Novel potent and selective calcium-release-activated calcium (CRAC) channel inhibitors. Part 1: Synthesis and inhibitory activity of 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-2-thiophenecarboxamides

Article abstract of DOI:10.1016/j.bmc.2006.03.024

We synthesized and evaluated a series of 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-2-thiophenecarboxamides to identify potent inhibitors of calcium-release-activated calcium (CRAC) channels with greater selectivity than voltage-operated calcium (VOC) channels. These efforts resulted in identification of compounds 22 and 24. The former exhibits highly potent and selective CRAC channel inhibitory activity, and the latter inhibited phytohemagglutinin-induced interleukin-2 production by Jurkat T lymphocytes and concanavalin A-induced hepatitis in mice.

Full text of DOI:10.1016/j.bmc.2006.03.024

Bioorganic & Medicinal Chemistry 14 (2006) 4750–4760
Novel potent and selective calcium-release-activated calcium
(CRAC) channel inhibitors. Part 1: Synthesis and inhibitory
activity of 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-2-
thiophenecarboxamides
Yasuhiro Yonetoku,* Hirokazu Kubota, Yoshinori Okamoto, Akira Toyoshima,ꢀ
Masashi Funatsu, Jun Ishikawa, Makoto Takeuchi,
Mitsuaki Ohta and Shin-ichi Tsukamoto
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
Received 16 February 2006; revised 14 March 2006; accepted 15 March 2006
Available online 31 March 2006
Abstract—We synthesized and evaluated a series of 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-2-thiophenecarboxamides to
identify potent inhibitors of calcium-release-activated calcium (CRAC) channels with greater selectivity than voltage-operated cal-
cium (VOC) channels. These efforts resulted in identification of compounds 22 and 24. The former exhibits highly potent and selec-
tive CRAC channel inhibitory activity, and the latter inhibited phytohemagglutinin-induced interleukin-2 production by Jurkat T
lymphocytes and concanavalin A-induced hepatitis in mice.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
The early stages in T lymphocyte activation can be
divided into events pre- and post-intracellular Ca2+ re-
lease.3 Before Ca2+ release, the stimulation of cell sur-
face receptors induces the generation of IP3, followed
by the release of Ca2+ from the endoplasmic reticulum
(ER).4 After the depletion of Ca2+ from ER stores, cal-
cium-release-activated calcium (CRAC) channels are
activated and capacitative Ca2+ influx through these
channels sustains a high intracellular Ca2+ concentra-
tion ([Ca2+]i).5 This prolonged high [Ca2+]i is crucial
for cytosolic signal transduction which induces the pro-
duction of lipid mediators (e.g., LTD4), cytokines (e.g.,
interleukin-2 (IL-2)), and matrix metalloproteinases,
which are known to play important roles in the patho-
genesis of inflammation and autoimmune diseases.
Ca2+ channels in the plasma membrane are known to
regulate Ca2+ influx, and as a result they regulate a vari-
ety of cellular functions. Voltage-operated calcium
(VOC) channels, located in the nervous, endocrine, car-
diovascular, and skeletal systems, are known to be mod-
ulated by the membrane potential, and are classified into
L, N, P, Q, R, and T subtypes.1 Some of these molecules
have been well characterized, and their inhibitors, such
as nifedipine, have been used for the treatment of hyper-
tension1b and brain dysfunction.1c In contrast, Ca2+
channels in inflammatory cells, such as lymphocytes,
mast cells and neutrophils, are activated regardless of
their membrane potential2a and are known as store-
operated Ca2+ channels. They have been shown to play
important roles in the pathogenesis and exacerbation of
inflammatory and autoimmune diseases.2b–e
These facts suggested that CRAC channel inhibitors
would be useful for the treatment of diseases, such as
asthma and rheumatoid arthritis, caused by the activa-
tion of inflammatory cells.6 However, inhibitors which
affect both CRAC and VOC channels are predicted to
show undesirable side-effects in the nervous and cardio-
vascular systems. Therefore, it may be necessary for
CRAC channel inhibitors to exhibit sufficient selectivity
over VOC channels to be useful as anti-inflammatory
drugs.
Keywords: Anti-inflammatory; Channel inhibitors; Ca2+-release-acti-
vated Ca2+ channel; CRAC channel; Interleukin-2; Quantitative stru-
cture–activity relationship.
*
Corresponding author. Tel.: +81 0 29 847 8611; fax: +81 0 29 847
8313; e-mail: yasuhiro.yonetoku@jp.astellas.com
ꢀ
Present address: Development Division, Astellas Pharma Inc., 3-17-1
Hasune, Itabashi, Tokyo 174-8612, Japan
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.024

Y. Yonetoku et al. / Bioorg. Med. Chem. 14 (2006) 4750–4760
4751
In the last decade, several compounds including SK&F
96365 (1),7 LOE 908 (2),8 and L-651582 (3,9 Fig. 1) have
been reported to inhibit CRAC channels. However,
their potency and selectivity are unlikely to be adequate
for their use as anti-inflammatory drugs. To find potent
and selective CRAC channel inhibitors, we screened our
chemical library and identified N-(4-chlorophenyl)-5-(1-
methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-2-thiophen-
ecarboxamide (4,10 Fig. 1) as a lead compound. We have
studied the structure–activity relationships (SARs) of
this compound and in this paper, we describe the synthe-
sis of a series of 5-(1-methyl-3-trifluoromethyl-1H-pyra-
zol-5-yl)-2-thiophenecarboxamides, and the SARs of
their 4-chlorophenyl moiety.
Schemes 1–3. In the presence of sodium methoxide
(NaOMe) and ethyl trifluoroacetate, 2-acetylthiophene
5 was converted to the dione 6,11 and regioselective
cyclization of compound 6 with methylhydrazine pro-
duced the pyrazole 7 (Scheme 1). The structure of com-
pound 7 was determined as illustrated in Scheme1, using
the nuclear Overhauser effect (NOE), observed between
the protons of the N-methyl group (4.01 ppm) and the
thiophene (7.21 ppm). Lithiation of compound 7 with
n-butyllithium (n-BuLi) in tetrahydrofuran (THF), fol-
lowed by treatment with ethyl chloroformate, gave the
ester 8, which was hydrolyzed with aqueous NaOH to
form the acid 9. Compound 9 was treated with oxalyl
chloride and the resultant acyl chloride 10 was allowed
to react with amines to form 5-(1-methyl-3-
trifluoromethyl-1H-pyrazol-5-yl)-2-thiophenecarboxa-
mides (11–35). The 4-hydroxyphenyl (36) and the
1-tert-butoxycarbonylpiperidin-4-yl (37) derivatives
were prepared by the reaction of compound 9 with the
corresponding amines12 in the presence of 1-ethyl-3-(3-
2. Chemistry
The 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-2-
thiophenecarboxamides were prepared as shown in
N
MeO
OMe
OMe
MeO
MeO
OMe
N
N
O
O
N
MeO
OMe
OMe
LOE 908 (2)
OMe SK&F 96365 (1)
O
Cl
O
Me
N
H2N
H
N
NH2
N
S
N
Cl
N
O
N
Cl
Cl
F3C
4
L-651582 (3)
Figure 1. Structures of SK&F 96365 (1), LOE 908 (2), L-651582 (3), and compound 4.
NOE
4.01
ppm
7.21 ppm
Me
F3C
a
b
c
N
N
S
S
S
O
O
O
F3C
5
6
7
Me
Me
N
Me
N
d
e
N
N
Cl
OEt
OH
N
N
S
S
S
O
O
O
F3C
F3C
F3C
9
10
8
g
f
Me
N
Me
N
H
N
H
N
N
N
R
R
S
S
O
O
F3C
F3C
11 35
36 R=
37 R=
OH
N
Boc
Scheme 1. Reagents: (a) CF3CO2Et, NaOMe, MeOH; (b) MeNHNH2, AcOH–EtOH; (c) n-BuLi, THF, then ClCO2Et; (d) 1 M NaOH, EtOH;
(e) (COCl)2, cat. DMF, THF; (f) R-NH2, base; (g) R-NH2, EDC Æ HCl, THF.

4752
Y. Yonetoku et al. / Bioorg. Med. Chem. 14 (2006) 4750–4760
Me
Me
N
H
H
N
a
N
N
N
N
S
S
O
O
NH2
NO2
F3C
F3C
38
15
Scheme 2. Reagents: (a) Zn, NH4Cl, EtOH–H2O.
channels on PC12-h5 cells.14 The results are summarized
in Tables 1–3.
Me
H
N
a
N
N
37
S
O
NH
.HCl
We first examined the effects of substitutions at the 4-po-
sition in the phenyl group of compound 4 on the inhib-
itory activity and selectivity (Table 1). Substituting
fluoro (11) and bromo (12) groups for the chloro group
did not change the compounds’ inhibitory effects on
CRAC channels, but reduced their inhibitory activity
against VOC channels. Substituting ethoxycarbonyl
(13), cyano (14), and nitro (15) groups reduced the
inhibitory effect on CRAC channels, suggesting that
substituting groups that were highly electron-withdraw-
ing at the 4-position would not favor increased CRAC
channel inhibitory activity. In contrast, removal of the
chloro group significantly increased CRAC channel
inhibitory activity, with compound 16 showing an IC50
value of 0.062 lM, and in addition exhibiting high selec-
tivity for CRAC channels with an index of over 31. The
methyl derivative 17 was almost equipotent to com-
pound 4, whereas the isopropyl derivative 18 was a less
effective inhibitor. From these results, we speculated
that the ability to inhibit CRAC channels was sensitive
to the bulkiness of the group at the 4-position. Hydroxy
(36) and methoxy (19) groups, which maintained the
potency, improved the selectivity for CRAC channels,
with indices of approximately 20. In contrast, substitut-
ing nitrogen-containing groups such as amino (38) and
dimethylamino (20) groups at this position dramatically
decreased the inhibitory activity.
F3C
39
Scheme 3. Reagents: (a) 4 M HCl (g), AcOEt.
Table 1. Biological properties of 5-(1-methyl-3-trifluoromethyl-1H-
pyrazol-5-yl)-N-(4-substituted-phenyl)-2-thiophenecarboxamides
Me
H
N
N
N
S
O
R
F3C
Compound
R
IC50 (lM)
CRAC channel
selectivity indexc
CRACa
VOCb
4
11
12
13
14
15
16
17
18
36
19
38
20
Cl
0.13
0.14
0.27
46%d
0.56
1.4
0.75
3.6
5.8
26
F
Br
2.6
10
—
CO2Et
CN
NO2
H
NTe
1.5
2.7
2.9
31
4.0
1.9
0.062
0.37
0.92
0.24
0.44
4.1
Me
iPr
2.4
1.2
6.5
1.3
18
OH
OMe
NH2
NMe2
4.3
24%f
69%f
NT
>23g
—
—
8%f
To investigate the SARs in detail, we performed a quan-
titative analysis using various parameters for side
groups, such as p,15 molecular refractivity,16 Swein
a Inhibition of Ca2+ influx through CRAC channels on Jurkat T
lymphocytes. See experimental section.
b Inhibition of Ca2+ influx through VOC channels on PC12-h5 cells.
See experimental section.
c IC50 for VOC channels/IC50 for CRAC channels.
d % inhibition at 0.3 lM.
Table 2. Biological properties of 5-(1-methyl-3-trifluoromethyl-1H-
pyrazol-5-yl)-N-(substituted-phenyl)-2-thiophenecarboxamides
e Not tested.
f % inhibition at 10 lM.
Me
g IC50 value for VOC channels was taken as >10 lM.
H
N
N
N
S
R
O
F3C
dimethylaminopropyl)carbodiimide hydrochloride (EDC Æ
HCl), as shown in Scheme 1. Reduction of the 4-nitro-
phenyl derivative 15 with zinc powder gave the 4-amino-
phenyl derivative 38 (Scheme 2), and the piperidin-4-yl
derivative 39 was obtained from compound 37 by treat-
ment with HCl (Scheme 1).
Compound
R
IC50 (lM)
CRAC channel
selectivity indexc
CRACa
VOCb
4
21
22
23
24
25
4-Cl
3-Cl
0.13
1.1
0.75
2.2
5.8
2.0
2-Cl
2-F
0.050
0.090
0.30
0.14
0%d
4.0
>200e
44
2-Me
2,4-diCl
10
18%d
33
>71e
3. Results and discussion
a–cSee the corresponding footnotes to Table 1.
The compounds synthesized as described above were
evaluated for their ability to inhibit Ca2+ influx through
CRAC channels on Jurkat T lymphocytes13 and VOC
d % inhibition at 10 lM.
e IC50 value for VOC channels was taken as >10 lM.

Y. Yonetoku et al. / Bioorg. Med. Chem. 14 (2006) 4750–4760
4753
Table 3. Biological properties of 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-N-substituted-2-thiophenecarboxamides
Me
H
N
N
N
R
S
O
F3C
Compound
R
IC50 (lM)
CRAC channel selectivity indexc
CRACa
VOCb
4
26
27
28
29
30
31
32
33
34
35
39
4-Chlorophenyl
Thiophen-2-yl
Thiophen-3-yl
1-Methylpyrrol-2-yl
Pyridin-3-yl
0.13
0.10
0.61
0.58
0.99
0.42
35%f
6%d
12%d
1.7
0.75
1.2
5.8
12
2.3
10
47%d
3.8
17
>10e
Pyridin-4-yl
Thiazol-2-yl
4.1
10
—
NTg
NTg
NTg
17%d
4.3
1,2,4-Triazol-4-yl
Tetrazol-5-yl
Ethyl
—
—
>6e
12
Cyclohexyl
Piperidin-4-yl
0.36
10%d
NTg
—
a–eSee the corresponding footnotes to Table 1.
f % inhibition at 1 lM.
g Not tested.
18
and Lupton F/R,17 rpara
molecular volume, molecular surface area, and Verloop
,
Taft Es,19 Verloop L,20
IC50 value of 0.050 lM, was 3-fold more potent than
compound 4 as a CRAC channel inhibitor. Surprisingly,
this compound showed no inhibition of VOC channels
at 10 lM, and an excellent selectivity for CRAC chan-
nels with an index of more than 200. Other substitutions
at the 2-position (23–25) also improved the selectivity of
the compounds for CRAC channels, implying that sub-
stitutions at this position would generally be expected to
reduce inhibitory effects on VOC channels and produce
CRAC channel-selective compounds.
B5.20 For the series of compounds shown in Table 1, the
para values of the group substituted at the 4-position in
the phenyl group showed the best correlation (r = 0.81)
with the ability to inhibit CRAC channels. These results
are shown in Figure 2 and described by the following
parabolic equation:
r
pIC50 ¼ ꢀ2:04ðꢁ0:44Þr2para þ 6:74 ðEq. 1; n ¼ 13;
r ¼ 0:81; s ¼ 0:37; F ¼ 21:46Þ.
Next, we investigated the effects of substituting heteroa-
romatic and aliphatic groups for the 4-chlorophenyl
moiety of compound 4. As shown in Table 3, in the ser-
ies of compounds containing heteroaromatics, the abili-
ty of the compound to inhibit CRAC channels was
unaffected by a 2-thienyl group (26), but was reduced
5-fold in the 3-thienyl derivative 27. The 1-methylpyrrol-
yl (28) and pyridyl (29, 30) derivatives were also less po-
tent than compound 4, and the introduction of thiazolyl
(31), 1,2,4-triazolyl (32), and tetrazolyl (33) groups
resulted in dramatic reductions in the inhibition of
CRAC channels. Among the compounds with aliphatic
groups (34, 35, and 39), the cyclohexyl derivative 35 was
found to show the most potent CRAC channel inhibito-
ry activity and was almost as potent as compound 4.
These results implied that aromatic groups were not
essential features of CRAC channel inhibitors.
These results showed that both highly electron-with-
drawing and highly electron-donating groups tended
to dramatically reduce the inhibitory effect on CRAC
channels. On the other hand, compounds containing
groups with rpara values of around 0 would be expected
to act as highly potent inhibitors.
In the series of compounds with chloro groups in the
phenyl group (4, 21, and 22), a 3-chloro group (21)
decreased both the activity and the selectivity (Table
2). In contrast, the 2-chloro derivative (22), with an
7.5
7
6.5
6
To investigate the SARs of this series in more detail, we
attempted to calculate logP (clogP) values for the acet-
amides 40–50 using the ACD/logP program.21 The clogP
values obtained were compared with the CRAC channel
inhibitory activity of the corresponding 5-(1-methyl-3-
trifluoromethyl-1H-pyrazol-5-yl)-2-thiophenecarboxa-
mides (4, 26–30, 32–35, 39, respectively), and the results
are shown in Table 4. Among the acetamides bearing
aromatic groups (40–47), the 4-chlorophenyl (40) and
5.5
5
4.5
-1
-0.5
0
0.5
1
para
σ
Figure 2. Correlation between Hammett constant (rpara) and pIC50
The pIC50 values of compounds 13 and 20 are plotted as 6.5 and 5.0,
.
respectively.

4754
Y. Yonetoku et al. / Bioorg. Med. Chem. 14 (2006) 4750–4760
Table 4. clogP values of acetamides 40–50 and CRAC channel inhibitory activity of corresponding 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-
2-thiophenecarboxamides
H
Me
N
R
O
clogPa
Compound
CRAC IC50 (lM)
b
Compound
R
40
41
49
42
43
45
44
48
50
46
47
4-Chlorophenyl
Thiophen-2-yl
Cyclohexyl
2.05
1.43
4
26
35
27
28
30
29
34
39
32
33
0.13
0.10
0.36
0.61
0.58
0.42
0.99
1.7
10%c
6%c
12%c
1.01
0.76
Thiophen-3-yl
1-Methylpyrrol-2-yl
Pyridin-4-yl
0.74
0.59
Pyridin-3-yl
Ethyl
0.26
ꢀ0.52
ꢀ0.85
ꢀ1.43
ꢀ1.47
Piperidin-4-yl
1,2,4-Triazol-4-yl
Tetrazol-5-yl
a ACD/logP values.
b See the footnote a of Table 1.
c % inhibition at 10 lM.
Table 5. Biological properties of 5-(1-methyl-3-trifluoromethyl-1H-
pyrazol-5-yl)-2-thiophenecarboxamides in vitro
thiophen-2-yl (41) derivatives gave clogP values greater
than 1, and the corresponding compounds 4 and 26 were
potent inhibitors of CRAC channels. In contrast, 1,2,4-
triazol-4-yl (46) and tetrazol-5-yl (47) groups produced
hydrophilic acetamides and the corresponding thio-
phene-2-carboxamides 32 and 33 failed to inhibit CRAC
channels. From these results, we hypothesized that a
hydrophobic acetamide moiety would be an essential
feature of potent inhibitors of CRAC channels. This
speculation was supported by the fact that compounds
27–30, whose corresponding acetamides possessed
clogP values of 0.2–0.8, showed moderate activity with
IC50 values of 0.4–1.0 lM. In addition, the clogP value
of the cyclohexyl derivative (49) was the highest in the
N-aliphatic acetamide series 48–50, and the N-cyclo-
hexylthiophene-2-carboxamide 35 was the most potent
CRAC channel inhibitor of compounds 34, 35, and 39
(Table 4).
Compound
IC50 (lM)
CRACa
IL-2b
4
11
12
16
17
36
22
23
24
25
26
28
29
35
0.13
0.14
0.27
0.062
0.37
0.24
0.050
0.09
0.30
0.14
0.10
0.58
0.99
0.36
0.53
0.75
1.8
0.85
0.54
1.5
0.14
0.023
0.053
0.026
0.70
0.20
1.4
0.076
a See the footnote a of Table 1.
b Inhibition of PHA-induced IL-2 production in Jurkat T lymphocytes.
See experimental section.
A selection of compounds which showed potent and
selective inhibitory activity for CRAC channels (11,
12, 16, 17, 22–26, 28, 29, 35, and 36) was tested for their
ability to inhibit phytohemagglutinin (PHA)-induced
IL-2 production in Jurkat T lymphocytes7 (Table 5).
Compound 4 inhibited IL-2 production with an IC50
value of 0.53 lM. Substitutions at the 2-position of the
phenyl group (22–25) increased the inhibitory activity,
with compounds 23 and 25 in particular inhibiting IL-
2 production with IC50 values of 0.023 and 0.026 lM,
respectively. The 1-methylpyrrolyl derivative 28 was also
a more potent inhibitor than compound 4 in this assay,
despite having shown a 4-fold lower potency as a CRAC
channel inhibitor. These results suggested that substitu-
tions adjacent to the amide bond would be likely to in-
crease the inhibitory effect of compounds on IL-2
production. The cyclohexyl derivative 35 inhibited IL-
2 production with an IC50 value of 0.076 lM, which
showed that hydrophobic groups in this moiety may
be crucial for inhibitory activity against both IL-2 pro-
duction and CRAC channels. Some compounds which
were potent inhibitors of both CRAC channel and IL-
2 production (16, 22–25, 35) were further tested for their
ability to inhibit concanavalin A (Con A)-induced hep-
atitis in mice (Table 6).22 Among the compounds tested,
Table 6. Effects of selected compounds on Con A-induced hepatitis in
mice
Compound
In vivo hepatitisa
ED50 (mg/kg p.o.) % inhibition at 30 mg/kg p.o.
16
22
23
24
25
35
48
46
32
12.7
67
0
9
a Inhibitory activities against Con A-induced liver injury in mice. See
experimental section.

Y. Yonetoku et al. / Bioorg. Med. Chem. 14 (2006) 4750–4760
4755
24 was found to be the most potent inhibitor with an
ED50 value of 12.7 mg/kg p.o.
(150 mL), and the whole mixture was stirred for
20 min at room temperature. It was cooled on ice-bath,
then added ethyl trifluoroacetate (25.0 g, 176 mmol),
and the whole mixture was heated to reflux for 19 h.
H2O (300 mL) was added to the mixture and extracted
with AcOEt, washed with brine, dried, and concentrated
in vacuo to give a pale brown solid (6, 34.7 g). This
crude compound (6, 20.0 g) was added to a mixture of
methylhydrazine (4.56 g, 99.0 mmol), AcOH (20 mL),
and EtOH (200 mL), and the whole mixture was heated
to reflux for 30 min. The mixture was concentrated in
vacuo, and saturated aqueous NaHCO3 was added to
the residue and extracted with AcOEt, washed with
brine, dried, and concentrated in vacuo. The residue
was purified by column chromatography (hexane/
4. Conclusion
We designed and synthesized novel 5-(1-methyl-3-trifluo-
romethyl-1H-pyrazol-5-yl)-2-thiophenecarboxamides
based on compound 4 and evaluated their ability to inhi-
bit CRAC and VOC channels. SAR study showed a rela-
tionship between the ability of compounds to inhibit
CRAC channels and the rpara values of the groups substi-
tuted at the 4-position on the phenyl ring, with values of
around 0 being most favorable for a high potency. In
addition, substitutions at the 2-position resulted in an
improvement of the selectivity of the CRAC channel
inhibitors and hydrophobic moieties such as chlorophe-
nyl and cyclohexyl groups were shown to be essential to
potent CRAC channel inhibitory activity. We identified
compound 22 as the most promising novel CRAC chan-
nel inhibitor, with an IC50 value of 0.050 lM and a selec-
tivity index of more than 200. Additionally, some other
compounds including compound 24 were found to inhibit
PHA-induced IL-2 production in vitro with IC50 values
of the order of 10ꢀ8 M, and Con A-induced hepatitis in vi-
vo, after oral administration. This study demonstrated
that T lymphocyte function can be inhibited by CRAC
channel inhibitors both in vitro and in vivo. These com-
pounds have potential uses as a new class of orally active
anti-inflammatory agents.
1
AcOEt, 20:1–10:1) to give 7 (11.8 g, 60%): H NMR
(CDCl3) d 4.01 (3H, s), 6.64 (1H, s), 7.15 (1H, dd,
J = 5.2, 3.7 Hz), 7.21 (1H, dd, J = 3.7, 1.3 Hz), 7.47
(1H, dd, J = 5.2, 1.3 Hz); FAB-MS m/z 233 [(M+H)+].
5.3. Ethyl 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-5-
yl)-2-thiophenecarboxylate (8)
A 1.6 M solution of n-BuLi (33.0 mL, 52.8 mmol) in
hexane was added dropwise to a solution of 1-methyl-
5-(2-thienyl)-3-trifluoromethyl-1H-pyrazole (7, 11.2 g,
48.2 mmol) in THF (150 mL) at ꢀ70 ꢁC. The mixture
was stirred for 1.5 h at ꢀ70 ꢁC and then ethyl chlorofor-
mate (10.5 g, 96.8 mmol) was added at the same temper-
ature. The mixture was stirred for 15 min and then
aqueous NH4Cl was added. The whole mixture was
extracted with AcOEt, washed with brine, dried, and
concentrated in vacuo. The residue was purified by col-
umn chromatography (hexane/AcOEt, 7:1) to give 8
5. Experimental
5.1. Chemistry
1
(9.58 g, 65%) as a pale yellow solid: H NMR (CDCl3)
d 1.40 (3H, t, J = 7.3 Hz), 4.05 (3H, s), 4.39 (2H, q,
J = 7.0 Hz), 6.71 (1H, s), 7.20 (1H, d, J = 4.5 Hz), 7.80
(1H, d, J = 3.5 Hz); FAB-MS m/z 305 [(M+H)+].
Melting points were determined with a Yanaco MP-
500D melting point apparatus and are uncorrected. H
1
NMR spectra were recorded on a JEOL JNM-EX90,
JEOL JNM-LA300, JEOL JNM-EX400 or JEOL
JNM-A500 spectrometer and were referenced to an
internal standard, tetramethylsilane. The abbreviations
used for the signal patterns are as follows: s, singlet;
br s, broad singlet; d, doublet; t, triplet; q, quartet; dd,
double doublet; dt, double triplet; tt, triple triplet; m,
multiplet. Mass spectra were recorded on a Hitachi M-
80 or JEOL JMS-DX300 mass spectrometer, and the
ionization method was chosen from EI and FAB.
High-resolution mass spectra (HRMS) were recorded
on VG ZAB-VSE mass spectrometers. The elemental
analyses were performed with a Yanako MT-5 microan-
alyzer (C, H, and N) and a Yokogawa IC-7000S ion
chromatographic analyzer (halogens and S). Drying of
organic solutions during workup was done over anhy-
drous MgSO4. Preparative column chromatography
was performed with Wakogel C-200 or Merck silica
gel 60.
5.4. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-2-
thiophenecarboxylic acid (9)
A mixture of ethyl 5-(1-methyl-3-trifluoromethyl-1H-
pyrazol-5-yl)-2-thiophenecarboxylate
(8,
3.07 g,
10.1 mmol), 1 M NaOH (15 mL), and EtOH (30 mL)
was stirred for 4 h at room temperature. One molar
HCl (18 mL) was added to the mixture and extracted
with AcOEt, washed with brine and dried, and then con-
centrated in vacuo to give 9 (2.79 g, 100%) as a pale
1
brown solid: H NMR (DMSO-d6) d 4.06 (3H, s), 7.18
(1H, s), 7.59 (1H, d, J = 3.9 Hz), 7.80 (1H, d,
J = 3.9 Hz), 13.44 (1H, br s); FAB-MS m/z 275
[(MꢀH)ꢀ].
5.5. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-2-
thiophenecarbonylchloride (10)
A mixture of 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-
5-yl)-2-thiophenecarboxylic acid (9, 2.21 g, 8.00 mmol),
oxalyl chloride (2.03 g, 16.0 mmol), and DMF (3 drops)
in ClCH2CH2Cl (30 mL) was stirred for 90 min at room
temperature. The mixture was concentrated in vacuo to
give 10 (2.23 g, 95%) as a brown solid: 1H NMR
5.2. 1-Methyl-5-(2-thienyl)-3-trifluoromethyl-1H-pyra-
zole (7)
NaOMe (10.3 g, 191 mmol) was added to a solution of
2-acetylthiophene (5, 18.5 g, 147 mmol) in MeOH

4756
Y. Yonetoku et al. / Bioorg. Med. Chem. 14 (2006) 4750–4760
(CDCl3) d 4.09 (3H, s), 6.78 (1H, s), 7.30 (1H, d,
J = 4.4 Hz), 8.00 (1H, d, J = 3.9 Hz); FAB-MS m/z 291
[(M+H)+].
5.10. N-(4-Isopropylphenyl)-5-(1-methyl-3-trifluorometh-
yl-1H-pyrazol-5-yl)-2-thiophenecarboxamide (18)
Yield 71%; mp 163–164 ꢁC (AcOEt–hexane); 1H
NMR (DMSO-d6) d 1.21 (6H, d, J = 6.8 Hz), 2.83–
2.92 (1H, m), 4.08 (3H, s), 7.17 (1H, s), 7.24 (2H,
d, J = 8.3 Hz), 7.63 (1H, d, J = 3.9 Hz), 7.63 (2H,
d, J = 8.8 Hz), 8.09 (1H, d, J = 3.9 Hz), 10.30 (1H,
s); FAB-MS m/z 394 [(M+H)+]. Anal. Calcd for
C19H18F3N3OS: C, 58.00, H, 4.61; N, 10.68; F,
14.49; S, 8.15. Found: C, 58.17; H, 4.59; N, 10.68;
F, 14.48; S, 8.16.
5.6. N-(4-Fluorophenyl)-5-(1-methyl-3-trifluoromethyl-
1H-pyrazol-5-yl)-2-thiophenecarboxamide (11)
To a mixture of 4-fluoroaniline (93 mg, 0.84 mmol), sat-
urated aqueous NaHCO3 (3 mL), and CH2Cl2 (2 mL),
5-(1-methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-2-thio-
phenecarbonylchloride (10, 195 mg, 0.662 mmol) was
added and the whole mixture was stirred for overnight
at room temperature. H2O was added to the mixture
and extracted with AcOEt, washed with brine and dried,
and then concentrated in vacuo. The residue was recrys-
tallized from AcOEt to give 11 (64 mg, 26%) as a color-
5.11. N-(4-Dimethylaminophenyl)-5-(1-methyl-3-trifluo-
romethyl-1H-pyrazol-5-yl)-2-thiophenecarboxamide (20)
1
1
less powder: mp 189–192 ꢁC; H NMR (CDCl3) d 4.07
Yield 60%; mp 202–203 ꢁC (EtOH–AcOEt); H NMR
(3H, s), 6.72 (1H, s), 7.08 (2H, t, J = 8.6 Hz), 7.23
(1H, d, J = 3.9 Hz), 7.55–7.61 (2H, m), 7.62 (1H, d,
J = 3.9 Hz), 7.69 (1H, br s); FAB-MS m/z 370
[(M+H)+]. Anal. Calcd for C16H11F4N3OS: C, 52.03;
H, 3.00; N, 11.38; F, 20.58; S, 8.68. Found: C, 51.98;
H, 2.84; N, 11.43; F, 20.75; S, 8.72.
(DMSO-d6) d 2.88 (6H, s), 4.08 (3H, s), 6.74 (2H, d,
J = 9.2 Hz), 7.15 (1H, s), 7.52 (2H, d, J = 9.3 Hz), 7.60
(2H, d, J = 4.4 Hz), 8.04 (1H, d, J = 4.4 Hz), 10.13
(1H, s); FAB-MS m/z 394 (M+). Anal. Calcd for
C18H17F3N4OS: C, 54.81, H, 4.34; N, 14.20; F, 14.45;
S, 8.13. Found: C, 54.73; H, 4.43; N, 14.19; F, 14.34;
S, 7.95.
The following compounds were prepared following the
same method. Among the corresponding amines, 2-ami-
no-1-methylpyrrole was prepared by the methods
reported previously.23
5.12. N-(3-Chlorophenyl)-5-(1-methyl-3-trifluoromethyl-
1H-pyrazol-5-yl)-2-thiophenecarboxamide (21)
1
Yield 39%; mp 127–129 ꢁC (AcOEt–hexane); H NMR
5.7. N-(4-Bromophenyl)-5-(1-methyl-3-trifluoromethyl-
1H-pyrazol-5-yl)-2-thiophenecarboxamide (12)
(DMSO-d6) d 4.09 (3H, s), 7.17–7.21 (2H, m), 7.41
(1H, t, J = 8.1 Hz), 7.65 (1H, d, J = 3.9 Hz), 7.66–7.70
(1H, m), 7.91 (1H, t, J = 2.0 Hz), 8.11 (1H, d,
J = 3.9 Hz), 10.51 (1H, s); FAB-MS m/z 386
[(M+H)+]. Anal. Calcd for C16H11ClF3N3OS: C,
49.81; H, 2.87; N, 10.89; Cl, 9.19; F, 14.77; S, 8.31.
Found: C, 49.74; H, 2.73; N, 10.94; Cl, 9.11; F, 14.73;
S, 8.29.
Yield 11%; mp 191–192 ꢁC (AcOEt); 1H NMR (CDCl3)
d 4.07 (3H, s), 6.73 (1H, s), 7.24 (1H, d, J = 3.9 Hz),
7.47–7.55 (4H, m), 7.62 (1H, d, J = 3.9 Hz), 7.67 (1H,
br s); FAB-MS m/z 430, 432 [(M+H)+]. Anal. Calcd
for C16H11BrF3N3OS: C, 44.67; H, 2.58; N, 9.77; Br,
18.57; F, 13.25; S, 7.45. Found: C, 44.56; H, 2.75; N,
9.80; Br, 18.62; F, 13.30; S, 7.36.
5.13. N-(1-Methyl-1H-pyrrol-2-yl)-5-(1-methyl-3-trifluo-
romethyl-1H-pyrazol-5-yl)-2-thiophenecarboxamide (28)
5.8. N-(4-Cyanophenyl)-5-(1-methyl-3-trifluoromethyl-
1H-pyrazol-5-yl)-2-thiophenecarboxamide (14)
1
Yield 36%; mp 158–159 ꢁC (AcOEt–hexane); H NMR
(DMSO-d6) d 3.44 (3H, s), 4.07 (3H, s), 5.92 (1H, br
s), 5.98 (1H, t, J = 3.2 Hz), 6.69 (1H, s), 7.16 (1H, s),
7.62 (1H, d, J = 3.9 Hz), 8.03 (1H, d, J = 3.9 Hz),
10.12 (1H, s); FAB-MS m/z 355 [(M+H)+]. Anal. Calcd
for C15H13F3N4OS: C, 50.84; H, 3.70; N, 15.81; F,
16.08; S, 9.05. Found: C, 50.90; H, 3.66; N, 15.77; F,
16.07; S, 9.01.
1
Yield 55%; mp 211–214 ꢁC (AcOEt–hexane); H NMR
(DMSO-d6) d 4.09 (3H, s), 7.20 (1H, s), 7.67 (1H, d,
J = 3.9 Hz), 7.85 (2H, d, J = 8.8 Hz), 7.95 (2H, d,
J = 9.3 Hz), 8.15 (1H, d, J = 3.9 Hz), 10.72 (1H, s);
FAB-MS m/z 377 [(M+H)+]. Anal. Calcd for
C17H11F3N4OS Æ 0.15H2O: C, 53.87; H, 3.00; N, 14.78;
F, 15.04; S, 8.46. Found: C, 53.49; H, 2.85; N, 14.53;
F, 15.41; S, 8.46.
5.14. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-N-
(pyridin-4-yl)-2-thiophenecarboxamide monohydrochlo-
ride (30)
5.9. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-N-
(4-nitrophenyl)-2-thiophenecarboxamide (15)
Yield 24%; mp 247 ꢁC (AcOEt–hexane); 1H NMR
(DMSO-d6) d 4.11 (3H, s), 7.25 (1H, s), 7.73 (1H, d,
J = 4.3 Hz), 8.39 (2H, d, J = 5.9 Hz), 8.53 (1H, d,
J = 3.9 Hz), 8.76 (2H, d, J = 6.4 Hz), 11.93 (1H, s);
FAB-MS m/z 353 [(M+H)+]. Anal. Calcd for
C15H11F3N4OS Æ HCl: C, 46.34; H, 3.11; N, 14.41; Cl,
9.12; F, 14.66; S, 8.25. Found: C, 46.19; H, 2.99; N,
14.39; Cl, 9.07; F, 14.72; S, 8.13.
1
Yield 24%; mp 183–184 ꢁC (AcOEt–hexane); H NMR
(DMSO-d6) d 4.10 (3H, s), 7.20 (1H, s), 7.68 (1H, d,
J = 3.9 Hz), 8.03 (2H, d, J = 9.3 Hz), 8.18 (1H, d,
J = 4.4 Hz), 8.29 (2H, d, J = 9.2 Hz), 10.88 (1H, s);
FAB-MS m/z 397 [(M+H)+]. HRMS m/z Calcd for
C16H11F3N4O3S
397.0601.
[(M+H)+]:
397.0582.
Found:

Y. Yonetoku et al. / Bioorg. Med. Chem. 14 (2006) 4750–4760
4757
5.15. N-Cyclohexyl-5-(1-methyl-3-trifluoromethyl-1H-
pyrazol-5-yl)-2-thiophenecarboxamide (35)
10.00; Cl, 16.87; F, 13.56; S, 7.63. Found: C, 45.65; H,
2.38; N, 9.99; Cl, 16.77; F, 13.40; S, 7.59.
1
Yield 68%; mp 140–141 ꢁC (AcOEt–hexane); H NMR
5.20. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-N-
(thiophen-2-yl)-2-thiophenecarboxamide (26)
(DMSO-d6) d 1.10–1.20 (1H, m), 1.24–1.36 (4H, m),
1.58–1.66 (1H, m), 1.69–1.87 (4H, m), 3.67–3.77 (1H,
m), 4.05 (3H, s), 7.10 (1H, s), 7.53 (1H, d, J = 3.9 Hz),
7.87 (1H, d, J = 3.9 Hz), 8.38 (1H, d, J = 7.8 Hz);
FAB-MS m/z 358 [(M+H)+]. Anal. Calcd for
C16H18F3N3OS: C, 53.77; H, 5.08; N, 11.76; F, 15.95;
S, 8.97. Found: C, 53.47; H, 4.94; N, 11.68; F, 16.25;
S, 8.97.
Yield 15%; mp 180–185 ꢁC (Et2O–hexane); 1H NMR
(CDCl3) d 4.07 (3H, s), 6.73 (1H, s), 6.83 (1H, dd,
J = 3.7, 1.2 Hz), 6.92 (1H, dd, J = 5.7, 3.7 Hz), 6.97–
7.00 (1H, m), 7.24 (1H, d, J = 3.9 Hz), 7.65 (1H, d,
J = 3.9 Hz), 8.37 (1H, br s); FAB-MS m/z 358
[(M+H)+]. Anal. Calcd for C14H10F3N3OS2 Æ 0.25H2O:
C, 46.47; H, 2.92; N, 11.61; F, 15.75; S, 17.72. Found:
C, 46.37; H, 2.71; N, 11.44; F, 15.65; S, 17.72.
The following compounds were prepared according to
this method, substituting Et3N for aqueous NaHCO3.
Among the corresponding amines, 2- and 3-aminothi-
ophene were prepared by the methods reported
previously.24
5.21. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-N-
(thiophen-3-yl)-2-thiophenecarboxamide (27)
1
Yield 62%; mp 166–168 ꢁC (AcOEt–hexane); H NMR
5.16. N-(4-Methylphenyl)-5-(1-methyl-3-trifluoromethyl-
1H-pyrazol-5-yl)-2-thiophenecarboxamide (17)
(CDCl3) d 4.07 (3H, s), 6.72 (1H, s), 7.13 (1H, dd,
J = 4.9, 1.5 Hz), 7.23 (1H, d, J = 3.9 Hz), 7.31 (1H, dd,
J = 4.7, 3.2 Hz), 7.60 (1H, d, J = 3.9 Hz), 7.68 (1H, dd,
J = 3.0, 1.4 Hz), 7.97 (1H, s); FAB-MS m/z 358
[(M+H)+]. Anal. Calcd for C14H10F3N3OS2: C, 47.05;
H, 2.82; N, 11.76; F, 15.95; S, 17.95. Found: C, 46.91;
H, 2.83; N, 11.68; F, 16.24; S, 17.91.
1
Yield 61%; mp 167–169 ꢁC (AcOEt–hexane); H NMR
(CDCl3) d 2.35 (3H, s), 4.06 (3H, s), 6.72 (1H, s), 7.19
(2H, d, J = 8.3 Hz), 7.22 (1H, d, J = 3.9 Hz), 7.49 (2H,
d, J = 8.3 Hz), 7.60 (1H, d, J = 3.9 Hz), 7.65 (1H, br
s); FAB-MS m/z 366 [(M+H)+]. Anal. Calcd for
C17H14F3N3OS Æ 0.25H2O: C, 55.20; H, 3.95; N, 11.36;
F, 15.41; S, 8.67. Found: C, 55.15; H, 3.77; N, 11.43;
F, 15.50; S, 8.87.
5.22. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-N-
(pyridin-3-yl)-2-thiophenecarboxamide (29)
1
Yield 62%; mp 186–187 ꢁC (AcOEt–hexane); H NMR
5.17. N-(2-Fluorophenyl)-5-(1-methyl-3-trifluoromethyl-
1H-pyrazol-5-yl)-2-thiophenecarboxamide (23)
(CDCl3) d 4.07 (3H, s), 6.73 (1H, s), 7.25 (1H, d,
J = 3.9 Hz), 7.35 (1H, dd, J = 8.3, 3.8 Hz), 7.68 (1H, d,
J = 3.9 Hz), 7.95 (1H, s), 8.24–8.28 (1H, m), 8.42 (1H,
dd, J = 4.7, 1.2 Hz), 8.68 (1H, d, J = 2.5 Hz); FAB-MS
m/z 353 [(M+H)+]. Anal. Calcd for C15H11F3N4OS: C,
51.13; H, 3.15; N, 15.90; F, 16.18; S, 9.10. Found: C,
51.03; H, 3.14; N, 15.84; F, 15.89; S, 9.04.
1
Yield 26%; mp 147–148 ꢁC (AcOEt–hexane); H NMR
(DMSO-d6) d 4.09 (3H, s), 7.18 (1H, s), 7.22–7.35 (3H,
m), 7.58–7.62 (1H, m), 7.64 (1H, d, J = 3.9 Hz), 8.10
(1H, d, J = 3.9 Hz), 10.33 (1H, s); FAB-MS m/z 370
[(M+H)+]. Anal. Calcd for C16H11F4N3OS: C, 52.03;
H, 3.00; N, 11.38; F, 20.58; S, 8.68. Found: C, 51.81;
H, 2.92; N, 11.41; F, 20.55; S, 8.69.
5.23. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-N-
(1H-tetrazol-5-yl)-2-thiophenecarboxamide (33)
5.18. N-(2-Methylphenyl)-5-(1-methyl-3-trifluoromethyl-
1H-pyrazol-5-yl)-2-thiophenecarboxamide (24)
Yield 72%; mp 285–287 ꢁC (acetone); 1H NMR
(DMSO-d6) d 4.09 (3H, s), 7.22 (1H, s), 7.69 (1H, d,
J = 3.9 Hz), 8.32 (1H, d, J = 4.4 Hz), 12.67 (1H, br s);
FAB-MS m/z 344 [(M+H)+]. Anal. Calcd for
C11H8F3N7OS Æ 0.2H2O: C, 38.09; H, 2.44; N, 28.26;
F, 16.43; S, 9.24. Found: C, 37.95; H, 2.40; N, 28.54;
F, 16.78; S, 9.24.
1
Yield 50%; mp 159–161 ꢁC (AcOEt–hexane); H NMR
(CDCl3) d 2.35 (3H, s), 4.06 (3H, s), 6.72 (1H, s),
7.13–7.18 (1H, m), 7.22 (1H, d, J = 3.9 Hz), 7.23–7.30
(2H, m), 7.58 (1H, s), 7.61 (1H, d, J = 3.9 Hz), 7.86
(1H, d, J = 7.8 Hz); FAB-MS m/z 366 [(M+H)+]. Anal.
Calcd for C17H14F3N3OS: C, 55.88; H, 3.86; N, 11.50;
F, 15.60; S, 8.78. Found: C, 55.77; H, 3.79; N, 11.64;
F, 15.56; S, 8.77.
The following compounds were prepared according to
this method, substituting pyridine for aqueous
NaHCO3.
5.19. N-(2,4-Dichlorophenyl)-5-(1-methyl-3-trifluoro-
methyl-1H-pyrazol-5-yl)-2-thiophenecarboxamide (25)
5.24. Ethyl 4-{[5-(1-methyl-3-trifluoromethyl-1H-pyrazol-
5-yl)-2-thiophenecarbonyl]amino}benzoate (13)
1
1
Yield 25%; mp 181–183 ꢁC (AcOEt–hexane); H NMR
Yield 45%; mp 183–185 ꢁC (AcOEt–hexane); H NMR
(CDCl3) d 4.08 (3H, s), 6.74 (1H, s), 7.26 (1H, d,
J = 3.9 Hz), 7.32 (1H, dd, J = 9.1, 2.2 Hz), 7.45 (1H, d,
J = 2.4 Hz), 7.66 (1H, d, J = 3.9 Hz), 8.25 (1H, s), 8.45
(1H, d, J = 8.8 Hz); FAB-MS m/z 420 [(M+H)+]. Anal.
Calcd for C16H10Cl2F3N3OS: C, 45.73; H, 2.40; N,
(DMSO-d6) d 1.33 (3H, t, J = 7.1 Hz), 4.09 (3H, s),
4.31 (2H, q, J = 7.2 Hz), 7.19 (1H, s), 7.66 (1H, d,
J = 3.9 Hz), 7.91 (2H, d, J = 8.7 Hz), 7.99 (2H, d,
J = 8.8 Hz), 8.16 (1H, d, J = 4.4 Hz), 10.65 (1H, s);
FAB-MS m/z 424 [(M+H)+]. Anal. Calcd for

4758
Y. Yonetoku et al. / Bioorg. Med. Chem. 14 (2006) 4750–4760
C19H16F3N3O3S: C, 53.90; H, 3.81; N, 9.92; F, 13.46; S,
7.57. Found: C, 53.89; H, 3.79; N, 9.87; F, 13.43; S, 7.48.
methyl-1H-pyrazol-5-yl)-2-thiophenecarbonylchloride
(10, 150 mg, 0.509 mmol) was added and the whole mix-
ture was stirred for 2 h at room temperature. H2O was
added to the mixture and extracted with AcOEt, washed
with brine and dried, and then concentrated in vacuo.
The residue was recrystallized from AcOEt–hexane to
give 34 (96 mg, 62%) as a colorless powder: mp 136–
5.25. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-N-
phenylthiophene-2-carboxamide (16)
1
Yield 86%; mp 129–130 ꢁC; H NMR (CDCl3) d 4.07
(3H, s), 6.72 (1H, s), 7.19 (1H, t, J = 7.6 Hz), 7.23
(1H, d, J = 3.4 Hz), 7.40 (2H, t, J = 8.1 Hz), 7.60–7.63
(3H, m), 7.66 (1H, br s); FAB-MS m/z 352 [(M+H)+].
Anal. Calcd for C16H12F3N3OS: C, 54.70; H, 3.44; N,
11.96; F, 16.22; S, 9.13. Found: C, 54.69; H, 3.22; N,
12.21; F, 16.10; S, 9.05.
137 ꢁC; 1H NMR (DMSO-d6)
d
1.14 (3H, t,
J = 7.1 Hz), 3.25–3.32 (2H, m), 4.05 (3H, s), 7.11 (1H,
s), 7.54 (d, J = 3.9 Hz), 7.81 (1H, d, J = 3.9 Hz), 8.65
(1H, t, J = 5.4 Hz); FAB-MS m/z 304 [(M+H)+]. Anal.
Calcd for C12H12F3N3OS: C, 47.52; H, 3.99; N, 13.85;
F, 18.79; S, 10.57. Found: C, 47.45; H, 3.98; N, 13.83;
F, 19.07; S, 10.46.
5.26. N-(4-Methoxyphenyl)-5-(1-methyl-3-trifluorometh-
yl-1H-pyrazol-5-yl)-2-thiophenecarboxamide (19)
5.31. N-(4-Hydroxyphenyl)-5-(1-methyl-3-trifluorometh-
yl-1H-pyrazol-5-yl)-2-thiophenecarboxamide (36)
1
Yield 49%; mp 166–167 ꢁC (AcOEt–hexane); H NMR
(DMSO-d6) d 3.75 (3H, s), 4.08 (3H, s), 6.95 (2H, d,
J = 9.3 Hz), 7.16 (1H, s), 7.62 (1H, d, J = 3.9 Hz), 7.62
(2H, d, J = 9.3 Hz), 8.06 (1H, d, J = 3.9 Hz), 10.27 (1H,
s); FAB-MS m/z 382 [(M+H)+]. Anal. Calcd for
C17H14F3N3O2S: C, 53.54, H, 3.70; N, 11.02; F, 14.94; S,
8.41. Found: C, 53.45; H, 3.67; N, 10.96; F, 14.88; S, 8.28.
A mixture of 5-(1-methyl-3-trifluoromethyl-1H-pyrazol-
5-yl)-2-thiophene (with Standard) carboxylic acid (9,
156 mg, 0.565 mmol), 4-aminophenol (62 mg, 0.57 mmol),
and EDC Æ HCl (114 mg, 0.595 mmol) in THF (2 mL)
was stirred for 3 days at room temperature. H2O
(10 mL) was added to the mixture and extracted with
AcOEt, washed with 1 M HCl, saturated aqueous NaH-
CO3, brine and dried, and then concentrated in vacuo.
The residue was purified by column chromatography
(hexane/AcOEt, 2:1) and recrystallized from AcOEt–
hexane to give 36 (113 mg, 54%) as a colorless powder:
5.27. N-(2-Chlorophenyl)-5-(1-methyl-3-trifluoromethyl-
1H-pyrazol-5-yl)-2-thiophenecarboxamide (22)
Yield 41%; mp 156 ꢁC (EtOH); 1H NMR (CDCl3) d 4.08
(3H, s), 6.74 (1H, s), 7.11 (1H, td, J = 7.8, 1.5 Hz), 7.26
(1H, d, J = 3.4 Hz), 7.35 (1H, td, J = 7.8, 1.4 Hz), 7.44
(1H, dd, J = 7.8, 1.5 Hz), 7.66 (1H, d, J = 3.9 Hz), 8.32
(1H, s), 8.48 (1H, dd, J = 8.3, 1.4 Hz); FAB-MS m/z 386
[(M+H)+]. Anal. Calcd for C16H11ClF3N3OS: C, 49.81;
H, 2.87; N, 10.89; Cl, 9.19; F, 14.77; S, 8.31. Found: C,
49.79; H, 2.80; N, 10.95; Cl, 9.18; F, 14.73; S, 8.30.
1
mp 185–186 ꢁC; H NMR (DMSO-d6) d 4.08 (3H, s),
6.76 (2H, d, J = 8.8 Hz), 7.15 (1H, s), 7.48 (2H, d,
J = 9.3 Hz), 7.61 (1H, d, J = 4.4 Hz), 8.04 (1H, d,
J = 3.9 Hz), 9.32 (1H, s), 10.17 (1H, s); FAB-MS m/z
368 [(M+H)+]. Anal. Calcd for C16H12F3N3O2S: C,
52.31, H, 3.29; N, 11.44; F, 15.52; S, 8.73. Found: C,
51.99; H, 3.37; N, 11.27; F, 15.76; S, 8.68.
5.28. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-N-
(thiazol-2-yl)-2-thiophenecarboxamide (31)
The following compound was prepared using similar
method.
1
Yield 58%; mp 235–238 ꢁC (EtOH); H NMR (DMSO-
5.32. tert-Butyl 4-{[5-(1-methyl-3-trifluoromethyl-1H-
pyrazol-5-yl)-2-thiophenecarbonyl]amino}piperidine-1-
carboxylate (37)
d6) d 4.09 (3H,s), 7.20 (1H, s), 7.29 (1H, d, J = 3.4 Hz),
7.57 (1H, d, J = 3.9 Hz), 7.65 (1H, d, J = 3.9 Hz), 8.28
(1H, br s), 12.94 (1H, br s); FAB-MS m/z 359
[(M+H)+]. Anal. Calcd for C13H9F3N4OS2: C, 43.57;
H, 2.53; N, 15.63; F, 15.90; S, 17.90. Found: C, 43.34;
H, 2.48; N, 15.64; F, 16.20; S, 17.82.
1H NMR (CDCl3) d 1.44–1.49 (2H, m), 1.47 (9H, s),
1.98–2.08 (2H, m), 2.83–2.97 (2H, m), 4.04 (3H, s),
4.08–4.17 (3H, m), 6.02–6.08 (1H, m), 6.68 (1H, s),
7.17 (1H, d, J = 3.6 Hz), 7.50 (1H, d, J = 3.9 Hz);
FAB-MS m/z 459 [(M+H)+].
5.29. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-N-
(4H-1,2,4-triazol-4-yl)-2-thiophenecarboxamide (32)
5.33. N-(4-Aminophenyl)-5-(1-methyl-3-trifluoromethyl-
1H-pyrazol-5-yl)-2-thiophenecarboxamide (38)
Yield 71%; mp 207–210 ꢁC (AcOEt); 1H NMR (DMSO-
d6) d 4.08 (3H, s), 7.22 (1H, s), 7.69 (1H, d, J = 3.9 Hz),
7.98 (1H, d, J = 3.9 Hz), 8.83 (2H, s), 12.34 (1H, s);
FAB-MS m/z 343 [(M+H)+]. Anal. Calcd for
C12H9F3N6OS: C, 42.11; H, 2.65; N, 24.55; F, 16.65; S,
9.37. Found: C, 41.77; H, 2.55; N, 24.20; F, 16.59; S, 9.17.
Zinc powder (240 mg, 3.67 mmol) and NH4Cl (196 mg,
3.66 mmol) was added to a solution of 5-(1-methyl-3-
trifluoromethyl-1H-pyrazol-5-yl)-N-(4-nitrophenyl)-2-
thiophenecarboxamide (15, 146 mg, 0.368 mmol) in
EtOH–H2O (2:1, 6 mL) at 5 ꢁC. The mixture was stirred
for 20 min at room temperature and filtered on Celite.
H2O was added to the filtrate and extracted with AcOEt,
washed with 0.1 M HCl, saturated aqueous NaHCO3,
brine and dried, and then concentrated in vacuo. The
residue was recrystallized from AcOEt–hexane to give
5.30. N-Ethyl-5-(1-methyl-3-trifluoromethyl-1H-pyrazol-
5-yl)-2-thiophenecarboxamide (34)
To a mixture of ethylamine (70% in H2O, 1 mL,
15.5 mmol) and THF (4 mL), 5-(1-methyl-3-trifluoro-

Y. Yonetoku et al. / Bioorg. Med. Chem. 14 (2006) 4750–4760
4759
38 (64 mg, 47%) as a yellow powder: mp 181–183 ꢁC; 1H
NMR (DMSO-d6) d 4.07 (3H, s), 4.99 (2H, s), 6.56 (2H,
d, J = 8.8 Hz), 7.14 (1H, s), 7.32 (2H, d, J = 8.8 Hz),
7.59 (1H, d, J = 3.9 Hz), 8.01 (1H, d, J = 3.9 Hz),
10.02 (1H, s); FAB-MS m/z 367 [(M+H)+]. Anal. Calcd
for C16H13F3N4OS: C, 52.45, H, 3.58; N, 15.29; F,
15.56; S, 8.75. Found: C, 52.65; H, 3.47; N, 15.04; F,
15.35; S, 8.60.
from the fluorescence ratio, using the standard equation.
The Rmax value was obtained from 25 lM ionomycin-
treated wells. The Rmin value was obtained from
25 lM ionomycin/50 mM EGTA-treated wells.
5.37. CRAC channel inhibition assay
CRAC channel inhibition was evaluated in Jurkat cells
(2 · 106 cells/mL). Fura-2 loaded Jurkat cells were stim-
ulated with 1 lM thapsigargin for 30 min, and measured
intracellular calcium concentration at the endpoint of
30 min. IC50 values on CRAC channel inhibition of each
compounds were calculated from percent inhibition of
thapsigargin-induced calcium influx in Jurkat cells.
5.34. 5-(1-Methyl-3-trifluoromethyl-1H-pyrazol-5-yl)-N-
(4-piperidyl)-2-thiophenecarboxamide monohydrochloride
(39)
A mixture of tert-butyl 4-{[5-(1-methyl-3-trifluorometh-
yl-1H-pyrazol-5-yl)-2-thenoyl]amino}piperidine-1-car-
boxylate (37, 65 mg, 0.142 mmol) and 4 M HCl–AcOEt
(1 mL) in AcOEt (1 mL) was stirred for 4 h at room tem-
perature. The mixture was concentrated in vacuo, and
the residue was recrystallized from EtOH to give 39
(34 mg, 61%) as a colorless powder: mp 265–266 ꢁC;
1H NMR (DMSO-d6) d 1.73–1.85 (2H, m), 1.93–2.01
(2H, m), 2.96–3.06 (2H, m), 3.28–3.32 (2H, m), 4.00–
4.08 (1H, m), 4.05 (3H, s), 7.12 (1H, s), 7.56 (1H, d,
J = 3.9 Hz), 7.97 (1H, d, J = 3.9 Hz), 8.74 (1H, d,
J = 7.4 Hz), 8.78–8.90 (1H, br s); FAB-MS m/z 359
[(M+H)+]. Anal. Calcd for C15H17F3N4OS Æ HCl: C,
45.63; H, 4.59; N, 14.19; Cl, 8.98; F, 14.43; S, 8.12.
Found: C, 45.32; H, 4.76; N, 14.20; Cl, 8.89; F, 14.63;
S, 8.10.
5.38. VOC channel inhibition assay
VOC channel inhibition was evaluated in murine neuro-
blastoma, PC12-h5 cells (1 · 106 cells/mL). Fura-2 load-
ed PC12-h5 cells were stimulated with 50 mM KCl for
20 min, and measured intracellular calcium concentra-
tion at the endpoint of 20 min. IC50 values on VOC
channel inhibition of each compounds were calculated
from percent inhibition of KCl-induced calcium influx
in PC12 cells.
5.39. IL-2 production assay
Jurkat T lymphocytes (5 · 106 cells/mL) were placed in a
96-well microplate and incubated with PHA (20 lg/mL)
for 20 h and supernatant was collected from these cells.
IL-2 concentration in each supernatant was measured
by human IL-2 ELISA system (DuoSeTTM, Genzyme).
5.35. QSAR analysis
Linear and multiple regression analyses were carried out
using TSAR 3.0 (Oxford Molecular Group). One or two
out of the following parameters (p, molecular refractiv-
ity, Swein and Lupton F/R, rpara ðr2paraÞ, Es, Verloop L,
molecular volume, molecular surface area, and Verloop
B5) were applied to the analyses. The following statisti-
cal parameters were determined for each regression
equation: the number of points: n; the correlation coef-
ficient: r; the significance of the regression model: F; the
standard error: s.
5.40. Con A-induced hepatitis
Con A (20 mg/kg) was intravenously injected to female
Balb/c mice in a volume of 10 mL/kg. Blood samples
were obtained 24 h after Con A injection, and serum
GOT and GPT levels were measured. For oral dosage
groups, compounds were suspended with 0.5% MC
and administered orally in a volume of 10 mL/kg 1 h be-
fore Con A injection. Cyclosporin A (CsA) was dis-
solved in physiological saline and subcutaneously
administered at 50 mg/kg in a volume of 10 mL/kg 2 h
before Con A injection.
5.36. Fura-2 loading and population intracellular calcium
measurements
Cells were suspended in Hepes buffered solution (pH
7.4) of the following composition: NaCl: 137 mM,
KCl: 5.8 mM, MgCl2: 1 mM, CaCl2: 2.5 mM, glucose:
5 mM, and Hepes: 10 mM. The cells were loaded with
1 lM Fura-2/AM at room temperature for 45 min, fol-
lowed by successive washes to remove unincorporated
dye, and resuspended in Hepes buffered solution. Cell
suspensions were studied in a 96-well black plate. Fluo-
rescence measurements for the determination of intra-
cellular calcium concentration were made in
fluorescence microplate reader (Fluostar, SLT Labin-
struments Ges.m.b.H, Austria) with excitation wave-
length of each 340 and 380 nm at emission
fluorescence detection of 500 nm. Then, self-fluorescence
of each compound was measured in a cell-free way and
was deducted from cell way fluorescence. Final intracel-
lular calcium concentrations in each well were calculated
Acknowledgments
The authors are grateful to the staff of the Division of
Analytical Science Laboratories for the elemental analy-
sis and spectral measurements.
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