3,5-Bis(trifluoromethyl)pyrazoles: A novel class of NFAT transcription factor regulator

Article abstract of DOI:10.1021/jm990615a

A series of bis(trifiuoromethyl)pyrazoles (BTPs) has been found to be a novel inhibitor of cytokine production. Identified initially as inhibitors of IL-2 synthesis, the BTPs have been optimized in this regard and even inhibit IL-2 production with a 10-fold enhancement over cyclosporine in an ex vivo assay. Additionally, the BTPs show inhibition of IL-4, IL-5, IL-8, and eotaxin production. Unlike the IL-2 inhibitors, cyclosporine and FK506, the BTPs do not directly inhibit the dephosphorylation of NFAT by calcineurin.

Full text of DOI:10.1021/jm990615a

J . Med. Chem. 2000, 43, 2975-2981
2975
Expedited Articles
3,5-Bis(tr iflu or om eth yl)p yr a zoles: A Novel Cla ss of NF AT Tr a n scr ip tion F a ctor
Regu la tor
Stevan W. Djuric,* Nwe Y. BaMaung, Anwer Basha, Huaqing Liu, J ay R. Luly, David J . Madar,
Richard J . Sciotti, Noah P. Tu, Frank L. Wagenaar, Paul E. Wiedeman, Xun Zhou, Stephen Ballaron, J oy Bauch,
Yung-Wu Chen, X. Grace Chiou, Thomas Fey, Donna Gauvin, Earl Gubbins, Gin C. Hsieh, Kennan C. Marsh,
Karl W. Mollison, Melissa Pong, Thomas K. Shaughnessy, Michael P. Sheets, Morey Smith,
J ames M. Trevillyan, Usha Warrior, Craig D. Wegner, and George W. Carter
Immunological Diseases Research, Abbott Laboratories, 200 Abbott Park Road, Abbott Park, Illinois 60064-6217
Received December 16, 1999
A series of bis(trifluoromethyl)pyrazoles (BTPs) has been found to be a novel inhibitor of cytokine
production. Identified initially as inhibitors of IL-2 synthesis, the BTPs have been optimized
in this regard and even inhibit IL-2 production with a 10-fold enhancement over cyclosporine
in an ex vivo assay. Additionally, the BTPs show inhibition of IL-4, IL-5, IL-8, and eotaxin
production. Unlike the IL-2 inhibitors, cyclosporine and FK506, the BTPs do not directly inhibit
the dephosphorylation of NFAT by calcineurin.
Ch a r t 1. High-Throughput Screening Leads
In tr od u ction
The interaction of T-lymphocytes with antigens trig-
gers a complex signaling cascade that switches on the
gene program leading to T-cell activation. During this
process, T-cells express the autocrine growth factor
interleukin-2 (IL-2), which promotes cell proliferation
by interacting with its receptor, also expressed by
activated T-cells. The transcriptional regulation of the
IL-2 gene has been extensively analyzed with the IL-2
promotor, a 275-bp region located upstream of the
transcriptional start site of the gene. Cis-acting ele-
ments for several transcription factors have been identi-
fied within this regulatory region. The factors which
bind to these motifs include AP-1, NF-κB, and nuclear
factor of activated T-cells (NFAT).1 The transcription
factor NFAT plays an essential role in IL-2 expression.
Binding sites for NFAT have also been found within the
promotor regions of several cytokines, including IL-3,
IL-4, and IL-5. NFAT is composed of a complex whose
binding specificity is mediated by a cytosolic subunit and
an inducible nuclear component comprised of AP-1
family members. The cytoplasmic subunit is encoded by
a family of genes constituted by at least four structurally
related NFAT members: NFATp/NFAT1,NFATc,NFAT3,
and NFATX/NFAT4/NFATc3. These members translo-
cate to the nucleus upon calcium mobilization during
T-cell activation by a mechanism involving the calcium
dependent dephosphorylation of the transcription factor
by calcineurin. The immunosuppressive drugs cyclospor-
in A (CsA) and FK506 block the phosphatase activity
of calcineurin, thus preventing the subsequent dephos-
phorylation and translocation of NFAT to the nucleus.
Hence, NFAT can be considered a secondary target of
the action of the immunosuppressive drugs, whose
Sch em e 1a
a
(a) CF3C(O)CH2C(O)CF3, HCl, EtOH, heat; (b) H2, Pd-C,
EtOAc; (c) RC(O)Cl, base, CH2Cl2 or RCO2H, EDC, DMAP.
inhibition accounts, at least in part, for the transcrip-
tional inhibitory activity of the immunosuppressants.2
Both CsA and FK506 have been shown to be effective
in preventing organ graft rejection in the clinic.3 How-
ever, side effects observed with the clinical use of both
of these compounds, notably nephrotoxicity, neurotox-
icity, diabetogenicity, and gastrointestinal toxicity, have
markedly reduced their impact.4 These side effects are
likely to be caused by the pleotropic metabolic effects
these agents exert through binding to immunophilins.5
* Address correspondence to: Dr. Stevan W. Djuric, Dept. 47N, Bldg.
AP52N-1, Abbott Laboratories, 200 Abbott Park Rd., Abbott Park, IL
60064-6217. Tel: (847)-935-4170. Fax: (847)-938-6603. E-mail: stevan.w.
djuric@abbott.com.
10.1021/jm990615a CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/21/2000

2976 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Djuric et al.
Sch em e 2a
a
(a) 4-Nitrophenylhydrazine, HCl, EtOH, heat; (b) KMnO4, t-BuOH, 0.5 N NaOH; (c) CH3NH(OCH3)‚HCl, HOBt, EDC, CH2Cl2; (d) Fe
powder, NH4Cl, EtOH, H2O, heat; (e) CH3Li, THF; (f) DPPA, Et3N, t-BuOH, toluene, heat; (g) 3-fluoroisonicotinic acid, EDC, DMAP,
CH2Cl2; (h) trifluoroacetic acid, CH2Cl2; (i) NaNO2, CuSO4, CH3CN, H2O.
Sch em e 3a
Sch em e 4a
a
(a) LiAlH4, Et2O; (b) HCl, EtOH, heat; (c) NCS, DMF; (d)
2-chloroacrylonitrile, Et3N, toluene, heat; (e) Fe powder, NH4Cl,
EtOH, H2O, heat.
a
(a) 4-Nitrophenylhydrazine, HCl, EtOH, reflux; (b) for Y )
OCH3: (CH3O)2SO2, K2CO3, CH3CN, for Y ) OCHF2: CHF2Cl,
K2CO3, DMF, heat, for Y ) Cl: PhP(O)Cl2, heat, for Y ) Br:
POBr3, heat; (c) Fe powder, NH4Cl, EtOH, H2O.
testing was done in a concanavalin A (Con A)-induced
proliferation assay using human and rat peripheral
blood mononuclear cells (PBMC proliferation, rat or
human) followed by an assay measuring IL-2 synthesis
inhibition in human whole blood stimulated by PMA/
ionomycin (IL-2 synthesis in human blood). These data
are shown in Tables 1 and 2.
The identification of NFAT as a molecular target in
T-cell activation suggests a more direct, molecular-based
approach to the development of immunosuppressive
agents with the potential for improved efficacy and
reduced side effects. In this article, we describe the
identification and characterization of a novel series of
NFAT regulators that exert their biological effects via
a mechanism that does not involve inhibition of the
Ca2+-dependent phosphatase calcineurin.6
We initially identified the bis(trifluoromethyl)pyrazole
(BTP) derivatives 1 and 2 (Chart 1) as leads from a high-
throughput reporter gene-based screen for IL-2 synthe-
sis inhibitors which featured PMA plus ionomycin
stimulation of the J urkat T-cell line transfected with
the luciferase gene under the transcriptional control of
a full-length IL-2 promotor.7,8 Subsequent in vitro
Ch em istr y
A 350-member focused amide library was prepared
by solution-phase parallel synthesis (Scheme 1) rapidly
providing analogues with optimized potency in the Con
A-stimulated human PBMC proliferation assay (Table
1). Subsequent SAR studies allowed for optimization of
the physicochemical and pharmacokinetic properties of
further analogues. The profiles of these compounds are
summarized in the biology section.
The general synthetic plan for this class of compounds
centered upon preparation of pyrazolyl anilines which

3,5-Bis(trifluoromethyl)pyrazoles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 2977
Ta ble 1. SAR for Terminal Amide Modifications
a
b
IC50 values are expressed as the mean ( SEM of n assays. Peripheral blood mononuclear cell (PBMC) proliferation stimulated by
Con A. c PMA/ionomycin-induced IL-2 synthesis in human whole blood. See ref 19 for a description of this assay.
d
were subsequently coupled with appropriate carboxylic
acids or acid chlorides. Two synthetic routes were used
to access the pyrazolyl anilines. The first consisted of
condensation between 4-nitrophenylhydrazine and ei-
ther a 1,3-diketone or a â-ketoester. These condensation
products were often further modified to provide the
requisitely functionalized pyrazole. The second method
invoked a 1,3-dipolar cycloaddition to construct the
pyrazole nucleus.
1,3-diketone was used to construct the majority of the
pyrazoles. Illustrative of this approach was the synthe-
sis of the BTP analogues as shown in Scheme 1.
4-Nitrophenylhydrazine was condensed with 1,1,1,5,5,5-
hexafluoro-2,4-pentanedione under acidic conditions to
provide the pyrazole.9 Reduction of the aromatic nitro
group, most frequently with either hydrogenation or
iron powder, to the corresponding aniline provided the
amine partner required for amide bond formation.
Coupling with acid chlorides in the presence of a base
Condensation between 4-nitrophenylhydrazine and a

2978 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Ta ble 2. Evaluation of Pyrazole Substituents
Djuric et al.
IC50 (nM)a
rat pharmacokinetics (4 mpk, po)b
PBMC proliferationc PBMC proliferationc IL-2 synthesis in
T1/2
(h)
Cmax
(µg/mL)
AUC
(µg‚h/mL)
compound
X
Y
human
rat
human bloodd
F%
11
12
13
14
15
16
17
18
19
20
21
C
N
C
N
N
C
N
N
N
N
N
CN
CN
C(O)CH3
NO2
79 ( 10 (4)
266 ( 14 (16)
170 ( 14 (4)
410 ( 86 (4)
-23%c ( 6 (2)
66 ( 16 (10)
185 ( 77 (4)
43 ( 8 (4)
613 (1)
1524 ( 418 (3)
869 ( 76 (2)
304 ( 68 (4)
240 ( 31 (20)
1575 ( 241 (8)
440 ( 112 (4)
16%e ( 6 (3)
570 ( 146 (6)
261 ( 59 (4)
944 ( 263 (10)
182 ( 19 (40)
158 ( 20 (23)
288 ( 92 (10)
6.2
4.3
31
78
5
0.28
1.09
0.12
0.22
3.24
10.90
1.11
1.7
18
0.31
OH
OCH3
OCH3
SCH3
OCHF2
Cl
284 ( 11 (2)
501 (1)
158 ( 35 (3)
146 (1)
149 ( 38 (2)
224 (1)
26
68
63
74
79
94
0.07
1.48
0.87
0.61
0.78
1.04
0.97
3.05
7.79
7.75
11.05
16.42
1.0
5.2
6.8
4.8
7.2
82 ( 18 (6)
75 ( 8 (12)
44 ( 2 (4)
Br
a
b
IC50 values are expressed as the mean ( SEM of n assays. Pharmacokinetic data were normalized to 4 mpk, po unless otherwise
specified. c Peripheral blood mononuclear cell (PBMC) proliferation stimulated by Con A. PMA/ionomycin-induced IL-2 synthesis in
d
human whole blood. e Response at maximum concentration tested (10 µM).
scavenger or with carboxylic acids activated with car-
bodiimides completed the synthesis. On some occasions,
solid-phase reagents or scavengers were used to simplify
reaction workup.10 For example, in the coupling with
acid chlorides, poly(4-vinylpyridine) was used as the acid
scavenger and a polymer-bound benzylamine was used
to remove excess acid chloride.
Unsymmetric diketones provided access to function-
alization at either C3 and/or C5 of the pyrazole depend-
ent on the nature of each diketone as depicted in
Scheme 2. For example, when 4,4,4-trifluoro-1-(2-furyl)-
1,3-butanedione was reacted with 4-nitrophenylhydra-
zine, the furan regiochemistry was nearly exclusively
at C5.11 This compound proved to be a useful intermedi-
ate to supply the C5 carboxylic acid which was obtained
by oxidation with potassium permanganate.12 Conver-
sion to the acetyl derivative was achieved by addition
of methyllithium to the corresponding Weinreb’s amide
after the nitro group had been reduced. The same
carboxylic acid underwent a Curtius rearrangement to
introduce a nitrogen functionality at C5. The nitro group
was obtained on the pyrazole by treatment with sodium
nitrite and copper sulfate following aniline production,
amide bond formation, and deprotection.13
One of the more versatile intermediates for introduc-
ing functionality at C5 on the pyrazole was the conden-
sation product of 4-nitrophenylhydrazine and the â-ke-
toester,ethyl4,4,4-trifluoroacetoacetate,which isillustrated
in Scheme 3. Although the C5 hydroxy analogues lacked
activity, alkylation14 or transformation to a halogen15
produced some of the more biologically interesting
analogues. The key intermediate was also accessed with
S-methyl 4,4,4-trifluoro-3-oxothiobutyrate which ad-
ditionally gave a limited amount of the C5 S-methyl
ether.
Dipolar cycloaddition provided a second alternative
path to the pyrazolyl anilines.16 This route gave com-
plete regiochemical control over the position of substit-
uents on the pyrazole ring and is shown in Scheme 4.
The hydrazone formed from reaction of trifluoroacetal-
dehyde and 4-nitrophenylhydrazine was chlorinated to
provide the dipolar precursor.17 Upon exposure to base
and a dipolarophile (2-chloroacrylonitrile is illustrated
here) the C5 nitrile was obtained in good yield.18 Once
again, the aniline and subsequent amide were formed
in the usual manner.
Biologica l Da ta a n d Con clu sion s
The profiling of the compounds began with the Con
A-stimulated human PBMC proliferation assay. The
leads 1 and 2 indicated that the terminal amide was
amenable to modification. Certainly the majority of
compounds made were synthesized in parallel by forma-
tion of the amide bond depicted in Scheme 1. Although
this terminal position proved accepting of a great deal
of modification, the most potent compounds were found
in the halogenated aromatic series. In particular, fluo-
rine in the ortho position boosted potency as is noted in
a comparison of 1, 4, and 6 (Table 1). Multiple fluorines
on the phenyl ring maximized potency in this assay as
shown with 7-9. Fluorinated aromatic heterocyles such
as 10 maintained this potency. Potency of compounds
observed in the Con A-stimulated human PBMC pro-
liferation assay was, in general, not maintained in
moving to the human whole blood paradigm using IL-2
synthesis as the readout, presumably due to extensive
plasma protein binding.19 The compounds shown in
Table 1 all exhibited a great deal of protein binding
(>99%) despite a clog P range of 3.2-5.6. As expected,
the heterocycles were at the lower end of the range.
Apparently, the BTP portion of these compounds was
overriding any benefit gained by heterocyclic substitu-
ents, and at least one of the lipophilic trifluoromethyl
groups on the pyrazole ring had to be replaced to reduce
protein binding. Further SAR studies focused on sub-
stituent modification of the pyrazole ring did produce
analogues with reduced protein binding affinity and
substantially greater potency in the assay measuring
inhibition of IL-2 in human whole blood (Table 2). As a
case in point, compounds 10 and 12 were compared. As
with all the BTPs, 10 was highly protein bound (99.7%)
despite a modest clog P (3.47). Substitution of a trifluo-
romethyl group with a nitrile supplied 12 with reduced
protein binding (96.2%) and clog P (2.05). Modifications

3,5-Bis(trifluoromethyl)pyrazoles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 2979
Ta ble 3. Efficacy in Primate Model of Acute Asthmaa
respiratory system resistancec
bronchoalveolar lavagesd
compound
dose (mg/kg)b
n
10 min
6 h
48 h eos
6 h IL-5
6 h IL-8
6 h eotaxin
prednisolone
cyclosporin A
19
1.0
25
30
8
6
6
24*
13
61**
57*
90**
69**
106
137
185*
94
92
73*
9
68
69*
67**
62*
56***
a
b
Data represent mean percentage inhibition versus crossover vehicle control. Compounds were administered by oral gavage at -26,
-2, and +22 h relative to antigen inhalation (QD). c Bronchoconstriction was assessed by measuring respiratory system resistance at
d
baseline, +10 min (immediate response), and +6 h (late-phase response). Bronchoalveolar lavages were collected at -5 day (baseline),
+6 h (for IL-5, IL-8, and eotaxin), and +48 h (for eosinophil influx, eos). Paired t-test vs vehicle controls: *p < 0.05, **p < 0.01, ***p )
0.054.
are required on both the pyrazole and terminal amide
portions of these molecules to reduce protein binding.
Although the protein binding remains high, there was
a sufficient free fraction of compound 12 to give com-
parable IC50 values between the proliferation assay (low-
protein environment) and the IL-2 synthesis inhibition
assay (whole blood environment). The other potent
compounds in Table 2 have a similar profile.
Although several of these compounds now displayed
excellent potency for inhibition of IL-2 production in
human PBMCs, rodent cells were much less sensitive
to inhibition by this structural class. In fact, potencies
were generally at least 4 times less in rodent than in
human cells. This confounding property made efficacy
evaluations in rodent models of autoimmune disease
particularly problematic. As a consequence, we elected
to evaluate the most promising compounds in a primate
ex vivo IL-2 synthesis inhibition model followed by
testing in a primate asthma model.20 For these studies
we chose to use the relatively optimized analogue 19
as an example. This compound displayed an excellent
PK profile in rodents (as shown in Table 2) and in
cynomolgus monkeys (t1/2 ∼ 8 h).
F igu r e 1. Inhibition of whole blood IL-2 production ex vivo
following oral administration of 19 and cyclosporin A (CsA)
in cynomolgus monkeys. The blood samples were drawn at 2
h
after drug administration and then analyzed for IL-2
production over a 24-h incubation. This gave adequate time
for drug inhibition to be maximal, regardless of whether the
modes of action have differing kinetics. Preliminary pretreat-
ment studies done in vitro with whole blood exposed to
compound prior to stimulation with PMA/ionomycin showed
a rapid onset of drug inhibition: bars, mean ( SEM; open bars,
IL-2 production of predrug controls; filled bars, IL-2 production
2 h postdrug; n ) 3 for 19 and n ) 6 for CsA; *p < 0.05.
and IL-8 generation than cyclosporine at similar doses
(data shown in Table 3).
To assess in vivo efficacy, the ability of orally admin-
istered 19 to block cytokine production of circulating
T-cells was measured and compared to that of cy-
closporin A (CsA). Blood samples obtained at 2 h after
dosing, the time of peak drug concentration for both
compounds, were stimulated ex vivo to produce IL-2,
and these levels were compared to baseline samples
from the same animals.21 As shown in Figure 1, cy-
closporine inhibited 76% at a dose of 30 mg/kg, po. This
is consistent with its potency when used in cynomolgus
macaques for preventing allograft rejection.22 Compound
19 was found to have an inhibitory potency approxi-
mately 10-fold better than that of cyclosporine. Com-
parable inhibitory effects on T-cell IL-2 production were
obtained with 19 and cyclosporine at doses of 3.0 and
30 mg/kg, po, respectively. The efficacies achieved in
monkeys in vivo for blocking T-cell cytokine production
suggest that 19, and the BTP class of cytokine inhibi-
tors, have potential similar to that of cyclosporine for
use in transplantation.
Mechanism of action studies24 on compounds 1 and 2
showed that the BTP compounds regulate NFAT activ-
ity by a mechanism distinct from the current immuno-
suppressive drugs cyclosporine and FK506. The activity
of NFAT proteins is tightly regulated by the calcium/
calmodulin-dependent phosphatase 2B (calcineurin).
Dephosphorylation of NFAT by calcineurin is required
for NFAT activation and transport from the cytosol to
the nucleus. The current immunosuppressive drugs
cyclosporin A and FK506 block calcineurin activity
leading to inhibition of NFAT nuclear translocation and
consequent cytokine gene transcription.6 Treatment of
intact T-cells with the BTP compounds, prior to calcium
ionophore-induced activation of calcineurin, caused
NFAT to remain in a phosphorylated state and blocked
nuclear translocation. However, the BTP compounds did
not directly inhibit the dephosphorylation of NFAT by
calcineurin in vitro, nor did the drugs block the in vitro
dephosphorylation of other calcineurin substrates in-
cluding the type II regulatory subunit of protein kinase
A and the transcription factor, Elk-1. The data suggest
that the BTP compounds cause NFAT to be maintained
in the cytosol in a phosphorylated state and block the
nuclear import of NFAT and hence NFAT-dependent
cytokine gene transcription by a mechanism other than
direct inhibition of calcineurin phosphatase activity.
Studies to identify a molecular target are continuing.
Ongoing studies in nonhuman primate models of
As 19 was able to inhibit IL-4 and IL-5 production in
human T-cell lines with similar potency to its effects
on IL-2 release (data not shown), it was evaluated in
an Ascaris-induced nonhuman primate model of asthma
where, like human asthma, IL-5 is proposed to be a
pathogenic mediator.23 Difluoromethoxy ether 19 showed
remarkable efficacy in this model at a dose of 30 mg/kg
(oral gavage), producing a more pronounced and sig-
nificant inhibition of the immediate bronchoconstriction

2980 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16
Djuric et al.
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I.; Fiorentino, D.; Nourse, J .; Crabtree, G. R. The mechanism of
action of Cyclosporin A and FK-506. Clin. Immunol. Immuno-
pathol. 1996, 80 (3), S40-S45.
(7) Deter m in a tion of IL-2 r ep or ter gen e exp r ession in J u r k a t
E2 cells: J urkat E2 cells were routinely cultured in RPMI 1640
plus 10% fetal bovine serum (FBS) containing 400 µg/mL
hygromycin and 500 µg/mL geneticin. Prior to assay, J urkat E2
cells were resuspended at 4 × 105 cells/mL in RPMI 1640 plus
5% FBS (lacking antibiotics), stimulated with 10 ng/mL 12-O-
tetradecanoylphorbol-13-acetate (TPA) and 2 µM ionomycin
(Northrop, J . P.; Crabtree, G. R.; Mattila, P. S. J . Exp. Med.
1992, 175, 1235-1245), and placed in 96-well microtiter plates
in a final volume of 200 µL/well. Cells were incubated for 18 h
at 37 °C, 10% CO2 in a humidified incubator. J urkat E2 cells
were pelleted by centrifugation and the media aspirated. Cells
were lysed by the addition of 20 µL of cell-lysing buffer
containing 25 mM Tris-phosphate, pH 7.8/2 mM dithiothreitol/2
mM 1,2-diaminocyclohexane N,N,N′,N′-tetraacetic acid/10% gly-
cerol/1% Triton X-100 and incubated at room temperature for
20 min. 100 µL of luciferase reaction buffer containing 20 mM
Tricine, pH 7.8/1.07 mM (MgCO3)4‚Mg(OH)2‚5H2O/2.67 mM
MgSO4/0.1 mM EDTA/33.3 mM dithiothreitol/270 µM coenzyme
A/470 µM luciferin/530 µM ATP was added to the cell lysate,
and chemiluminescence was measured with a luminometer. The
efficacy of compounds to inhibit IL-2 gene promoter activity was
determined by measuring the inhibition of TPA plus ionomycin-
induced luciferase expression in J urkat E2 cells. Con A a ssa y:
Serially diluted compounds were distributed to 96-well plates.
Human PBMCs, isolated by ficoll/hypaque gradient centrifuga-
tion, were added to effect 2.5 × 105 cells/mL in complete RPMI
1640 medium. Con A was added to effect 2.5 µg/mL final
concentration. Cells were incubated in a CO2 incubator for 3 days
and were labeled with [3H]thymidine for the last 6 h of culture.
[3H]Thymidine incorporation, after harvesting onto glass fiber
filters, was assessed by direct â-counting. Hu m a n w h ole blood
IL-2 in h ibition a ssa y: Serially diluted compounds were dis-
tributed to 96-well plates. Human heparinized blood was then
added to each well. PMA and ionomycin mixture was added to
effect 50 ng/mL for PMA and 3 mg/mL for ionomycin. Plasma
samples were collected 24 h later by centrifugation and were
assayed for IL-2 by an in-house ELISA.
(17) Tanaka, K.; Maeno, S.; Mitsuhashi, K. Preparation of trifluoro-
acetonitrile phenylimine and its reactions with some dipolaro-
philes. Chem. Lett. 1982, 543-546.
(18) (a) Benages, I. A.; Albonico, S. M. 2-Chloroacrylonitrile as a
cyclodipolarophile in 1,3-cycloadditions. 3-Cyanopyrroles. J . Org.
Chem. 1978, 43, 4273-4276. (b) J ones, R. C. F.; Nichols, J . R.;
Cox, M. T. Annulation of imidazolines: A 1,3-dipolar cycload-
dition route to pyrroloimidazoles, pyrrolidines and pyrroles.
Tetrahedron Lett. 1990, 31, 2333-2336. (c) Hassaneen, H. M.;
Mousa, H. A. H.; Abed, N. M.; Shawali, A. S. Chemistry of
C-heteroarylhydrazidoyl halides. Synthesis and reactions of
N-(p-nitrophenyl)-C-(2-thienyl)-formohydrazidoyl halides. Het-
erocycles 1988, 27, 695-706.
(19) In vitro protein binding of BTP analogues was assayed in human
serum at 5 µg/mL. After incubation at room temperature for 30
min, samples were ultrafiltered through a centrifree micropar-
tition device with a 30 000 molecular weight cutoff (Amicron
Corp., Danvers, MA) by centrifugation at 2000g for 25 min at
room temperature. The drug in the resultant filtrate was
analyzed by HPLC.
(20) Wegner, C. D.; Gundel, R. H.; Abraham, W. M.; Schulman, E.
S.; Kontny, M. J .; Lazer, E. S., et al. The role of 5-lipoxygenase
products in preclinical models of asthma. J . Allergy Clin.
Immunol. 1993, 91, 917-929.
(21) Cynomolgus monkeys were bled to obtain heparinized baseline
samples for measuring predrug cytokine production and then
briefly intubated for intragastric dosing. Cyclosporine was
administered in the Neoral formulation. Compound 19 was given
in a vehicle consisting of 20% ethanol, 30% propylene glycol, 2%
cremophor EL, and 48 vol % of 5% dextrose in water. Postdrug
blood samples were similarly obtained 2 h later, which was the
approximate Tmax for peak blood level with both compounds. The
samples were stimulated by spiking undiluted blood with PMA
(50 ng/mL) and ionomycin (1µg/mL) and incubating for 24 h.
Plasma samples were collected by centrifugation, and IL-2
concentrations were determined by ELISA using recombinant
human IL-2 as standard. Percent of postdrug IL-2 inhibition was
calculated in reference to the respective baseline sample for each
monkey and tested for statistical significance by t-test.
(8) During the preparation of this manuscript, the authors became
aware of related work: (a) Kobuta, H.; Yonetoku, Y.; Sugasawa,
K.; Funatsu, M.; Kawazoe, S.; Toyoshima, A.; Okamoto, Y.;
Ishikawa, J .; Takeuchi, M. WO 9919303 A1, 1999. (b) Betageri,
R.; Cywin, C. L.; Hargrave, K.; Hoermann, M. A.; Kirrane, T.
M.; Parks, T. M.; Patel, U. R.; Proudfoot, J . R.; Sharma, R.; Sun,
S.; Wang, X.-J . WO 9962885.

3,5-Bis(trifluoromethyl)pyrazoles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 16 2981
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Schuler, W.; J onker, M. Neoral pharmacokinetics in cynomolgus
monkeys: Relation to efficacy in renal allografting. Transplant.
Proc. 1996, 28, 3142-3144.
(23) (a) Wegner, C. D.; Gundel, R. H.; Rothlein, R.; Letts, L. G.
Expression and probable roles of cell adhesion molecules in lung
inflammation. Chest 1992, 101, 34S-39S. (b) Wegner, C. D.;
Gundel, R. H.; Letts, L. G. Efficacy of monoclonal antibodies
against adhesion molecules in animal models of asthma. Agents
Actions Suppl. 1993, 43, 151-162. (c) Mauser, P. J .; Pitman, A.
M.; Fernandez, X.; Foran, S. K.; Adams, G. K., 3rd; Kreutner,
W.; Egan, R. W.; Chapman, R. W. Effects of an antibody to
interleukin-5 in a monkey model of asthma. Am. J . Respir. Crit.
Care Med. 1995, 152, 467-472.
(24) Trevillyan, J . M.; Djuric, S. W.; et al. J . Biol. Chem., manuscript
submitted for publication. NFAT proteins are expressed in most
immune system cells and play a pivotal role in the transcription
of cytokine genes critical for the immune response. The activity
of NFAT proteins is tightly regulated by the calcium/calmodulin-
dependent phosphatase 2B (calcineurin). Dephosphorylation of
NFAT by calcineurin is required for NFAT activation and
nuclear localization. The BTP compounds block the activation-
dependent nuclear localization of NFAT as determined by
electrophoretic mobility shift assays. Confocal microscopy of cells,
expressing green fluorescent protein-tagged NFAT, confirms that
the BTP compounds block calcium-induced movement of NFAT
from the cytosol to the nucleus. Inhibition of NFAT is apparently
selective since the BTP compounds do not effect the activation
of other transcription factors including NF-kB and AP-1. Treat-
ment of intact T-cells with the BTP compounds, prior to calcium
ionophore-induced activation of calcineurin, causes NFAT to
remain in a phosphorylated state. However, the BTP compounds
do not directly inhibit the dephosphorylation of NFAT by
calcineurin in vitro, nor do the drugs block the dephosphorylation
of other calcineurin substrates including the type II regulatory
subunit of protein kinase A and the transcription factor, Elk-1.
The data suggest that the BTP compounds cause NFAT to be
maintained in the cytosol in a phosphorylated state and block
the nuclear import of NFAT and hence NFAT-dependent cyto-
kine gene transcription by a mechanism other than direct
inhibition of calcineurin phosphatase activity.
J M990615A