Novel potent and selective Ca2+ release-activated Ca2+ (CRAC) channel inhibitors. Part 3: Synthesis and CRAC channel inhibitory activity of 4′-[(trifluoromethyl)pyrazol-1-yl]carboxanilides

Article abstract of DOI:10.1016/j.bmc.2008.09.047

From a series of 4'-[(trifluoromethyl)pyrazol-1-yl]carboxanilides derived from 4-methyl-4'-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3-thiadiazole-5-carboxanilide, one inhibited thapsigargin-induced Ca²⁺ influx in Jurkat T cells (IC₅₀ = 77 nM) and exhibited high selectivity for the CRAC channel over the VOC channel (index: >130). Another acted as an inhibitor for both T lymphocyte activation-induced diseases and ovalbumin-induced airway eosinophilia in rats (ED₅₀ = 1.3 mg/kg) p.o.

Full text of DOI:10.1016/j.bmc.2008.09.047

Bioorganic & Medicinal Chemistry 16 (2008) 9457–9466
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel potent and selective Ca2+ release-activated Ca2+ (CRAC) channel inhibitors.
Part 3: Synthesis and CRAC channel inhibitory activity of
40-[(trifluoromethyl)pyrazol-1-yl]carboxanilides
*
Yasuhiro Yonetoku , Hirokazu Kubota, Yoji Miyazaki, Yoshinori Okamoto, Masashi Funatsu,
Noriko Yoshimura-Ishikawa, Jun Ishikawa, Taiji Yoshino, Makoto Takeuchi, Mitsuaki Ohta
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
From a series of 4’-[(trifluoromethyl)pyrazol-1-yl]carboxanilides derived from 4-methyl-4’-[3,5-bis(tri-
fluoromethyl)-1H-pyrazol-1-yl]-1,2,3-thiadiazole-5-carboxanilide, one inhibited thapsigargin-induced
Ca2+ influx in Jurkat T cells (IC50 = 77 nM) and exhibited high selectivity for the CRAC channel over the
VOC channel (index: >130). Another acted as an inhibitor for both T lymphocyte activation-induced dis-
eases and ovalbumin-induced airway eosinophilia in rats (ED50 = 1.3 mg/kg) p.o.
Received 1 August 2008
Revised 16 September 2008
Accepted 17 September 2008
Available online 20 September 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Anti-inflammatory
Channel inhibitors
Ca2+ release-activated Ca2+ channel
CRAC channel
Interleukin-2
1. Introduction
Ca2+ (CRAC) channels, and capacitative Ca2+ influx through the
CRAC channel sustains high intracellular Ca2+ concentration
It is well-established that intracellular Ca2+ plays an important
role in various cellular functions, and that its concentration is reg-
ulated by Ca2+ influx through Ca2+ channels on the cell membrane.
Ca2+ channels, which are located in the nervous, endocrine, cardio-
vascular, and skeletal systems and are modulated by membrane
potential, are called voltage-operated Ca2+ (VOC) channels. These
channels are classified into L, N, P, Q, R, and T subtypes.1 Excessive
Ca2+ influx through the VOC channels causes hypertension and
brain dysfunction.
([Ca2+]i).5 This prolonged high [Ca2+
] activates cytosolic signal
i
transduction to produce lipid mediators (e.g., LTD4), cytokines
[e.g., interleukin-2 (IL-2)], and matrix metalloproteinases, which
participate in the pathogenesis of inflammation and autoimmune
diseases.
These facts suggest that CRAC channel inhibitors can be useful
for the treatment of diseases caused by the activation of inflamma-
tory cells without side effects observed in steroids.6 Since VOC
channel inhibitors would cause adverse events in the nervous
and cardiovascular systems, it may be necessary for CRAC channel
inhibitors to exhibit sufficient selectivity over VOC channels if they
are to be used as anti-inflammatory drugs. In the last decade, SK&F
96365 (1),7 econazole (2),8 and L-651582 (3)9 have been reported
to inhibit the CRAC channel (Fig. 1); however, their potency and
selectivity over VOC channels was not sufficient for use as anti-
inflammatory drugs.
In contrast, Ca2+ channels on inflammatory cells, including lym-
phocytes, mast cells, and neutrophils, can be activated regardless
of their membrane potential. This type of Ca2+ channel has been re-
ported to act in the crisis and exacerbation of inflammation and
autoimmune diseases.2 In the T cells, it has been reported that
the early stages of activation consist of pre- and post-Ca2+ events.3
The stimulation of T cell receptors induces pre-Ca2+ events, includ-
ing the generation of IP3, followed by the release of Ca2+ from the
endoplasmic reticulum (ER).4 In post-Ca2+ events, depletion of
Ca2+ in the ER induces the activation of Ca2+ release-activated
Our effort to discover novel CRAC channel inhibitors led to the
discovery of a series of aryl-3-trifluoromethylpyrazoles, including
compounds 4–6 (Fig. 2), that showed potent and selective CRAC
channel inhibitory activity.10 Among these, 4-methyl-4’-[3,
5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3-thiadiazole-5-carbox-
* Corresponding author at present address: Drug Discovery Research, Astellas
Pharma Inc., 5-2-3 Tokodai, Tsukuba-shi, Ibaraki 300-2698, Japan. Tel.: +81 (0)29
847 8611; fax: +81 (0)29 847 8313.
anilide (6)10,11 was found to have inhibitory activity (IC50: 0.15
lM,
selectivity index: 31) selective for the CRAC channel over the VOC
E-mail address: yasuhiro.yonetoku@jp.astellas.com (Y. Yonetoku).
channel. Although similar carboxanilide derivatives, including
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.047

9458
Y. Yonetoku et al. / Bioorg. Med. Chem. 16 (2008) 9457–9466
followed by cyclization with 4-nitrophenylhydrazine (Scheme 1).
N
N
The structure of compound 8 was determined by observing the nu-
clear Overhauser effect (NOE) between the protons of the methy-
lene group (3.12 ppm) and the benzene (7.86 ppm, Scheme 1).
Cl
OMe
N
N
O
O
The hydrogenation of compound
8 and 5-methyl-1-(4-nitro-
Cl
phenyl)-3-(trifluoromethyl)pyrazole (10)13 yielded the anilines
11a and b, respectively (Scheme 2).
econazole (2)
The desired 4’-[(trifluoromethyl)pyrazol-1-yl]carboxanilides
(13) were prepared from the anilines (11) via the methods de-
picted in Schemes 3–7. The anilines (11) were converted into com-
pounds 13a–z via treatment with the carboxylic acids (12) in the
presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimideꢀHCl
(EDC HCl) (Method A, Scheme 3) or EDC HCl and 1-hydroxybenzo-
triazole hydrate (HOBT) (Method B). The acid chlorides were pre-
pared from the corresponding carboxylic acids (12) via treatment
with oxalyl chloride and catalytic amounts of dimethylformamide
(DMF), after which they were coupled with the anilines (11) in the
presence of triethylamine (Et3N) to yield the desired anilides (13)
(Method C). The coupling reaction of the corresponding acids
(12) and anilines (11) afforded the anilides (13) in the presence
of O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluoro-
phosphate (HBTU) and Et3N (Method D). The imidazole-4-carbox-
anilide (14) and 5-aminonicotinanilide (15) derivatives were
obtained by deprotecting the trityl and t-butoxycarbonyl groups
in compounds 13y14 and 13z,15 respectively (Schemes 4 and 5).
The 4-chloronicotinanilide derivative (13n) was reacted with
methanol (MeOH) under acidic conditions to yield the 4-methoxy
derivative (16), and amination of compound 13n in liquid ammo-
nia yielded the 4-amino derivative (17) (Scheme 6). Acidic treat-
ment of compounds 13n and 13p yielded the 4-hydroxy (18,
Scheme 6) and 2-hydroxy (19, Scheme 7) derivatives, respectively.
Cl
OMe SK&F 96365 (1)
O
Cl
O
H2N
NH2
N
N
N
Cl
Cl
L-651582 (3)
Figure 1. Structures of SK&F 96365 (1), econazole (2), L-651582 (3).
Cl
Cl
H
N
Me
N
Me
N
H
N
N
N
S
O
O
F3C
F3C
5
4
Me
N
H
N
N
N
F3C
S
N
O
CF3
6
Figure. 2. Pyrazole derivatives 4–6 identified as CRAC channel inhibitors.
compound 6, were recently reported to inhibit the transcriptional
activity of nuclear factor in activated T cells,12 inhibition of CRAC
channels was not observed. In addition, compound 6 inhibited con-
canavalin A-induced liver injury and trinitrochlorobenzene
(TNCB)-induced ear swelling in mice with ED50 values of 0.61
and 1.1 mg/kg p.o., respectively.10 We believe that compound 6
should be considered a lead compound in the discovery of potent
and selective CRAC channel inhibitors, and designed some
carboxanilides substituting 3,5-bis(trifluoromethyl)-1H-pyrazole
and 4-methyl-1,2,3-thiadiazole moieties as other pyrazoles and
heteroaryl groups, respectively. In this paper, we describe the
synthesis and the structure–activity relationships (SARs) of
4’-[(trifluoromethyl)pyrazol-1-yl]carboxanilide derivatives as
novel CRAC channel inhibitors.
3. Results and discussion
The synthesized compounds were evaluated for their inhibition
of Ca2+ influx through the CRAC and VOC channels on Jurkat T
cells16 and PC12-h5 cells,17 respectively. The results are summa-
rized in Table 1.
We first investigated the effect of substituents in the pyrazole
moiety of compound 6 on CRAC channel inhibitory activity and
selectivity over VOC channels (Table 1). Removal of the 5-trifluoro-
methyl group (13a) dramatically reduced CRAC channel inhibitory
activity. In contrast, the 5-methyl derivative 13b was equipotent to
compound 6, which suggested that the trifluoromethyl and methyl
groups would be interchangeable at this position. These results
indicated that the substituents at the 5-position, neighboring the
benzene ring, might be essential for CRAC channel inhibition.
Introduction of the larger tolyl group (13c) reduced both the activ-
ity and selectivity of the compounds. The cyclopentapyrazole
derivative (13d) showed less potent activity and lower selectivity
than compound 6. This indicated that the trifluoromethyl or small
alkyl groups at the 5-position on the pyrazole ring were favorable
for inhibiting CRAC channels, and that bulky groups may interfere
with interactions with target molecules. In contrast, compounds
13e–g, which were substituted with electron-withdrawing groups
for the 5-trifluoromethyl group, exhibited potent and selective
CRAC channel inhibitory activity. In particular, the chloro (13e)
and bromo (13f) derivatives potently inhibited the CRAC channel
2. Chemistry
The routes of synthesis for intermediates 11a and b are shown
in Schemes 1 and 2. The cyclopentapyrazoles 8 and 9 were ob-
tained via the trifluoroacetylation of cyclopentanone 7 with ethyl
trifluoroacetate in the presence of sodium methoxide (NaOMe),
NO2
NO2
O
a
N
N
N
F3C
+
N
7.86
ppm
with IC50 values of 0.082 and 0.093 lM, respectively.
To investigate the SARs in detail, we attempted to calculate the
torsion angles between the pyrazole and benzene rings for com-
pounds 6, 13a–g using the MOPAC/AM1 program, and then com-
pared these values with the IC50 values of the CRAC channel
(Table 2). The torsion angle of the inactive compound (13a) was
30 degrees. In contrast, the substituents at the 5-position in com-
CF3
NOE
7
9
3.12 ppm
8
Scheme 1. Reagents: (a) i—CF3CO2Et, NaOMe, EtOH; ii—4-NO2C6H4NHNH2, concd
HCl, EtOH.

Y. Yonetoku et al. / Bioorg. Med. Chem. 16 (2008) 9457–9466
9459
NO2
NH2
a
N
N
N
F3C
N
F3C
b: R=
a: R=
R
R
Me
8, 10
11
Scheme 2. Reagents: (a) H2, Pd–C, EtOH.
torsion between the pyrazole and benzene rings is necessary for
CRAC channel inhibition.
Second, we investigated the effect of substituting various het-
eroaryl groups for the 4-methyl-1,2,3-thiadiazole moiety of com-
H
N
R2
NH2
a
R2 CO2H
+
R1
O
R1
12
13
11
pound
6 (Table 3). In the series of compounds possessing
5-membered heteroaryl rings (13h, 13i, 14), the thiazolyl derivatives
(13h, 13i) were less active than compound 6, and the imidazolyl
group (14) dramatically decreased the activity. Although the 2-pyr-
Scheme 3. Reagents: (a) Method A: EDCꢀHCl, THF; Method B: EDCꢀHCl, HOBT, DMF;
Method C: (COCl)2, cat. DMF, CH2Cl2, and then Et3N, CH2Cl2; Method D: HBTU, Et3N,
DMF.
Tr
H
N
H
N
N
H
N
a
N
N
O
N
O
N
N
F3C
N
F3C
CF3
CF3
14
13y
Scheme 4. Reagents: (a) concd HCl, acetone.
NHBoc
NH2
H
H
N
a
N
N
N
O
N
O
N
.HCl
N
F3C
N
F3C
CF3
CF3
13z
15
Scheme 5. Reagents: (a) CF3CO2H, CH2Cl2, and then HCl, AcOEt.
MeO
Cl
H
H
N
N
N
N
N
a
O
O
N
N
N
N
N
N
F3C
F3C
CF3
CF3
16
13n
c
H2N
H
b
N
HO
O
H
F3C
N
N
CF3
O
N
17
N
F3C
CF3
18
Scheme 6. Reagents: (a) HCl, AcOEt, MeOH; (b) liq. NH3; (c) aq HCl, dioxane.
pounds 6, 13b–g, which showed CRAC channel inhibitory activity
with IC50 values less than 10ꢁ6 M, generated torsion angles greater
than 40 degrees. The results indicated that these substituents may
be sterically repulsed by the phenyl group, and that the resultant
idyl derivative 13j showed no inhibitory activity against the CRAC
channel, substitution of the 3- and 4-pyridyl groups (13k, 13l) for
the thiadiazole group retained the activity and improved selectiv-
ity for the CRAC channel over the VOC channel. Compound 13k, in

9460
Y. Yonetoku et al. / Bioorg. Med. Chem. 16 (2008) 9457–9466
NHBoc
NH2
H
H
N
a
N
N
N
O
N
O
N
.HCl
N
F3C
N
F3C
CF3
CF3
13z
15
Scheme 7. Reagents: (a) HCl, AcOEt.
particular, showed almost no VOC channel inhibitory activity,
which meant that CRAC channel selectivity was high (index:
>40). Given this, we speculated that the amide moiety and the pyr-
idine ring of compound 13j would settle into a co-planar confor-
mation through hydrogen bonding between the amide proton
and the nitrogen atom of the pyridine ring, and that this conforma-
tion may be unfavorable for CRAC channel inhibition. This specula-
tion is supported by previous results.10
Next, we examined the effect of substituents in the 3-pyridyl
group of compound 13k. In the series of compounds possessing
substituents at the 4-position (13m–o, 16–18), the methyl
(13m), chloro (13n), and methoxy (16) derivatives exhibited po-
tent and selective CRAC channel inhibitory activity. The trifluoro-
methyl derivative (13o) was almost as potent as compound 13k;
however, its selectivity for the CRAC channel was not sufficient.
Substitution with the amino group (17) resulted in decreased
activity, and the hydroxy derivative (18) exhibited no CRAC chan-
nel inhibitory activity. Similar substituent effects were observed in
the series of compounds substituted at the 2-position (13p, 13q,
19) in the 3-pyridyl group. The chloro (13p) and methoxy (13q)
derivatives exhibited potent CRAC channel inhibitory activity. In
contrast, a more than 10-fold decrease in activity was observed
for the hydroxy derivative 19. We speculated that hydrogen bond-
ing would exist between the carbonyl groups of the amide moiety
and the protons of the hydroxy or amino groups in compounds 17–
19, and that the resultant conformations may cause a loss of CRAC
channel inhibitory activity. This speculation is supported by the
fact that the 5-amino derivative (15) was equipotent to compound
13k. Among the 5-substituted nicotinanilides (13r-u, 15), the 5-
Table 1
Biological Properties of the 4-methyl-1,2,3-thiadiazole-5-carboxanilides
Me
N
H
N
N
S
O
R
Compound
R
IC50
CRACa
(l
M)
CRAC channel
selectivityc
VOCb
N
N
F3C
6
0.15
40%d
0.22
4.7
31
—
fluoro derivative 13s (IC50 = 0.077
CRAC channel inhibitor than compound 13k. This compound also
showed no inhibitory activity against the VOC channel at 10 M,
lM) was a 3-fold more potent
CF3
Me
N
N
l
N
N
13a
13b
NTe
46%d
F3C
F3C
which resulted in excellent selectivity for the CRAC channel (index:
>130). The chloro (13t) and bromo (13u) groups also improved
CRAC channel inhibitory activity and selectivity. These results im-
plied that the introduction of halogens at the 5-position would re-
sult in potent and selective CRAC channel inhibitory activity. This
activity was maintained in compounds 13r and 13v after introduc-
tion of a methyl group at the 5- and 6-positions, respectively. In
compounds with further substitutions, the 4,6-dimethyl derivative
(13w) maintained activity, but compound 13x, which possessed
two methyl groups at the 2- and 4-positions, was less potent than
the other methylated derivatives.
>45
N
N
N
N
F3C
F3C
13c
13d
0.72
0.40
2.5
3.5
3.5
8.8
Me
Selected compounds with potent and selective CRAC channel
inhibitory activity were evaluated for their ability to inhibit phyto-
hemagglutinin (PHA)-induced IL-2 production in Jurkat T cells7
(Table 4). Compound 6 showed potency with an IC50 value of
17 nM. The methyl (13b), chloro (13e), and bromo (13f) derivatives
also demonstrated potent inhibitory activity against IL-2 produc-
tion; in particular, the 5-bromo-3-trifluoromethylpyrazole deriva-
tive (13f) exhibited highly potent activity with an IC50 value of
5.9 nM. In the series of compounds possessing pyridine rings, the
4-pyridyl derivative (13l) was as potent as compound 6; however,
the 3-pyridyl derivative 13k was 5-fold less potent than compound
6. In the 3-pyridyl group, substituents such as halogens (13n, 13s–
u) and methyl groups (13w) improved the activity, which resulted
in compounds as potent as compound 6. In contrast, the introduc-
tion of an amino group (15) decreased in the activity. It is known
that IL-2 plays an important role in the activation, cell cycle pro-
gression, and proliferation of T cells; thus, these results imply that
the function of T lymphocytes would be inhibited by CRAC channel
inhibitors in vitro.
N
N
N
N
F3C
F3C
13e
13f
0.082
0.092
2.9
2.6
35
28
Cl
Br
N
N
F3C
13g
0.22
45%d
>45
CN
The inhibition of Ca2+ influx through the CRAC channels on Jurkat T-cells. See
a
Section 5.
The inhibition of Ca2+ influx through the VOC channels on PC12-h5 cells. See
b
Section 5.
c
IC50 to VOC channel/IC50 to CRAC channel.
d
e
% inhibition at 10
Not tested.
lM.

Y. Yonetoku et al. / Bioorg. Med. Chem. 16 (2008) 9457–9466
9461
Table 2
effectively at a dose of 30 mg/kg p.o. Selected compounds (13k,
13w, 15) were further evaluated for their inhibitory activity
against ovalbumin (OA)-induced airway eosinophilia in rats.20
Among the compounds tested, 13w was found to be the most po-
tent inhibitor, with an ED50 value of 1.3 mg/kg p.o. Airway eosino-
philia is widely studied as a disease model of asthma, and
compounds 6 and 13w, which are expected to exhibit potent inhib-
itory activity in this model, could become candidates for novel
anti-asthmatic agents.21
Torsion angle and CRAC channel inhibitory activity of the 4-methyl-1,2,3-thiadiazole-
5-carboxanilides
Me
N
H
N
N
S
O
N
N
F3C
R
4. Conclusion
Compound
CRAC IC50
(
l
M)a
Torsion angle (deg.)b
Novel 4’-[(trifluoromethyl)pyrazol-1-yl]carboxanilides derived
from compound 6 were designed and evaluated for their CRAC
and VOC channel inhibitory activity. SARs on the pyrazole ring
indicated that substituents would be required at the 5-position
in order to inhibit the CRAC channel, and that the introduction of
electron-withdrawing groups at this position (13e–g) would in-
crease the activity. It was found that the steric repulsion between
the pyrazole and benzene rings caused by introducing these sub-
stituents at the 5-position would be suitable for showing potent
CRAC channel inhibitory activity. On the other hand, replacement
of the 4-methyl-1,2,3-thiadiazole moiety of compound 6 with the
3- and 4-pyridyl groups (13k, 13l) resulted in the inhibition of a
potent and selective CRAC channel. In particular, the 5-haloge-
nated nicotinanilides (13s–u) showed favorable potency and selec-
tivity. Potent and highly selective CRAC channel inhibitors were
also found to inhibit PHA-induced IL-2 production with IC50 values
at the level of 10ꢁ8 to 10ꢁ9 M. In addition, some compounds were
orally available and effective inhibitors in DTH models associated
with T cell activation. Moreover, compound 13w inhibited anti-
gen-induced airway eosinophilia in rats as potently as compound
6. We expect these CRAC channel inhibitors to become a new class
of anti-inflammatory and immunosuppressive agents.
6
0.15
40%d
0.22
0.72
0.40
0.082
0.092
0.22
55
30
43
46
60
52
57
42
13a
13b
13c
13d
13e
13f
13g
a
See footnote a in Table 1.
Torsion angle between the N–N bond in the pyrazole and the C–C bond in the
b
benzene (bold in scheme).
These compounds were also tested in a delayed-type hypersen-
sitivity (DTH) model19 (Table 3). Substitution of the methyl (13b),
chloro (13e), and bromo (13f) groups for the trifluoromethyl (6)
group at the 5-position in the pyrazole moiety tended to reduce
activity in vivo. However, several nicotinanilide derivatives were
found to inhibit TNCB-induced ear swelling in sensitized mice
Table 3
Biological Properties of the 40-[3,5-bis(trifluoromethyl)pyrazol-1-yl]carboxanilides
H
N
R
5. Experimental
O
N
N
Melting points were determined without correction using a
Yanagimoto MP-3 melting point apparatus. 1H NMR spectra were
recorded on a JEOL JNM-LA300, a JEOL JNM-EX400, or a JEOL
JNM-A500 spectrometer, and chemical shifts were measured in
ppm using tetramethylsilane as an internal standard. The signal
pattern abbreviations are as follows: s: singlet, br s: broad singlet,
d: doublet, t: triplet, dd: double doublet, dt: double triplet, and m:
multiplet. FAB-MS were recorded on a JEOL JMS-DX300, a JEOL
JMS-DX2000, a HP 5970-MSD, a Fisons TRIO-1000, or a Finnigan
mat TSQ 700 mass spectrometer. The elemental analyses were per-
formed with a Yanako MT-5 microanalyzer and a Yokogawa IC-
7000S ion chromatographic analyzer. The drying of organic solu-
tions during workup was done over anhydrous MgSO4. Column
chromatography was performed with Wakogel C-200 or Merck sil-
ica gel 60.
F3C
CF3
Compound.
R
IC50
(l
M)
CRAC channel
selectivityc
CRACa
VOCb
6
4-Methyl-1,2,3-thiadiazol-5-yl
Thiazol-5-yl
0.15
0.96
0.46
29%d
14%d
0.25
0.24
0.29
0.16
0.43
0.23
0.78
21%d
0.24
0.12
3.3
0.21
0.077
0.11
0.15
0.27
0.22
0.29
1.6
4.7
10
31
10
>22
—
—
>40
42
20
32
5
21
3.1
—
19
24
13h
13i
14
13j
13k
13l
13m
13n
13o
16
Thiazol-2-yl
1H-imidazol-4-yl
Pyridin-2-yl
Pyridin-3-yl
37%d
NTe
NTe
30%d
10
Pyridin-4-yl
4-Methylpyridin-3-yl
4-Chloropyridin-3-yl
4-Trifluoromethylpyridin-3-yl
4-Methoxypyridin-3-yl
4-Aminopyridin-3-yl
4-Hydroxypyridin-3-yl
2-Chloropyridin-3-yl
2-Methoxypyridin-3-yl
2-Hydroxypyridin-3-yl
5-Methylpyridin-3-yl
5-Fluoropyridin-3-yl
5-Chloropyridin-3-yl
5-Bromopyridin-3-yl
5-Aminopyridin-3-yl
6-Methylpyridin-3-yl
4,6-Dimethylpyridin-3-yl
2,4-Dimethylpyridin-3-yl
6.3
5.1
2.0
4.8
17
18
2.4
5.1. 1-(4-Nitrophenyl)-3-(trifluoromethyl)-1,4,5,6-tetrahydro-
cyclopenta[c]pyrazole (8 ) and 2-(4-nitrophenyl)-3-(trifluoro-
methyl)-2,4,5,6-tetrahydrocyclopenta[c]pyrazole (9)
NTe
4.5
13p
13q
19
13r
13s
13t
13u
15
2.9
NTe
3.1
—
15
Ethyl trifluoroacetate (4.26 g, 30.0 mmol) was added to a mix-
ture of NaOMe (1.78 g, 33.0 mmol) in Et2O (30 mL) dropwise below
30 °C, then cyclopentanone (7, 2.52 g, 30.0 mmol) was added drop-
wise. The mixture was stirred for 24 h at room temperature, and
then 1 M HCl was added. This mixture was extracted with AcOEt,
washed with H2O and brine, and then dried and concentrated
27%d
31%d
29%d
14%d
27%d
41%d
NTe
>130
>91
>67
>37
>45
>34
—
13v
13w
13x
in vacuo. The residue (4.71 g) was added to
a mixture of
a–e
34-nitrophenylhydrazine (4.59 g, 30.0 mmol), concentrated HCl
See the corresponding footnotes in Table 1.

9462
Y. Yonetoku et al. / Bioorg. Med. Chem. 16 (2008) 9457–9466
Table 4
and washed with 1 M HCl, saturated aqueous NaHCO3, and brine.
The extract was dried and concentrated in vacuo, and the resi-
Biological Properties of Selected Compounds
Compound
IC50
(
lM)
IC50 (nM)
IL-2b
DTHc
% inhibition at
30 mg/kg p.o.
Eosinophiliad
ED50
(mg/kg p.o.)
due was recrystallized from AcOEt/hexane to yield 13h (77 mg,
CRACa
33%) as
a
pale brown powder. mp 194–196 °C; 1H NMR
(DMSO-d6) d 7.64 (2H, d, J = 9.0 Hz), 7.83 (1H, s), 7.94 (2H, d,
J = 9.0 Hz), 8.74 (1H, s), 9.35 (1H, s), 10.76 (1H, s); FAB-MS m/z
407 [(M+H)+]. Anal. calcd for C15H8F6N4OS: C, 44.34; H, 1.98;
N, 13.79; F, 28.05; S, 7.89. Found: C, 44.18; H, 2.05; N, 13.77;
F, 27.89; S, 7.94.
The compounds described below were prepared using the same
method. Among the corresponding amines, 4-[5-(4-methyl-
phenyl)-3-trifluoromethyl-1H-pyrazol-1-yl]aniline18 was prepared
using the method reported previously.
6
0.15
0.22
0.082
0.092
0.25
0.24
0.16
0.077
0.11
0.15
0.27
0.22
0.29
17
18
15
5.9
93
16
18
18
34
24
160
94
33
ca.100e
24
42
8.3
62
25
-18
44
42
17
59
2.4
NTf
NTf
NTf
4.7
NTf
NTf
NTf
NTf
NTf
4.4
NTf
1.3
13b
13e
13f
13k
13l
13n
13s
13t
13u
15
13v
13w
46
64
5.1.6. 4-Methyl-4’-(3-trifluoromethyl-1H-pyrazol-1-yl)-1,2,3-
thiadiazole-5-carboxanilide (13a)
a
(66%): mp 158–161 °C (AcOEt-Hexane); 1H NMR (DMSO-d6) d
2.84 (3H, s), 7.05 (1H, d, J = 2.5 Hz), 7.84-7.94 (4H, m), 8.70 (1H,
dd, J = 2.5, 1.0 Hz), 10.92 (1H, s); FAB-MS m/z 354 [(M+H)+]. Anal.
calcd for C14H10F3N5OS: C, 47.59; H, 2.85; N, 19.82; F, 16.13; S,
9.08. Found: C, 47.43; H, 3.03; N, 19.96; F, 16.20; S, 9.09.
See footnote c in Table 1.
b
c
d
e
f
Inhibition of PHA-induced IL-2 production in Jurkat T-cells. See Section 5.
Inhibition of TNCB-induced contact hypersensitivity in mice. See Section 5.
The inhibition of OA-induced airway eosinophilia in rats. See Section 5.
ED50 = 1.1 mg/kg p.o.
Not tested.
5.1.7. 4-Methyl-4’-(5-methyl-3-trifluoromethyl-1H-pyrazol-1-
yl)-1,2,3-thiadiazole-5-carboxanilide (13b)
(30 mL) and EtOH (30 mL). This mixture was heated at 80 °C for
4 h, and then H2O was added and extracted with AcOEt, washed
with 1 M HCl, brine, dried, and concentrated in vacuo. The residue
was purified via column chromatography (hexane/AcOEt 19:1–
9:1) to yield 8 (1.65 g, 19%) and 9 (178 mg, 2%) as light yellow nee-
dle crystals.
(76%): mp 75–76 °C (EtOH-Et2O); 1H NMR (DMSO-d6) d 2.35
(3H, s), 2.84 (3H, s), 6.76 (1H, s), 7.60 (2H, d, J = 8.8 Hz), 7.88 (2H,
d, J = 8.8 Hz), 10.97 (1H, s); FAB-MS m/z 368 [(M+H)+]. Anal. calcd
for C15H12F3N5OSꢀ0.3C2H6Oꢀ0.5H2O: C, 48.02; H, 3.82; N, 17.95; F,
14.61; S, 8.22. Found: C, 47.97; H, 3.84; N, 17.61; F, 14.26; S, 7.95.
5.1.1. 1-(4-Nitrophenyl)-3-(trifluoromethyl)-1,4,5,6-tetrahydro-
cyclopenta[c]pyrazole (8)
5.1.8. 4-Methyl-4’-[5-(4-methylphenyl)-3-trifluoromethyl-1H-
pyrazol-1-yl]-1,2,3-thiadiazole-5-carboxanilide (13c)
1H NMR (CDCl3) d 2.60–2.85 (4H, m), 3.12 (2H, t, J = 6.9 Hz), 7.86
(2H, d, J = 9.2 Hz), 8.33 (2H, d, J = 9.2 Hz); GC–MS m/z 297 (M+).
(77%): 1H NMR (DMSO-d6) d 2.31 (3H, s), 2.82 (3H, s), 7.13 (1H,
s), 7.16–7.22 (4H, m), 7.37 (2H, d, J = 8.8 Hz), 7.76 (2H, d, J = 8.8 Hz),
10.91 (1H, s); FAB-MS m/z 444 [(M+H)+]. Anal. calcd for
C21H16F3N5OS: C, 56.88; H, 3.64; N, 15.79; F, 12.85; S, 7.23. Found:
C, 56.72; H, 3.61; N, 15.61; F, 12.62; S, 7.06.
5.1.2. 2-(4-Nitrophenyl)-3-(trifluoromethyl)-2,4,5,6-tetrahydro-
cyclopenta[c]pyrazole (9)
1H NMR (CDCl3) d 2.46-2.57 (2H, m), 2.80-2.92 (4H, m), 7.70
(2H, d, J = 9.0 Hz), 8.34 (2H, d, J = 9.0 Hz); GC-MS m/z 297 (M+).
5.1.9. 4-Methyl-40-(3-trifluoromethyl-1,4,5,6-tetrahydrocyclo-
pentapyrazol-1-yl)-1,2,3-thiadiazole-5-carboxanilide (13d)
(35%): mp 155–156 °C (AcOEt–hexane); 1H NMR (DMSO-d6) d
2.57–2.65 (2H, m), 2.72 (2H, t, J = 6.8 Hz), 2.83 (3H, s), 3.08 (2H, t,
J = 7.1 Hz), 7.73 (2H, d, J = 9.1 Hz), 7.85 (2H, d, J = 9.1 Hz), 10.91
(1H, s); FAB-MS m/z 394 [(M+H)+]. Anal. calcd for C17H14F3N5OS:
C, 51.90; H, 3.59; N, 17.80; F, 14.49; S, 8.15. Found: C, 51.70; H,
3.51; N, 17.89; F, 14.45; S, 8.08.
5.1.3. 4-[3-(Trifluoromethyl)-5,6-dihydrocyclopenta[c]pyrazol-
1(4H)-yl]aniline (11a)
1-(4-Nitrophenyl)-3-(trifluoromethyl)-1,4,5,6-tetrahydrocyclo-
penta[c]pyrazole (8, 1.00 g, 3.36 mmol) was hydrogenated at room
temperature for 1 h in the presence of 10% Pd–C in EtOH (10 mL)
and THF (10 mL). After the catalyst was removed via filtration,
the filtrate was concentrated in vacuo and recrystallized from
AcOEt/hexane to yield 11a (583 mg, 65%) as a gray powder: 1H
NMR (CDCl3) d 2.57–2.69 (2H, m), 2.76 (2H, t, J = 7.1 Hz), 2.93
(2H, t, J = 7.1 Hz), 3.75 (2H, br s), 6.71 (2H, d, J = 8.8 Hz), 7.38
(2H, d, J = 8.8 Hz); FAB-MS m/z 268 [(M+H)+].
5.1.10. 40-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]thiazole-2-
carboxanilide (13i)
(49%): mp 174–175 °C (AcOEt–hexane); 1H NMR (DMSO-d6) d
7.63 (2H, d, J = 8.8 Hz), 7.83 (1H, s), 8.10 (2H, d, J = 8.8 Hz), 8.15
(1H, d, J = 3.2 Hz), 8.19 (1H, d, J = 3.2 Hz), 11.17 (1H, s); FAB-MS
m/z 407 [(M+H)+]. Anal. calcd for C15H8F6N4OS: C, 44.34; H, 1.98;
N, 13.79; F, 28.05; S, 7.89. Found: C, 44.15; H, 1.85; N, 13.87; F,
27.83; S, 7.84.
The compound described below was prepared similarly.
5.1.4. 4-[5-Methyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]aniline
(11b)
1H NMR (DMSO-d6) d 2.24 (3H, s), 5.47 (2H, s), 6.59-6.68 (3H,
m), 7.10–7.15 (2H, m); FAB-MS m/z 242 [(M+H)+].
5.1.11. 40-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]pyridine-2-
carboxanilide (13j)
5.1.5. 40-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]thiazole-5-
carboxanilide (13h)
A mixture of thiazole-5-carboxylic acid (75 mg, 0.581 mmol),
4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]aniline (11c, 171 mg,
0.579 mmol), EDC HCl (117 mg, 0.610 mmol), and THF (3 ml)
were stirred together for 5 h at room temperature, and then
H2O (10 ml) was added. This mixture was extracted with AcOEt
(55%): mp 171–173 °C (AcOEt–hexane); 1H NMR (DMSO-d6) d
7.62 (2H, d, J = 9.0 Hz), 7.70-7.74 (1H, m), 7.82 (1H, s), 8.10 (1H,
td, J = 7.8, 1.5 Hz), 8.16 (2H, d, J = 9.0 Hz), 8.20 (1H, d, J = 7.8 Hz),
8.78 (1H, dd, J = 4.9, 1.5 Hz), 11.20 (1H, s); FAB-MS m/z 401
[(M+H)+]. Anal. calcd for C17H10F6N4O: C, 51.01; H, 2.52; N,
14.00; F, 28.48. Found: C, 50.82; H, 2.50; N, 14.04; F, 28.72.

Y. Yonetoku et al. / Bioorg. Med. Chem. 16 (2008) 9457–9466
9463
5.1.12. 40-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]nicotinan-
ilide (13k)
5.1.18. 40-(5-Bromo-3-trifluoromethyl-1H-pyrazol-1-yl)-4-methyl-
1,2,3-thiadiazole-5-carboxanilide (13f)
(53%): mp 188–190 °C (AcOEt–hexane); 1H NMR (DMSO-d6) d
7.58–7.62 (1H, m), 7.64 (2H, d, J = 9.0 Hz), 7.83 (1H, s), 8.00 (2H,
d, J = 9.0 Hz), 8.33 (1H, dt, J = 7.8, 1.8 Hz), 8.80 (1H, dd, J = 4.7,
1.8 Hz), 9.14 (1H, d, J = 1.8 Hz), 10.77 (1H, s); FAB-MS m/z 401
[(M+H)+]. Anal. calcd for C17H10F6N4O: C, 51.01; H, 2.52; N,
14.00; F, 28.48. Found: C, 50.91; H, 2.33; N, 14.02; F, 28.77.
(73%): mp 150 °C (MeOH–H2O); 1H NMR (DMSO-d6) d 2.84
(3H, s), 7.32 (1H, s), 7.64 (2H, d, J = 9.0 Hz), 7.90 (2H, d,
J = 9.0 Hz), 11.02 (1H, s); FAB-MS m/z 432, 434 [(M+H)+]. Anal.
calcd for C14H9BrF3N5OS: C, 38.90; H, 2.10; N, 16.20; Br, 18.49;
F, 13.19; S, 7.42. Found: C, 38.81; H, 1.99; N, 16.29; Br, 18.41;
F, 13.11; S, 7.38.
5.1.19. 40-(5-Cyano-3-trifluoromethyl-1H-pyrazol-1-yl)-4-methyl-
1,2,3-thiadiazole-5-carboxanilide (13g)
5.1.13. 4’-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]isonicotin-
anilide monohydrochloride (13l)
(66%): mp 170–172 °C (EtOH–MeCN); 1H NMR (DMSO-d6) d
7.66 (2H, d, J = 9.0 Hz), 7.84 (1H, s), 8.04 (2H, d, J = 9.0 Hz), 8.19
(2H, d, J = 5.9 Hz), 8.94–9.02 (2H, m), 11.13 (1H, s); FAB-MS m/z
401 [(M+H)+]. Anal. calcd for C17H10F6N4OꢀHCl: C, 46.75; H, 2.54;
N, 12.83; Cl, 8.12; F, 26.10. Found: C, 46.71; H, 2.46; N, 12.98; Cl,
8.09; F, 26.15.
(60%): mp 215–216 °C (MeOH); 1H NMR (DMSO-d6) d 2.84 (3H,
s), 7.83 (2H, d, J = 8.8 Hz), 7.95 (2H, d, J = 8.8 Hz), 8.08 (1H, s), 11.06
(1H, s); FAB-MS m/z 379 [(M+H)+]. Anal. calcd for C15H9F3N6OS: C,
47.62; H, 2.40; N, 22.21; F, 15.06; S, 8.48. Found: C, 47.60; H, 2.37;
N, 22.43; F, 15.06; S, 8.51.
5.1.20. 4-Chloro-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-
nicotinanilide (13n)
A mixture of 4-chloronicotinic acid (564 mg, 3.58 mmol), oxa-
lyl chloride (0.26 ml, 2.98 mmol), and DMF (5 ll) in dichloro-
5.1.14. 5-Methyl-4’-[3,5-bis(trifluoromethyl)-1>H-pyrazol-1-yl]
nicotinanilide (13r)
(10%): mp 178–181 °C (AcOEt–hexane); 1H NMR (DMSO-d6) d
2.42 (3H, s), 7.63 (2H, d, J = 8.8 Hz), 7.83 (1H, s), 8.00 (2H, d,
J = 8.8 Hz), 8.14 (1H, s), 8.64 (1H, d, J = 1.5 Hz), 8.94 (1H, d,
J = 1.5 Hz), 10.73 (1H, s); FAB-MS m/z 415 [(M+H)+]. Anal. calcd
for C18H12F6N4O: C, 52.18; H, 2.92; N, 13.52; F, 27.51. Found: C,
52.10; H, 2.80; N, 13.57; F, 27.66.
methane (6 ml) was stirred for 3 h at room temperature and
concentrated in vacuo. The residue was added to dichlorometh-
ane (6 ml), 4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]aniline
(11c12, 590 mg, 2.00 mmol), and Et3N (0.42 ml, 3.01 mmol) at
0 °C, and this mixture was stirred for 2 h at room temperature.
The reaction mixture was diluted with AcOEt and washed with
saturated aqueous NaHCO3 and brine. The organic layer was dried
and concentrated in vacuo. The residue was recrystallized from
EtOH–H2O to yield 13n (470 mg, 54%) as a pink powder. mp
170–171 °C; 1H NMR (DMSO-d6) d 7.64 (2H, d, J = 8.8 Hz), 7.74
(1H, d, J = 5.6 Hz), 7.83 (1H, s), 7.93 (2H, d, J = 8.8 Hz), 8.68 (1H,
d, J = 5.6 Hz), 8.85 (1H, s), 11.06 (1H, s); FAB-MS m/z 435
[(M+H)+]. HRMS m/z calcd for C17H10ClF6N4O [M+H]+: 435.0447.
Found: 435.0440.
5.1.15. 5-Fluoro-4’-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-
nicotinanilide (13s)
(49%): mp 176–178 °C (AcOEt–hexane); 1H NMR (DMSO-d6) d
7.64 (2H, d, J = 8.8 Hz), 7.82 (1H, s), 7.99 (2H, d, J = 8.8 Hz), 8.25
(1H, dt, J = 9.3, 1.9 Hz), 8.83 (1H, d, J = 3.0 Hz), 9.02 (1H, s), 10.81
(1H, s); FAB-MS m/z 419 [(M+H)+]. Anal. calcd for C17H9F7N4O: C,
48.82; H, 2.17; N, 13.39; F, 31.80. Found: C, 48.62; H, 2.13; N,
13.43; F, 31.64.
The compounds described below were prepared following the
same method.
5.1.16. 5-Chloro-4’-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-
nicotinanilide (13t)
(25%): mp 175–177 °C (AcOEt–hexane); 1H NMR (DMSO-d6) d
7.64 (2H, d, J = 8.8 Hz), 7.82 (1H, s), 7.99 (2H, d, J = 8.8 Hz), 8.45
(1H, t, J = 2.2 Hz), 8.86 (1H, d, J = 2.2 Hz), 9.07 (1H, d, J = 2.2 Hz),
10.82 (1H, s); FAB-MS m/z 435 [(M+H)+]. Anal. calcd for
C17H9ClF6N4O: C, 46.97; H, 2.09; N, 12.89; Cl, 8.16; F, 26.22. Found:
C, 46.96; H, 2.02; N, 12.97; Cl, 8.17; F, 26.08.
5.1.21. 4-Trifluoromethyl-40-[3,5-bis(trifluoromethyl)-1H-pyra-
zol-1-yl]nicotinanilide (13o)
(91%): mp 179–180 °C (EtOH–H2O); 1H NMR (DMSO-d6) d 7.65
(2H, d, J = 8.8 Hz), 7.83 (1H, s), 7.90 (2H, d, J = 8.8 Hz), 7.94 (1H,
d, J = 5.1 Hz), 9.02 (1H, d, J = 5.1 Hz), 9.10 (1H, s), 11.14 (1H, s);
FAB-MS m/z 469 [(M+H)+]. Anal. calcd for C18H9F9N4O: C, 46.17;
H, 1.94; N, 11.96; F, 36.51. Found: C, 46.14; H, 1.64; N, 12.21; F,
36.57.
5.1.17. 40-(5-Chloro-3-trifluoromethyl-1H-pyrazol-1-yl)-4-methyl-
1,2,3-thiadiazole-5-carboxanilide (13e)
A
mixture of 1,2,3-thiadiazole-5-carboxylic acid (166 mg,
5.1.22. 2-Methoxy-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]nicotinanilide (13q)
1.15 mmol), 4-(5-chloro-3-trifluoromethyl-1H-pyrazol-1-yl)ani-
line12 (11d, 250 mg, 0.956 mmol), EDC HCl (220 mg, 1.15 mmol),
HOBT (155 mg, 1.15 mmol), and DMF (5 ml) was stirred overnight
at room temperature, and then H2O was added. This mixture was
extracted with AcOEt and washed with brine. The extract was
dried and concentrated in vacuo, and the residue was recrystallized
from AcOEt–hexane to yield 13e (282 mg, 76%) as a colorless pow-
der. mp 148 °C; 1H NMR (DMSO-d6) d 2.84 (3H, s), 7.30 (1H, s), 7.66
(2H, d, J = 8.8 Hz), 7.91 (2H, d, J = 8.8 Hz), 11.00 (1H, s); FAB-MS m/z
388 [(M+H)+]. Anal. calcd for C14H9ClF3N5OS: C, 43.36; H, 2.34; N,
18.06; Cl, 9.14; F, 14.70; S, 8.27. Found: C, 43.41; H, 2.21; N,
18.30; Cl, 8.90; F, 14.72; S, 8.24.
The compounds described below were prepared following the
same method. Among the corresponding amines, 4-(5-bromo-
3-trifluoromethyl-1H-pyrazol-1-yl)aniline and 4-(5-cyano-3-tri-
fluoromethyl-1H-pyrazol-1-yl)aniline were prepared using the
methods reported previously.12
(44%): mp 128 °C (hexane); 1H NMR (DMSO-d6) d 4.00 (3H, s),
7.14–7.20 (1H, m), 7.61 (2H, d, J = 8.6 Hz), 7.82 (1H, s), 7.94 (2 H,
d, J = 8.6 Hz), 8.07 (1H, d, J = 7.3 Hz), 8.36 (1H, d, J = 4.8 Hz), 10.56
(1H, s); FAB-MS m/z 431 [(M+H)+]. Anal. calcd for C18H12F6N4O2:
C, 50.24; H, 2.81; N, 13.02; F, 26.49. Found: C, 50.25; H, 2.97; N,
13.06; F, 26.33.
5.1.23. 6-Methyl-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]nicotinanilide monohydrochloride (13v)
(27%): mp 215–216 °C (EtOH–H2O); 1H NMR (DMSO-d6) d 2.74
(3H, s), 7.65 (2H, d, J = 9.0 Hz), 7.84 (1H, s), 7.82-7.86 (1H, m),
8.05 (2H, d, J = 9.0 Hz), 8.70 (1H, dd, J = 8.3, 1.7 Hz), 9.30 (1H, d,
J = 1.7 Hz), 11.13 (1H, s); FAB-MS m/z 415 [(M+H)+]. Anal. calcd
for C18H12F6N4OꢀHCl: C, 47.96; H, 2.91; N, 12.43; Cl, 7.87; F,
25.29. Found: C, 48.01; H, 2.87; N, 12.43; Cl, 7.67; F, 25.02.

9464
Y. Yonetoku et al. / Bioorg. Med. Chem. 16 (2008) 9457–9466
5.1.24. 4,6-Dimethyl-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]nicotinanilide (13w)
was purified via column chromatography (hexane/AcOEt 1:1–2:3)
and recrystallized from AcOEt–hexane to yield 14 (35 mg, 13%) as a
colorless powder. mp 200 °C (decomposed); 1H NMR (DMSO-d6) d
7.55 (2H, d, J = 9.0 Hz), 7.80 (1H, s), 7.86 (1H, s), 7.87 (1H, s), 8.07
(2H, d, J = 9.0 Hz), 10.23 (1H, s), 12.64-12.76 (1H, br s); FAB-MS
m/z 390 [(M+H)+]. Anal. calcd for C15H9F6N5Oꢀ0.25H2O: C, 45.75;
H, 2.43; N, 17.79; F, 28.95. Found: C, 45.84; H, 2.34; N, 17.96; F,
28.75.
(9%): mp 194-196 °C (AcOEt–hexane); 1H NMR (DMSO-d6) d
2.41 (3H, s), 2.50 (3H, s), 7.25 (1H, s), 7.61 (2H, d, J = 8.8 Hz), 7.82
(1H, s), 7.95 (2H, d, J = 8.8 Hz), 8.59 (1H, s), 10.76 (1H, s); FAB-MS
m/z 429 [(M+H)+]. Anal. calcd for C19H14F6N4O: C, 53.28; H, 3.29;
N, 13.08; F, 26.61. Found: C, 53.16; H, 3.19; N, 13.22; F, 26.71.
5.1.25. 2,4-Dimethyl-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]nicotinanilide monohydrochloride (13x)
5.1.30. 5-Amino-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]nicotinanilide monohydrochloride dihydrate (15)
(35%): mp 190 °C (decomposed) (EtOH–AcOEt–hexane); 1H
NMR (DMSO-d6)
d
2.54 (3H, s), 2.71 (3H, s), 7.66 (2H, d,
A mixture of t-butyl 5-{4-[3,5-bis(trifluoromethyl)-1H-pyrazol-
1-yl]phenylcarbamoyl}pyridin-3-ylcarbamate (13z, 147 mg,
0.285 mmol), trifluoroacetic acid (2 ml) and CH2Cl2 (2 ml) was stir-
red for 2 h at room temperature, and then concentrated in vacuo.
This mixture was diluted with AcOEt and washed with aqueous
NaHCO3 and brine. The organic layer was dried and concentrated
in vacuo. The residue was added to AcOEt (10 ml) and 4 M HCl–
AcOEt (1 ml) to yield the crude hydrochloride, and the crude prod-
uct was recrystallized from EtOH–AcOEt to yield 15 (116 mg, 83%)
as colorless needle crystals. mp 260 °C (sublimated); 1H NMR
(DMSO-d6) d 7.64 (2H, d, J = 8.8 Hz), 7.82 (1H, s), 7.97 (1H, s),
8.00 (2H, d, J = 8.8 Hz), 8.18 (1H, s), 8.58 (1H, s), 11.02 (1H, s);
FAB-MS m/z 416 [(M+H)+]. Anal. calcd for C17H11F6N5OꢀHClꢀ2H2O:
C, 41.86; H, 3.31; N, 14.36; Cl, 7.27; F, 23.37. Found: C, 41.82; H,
3.02; N, 14.47; Cl, 7.09; F, 23.42.
J = 8.8 Hz), 7.81 (1H, d, J = 5.9 Hz), 7.84 (1H, s), 7.96 (2H, d,
J = 8.8 Hz), 8.72 (1H, d, J = 5.9 Hz), 11.42 (1H, s); FAB-MS m/z 429
[(M+H)+]. Anal. calcd for C19H14F6N4OꢀHCl: C, 49.10; H, 3.25; N,
12.05; Cl, 7.63; F, 24.53. Found: C, 49.03; H, 3.22; N, 12.10; Cl,
7.65; F, 24.43.
5.1.26. 4-Methyl-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-
nicotinanilide monohydrochloride (13m)
Et3N (1.90 ml, 13.6 mmol) and HBTU (2.46 g, 6.49 mmol) were
added to a chilled solution of 4-methylnicotinic acid monohydro-
chloride (1.04 g, 5.99 mmol) in DMF (15 ml), and the mixture
was stirred for 3 h at room temperature. The mixture was then
added to 4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]aniline (11c,
1.47 g, 4.99 mmol) and stirred for 6 days at room temperature,
after which H2O was added. This mixture was extracted with
AcOEt and washed with H2O, saturated aqueous NaHCO3 and brine.
The organic layer was dried and concentrated in vacuo. The residue
was purified via column chromatography (CHCl3/MeOH 99: 1), and
added AcOEt (30 ml) and 4 M HCl–AcOEt (3 ml) to yield the crude
hydrochloride, and the crude product was recrystallized from EtOH
to yield 13m (735 mg, 33%) as a colorless powder. mp 181 °C
(decomposed); 1H NMR (DMSO-d6) d 2.62 (3H, s), 7.65 (2H, d,
J = 8.8 Hz), 7.83 (1H, s), 7.85 (1H, d, J = 5.9 Hz), 7.98 (2H, d,
J = 8.8 Hz), 8.81 (1H, d, J = 5.9 Hz), 9.04 (1H, s), 11.24 (1H, s);
FAB-MS m/z 415 [(M+H)+]. Anal. calcd for C18H12F6N4OꢀHCl: C,
47.96; H, 2.91; N, 12.43; F, 25.29; Cl, 7.87. Found: C, 47.74; H,
2.74; N, 12.42; F, 25.18; Cl, 7.86.
5.1.31. 4-Methoxy-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]nicotinanilide monohydrochloride (16)
A mixture of 4-chloro-4’-[3,5-bis(trifluoromethyl)-1H-pyrazol-
1-yl]-nicotinanilide (13n, 100 mg, 0.230 mmol), 4 M HCl–AcOEt
(0.115 ml, 0.460 mmol) and MeOH (2 ml) was stirred for 5 h at
room temperature, and then concentrated in vacuo, and the resi-
due was recrystallized from MeOH–Et2O to yield 16 (70 mg, 65%)
as a colorless powder. mp 272–275 °C; 1H NMR (DMSO-d6) d 4.13
(3H, s), 7.64 (2H, d, J = 8.8 Hz), 7.67 (1H, d, J = 6.9 Hz), 7.82 (1H,
s), 7.93 (2H, d, J = 8.8 Hz), 8.86 (1H, d, J = 6.9 Hz), 8.95 (1H, s),
10.91 (1H, s); FAB-MS m/z 431 [(M+H)+]. Anal. calcd for
C18H12F6N4O2ꢀHCl: C, 46.32; H, 2.81; N, 12.00; Cl, 7.60; F, 24.42.
Found: C, 45.97; H, 2.66; N, 11.94; Cl, 7.57; F, 24.15.
The compounds described below were prepared following the
same method.
5.1.32. 4-Amino-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]nicotinanilide (17)
5.1.27. 2-Chloro-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]nicotinanilide (13p)
A mixture of 4-chloro-4’-[3,5-bis(trifluoromethyl)-1H-pyrazol-
1-yl]-nicotinanilide (13n, 450 mg, 1.04 mmol) and liquid ammonia
(10 ml) was stirred overnight at 80 °C in a sealed tube, and then
concentrated in vacuo. The residue was purified via column chro-
matography (CHCl3/MeOH 50:1–20:1) and recrystallized from
AcOEt–hexane to yield 17 (72 mg, 17%) as a pale yellow powder.
mp 241 °C (decomposed); 1H NMR (DMSO-d6) d 6.69 (1H, d,
J = 5.6 Hz), 7.09 (2H, s), 7.59 (2H, d, J = 8.8 Hz), 7.82 (1H, s), 7.94
(2H, d, J = 8.8 Hz), 8.09 (1H, d, J = 5.6 Hz), 8.68 (1H, s), 10.49 (1H,
s); FAB-MS m/z 416 [(M+H)+]. Anal. calcd for C17H11F6N5Oꢀ0.25H2O:
C, 48.64; H, 2.76; N, 16.68; F, 27.15. Found: C, 48.85; H, 2.54; N,
16.77; F, 27.42.
(55%): mp 154–157 °C (EtOH–H2O); 1H NMR (DMSO-d6) d 7.60
(1H, dd, J = 7.8, 4.8 Hz), 7.64 (2H, d, J = 9.0 Hz), 7.83 (1H, s), 7.92
(2H, d, J = 9.0 Hz), 8.15 (1H, dd, J = 7.8, 2.0 Hz), 8.57 (1H, dd,
J = 4.8, 2.0 Hz), 11.02 (1H, s); FAB-MS m/z 435 [(M+H)+]. Anal. calcd
for C17H9ClF6N4O: C, 46.97; H, 2.09; N, 12.89; Cl, 8.16; F, 26.22.
Found: C, 46.98; H, 2.02; N, 12.97; Cl, 8.16; F, 26.01.
5.1.28. 5-Bromo-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]nicotinanilide (13u)
(48%): mp 169–170 °C (AcOEt–hexane); 1H NMR (DMSO-d6) d
7.64 (2H, d, J = 9.0 Hz), 7.83 (1H, s), 7.98 (2H, d, J = 9.0 Hz), 8.58
(1H, t, J = 2.0 Hz), 8.94 (1H, d, J = 2.0 Hz), 9.09 (1H, d, J = 2.0 Hz),
10.82 (1H, s); FAB-MS m/z 479, 481 [(M+H)+]. Anal. calcd for
C17H9BrF6N4O: C, 42.61; H, 1.89; N, 11.69; Br, 16.68; F, 23.79.
Found: C, 42.63; H, 1.75; N, 11.73; Br, 16.76; F, 23.55.
5.1.33. 4-Hydroxy-40-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]nicotinanilide (18)
A mixture of 4-chloro-4’-[3,5-bis(trifluoromethyl)-1H-pyrazol-
1-yl]-nicotinanilide (13n, 350 mg, 0.806 mmol), 1 M HCl (4 ml),
and dioxane (4 ml) was stirred overnight at 50 °C, and then 1 M
NaOH was added. This mixture was extracted with AcOEt and
washed with brine. The organic layer was dried and concentrated
in vacuo. The residue was recrystallized from AcOEt–hexane to
yield 18 (110 mg, 33%) as a colorless powder. mp >300 °C; 1H
NMR (DMSO-d6) d 6.53–6.57 (1H, m), 7.60 (2H, d, J = 8.8 Hz), 7.81
5.1.29. 40-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]-1H-
imidazole-4-carboxanilide (14)
A mixture of 4’-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1-
trityl-1H-imidazole-4-carboxanilide (13y, 180 mg, 0.285 mmol),
concentrated HCl (0.1 ml) and acetone (3 ml) was stirred for 17 h
at room temperature, and then concentrated in vacuo. The residue

Y. Yonetoku et al. / Bioorg. Med. Chem. 16 (2008) 9457–9466
9465
(1H, s), 7.89 (2H, d, J = 8.8 Hz), 7.89–7.93 (1H, m), 8.60-8.63 (1H,
was measured, and 10
lL of 0.25% TNCB was applied to both the
m), 12.25-12.55 (1H, br s), 13.21 (1H, s); FAB-MS m/z 417
[(M+H)+]. Anal. calcd for C17H10F6N4O2ꢀ0.25H2O: C, 48.52; H,
2.52; N, 13.31; F, 27.09. Found: C, 48.51; H, 2.28; N, 13.36; F, 27.20.
The compounds described below were prepared following the
same method.
inside and outside of the ear pinnas. Only the solvent was applied
to negative control mice. Ear thickness was measured 24 h after
the TNCB challenge using a dial thickness gauge, and the change
due to swelling were calculated from the pre-exposure value. The
compounds were administered orally 1 h before exposure to TNCB,
and 0.5% MC was administered orally to the negative control and
control animals.
5.1.34. 2-Hydroxy-4’-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-
yl]nicotinanilide (19)
(5%): mp >300 °C (MeOH); 1H NMR (DMSO-d6) d 6.61 (1H, t,
J = 6.7 Hz), 7.61 (2H, d, J = 9.0 Hz), 7.82 (1H, s), 7.86 (1H, dd,
J = 6.7, 2.4 Hz), 7.91 (2H, d, J = 9.0 Hz), 8.50 (1H, dd, J = 6.7,
2.4 Hz), 12.48 (1H, s), 12.82 (1H, s); FAB-MS m/z 417 [(M+H)+].
Anal. calcd for C17H10F6N4O2: C, 49.05; H, 2.42; N, 13.46; F,
27.38. Found: C, 49.07; H, 2.38; N, 13.51; F, 27.45.
5.1.40. Antigen-induced airway eosinophilia in rats
Four-week-old female BN rats were sensitized via intraperito-
neal injections of OA (1 mg) and Al(OH)3 (20 mg) once daily for
three consecutive days. Three weeks after sensitization, the rats
were exposed to an aerosol of 1% (w/v) OA solution for 15 min. Test
compounds were administered orally 1 h before the start of anti-
gen exposure. Bronchoalveolar lavage (BAL) was performed 24 h
after antigen exposure. The number of white blood cells in the
BAL fluid from each animal was determined using a blood cell
counter. A cell smear was prepared, and then observed microscop-
ically to identify and classify the cells as eosinophils, neutrophils,
or monocytes according to their morphological characteristics.
The number of eosinophils in each animal’s BAL fluid was then
calculated.
5.1.35. Fura-2 loading and population intracellular calcium
measurements
Cells were suspended in HEPES buffered solution (pH 7.4) of the
following composition (mM). NaCl: 137, KCl: 5.8, MgCl2: 1, CaCl2:
2.5, glucose: 5, and HEPES: 10. The cells were loaded with 1 lM
Fura-2 /AM at room temperature for 45 min followed by successive
washes to remove unincorporated dye, and resuspended in HEPES
buffered solution. Cell suspensions were studied in a 96-well black
plate. Fluorescence measurements for the determination of intra-
cellular calcium concentration were made using a fluorescence
microplate reader (Fluostar, SLT Labinstruments GmbH, Austria)
with excitation wavelengths of 340 nm and 380 nm and an emis-
sion fluorescence detection wavelength of 500 nm. The cell-free
fluorescence of each compound was then measured, after which
it was deducted from the cell fluorescence value. The final intracel-
lular calcium concentration in each well was calculated from the
fluorescence ratio using the standard equation. Wells treated with
Acknowledgments
The authors are grateful to the staff of the Division of Analytical
Science Laboratories for the elemental analysis and spectral
measurements.
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T
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some inhibitors including 6 exerted no effects on blood pressure and
heart rate at doses of which exhibit anti-inflammatory activities (data
not shown).