmol) and Ph3PCHCO2C2H5 (248 mg, 1.0 mmol). Yellow
solid (217 mg, yield 72%): Rf = 0.5 (30% ethyl acetate/hexane);
1H NMR (300 MHz, CDCl3); δ 1.34 (t, 3H, J = 7.1 Hz, –CH3),
3.94 (s, 3H, –OCH3), 3.95 (s, 3H, –OCH3), 4.10–4.31 (q, 2H, J1 =
7.0 Hz, J2 = 7.1 Hz, –CH2), 6.12 (d, 1H, transH J = 16.1 Hz), 6.42
(d, 1H, transH J = 16.0 Hz), 6.74 (s, 2H, ArH), 7.19–7.21 (m, 2H,
ArH) ppm; 13C NMR (75 MHz, CDCl3): 12.1, 53.7, 58.7, 94.4,
106.8, 109.7, 116.9, 117.4, 123.8, 129.3, 147.1, 148.8, 158.1,
163.2, 168.2 ppm; MS (ESI) m/z 303 [M + H]; HR-MS (ESI) m/z
for C16H19O4N2 calculated m/z: 303.1305, found m/z: 303.1312.
(E)-Ethyl 3-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)acrylate
12c. This compound was prepared using the above-mentioned
method by the addition of 3-(4-methoxyphenyl)-1H-pyrazole-
5-carbaldehyde 11c (202 mg 1.0 mmol) and Ph3PCHCO2C2H5
(248 mg, 1.0 mmol). Yellow solid (204 mg, yield 75%): Rf = 0.5
Experimental section
General
All the chemicals and reagents used in this study were
purchased from Aldrich (Sigma-Aldrich, St. Louis, MO, USA),
Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill,
MA, USA), or Spectrochem Pvt. Ltd (Mumbai, India) and were
used as such without purification. The reactions were moni-
tored by TLC performed on silica gel glass plates containing
60 GF-254, and visualized by using a UV light or iodine indi-
cator. Column chromatography was performed using Merck
60–120 mesh silica gel. 1H NMR spectra were recorded on
Bruker UXNMR/XWIN-NMR (300 MHz) or Inova Varian-VXR-
unity (400, 500 MHz) instruments. 13C NMR spectra were
recorded on a Bruker UXNMR/XWIN-NMR (75 MHz) instru-
ment. Chemical shifts (δ) were expressed as ppm downfield
from an internal standard TMS. ESI spectra were recorded on a
Micro mass Quattro LC using ESI+ software with a capillary
voltage of 3.98 kV and an ESI mode positive ion trap detector.
High-resolution mass spectra (HRMS) were recorded on a
QSTAR XL hybrid MS–MS mass spectrometer.
1
(30% ethyl acetate/hexane); H NMR (300 MHz, CDCl3); δ 1.29
(t, 3H, J = 7.1 Hz, –CH3), 3.95 (s, 3H, –OCH3), 4.08–4.23 (q, 2H,
J1 = 7.0 Hz, J2 = 7.1 Hz, –CH2), 6.32 (d, 1H, transH J = 16.1 Hz),
6.50 (s, 2H, ArH), 6.96 (d, 1H, J = 8.6 Hz, ArH), 6.65 (d, 1H,
transH J = 16.2 Hz), 7.34–7.40 (m, 2H, ArH) ppm; 13C NMR
(75 MHz, CDCl3): 13.9, 55.0, 60.7, 95.2, 114.1, 119.3, 125.1,
127.1, 128.9, 131.5, 159.8, 161.0, 165.3, 170.4 ppm; MS (ESI)
m/z 273 [M + H]; HR-MS (ESI) m/z for C15H17O3N2 calculated
m/z: 273.1148, found m/z: 273.1150.
Melting points were determined with an electrothermal
melting point apparatus, and are uncorrected.
(E)-ethyl 3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)acryl-
ate 12d. This compound was prepared by the addition of
3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 11d
General preparation of (E)-ethyl 3-(3-aryl-1H-pyrazol-5-yl)-
acrylates 12a–d
The different pyrazole carbaldehydes (11a–d) used in this (216 mg 1.0 mmol) and Ph3PCHCO2C2H5 (248 mg, 1.0 mmol).
study were taken from our previous report.23 To the each Yellow solid (208 mg, yield 73%): Rf = 0.4 (30% ethyl acetate/
1
11a–d was added equimolar amount of (carbethoxymethylene)- hexane); H NMR (300 MHz, CDCl3); δ 0.85 (t, 3H, J = 7.3 Hz,
triphenylphosphine, Ph3PCHCO2C2H5, C2-wittig reagent in –CH3), 4.13–4.30 (q, 2H, J1 = 5.3 Hz, –CH2), 6.02 (s, 2H,
toluene at room temperature and continued stirring for 3–4 h. –OCH2O–), 6.45 (d, 1H, transH J = 15.8 Hz), 6.74–6.91 (m, 1H,
After performing TLC toluene was evaporated and an appropri- ArH), 6.92 (d, 1H, J = 8.6 Hz, ArH), 7.06–7.32 (m, 2H, ArH),
ate amount of water was added. The crude compounds were 7.56 (d, 1H, transH J = 15.8 Hz) ppm; 13C NMR (75 MHz,
extracted by ethyl acetate (50 ml × 4). The organic layer was CDCl3): 12.7, 58.7, 99.6, 100.2, 104.3, 107.0, 117.3, 117.0,
dried on anhydrous Na2SO4 and ethyl acetate was evaporated 123.4, 126.5, 130.8, 132.8, 139.2, 142.9 ppm; MS (ESI) m/z 287
to obtain the corresponding pure α,β-unsaturated pyrazole [M + H]; HR-MS (ESI) m/z for C15H15O4N2 calculated m/z:
esters (12a–d) in good yields (70–75%).
287.0988, found m/z: 287.0985.
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Preparation of (E)-3-(3-phenyl-1H-pyrazol-5-yl)acrylic acid 13a–d
CDCl3 + DMSO-d6); δ 3.86 (s, 6H, –OCH3), 3.96 (s, 9H, –OCH3),
7.01(s, 1H, ArH), 7.07 (s, 1H, ArH), 7.55 (s, 1H, ArH), 7.58–7.68
(m, 1H, –ArH), 7.72 (d, 1H, transH, J = 15.8 Hz), 7.85 (t, 1H,
ArH, J = 7.3 Hz), 8.03 (d, 1H, ArH, J = 8.4 Hz), 8.08 (d, 1H,
transH, J = 15.6 Hz), 8.55 (brs, 1H, –NH) ppm; 13C NMR
(75 MHz, CDCl3 + DMSO-d6): 58.6, 95.4, 99.1, 107.2, 108.1,
109.9, 116.2, 120.8, 122.8, 124.6, 126.9, 130.0, 131.9, 133.8,
147.1, 147.2, 151.0, 162.0 IR (KBr) (νmax/cm−1): ν = 3253, 2932,
2926, 1693, 1622, 1589, 1460, 1460, 1426, 1342, 1084,
971 cm−1; MS (ESI) m/z 440 [M + H]; HR-MS (ESI) m/z for
C23H26O6N3 calculated m/z: 440.1823, found m/z: 440.1823.
(E)-N-(4-Methoxyphenyl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-5-yl)acrylamide 15c. This compound was prepared by
the addition of 13a (304 mg, 1.0 mmol) and 4-methoxyaniline
14c (123 mg 1.0 mmol). Pale yellow solid, yield: 285 mg (69%);
mp: 208–210 °C; 1H NMR (300 MHz, CDCl3 + DMSO-d6); δ 3.79
(s, 3H, –OCH3), 3.81 (s, 3H, –OCH3), 3.92 (s, 6H, –OCH3), 6.78
(d, 1H, transH, J = 15.6 Hz), 6.85 (d, 1H, ArH, J = 8.8 Hz), 7.05
To each of the α,β-unsaturated pyrazole esters (12a–d) obtained
in the above step was added equimolar lithium hydroxide
(LiOH·H2O) to
a mixture of solvents THF : MeOH : H2O
(3 : 1 : 1) at room temperature and continued stirring for 3–4 h.
After performing TLC the solvent mixture was evaporated and
an appropriate amount of water was added. The crude com-
pounds were extracted by ethyle acetate (50 ml × 4). The indi-
vidual part of the aqueous layer was acidified using dil. HCl
and then extracted by ethyl acetate (50 ml × 4) once again. The
organic layer was dried on anhydrous Na2SO4 and then ethyl
acetate was evaporated to obtain the corresponding pure (E)-3-
(3-phenyl-1H-pyrazol-5-yl)acrylic acids (13a–d) in good yields
(80–85%).
General procedure for synthesis of pyrazole linked
arylcinnamides 15a–o
To the (E)-3-(3-phenyl-1H-pyrazol-5-yl)acrylic acid (13a–d) pre- (s, 3H, ArH), 7.57 (d, 1H, transH, J = 15.8 Hz), 7.64 (d, 1H, ArH,
pared in the above step was added equimolar EDCI (1-ethyl-3- J = 8.6 Hz), 7.83 (s, 2H, ArH, J = 8.6 Hz), 9.88 (brs, 1H, –NH)
(3-dimethylaminopropyl)carbodiimide) and a catalytic amount ppm; 13C NMR (75 MHz, CDCl3 + DMSO-d6): 53.6, 54.4, 58.6,
of Hobt (hydroxybenzotriazole) in ice cold dichloromethane. 100.0, 100.1, 112.3, 119.5, 121.2, 130.4, 135.5, 151.6, 153.9,
After 10 min appropriate amounts of aryl amines 14a–d at 0 °C 161.9 ppm; IR (KBr) (νmax/cm−1): ν = 3422, 3061, 3606, 2927,
were added and stirring was continued at room temperature 1657, 1604, 1509, 1466, 1408, 1346, 1237, 1127, 1024, 1000,
for 8 h. The progress of the reaction was monitored by TLC. 968 cm−1; MS (ESI) m/z 410 [M + H]; HR-MS (ESI) m/z for
After completion of the reaction an appropriate amount of C22H24O5N3 calculated m/z: 410.1710, found m/z: 410.1731.
sodium bicarbonate (NaHCO3) solution was added and sub-
(E)-N-(3,4-Difluorophenyl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-
sequently the final compounds were extracted with ethyl pyrazol-5-yl)acrylamide 15d. This compound was prepared by
acetate (50 ml × 4). The organic layers so obtained were the addition of 13a (304 mg 1.0 mmol) and 3,4-difluoroaniline
washed with 5% HCl solution and the organic solvent was 14d (130 mg 1.0 mmol). Pale yellow solid, yield: 290 mg (70%);
evaporated, affording crude compounds. Further these com- mp: 216–218 °C; 1H NMR (300 MHz, CDCl3 + DMSO-d6); δ 3.82
pounds were purified using column chromatography by using (s, 3H, –OCH3), 3.94 (s, 6H, –OCH3), 6.75 (d, 2H, J = 15.8 Hz,
an ethyl acetate and hexane solvent system to get pure com- transH), 7.05 (s, 1H, ArH), 7.06–7.18 (m, 1H, ArH), 7.36 (d, 1H,
pounds in good yields (60–80%).
ArH J = 9.2 Hz), 7.63 (d, 1H, J = 15.8 Hz, transH), 7.77 (s, 1H,
(E)-N-(3,4,5-Trimethoxyphenyl)-3-(3-(3,4,5-trimethoxyphenyl)- ArH), 7.83–7.94 (m, 1H, ArH), 10.15 (brs, 1H, NH); 13C NMR
1H-pyrazol-5-yl)acrylamide 15a. This compound was prepared (75 MHz, CDCl3 + DMSO-d6): δ 54.2, 58.4, 99.8, 101.0, 106.8,
by the addition of (E)-3-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol- 106.8, 107.8, 113.6, 11501, 11504, 120.3, 134.4, 135.8, 142.2,
5-yl)acrylic acid (304 mg 1.0 mmol) (13a) and 3,4,5-trimeth- 151.5, 162.1 ppm; IR (KBr) (νmax/cm−1): ν = 3218, 2928, 1623,
oxyaniline (183 mg, 1.0 mmol) (14a). The compound was 1532, 1512, 1468, 1424, 1345, 1247, 1132, 1087, 996 cm−1
;
obtained as a pale yellow solid yield: 285 mg (60%); mp: MS (ESI) m/z 416 [M + H]; HR-MS (ESI) m/z for C21H20F2O4N3
175–177 °C; 1H NMR (300 MHz, CDCl3 + DMSO-d6); δ 3.80 calculated m/z: 416.1419 found m/z: 416.1420.
(s, 6H, –OCH3), 3.91 (s, 6H, –OCH3), 3.96 (s, 6H, –OCH3), 6.70
(E)-3-(3-(3,4-Dimethoxyphenyl)-1H-pyrazol-5-yl)-N-(3,4,5-
(s, 1H, –NH), 6.76 (d, 1H, J = 15.8 Hz, transH), 6.85 (d, 1H, trimethoxyphenyl)acrylamide 15e. This compound was pre-
ArH), 6.94 (d, 1H, ArH), 7.35 (s, 1H transH J = 15.8 Hz) pared by the addition of (E)-3-(3-(3,4-dimethoxyphenyl)-1H-
7.51–7.57 (m, 1H, ArH) 7.60–7.68(m, 1H, ArH), 9.54 (brs, 1H, pyrazol-5-yl)acrylic acid 13b (274 mg 1.0 mmol) and 3,4,5-tri-
–NH); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 54.1, 58.3, methoxyaniline 14a (183 mg, 1.0 mmol). Pale yellow solid,
100.8, 107.8, 117.1, 117.7, 125.1, 122.5, 126.0, 135.6, 140.9, yield: 310 mg (70%); mp: 204–206 °C; 1H NMR (500 MHz,
151.4, 165.8 ppm; IR (KBr) (νmax/cm−1): ν = 3235, 2938, 1695, DMSO-d6); δ 3.87 (s, 6H, –OCH3), 3.96 (s, 12H, OCH3), 7.01
1623, 1592, 1500, 1462, 1427, 1368, 1246, 1129, 1086, (s, 1H, –ArH), 7.07 (s, 1H, ArH), 7.54 (s, 1H, ArH), 7.60–7.68
977 cm−1; MS (ESI) m/z 471 [M + H]; HR-MS (ESI) m/z for (m, 1H, ArH) 7.72 (d, 1H, transH, J = 15.6 Hz), 7.82–7.89
C24H28O7N2 calculated m/z: 470.1849, found m/z: 470.1850.
(m, 1H, ArH), 8.09 (d, 1H, transH, J = 15.8 Hz), 8.54 (d, 1H,
(E)-N-(3,4-Dimethoxyphenyl)-3-(3-(3,4,5-trimethoxyphenyl)- ArH, J = 8.4 Hz), 9.67 (brs, 1H, NH), 13.03 (brs, 1H, NH);
1H-pyrazol-5-yl)acrylamide 15b. This compound was pre- 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 54.5, 58.7, 96.3, 100.2,
pared by the addition of 13a (304 mg, 1.0 mmol) and 3,4- 101.2, 108.0, 117.3, 117.9, 122.7, 125.2, 136.0, 151.7,
dimethoxyaniline (15b) (153 mg 1.0 mmol). Colorless solid, 166.2 ppm; IR (KBr) (νmax/cm−1): ν = 3230, 3010, 1685, 1635,
yield: 276 mg (62%); mp: 191–193 °C; 1H NMR (500 MHz, 1596, 1575, 1500, 1487, 1468, 1368, 1290, 1225, 1180, 1115,
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1026, 998 cm−1; MS (ESI) m/z 440 [M + H]; HR-MS (ESI) m/z for 133.3, 135.0, 152.4, 158.8, 163.7 ppm; IR (KBr) (νmax/cm−1): ν =
C23H26O6N3 calculated m/z: 440.1816 found m/z: 440.1807.
3220, 3016, 2960, 1680, 1650, 1600, 1584, 1536, 1491, 1437,
(E)-N-(3,4-Dimethoxyphenyl)-3-(3-(3,4-dimethoxyphenyl)-1H- 1366, 1244, 1215, 1173, 1105, 1030, 985 cm−1; MS (ESI) m/z 410
pyrazol-5-yl)acrylamide 15f. This compound was prepared by [M + H]; HR-MS (ESI) m/z for C22H24O5 N3 calculated m/z:
the addition of 13b (274 mg, 1.0 mmol) and 3,4-dimethoxyani- 410.1714, found m/z: 410.1713.
line 14b (153 mg 1.0 mmol). Yellow crystals, yield: 260 mg
(E)-N-(3,4-Dimethoxyphenyl)-3-(3-(4-methoxyphenyl)-1H-
(68%); mp: 214–216 °C; 1H NMR (500 MHz, CDCl3 + DMSO- pyrazol-5-yl)acrylamide 15j. This compound was prepared
d6); δ 3.87 (s, 3H, –OCH3), 3.91 (s, 6H, –OCH3), 3.95 (s, 3H, by the addition of 13c (244 mg, 1.0 mmol) and 3,4-dimethoxy-
–OCH3), 6.69 (s, 1H, ArH), 6.72 (d, 1H, transH, J = 16.0 Hz), aniline 14b (153 mg 1.0 mmol). Yellow solid, yield: 260 mg
6.82 (d, 1H, ArH, J = 8.6 Hz), 6.91 (d, 1H, ArH, J = 9.0 Hz), 7.33 (68%); mp: 218–220 °C; 1H NMR (300 MHz, CDCl3 + DMSO-d6);
(s, 3H, ArH), 7.61 (s, 1H, ArH), 7.66 (d, 1H, transH, J = 15.7 Hz), δ 3.78 (s, 6H, –OCH3), 3.84 (s, 3H, –OCH3), 6.19 (s, 1H, ArH),
8.99 (brs, 1H, –NH); 13C NMR (75 MHz, DMSO-d6): δ 54.0, 6.69 (s, 1H, ArH), 6.75 (d, 1H, J = 15.6 Hz, transH), 6.91–6.97
54.2, 54.4, 99.1, 103.1, 107.5, 110.0, 110.2, 116.5, 121.2, 131.6, (m, 3H, ArH), 7.01 (s, 1H, ArH), 7.62 (d, 1H, J = 15.4 Hz,
143.5, 147.3, 147.5, 147.3, 162.1, 165.5 ppm; IR (KBr) (νmax
cm−1): ν = 2934, 1660, 1510, 1463, 1339, 1234, 1169, 1026, (νmax/cm−1): ν = 3241, 3112, 3060, 1680, 1640, 1591, 1555,
805 cm−1; MS (ESI) m/z 410 [M + H].
1484, 1430, 1316, 1288, 1261, 1190, 1180, 1115, 1020,
(E)-3-(3-(3,4-Dimethoxyphenyl)-1H-pyrazol-5-yl)-N-(4-methoxy- 987 cm−1; MS (ESI) m/z 380 [M + H].
/
transH), 7.66–7.74 (m, 2H, ArH), 9.82 (brs, 1H, –NH); IR (KBr)
phenyl)acrylamide 15g. This compound was prepared by the
(E)-N-(3,4-Difluorophenyl)-3-(3-(4-methoxyphenyl)-1H-pyrazol-
addition of 13b (274 mg, 1.0 mmol) and 4-methoxyaniline 14c 5-yl)acrylamide 15k. This compound was prepared by the
(123 mg 1.0 mmol). The compound was obtained as a colorless addition of 13c (244 mg, 1.0 mmol) and 3,4-difluoroaniline
solid, yield: 290 mg (76%); mp: 228–230 °C; 1H NMR 14d (130 mg 1.0 mmol). Yellow solid, yield: 310 mg (78%); mp:
(500 MHz, CDCl3 + DMSO-d6); δ 3.80 (s, 3H, –OCH3), 3.91 214–216 °C; 1H NMR (500 MHz, CDCl3 + DMSO-d6); δ 3.84
(s, 3H, –OCH3), 3.95 (s, 3H, –OCH3), 6.72 (s, 1H, ArH), 6.77 (s, 3H, –OCH3), 6.73 (s, 1H, ArH), 6.73 (d, 1H, J = 15.8 Hz
(d, 1H, J = 15.6 Hz, transH), 6.84 (s, 1H, ArH), 6.89 (s, 1H, transH), 6.95 (d, 2H, J = 8.3 Hz, ArH), 7.07–7.20 (m, 1H, ArH),
ArH), 6.94 (d, 1H, J = 8.3 Hz, ArH), 7.35 (d, 1H, transH, J = 7.31–7.40 (m, 1H, ArH), 7.61 (d, 1H, J = 15.6 Hz, transH), 7.69
16.2 Hz), 7.57–7.69 (m, 4H, ArH), 8.72 (brs, 1H, –NH); (d, 2H, J = 8.3 Hz, ArH), 6.73 (s, 1H, ArH), 10.2 (brs, 1H, –NH);
13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 54.4, 54.9, 100.1, 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 54.6, 95.4, 97.5, 100.3,
108.0, 110.6, 112.9, 117.2, 120.3, 121.9, 131.4, 147.9, 148.1, 113.5, 122.0, 123.8, 126.2, 130.3, 130.9, 140.3, 158.8,
154.8, 162.9 ppm; IR (KBr) (νmax/cm−1): ν = 3311, 3135, 2940, 160.1 ppm; IR (KBr) (νmax/cm−1): ν = 3223, 3018, 2950, 1679,
2841, 1729, 1676, 1610, 1509, 1449, 1419, 1307, 1250, 1181, 1610, 1580, 1515, 1486, 1441, 1353, 1325, 1273, 1260, 1147,
1140, 995 cm−1; MS (ESI) m/z 380 [M + H]; HR-MS (ESI) m/z for 1110, 1065, 998 cm−1; MS (ESI) m/z 356 [M + H].
C21H22O4N3 calculated m/z: 380.1602 found m/z: 380.1614.
(E)-3-(3-(Benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-N-(3,4,5-
(E)-N-(3,4-Difluorophenyl)-3-(3-(3,4-dimethoxyphenyl)-1H- trimethoxyphenyl)acrylamide 15l. This compound was pre-
pyrazol-5-yl)acrylamide 15h. This compound was prepared pared by the addition of (E)-3-(3-(benzo[d][1,3]dioxol-5-yl)-1H-
by the addition of 13b (274 mg, 1.0 mmol) and 3,4-difluoroani- pyrazol-5-yl)acrylic acid 13d (258 mg 1.0 mmol) and 3,4,5-tri-
line 14d (130 mg 1.0 mmol). Yellow solid, yield: 300 mg (77%); methoxyaniline 14a (183 mg, 1.0 mmol). Brown solid, yield:
1
mp: 188–189 °C; 1H NMR (500 MHz, CDCl3 + DMSO-d6); δ 3.91 310 mg (73%); mp: 198–200 °C; H NMR (300 MHz, CDCl3 +
(s, 3H, –OCH3), 3.96 (s, 3H, –OCH3), 6.75 (d, 1H, transH, J = DMSO-d6); δ 3.87 (s, 6H, –OCH3), 3.70 (s, 3H, –OCH3), 6.09
15.6 Hz), 6.93 (d, 1H, ArH, J = 8.3 Hz), 7.05–7.22 (m, 1H, ArH), (s, 2H, –OCH2O–), 6.97 (d, 1H, J = 15.8 Hz, transH), 7.12 (s, 1H,
7.29–7.41 (m, 2H, ArH), 7.55 (s, 2H, ArH), 7.65 (d, 1H, transH, ArH), 7.31–7.35 (m, 1H, ArH), 7.37 (s, 1H, ArH), 7.41 (d, 1H, J =
J = 15.8 Hz), 7.81–7.93 (m, 1H, ArH), 10.05 (brs, 1H, –NH); 8.0 Hz, ArH), 7.52 (d, 1H, J = 15.8 Hz, transH), 7.61 (d, 1H, J =
IR (KBr) (νmax/cm−1): ν = 3230, 3010, 1640, 1610, 1586, 1550, 8.4 Hz, ArH), 7.86 (d, 1H, J = 8.4 Hz, ArH), 10.3 (brs, 1H, –NH);
1496, 1480, 1410, 1380, 1254, 1200, 1175, 1110, 1026, IR (KBr) (νmax/cm−1): ν = 3208, 2933, 1669, 1609, 1550, 1506,
993 cm−1; MS (ESI) m/z 386 [M + H]; HR-MS (ESI) m/z for 1455, 1386, 1234, 1187, 1127, 1105, 1036, 975 cm−1; MS (ESI)
C20H18F2O3N3 calculated m/z: 386.1338, found m/z: 386.1320.
m/z 424 [M + H]; HR-MS (ESI) m/z for C22H22O6N3 calculated
(E)-3-(3-(4-Methoxyphenyl)-1H-pyrazol-5-yl)-N-(3,4,5-trimethoxy- m/z: 424.1502, found m/z: 424.1502.
phenyl)acrylamide 15i. This compound was prepared by the
(E)-3-(3-(Benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-N-(3,4-
addition of (E)-3-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)acrylic dimethoxyphenyl)acrylamide 15m. This compound was pre-
acid 13c (244 mg 1.0 mmol) and 3,4,5-trimethoxyaniline 14a pared by the addition of 13d (258 mg, 1.0 mmol) and 3,4-
(183 mg, 1.0 mmol). Yellow solid, yield: 298 mg (73%); mp: dimethoxyaniline 14b (153 mg 1.0 mmol). Pale yellow solid,
208–210 °C; 1H NMR (500 MHz, CDCl3 + DMSO-d6); δ 3.76 yield: 280 mg (71%); mp: 180–182 °C; 1H NMR (300 MHz,
(s, 3H, –OCH3), 3.85 (s, 9H, –OCH3), 6.75 (d, 1H, transH, J = CDCl3 + DMSO-d6); δ 3.78 (s, 3H, –OCH3), 3.80 (s, 3H, –OCH3),
15.4 Hz), 6.78 (s, 1H, ArH), 6.96 (d, 2H, J = 8.4 Hz, ArH), 7.12 5.98 (s, 2H, –OCH2O–), 6.22 (s, 1H, ArH), 6.60–6.72 (m, 2H,
(s, 2H, ArH), 7.59 (d, 1H, transH, J = 15.6 Hz), 7.70 (d, 2H, ArH, ArH, J = 15.8 Hz, transH), 6.84 (t, 1H, J = 8.2 Hz, ArH), 6.95
J = 8.4 Hz), 9.98 (brs, 1H, –NH); 13C NMR (75 MHz, DMSO-d6): (s, 2H, ArH), 7.20–7.28 (m, 2H, ArH), 7.63 (d, 1H, J = 15.5 Hz,
δ 54.8, 55.4, 60.1, 96.8, 100.4, 113.7, 122.1, 123.2, 126.3, 130.3, transH), 8.87 (brs, 1H, –NH); 13C NMR (75 MHz, CDCl3
+
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DMSO-d6): δ 53.9, 96.8, 99.8, 104.7, 107.2, 117.9, 121.4, 130.6, acetate/hexane); 1H NMR (300 MHz, CDCl3); δ 1.21–1.31 (t, 3H,
132.2, 139.6, 146.1, 146.7, 159.4 ppm; IR (KBr) (νmax/cm−1): ν = J = 7.4 Hz, –CH3), 3.87 (s, 3H, –OCH3), 3.94 (s, 3H, –OCH3)
3208, 2927, 1614, 1558, 1462, 1322, 1246, 1204, 1153, 1037, 4.10–4.22 (q, 2H, J = 7.0 Hz, –CH2), 6.85 (d, 1H, J = 7.2 Hz,
972 cm−1; MS (ESI) m/z 394 [M + H].
–ArH), 6.97 (s, 1H, ArH), 7.23–7.27 (m, 1H, ArH) 7.30–7.33
(E)-3-(3-(Benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-N-(4-meth- (m, 1H, ArH) ppm; MS (ESI) m/z 278 [M + H].
oxyphenyl)acrylamide 15n. This compound was prepared by Ethyl 3-(4-methoxyphenyl)isoxazole-5-carboxylate 16c. Yellow
1
the addition of 13d (258 mg, 1.0 mmol) and 4-methoxyaniline solid (yield 80%); Rf = 0.5 (15% ethyl acetate/hexane); H NMR
14c (123 mg 1.0 mmol). Brown solid, yield: 291 mg (80%); (500 MHz, CDCl3); δ 1.23–1.37 (t, 3H, J = 6.7 Hz,–CH3), 3.87
196–198 °C; 1H NMR (300 MHz, DMSO-d6); δ 3.78 (s, 3H, (s, 3H, –OCH3), 4.15–4.35 (q, 2H, J1 = 6.7 Hz, J2 = 7.5 Hz, CH2),
–OCH3), 6.02 (s, 2H, –OCH2O–), 6.73 (s, 1H, ArH), 6.75 (d, 1H, 6.85 (s, 1H, ArH), 7.01 (d, 2H, J = 7.2 Hz, ArH), 7.68 (d, 2H, J =
ArH, J = 15.6 Hz, transH), 6.86 (t, 3H, J = 9.0 Hz, ArH), 8.9 Hz, ArH) ppm; MS (ESI) m/z 248 [M + H].
7.21–7.34 (m, 2H, ArH), 7.52 (d, 1H, J = 15.6 Hz, transH), 7.63
Ethyl 3-(benzo[d][1,3]dioxol-5-yl)isoxazole-5-carboxylate 16d.
(d, 2H, ArH, J = 8.8 Hz) 7.96 (brs, 1H, –NH); 13C NMR (75 MHz, Pale yellow solid (yield 75.0%): Rf = 0.6 (30% ethyl acetate/
1
DMSO-d6): δ 53.4, 99.4, 103.9, 106.8, 112.1, 117.4, 119.3, 121.0, hexane); H NMR (500 MHz, CDCl3); δ 1.44 (t, 3H, J1 = 7.1 Hz,
130.5, 145.4, 146.0, 153.7, 161.6 ppm; IR (KBr) (νmax/cm−1): ν = –CH3), 4.47 (q, 2H, J = 7.1 Hz, –CH2), 5.98 (s, 2H, –OCH2O),
3216, 3100, 3096, 1688, 1651, 1596, 1542, 1505, 1473, 1450, 6.79 (s, 1H, ArH), 6.81 (s, 1H, ArH), 6.86 (d, 1H, J = 7.1 Hz,
1330, 1311, 1285, 1215, 1167, 1108, 1035, 998 cm−1; MS (ESI) ArH), 7.19 (s, 1H, ArH) ppm; MS (ESI) m/z 218 [M + H].
m/z 364 [M + H]; HR-MS (ESI) m/z for C20H18O4N3 calculated
Preparation of (3-substituted phenylisoxazol-5-yl)methanol
17(a–d)
m/z: 364.1298, found m/z: 364.1298.
(E)-3-(3-(Benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-N-(3,4-
difluorophenyl)acrylamide 15o. This compound was pre- To the 3-substituted phenylisoxazole-5-carboxylates 16(a–d),
pared by the addition of 13d (258 mg, 1.0 mmol) and 3,4- obtained in the above step was added LiAlH4 (0.5 mol) in dry
difluoroaniline 14d (130 mg 1.0 mmol). Pale yellow solid, THF at 0 °C and stirred for 1 h at room temperature. Saturated
yield: 270 mg (73%); mp: 185–187 °C; 1H NMR (300 MHz, NH4Cl solution was added dropwise to quench the unreacted
CDCl3 + DMSO-d6); δ 6.03 (s, 2H, –OCH2O–), 6.72 (d, 1H, ArH, LiAlH4 and THF was removed under vacuum and then
J = 15.6 Hz, transH), 6.78 (s, 1H, ArH), 6.89 (d, 1H, J = 9.0 Hz, extracted with ethyl acetate (100 ml × 4). The organic layer was
ArH), 7.12–7.43 (m, 4H, ArH), 7.55 (d, 1H, J = 15.6 Hz, transH), dried on anhydrous Na2SO4 and ethyl acetate was evaporated
7.83–7.96 (m, 1H, ArH), 8.06 (brs, 1H, –NH); 13C NMR to obtain colorless solid products of (3-substituted phenyl-
(75 MHz, CDCl3+DMSO-d6): δ 99.03, 103.9, 106.5, 106.7, 115.1, isoxazol-5-yl)methanols 17(a–d) (yield 70–80%). The alcohols
113.6, 115.3, 117.2, 120.2, 134.5, 145.4, 146.0, 162.0 ppm; produced in this step were pure and taken as such for the next
IR (KBr) (νmax/cm−1): ν = 3302, 3163, 3016, 1690, 1645, 1610, step.
1550, 1515, 1473, 1441, 1361, 1322, 1241, 1201, 1116, 1026,
Preparation of 3-substituted phenylisoxazole-5-carbaldehydes
18a–d
979 cm−1; MS (ESI) m/z 370 [M + H]; HR-MS (ESI) m/z for
C19H14O3N3F2 calculated m/z: 370.1003, found m/z: 370.1002.
To each (3-substituted phenylisoxazol-5-yl) of the methanols
17(a–d) produced in the above step was added IBX (1.2 mol) in
DMSO and stirred for 1 h at room temperature. Ice cold water
Preparation of ethyl 3-substituted phenylisoxazole-
5-carboxylates 16a–d
To each of the ethyl 2,4-dioxo-4-(substituted phenyl) butano- was added to the reaction mixture and extracted with ethyl-
ates 8(a–d) (1.0 mol) which were obtained in the earlier step acetate (50 ml × 4). The organic layer was dried on anhydrous
was added hydroxylamine hydrochloride (NH2–OH·HCl) Na2SO4 and ethyl acetate was evaporated to obtain the corres-
(1.5 mol) in ethanol and heated to reflux for 3 h. The solvent ponding pure 3-sustituted phenylisoxazole-5-carbaldehydes 18
was removed under vacuum and then water was added to the (a–d) in good yields (80–85%). The obtained carbaldehydes
residue and the compound was extracted with ethyl acetate were taken as such in the next step.
(50 ml × 4). The organic layer was dried on anhydrous Na2SO4
3-(3,4,5-Trimethoxyphenyl)isoxazole-5-carbaldehyde
18a.
and the solvent was evaporated to obtain the crude product This compound was prepared by the addition of (3-(3,4,5-tri-
that was further purified by column chromatography using methoxyphenyl)isoxazol-5-yl)methanol 17a (2.62 g 10 mmol)
ethyl acetate and hexane. The pure compounds 16(a–d) were and IBX (3.36 g 1.2 mmol). Yellow solid (2.13 g, yield 81%):
eluted at 20–25% ethyl acetate with good yields (75–80%).
Rf = 0.3 (40% ethyl acetate/hexane); 1H NMR (400 MHz,
Ethyl 3-(3,4,5-trimethoxyphenyl)isoxazole-5-carboxylate CDCl3); δ 3.89 (s, 3H, –OCH3), 3.95 (s, 6H, –OCH3), 6.96 (s, 1H,
16a. Pale yellow solid (yield 75.0%): Rf = 0.5 (20% ethyl ArH), 7.08 (s, 2H, ArH), 10.18 (s, 1H, –CHO) ppm; MS (ESI) m/z
1
acetate/hexane); H NMR (300 MHz, CDCl3); δ 1.45 (t, 3H, J = 264 [M + H].
7.1 Hz, –CH3), 3.92 (s, 3H, –OCH3), 3.93 (s, 6H, –OCH3) 4.48
(q, 2H, J1 = 7.0 Hz, –CH2), 6.88 (s, 1H, ArH), 7.02 (s, 2H, ArH) compound was prepared by the addition of (3-(3,4-dimethoxy-
ppm; MS (ESI) m/z 308 [M + H]. phenyl)isoxazol-5-yl)methanol 17b (2.35 g 10 mmol) and IBX
ethyl 3-(3,4-dimethoxyphenyl)isoxazole-5-carboxylate (3.36 g 1.2 mmol). Yellow solid (1.93 g, yield 83%): Rf
16b. Pale yellow solid (yield 80.0%): Rf = 0.5 (15% ethyl 0.4 (30% ethyl acetate/hexane); 1H NMR (300 MHz, CDCl3); δ 3.89
3-(3,4-Dimethoxyphenyl)isoxazole-5-carbaldehyde 18b. This
2
=
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(s, 3H, –OCH3), 3.90 (s, 3H, –OCH3), 6.90 (s, 1H, ArH), 110.1, 117.4, 117.8, 124.2, 129.8, 147.5, 149.2, 158.6, 163.6,
7.26–7.49 (m, 2H, ArH), 7.88–7.97 (m, 1H, ArH) 10.18 (s, 1H, 168.6 ppm; MS (ESI) m/z 304 [M + H]; HR-MS (ESI) m/z for
–CHO) ppm; MS (ESI) m/z 234 [M + H].
3-(4-Methoxyphenyl)isoxazole-5-carbaldehyde 18c. This
C16H18O5N calculated m/z: 304.1106, found m/z: 304.1109.
(E)-Ethyl 3-(3-(4-methoxyphenyl)isoxazol-5-yl)acrylate 19c.
compound was prepared by the addition of (3-(4-methoxy- This compound was prepared by the addition of 3-(4-methoxy-
phenyl)isoxazol-5-yl)methanol 17c (2.05 g. 10 mmol) and IBX phenyl)isoxazole-5-carbaldehyde 18c (203 mg 1.0 mmol) and
(3.36 g 1.2 mmol). Yellow solid (1.72 g, yield 85%): Rf
=
Ph3PCHCO2C2H5 (248 mg 1.0 mmol). Yellow solid (204.7 mg,
0.5 (25% ethyl acetate/hexane); 1H NMR (500 MHz, CDCl3); yield 75%): Rf = 0.4 (30% ethyl acetate/hexane); 1H NMR
δ 3.87 (s, 3H, –OCH3), 6.79 (s, 1H, ArH), 7.02 (d, 2H, J = 8.8 Hz, (300 MHz, CDCl3); δ 1.35 (t, 3H, J = 7.1 Hz, –CH3), 3.87 (s, 3H,
ArH), 7.77 (d, 2H, J = 9.0 Hz, ArH), 10.19 (s, 1H, –CHO) ppm; –OCH3), 4.23–4.34 (q, 2H, J1 = 7.0 J2 = 7.1 Hz, –CH2), 6.54
MS (ESI) m/z 204 [M + H].
(d, 1H, transH J = 16.1 Hz), 6.58 (s, 1H, ArH), 6.99 (d, 2H, J =
3-(Benzo[d][1,3]dioxol-5-yl)isoxazole-5-carbaldehyde
18d. 8.3 Hz, ArH), 7.68 (d, 1H, transH J = 16.1 Hz), 7.73 (d, 2H, J =
This compound was prepared by the addition of (3-(benzo[d]- 8.8 Hz, ArH) ppm; 13C NMR (75 MHz, CDCl3): 14.1, 55.3, 60.9,
[1,3]dioxol-5-yl)isoxazol-5-yl)methanol 17d (2.19 g 10 mmol) 95.4, 114.4, 119.6, 125.3, 129.1, 127.4, 127.9, 131.8, 160.1,
and IBX (3.36 g 1.2 mmol). Yellow solid (1.8 g, yield 83%): Rf = 161.3, 165.6, 170.6 ppm; MS (ESI) m/z 274 [M + H]; HR-MS
0.6 (30% ethyl acetate/hexane); 1H NMR (300 MHz, CDCl3); (ESI) m/z for C15H16O4N calculated m/z: 274.1001, found m/z:
δ 6.06 (s, 2H, –OCH2O), 6.75 (s, 1H, ArH), 6.92 (d, 1H, J = 274.1002.
8.2 Hz, ArH), 7.29 (s, 1H, ArH), 7.36 (d, 1H, J = 8.0 Hz, ArH),
10.16 (s, 1H, –CHO) ppm; MS (ESI) m/z 218 [M + H].
(E)-Ethyl 3-(3-(benzo[d][1,3]dioxol-5-yl)isoxazol-5-yl)acrylate
19d. This compound was prepared by the addition of 3-(benzo-
[d][1,3]dioxol-5-yl)isoxazole-5-carbaldehyde 18d (217 mg
1.0 mmol) and Ph3PCHCO2C2H5 (248 mg 1.0 mmol). Yellow
Preparation of (E)-ethyl 3-(3-arylisoxazol-5-yl)acrylate 19a–d
To each of the isoxazole carbaldehydes obtained in the above solid (212 mg, yield 75%): Rf = 0.5 (30% ethyl acetate/hexane);
step was added an equimolar amount of (carbethoxymethyl- 1H NMR (300 MHz, CDCl3); δ 1.23 (t, 3H, J = 7.0 Hz, –CH3),
ene)triphenylphosphine (Ph3PCHCO2C2H5, C2-wittig reagent) 4.15–4.33 (q, 2H, J = 6.0 Hz, –CH2), 6.01 (s, 2H, –OCH2O–), 6.45
in toluene at room temperature and continued stirring for (s, 1H, ArH), 6.67 (d, 1H, transH J = 16.0 Hz), 6.72–6.78 (m, 1H,
3–4 h. After performing TLC toluene was evaporated and ArH), 6.92 (s, 1H, ArH), 7.11–7.35 (m, 1H, ArH), 7.63 (d, 1H,
appropriate amounts of water were added. The crude com- transH J = 15.9 Hz) ppm; 13C NMR (75 MHz, CDCl3): 14.1, 56.2,
pounds were extracted by ethyle acetate (50 ml × 4). The 61.0, 96.6, 103.1, 122.1, 125.6, 131.6, 140.1, 153.6, 160.2, 165.5,
organic layer was dried on anhydrous Na2SO4 and ethyl acetate 170.5 ppm; MS (ESI) m/z 288 [M + H]; HR-MS (ESI) m/z for
was evaporated to obtain crude α,β-unsaturated isoxazole C15H17O3N2 calculated m/z: 288.0793, found m/z: 288.0796.
esters 19a–d in good yields (70–75%).
Preparation of (E)-3-(3-arylisoxazol-5-yl)acrylic acids 20a–d
(E)-Ethyl 3-(3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl)acrylate
19a. This compound was prepared by the addition of 3-(3,4,5- To each of the (E)-ethyl 3-(3-arylisoxazol-5-yl)acrylates 19a–d
trimethoxyphenyl)isoxazole-5-carbaldehyde 18a (263 mg obtained in the above step was added equimolar amounts of
1.0 mmol) and Ph3PCHCO2C2H5 (248 mg 1.0 mmol). Pale lithium hydroxide (LiOH·H2O) in a mixture of solvents THF :
yellow solid (233 mg, yield 70%): Rf = 0.5 (30% ethyl acetate/ MeOH : H2O (3 : 1 : 1) at room temperature and continued stir-
1
hexane); H NMR (300 MHz, CDCl3); δ 1.36 (t, 3H, J = 7.1 Hz, ring for 3–4 h. After performing TLC the solvent mixture was
–CH3), 3.91 (s, 3H, –OCH3), 3.94 (s, 6H, –OCH3), 4.30 (q, 2H, evaporated and appropriate amounts of water were added. The
J1 = 7.1 Hz, J2 = 6.9 Hz, –CH2), 6.57 (d, 1H, transH J = 16.0 Hz), crude compounds were extracted by ethyle acetate (50 ml × 4).
6.65 (s, 1H, ArH), 7.02 (s, 2H, ArH), 7.70 (d, 1H, transH J = The aqueous layer was acidified using dil. HCl, and then again
16.2 Hz) ppm; 13C NMR (75 MHz, CDCl3): 12.5, 54.6, 58.8, extracted by ethyl acetate (50 ml × 4). The organic layer was
59.1, 96.2, 101.6, 104.1, 107.9, 120.5, 124.4, 129.7, 138.0, 151.8, dried on anhydrous Na2SO4 and ethyl acetate was evaporated
158.7, 163.2, 163.6, 168.5 ppm; MS (ESI) m/z 334 [M + H]; to obtain the corresponding pure α,β-unsaturated isoxazole
HR-MS (ESI) m/z for C17H20O6N calculated m/z: 334.1212, acids 20a–d in good yields (80–85%).
found m/z: 334.1206.
General procedure for the synthesis of isoxazole linked
arylcinnamides 21a–n
(E)-Ethyl
3-(3-(3,4-dimethoxyphenyl)isoxazol-5-yl)acrylate
19b. This compound was prepared by the addition of 3-(3,4-
dimethoxyphenyl)isoxazole-5-carbaldehyde 18b (233 mg To the (E)-3-(3-arylisoxazol-5-yl)acrylic acid 20a–d prepared in
1.0 mmol) and Ph3PCHCO2C2H5 (248 mg 1.0 mmol). Yellow the above step was added an equimolar amount of EDCI and
solid (218 mg, yield 72%): Rf = 0.5 (30% ethyl acetate/hexane); catalytic amount of hydroxybenzotriazole (Hobt) in ice cold
1H NMR (300 MHz, CDCl3); δ 1.35 (t, 3H, J = 7.1 Hz, –CH3), dichloromethane. After 10 min appropriate amounts of aryl
3.93 (s, 3H, –OCH3), 3.94 (s, 3H, –OCH3), 4.15–4.31 (q, 2H, J1 = amines were added at 0 °C and stirring was continued at room
7.0 Hz, J2 = 7.1 Hz, –CH2), 6.23 (s, 1H, ArH), 6.62 (s, 1H, ArH), temperature for 8 h. The progress of the reaction was moni-
6.72 (d, 1H, transH J = 16.2 Hz), 6.92 (d, 1H, J = 8.4 Hz, ArH), tored by TLC. After completion of the reaction an appropriate
6.42 (d, 1H, transH J = 16.2 Hz), 7.20–7.28 (m, 1H, ArH) ppm; amount of sodium bicarbonate (NaHCO3) solution was added
13C NMR (75 MHz, CDCl3): 12.5, 54.2, 54.2, 59.1, 95.1, 107.3, and subsequently the final compounds were extracted with
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ethyl acetate (50 ml × 4). The organic layers so obtained were mp: 192–194 °C; 1H NMR (500 MHz, CDCl3 + DMSO-d6); δ 3.84
washed with 5% HCl solution and the organic solvent was (s, 3H, –OCH3), 3.96 (s, 6H, –OCH3), 6.91–7.05 (m, 2H, ArH),
evaporated to afford crude compounds. Further these com- 7.06–7.23 (m, 2H, ArH), 7.37 (s, 2H, ArH), 7.61 (d, 1H, transH
pounds were purified using column chromatography by using J = 16.0 Hz), 7.88 (d, 1H, transH J = 16.4 Hz, ArH) 10.40 (brs,
ethyl acetate and a hexane solvent system to get pure com- 1H, –NH); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 55.0, 59.4,
pounds in good yields (65–78%).
96.6, 102.0, 107.7, 108.0, 114.4, 115.6, 115.9, 121.0, 126.7,
(E)-N-(3,4,5-Trimethoxyphenyl)-3-(3-(3,4,5-trimethoxyphenyl)- 127.9, 152.3, 159.2, 161.7, 168.8 ppm; IR (KBr) (νmax/cm−1):
isoxazol-5-yl)acrylamide 21a. This compound was prepared by
ν = 3309, 2940, 1669, 1624, 1574, 1518, 1505, 1424,
the addition of (E)-3-(3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl)- 1241, 967 cm−1; MS (ESI) m/z 417 [M + H]; HR-MS (ESI)
acrylic acid 20a (305 mg 1.0 mmol) and 3,4,5-trimethoxyaniline m/z for C21H19F2O5N2 calculated m/z: 417.1256, found m/z:
14a (183 mg 1.0 mmol). Pale yellow solid, yield: 340 mg (75%); 417.1247.
mp: 202–204 °C; 1H NMR (300 MHz, CDCl3 + DMSO-d6); δ 3.84
(E)-3-(3-(3,4-Dimethoxyphenyl)isoxazol-5-yl)-N-(3,4,5-trimeth-
(s, 3H, –OCH3), 3.87 (s, 6H, –OCH3), 3.91 (s, 3H, –OCH3), 3.94 oxyphenyl)acrylamide 21e. This compound was prepared by
(s, 6H, –OCH3), 6.61 (s, 1H, ArH), 6.78 (d, 1H, transH J = the addition of (E)-3-(3-(3,4-dimethoxyphenyl)isoxazol-5-yl)-
15.8 Hz), 6.96 (s, 1H, J = 7.5 Hz, ArH) 7.00 (s, 2H, J = 8.8 Hz, acrylic acid 20b (275 mg 1.0 mmol) and 3,4,5-trimethoxyaniline
ArH) 7.69 (d, 1H, transH, J = 15.4 Hz), 7.67(s, 1H, ArH); 13C 14a (183 mg 1.0 mmol). Yellow solid, yield: 300 mg (68%);
NMR (75 MHz, CDCl3 + DMSO-d6): δ 54.4, 54.8, 59.0, 96.0, mp: 212–214 °C; 1H NMR (300 MHz, CDCl3 + DMSO-d6); δ 3.69
96.4, 101.8, 120.8, 125.4, 128.2, 132.7, 133.6, 138.3, 151.4, (s, 3H, –OCH3), 3.80 (s, 6H, –OCH3), 3.87 (s, 3H, –OCH3), 3.91
152.1, 159.1, 161.2, 168.6 ppm; IR (KBr) (νmax/cm−1): ν = 3315, (s, 3H, –OCH3), 7.00 (d, 1H, transH J = 15.6 Hz), 7.07 (s, 1H,
2997, 1680, 1549, 1520, 1484, 1454, 1420, 1376, 1312, 1273, ArH), 7.13 (s, 1H, ArH), 7.31 (d, 1H, transH J = 14.9 Hz),
1204, 1181, 1142, 1026 cm−1; MS (ESI) m/z 471; HR-MS (ESI) 7.34–7.44 (m, 1H, ArH), 7.45–7.51 (m, 1H, ArH), 7.52–7.60
m/z for C24H27O8N2 calculated m/z: 471.1742, found m/z: (m, 1H, ArH), 7.84 (d, 1H, ArH J = 8.3 Hz), 10.43 (brs, 1H,
471.1761.
–NH); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 55.5, 60.1, 96.4,
(E)-N-(3,4-Dimethoxyphenyl)-3-(3-(3,4,5-trimethoxyphenyl)- 97.1, 108.5, 109.7, 111.2, 118.2, 118.7, 119.3, 123.6, 125.4,
isoxazol-5-yl)acrylamide 21b. This compound was prepared by 127.1, 133.7, 134.7, 148.8, 152.5, 160.1, 162.4, 169.7 ppm;
the addition of 20a (150 mg 1.0 mmol) and 3,4-dimethoxy- IR (KBr) (νmax/cm−1): ν = 3320, 2940, 1687, 1610, 1596, 1502,
aniline 14b (153 mg 1.0 mmol). Colourless solid, yield: 295 mg 1465, 1427, 1416, 1283, 1133, 1019, 996 cm−1; MS (ESI) m/z 441
(67%); mp: 182–184 °C; 1H NMR (500 MHz, CDCl3 + DMSO- [M + H]; HR-MS (ESI) m/z for C23H25O7N2 calculated m/z:
d6); δ 3.80 (s, 6H, –OCH3), 3.89 (s, 3H, –OCH3), 3.95 (s, 6H, 441.1656, found m/z: 441.1644.
–OCH3), 6.23 (s, 1H, ArH), 6.78 (s, 1H, ArH), 6.97 (d, 1H,
(E)-N-(3,4-Dimethoxyphenyl)-3-(3-(3,4-dimethoxyphenyl)-
transH J = 15.6 Hz), 7.02 (s, 1H, ArH), 7.05 (s, 2H, ArH), 7.54 isoxazol-5-yl)acrylamide 21f. This compound was prepared
(s, 1H, ArH), 7.62 (d, 1H, transH J = 15.6 Hz) 9.92 (brs, 1H, by the addition of 20b (275 mg 1.0 mmol) and 3,4-dimethoxy-
–NH); IR (KBr) (νmax/cm−1): ν = 3314, 2937, 1686, 1611, 1507, aniline 14b (153 mg 1.0 mmol). The compound obtained as
1
1458, 1425, 1412, 1242, 1131, 999 cm−1; MS (ESI) m/z 441 yellow crystals, yield: 305 mg (74%); mp: 196–198 °C; H NMR
[M + H]; HR-MS (ESI) m/z for C23H25O7N2 calculated m/z: (400 MHz, CDCl3 + DMSO-d6); δ 3.79 (s, 6H, –OCH3), 3.94
441.1656, found m/z: 441.1641.
(s, 3H, –OCH3), 3.97 (s, 3H, –OCH3), 6.22 (s, 1H, ArH),
(E)-N-(4-Methoxyphenyl)-3-(3-(3,4,5-trimethoxyphenyl)isoxazol- 6.80–6.90 (m, 1H, ArH), 6.96 (s, 1H, ArH), 6.99–7.04 (m, 3H,
5-yl)acrylamide 21c. This compound was prepared by the ArH), 7.33 (d, 1H, transH J = 15.1 Hz), 7.42 (d, 1H, ArH J =
addition of 20a (150 mg 1.0 mmol) and 4-methoxyaniline 14c 9.8 Hz), 7.61 (d, 1H, transH J = 15.6 Hz), 7.66–7.71 (m, 1H,
(123 mg 1.0 mmol). Pale yellow solid, yield: 300 mg (73%); mp: ArH), 10.71 (brs, 1H, –NH); IR (KBr) (νmax/cm−1): ν = 3330,
186–188 °C; 1H NMR (300 MHz, DMSO-d6); δ 3.79 (s, 3H, 3010, 2910, 1671, 1620, 1566, 1511, 1469, 1443, 1383, 1221,
–OCH3), 3.87 (s, 3H, –OCH3), 3.95 (s, 6H, –OCH3), 6.87 (d, 2H, 1116, 1026, 989 cm−1; MS (ESI) m/z 411 [M + H]; HR-MS (ESI)
J = 8.4 Hz, ArH), 6.99 (d, 1H, transH J = 15.6 Hz), 7.08 (s, 2H, m/z for C22H23O6N2 calculated m/z: 411.1550, found m/z:
ArH), 7.53–7.74 (m, 3H, ArH), 7.66 (d, 1H, transH J = 15.6 Hz), 411.1547.
10.03 (brs, 1H, –NH); 13C NMR (75 MHz, DMSO-d6):
(E)-3-(3-(3,4-Dimethoxyphenyl)isoxazol-5-yl)-N-(4-methoxy-
δ 53.7, 54.7, 58.9, 96.4, 101.7, 112.3, 119.7, 120.7, 125.4, phenyl)acrylamide 21g. This compound was prepared by the
128.3, 130.5, 138.1, 151.9, 154.3, 159.0, 160.8, 168.4 ppm; addition of 20b (275 mg 1.0 mmol) and 4-methoxyaniline 14c
IR (KBr) (νmax/cm−1): ν = 3286, 3040, 1680, 1630, 1573, (123 mg 1.0 mmol). Brown solid, yield: 290 mg (76%); mp:
1544, 1512, 1475, 1426, 1390, 1315, 1227, 1210, 1184, 208–210 °C; 1H NMR (300 MHz, CDCl3 + DMSO-d6); δ 3.79
1112, 1036, 990 cm−1; MS (ESI) m/z 411 [M + H]; HR-MS (ESI) (s, 3H, –OCH3), 3.91 (s, 3H, –OCH3), 3.94 (s, 3H, –OCH3), 6.86
m/z for C22H23O6N2 calculated m/z: 411.1550, found m/z: (d, 2H, ArH J = 8.6 Hz), 6.97–7.04 (m, 1H, ArH), 7.37 (s, 1H,
411.1542.
ArH), 7.45 (d, 1H, ArH J = 8.3 Hz), 7.55 (d, 1H, transH J = 15.6
(E)-N-(3,4-Difluorophenyl)-3-(3-(3,4,5-trimethoxyphenyl)- Hz), 7.65 (d, 2H, J = 8.8 Hz ArH), 7.92 (d, 1H, transH J = 15.6
isoxazol-5-yl)acrylamide 21d. This compound was prepared Hz), H J = 9.8 Hz), 7.91 (s, 1H, –ArH), 10.17 (brs, 1H, –NH);
by the addition of 20a (305 mg 1.0 mmol) and 3,4-difluoro- 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 54.9, 55.4, 96.7, 108.7,
aniline 14d (130 mg 1.0 mmol). Brown solid, yield: 290 mg (70%); 111.6, 113.6, 118.7, 119.2, 120.8, 126.5, 129.6, 148.9, 150.5,
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155.4, 160.2, 161.9, 169.7 ppm; IR (KBr) (νmax/cm−1): ν = 3290, 15.6 Hz), 7.02 (d, 2H, J = 9.0 Hz, ArH), 7.07–7.23 (m, 1H, ArH),
3033, 3010, 1682, 1615, 1574, 1526, 1419, 1376, 1320, 1026, 7.39 (d, 1H, J = 8.4 Hz, ArH), 7.61 (d, 1H, J = 15.6 Hz, transH),
993 cm−1; MS (ESI) m/z 381 [M + H]. HR-MS (ESI) m/z for 7.77 (d, 2H, J = 8.4 Hz, ArH), 7.82–7.97 (m, 1H, ArH), 10.47
C21H21O5N2 calculated m/z: 381.1445, found m/z: 381.1454.
(brs, 1H, –NH); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 55.1,
(E)-N-(3,4-Difluorophenyl)-3-(3-(3,4-dimethoxyphenyl)isoxazol- 96.3, 108.4, 108.6, 114.3, 115.4, 116.8, 117.0, 119.1, 127.1,
5-yl)acrylamide 21h. This compound was prepared by the 127.7, 128.8, 135.6, 160.1, 160.8, 162.5, 169.8 ppm; IR (KBr)
addition of 20b (275 mg 1.0 mmol) and 3,4-difluoroaniline (νmax/cm−1): ν = 3233, 3060, 2961, 1663, 1620, 1543, 1518,
14d (130 mg 1.0 mmol). Yellow solid, yield: 295 mg (75%); mp: 1437, 1305, 1253, 1214, 1175, 1030, 968 cm−1; MS (ESI) m/z 357
216–218 °C; 1H NMR (400 MHz, CDCl3 + DMSO-d6); δ 3.91 [M + H]; HR-MS (ESI) m/z for C19H15O3F2N2 calculated m/z:
(s, 3H, –OCH3), 3.96 (s, 3H, –OCH3), 6.75 (d, 1H, transH, J = 357.1045, found m/z: 357.1041.
15.6 Hz), 6.93 (d, 1H, ArH, J = 8.3 Hz), 7.05–7.22 (m, 1H, ArH),
(E)-3-(3-(Benzo[d][1,3]dioxol-5-yl)isoxazol-5-yl)-N-(3,4,5-tri-
7.29–7.41 (m, 2H, ArH), 7.55 (s, 2H, ArH), 7.65 (d, 1H, transH, methoxyphenyl)acrylamide 21l. This compound was prepared
J = 15.8 Hz), 7.81–7.93 (m, 1H, ArH), 10.05 (brs, 1H, –NH); by the addition of (E)-3-(3-(benzo[d][1,3]dioxol-5-yl)isoxazol-5-
13C NMR (75 MHz, CDCl3 + DMSO-d6): δ ppm; IR (KBr) yl)acrylic acid 20d (258 mg 1.0 mmol) and 3,4,5-trimethoxy-
(νmax/cm−1): ν = 3230, 3010, 1640, 1610, 1586, 1550, 1496, aniline (183 mg 1.0 mmol) (14a). The compound was obtained
1480, 1410, 1380, 1254, 1200, 1175, 1110, 1026, 993 cm−1
;
as a yellow solid, yield: 315 mg (74%); mp: 165–167 °C; 1H NMR
MS (ESI) m/z 387 [M + H]; HR-MS (ESI) m/z for C20H17F2O4N2 (400 MHz, CDCl3 + DMSO-d6); δ 3.80 (s, 3H, –OCH3), 3.89
calculated m/z: 387.1150, found m/z: 387.1147. (s, 6H, –OCH3), 6.07 (s, 2H, –OCH2O–), 6.75 (s, 1H, ArH),
(E)-3-(3-(4-Methoxyphenyl)isoxazol-5-yl)-N-(3,4,5-trimeth- 6.86–6.97 (m, 1H, ArH), 7.04 (d, 1H, J = 16.0 Hz, transH), 7.13
oxyphenyl)acrylamide 21i. This compound was prepared by (s, 1H, ArH), 7.30–7.47 (m, 2H, ArH), 7.62 (d, 1H, J = 15.8 Hz,
the addition of (E)-3-(3-(4-methoxyphenyl)isoxazol-5-yl)acrylic transH), 7.88 (d, 1H, J = 8.1 Hz, ArH), 10.05 (brs, 1H, –NH); IR
acid 20c (245 mg 1.0 mmol) and 3,4,5-trimethoxyaniline 14a (KBr) (νmax/cm−1): ν = 3339, 3128, 2937, 1667, 1636, 1599,
(183 mg 1.0 mmol). Pale yellow solid yield: 305 mg (74%); mp: 1490, 1505, 1431, 1410, 1233, 1127, 1039, 806 cm−1; MS (ESI)
168–170 °C; 1H NMR (500 MHz, CDCl3 + DMSO-d6); δ 3.84 m/z 425 [M + H]; HR-MS (ESI) m/z for C22H21O7N2 calculated
(s, 3H, –OCH3), 3.88 (s, 9H, –OCH3), 6.55 (s, 1H, ArH), 6.73 m/z: 425.1343, found m/z: 425.1350.
(d, 1H, J = 16.1 Hz, transH), 6.93 (s, 1H, ArH), 7.00 (d, 2H, J =
(E)-3-(3-(Benzo[d][1,3]dioxol-5-yl)isoxazol-5-yl)-N-(3,4-dimethoxy-
8.3 Hz, ArH), 7.41 (s, 1H, ArH), 7.69 (d, 1H, J = 16.1 Hz, phenyl)acrylamide 21m. This compound was prepared by the
transH), 7.73 (d, 2H, J = 9.0 Hz, ArH); 13C NMR (75 MHz, addition of 20d (258 mg 1.0 mmol) and 3,4-dimethoxyaniline
CDCl3 + DMSO-d6): δ 55.0, 55.5, 60.0, 96.1, 97.1, 114.2, 119.1, 14b (153 mg 1.0 mmol). Brown solid, yield: 287 mg (73%); mp:
127.0, 129.2, 133.7, 134.7, 152.5, 160.1, 162.3, 169.7 ppm; 182–184 °C; 1H NMR (500 MHz, CDCl3 + DMSO-d6); δ 3.77
IR (KBr) (νmax/cm−1): ν = 3292, 3060, 1685, 1650, 1584, 1524, (s, 6H, –OCH3), 6.07 (s, 2H, –OCH2O–), 6.73–7.02 (m, 4H, ArH),
1473, 1444, 1363, 1315, 1296, 1145, 1064, 996 cm−1; MS (ESI) 7.25–7.42 (m, 2H, ArH), 7.34 (d, 1H, transH J = 14.8 Hz), 7.55
m/z 411 [M + H]; HR-MS (ESI) m/z for C22H23O6N2 calculated (d, 1H, transH J = 15.8 Hz), 7.76–7.91 (m, 1H, ArH), 10.1
m/z: 411.0953, found m/z: 411.0950.
(brs, 1H, –NH); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 53.4,
(E)-N-(3,4-Dimethoxyphenyl)-3-(3-(4-methoxyphenyl)isoxazol- 94.2, 95.3, 96.3, 100.0, 104.1, 107.1, 118.6, 119.0, 125.7, 127.9,
5-yl)acrylamide 21j. This compound was prepared by the 138.8, 146.4, 147.6, 158.7, 158.9, 158.8, 159.4, 160.9,
addition of 20c (245 mg 1.0 mmol) and 3,4-dimethoxyaniline 168.0 ppm; IR (KBr) (νmax/cm−1): ν = 3326, 3112, 2940, 1670,
14b (153 mg 1.0 mmol). Yellow solid, yield: 296 mg (78%); mp: 1646, 1587, 1541, 1467, 1414, 1309, 1284, 1256, 1145, 1048,
196–198 °C; 1H NMR (500 MHz, CDCl3 + DMSO-d6); δ 3.78 908 cm−1; MS (ESI) m/z 395 [M + H]; HR-MS (ESI) m/z for
(s, 6H, –OCH3), 3.87 (s, 3H, –OCH3), 6.21 (s, 1H, ArH), C21H19O6N2 calculated m/z: 395.1246, found m/z: 395.1245.
6.90–7.09 (m, 4H, ArH), 6.64 (d, 1H, J = 16.0 Hz, transH),
(E)-3-(3-(Benzo[d][1,3]dioxol-5-yl)isoxazol-5-yl)-N-(3,4-difluoro-
7.46–7.58 (m, 1H, ArH), 7.66 (d, 1H, J = 16.0 Hz, transH), phenyl)acrylamide 21n. This compound was prepared by the
7.74–7.82 (m, 1H, ArH), 7.86–7.99 (m, 1H, ArH), 10.27 (brs, 1H, addition of 20d (258 mg 1.0 mmol) and 3,4-difluoroaniline
–NH); 13C NMR (75 MHz, CDCl3 + DMSO-d6): δ 54.7, 54.9, 95.7, 14d (130 mg 1.0 mmol). Pale yellow solid, yield: 275 mg (75%);
95.8, 96.0, 97.7, 109.3, 114.1, 118.4, 119.1, 124.1, 126.5, 126.4, mp: 196–198 °C; 1H NMR (300 MHz, CDCl3 + DMSO-d6); δ 6.08
127.0, 127.3, 130.7, 140.1, 159.9, 160.3, 160.8, 162.5, 166.5, (s, 2H, –OCH2O–), 6.75 (s, 4H, ArH), 7.93 (d, 1H, J = 15.8 Hz,
169.7 ppm; IR (KBr) (νmax/cm−1): ν = 3290, 3033, 3010, 1682, transH), 6.94 (s, 1H, ArH), 7.05–7.20 (m, 1H, ArH), 7.32–7.45
1615, 1574, 1526, 1419, 1376, 1320, 1026, 993 cm−1; MS (ESI) (m, 2H, ArH), 7.61 (d, 1H, J = 15.6 Hz, transH), 7.64 (s, 1H,
m/z 381 [M + H]; HR-MS (ESI) m/z for C21H21O5N2 calculated ArH), 7.81–7.94 (m, 1H, ArH), 10.1 (brs, 1H, –NH); 13C NMR
m/z: 381.1445, found m/z: 381.1454.
(75 MHz, CDCl3 + DMSO-d6): δ 95.3, 100.0, 104.1, 107.1, 113.9,
(E)-N-(3,4-Difluorophenyl)-3-(3-(4-methoxyphenyl)isoxazol- 117.0, 118.7, 119.0, 126.1, 127.4, 134.0, 146.4, 147.6, 158.6,
5-yl)acrylamide 21k. This compound was prepared by the 161.0, 168.1 ppm; IR (KBr) (νmax/cm−1): ν = 3315, 3097, 3014,
addition of 20c (245 mg 1.0 mmol) and 3,4-difluoroaniline 14d 1680, 1656, 1593, 1533, 1476, 1440, 1339, 1308, 1274, 1215,
(130 mg 1.0 mmol). Pale yellow solid, yield: 265 mg (74%); mp: 1145, 1026, 924 cm−1; MS (ESI) m/z 371 [M + H]; HR-MS (ESI)
186–188 °C; 1H NMR (300 MHz, CDCl3 + DMSO-d6); δ 3.88 m/z for C19H13O4F2N2 calculated m/z: 371.0837, found m/z:
(s, 3H, –OCH3), 6.84 (s, 1H, ArH), 6.98 (d, 1H, transH J = 371.0846.
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polymerization assay kit (BK011, Cytoskeleton, Inc.) according
to the manufacturer’s protocol. The reaction mixture in a final
Cell cultures, maintenance and antiproliferative evaluation. volume of 10 µl in PEM buffer (80 mM PIPES, 0.5 mM EGTA,
Cell lines used in this study were purchased from the Ameri- 2 mM MgCl2, pH 6.9) in 384 well plates contained 2 mg per
can Type Culture Collection (ATCC, United States). A549, mL of bovine brain tubulin, 10 µM fluorescent reporter, 1 mM
MDA-MB231, and HeLa cells were grown in Dulbecco’s modi- GTP in the presence or absence of test compounds at 37 °C.
fied Eagle’s medium (containing 10% FBS under a humidified Tubulin polymerization was followed by monitoring the fluo-
atmosphere of 5% CO2 at 37 °C). DU145 cells were cultured in rescence enhancement due to the incorporation of a fluo-
Eagle’s minimal essential medium (MEM) containing non- rescence reporter into microtubules as polymerization
essential amino acids, 1 mM sodium pyruvate, 10 mg per mL proceeds. Fluorescence emission at 420 nm (excitation wave-
bovine insulin, and 10% FBS. Cells were trypsinized when sub- length is 360 nm) was measured for 1 h at 1 min intervals in a
confluent from T25 flasks/60 mm dishes and seeded in 96-well multimode plate reader (Tecan M200). To determine the IC50
plates. The synthesized test compounds were evaluated for values of the compounds against tubulin polymerization, the
their in vitro antiproliferative effects in four different human compounds were pre-incubated with tubulin at varying con-
cancer cell lines. A protocol of 48 h continuous drug exposure centrations (1, 5, 10 and 20 μM). Assays were performed under
was used, and an MTT cell proliferation assay was used to esti- similar conditions to those employed for polymerization
mate cell viability or growth. The cell lines were grown in their assays as described above.15
respective media containing 10% fetal bovine serum and were
Western blot analysis of soluble versus polymerized tubulin
seeded into 96-well microtiter plates in 200 μL aliquots at and cyclin B1. Cells were seeded in 12-well plates at 1 × 105
plating densities depending on the doubling time of indivi- cells per well in complete growth medium. Following treat-
dual cell lines. The microtiter plates were incubated at 37 °C, ment of cells with respective compounds (15a, 15b and 15e)
5% CO2, 95% air, and 100% relative humidity for 24 h prior to for a duration of 24 h, cells were washed with PBS and sub-
the addition of experimental drugs. Aliquots of 2 μL of the test sequently soluble and insoluble tubulin fractions were col-
compounds were added to the wells already containing 198 μL lected. To collect the soluble tubulin fractions, cells were
of cells, resulting in the required final drug concentration. For permeabilized with 200 µL of pre-warmed lysis buffer [80 mM
each compound, four concentrations (1, 10, 100, and 1000 μM) Pipes-KOH (pH 6.8), 1 mM MgCl2, 1 mM EGTA, 0.2% Triton
were evaluated, and each evaluation was done in triplicate X-100, 10% glycerol, 0.1% protease inhibitor cocktail (Sigma-
wells. Plates were incubated further for 48 h, and the assay was Aldrich)] and incubated for 3 min at 30 °C. Lysis buffer was
terminated by the addition of 10 μL of 5% MTT and incubated gently removed, and mixed with 100 µL of 3 × Laemmli’s
for 60 min at 37 °C. Later, the plates were air-dried. The bound sample buffer (180 mM Tris-Cl pH 6.8, 6% SDS, 15% glycerol,
stain was subsequently eluted with 100 μL of DMSO, and the 7.5% β-mercaptoethanol and 0.01% bromophenol blue).
absorbance was read on a multimode plate reader (Tecan Samples were immediately heated to 95 °C for 3 min. To
M200) at a wavelength of 560 nm. Percent growth was calcu- collect the insoluble tubulin fraction, 300 μL of 1 × Laemmli’s
lated on a plate by plate basis for test wells relative to control sample buffer was added to the remaining cells in each well,
wells. The above procedure was repeated thrice. The growth and the samples were heated to 95 °C for 3 min. Equal
inhibitory effects of the compounds were analyzed by generat- volumes of samples were run on an SDS-10% polyacrylamide
ing dose response curves as a plot of the percentage surviving gel and were transferred to a nitrocellulose membrane employ-
cells versus compound concentration. The sensitivity of the ing semidry transfer at 50 mA for 1 h. Blots were probed with
cancer cells to the test compound was expressed in terms of mouse anti-human α-tubulin diluted 1 : 2000 ml (Sigma) and
IC50, a value defined as the concentration of a compound that stained with a rabbit anti-mouse secondary antibody coupled
produced 50% reduction as compared to the control absor- with horseradish peroxidase, diluted 1 : 5000 ml (Sigma).
bance. IC50 values are indicated as mean SD of the three Bands were visualized using an enhanced chemiluminescence
independent experiments.15
protocol (Pierce) and radiographic film (Kodak). For cyclin B1
Analysis of cell cycle. HeLa cells in 60 mm dishes were incu- immunoblots, cells were seeded in 12-well plates at 1 × 105
bated for 24 h in the presence or absence of test compounds cells per well in complete medium and treated with different
15a, 15b and 15e at 2 μM concentration. Cells were harvested concentrations of 15a, 15b and 15e for 24 h. After treatment,
with Trypsin-EDTA, fixed with ice-cold 70% ethanol at 4 °C for cells were washed twice with phosphate-buffered saline and
30 min, ethanol was removed by centrifugation and cells were lysed in 1× SDS sample buffer. Proteins were separated, trans-
stained with 1 mL of DNA staining solution [0.2 mg of Propi- ferred, probed and analyzed similar to tubulin. The primary
dium Iodide (PI) and 2 mg RNase A] for 30 min as described anti-cyclin B1 antibody was employed at 1 : 1500 (Sigma) and
earlier. The DNA contents of 20 000 events were measured by a horseradish peroxidase coupled goat anti-rabbit secondary
flow cytometer (BD FACSCanto II). Histograms were analyzed antibody diluted 1 : 5000 (Sigma).24
using FCS express 4 plus.15
RT-PCR analysis. Total RNA was isolated using the TRIzol
Tubulin polymerization assay. An in vitro assay for monitor- reagent (Invitrogen). Semiquantitative RT-PCR was carried
ing the time-dependent polymerization of tubulin to micro- out essentially as described previously.27 RNA was reverse tran-
tubules was performed employing a fluorescence-based tubulin scribed using reagents from the first-strand cDNA synthesis
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Organic & Biomolecular Chemistry
kit (Fermentas). Primers p21F (5′-GCACCCTAGTTCTACCTCAG- picked based on the best stabilization energy. The final figures
GCAGCTC-3′) and p21R (5′-GACACAGAACAGTACAGGGTGTG- for molecular modeling were generated by using PyMol.37
GTCC-3′) were used for the amplification of human
p21 mRNA. Primers GAPDHF (5′-GCCAACGTGTCAGTGGT-
GGACCTG-3′) and GAPDHR (5′-CAGCAGTGAGGGTCTCTCT-
CTTCC-3′) were used for the amplification of human GAPDH.
Acknowledgements
The PCR conditions for p21 and GAPDH were: 1 cycle of 3 min
A. B. S. thanks CSIR, New Delhi and I. K. thanks the UGC, New
at 95 °C; 37 cycles of 1 min at 95 °C, 1 min at 60 °C and 1 min
Delhi for the award of senior research fellowship. We thank
at 72 °C; and 1 cycle of 7 min at 72 °C for 22 cycles.
CSIR for financial support under the 12th Five Year plan
Immunohistochemistry of tubulin and analysis of nuclear
project “Affordable Cancer Therapeutics” (CSC0301). This
morphology. HeLa cells were seeded on a glass cover slip,
project was also supported by King Saud University, Deanship
incubated for 24 h in the presence or absence of test com-
of Scientific Research, Research Chair.
pounds 15a, 15b and 15e at a concentration of 2 μM. Cells
grown on coverslips were fixed in 3.5% formaldehyde in phos-
phate-buffered saline (PBS) pH 7.4 for 10 minutes at room
temperature. Cells were permeabilized for 6 minutes in PBS
containing 0.5% Triton X-100 (Sigma) and 0.05% Tween-20
Notes and references
(Sigma). The permeabilized cells were blocked with 2% BSA
(Sigma) in PBS for 1 h. Later, the cells were incubated with the
primary antibody for tubulin from Sigma at 1 : 200 diluted in
blocking solution for 4 h at room temperature. Subsequently
the antibodies were removed and the cells were washed thrice
with PBS. Cells were then incubated with the FITC labeled
anti-mouse secondary antibody (1 : 500) for 1 h at room temp-
erature. Cells were washed thrice with PBS and mounted in
medium containing DAPI. Images were captured using an
Olympus confocal microscope and analyzed with Provision
software.
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ligand complex structure were derived from the crystal struc-
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pared for molecular docking simulation by removing water
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