3,5-Diaryl-1H-pyrazole as a molecular scaffold for the synthesis of apoptosis-inducing agents

Article abstract of DOI:10.1016/j.bmc.2010.03.016

The scaffold of 3,5-diaryl-1H-pyrazole was selected as a molecular template to synthesize novel growth-inhibitory agents in the present study. Our findings suggested that analogs bearing electron-withdrawing groups on one ring while electron-donating grou

Full text of DOI:10.1016/j.bmc.2010.03.016

Bioorganic & Medicinal Chemistry 18 (2010) 3270–3278
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
3,5-Diaryl-1H-pyrazole as a molecular scaffold for the synthesis
of apoptosis-inducing agents
a
b
c
b
d
Arthur Y. Shaw a,e, , Hao-Han Liau , Pei-Jung Lu , Chia-Ning Yang , Chien-Hsing Lee , Jun-Yan Chen ,
*
Zhigang Xu e, Gary Flynn e
a Department of Chemistry, Tamkang University, Taipei 251, Taiwan
b Institute of Clinical Medicine, National Cheng Kung University, Tainan 701, Taiwan
c Institute of Biotechnology, National University of Kaohsiung, Kaohsiung 700, Taiwan
d Department of Life Science, National University of Kaohsiung, Kaohsiung 700, Taiwan
e Department of pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85724, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The scaffold of 3,5-diaryl-1H-pyrazole was selected as a molecular template to synthesize novel
growth-inhibitory agents in the present study. Our findings suggested that analogs bearing electron-
withdrawing groups on one ring while electron-donating groups on another reveal significant activities.
In particular, 26 bearing a 1,10-biphenyl moiety displayed the most potent activity against OVCA, SW620,
Received 13 January 2010
Revised 8 March 2010
Accepted 9 March 2010
Available online 16 March 2010
H460 and AGS cells with GI50 values of 0.67, 0.89, 0.73 and 0.79 lM, respectively. The mechanistic study
revealed that 26-mediated apoptosis-inducing effect on OVCA cells was, in part, attributed to the inhibi-
tion of protein kinase B/Akt activity, accompanied by the mitochondrial apoptotic pathway through the
activation of caspase-9, caspase-3, as well as the cleavage of protein poly(ADP-ribose) polymerase (PARP)
and DNA fragmentation. Further structure–activity relationship study employed by Comparative Molec-
ular Field Analysis (CoMFA) was carried out with q2 and R2 values of 0.671 and 0.846, respectively.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
3,5-Diaryl-1H-pyrazole
Molecular scaffold
Apoptosis-inducing agent
Mitochondrial apoptotic pathway
Structure–activity relationship
1. Introduction
drugs (NSAIDs).6 Celecoxib, a selective cyclooxygenase-2 (COX-2)
inhibitor, is clinically used for the therapy of osteoarthritis, rheu-
Apoptosis, or programmed cell death, is the prevalent mecha-
nism for cellular development and differentiation. Deregulation of
apoptosis is widely believed to be involved in a variety of human dis-
eases, including cancer, autoimmune diseases, and neurodegenera-
tion disorders.1,2 Appreciable progress in elucidating molecular
components of the apoptotic machinery have resulted in the identi-
fication of many molecular targets for the development of new drugs
with pro- and anti-apoptotic activity to treat different diseases
including cancer. So far, two principle apoptotic pathways are iden-
tified, the death receptor (or extrinsic) and the mitochondria (or
intrinsic) pathways, both of which involve the activation of a family
of cysteine proteases with aspartate specificity, called caspases, as
the executioners of apoptosis.3,4 Based on the urgent need for cancer
patients, developing small molecules that activate and induce apop-
tosis is a promising strategy for the treatment of cancer.
matoid arthritis, acute pain and so on (Fig. 1).7 On the other hand,
N-substituted pyrazoles have also been developed as inhibitors of
p38 mitogen-activated protein kinase, SERMs (selective estrogen
receptor modulators) for the treatment of cancer cells.8,9 Recently,
several 3,5-diary-1H-pyrazoles were reported as inhibitors of
monoamine oxidases.10 In light of our research on the develop-
ment of small molecules targeting cancer cells, the present study
we take advantage of 3,5-diaryl-1H-pyrazole (Fig. 1) as a molecular
scaffold to synthesize a series of derivatives for the evaluation of
their growth-inhibitory effect against carcinoma cell lines.
2. Results and discussion
2.1. Synthesis of 3,5-diaryl-1H-pyrazoles 1–33
The pyrazole scaffold has drawn a great deal of attention due to
its contributions in biological and pharmacological fields regard-
less of scarcity in nature.5 For example, N-substituted pyrazoles
such as pirazolac is known as non-steroidal anti-inflammatory
The preparation of 3,5-diaryl-1H-pyrazole was originally from
the cyclization of 1,3-diketone and hydrazine in 1893.11 Although
several methods have been developed, some tedious procedures or
more reagents are required to handle with. In the present study,
we initially carried out the nucleophilic substitution by the treat-
ment of commercially available acetophenone and benzoyl
chloride in the presence of strong base n-BuLi that afforded
* Corresponding author at: 1703 E. Mabel St., Tucson, AZ 85721, USA.
E-mail address: shaw.299@gmail.com (A.Y. Shaw).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.016

A. Y. Shaw et al. / Bioorg. Med. Chem. 18 (2010) 3270–3278
3271
SO2NH2
N
NH
R
N
N
F3C
R'
CH3
Celecoxib
3,5-diaryl-1H-pyrazole
Figure 1. Chemical structures of pyrazole derivatives.
1,3-diketone. Without further purification, 1,3-diketone was sub-
jected to undergo cyclization with hydrazine in refluxed ethanol to
give 3,5-diaryl-1H-pyrazole.12 The acetophenone was deprotonated
by n-BuLi to generate a nucleophilic enolate, followed by the nucle-
ophilic substitution with benzoyl chloride to give 1,3-diketone. Nev-
ertheless, some side products have been found during the
preparation of 1,3-diketone. We assumed that side products might
be attributed to the self-condensation of acetophenone. Moreover,
n-BuLi was also found to serve as a nucleophile to attack benzoyl
chloride that afforded 1-aryl-pentan-1-ones. As a result, we took
advantageof a moreupdated synthetic methodwith slight modifica-
tions according to the report in the literature.13 As shown in Scheme
1, both 2 equiv of acetophenone and lithium bis(trimethyl-
silyl)amide (LiHMDS) and 1 equiv of benzoyl chloride were charged
in the toluene. Without further purification, the reaction solution
was evaporated in vacuo to obtain 1,3-diketone, followed by the
addition of hydrazine in refluxed ethanol to synthesize 3,5-diaryl-
1H-pyrazoles. The strong base LiHMDS used in this method is more
advantageous than n-BuLi thanks to its poor nucleophilicity. Conse-
quently, we synthesized a series of 3,5-diaryl-1H-pyrazoles, some of
which were subjected to proceed hydrogenation or demethylation
reaction to afford the corresponding products (Scheme 2).
N
HN
N
HN
Ar
a
Ar
O2N
H2N
12-17, 33
1, 3-11, 32
N
N
HN
HN
Ar
Ar
b
MeO
HO
21, 30, 31
20, 26, 28
Scheme 2. Reagents and conditions: (a) Pd/C, H2, MeOH, rt, 16 h; (b) BBr3, DCM.
not show any growth-inhibitory effects. Nevertheless, 17 (4-NH2)
showed an improved growth-inhibitory effect against OVCA and
H460 cell lines in comparison with the counterpart 11 (4-NO2)
with GI50 values of 18.6 and 21.5 lM, respectively (Table 2). These
results indicated that either electron-donating or electron-with-
drawing group simultaneously present on both sides would result
in a precipitous loss in potency. On the other hand, as the amino
group in 17 (4-NH2) was replaced with a dimethylamino group
in 18 (4-NMe2), a dramatic decrease in activity was observed. This
finding suggests that the amino group bearing hydrogen bond-
donating character plays an important role for activity.
It is worthwhile to notice that 19 without any substituents on
one side exhibited more activity than 22–24 with substituents on
both sides. In addition to the electronic modulations, the steric fac-
tor demanded modification was further investigated based on 19.
As shown in Table 3, a series of 1,10-biphenyl analogs 26–33 intro-
duced a phenyl at 2-position on one side revealed a tremendous
enhancement in potency. Remarkably, 26 displayed the most po-
tent activity against OVCA, SW620, H460 and AGS cell lines with
2.2. Growth inhibition of 1–33 against four carcinoma cells
The evaluation of growth-inhibitory activity of 3,5-diaryl-1H-
pyrazoles 1–33 was examined on a panel of human carcinoma cell
lines, including OVCA (ovarian carcinoma cell), SW620 (colorectal
adenocarcinoma cell), H460 (large lung carcinoma cell) and AGS
(gastric carcinoma cell). The MTT assay was employed for growth
inhibition studies and the GI50 values are summarized in Tables
1–3. The tested compound concentration causing a 50% cell growth
inhibition (GI50) was determined by interpolation from dose–re-
sponse curves14
.
As shown in Table 1, analogs 1–11 contain an electron-with-
drawing nitro group at 4- or 3-position on one benzene ring while
various substituents on another. Among four carcinoma cell lines,
OVCA and H460 cell lines seemed to be more sensitive in response
GI50 values of 0.67, 0.89, 0.73 and 0.79 lM, respectively. On the
contrary, upon the replacement of the methoxy group in 26 with
a hydroxyl group in 31, a 10-fold decrease in activity was clearly
observed. Similarly, as the nitro group in 32 was transformed into
an amino group to generate the counterpart 33, a significant loss in
potency was demonstrated as well. Together, these results indicate
that a hydrogen-bond donor such as hydroxyl and amino groups at
4-position of benzene ring potentially counteract their growth-
inhibitory effect. On the other hand, as 4-methoxygroup was
replaced with 2- or 3-methoxy group, a precipitous decreases in
to the growth inhibition with GI50 values between 5.8 and 28.9 lM.
Compared to the mono-substituted counterparts, disubstituted 6
(3,4-dimethoxy) and 7 (3,4-methoxydioxy) displayed higher activ-
ities against most cell lines. Interestingly, substituents at 4-posi-
tion such as 6–10 showed better activity than the counterparts
1–5 against SW620 cell line.
Upon the reduction of the nitro group in 1 and 3–7, the corre-
sponding counterparts 12–17 were generated, most of which did
O
N
O
O
O
HN
b
a
Ar'
+
Ar'
Cl
Ar
Ar
Ar
Ar
I
II
III
1 11 18 20 22 29, 32
-
,
-
,
-
Scheme 1. Reagents and conditions: (a) LiHMDS, toluene, 0 °C, 2 h; (b) NH2NH2ꢀHCl, EtOH, reflux, 16 h.

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A. Y. Shaw et al. / Bioorg. Med. Chem. 18 (2010) 3270–3278
Table 1
Table 3
Chemical structures and GI50 values of 3,5-diarylpyrazoles 1–11
Chemical structures and GI50 values of 3,5-diaryl-1H-pyrazoles 26–33
N
N
R'
HN
HN
R
R
R = 4-NO 1, 3-11
2
R = 3-NO
2
26-33
2
a
a
Entry R0
GI50
(lM)
Entry
R
GI50
(lM)
OVCA
SW620
H460
AGS
OVCA
SW620
H460
AGS
7.2 1.8 NDb
5.8 0.4 ND
11.7 0.9 ND
19.1 5.1 ND
27.3 4.3 14.3 1.8 18.3 5.6 ND
28.9 5.3 26.4 1.1 17.9 4.8 ND
10.2 1.9 ND
6.1 2.6 11.4 0.9
20.5 4.7 ND
11.2 2.4 ND
26
27
28
29
30
31
32
33
4-OMe
4-CF3
2-OMe
3-OMe
2-OH
4-OH
4-NO2
4-NH2
0.67 0.1
2.75 0.1
14.2 1.1
5.8 1.3
5.8 0.4
6.2 0.6
3.4 0.4
17.9 1.1
0.89 0.12
NDb
ND
0.73 0.11
ND
0.79 0.08
6.9 0.7
ND
7.5 0.8
7.5 0.5
9.2 0.6
1.2 0.1
8.4 0.6
1
2
3
4
5
6
7
Hydrogen
Hydrogen
2-Methoxy
3-Methoxy
4-Methoxy
3,4-Dimethoxy
3,4-
Methylenedioxy
4-Fluoro
4-Chloro
9.1 2.5
8.7 1.4
4.6 1.2
6.9 1.5
2.7 1.1
8.2 1.3
ND
10.6 0.7
25.9 4.6
12.2 1.2
ND
10.8 1.8 23.5 3.4
8.9 2.2
13.1 1.3
8
9
10
11
ND
ND
18.1 1.2 ND
12.2 1.8 20.7 6.2 24.1 0.8
18.3 0.3
a
GI50 values are presented as the mean SEM (standard error of the mean) from
four to six separated experiments.
ND indicated that no appreciable growth inhibition was observed upon treat-
ment of maximal concentration at 40 lM.
4-Bromo
4-Trifluoromethyl
17.5 3.2 28.6 2.9 ND
ND ND ND
14.6 0.5
ND
b
a
GI50 values are presented as the mean SEM (standard error of the mean) from
four to six separated experiments.
b
ND indicated that no appreciable growth inhibition was observed upon treat-
2.3. Induction of apoptosis by 26 and 27 through caspase-
dependent pathways and inactivation of protein kinase B/Akt
ment of maximal concentration at 40 lM.
To demonstrate whether the cell growth inhibition was related
to cell cycle regulation and apoptosis, 26 and 27 were selected for
further mechanistic study on OVCA cells. As indicated in the flow
cytometric analysis (Fig. 2), accumulation of sub-G1 arrest in re-
sponse to 26- and 27-treated cells was clearly observed in a
dose-dependent manner. Upon the exposure of OVCA cells to 26
potency was also observed, suggesting that substituents at 4-posi-
tion exhibit a favorable interaction for growth inhibitor. In addi-
tion, 2-hydroxy substituted 30 showed at least twofold more
potent than the counterpart 28 with 2-methoxy group. We spec-
ulate the improved activity might due to the intramolecular
hydrogen bonding between the hydroxyl group and nitrogen atom
on the pyrazole core that maintains the rigidity of structure for
activity.
at 1.2, 2.5 and 5.0
lM, the substantial sub-G1 arrest was detected
with 14.97%, 20.53% and 32.05%, respectively. Similarly, OVCA
treated with 27 at 1.2, 2.5 and 5.0
lM also led to the increased
Table 2
Chemical structures and GI50 values of 3,5-diarylpyrazoles 12–25
N
HN
Ar
R
R = 4-NH
12-17
R = 4-NMe 18
2
2
R = 4-OMe 19, 20, 22-25 R = 4-OH
21
a
Entry
Ar
GI50 (lM)
OVCA
SW620
H460
AGS
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Phenyl
NDb
ND
ND
ND
ND
18.6 5.5
ND
9.9 1.9
22.8 3.5
9.2 1.0
14.0 2.7
12.8 2.9
20.7 2.1
17.5 1.6
25.4 2.4
21.1 1.2
ND
ND
ND
ND
ND
27.2 3.8
11.2 1.1
19.6 1.8
ND
26.7 0.9
ND
ND
ND
ND
21.5 3.1
ND
10.4 3.4
ND
20.1 2.4
ND
11.6 4.1
27.1 3.9
13.2 1.0
ND
ND
ND
ND
ND
ND
ND
11.3 0.7
14.8 0.3
9.8 0.9
ND
ND
ND
2-Methoxyphenyl
3-Methoxyphenyl
4-Methoxyphenyl
3,4-Dimethoxyphenyl
4-Trifluoromethylphenyl
4-Trifluoromethylphenyl
Phenyl
4-trifluoromethylphenyl
4-Trifluoromethylphenyl
4-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
2-Naphthyl
ND
ND
26.9 2.3
19.0 0.4
a
GI50 values are presented as the mean SEM (standard error of the mean) from four to six separated experiments.
ND indicated that no appreciable growth inhibition was observed upon treatment of maximal concentration at 40
b
l
M.

A. Y. Shaw et al. / Bioorg. Med. Chem. 18 (2010) 3270–3278
3273
Figure 2. Flow cytometric analysis of OVCA cells. Cells were harvested after 24 h treatment and followed by fixation, propidium iodide staining prior to the flow cytometric
analysis. Sub-G1 phase indicated the DNA fragmentation and cell death. Exposure of OVCA cells to 26 at 1.2, 2.5 and 5.0 M caused the sub-G1 phase arrest with 14.97%,
20.53% and 32.05%, respectively. Similarly, OVCA cells exposed to 26 at 1.2, 2.5 and 5.0 M caused the sub-G1 phase arrest with 11.43%, 15.75% and 23.51%, respectively. (A)
Untreated OVCA cells were taken as blank group; (B) cells were treated with 0.5% DMSO as the control; (C) treatment of 26 at 1.2 M; (D) treatment of 26 at 2.5 M; (E)
treatment of 26 at 5.0 M; (F) treatment of 27 at 1.2 M; (G) treatment of 27 at 2.5 M; (H) treatment of 27 at 5.0 M.
l
l
l
l
l
l
l
l
sub-G1 arrest with 11.43%, 15.75% and 23.51%, respectively. These
findings suggested that growth inhibition of OVCA cells mediated
by 26 and 27 may cause DNA fragmentation and cell death.
On the other hand, Western blot of cytosolic extracts prepared
from 26- and 27-treated OVCA cells for 16 h revealed cleavage of
the 116-kDa protein poly(ADP-ribose) polymerase (PARP) to gen-
erate 89-kDa fragment (Fig. 3). The results were consistent with
the activation of pro-apoptotic caspase-3, as shown by the in-
creased level of 17/19-kDa cleaved caspase-3. Moreover, the acti-
vation of the upstream caspase-9 was also monitored by the
elevated levels of 35/37-kDa cleaved caspase-9. Taken together,
we concluded that the apoptosis-inducing effect on OVCA cells
mediated by 26 and 27 was through the mitochondrial apoptotic
pathway.
In addition to the elucidation of mitochondrial apoptotic pathway,
we attempted to examine the effect on the phosphorylation state
of Akt due to its significance in OVCA cell survival. As indicated
in Figure 3, the reduction of phospho-Akt level was clearly ob-
served, indication deactivation of Akt was in response to 26 and
27 treatment. Additionally, the more potent inhibitory effect on
Akt activity of 26 over 27 coincided with growth-inhibitory activity
with GI50 values of 0.67 and 2.75 lM, respectively. These results
suggested the induction of apoptosis by 26 and 27 in OVCA ovarian
cancer cells was, in part, attributable to the inhibition of Akt
activity.
2.4. Structure–activity relationship study on OVCA cells
Aberrant function of PI3K/Akt signaling pathway contributes to
cell proliferation and survival in a variety of neoplasms including
ovarian cancer. Akt is overexpressed in a substantial number of
ovarian cancers, resulting in the constitutive activation of signaling
pathway indicated by phosphorylation of Akt in vitro and in vivo.15
Structure–activity relationship analysis was further examined
on OVCA cells. Comparative Molecular Field Analysis (CoMFA)
was employed by software Sybyl 8.1 to characterize structure–
activity relationships in this study. Our final training set model
and coefficients such as non-validation R2 and F-value were ob-

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A. Y. Shaw et al. / Bioorg. Med. Chem. 18 (2010) 3270–3278
Figure 3. Induction of apoptosis in OVCA cells by 26 and 27. Induction of poly(ADP-
ribose) polymerase (PARP) cleavage by 26 and 27 at the indicated concentrations
after 16 h of treatment. PARP proteolysis to the apoptosis-specific 85-kDa fragment
was monitored by western blotting. The appearance of a 89-kDa fragment coincided
with the activation of caspase-3, as shown by the increased level of the 17/19-kDa
cleaved caspase-3. The activation of the upstream caspase-9 was also observed with
the increased level of the 35/37-kDa cleaved caspase-9, indicating the mitochon-
drial apoptotic pathway was involved. The phosphorylation status of Akt was also
determined by immunoblot analysis with phosphospecific antibody. Unphosphor-
ylated Akt, as immunostained by anti-Akt antibody, was used as internal standard
for the comparison of phospho-Akt level among samples of different preparations.
tained by using the optimal number of components and all the data
points in the training set. The cross-validated q2 value with 0.671
and the subsequent non-validation R2 value with 0.846 meet the
criteria (q2 = 0.5; R2 = 0.8) that indicate the constructed model is
internally predictive and accountable. The contributions for steric
and electrostatic fields are 0.459 and 0.561, respectively. As shown
in Figure 4A, all analogs were aligned to the pyrazole core as spec-
ified in stick. The steric field (in yellow/green for unfavorable/
favorable contours, Fig. 4B) and electrostatic field (in blue/red for
positive/negative charge favorable contours, Fig. 4C) projected onto
32. In steric field model, three steric unfavorable contours near C-
20, C-30, and C-40 greatly restrict the modifications around the ring
A moiety. Accordingly, 1 shows a higher activity (GI50, 7.2
compared to 3 (GI50, 11.7
M) bearing a methoxy group on C-20
and 6 (GI50, 28.9
M) with methoxy groups on C-30 and C-40. The
steric favorable contour centering on C-60 is amenable for modifi-
cations evidenced by comparing 25 (GI50, 17.5 M) and 26 (GI50
0.67
lM)
l
l
l
,
l
M) with a bulky phenyl group on C-60. A steric unfavorable
Figure 4. 3D-QSAR study on OVCA cells. Comparative Molecular Field Analysis
(CoMFA) was employed by software Sybyl 8.1 to characterize structure–activity
relationships. (A) Alignment of 3,5-diaryl-1H-pyrazoles 1–33 to the pyrazole core
specified in stick. (B) Steric field projected on 32: yellow contour indicate bulky
groups decrease activity (contribution level of 80%), whereas green contours
indicate bulky groups increase activity (contribution level of 20%); (C) Electrostatic
field projected on 32: red contours indicate negatively charged groups increase
activity (contribution level of 20%), whereas blue contours indicate positively
charged groups increase activity (contribution level of 80%).
contour is located at C-400 position on the ring B moiety, which ex-
plains the different location of a nitro group, such as C-400 in 1 and
C-300 in 2, responsible for different activities with GI50 values of 7.2
and 5.8
blue contour around C-20 suggests a positively charged substituent
to increase activity. As a consequence, 3 (GI50, 11.7 M) with a par-
lM, respectively. In electrostatic field model (Fig. 4C), the
l
tial negatively charged methoxy oxygen atom nearby the blue con-
tour has a lower activity compared to 1 without substituent. The
mixed one blue contour and two red contours around C-400 corre-
spondingly match a nitro group composed of a positively charged
nitrogen atom and two negatively charged oxygen atoms. It inter-
observed upon the introduction of a phenyl moiety at 2-position
on one side. Particularly, 26 bearing a 1,10-biphenyl moiety exhib-
ited the most potent activity against OVCA, SW620, H460 and AGS
prets the dramatic discrepancy in activity between 1 (4-NO2, GI50
,
7.2 lM) and 12 (4-NH2, GI50, ND) with distinct charge distribution
on C-400.
cells with GI50 values of 0.67, 0.89, 0.73 and 0.79 lM, respectively.
Furthermore, the mechanistic study on OVCA cells demonstrated
that growth inhibition mediated by 26 and 27 was, in part, attrib-
uted to the inhibition of intracellular Akt activity. The mitochon-
drial apoptotic pathway was also evidenced by the activation of
caspase-9, caspase-3, cleavage of protein poly(ADP-ribose) poly-
merase (PARP) and DNA fragmentation. The structure–activity
relationship analysis employed by Comparative Molecular Field
Analysis (CoMFA) model was generated with q2 and R2 values of
0.671 and 0.846, respectively, suggesting its predictivity and
accountability warranted for further study.
3. Conclusions
In the present study, a more updated two-step synthesis of 3,5-
diaryl-1H-pyrazoles was carried out. With 33 analogs in hand,
growth inhibition evaluation on a panel of four carcinoma cells
was examined. Our findings suggest that analogs bearing an elec-
tron-withdrawing group on one side while an electron-donating
group on another exhibited better growth-inhibitory effect. In
addition, we found that dramatic enhancement in potency was

A. Y. Shaw et al. / Bioorg. Med. Chem. 18 (2010) 3270–3278
3275
4.1.5. 3-(4-Methoxyphenyl)-5-(4-nitrophenyl)-1H-pyrazole (5)
4. Experimental
4.1. Synthesis
Compound 5 was synthesized from the procedure described for
1. Mp 205 °C. 1H NMR (300 MHz, DMSO-d6) d 3.79 (s, 3H), 7.04 (d,
J = 8.6 Hz, 2H), 7.30 (s, 1H), 7.76 (d, J = 8.6 Hz, 2H), 8.10 (d,
J = 8.8 Hz, 2H), 8.30 (d, J = 8.8 Hz, 2H), 13.54 (br s, NH) ppm. 13C
NMR (75 MHz, DMSO-d6) d 55.22, 100.09, 114.45, 124.10, 125.77,
126.61, 146.35, 159.36 ppm. HRMS (M)+ calcd for C16H13N3O3
295.0957; found 295.0963.
Chemical reagents and organic solvents were purchased from
TCI and Alfa Aesar unless otherwise mentioned. Melting points
were determined by Fargo MP-2D. Nuclear magnetic resonance
spectra (1H and 13C NMR) were measured on a Bruker AC-300
instrument. Chemical shifts (d) are reported in ppm relative to
the TMS peak. Mass spectra were obtained by FAB on a Jeol JMS-
700 instrument. Flash column chromatography was performed
with silica gel (230–400 mesh).
4.1.6. 3-(3,4-Dimethoxyphenyl)-5-(4-nitrophenyl)-1H-pyrazole
(6)
Compound 6 was synthesized from the procedure described for
1. Mp 194 °C. 1H NMR (300 MHz, DMSO-d6) d 3.79 (s, 3H), 3.85 (s,
3H), 7.04 (d, J = 8.2 Hz, 1H), 7.32 (s, 1H), 7.36 (d, J = 8.2 Hz, 1H),
7.43 (s, 1H), 8.10 (d, J = 8.4 Hz, 2H), 8.28 (d, J = 8.4 Hz, 2H), 13.58
(br s, NH) ppm. 13C NMR (75 MHz, DMSO-d6) d 55.56, 55.65,
100.29, 109.11, 112.10, 117.75, 121.68, 124.11, 125.75, 140.21,
144.23, 146.34, 149.04, 149.19 ppm. HRMS (M)+ calcd for
C17H15N3O4 325.1063; found 325.1060.
4.1.1. 5-(4-Nitrophenyl)-3-phenyl-1H-pyrazole (1)
To an ice-cooled solution of acetophenone (0.48 g, 4.0 mmol) in
toluene (20 ml) was added a solution of LiHMDS (4.2 ml, 1.0 M in
THF, 4.2 mmol), the resulting mixture was stirred for 20 min, fol-
lowed by the addition of 4-nitrobenzoyl chloride (0.37 g,
2.0 mmol) then allowed to stir at room temperature for 2 h. The
reaction mixture was diluted with ethyl acetate (40 ml), washed
with water (50 ml), brine (50 ml), and dried over MgSO4(s) and
evaporated in vacuo to obtain the crude 1,3-diketone intermediate.
Without further purification, the 1,3-diketone and hydrazine
hydrochloric acid (NH2NH2ꢀHCl, 10.0 mmol) were added to ethanol
(80 ml) solution and the resulting mixture was stirred at reflux for
16 h. The reaction mixture was evaporated in vacuo to obtain the
residue, followed by the addition of ethyl acetate (50 ml), washed
with water (50 ml), brine (50 ml). The organic layer was dried over
MgSO4, evaporated in vacuo and purified with silica gel
chromatography (ethyl acetate/hexane = 1:9) to afford 5-(4-nitro-
phenyl)-3-phenyl-1H-pyrazole (1). Mp 281 °C. 1H NMR (300 MHz,
DMSO-d6) d 7.38 (t, J = 7.3 Hz, 1H), 7.42 (s, 1H), 7.49 (dd, J = 7.6,
7.3 Hz, 2H), 7.84 (d, J = 7.6 Hz, 2H), 8.12 (d, J = 8.6 Hz, 2H), 8.31
(d, J = 8.6 Hz, 2H) ppm. 13C NMR (75 MHz, DMSO-d6) d 101.21,
124.19, 125.18, 125.83, 128.22, 128.95, 146.43 ppm. HRMS
(M+1)+ calcd for C15H11N3O2 266.0885; found 266.0931.
4.1.7. 3-(Benzo[d][1,3]dioxol-5-yl)-5-(4-nitrophenyl)-1H-
pyrazole (7)
Compound 7 was synthesized from the procedure described for
1. Mp 249 °C. 1H NMR (300 MHz, DMSO-d6) d 6.06 (s, 2H), 7.01 (d,
J = 7.6 Hz, 1H), 7.27 (s, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.37 (s, 1H), 8.07
(d, J = 7.9 Hz, 2H), 8.23 (d, J = 7.9 Hz, 2H), 13.53 (br s, NH) ppm. 13C
NMR (75 MHz, DMSO-d6) d 100.54, 101.38, 105.66, 108.82, 119.13,
123.04, 124.14, 125.78, 140.11, 143.98, 146.41, 147.40, 147.90,
149.26 ppm. HRMS (M)+ calcd for C16H11N3O4 309.0750; found
309.0749.
4.1.8. 3-(4-Fluorophenyl)-5-(4-nitrophenyl)-1H-pyrazole (8)
Compound 8 was synthesized from the procedure described for
1. Mp 302 °C. 1H NMR (300 MHz, DMSO-d6) d 7.31 (d, J = 5.2 Hz,
2H), 7.35 (s, 1H), 7.85 (s, J = 5.2 Hz, 2H), 8.09 (d, J = 8.4 Hz, 2H),
8.27 (d, J = 8.4 Hz, 2H), 13.70 (br s, NH) ppm. 13C NMR (75 MHz,
DMSO-d6)
d 101.04, 115.88, 124.11, 125.81, 127.29, 143.12,
163.62 ppm. HRMS (M)+ calcd for C15H10FN3O2 283.0757; found
283.0761.
4.1.2. 5-(3-Nitrophenyl)-3-phenyl-1H-pyrazole (2)
Compound 2 was synthesized from the procedure described for
1. Mp 214 °C. 1H NMR (300 MHz, DMSO-d6) d 7.36 (t, J = 7.3 Hz,
1H), 7.42 (s, 1H), 7.47 (dd, J = 7.5, 7.3 Hz, 2H), 7.76 (dd, J = 8.0,
7.6 Hz, 1H), 7.84 (d, J = 7.5 Hz, 2H), 8.16 (d, J = 8.0 Hz, 1H), 8.28
(d, J = 7.6 Hz, 1H), 8.66 (s, 1H), 13.61 (br s, NH) ppm. 13C NMR
(75 MHz, DMSO-d6) d 100.38, 119.20, 122.01, 125.17, 128.35,
129.02, 130.30, 131.32, 135.32, 143.95, 148.35, 149.28 ppm. HRMS
(M)+ calcd for C15H11N3O2 265.0851; found 265.0848.
4.1.9. 3-(4-Chlorophenyl)-5-(4-nitrophenyl)-1H-pyrazole (9)
Compound 9 was synthesized from the procedure described for
1. Mp 280 °C. 1H NMR (300 MHz, DMSO-d6) d 7.41 (s, 1H), 7.51 (d,
J = 8.4 Hz, 2H), 7.85 (d, J = 8.4 Hz, 2H), 8.08 (d, J = 8.7 Hz, 2H), 8.28
(d, J = 8.7 Hz, 2H), 13.74 (br s, NH) ppm. 13C NMR (75 MHz, DMSO-
d6) d 101.59, 124.22, 125.86, 126.90, 128.96, 132.66, 146.48 ppm.
HRMS (M)+ calcd for C15H10ClN3O2 299.0462; found 299.0459.
4.1.3. 3-(2-Methoxyphenyl)-5-(4-nitrophenyl)-1H-pyrazole (3)
Compound 3 was synthesized from the procedure described for
1. Mp 226 °C. 1H NMR (300 MHz, DMSO-d6) d 3.92 (s, 3H), 7.05 (dd,
J = 7.5, 7.2 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 7.34 (s, 1H), 7.36 (dd,
J = 8.3, 7.2 Hz,1H), 7.77 (d, J = 7.5 Hz, 1H), 8.11 (d, J = 8.8 Hz, 2H),
8.28 (d, J = 8.8 Hz, 2H), 13.27 (br s, NH) ppm. 13C NMR (75 MHz,
4.1.10. 3-(4-Bromophenyl)-5-(4-nitrophenyl)-1H-pyrazole (10)
Compound 10 was synthesized from the procedure described
for 1. Mp 258 °C. 1H NMR (300 MHz, DMSO-d6) d 7.36 (s, 1H),
7.62 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 8.5 Hz, 2H), 8.06 (d, J = 8.7 Hz,
2H), 8.25 (d, J = 8.7 Hz, 2H), 13.80 (br s, NH) ppm. 13C NMR
(75 MHz, DMSO-d6) d 101.54, 121.21, 124.17, 125.87, 127.20,
131.83, 146.45 ppm. HRMS (M)+ calcd for C15H10ClN3O2
299.0462; found 299.0459.
DMSO-d6)
d 55.53, 103.27, 111.95, 120.72, 124.17, 125.79,
127.74, 129.70, 139.79, 146.33, 155.78 ppm. HRMS (M)+ calcd for
C16H13N3O3 295.0957; found 295.0955.
4.1.4. 3-(3-Methoxyphenyl)-5-(4-nitrophenyl)-1H-pyrazole (4)
Compound 4 was synthesized from the procedure described for
1. Mp 233 °C. 1H NMR (300 MHz, DMSO-d6) d 3.81 (s, 3H), 6.93 (d,
J = 6.7 Hz, 1H), 7.38 (dd, J = 7.2, 6.7 Hz,1H), 7.39 (d, J = 7.2 Hz, 1H),
7.40 (s, 1H), 7.44(s, 1H), 8.11 (d, J = 8.8 Hz, 2H), 8.31 (d,
J = 8.8 Hz, 2H), 13.70 (br s, NH) ppm. 13C NMR (75 MHz, DMSO-
4.1.11. 5-(4-Nitrophenyl)-3-(4-(trifluoromethyl)phenyl)-1H-
pyrazole (11)
Compound 11 was synthesized from the procedure described
for 1. Mp 244 °C. 1H NMR (300 MHz, DMSO-d6) d 7.58 (s, 1H),
7.84 (d, J = 8.3 Hz, 2H), 8.06 (d, J = 8.3 Hz, 2H), 8.12 (d, J = 8.8 Hz,
2H), 8.33 (d, J = 8.8 Hz, 2H), 13.93 (br s, NH) ppm. 13C NMR
(75 MHz, DMSO-d6) d 102.45, 124.29, 125.72, 125.93, 128.19,
146.61 ppm. HRMS (M)+ calcd for C16H10F3N3O2 333.0725; found
333.0730.
d6)
d 55.21, 101.41, 110.60, 113.92, 117.53, 124.19, 125.82,
125.97, 130.11, 146.44, 159.72 ppm. HRMS (M)+ calcd for
C16H13N3O3 295.0957; found 295.0959.

3276
A. Y. Shaw et al. / Bioorg. Med. Chem. 18 (2010) 3270–3278
4.1.12. 4-(3-Phenyl-1H-pyrazol-5-yl)aniline (12)
4.1.18. N,N-Dimethyl-4-(3-(4-(trifluoromethyl)phenyl)-1H-
pyrazol-5-yl)aniline (18)
To a solution of 1 (0.265 g, 1.0 mmol) in methanol (5 ml), palla-
dium on charcoal (5 mg, 10% w/w) was added. The mixture was
treated with hydrogen under atmospheric pressure for 16 h and fil-
tered. The filtrate was evaporated in vacuo and purified with silica
gel chromatography (ethyl acetate/hexane = 1:4) to yield 12. Mp
163 °C. 1H NMR (300 MHz, DMSO-d6) d 5.58 (br s, NH2), 6.64 (d,
J = 8.4 Hz, 2H), 6.90 (s, 1H), 7.29 (t, J = 7.3 Hz, 1H), 7.34 (dd,
J = 7.7, 7.3 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 7.7 Hz, 2H),
12.83 (br s, NH) ppm. 13C NMR (75 MHz, DMSO-d6) d 97.70,
111.67, 114.21, 125.02, 126.15, 127.42, 127.78, 128.67, 128.82,
148.08 ppm. HRMS (M)+ calcd for C15H13N3 235.1109; found
235.1113.
Compound 18 was synthesized from the procedure described
for 1. Mp 248 °C. 1H NMR (300 MHz, DMSO-d6) d 2.92 (s, 6H),
6.77 (d, J = 8.2 Hz, 2H), 7.07 (s, 1H), 7.63 (d, J = 8.2 Hz, 2H), 7.75
(d, J = 7.8 Hz, 2H), 8.04 (d, J = 7.8 Hz, 2H), 13.26 (br s, NH) ppm.
13C NMR (75 MHz, DMSO-d6) d 39.97, 98.52, 112.32, 116.94,
122.67, 125.60, 126.19, 127.62, 129.87, 137.94, 144.65, 149.76,
150.25 ppm.HRMS (M)+ calcd for C18H16F3N3 331.1296; found
331.1299.
4.1.19. 5-(4-Methoxyphenyl)-3-phenyl-1H-pyrazole (19)
Compound 19 was synthesized from the procedure described
for 1. Mp 178 °C. 1H NMR (300 MHz, DMSO-d6) d 3.77 (s, 3H),
7.00 (d, J = 8.0 Hz, 2H), 7.06 (s, 1H), 7.32 (t, J = 7.0, 1H), 7.43 (dd,
J = 7.2, 7.0, 2H), 7.77 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 7.2, 2H), 13.30
(br s, NH) ppm. 13C NMR (75 MHz, DMSO-d6) d 55.13, 98.80,
114.21, 125.08, 126.48, 127.62, 128.73, 158.99 ppm. HRMS (M)+
calcd for C16H14N2O 250.1106; found 250.1101.
4.1.13. 4-(3-(2-Methoxyphenyl)-1H-pyrazol-5-yl)aniline (13)
Compound 13 was synthesized from the procedure described
for 12. Mp 158 °C. 1H NMR (300 MHz, DMSO-d6) d 3.89 (s, 3H),
7.00 (d, J = 8.3 Hz, 2H), 7.01 (dd, J = 7.8, 7.3 Hz, 1H), 7.02 (s, 1H),
7.11 (d, J = 8.3 Hz, 1H), 7.31 (dd, J = 8.3, 7.8 Hz, 1H), 7.70 (d,
J = 8.3 Hz, 2H), 7.79 (d, J = 7.3 Hz, 1H) ppm. 13C NMR (75 MHz,
DMSO-d6)
d
55.48, 101.41, 111.89, 114.15, 118.30, 120.62,
4.1.20. 3-(4-Methoxyphenyl)-5-(4-(trifluoromethyl)phenyl)-1H-
pyrazole (20)
126.15, 127.66, 129.13, 155.87 ppm. HRMS (M)+ calcd for
C16H15N3O 265.1215; found 265.1218.
Compound 20 was synthesized from the procedure described
for 1. Mp 221 °C. 1H NMR (300 MHz, DMSO-d6) d 3.78 (s, 3H),
7.03 (d, J = 8.6 Hz, 2H), 7.20 (s, 1H), 7.75 (d, J = 8.6 Hz, 2H), 7.77
(d, J = 8.0 Hz, 2H), 8.04 (d, J = 8.0 Hz, 2H), 13.43 (br s, NH) ppm.
13C NMR (75 MHz, DMSO-d6) d 55.18, 99.43, 114.03, 114.41,
121.70, 125.51, 125.88, 126.55, 127.51, 137.66, 143.69, 149.74,
159.27 ppm. HRMS (M)+ calcd for C17H13F3N2O 318.0980; found
318.0987.
4.1.14. 4-(3-(3-Methoxyphenyl)-1H-pyrazol-5-yl)aniline (14)
Compound 14 was synthesized from the procedure described
for 12. Mp 86 °C. 1H NMR (300 MHz, CDCl3) d 3.64 (s, 3H), 5.80
(br s, NH2), 6.57 (d, J = 8.3 Hz, 2H), 6.63 (s, 1H), 6.81 (d, J = 7.7 Hz,
1H), 7.26 (dd, J = 7.7, 4.8 Hz, 1H), 7.27 (s, 1H), 7.27 (d, J = 4.8 Hz,
1H), 7.42 (d, J = 8.3 Hz, 2H), 12.82 (br s, NH) ppm. 13C NMR
(75 MHz, CDCl3) d 55.27, 99.11, 110.67, 114.24, 115.43, 118.36,
121.41, 126.96, 129.85, 133.55, 146.68, 147.94, 149.72,
160.01 ppm. HRMS (M)+ calcd for C16H15N3O 265.1215; found
265.1217.
4.1.21. 4-(3-(4-(Trifluoromethyl)phenyl)-1H-pyrazol-5-
yl)phenol (21)
A solution of 20 (0.32 g, 1.0 mmol) in CH2Cl2 (10 ml) was cooled
to ꢁ70 °C under argon, and boron tribromide (1.0 g, 2.0 mmol) was
added. The mixture was allowed to warm up to room temperature
over a period of 16 h and was then cooled to ꢁ70 °C. Methanol
(10 ml) was added to the reaction mixture, and the solution was
evaporated in vacuo and purified with silica gel chromatography
(ethyl acetate/hexane = 1:4) to afford 21. Mp 207 °C. 1H NMR
(300 MHz, DMSO-d6) d 6.84 (d, J = 8.5 Hz, 2H), 7.13 (s, 1H), 7.62
(d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.2 Hz, 2H), 8.03 (d, J = 8.2 Hz, 2H),
9.65 (br s, OH), 13.30 (br s, NH) ppm. 13C NMR (75 MHz, DMSO-
d6) d 99.28, 115.65, 125.51, 125.61, 125.66, 126.65, 126.59,
157.53 ppm. HRMS (M)+ calcd for C16H11F3N2O 304.0823; found
304.0825.
4.1.15. 4-(3-(4-Methoxyphenyl)-1H-pyrazol-5-yl)aniline (15)
Compound 15 was synthesized from the procedure described
for 12. Mp 173 °C. 1H NMR (300 MHz, DMSO-d6) d 3.77 (s, 3H),
5.25 (br s, NH2), 6.61 (d, J = 8.5 Hz, 2 H), 6.78 (s, 1H), 6.97
(d, J = 8.7 Hz, 2H), 7.46 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.7 Hz,
2H), 12.84 (br s, NH) ppm. 13C NMR (75 MHz, DMSO-d6) d
55.14, 97.02, 113.88, 114.09, 126.12, 126.35, 148.61,
158.75 ppm. HRMS (M)+ calcd for C16H15N3O 265.1215; found
265.1218.
4.1.16. 4-(3-(3,4-Dimethoxyphenyl)-1H-pyrazol-5-yl)aniline
(16)
4.1.22. 3-(4-Fluorophenyl)-5-(4-methoxyphenyl)-1H-pyrazole
(22)
Compound 16 was synthesized from the procedure described
for 12. Mp 105 °C. 1H NMR (300 MHz, DMSO-d6) d 3.77 (s, 3H),
3.80 (s, 3H), 5.27 (br s, NH2), 6.60 (d, J = 8.3 Hz, 2H), 6.84 (s, 1H),
6.98 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.38 (s, 1H), 7.46
(d, J = 8.3 Hz, 2H), 12.82 (br s, NH) ppm. 13C NMR (75 MHz,
DMSO-d6) d 55.54, 97.30, 108.93, 112.01, 113.88, 117.52, 126.13,
148.41, 148.58, 148.90 ppm. HRMS (M)+ calcd for C17H17N3O2
295.1321; found 295.1325.
Compound 22 was synthesized from the procedure described
for 1. Mp 203 °C. 1H NMR (300 MHz, DMSO-d6) d 3.78 (s, 3H),
7.01 (d, J = 8.4 Hz, 2H), 7.04 (s, 1H), 7.26 (dd, J = 7.6, 2.1 Hz, 2H),
7.74 (d, J = 8.4 Hz, 2H), 7.85 (dd, J = 7.6, 2.1 Hz, 2H), 13.22 (br s,
NH) ppm. 13C NMR (75 MHz, DMSO-d6) d 55.14, 98.71, 114.31,
121.98, 126.48, 126.99, 127.09, 159.10 ppm. HRMS (M)+ calcd for
C16H13FN2O 268.1012; found 268.1016.
4.1.17. 4-(3-(4-(Trifluoromethyl)phenyl)-1H-pyrazol-5-
yl)aniline (17)
4.1.23. 3-(4-Chlorophenyl)-5-(4-methoxyphenyl)-1H-pyrazole
(23)
Compound 17 was synthesized from the procedure described
for 12. Mp 266 °C. 1H NMR (300 MHz, DMSO-d6) d 5.33 (br s,
NH2), 6.61 (d, J = 8.4 Hz, 2H), 7.02 (s, 1 H), 7.46 (d, J = 8.4 Hz, 2H),
7.76 (d, J = 8.2 Hz, 2H), 8.02 (d, J = 8.2 Hz, 2H), 13.18 (br s, NH)
ppm. 13C NMR (75 MHz, DMSO-d6) d 98.27, 113.92, 122.61,
125.48, 126.27, 127.52, 148.98 ppm. HRMS (M)+ calcd for
C16H12F3N3 303.0983; found 303.0984.
Compound 23 was synthesized from the procedure described
for 1. Mp 221 °C. 1H NMR (300 MHz, DMSO-d6) d 3.78 (s, 3H),
7.01 (d, J = 7.9 Hz, 2H), 7.09 (s, 1H), 7.49 (d, J = 7.2 Hz, 2H), 7.73
(d, J = 7.9 Hz, 2H), 7.84 (d, J = 7.2 Hz, 2H), 13.26 (br s, NH) ppm.
13C NMR (75 MHz, DMSO-d6) d 55.33, 99.11, 114.47, 121.96,
126.68, 126.92, 128.86, 159.27 ppm. HRMS (M)+ calcd for
C16H13FN2O 284.0716; found 284.0713.

A. Y. Shaw et al. / Bioorg. Med. Chem. 18 (2010) 3270–3278
3277
4.1.24. 3-(4-Bromophenyl)-5-(4-methoxyphenyl)-1H-pyrazole
(24)
4.1.30. 2-(3-(Biphenyl-2-yl)-1H-pyrazol-5-yl)phenol (30)
Compound 30 was synthesized from the procedure described
for 21. Mp 135 °C. 1H NMR (300 MHz, DMSO-d6) d 6.28 (s, 1H),
6.81 (dd, J = 7.4, 7.3 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 7.12 (dd,
J = 8.0, 7.4 Hz, 1H), 7.22–7.24 (m, 2H), 7.31–7.33 (m, 3H), 7.42–
7.48 (m, 3H), 7.46 (d, J = 7.3 Hz, 1H), 7.66 (s, 1H), 10.83 (br s,
OH), 13.21 (br s, NH) ppm. 13C NMR (75 MHz, DMSO-d6) d
102.96, 116.42, 116.98, 119.27, 126.59, 127.20, 127.66, 128.18,
128.82, 129.07, 129.65, 130.56, 140.71, 142.68, 150.29,
155.21 ppm. HRMS (M)+ calcd for C21H16N2O 312.1263; found
312.1260.
Compound 24 was synthesized from the procedure described
for 1. Mp 223 °C. 1H NMR (300 MHz, DMSO-d6) d 3.78 (s, 3H),
7.01 (d, J = 8.7 Hz, 2H), 7.08 (s, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.74
(d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.4 Hz, 2H), 13.29 (br s, NH) ppm.
13C NMR (75 MHz, DMSO-d6) d 55.17, 99.02, 114.29, 120.54,
126.49, 127.04, 131.64, 159.10 ppm. HRMS (M)+ calcd for
C16H13BrN2O 328.0211; found 328.0208.
4.1.25. 3-(4-Methoxyphenyl)-5-(naphthalen-2-yl)-1H-pyrazole
(25)
Compound 25 was synthesized from the procedure described
for 1. Mp 266 °C. 1H NMR (300 MHz, DMSO-d6) d 3.80 (s, 3H),
7.03 (d, J = 8.4 Hz, 2H), 7.22 (s, 1H), 7.51 (d, J = 5.9 Hz, 1H), 7.55
(d, J = 5.9 Hz, 1H), 7.78 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 7.3 Hz, 2H),
7.97 (d, J = 7.3 Hz, 2H), 8.35 (s, 1H), 13.30 (br s, NH) ppm. 13C
NMR (75 MHz, DMSO-d6) d 55.17, 99.04, 114.41, 123.33, 125.73,
126.49, 127.63, 127.91, 132.45, 133.20, 143.47, 151.12,
159.17 ppm. HRMS (M)+ calcd for C20H16N2O 300.1263; found
300.1264.
4.1.31. 4-(3-(Biphenyl-2-yl)-1H-pyrazol-5-yl)phenol (31)
Compound 31 was synthesized from the procedure described
for 21. Mp 119 °C. 1H NMR (300 MHz, DMSO-d6) d 5.84 (s, 1H),
6.74 (d, J = 8.3 Hz, 2H), 7.21–7.23 (m, 2H), 7.27–7.36 (m, 3H),
7.34 (d, J = 8.3 Hz, 2H), 7.41–7.45 (m, 3H), 7.69 (s, 1H), 9.59 (br s,
OH), 12.75, 13.00 (br s, NH) ppm. 13C NMR (75 MHz, DMSO-d6) d
101.50, 115.52, 126.27, 126.93, 127.45, 128.01, 129.09, 129.18,
130.38, 140.27, 157.14 ppm. HRMS (M)+ calcd for C21H16N2O
312.1263; found 312.1264.
4.1.26. 3-(Biphenyl-2-yl)-5-(4-methoxyphenyl)-1H-pyrazole
(26)
4.1.32. 3-(Biphenyl-2-yl)-5-(4-nitrophenyl)-1H-pyrazole (32)
Compound 32 was synthesized from the procedure described
for 1. Mp 213 °C. 1H NMR (300 MHz, DMSO-d6) d 6.31 (s, 1H),
7.20–7.24 (m, 2H), 7.31–7.36 (m, 3H), 7.44(m, 1H), 7.50–7.52 (m,
2H), 7.60 (s, 1H), 7.87 (d, J = 8.7 Hz, 2H), 8.22 (d, J = 8.7 Hz, 2H),
13.29 (br s, NH) ppm. 13C NMR (75 MHz, DMSO-d6) d 103.84,
124.11, 125.59, 125.98, 127.20, 127.64, 128.14, 128.99, 129.54,
130.51, 140.00, 140.38, 140.60, 143.45, 146.28, 148.24 ppm. HRMS
(M)+ calcd for C21H15N3O2 341.1164; found 341.1165.
Compound 26 was synthesized from the procedure described
for 1. Mp 159 °C. 1H NMR (300 MHz, DMSO-d6) d 3.74 (s, 3H),
5.89 (s, 1H), 6.92 (d, J = 8.4 Hz, 2H), 7.21–7.24 (m, 2H), 7.30–7.35
(m, 4H), 7.45(d, J = 8.4 Hz, 2H), 7.45(m, 2H), 7.68 (s, 1H), 12.80,
13.07 (br s, NH) ppm. 13C NMR (75 MHz, DMSO-d6) d 55.09,
101.39, 114.18, 126.18, 126.94, 127.46, 128.01, 129.08, 129.17,
130.38, 140.30, 158.84 ppm. HRMS (M)+ calcd for C22H18N2O
326.1419; found 326.1425.
4.1.33. 4-(3-(Biphenyl-2-yl)-1H-pyrazol-5-yl)aniline (33)
Compound 33 was synthesized from the procedure described
for 12. Mp 93 °C. 1H NMR (300 MHz, DMSO-d6) d 5.22 (br s, NH2),
5.74(s, 1H), 6.52 (d, J = 8.4 Hz, 2H), 7.19–7.24 (m, 2H), 7.24 (d,
J = 8.4 Hz, 2H), 7.27–7.33 (m, 4H), 7.38–7.43 (m, 2H), 7.70 (s, 1H),
12.81 (br s, NH) ppm. 13C NMR (75 MHz, DMSO-d6) d 100.70,
113.81, 125.86, 126.84, 127.37, 127.66, 127.96, 129.09, 130.34,
140.19, 141.55, 148.52 ppm. HRMS (M)+ calcd for C21H17N3
311.1422; found 311.1418.
4.1.27. 3-(Biphenyl-2-yl)-5-(4-(trifluoromethyl)phenyl)-1H-
pyrazole (27)
Compound 27 was synthesized from the procedure described
for 1. Mp 170 °C. 1H NMR (300 MHz, DMSO-d6) d 6.19 (s, 1H),
7.20–7.24 (m, 2H), 7.31–7.36 (m, 3H), 7.37(m, 1H), 7.40 (m, 2H),
7.64 (s, 1H), 7.72 (d, J = 7.8 Hz, 2H), 7.79 (d, J = 7.8 Hz, 2H), 13.29
(br s, NH) ppm. 13C NMR (75 MHz, DMSO-d6) d 103.22, 125.35,
125.58, 125.99, 126.85, 127.18, 127.43, 127.64, 128.84, 129.00,
129.43, 130.51, 137.51, 140.50, 140.57, 143.14, 148.74 ppm. HRMS
(M)+ calcd for C20H16N2O 364.1187; found 364.1197.
4.2. Cell culture
4.1.28. 3-(Biphenyl-2-yl)-5-(2-methoxyphenyl)-1H-pyrazole
(28)
Cancer cells were purchased from Bioresource Collection and
Research Center in, Hsinchu, Taiwan. Each cell line was maintained
in the standard medium and grown as a monolayer in Dulbecco’s
Modified Eagle Medium (DMEM) containing 10% fetal bovine ser-
um, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml strep-
tomycin. Cultures were maintained at 37 °C with 5% CO2 in a
humidified atmosphere.
Compound 28 was synthesized from the procedure described
for 1. Mp 122 °C. 1H NMR (300 MHz, DMSO-d6) d 3.72 (s, 3H),
6.00 (s, 1H), 6.94 (dd, J = 7.4, 7.3 Hz, 1H), 7.03 (d, J = 8.2 Hz, 1H),
7.22 (dd, J = 8.2, 7.3 Hz, 1H), 7.22–7.25 (m, 2H), 7.34 (m, 4H),
7.42 (d, J = 7.4 Hz, 1H), 7.42 (m, 2H), 7.78 (s, 1H), 12.93 (br s, NH)
ppm. 13C NMR (75 MHz, DMSO-d6) d 55.14, 105.13, 111.88,
117.78, 120.57, 126.80, 127.04, 127.41, 128.03, 128.50, 129.12,
130.37, 132.63, 138.60, 140.17, 141.82, 150.21, 155.41 ppm. HRMS
(M)+ calcd for C22H18N2O 326.1419; found 326.1424.
4.3. MTT assay for cell viability
OVCA, SW620, H460 and AGS cells were plated in 96-well
microtiter plates at a density of 3000, 3000, 3000 and 5000 cells/
well, respectively. After 24 h incubation, cells were treated with
vehicle alone (control) or compounds (drugs were dissolved in
DMSO previously) at the concentrations indicated. Treated cells
were further incubated for 48 h. Cell survival is expressed as per-
centage of control cell growth. The 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide (MTT, 2 mg/ml) dye reduction
assay in 96-well microplates was used. The assay is dependent
on the reduction of MTT by mitochondrial dehydrogenases of
viable cell to a blue formazan product, which come be measured
spectrophotometrically. Tumor cells were incubated in each well
4.1.29. 3-(Biphenyl-2-yl)-5-(3-methoxyphenyl)-1H-pyrazole (29)
Compound 29 was synthesized from the procedure described
for 1. Mp 63 °C. 1H NMR (300 MHz, DMSO-d6) d 3.75 (s, 3H), 6.00
(s, 1H), 6.84 (d, J = 7.2 Hz, 1H), 7.15 (s, 1H), 7.15 (d, J = 6.8 Hz,
1H), 7.21–7.27 (m, 2H), 7.24 (dd, J = 7.2, 6.8 Hz, 1H), 7.30–7.37
(m, 4H), 7.46 (m, 2H), 7.69 (s, 1H), 12.93, 13.25 (br s, NH) ppm.
13C NMR (75 MHz, DMSO-d6) d 55.04, 102.77, 110.32, 113.07,
117.30, 126.98, 127.50, 128.05, 129.09, 129.22, 129.88, 130.39,
140.37, 159.55 ppm. HRMS (M)+ calcd for C22H18N2O 326.1419;
found 326.1421.

3278
A. Y. Shaw et al. / Bioorg. Med. Chem. 18 (2010) 3270–3278
with serial dilutions of the tested compounds. After two days of
incubation (37 °C, 5% CO2 in a humid atmosphere) 100 l of MTT
(2 mg/ml in PBS) was added to each well and the plate was incu-
used the Bonferroni t-test after ANOVA for multi-group compari-
son and Student’s t-test for two-group comparison. P <0.05 was
considered significant.
l
bated for a further 2 h (37 °C). The resulting formazan was dis-
solved in 100 ll DMSO and read at 570 nm. The percentage of
4.7. CoMFA 3D-QSAR analysis
growth inhibition was calculated by the following equation: per-
centage growth inhibition = (1 ꢂ At/Ac) ꢂ 100, where At and Ac
represent the absorbance in treated and control cultures, respec-
tively. The drug concentration causing a 50% cell growth inhibition
CoMFA (Comparative Molecular Field Analysis) was carried out
by software Sybyl 8.1 (Tripos International, St. Louis, Missouri,
USA). MMFF94 charge assignment and force field were applied to
prepare compound structures. The geometries were minimized
by Powell algorithm to an energy convergence criterion of
0.01 kcal/mol Å, within 100,000 iterations. Each compound in its
energetically minimized geometry was aligned. Two CoMFA
descriptors including steric (Lennard–Jones 6–12 potential) and
electrostatic (Coulombic potential) field energies were calculated
with a sp3 carbon atom carrying +1.0 charge to serve as a probe
atom using Sybyl default parameters. The optimal number of com-
ponents (ONC = 2) was recommended after a leave-one-out cross-
validated run. The final training set model and coefficients such as
non-validation R2 and F-value were obtained by using the optimal
number of components and all the data points in the training set.
The cross-validated q2 = 0.671 and the subsequent non-validation
R2 = 0.846 meet the criteria q2 = 0.5 and R2 = 0.8. The contribu-
tions for steric and electrostatic fields are 0.459 and 0.561,
respectively.
(GI50
) was determined by interpolation from dose–response
curves. All determinations were carried out in four to six separated
experiments.
4.4. Determination of apoptosis by flow cytometry
Apoptosis and cell cycle profile were assessed by DNA fluores-
cence flow cytometry. OVCA cells treated with 0.5% DMSO or
tested compounds at indicated concentrations for 24 h were har-
vested, rinsed in PBS, resuspended and fixed in 80% ethanol, and
stored at ꢁ20 °C in fixation buffer until ready for analysis. Then
the pellets were suspended in 1 ml of fluorochromic solution
(0.08 mg/ml PI (propidium iodide), 0.1% TritonX-100 and 0.2 mg/
ml RNase A in 1ꢂ PBS) at room temperature in the dark for
30 min. The DNA content was analyzed by FACScan flow cytometer
(Becton Dickinson, Mountain View, CA) and CELLQUEST software
(Becton Dickinson). The population of apoptotic nuclei (subdiploid
DNA peak in the DNA fluorescence histogram) was expressed as
the percentage in the entire population.
Acknowledgments
Financial support from the National Science Council of the
Republic of China and Tamkang University to A. Y. Shaw is grate-
fully acknowledged.
4.5. Protein extraction and Western blotting
After the treatment of cells with vehicle (1% DMSO), 26 and 27
for indicated time treatment, the cells were washed twice with PBS
and reaction was terminated by the addition of 100
For Western blot analysis, the amount of proteins (50
l
l lysis buffer.
g) were
References and notes
l
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Data are presented as means SEM (standard error of the
mean) from three independent experiments. Statistical analyses