Copper triflate-mediated synthesis of 1,3,5-triarylpyrazoles in [bmim][PF6] ionic liquid and evaluation of their anticancer activities

Article abstract of DOI:10.1039/c3ra41830h

A simple, efficient, and environment friendly protocol for the synthesis of 1,3,5-triarylpyrazoles and 1,3,5-triarylpyrazolines in [bmim][PF₆] ionic liquid mediated by Cu(OTf)₂ is described. The reaction protocol gave 1,3,5-triarylpyrazoles in good to high yields (71-84%) via a one-pot addition-cyclocondensation between chalcones and arylhydrazines, and oxidative aromatization without the requirement for an additional oxidizing reagent. The catalyst can be reused for up to four cycles without much loss in the catalytic activity. The pyrazoles (4a-o) and pyrazolines (3a-n) were evaluated for their antiproliferative activity in SK-OV-3, HT-29, and HeLa human cancer cells lines. Among all the compounds, 3b inhibited cell proliferation of HeLa cells by 80% at a concentration of 50 μM. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra41830h

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Copper triflate-mediated synthesis of 1,3,5-
triarylpyrazoles in [bmim][PF6] ionic liquid and
Cite this: DOI: 10.1039/c3ra41830h
evaluation of their anticancer activities3
V. Kameshwara Rao,a Rakesh Tiwari,b Bhupender S. Chhikara,b Amir
Nasrolahi Shirazi,b Keykavous Parangb and Anil Kumar*a
A simple, efficient, and environment friendly protocol for the synthesis of 1,3,5-triarylpyrazoles and 1,3,5-
triarylpyrazolines in [bmim][PF6] ionic liquid mediated by Cu(OTf)2 is described. The reaction protocol gave
1,3,5-triarylpyrazoles in good to high yields (71–84%) via a one-pot addition–cyclocondensation between
chalcones and arylhydrazines, and oxidative aromatization without the requirement for an additional
oxidizing reagent. The catalyst can be reused for up to four cycles without much loss in the catalytic
activity. The pyrazoles (4a–o) and pyrazolines (3a–n) were evaluated for their antiproliferative activity in
SK-OV-3, HT-29, and HeLa human cancer cells lines. Among all the compounds, 3b inhibited cell
proliferation of HeLa cells by 80% at a concentration of 50 mM.
Received 16th April 2013,
Accepted 13th June 2013
DOI: 10.1039/c3ra41830h
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results in the formation of 4,5-dihydro-1H-pyrazoles (pyrazo-
lines) that need to be further oxidized to their corresponding
Introduction
Pyrazoles and their derivatives are well recognized as an
important class of heterocyclic compounds that have found
extensive use in the pharmaceutical, material, and agrochem-
ical industries.1 Compounds containing the pyrazole moiety
have exhibited diverse biological activities. For example,
4-substituted 1,5-diaryl-1H-pyrazole-3-carboxylate derivatives
can act as cannabinoid-1 (CB1) receptor antagonists,2–7 Ikb
kinase b (IKKb or IKK-2) inhibitors,8 and anti-inflammatory
agents.9 Pyrazole derivatives have been shown to have a good
binding affinity towards estrogen receptors.10–12 Some of the
pyrazole derivatives have been reported to possess anti-
depressant, anti-convulsant,13 anti-inflammatory, and anti-
arthritic14 activities. Pyrazole scaffolds constitute the basic
framework of several drug molecules such as celecoxib (a non-
steroidal anti-inflammatory drug)14 and rimonabant (an
anorectic antiobesity drug) (Fig. 1).
pyrazoles. For this oxidative aromatization of pyrazolines to
pyrazoles, various reagents have been employed such as I2,17
Bi(NO3)3?5H2O,18
MnO2,19
DDQ,20
Pd/C,21
NaOEt,22
PhI(OAc)2,23 TBBDA,24 and ionic liquids.25,26 However, many
of these oxidative methods suffer from relatively high oxidant
loading, the use of strong oxidants and chlorinated organic
solvents, harsh conditions, poor yields, and a longer reaction
time. Thus, the development of an environmentally benign
process with the use of alternative solvents such as ionic
liquids in place of organic solvents, and a catalytic amount of
ecofriendly catalyst to avoid harsh oxidizing reagents, is highly
desirable.
As part of our ongoing work on the development of novel
reaction methodologies using metal triflates,27–29 and evalua-
tion of small molecules as anticancer agents,29–32 herein we
report a copper triflate-mediated protocol for the synthesis of
pyrazoles by the reaction of hydrazines with a,b-unsaturated
Pyrazoles have received considerable attention from che-
mists because of their diverse bioactivities. Thus, a number of
synthetic strategies have been developed for their synth-
esis.15,16 The most common approach for the synthesis of
substituted pyrazoles is the condensation of a,b-unsaturated
carbonyl compounds with hydrazines. However, this strategy
ketones in
a 1-butyl-3-methylimidazolium hexafluoropho-
aDepartment of Chemistry, Birla Institute of Technology and Science, Pilani 333031,
Rajasthan, India. E-mail: anilkumar@pilani.bits-pilani.ac.in
bDepartment of Biomedical and Pharmaceutical Sciences, College of Pharmacy,
University of Rhode Island, Kingston, RI, 02881, USA
Electronic supplementary information (ESI) available: Copies of 1H and 13C
3
Fig. 1 Chemical structure of drug molecules, celecoxib and rimonabant
NMR for 4a–o and 3a–n can be found in the online version of this article. See
containing a pyrazole scaffold.
DOI: 10.1039/c3ra41830h
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entry 11 and 12). There was not much increase in the yield of
4a on changing the amount of Cu(OTf)2 from 20 mol% to 30
mol%. However, reducing the amount of Cu(OTf)2 to 10 mol%
decreased the yield of 4a to 64% along with the formation of
15% 3a (Table 1, entries 1–3). These data indicate that
Cu(OTf)2 was involved in the aerobic oxidation of 3a to 4a. It
is necessary to mention that 4a was not formed in the absence
of Cu(OTf)2 in [bmim][PF6] ionic liquids, and only 3a was
isolated in 20% yield along with the starting material; the yield
of 3a did not increase with increasing the time up to 2 h.
Scheme 1 Synthesis of substituted 1,3,5-triarylpyrazoles.
1
The structure of 4a was confirmed by H NMR, 13C NMR,
and high-resolution mass spectrometry. In the 1H NMR
spectrum, a singlet was observed at d 6.81 for the proton at
the C4-position of the pyrazole ring along with other protons
on aryl substituents. In the 13C NMR, a peak appeared at d
104.97 for the C4-carbon of the pyrazole ring. The presence of a
peak at m/z 355.2173 for the [M + H]+ ion with molecular
sphate ([bmim][PF6]) ionic liquid (Scheme 1) and evaluation of
their anti-proliferative activity against different cancer cell
lines.
Results and discussion
+
formula C25H27N2 confirmed the structure of 4a.
In the standardization experiment, when 1,2-diphenylprop-2-
en-1-one (1) and 4-tert-butylphenylhydrazine hydrochloride (2)
were reacted in ethanol under reflux in the presence of
Cu(OTf)2 (20 mol%), 1-(4-tert-butylphenyl)-3,5-diphenyl-4,5-
dihydro-1H-pyrazole (3a) was obtained in 62% yield (Table 1,
entry 8). Further optimization of the reaction conditions was
carried out by changing solvents, catalysts, and catalyst
loading. As shown in Table 1, the use of 20 mol% Cu(OTf)2
in [bmim][PF6] gave the desired product 1-(4-tert-butylphenyl)-
3,5-diphenyl-1H-pyrazole (4a) in excellent yield (82%) (Table 1,
entry 2). When Cu(OTf)2 was replaced with other catalysts such
as pTSA, Sc(OTf)3, Ce(OTf)3, Zn(OTf)2, AgOTf, or Yb(OTf)3, a
mixture of 3a and 4a was observed. The use of Ce(OTf)3 in
[bmim][PF6] resulted in 75% yield of 3a along with 10% of 4a,
whereas use of pTSA in [bmim][PF6] gave 69% of 3a (Table 1,
To explore the synthetic scope and versatility of the
protocol, a series of arylhydrazines (2) were reacted with
different a,b-carbonyl compounds (1) under the optimal
reaction conditions. The results are summarized in Table 2.
Various functional groups, such as F, Cl, NO2, OCH3, CH3 and
–C(CH3)3 on arylhydrazines and chalcones were well tolerated
under these conditions, affording corresponding 1,3,5-sub-
stituted pyrazoles (4a–o) in good to high yields (71–84%).
By monitoring the model reaction between 1,2-diphenyl-
prop-2-en-1-one (1) and tert-butylphenylhydrazine hydrochlor-
ide (2) in the presence of 20 mol% Cu(OTf)2 in [bmim][PF6] at
different time intervals, it was found that in the first 30 min
pyrazoline (3a) was the major product, which was oxidized to
pyrazole in the reaction as time progressed. We thus decided
Table 2 Synthesized 1,3,5-triarylpyrazoles (4a–o)
Table 1 Optimization of reaction conditions for the synthesis of 4aa
Yield
Yield
Subst. no. Catalyst
Mol% Solvent
(3a) (%)b (4a) (%)b
1
2
3
4
5
6
7
8
Cu(OTf)2 10
Cu(OTf)2 20
[bmim][PF6] 15
64c
82de
84
50
21
15
33
—
[bmim][PF6]
[bmim][PF6]
—
—
Cu(OTf)2 30
Cu(OTf)2 20
Cu(OTf)2 20
Cu(OTf)2 20
Cu(OTf)2 20
Cu(OTf)2 20
Cu(OTf)2 20
Compd.
R1
R2
R3
Time (h)
Yield (%)a
[bmim][BF4] 35
[bmim][Br]
DMSO
DMF
Ethanol
PEG
50
—
—
62f
60
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
H
H
H
H
4-OMe
4-OMe
3-OMe
4-CH3
4-CH3
4-CH3
2-F
H
H
H
H
H
H
4-CH3
4-CH3
4-CH3
4-CH3
4-Cl
4-Cl
4-OMe
4-OMe
H
4-C(CH3)3
2-CH3
3,4-Cl
3-Cl, 4-CH3
3,4-Cl
3-Cl, 4-CH3
3,4-Cl
2
2
3
2.5
2
2
2
2
2
1.5
2
2
1
1
82b
81
80
72
71
77
79
74
77
77
84
82
75
81
78
9
—
10
11
12
13
14
15
Sc(OTf)3
Ce(OTf)3 20
pTSA 20
Zn(OTf)2 20
AgOTf 20
Yb(OTf)3 20
20
[bmim][PF6] 61
[bmim][PF6] 75
[bmim][PF6] 69
[bmim][PF6] 55
[bmim][PF6] 15
[bmim][PF6] 58
19
10
—
30
65
22
3,4-Cl
3-Cl, 4-CH3
4-C(CH3)3
4-C(CH3)3
2-CH3
4-C(CH3)3
3-Cl, 4-CH3
4-OMe
a
Reaction conditions: chalcone (1.0 mmol), arylhydrazine (1.2
2-F
b
mmol), catalyst (x mol%), solvent (2 mL), 130 uC, 2 h. Isolated
yield. Only 20% of 3a was formed after 30 min in the absence of
4-NO2
4-NO2
H
c
d
Cu(OTf)2 under similar conditions. At 100 uC, complete conversion
1.5
of 3a to 4a was not observed and it requires a longer reaction time.
e
f
a
b
When isolated 3a was used, 95% yield of 4a was obtained. Reflux
Isolated yield. In four consecutive recycling experiments 4a was
conditions.
observed in 82, 80, 78, and 79% yield, respectively.
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4a when isolated 3a was treated with Cu(OTf)2 under nitrogen
atmosphere. This further confirms that 3a is converted to 4a
via oxidation with atmospheric oxygen in the presence of
Cu(OTf)2. It appears that ionic liquid helps in the stabilization
of charged intermediates generated by the coordination of
Cu(OTf)2 to the carbonyl of chalcone and thereby increases the
electrophilicity of chalcone.
Table 3 Synthesized 1,3,5-triarylpyrazolines (3a–n)
Compd. R1
R2
R3
Time (Min.) Yield (%)a
Further, we investigated the possibility of recycling the
catalyst. After performing the first cycle, the product was
extracted with an ethyl acetate–hexane mixture, and Cu(OTf)2
in ionic liquid was properly dried under vacuum. The fresh
chalcone and 4-tert-butyl phenylhydrazine hydrochloride were
added to the recovered ionic liquid containing Cu(OTf)2 and
the reaction was carried out under the same conditions. The
above procedure was repeated four times to give 4a in high
yields (82, 80, 78, and 79%) without much loss of catalytic
activity (Table 2, footnote b).
To evaluate the anti-cancer activity of the synthesized
compounds, all derivatives (4a–o and 3a–n) were evaluated for
their inhibitory activity on the proliferation of human ovarian
adenocarcinoma (SK-OV-3), human colon adenocarcinoma
(HT-29), and human cervical adenocarcinoma (HeLa) cells.
Doxorubicin (Dox) and DMSO were used as positive and
negative controls, respectively. The antiproliferative activity
results of compounds 4a–o and 3a–n at 50 mM after 72 h
incubation are shown in Fig. 2 and 3, respectively. Fig. 2 shows
that among all the 1,3,5-triarylpyrazoles derivatives (4a–o), 4c,
4e, 4f, 4g, 4h, 4i, and 4k inhibit the proliferation of HeLa cells
by 50%, 55%, 45%, 39%, 54%, 42%, and 50%, respectively.
However, they did not exhibit significant inhibitory potency in
HT-29 and SK-OV-3 cells. 1,3,5-Triarylpyrazoline derivatives
(3a–n) showed high to weak antiproliferative activity against
HeLa cells after 72 h incubation. Compounds 3c, 3d, 3e, 3k, 3l,
and 3m inhibited the proliferation of HeLa cells by 62%, 50%,
35%, 58%, 23%, and 40%, respectively. The 2-methylsubsti-
tuted compound 3b showed the highest potency with 80%
inhibition of HeLa cells. They showed modest to weak potency
in SK-OV-3 and HT-29 cells. Among all derivatives, compound
3b showed comparable potency to doxorubicin (10 mmol) in
HeLa cells. Further modification on the chemical structure of
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
H
H
H
H
H
H
H
H
H
H
4-C(CH3)3
2-CH3
3,4-Cl
3-Cl, 4-CH3 30
3,4-Cl 30
3-Cl, 4-CH3 30
3,4-Cl
3,4-Cl
30
30
30
84
66
77
68
72
78
60
72
74
79
63
65
72
80
4-OMe
4-OMe
3-OMe 4-CH3
4-CH3
4-CH3
4-CH3
2-F
2-F
4-NO2
4-NO2
30
30
4-CH3
4-CH3
4-CH3
4-Cl
3-Cl, 4-CH3 30
4-C(CH3)3
4-C(CH3)3
2-CH3
30
30
30
20
4-Cl
4-OMe 4-C(CH3)3
4-OMe 3-Cl, 4-CH3 20
a
Isolated yield.
to synthesize the pyrazolines using this protocol in order to
evaluate them in our biological assay. The reaction of 1 and 2
afforded 1,3,5-triarylpyrazolines (3a–o) via a one-pot addition–
cyclocondensation process in good to high yields (60–84%).
Several a,b-unsaturated carbonyl compounds with both
electron-rich and electron-deficient arenes were successfully
applied to this reaction. The results of pyrazoline synthesis are
summarized in Table 3. The chemical structures of all the
synthesized compounds were elucidated by H NMR and 13C
NMR spectroscopic data (ESI ).
1
3
Based on the intermediate formed as pyrazoline 3a and
structure of the product 4a, the reaction is proposed to
proceed through the sequential steps as shown in Scheme 2.
The first step is believed to be the 1,2-addition of hydrazine to
chalcone mediated by Cu(OTf)2. 3-Hydroxypyrazoline (C)
undergoes elimination in the presence of Cu(OTf)2 to give
the 1,3,5-triarylpyrazoline derivative (3). Oxidative aromatiza-
tion of 3 in the presence of Cu(OTf)2 yields the corresponding
1,3,5-triarylpyrazole (4). We did not observe the formation of
Scheme 2 Proposed mechanism for the synthesis of 1,3,5-triarylpyrazole.
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Fig. 2 Antiproliferative activity of 4a–o.
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125.71, 124.81, 113.14, 65.06, 43.70, 33.96, 31.48; HRMS (ESI)
calcd for C25H27N2 355.2169, found 355.2176 [M + H]+.
+
4,5-Dihydro-3,5-diphenyl-1-o-tolyl-1H-pyrazole (3b). Brown
1
liquid; H NMR (300 MHz, CDCl3) d 7.70 (d, J = 7.3 Hz, 2H),
7.41–7.30 (m, 5H), 7.29–7.19 (m, 3H), 7.12 (d, J = 7.4 Hz, 1H),
7.05–6.85 (m, 3H), 5.26 (t, J = 10.8 Hz, 1H), 3.69 (dd, J = 16.5,
11.1 Hz, 1H), 3.19 (dd, J = 16.4, 10.5 Hz, 1H), 2.43 (s, 3H); 13C
NMR (75 MHz, CDCl3) d 148.13, 144.28, 141.00, 132.99, 131.47,
131.25, 128.69, 128.55, 127.66, 126.93, 126.01, 125.65, 123.28,
119.27, 67.66, 42.67, 20.46; HRMS (ESI) calcd for C22H20N2Na+
335.1519, found 335.1532 [M + Na]+.
1-(3,4-Dichlorophenyl)-4,5-dihydro-3,5-diphenyl-1H-pyrazole
(3c). Pale yellow solid, m.p. 133–134 uC; 1H NMR (300 MHz,
CDCl3) d 7.71 (d, J = 7.4 Hz, 2H), 7.42–7.31 (m, 5H), 7.30–7.23
(m, 4H), 7.14 (d, J = 8.9 Hz, 1H), 6.73 (dd, J = 8.9, 2.5 Hz, 1H),
5.22 (dd, J = 12.2, 6.7 Hz, 1H), 3.85 (dd, J = 17.3, 12.3 Hz, 1H),
3.16 (dd, J = 17.3, 6.7 Hz, 1H); 13C NMR (75 MHz, CDCl3) d
148.22, 144.02, 141.52, 132.65, 132.15, 130.30, 129.35, 129.15,
128.65, 127.98, 125.95, 125.75, 121.59, 114.94, 112.47, 64.18,
Fig. 3 Antiproliferative activity of 3a–n.
this derivative could lead to the synthesis of a promising
candidate that selectively targets HeLa cells.
+
43.78; HRMS (ESI) calcd for C21H17Cl2N2 367.0763, found
367.0782 [M + H]+.
Experimental
1-(3-Chloro-4-methylphenyl)-4,5-dihydro-3,5-diphenyl-
1
1H-pyrazole (3d). Pale yellow solid, m.p. 121–122 uC; H NMR
General
(300 MHz, CDCl3) d 7.71 (d, J = 7.2 Hz, 2H), 7.41–7.27 (m, 8H),
7.21 (d, J = 2.1 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.71 (dd, J = 8.3,
2.2 Hz, 1H), 5.20 (dd, J = 12.3, 7.2 Hz, 1H), 3.82 (dd, J = 17.1,
12.3 Hz, 1H), 3.12 (dd, J = 17.1, 7.2 Hz, 1H), 2.23 (s, 3H); 13C
NMR (75 MHz, CDCl3) d 147.15, 143.90, 142.17, 134.68, 132.52,
130.92, 129.22, 128.77, 128.58, 127.73, 125.93, 125.85, 125.81,
114.08, 111.56, 64.51, 43.65, 19.03; HRMS (ESI) calcd for
All chemicals were obtained from commercial suppliers and
used without further purification. Melting points were
determined in open capillary tubes on a MPA120-Automated
Melting Point apparatus and are uncorrected. NMR spectra
were recorded on a Bruker (300 MHz) NMR spectrometer using
CDCl3 as the solvent and the chemical shifts were expressed in
ppm. Metal triflates were purchased from Sigma-Aldrich and
used as received. All other reagents and solvents were
purchased from Merck (India), Spectrochem Chemicals, S. D.
Fine Chemicals, India and used without further purification
unless otherwise specified. Reactions were monitored by using
thin layer chromatography (TLC) on 0.2 mm silica gel F254
plates (Merck). The a,b-unsaturated ketones (chalcones) 1 were
prepared by the treatment of an appropriate acetophenone
with benzaldehydes in the presence of sodium hydroxide, as
reported in literature.33
+
C22H20ClN2 347.1310, found 347.1321 [M + H]+.
1-(3,4-Dichlorophenyl)-4,5-dihydro-5-(4-methoxyphenyl)-3-
phenyl-1H-pyrazole (3e). Yellow solid, m.p. 133–134 uC; 1H
NMR (300 MHz, CDCl3) d 7.71 (d, J = 6.5 Hz, 2H), 7.38 (d, J = 7.5
Hz, 3H), 7.29–7.24 (m, 1H), 7.16 (t, J = 8.6 Hz, 3H), 6.85 (d, J =
8.6 Hz, 2H), 6.75 (dd, J = 8.7, 2.7 Hz, 1H), 5.18 (dd, J = 12.1, 6.7
Hz, 1H), 3.83 (dd, J = 9.9, 7.3 Hz, 1H), 3.77 (s, 3H), 3.13 (dd, J =
17.3, 6.7 Hz, 1H); 13C NMR (75 MHz, CDCl3) d 159.23, 148.23,
144.06, 133.53, 132.60, 132.23, 130.26, 129.10, 128.64, 126.96,
125.92, 121.51, 114.95, 114.67, 112.54, 63.72, 55.29, 43.81,
HRMS (ESI) calcd for C22H19Cl2N2O+ 397.0869, found 397.0874
[M + H]+.
Experimental procedure for the synthesis of 3 and 4
Chalcone (1.0 mmol), arylhydrazine hydrochloride (1.2 mmol)
and Cu(OTf)2 (0.2 mmol, 20 mol%) were added to a 10 mL
round bottom flask containing [bmim][PF6] ionic liquid (2
mL). The reaction mixture was heated at 130 uC with stirring
for 30 min (for 3) or 1–2.5 h (for 4). After completion of the
reaction as indicated by TLC, the reaction mixture was
extracted with an ethyl acetate–hexane mixture and the solvent
was removed under vacuum. The crude compound was
purified by passing it through a bed of silica gel (100–200
mesh) to give pure 3 or 4.
1-(4-tert-Butylphenyl)-4,5-dihydro-3,5-diphenyl-1H-pyrazole
(3a). Pale green solid, m.p. 148–150 uC; 1H NMR (300 MHz,
CDCl3) d 7.71 (d, J = 7.2 Hz, 2H), 7.39–7.29 (m, 8H), 7.20 (d, J =
8.7 Hz, 2H), 7.01 (d, J = 8.6 Hz, 2H), 5.19 (dd, J = 12.2, 8.0 Hz,
1H), 3.80 (dd, J = 17.0, 12.4 Hz, 1H), 3.11 (dd, J = 17.0, 8.0 Hz,
1H), 1.25 (s, 9H); 13C NMR (75 MHz, CDCl3) d 146.30, 142.97,
142.90, 141.92, 132.88, 129.14, 128.53, 128.46, 127.54, 126.00,
1-(3-Chloro-4-methylphenyl)-4,5-dihydro-5-(4-methoxyphe-
nyl)-3-phenyl-1H-pyrazole (3f). Off white solid, m.p. 120–121
1
uC; H NMR (300 MHz, CDCl3) d 7.71 (d, J = 7.1 Hz, 2H), 7.37
(dd, J = 12.8, 5.3 Hz, 3H), 7.23–7.17 (m, 3H), 6.96 (d, J = 8.3 Hz,
1H), 6.85 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 6.2 Hz, 1H), 5.16 (dd, J
= 12.1, 7.1 Hz, 1H), 3.82 (d, J = 4.0 Hz, 1H), 3.76 (s, 3H), 3.10
(dd, J = 17.1, 7.1 Hz, 1H), 2.23 (s, 3H); 13C NMR (75 MHz,
CDCl3) d 159.07, 147.17, 143.94, 134.63, 134.21, 132.60, 130.90,
130.05, 128.73, 128.57, 127.05, 125.86, 125.79, 114.55, 114.09,
111.63, 64.04, 55.27, 43.68, 19.03; HRMS (ESI) calcd for
C23H22ClN2O+ 377.1415, found 377.1427 [M + H]+.
1-(3,4-Dichlorophenyl)-4,5-dihydro-5-(3-methoxyphenyl)-3-
1
p-tolyl-1H-pyrazole (3g). Off white solid, m.p. 184–185 uC; H
NMR (300 MHz, CDCl3) d 7.59 (d, J = 8.1 Hz, 2H), 7.30–7.10 (m,
5H), 6.89–6.75 (m, 3H), 6.73 (dd, J = 8.9, 2.6 Hz, 1H), 5.13 (dd, J
= 12.2, 6.9 Hz, 1H), 3.89–3.78 (m, 1H), 3.75 (s, 3H), 3.13 (dd, J =
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17.2, 6.9 Hz, 1H), 2.37 (s, 3H); 13C NMR (75 MHz, CDCl3) d
160.36, 148.48, 144.25, 143.36, 139.34, 132.59, 130.44, 130.26,
129.35, 125.93, 121.42, 117.96, 114.88, 113.09, 112.42, 111.36,
64.14, 55.26, 43.85, 21.44; HRMS (ESI) calcd for C23H21Cl2N2O+
411.1025, found 411.1043 [M + H]+.
41.0, 20.42; HRMS (ESI) calcd for C22H19ClFN2 365.1215,
+
found 365.1208 [M + H]+.
1-(4-tert-Butylphenyl)-4,5-dihydro-3-(4-methoxyphenyl)-5-(4-
nitrophenyl)-1H-pyrazole (3m). Dark red solid, m.p. 124–125
uC; H NMR (300 MHz, CDCl3) d 8.20 (d, J = 8.6 Hz, 2H), 7.64
1
1-(3,4-Dichlorophenyl)-4,5-dihydro-3,5-di-p-tolyl-1H-pyrazole
(3h). Off white solid, m.p. 115–116 uC; 1H NMR (300 MHz,
CDCl3) d 7.60 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 2.6 Hz, 1H), 7.21–
7.16 (m, 2H), 7.13–7.11 (m, 4H), 6.73 (dd, J = 8.9, 2.5 Hz, 1H),
5.16 (dd, J = 12.2, 6.7 Hz, 1H), 3.80 (dd, J = 17.2, 12.2 Hz, 1H),
3.11 (dd, J = 17.2, 6.7 Hz, 1H), 2.37 (s, 3H), 2.31 (s, 3H); 13C
NMR (75 MHz, CDCl3) d 148.41, 144.20, 139.27, 138.67, 137.64,
132.57, 130.23, 129.96, 129.44, 129.34, 125.90, 125.69, 121.27,
114.84, 112.41, 63.90, 43.91, 21.43, 21.11; HRMS (ESI) calcd for
(d, J = 8.7 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 7.21 (d, J = 8.7 Hz,
2H), 6.92 (dd, J = 8.7, 3.7 Hz, 4H), 5.25 (dd, J = 12.2, 8.0 Hz, 1H),
3.90–3.79 (m, 1H), 3.83 (s, 3H), 3.06 (dd, J = 17.0, 7.9 Hz, 1H),
1.25 (s, 9H); 13C NMR (75 MHz, CDCl3) d 160.33, 150.33,
147.43, 146.46, 142.70, 142.39, 127.27, 127.04, 125.87, 125.04,
124.51, 114.09, 113.08, 64.35, 55.36, 43.59, 33.98, 31.44; HRMS
(ESI) calcd for C26H28N3O3+ 430.2125, found 430.2136 [M + H]+.
1-(3-Chloro-4-methylphenyl)-4,5-dihydro-3-(4-methoxyphe-
nyl)-5-(4-nitrophenyl)-1H-pyrazole (3n). Dark red solid, m.p.
+
C23H21Cl2N2 395.1076, found 395.1102 [M + H]+.
1
120–122 uC; H NMR (400 MHz, CDCl3) d 8.19 (d, J = 6.7 Hz,
1-(3-Chloro-4-methylphenyl)-4,5-dihydro-3,5-di-p-tolyl-
2H), 7.64 (d, J = 6.9 Hz, 2H), 7.46 (d, J = 6.9 Hz, 2H), 7.12 (s,
1H), 6.94 (dd, J = 22.2, 7.2 Hz, 3H), 6.62 (d, J = 6.0 Hz, 1H), 5.26
(dd, J = 9.3, 6.0 Hz, 1H), 4.07–3.87 (s, 1H), 3.83 (s, 3H), 3.07 (dd,
J = 15.8, 5.5 Hz, 1H), 2.23 (s, 3H); 13C NMR (101 MHz, CDCl3) d
160.56, 149.55, 147.52, 147.27, 143.74, 134.87, 131.09, 127.43,
126.93, 126.41, 124.66, 124.59, 114.15, 114.02, 111.43, 63.80,
1
1H-pyrazole (3i). Pale yellow solid, m.p. 135–136 uC; H NMR
(300 MHz, CDCl3) d 7.59 (d, J = 8.1 Hz, 2H), 7.20–7.18 (m, 2H),
7.17–7.14 (m, 3H), 7.13–7.09 (m, 2H), 6.94 (d, J = 8.4 Hz, 1H),
6.74–6.68 (m, 1H), 5.14 (dd, J = 12.1, 7.2 Hz, 1H), 3.77 (dd, J =
17.1, 12.2 Hz, 1H), 3.08 (dd, J = 17.1, 7.2 Hz, 1H), 2.36 (s, 3H),
2.31 (s, 3H), 2.22 (s, 3H); 13C NMR (75 MHz, CDCl3) d 147.37,
144.12, 139.33, 138.83, 137.33, 134.62, 130.88, 129.84, 129.81,
129.39, 129.28, 128.61, 125.79, 125.78, 125.64, 114.00, 111.51,
64.25, 43.81, 21.41, 21.11, 19.02; HRMS (ESI) calcd for
+
55.38, 43.58, 19.02; HRMS (ESI) calcd for C23H20ClN3NaO3
444.1085, found 444.1108 [M + Na]+.
1-(4-tert-Butylphenyl)-3,5-diphenyl-1H-pyrazole (4a). Off
white solid, m.p. 149–150 uC; 1H NMR (400 MHz, CDCl3) d
7.92 (d, J = 7.3 Hz, 2H), 7.42 (t, J = 7.5 Hz, H), 7.37–7.25 (m,
10H), 6.81 (s, 1H), 1.31 (s, 9H); 13C NMR (101 MHz, CDCl3) d
151.74, 150.54, 144.30, 137.68, 133.17, 130.73, 128.76, 128.63,
128.46, 128.21, 127.92, 125.87, 125.80, 124.79, 104.97, 34.65,
+
C24H24ClN2 375.1623, found 375.1635 [M + H]+.
1-(4-tert-Butylphenyl)-4,5-dihydro-3,5-di-p-tolyl-1H-pyrazole
(3j). Pale green solid, m.p. 108–109 uC; 1H NMR (300 MHz,
CDCl3) d 7.59 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 11.0 Hz, 1H), 7.26–
7.20 (m, 3H), 7.19–7.13 (m, 4H), 7.01 (d, J = 8.7 Hz, 2H), 5.13
(dd, J = 12.2, 8.1 Hz, 1H), 3.76 (dd, J = 17.0, 12.3 Hz, 1H), 3.06
(dd, J = 17.0, 8.1 Hz, 1H), 2.36 (s, 3H), 2.32 (s, 3H), 1.25 (s, 9H);
13C NMR (75 MHz, CDCl3) d 146.54, 143.15, 141.66, 140.13,
138.46, 137.11, 129.76, 129.22, 128.60, 125.93, 125.66, 124.97,
113.07, 64.82, 43.87, 33.94, 31.48, 21.39, 21.12; HRMS (ESI)
+
31.35; HRMS (ESI) calcd for C25H27N2 355.2169, found
355.2173 [M + H]+.
3,5-Diphenyl-1-o-tolyl-1H-pyrazole (4b). Off white solid, m.p.
95–96 uC; 1H NMR (300 MHz, CDCl3) d 7.92 (d, J = 7.4 Hz, 2H),
7.42 (t, J = 7.5 Hz, 2H), 7.36–7.28 (m, 3H), 7.27–7.18 (m, 7H),
6.87 (s, 1H), 2.03 (s, 3H); 13C NMR (75 MHz, CDCl3) d 151.76,
145.48, 139.56, 135.71, 133.24, 131.11, 130.27, 129.01, 128.65,
128.46, 128.23, 128.14, 127.91, 127.88, 126.68, 125.80, 103.20,
calcd for C27H31N2 383.2482, found 383.2503 [M + H]+.
+
1-(4-tert-Butylphenyl)-3-(4-chlorophenyl)-5-(2-fluorophenyl)-
4,5-dihydro-1H-pyrazole (3k). Pale green solid, m.p. 81–82 uC;
1H NMR (300 MHz, CDCl3) d 7.63 (d, J = 8.5 Hz, 2H), 7.33 (d, J =
8.5 Hz, 2H), 7.23 (d, J = 9.1 Hz, 4H), 7.09 (dd, J = 20.6, 9.0 Hz,
2H), 6.99 (d, J = 8.7 Hz, 2H), 5.52 (dd, J = 12.4, 7.5 Hz, 1H), 3.83
(dd, J = 17.1, 12.4 Hz, 1H), 3.05 (dd, J = 17.1, 7.5 Hz, 1H), 1.26
(s, 9H); 13C NMR (75 MHz, CDCl3) d 161.34 (d, JC,F = 248.25
Hz), 145.64, 142.36, 134.25, 131.26, 129.22, 128.75, 127.64 (d,
+
17.72; HRMS (ESI) calcd for C22H19N2 311.1543, found 311.
1536 [M + H]+.
1-(3,4-Dichlorophenyl)-3,5-diphenyl-1H-pyrazole (4c). Pale
yellow liquid; 1H NMR (300 MHz, CDCl3) d 7.90 (d, J = 7.3
Hz, 2H), 7.62 (d, J = 2.4 Hz, 1H), 7.44 (t, J = 7.4 Hz, 2H), 7.39–
7.35 (m, 4H), 7.33 (s, 1H), 7.29 (dd, J = 6.6, 2.9 Hz, 2H), 7.08
(dd, J = 8.6, 2.4 Hz, 1H), 6.81 (s, 1H); 13C NMR (75 MHz, CDCl3)
d 152.62, 144.57, 139.34, 132.93, 132.60, 131.19, 130.30, 130.05,
128.84, 128.79, 128.75, 128.35, 126.71, 125.87, 123.95, 106.05;
JC,F = 3.9 Hz), 126.88, 125.87 (d, JC,F = 3.8 Hz), 124.84 (d, JC,F
=
3.5 Hz), 123.99, 115.80, 115.52, 113.00, 58.19 (d, JC,F = 3.0 Hz),
+
+
42.11, 33.99, 31.46; HRMS (ESI) calcd for C25H25ClFN2
HRMS (ESI) calcd for C21H15Cl2N2 365.0607, found 365. 0589
407.1685, found 407.1712 [M + H]+.
[M + H]+.
3-(4-Chlorophenyl)-5-(2-fluorophenyl)-4,5-dihydro-1-o-tolyl-
1H-pyrazole (3l). Off white solid, m.p. 153–154 uC; 1H NMR
(300 MHz, CDCl3) d 7.62 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.2 Hz,
3H), 7.22–7.12 (m, 2H), 7.05–6.97 (m, 3H),6.95–6.87 (m, 2H),
5.65 (t, J = 10.8 Hz, 1H), 3.73 (dd, J = 16.4, 11.3 Hz, 1H), 3.11
(dd, J = 16.4, 10.3 Hz, 1H), 2.48 (s, 3H); 13C NMR (75 MHz,
CDCl3) d 161.99 (d, JC,F = 244.5 Hz), 147.05, 143.62, 134.29,
131.52 (d, JC,F = 18.7 Hz), 131.08, 129.20 (d, JC,F = 8.2 Hz),
1-(3-Chloro-4-methylphenyl)-3,5-diphenyl-1H-pyrazole (4d).
Off white solid; m.p. 113–114 uC; H NMR (300 MHz, CDCl3)
1
d 7.91 (d, J = 7.3 Hz, 2H), 7.50 (s, 1H), 7.43 (t, J = 7.3 Hz, 2H),
7.36–7.27 (m, 6H), 7.13 (d, J = 8.1 Hz, 1H), 7.04 (d, J = 8.0 Hz,
1H), 6.80 (s, 1H), 2.37 (s, 3H); 13C NMR (75 MHz, CDCl3) d
152.15, 144.43, 138.87, 135.29, 134.51, 132.90, 130.78, 130.35,
128.76, 128.69, 128.60, 128.52, 128.12, 125.84, 125.69, 123.26,
+
105.41, 19.74; HRMS (ESI) calcd for C22H18ClN2 345.1153,
found 345. 1138 [M + H]+.
128.75, 128.23 (d, JC,F = 4.0 Hz), 126.82, 126.08, 124.54 (d, JC,F
=
3.5 Hz), 123.40, 118.36, 115.60, 115.31, 60.09 (d, JC,F = 2.5 Hz),
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1-(3,4-Dichlorophenyl)-5-(4-methoxyphenyl)-3-phenyl-
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1-(4-tert-Butylphenyl)-3-(4-chlorophenyl)-5-(2-fluorophenyl)-
1H-pyrazole (4k). Off white solid, m.p. 141–142 uC; 1H NMR
(300 MHz, CDCl3) d 7.85 (d, J = 8.2 Hz, 2H), 7.36 (dd, J = 14.7,
8.4 Hz, 5H), 7.29–7.18 (m, 3H), 7.16–7.03 (m, 2H), 6.83 (s, 1H),
1.30 (s, 9H); 13C NMR (75 MHz, CDCl3) d 161.19 (d, JC,F = 244.5
Hz), 150.65 (d, JC,F = 2.3 Hz), 138.05, 137.58, 133.65, 131.75,
131.20, 130.61 (d, JC,F = 8.1 Hz), 128.80, 127.04, 125.87, 124.33,
123.78, 118.78 (d, JC,F = 14.8 Hz), 116.28, 116.00, 106.44 (d, JC,F
= 2.0 Hz), 34.62, 31.29; HRMS (ESI) calcd for C25H22ClFN2Na+
427.1348, found 427.1351 [M + Na]+.
1H-pyrazole (4e). Off white solid, m.p. 99–100 uC; 1H NMR (300
MHz, CDCl3) d 7.89 (d, J = 7.4 Hz, 2H), 7.64 (d, J = 2.3 Hz, 1H),
7.43 (t, J = 7.4 Hz, 2H), 7.38–7.21 (m, 2H), 7.21 (d, J = 8.6 Hz,
2H), 7.09 (dd, J = 8.6, 2.3 Hz, 1H), 6.89 (d, J = 8.6 Hz, 2H), 6.75
(s, 1H), 3.83 (s, 3H); 13C NMR (75 MHz, CDCl3) d 159.98,
152.53, 144.43, 139.47, 132.90, 132.70, 131.07, 130.28, 130.09,
128.72, 128.28, 126.72, 125.84, 123.96, 122.34, 114.23, 105.57,
55.34; HRMS (ESI) calcd for C22H17Cl2N2O+ 395.0712, found
395.0696 [M + H]+.
1-(3-Chloro-4-methylphenyl)-5-(4-methoxyphenyl)-3-phenyl-
1H-pyrazole (4f). Off white solid, m.p. 147–148 uC; 1H NMR
(300 MHz, CDCl3) d 7.90 (d, J = 7.3 Hz, 2H), 7.51 (d, J = 1.6 Hz,
1H), 7.42 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.2 Hz, 1H), 7.26–7.12
(m, 3H), 7.04 (dd, J = 8.1, 1.7 Hz, 1H), 6.87 (d, J = 8.6 Hz, 2H),
6.74 (s, 1H), 3.82 (s, 3H), 2.37 (s, 3H); 13C NMR (75 MHz,
CDCl3) d 159.74, 152.05, 144.28, 138.99, 135.17, 134.49, 132.99,
130.77, 130.04, 128.65, 128.04, 125.82, 125.71, 123.28, 122.71,
114.04, 104.91, 55.30, 19.73; HRMS (ESI) calcd for
C23H20ClN2O+ 375.1259, found 375.1273 [M + H]+.
1-(3,4-Dichlorophenyl)-5-(3-methoxyphenyl)-3-p-tolyl-
1H-pyrazole (4g). Off white solid, m.p. 135–136 uC; 1H NMR
(300 MHz, CDCl3) d 7.79 (d, J = 7.8 Hz, 2H), 7.64 (d, J = 1.7 Hz,
1H), 7.35 (d, J = 8.6 Hz, 1H), 7.29–7.21 (m, 3H),7.10 (dd, J = 8.5,
1.8 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.87–6.74 (m, 3H), 3.76 (s,
3H), 2.39 (s, 3H); 13C NMR (75 MHz, CDCl3) d 159.69, 152.65,
144.29, 139.37, 138.20, 132.87, 131.36, 131.07, 130.24, 129.85,
129.75, 129.44, 126.62, 125.75, 123.88, 121.23, 114.38, 114.32,
105.96, 55.33, 21.35; HRMS (ESI) calcd for C23H19Cl2N2O+
409.0869, found 409.0882 [M + H]+.
3-(4-Chlorophenyl)-5-(2-fluorophenyl)-1-o-tolyl-1H-pyrazole
(4l). Dark red solid, m.p. 134–135 uC; 1H NMR (300 MHz,
CDCl3) d 7.83 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 7.25–
7.12 (m, 5H), 7.08–6.92 (m, 3H), 6.86 (d, J = 1.2 Hz, 1H), 2.08 (s,
3H); 13C NMR (75 MHz, CDCl3) d 161.15 (d, JC,F = 248.25 Hz),
150.72, 139.42, 139.14, 135.55, 133.67, 131.79, 131.16, 130.66
(d, JC,F = 8.23 Hz), 129.02, 128.94 (d, JC,F = 12.2 Hz), 127.81,
127.12, 126.47, 124.04 (d, JC,F = 3.7 Hz), 118.31 (d, JC,F = 14.3
Hz), 116.23, 115.94, 105.35 (d, JC,F = 3.2 Hz), 17.69; HRMS (ESI)
calcd for C22H16ClFN2Na+ 385.0878, found 385.0885 [M + Na]+.
1-(4-tert-Butylphenyl)-3-(4-methoxyphenyl)-5-(4-nitrophe-
nyl)-1H-pyrazole (4m). Pale green solid, m.p. 134–135 uC; 1H
NMR (300 MHz, CDCl3) d 8.16 (d, J = 8.7 Hz, 2H), 7.84 (d, J = 8.7
Hz, 2H), 7.42 (dd, J = 12.6, 8.6 Hz, 4H), 7.28–7.21 (m, 2H), 6.96
(d, J = 8.7 Hz, 2H), 6.85 (s, 1H), 3.85 (s, 3H), 1.33 (s, 9H); 13C
NMR (75 MHz, CDCl3) d 159.79, 152.04, 151.37, 147.21, 141.81,
137.11, 136.94, 129.18, 127.07, 126.26, 125.29, 124.92, 123.79,
114.12, 105.54, 55.32, 34.74, 31.30; HRMS (ESI) calcd for
+
C
26H25N3NaO3 450.1788, found 450.1793 [M + Na]+.
1-(3-Chloro-4-methylphenyl)-3-(4-methoxyphenyl)-5-(4-nitro-
phenyl)-1H-pyrazole (4n). Pale green solid, m.p. 109–110 uC; 1H
NMR (300 MHz, CDCl3) d 8.19 (d, J = 8.0 Hz, 2H), 7.83 (d, J = 7.9
Hz, 2H), 7.57–7.38 (m, 3H), 7.19 (d, J = 7.6 Hz, 1H), 6.97 (d, J =
7.6 Hz, 3H), 6.84 (s, 1H), 3.85 (s, 3H), 2.40 (s, 3H); 13C NMR (75
MHz, CDCl3) d 159.94, 152.44, 147.38, 141.92, 138.32, 136.55,
136.16, 134.94, 131.14, 129.25, 127.13, 125.86, 125.01, 123.91,
1-(3,4-Dichlorophenyl)-3,5-di-p-tolyl-1H-pyrazole (4h). Off
white solid, m.p. 129–130 uC; 1H NMR (300 MHz, CDCl3) d
7.78 (d, J = 7.8 Hz, 2H), d 7.63 (d, J = 2.7 Hz, 2H), 7.34 (d, J = 8.6
Hz, 1H), 7.24 (d, J = 7.4 Hz, 2H), 7.20–7.13 (m, 4H), 7.08 (dd, J =
9.0, 2.7 Hz, 1H), 6.74 (s, 1H), 2.39 (s, 3H), 2.38 (s, 3H); 13C NMR
(75 MHz, CDCl3) d 152.62, 144.56, 139.50, 138.81, 138.12,
132.87, 130.99, 130.23, 129.85, 129.46, 129.42, 128.63, 127.18,
126.71, 125.75, 123.96, 105.68, 21.34, 21.33; HRMS (ESI) calcd
123.38, 114.18, 106.02, 55.34, 19.78; HRMS (ESI) calcd for
+
C
23H19ClN3O3 420.1109, found 420.1121 [M + H]+.
for C23H19Cl2N2 393.0920, found 393.0897 [M + H]+.
+
1-(4-Methoxyphenyl)-3,5-diphenyl-1H-pyrazole (4o). Off
white solid, m.p. 121–122 uC; 1H NMR (300 MHz, CDCl3) d
7.91 (d, J = 7.6 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.36–7.25 (m,
8H), 6.86 (d, J = 8.9 Hz, 2H), 6.81 (s, 1H), 3.81 (s, 3H); 13C NMR
(75 MHz, CDCl3) d 158.85, 151.63, 144.34, 133.45, 133.17,
130.62, 128.84, 128.63, 128.45, 128.17, 127.89, 126.75, 125.78,
114.11, 104.64, 55.50; HRMS (ESI) calcd for C22H18N2NaO+
349.1311, found 349.1298 [M + Na]+.
1-(3-Chloro-4-methylphenyl)-3,5-di-p-tolyl-1H-pyrazole (4i).
Off white solid, m.p. 96–97 uC; H NMR (300 MHz, CDCl3) d
1
7.79 (d, J = 7.7 Hz, 2H), 7.52 (s, 1H), 7.23 (d, J = 8.2 Hz, 2H),
7.15 (q, J = 8.5 Hz, 5H), 7.03 (d, J = 8.0 Hz, 1H), 6.73 (s, 1H),
2.38 (s, 3H), 2.36 (s, 6H); 13C NMR (75 MHz, CDCl3) d 152.15,
144.41, 139.03, 138.41, 137.84, 135.10, 134.46, 130.72, 130.15,
129.35, 129.27, 128.60, 127.48, 125.72, 123.29, 105.03, 21.33,
+
21.30, 19.73; HRMS (ESI) calcd for C24H22ClN2 373.1466,
Cell culture and cell proliferation assay
found 373.1483 [M + H]+.
Cell culture. Human ovarian adenocarcinoma (SK-OV-3),
colon adenocarcinoma (HT-29) and cervical adenocarcinoma
(HeLa) cells were obtained from the American Type Culture
Collection. Cells were grown on 75 cm2 cell culture flasks with
EMEM (Eagle’s minimum essential medium), supplemented
with 10% fetal bovine serum, and a 1% penicillin–streptomy-
cin solution (10 000 units of penicillin and 10 mg of
streptomycin in 0.9% NaCl) in a humidified atmosphere of
5% CO2, 95% air at 37 uC.
1-(4-tert-Butylphenyl)-3,5-di-p-tolyl-1H-pyrazole (4j). Off
white solid, m.p. 160–161 uC; 1H NMR (300 MHz, CDCl3) d
7.80 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 8.7
Hz, 2H), 7.21–7.16 (m, 4H), 7.11 (d, J = 8.1 Hz, 2H), 6.74 (s, 1H),
2.37 (s, 3H), 2.35 (s, 3H), 1.31 (s, 9H); 13C NMR (75 MHz,
CDCl3) d 151.73, 150.35, 144.26, 138.04, 137.81, 137.59, 130.41,
129.30, 129.15, 128.59, 127.87, 125.82, 125.69, 124.79, 104.56,
+
31.35, 21.33, 21.30; HRMS (ESI) calcd for C27H29N2 381.2325,
found 381.2327 [M + H]+.
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Cell proliferation assay. The cell proliferation assay was
carried out using a CellTiter 96 aqueous one solution cell
proliferation assay kit (Promega, USA). Briefly, upon reaching
about 75–80% confluency, 5000 cells/well were plated in a 96-
well microplate in 100 mL media. After seeding for 72 h, the
cells were treated with 50 mM compound in triplicate.
Doxorubicin (10 mM) was used as the positive control. At the
end of the sample exposure period (72 h), 20 mL CellTiter 96
aqueous solution was added. The plate was returned to the
incubator for 1 h in a humidified atmosphere at 37 uC. The
absorbance of the formazan product was measured at 490 nm
using a microplate reader. The blank control was recorded by
measuring the absorbance at 490 nm with wells containing the
medium mixed with CellTiter 96 aqueous solution but no cells.
Results were expressed as a percentage of the control (without
compound set at 100%).
Conclusions
In summary, we have developed a simple, efficient and
environmentally friendly protocol for the synthesis of 1,3,5-
triarylpyrazoles and 1,3,5-triarylpyrazolines in [bmim][PF6]
ionic liquid mediated by Cu(OTf)2. The reaction protocol
exhibited tolerance with different functional groups, generat-
ing pyrazoles in good to high yields (71–82%) without any
requirement for additional reagents for the oxidation of in situ
generated pyrazolines. The catalyst can be reused up to four
cycles without much loss in catalytic activity. The pyrazoles
(4a–o) and pyrazolines (3a–n) were evaluated for antiprolifera-
tive activity. Compound 3b inhibited cell proliferation of HeLa
cells by 80% at a concentration of 50 mM. All other synthesized
derivatives exhibited a modest inhibition against the prolif-
eration of SK-OV-3, HT-29 and HeLa cells. Further structure–
activity relationship studies are required for optimizing the
antiproliferative activities of these classes of compounds.
¨
¨
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Acknowledgements
We thank University Grant Commission, New Delhi Project #
39-733/2010 (SR) and the National Science Foundation, Grant
Number CHE 0748555, and American Cancer Society grant
number RSG-07-290-01-CDD American Cancer Society Grant
#RSG-07-290-01-CDD for their financial support. VKR is
thankful to CSIR, New Delhi for SRF. We also thank the
National Institute of General Medical Sciences of the National
Institutes of Health under grant number 8 P20 GM103430-12
for sponsoring the core facility.
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