Pyrazole bioisosteres of leflunomide as B-cell immunosuppressants for xenotransplantation and chronic rejection: Scope and limitations

Article abstract of DOI:10.1021/jm981028c

T-cell immunosuppressant-based therapies efficiently control early graft rejection in allotransplantations settings. They fail, however, to prevent those rejection events which are mediated by transplant-induced antibody (Ab) responses such as those invol

Full text of DOI:10.1021/jm981028c

3530
J . Med. Chem. 1998, 41, 3530-3538
P yr a zole Bioisoster es of Leflu n om id e a s B-Cell Im m u n osu p p r essa n ts for
Xen otr a n sp la n ta tion a n d Ch r on ic Rejection : Scop e a n d Lim ita tion s
Christos Papageorgiou,*,† Rainer Albert,† Philipp Floersheim,‡ Michel Lemaire,§ Francis Bitch,|
Hans-Peter Weber, Elsebeth Andersen,† Valerie Hungerford,† and Max H. Schreier†
NOVARTIS Pharma AG, BAS-350.314, CH-4002 Basel, Switzerland, and Nova Research Services,
CH-4143 Dornach, Switzerland
Received April 21, 1998
T-cell immunosuppressant-based therapies efficiently control early graft rejection in allotrans-
plantation settings. They fail, however, to prevent those rejection events which are mediated
by transplant-induced antibody (Ab) responses such as those involved in xenograft and chronic
allograft rejection. This is mainly due to their inability to block T-cell-independent Ab
production against the transplanted organs. The bioactive metabolite 2(Z) of leflunomide (1)
inhibits the formation of such Ab, but the drug has pharmacokinetic properties and a therapeutic
window incompatible with transplantation indications. Pyrazole 3, a constrained analogue of
2(Z), was designed and shown to be conformationally and biologically similar to 2(Z). Further
investigations with derivatives of 3 demonstrated that the pyrazoles had very tight structure-
activity relationships, the only equipotent compound being 3o. However, in contrast to 2(Z),
both 3 and 3o were inactive in vivo due to short half-life and drug concentrations lower than
the in vitro obtained IC50 values. Compound 3o inhibits T-cell-independent Ab production by
a different biochemical mechanism from that of 2(Z) and 3 and may therefore represent a
valuable tool for the identification of new targets for B-cell inhibition.
In tr od u ction
transplanted xenograft.6 Therefore, Ab production by
B cells is a key event both during the hyperacute
rejection phase and during the acute rejection phase of
xenotransplantation. No participation of T cells is
required for these phenomena to occur, as demonstrated
by the failure of T-cell immunosuppressants such as
cyclosporin in xenotransplantation and the rejection of
hamster heart xenografts by T-cell-deficient nu/nu rats.7
A second syndrome associated with the presence of
Ab is chronic rejection (graft vessel disease, late graft
rejection), a disease leading to organ loss due to the
obstruction of arterial vessels of the transplanted al-
lograft. Persistent endothelial injury leads to intima
proliferation, smooth muscle cell migration, and matrix
deposition as key features of this complex process which
proceeds over many years after transplantation.8 Among
the large number of contributing factors leading to
endothelial cell damage, Ab and complement are the
most critical components. Evidence that humoral activ-
ity is responsible for graft vessel disease includes,
among others, the presence of anti-HLA Ab in rejecting
patients’ sera and the vascular deposition of comple-
ment-fixing immunoglobulin in areas of intimal thick-
ening.9 The importance of B cells in graft vessel disease
has also been demonstrated in in vivo models; only
minimal neointima formation is seen in carotid arteries
transplanted into B-cell-deficient mice.10
In contrast to allotransplantation where current
immunosuppressive therapies overcome the effects of
T-cell-mediated graft rejection1 and antibody (Ab) for-
mation against the graft,2 antibody-mediated rejection
is the first major obstacle toward clinical solid organ
xenotransplantation,3 the transplantation of organs
between individuals of different species. Indeed, xeno-
geneic immune responses mediated by preexisting,
naturally occurring antibodies and complement lead to
hyperacute and acute rejection of vascularized organ
grafts. These issues have been investigated in the pig
to non-human primate model of heart xenotransplan-
tation, where the presence of monkey xenoreactive
natural antibodies to pig antigens and their attachment
to the vascular endothelium of pig organs stimulated
the classical pathway of complement activation and led
to the irreversible loss of the heart because of hyper-
acute rejection.4 On the basis of genetic engineering,
significant progress has been made to avert hyperacute
rejection as demonstrated in monkey transplantation
studies with pig transgenic hearts expressing human
decay accelerating factor (DAF), a species-specific in-
hibitor of complement activation.5 However, if comple-
ment activation is blocked in the absence of immuno-
suppression and the xenografts survive hyperacute
rejection, the graft is still lost due to acute vascular
rejection since excessive Ab production is induced by the
Drugs which inhibit Ab formation and are effective
in xenotransplantation models can also be expected to
alter the progression of chronic rejection. Confirmation
has been obtained with leflunomide (1), a compound
which interferes with T-cell and B-cell function through
the selective inhibition of dihydroorotate dehydrogenase
(DHODH), a rate-limiting step of the de novo pyrimidine
biosynthetic pathway of cells.11 Indeed, data obtained
* Corresponding author. Tel: 0041-61-3246188. Fax: 0041-61-
3243036. E-mail: christos.papageorgiou@pharma.novartis.com.
† Transplantation Research, NOVARTIS.
‡ Nervous System, NOVARTIS.
§ Drug Safety, NOVARTIS.
| Central Functions, NOVARTIS.
Nova Research Services.
S0022-2623(98)01028-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/12/1998

Pyrazole Leflunomides as B-Cell Immunosuppressants
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3531
Sch em e 1a
F igu r e 1. Structural relationships between leflunomide (1),
its bioactive metabolite 2(Z) or 2(E), and the corresponding
pyrazoles 3 and 4, respectively.
a
(a) RbNCS (1.0 equiv), dioxane, reflux, 3-6 h; (b) H2NNH2
(1.2 equiv), EtOH, reflux, 2-3 h.
Sch em e 2a
with leflunomide in animal models demonstrate that the
drug can prevent Ab-mediated rejection in heart xe-
notransplantation12 and that it can control the progres-
sion of chronic rejection.13
Leflunomide’s potential for transplantation indica-
tions is clearly limited by its undesirable pharmacoki-
netics (mean t1/2 in humans: 300 h)14 that do not allow
easy adjustment of the immunosuppressive regime.
Moreover, when 1 is given to rats in combination with
the standard antirejection drug cyclosporin A (CsA), it
has a very narrow therapeutic window. This is also the
case for the published leflunomide analogues which
have a shorter half-life in humans than leflunomide.15,16
a
(a) (i) NaH (1.2 equiv), (ii) (p-CF3)PhNCS (1.2 equiv) then MeI
(1.2 equiv); (b) H2NNH2 (1.2 equiv), EtOH, reflux, 3 h.
Design Con cep t
Searching for drugs which prevent Ab responses, we
desired a lead compound with a pharmacological profile
similar to that of leflunomide and shorter half-life. To
this end, the bioactive metabolite of leflunomide was
considered (Figure 1). Indeed, 1 is a prodrug whose
isoxazole ring is quickly and quantitatively opened in
vivo to give the bioactive hydroxycyanopropenamide 2.
Among the two possible geometric isomers, 2(Z) and 2-
(E), the former having an enol structure stabilized by
a strong hydrogen bond between the OH and the amide
carbonyl was observed in the solid state by X-ray
crystallographic analysis.17 Moreover, 2(Z) is believed
to be the bioactive isomer on the basis of physicochem-
ical investigations of this compound class in solution18
and molecular modeling calculations.19 Consequently,
two substituted pyrazoles (3 and 4) were designed as
conformationally constrained analogues of the corre-
sponding possible structures of leflunomide’s bioactive
metabolite, 2(Z) and 2(E), respectively, and only one
was expected to be biologically active (Figure 1). Care
was taken in the design to not only keep the conjugated
system unchanged but also keep the molecular volumes.
noacrylonitriles 5 were employed as starting materials
which were obtained either via treatment of alkyl-â-
ketoacetonitriles with NH3 in a sealed vessel23 or from
the reaction of the sodium salt of acetonitrile with the
appropriate arylnitriles.24
Additional pyrazole derivatives required for structure-
activity investigations were synthesized according to
Schemes 3-5. The tetrahydro-4H-indazolone 10 was
obtained upon reaction of the commercially available
2-aminocyclohexenone with (p-CF3)PhNCS followed by
cyclocondensation of the resulting thioamide 9 with
hydrazine.25 The corresponding indazole derivative 11
was prepared through reduction of 10 with AlH3, the
intermediate allylic alcohol being too reactive to isolate.
The influence of the 4-cyano substituent was probed via
its replacement with a phenyl and ethoxycarbonyl
electron-withdrawing groups as well as with hydrazino
and alkyl moieties (Scheme 4). The 4-Ph-substituted
pyrazole 14 was prepared via acylation of the sodium
salt of benzyl methyl ketone with (p-CF3)PhNCS and
subsequent reaction with hydrazine.26 The analogous
reaction of the thioamide 13 furnished the 4-hydrazino
compound 15 together with an equal amount of 16, the
des-CN derivative of 3, whose formation is the conse-
quence of the elimination of the acetyl moiety of 13. No
trace of the corresponding methyl ketone could be
detected under a variety of experimental conditions.
The key reaction leading to 17 was the cycloconden-
sation of the commercially available (p-CF3)phenylthio-
semicarbazide with ethyl 2-chloroacetoacetate.27 Sub-
sequent reduction of the ester functionality under mild
conditions led to the allylic alcohol 18, while the use of
LiAlH4 yielded exclusively the corresponding 4-meth-
ylpyrazole 19 (Scheme 5). Both reactions proceeded in
Syn th esis
The synthesis of the required pyrazoles was based on
the reaction of acidic methylene compounds with isothio-
cyanates followed by treatment of the resulting thioa-
mides with hydrazine (Schemes 1 and 2).20,21 Starting
from ethyl cyanoacetate, the pyrazole 4 was obtained
together with the 4-ethoxycarbonyl-5-amino derivative
8 in a 20/80 ratio as a consequence of the two possible
ring closures of the thiomethyl intermediate 7.22 For
compound 3 as well as for the preparation of the closely
related analogues 3a -u , the corresponding 3-ami-

3532 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Papageorgiou et al.
Sch em e 3a
Sch em e 5a
a
(a) Concentrated HCl, 10 min then MeCOCH(Cl)COOEt (1.0
equiv); (b) LiAlH4 (4.0 equiv), H2SO4 (2.0 equiv), THF, -78 °C to
rt; (c) LiAlH4, THF, reflux, 2 h.
a
(a) (p-CF3)PhNCS (1.1 equiv), MeCN, reflux, 18 h; (b) H2NNH2
(1.2 equiv), EtOH, reflux, 18 h; (c) LiAlH4 (3.0 equiv), H2SO4 (1.5
equiv), THF, -78 °C to rt.
characterized by the coplanarity of the pyrazole and
phenyl rings and the cis arrangement of N(2)-C(3)-
N(6)-C(7). For this highly compact structure to be
formed, an unprotonated sp2-N(2) is required in order
to avoid the interaction of the N(2) hydrogen with that
of C(8). The driving force for the crystal packing
observed is not due to the energetic contribution of a
H-bond between the acidic H-C(8) and the imine N(2)28
since the corresponding distance is 2.2 Å. If, alterna-
tively, N(2)-C(3)-N(6)-C(7) were trans, the steric
hindrance between the phenyl and the cyano group
would again require loss of coplanarity. In conclusion,
a planar structure of the pyrazole is energetically the
most favorable one in the solid state, and it is only
compatible with the tautomeric form of 3. Clearly, the
conformation observed for this molecule in the solid
state does not provide any direct information on its
conformational preferences in solution, but the presence
of this folding pattern in the crystals suggests that a
similar folded conformation is energetically accessible
in solution too. Moreover, the recovery of the diffracting
crystals out of a physiologically compatible solvent
suggests that the experimentally determined conforma-
tion of 3 could indeed be identical with its bioactive one.
Sch em e 4a
a
(a) (i) (p-CF3)PhNCS (1.2 equiv), THF, (ii) NaH (1.2 equiv);
(b) H2NNH2 (1.3 equiv), EtOH, reflux, 18 h.
low yields and were accompanied by extensively decom-
posed starting material.
1
Interestingly, H NMR experiments conducted with 3
in DMSO-d6 also indicate the presence of the same
tautomer. Indeed, although a tautomeric exchange of
the proton cannot be excluded, a NOE is observed
between the methyl substituent and the H of N(1).
Str u ctu r a l Resu lts
Among the two designed isomeric pyrazoles 3 and 4,
only 3 was biologically active (see biological results).
Consequently and in order to check the structural
similarity of 2(Z) with 3, the conformation of the latter
in the solid state was investigated and compared with
that of 2(Z).17 To this end, triclinic and monoclinic
crystal forms were obtained from a MeOH/water solu-
tion which were initially thought to correspond to the
two possible tautomeric forms of 3. Subsequent X-ray
analysis of each crystal form demonstrated that both
corresponded to the same tautomer and that they
adopted identical spatial arrangements. Indeed, 3 is
The structural similarity of the bioactive pyrazole 3
and 2(Z) is shown in Figure 2. Clearly the pyrazole ring
is a bioisosteric replacement for the hydroxycyanopro-
penamide moiety of leflunomide’s metabolite, and the
two molecules share similar conformations and molec-
ular volumes (185.1 and 187.9 Å3 for 2(Z) and 3,
respectively).29 Despite the difference in ring size, a five
ring versus a pseudo-six ring, the conjugated systems
O-CdC-CdO in 2(Z) and N-CdC-CdN in 3 super-
impose with a RMS of 0.36 Å.

Pyrazole Leflunomides as B-Cell Immunosuppressants
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3533
ratio than 2(Z) indicating that it may have a lower
general toxicity. These effects of 3 are at most partially
mediated through DHODH inhibition since it has a 13-
fold higher IC50 than 2(Z). The lower affinity of the
pyrazole and the associated ∆∆G ) 1.56 kcal/mol (T )
298 K) indicate the possible loss of a H-bond interaction
with the enzyme.33 The isomeric pyrazole 4 is devoid
both of B-cell activity and of enzymatic activity up to
high micromolar concentrations. Importantly, the en-
semble of the biological data establishes unequivocally
that the bioactive metabolite of leflunomide (1) has a
Z-enol structure.
F igu r e 2. ORTEP plots and interatomic distances (Å) of
hydroxycyanopropenamide 2(Z) and pyrazole 3.
Having demonstrated that 3 behaves as an isostere
of leflunomide’s bioactive metabolite 2(Z), an extensive
investigation of structure-activity relationships was
undertaken with the hope that more active derivatives
of 3 could be identified. Consequently, the influence of
substitution at positions 1, 2, 4, and 5 of the pyrazole
ring as well as that of variations of the aromatic moiety
was studied, and the outcome is summarized in Table
2. Attempts to replace the anilinic nitrogen of 3 by
carbon or oxygen were fruitless due to synthetic issues.
Ta ble 1. In Vitro Profiles of Reference and Designed
Compounds
compd
type Ia,b
MLRa,c
J urkata,d
DHODHa,e
CsA
1
2(Z)
3
4
>100
5.1
4.4
5.3
>50
0.006
7.4
6.3
2.1
ND
>10
41.4
38.5
>50
ND
>50
13
0.2
2.6
>50
a
IC50 values in µM. Mean of at least three independent
b
The data of Table 2a show that a para electron-
withdrawing group on the aniline is necessary for B-cell
inhibition in the low-micromolar range, while for en-
zyme inhibition a para hydrophobic group is required,
the best being compound 3f with an IC50 ) 0.5 µM. An
analysis of the impact of the position of the aromatic
substituent on the DHODH activity in the pyrazole and
leflunomide series indicates that ortho and para sub-
stituents are detrimental for enzyme inhibition in both
substance classes.15,17 Overall, pyrazole 3 has the best
in vitro profile among the derivatives 3a -m since it
combines potent B-cell inhibitory activity and immu-
nosuppression, as indicated by the MLR data, and
shows no cytotoxic effects as assessed in the human
transformed J urkat cell line. The focus of further
structure-activity work was the elucidation of the role
of the 5-Me of 3 via its substitution by aromatic (3u ),
heteroaromatic (3q-u ), amino (3v,w ), and alkyl groups
(3n -p ). With the exception of the latter modifications,
all the others were detrimental for both the enzyme and
B-cell inhibition (Table 2b). Furthermore, among the
alkyl derivatives, only 3p , the 5-cyclopentyl one, is
inactive. This result together with the results obtained
with the aromatic derivatives 3q-u indicates that steric
bulk is not tolerated at position 5. However, the 5-iPr
and 5-tBu derivatives 3n ,o, respectively, are equipotent
to 3 concerning the B-cell inhibitory activity, have
comparable immunosuppressive potencies, and are not
cytotoxic. Interestingly, in contrast to 3, which medi-
ates its effects through blockade of the DHODH activity,
3o is not an enzyme inhibitor. In the hydroxypropena-
mide series, the analogous tBu derivative is a 70-fold
weaker DHODH inhibitor (IC50 ) 16.2 µM) than the
parent compound 2(Z) and is devoid of type I activity
up to 50 µM.17 This unexpected data indicates that
inhibition of B-cell responses to the TNP-LPS antigen
and overall immunosuppression can be achieved by a
mechanism which operates independently of DHODH
inhibition. Next, the influence of the 4-CN moiety of 3
was investigated by replacing it by a Ph and ester
electron-withdrawing groups as well as by alkyl groups.
The outcome of these studies indicates that 3 is by far
determinations. Inhibition of the in vitro murine antibody re-
sponse to T-cell-independent B-cell antigen TNP-LPS (type I).
d
c Inhibition of the mouse mixed lymphocyte reaction. Inhibition
of J urkat cell proliferation. e Inhibition of human dihydroorotate
dehydrogenase. ND, not determined.
In Vitr o Biologica l Resu lts a n d Discu ssion . The
activation of B cells can proceed by at least two
fundamentally different mechanisms, one that requires
B-cell contact with CD4+ T cells and one that is induced
by antigen contact with B cells in the absence of CD4+
T cells. Two types of T-independent B-cell responses
can be elicited depending on the antigen used. Type I
responses are elicited by antigens such as TNP-LPS,
whereas type II responses are elicited by antigens such
as DNP-Ficoll. A murine assay for the evaluation of
T-cell-independent B-cell responses was established,
and it was found that leflunomide inhibits type I
responses in vitro and in vivo while CsA does not (Table
1). Both compounds were inhibitors of the type II
responses, with IC50 of 5.5 µM and 4.0 nM, respectively.
The failure of CsA, a T-cell immunosuppressant,30 in
animal models of xenotransplantation and the inef-
fectiveness of therapeutically relevant CsA doses to
control the decline of long-functioning grafts in humans
underline the importance of the T-cell-independent
B-cell component and, in particular, the inhibition of
the CsA-resistant type I responses. Consequently, this
cellular assay was fundamental for the screening of the
B-cell inhibitors required. In addition, the immuno-
suppressive activity of the compounds as well as their
cytotoxicity was assessed in the mouse mixed lympho-
cyte reaction (MLR)31 and J urkat proliferation assays,
respectively, as an estimate of their therapeutic window.
The biochemical mechanism of action of the compounds
was compared to that of leflunomide using the human
DHODH enzymatic assay.32
The results of Table 1 demonstrate that, among the
two designed pyrazoles, only compound 3 is immuno-
suppressive. Indeed, it inhibits both type I responses
and the mixed lymphocyte reaction with IC50 values
comparable to those of leflunomide’s metabolite 2(Z).
In addition, the new compound has a better MLR/J urkat

3534 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Ta ble 2. Structure-Activity Relationships
Papageorgiou et al.
compd
R
type Ia,b MLRa,c J urkata,d DHODHa,e
a. Influence of the 3-Aniline Substituent
3
p-CF3
m-CF3
o-CF3
Ph
p-F
5.3
2.1
ND
ND
>50
ND
ND
2.6
3a
3b
3c
3d
3e
3f
3g
3h
3i
>50
>50
>50
9.2
11.6
20
6.5
30.6
>50
>50
>50
19.4
3.2
0.5
9.0
3.6
>50
>50
F igu r e 3. Concentration of [3H]-3 in blood after oral and
intravenous administration. Results are mean ( SE of three
mice.
7.4
6
>50
>50
p-CN
p-OCF3
p-NO2
p-tBu
0.45
4.3
7.6
ND
ND
ND
6.7
ND
16.1
>50
12.3
ND
ND
ND
16.2
ND
the most potent compound (Table 2c). Methylation of
the aniline N or of the N(2) of 3 or replacement of the
(p-CF3)aniline moiety by a (p-CF3)benzylamino one
resulted in total loss of the enzymatic activity and 3-5-
fold reduction in the B-cell activity (Table 2d). In an
attempt to include both an electron-withdrawing sub-
stituent and an alkyl substituent in a cyclic structure,
the indazole 10 was obtained which represents one of
the rare cytostatic compounds of the series. Taken
together, the structure-activity relationships indicate
that, in addition to compound 3, only the corresponding
5-isopropyl and 5-tBu derivatives 3n ,o fulfill the re-
quirement set, that is, potent inhibition of the cy-
closporin-resistant type I B-cell responses, potent im-
munosuppression, and lack of cytotoxicity.
p-Me
>50
12.1
3j
3k
3l
p-NH2
o,o′,p-tri-F
m,m′-di-CF3
p-CF3, m-Br
>50
>50
22.4
>50
>50
35
28.0
20.9
3m
b. Influence of the 5-Substituent
3
Me
iPr
tBu
5.3
4.7
5.8
22.4
27
13.6
2.1
3.1
5.3
2.9
ND
3.6
ND
ND
ND
3.7
7.5
>50
>50
>50
2.6
4.0
>50
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
cyclopentyl
3-pyridyl
4-pyridyl
2-thienyl
2-furanyl
Ph
31.8
>50
>50
>50
>50
>50
>50
>50
>50
In Vivo Eva lu a tion /P h a r m a cok in etics. Among
the three potent in vitro B-cell inhibitors (3, 3n ,o)
discovered, a DHODH modulating compound and a
compound acting independently of enzyme inhibition
were chosen for further investigations. Consequently,
3 and 3o were tested in vivo for inhibition of murine
TNP-LPS responses after oral and subcutaneous ad-
ministration of 100 and 50 mg/kg, respectively, and were
found inactive as assessed by the lack of inhibition of
TNP-specific immunoglobulin M (IgM) and G (IgG)
titers. At this dose, leflunomide inhibited IgM and IgG
by 61% and 97%, respectively. As expected, cyclosporin
A was inactive in this model. To gain insight into
possible reasons for the discrepancy between the in vitro
and in vivo results obtained with the two pyrazoles, 3
ND
10.8
ND
ND
ND
>50
38.7
>50
>50
32
H2N
EtNH
14.7
16.9
c. Influence of the 4-Substituent
3
14
15
16
17
18
19
CN
Ph
5.3
29.4
17.4
26.5
23.8
14.6
21.3
2.1
ND
20.3
16.5
ND
18.7
ND
>50
ND
41.0
2.6
>50
C(Me)NNH2
H
>50
3
was labeled with H at the meta position of the aniline
>50
16.5
3
EtOOC
Me
CH2OH
ND
43.0
ND
>50
22.5
via H2 reduction of the corresponding bromo derivative
and employed for pharmacokinetic studies. The radio-
labeled compound was administered to male mice orally
(100 mg/kg) or intravenously (10 mg/kg), and pharma-
cokinetic parameters were analyzed. Maximum blood
concentrations of [3H]-3 were achieved 0.5 h after oral
application and persisted up to 4 h postdose (1-2.5 µM).
The dose-normalized AUC ratio (po/iv) of radioactivity
pointed out to a very good absorption (81%) of the
pyrazole. The blood-concentration time profile (Figure
3) suggested a saturable presystemic first-pass and
biphasic kinetics with a main half-life ) 0.4 h. The
compound was strongly bound to plasma proteins (frac-
tion free ≈ 1.5%) but moderately distributed to tissues
(Vss ) 1.4 L/kg). The total body clearance (CL) amounted
to 1.0 mL/min after iv administration; the blood levels
of total radioactivity were about double those of [3H]-3
itself, pointing to an important metabolic clearance.
2.7
d. Miscellaneous
3
10
11
20
21
22
5.3
16.7
13.8
19.1
23.1
17.1
2.1
8.6
3.4
23.6
16.1
40.5
>50
2.6
6.5
6.2
17.7
31.2
>50
47.2
>50
>50
>50
>50
-eSee corresponding footnotes in Table 1.
a

Pyrazole Leflunomides as B-Cell Immunosuppressants
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3535
On the basis of the above results and hypothesizing
that 3o might be metabolically more stable than 3 due
to the absence of the allylic methyl moiety and the
presence of the tBu group which could shield the NH
from enzyme-mediated conjugation, its plasma concen-
tration as function of time was investigated. For this
study 10 mg of 3o was given orally to mice and the
concentration of the compound was quantified by LC-
MS/MS after 0.5, 1, 2, 4, and 8 h. The results obtained
were similar to those seen with 3. Indeed, 3o had a
short half-life (t1/2 ) 0.5 h), and plasma concentrations
in the 1 µM range persisted for approximately 4 h.
When comparing these data with those obtained after
po administration of 100 mg/kg 3, it appears that 3o
has a 10-fold increased stability as regards enzymatic
degradation.
a plausible explanation for the failure of the compounds
in vivo. Not only were drug levels less than the
measured IC50 values obtained, but they also persisted
for a relatively short time period thus allowing the fast
onset of antibodies against the TNP-LPS antigen.
In conclusion, despite the successful drug design
approach and the in vitro efficacy of pyrazoles 3 and
3o, the compounds fail to attain therapeutically relevant
concentrations in vivo. The very tight structure-
activity relationships with this compound class repre-
sent a severe limitation for the identification of further
compounds with improved pharmacokinetic properties.
Taken together, the results of this work illustrate the
importance of the combination of drug design with
absorption, distribution, and metabolic studies in order
to ensure the quality of the compounds involved in drug
discovery programs.
Con clu sion s
Exp er im en ta l Section
The structural and the in vitro biological results
obtained with 3 emphasize its bioisosteric relationship
with leflunomide’s bioactive metabolite 2(Z). Indeed,
both compounds inhibit T-cell-independent B-cell re-
sponses to the TNP-LPS antigen (type I), are immuno-
suppressive, and share a common biochemical target.
Therefore, 3 fulfills the requirement for a B-cell inhibitor
modulating cyclosporin A-resistant effects such as those
involved in xenotransplantation and chronic rejection.
Interestingly, the outcome of the structure-activity
investigations with derivatives of 3 revealed that there
is no correlation between inhibition of Ab formation and
DHODH inhibition. Since enzyme inhibition is not a
prerequisite for type I inhibition, the tBu derivative 3o
provides a valuable tool for the identification of new
targets for B-cell intervention.
A 2-fold evidence is provided that the above conclu-
sions are valid despite cellular data generated with
murine systems and enzymatic data with human en-
zyme. First, the rat versus human sequence alignment
of the enzymes indicates a 86.8% identity which indi-
cates that no large discrepancies should be expected
between human and the mouse enzyme which has not
as yet been cloned. Second, and most important,
competition mouse MLR experiments done with 3 and
3o in the absence and presence of uridine showed that
only the inhibitory activity of 3 could be affected by the
nucleoside. Indeed, addition of 50 and 100 µM of uridine
in the MLR culture led to a reduction of the IC50 of 3 by
30% and 44%, respectively, as compared to the control.
In contrast to leflunomide (1), the two most potent
pyrazoles (3 and 3o) failed to inhibit murine IgM and
IgG antibody responses in the TNP-LPS model in vivo
after either oral or subcutaneous administration. The
absorption and disposition studies carried out with
radiolabeled 3 indicated that absorption was not a
drawback and that high blood concentrations persisted
for 4 h. Given the highest dose applied in the in vivo
pharmacological studies (100 mg/kg, po), the extrapola-
tion of the pharmacokinetic results indicates that ap-
proximately 1 µM concentrations of 3 must have been
obtained. These concentrations are 5-fold lower than
the IC50 values of the compound in B-cell inhibition
(Table 1). Analogous results were obtained after expo-
sure studies conducted with the metabolically more
stable derivative 3o. The pharmacokinetic studies offer
(1) Ch em istr y. Compounds were characterized by 400-
MHz proton NMR at room temperature using a Brucker MX-
400 or DPX-400 spectrometer. Chemical shifts are expressed
as ppm downfield from tetramethylsilane; J values are ex-
pressed in Hz. Fast atom bombardment mass spectroscopy
(Xe, 8 keV) on a VG70-SE mass spectrometer was used for
the characterization of all reported compounds. C, H, and N
analyses were carried out with all substances biologically
evaluated, and (0.4% was acceptable. For chromatographic
purifications, the flash chromatography technique was applied
using 230-400 mesh silica gel.
2-Cya n o-3-im in o-N-(Rb)th iobu tyr a m id es 6-6u : Gen -
er a l P r oced u r e. 3-Iminoalkyl(aryl)onitrile (1 equiv) and the
respective aromatic isothiocyanate (1 equiv) were dissolved in
dioxane (∼10 mmol in 40 mL) and refluxed for 3-6 h. After
removal of the solvent under reduced pressure the title
compounds (6-6u ) were isolated as amorphous solids after
precipitation with hexane and/or diethyl ether. Confirmation
of the structure of the thioamides was obtained by mass
spectroscopy.
2-Cya n o-3-im in o-N-(4-tr iflu or om eth yl-p h en yl)th iobu -
tyr a m id e, 6. 3-Iminobutyronitrile (1.0 g, 12.2 mmol) and
4-trifluoromethyl-phenyl isothiocyanate (2.5 g, 12.2 mmol)
were refluxed in dioxane (50 mL) for 3 h. After removal of
the solvent, the crude was taken up with hexane and diethyl
ether. The resulting amorphous solid was filtered off and dried
to give 6 as a tautomeric mixture (3.1 g, 89%): 1H NMR
(DMSO-d6) δ 9.3 (s, NH), 11.43 (s, NH2), 7.7 (d, J ) 8.3, 2H),
7.6 (d, J ) 8.3, 2H), 2.3 (s, 3H). Anal. (C12H10F3N3S) C, H, N.
5-Meth yl-3-(Rb)-1H-pyr azole-4-car bon itr iles 3-3u : Gen -
er a l P r oced u r e. Thioamide 6-6u (1 equiv) and hydrazine
hydrate (1.2 equiv) were dissolved in EtOH (5 mmol of starting
material in ∼50 mL) and refluxed for 2-3 h. After removal
of the solvent, the crude was treated with hexane and/or
diethyl ether and then purified by chromatography. Elution
with EtOAc afforded analytically pure 3-3u (yields 70-85%).
Melting points (°C) are as follows:
3a
3b
3c
3d
3e
3f
3g
261-265 156-157 214-215 210-211 231-234 224-226
>265
3h 3i 3j 3k 3l 3m
3n
232-236 240-244 207-210 225-228 217-219 213-217 217-220
3o 3p 3q 3r 3s 3t 3u
219-222 205-207 223-224 254-256 267-269 294-295 amorph
5-Meth yl-3-(4-tr iflu or om eth yl-p h en yl)-1H-p yr a zole-4-
ca r bon itr ile, 3. A solution of 6 (1.5 g, 5.7 mmol) and
hydrazine hydrate (340 µL, 6.8 mmol) in ethanol (50 mL) was
refluxed for 2 h. After cooling at room temperature, the
solvent was evaporated, and crude 3 was isolated after
trituration with hexane. Chromatographic purification (elu-
ent: EtOAc) gave pure 3 (1.0 g, 70%): mp 258-260 °C; 1H NMR

3536 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18
Papageorgiou et al.
(DMSO-d6) δ 12.35 (s, NH), 9.23 (s, NH), 7.55 (m, 4H), 2.35 (s,
room temperature, and the resulting precipitate was filtered
and washed with diethyl ether to give 10 (2.4 g, 65%): mp 262-
263 °C; 1H NMR (DMSO-d6) δ 12.62 (s, NH), 8.40 (s, NH), 7.80
(d, J ) 7.8, 2H), 7.57 (t, J ) 7.8, 2H), 2.82 (d, J ) 6.0, 2H),
2.49 (t, J ) 6.2, 2H), 2.05 (dd, J ) 6.0, J ) 6.2, 2H). Anal.
(C14H12F3N3O) C, H, N.
(4,5,6,7-Tetr a h ydr o-1H-in da zol-3-yl)(4-tr iflu or om eth yl-
p h en yl)a m in e, 11. To a cooled (-78 °C) suspension of LiAlH4
(0.39 g, 7.5 mmol) in dry THF (50 mL) was added a solution
of concentrated H2SO4 (0.27 mL) in THF (2.7 mL) dropwise,
and the reaction was allowed to reach room temperature.
After addition of the ketone 10 (0.74 g, 2.5 mmol) in THF (7.4
mL) and further stirring for 15 min, the reaction was quenched
with a saturated solution of Na2SO4 (1.1 mL). Evaporation to
dryness and purification of the crude by chromatography
(eluent: hexane/EtOAc, 2/1) afforded 11 (0.5 g, 60%): mp 161-
163 °C; 1H NMR (DMSO-d6) δ 11.71 (s, NH), 8.37 (s, NH), 7.42
(s, 4H), 2.52 (t, J ) 6.1, 2H), 2.31 (t, J ) 6.1, 2H), 1.69 (m,
4H). Anal. (C14H14F3N3) C, H, N.
3-Oxo-2-p h en yl-N-(4-t r iflu or om et h yl-p h en yl)t h iob u -
tyr a m id e, 12. To a solution of (4-CF3)PhNCS (2.0 g, 9.7
mmol) in dry THF (10 mL) was added NaH (0.58 g, 14.5 mmol).
To the cooled (0 °C) mixture was dropwise added a solution of
benzyl methyl ketone (1.95 mL, 14.5 mmol) in THF (10 mL)
at such a rate that the temperature did not exceed 40 °C. The
reaction was further stirred for 12 h at room temperature and
then allowed to rest. To the crystalline mass formed was
added water (100 mL) carefully, and the resulting solution was
extracted with diethyl ether. The organic phase was discarded
and the aqueous one acidified to pH 5 with 2 N HCl. To the
oily layer which separated was added CH2Cl2 (15 mL), and
the solution was dried over Na2SO4 and concentrated in a
vacuum to give 12 as a red oil (3.05 g, 93%) which was used
in the next step without further purification: 1H NMR (DMSO-
d6) δ 11.22 (s, NH), 7.47 (m, 9H), 5.51 (s, 1H), 2.34 (s, 3H).
1H). Anal. (C12H9F3N4) C, H, N.
5-Am in o-3-((4-tr iflu or om eth yl-ph en yl)am in o)-1H-pyr a-
zole-4-ca r bon itr ile, 3v. An analogous procedure to that used
for the synthesis of compound 8 in which malononitrile was
used instead of ethyl cyanoacetate gave 3v (0.29 g, 77%): mp
210-213 °C; 1H NMR (DMSO-d6) δ 11.35 (s, 1H, NH), 8.88 (s,
1H, NH), 7.60 (d, J ) 8.3, 2H), δ 7.50 (d, J ) 8.3, 2H), 6.35 (s,
2H, NH2). Anal. (C11H8F3N5) C, H, N.
5-Eth ylam in o-3-((4-tr iflu or om eth yl-ph en yl)am in o)-1H-
p yr a zole-4-ca r bon itr ile, 3w . To a solution of 3v (0.30 g, 1.23
mmol) and acetaldehyde (125 µL, 2.25 mmol) in 20 mL of
dioxane was added NaCNBH3 (0.14 g, 2.25 mmol) under
continuous stirring. After adjustment of the pH in the range
of 4.6-5.0 (10% aqueous H3PO4), the reaction mixture was
kept at room temperature for 90 min. Additional acetaldehyde
(192 µL, 3.37 mmol) and NaCNBH3 (0.21 g, 3.37 mmol) was
added and the reaction mixture was kept again at room
temperature for a further 2 h. Dioxane was removed under
reduced pressure, and the aqueous phase was extracted twice
with EtOAc. The organic layer was dried over Na2SO4 and
concentrated. Chromatographic purification of the crude
(eluent: toluene/EtOAc, 1/1) afforded 3w (0.12 g, 33%): mp
217-222 °C; 1H NMR (DMSO-d6) δ 11.63 (s, NH), 8.93 (s, NH),
7.64 (d, J ) 8.3, 2H), δ 7.52 (d, J ) 8.3, 2H), 6.94 (t, J ) 6.2,
2H, NH2), 3.15 (q, J ) 6.2, 2H), 1.15 (t, J ) 6.2, 3H). Anal.
(C13H12F3N5) C, H, N.
2-Cyan o-3-m eth ylsu lfan yl-3-((4-tr iflu or om eth yl-ph en yl)-
a m in o)a cr ylic Acid Eth yl Ester , 7. To a stirred solution
of ethyl cyanoacetate (2.26 g, 20 mmol) in DMF (20 mL) was
added NaH (0.8 g, 25 mmol) in portions under argon over a
period of 30 min. The resulting yellow solution was further
stirred for 30 min at room temperature, and 4-trifluoromethyl-
phenyl isothiocyanate (4.8 g, 23.6 mmol) dissolved in DMF (2
mL) was added dropwise within 5 min. Subsequently, MeI
(1.26 mL, 24.4 mmol) in DMF (3 mL) was added, and stirring
was continued overnight. The reaction was quenched by
carefully pouring it onto water (400 mL) and was extracted
with EtOAc. The organic layer was dried over Na2SO4 and
concentrated under reduced pressure to a semicrystalline
residue which after two recrystallizations from a small amount
of EtOH afforded 7 (4.0 g, 61%): mp 84-85 °C; 1H NMR
(DMSO-d6) δ 11.9 (s, NH), 7.63 (m, 4H), 4.1 (q, J ) 6.3, 2H),
2.32 (s, 3H), 1.17 (t, J ) 6.3, 3H). Anal. (C14H13F3N2O2S) C,
H, N.
5-H yd r oxy-3-((4-t r iflu or om et h yl-p h en yl)a m in o)-1H -
p yr a zole-4-ca r bon itr ile, 4, a n d 5-Am in o-3-((4-tr iflu or o-
m eth yl-ph en yl)am in o)-1H-pyr azole-4-car boxylic Acid Eth -
yl Ester , 8. A methanolic solution (40 mL) of 7 (0.46 g, 1.4
mmol) containing hydrazine hydrate (84 µL, 1.7 mmol) was
refluxed for 3 h. After cooling to room temperature, MeOH
was removed under vacuum and the crude was chromato-
graphed (eluent gradient: toluene/EtOAc, 1/1, f EtOAc) to give
8 (0.33 g, 73%): mp 239-242 °C; 1H NMR (DMSO-d6) δ 11.24
(s, 1H, NH), 8.39 (s, 1H, NH), 7.75 (d, J ) 8.3, 2H), 7.56 (d, J
) 8.3, 2H), 6.14 (s, 2H), 4.22 (q, J ) 6.3, 2H), 1.30 (t, J ) 6.3,
3H). Anal. (C13H13F3N4O2) C, H, N. 4 was also obtained (80
mg, 20%): amorphous; 1H NMR (DMSO-d6) δ 9.68 (s, 1H, NH),
8.33 (s, 1H, OH), 7.54 (d, J ) 8.3, 2H), 7.42 (d, J ) 8.3, 2H).
Anal. (C11H7F3N4O) C, H, N.
2-(1-Im in oet h yl)-3-oxo-N-(4-t r iflu or om et h yl-p h en yl)-
th iobu tyr a m id e, 13. 4-Aminopent-3-en-2-one (0.50 g, 5.0
mmol) and (4-CF3)PhNCS (0.84 g, 5.5 mmol) were dissolved
in dry dioxane (5 mL), and the solution was refluxed for 4 h.
After evaporation of the solvent to dryness and addition of a
small amount of CH2Cl2, the crude crystallized. The crystals
were collected and thoroughly washed with CH2Cl2 to give 13
1
(0.74 g, 48%): mp 168-170 °C; H NMR (DMSO-d6) δ 12.00
(s, NH), 10.19 (s, NH), 8.19 (d, J ) 7.8, 2H), 7.82 (br s, OH),
7.78 (d, J ) 7.8, 2H), 2.32 (s, 3H), 2.383 (s, 3H). Anal.
(C13H13F3N3OS) C, H, N.
(5-Meth yl-4-ph en yl-1H-pyr azol-3-yl)(4-tr iflu or om eth yl-
p h en yl)a m in e, 14. A solution of 12 (2.0 g, 9.6 mmol) in EtOH
(30 mL) containing hydrazine hydrate (0.34 mL, 7.1 mmol) was
refluxed overnight and then allowed to reach room tempera-
ture. Upon concentration of the solvent to approximately 5
mL, crystallization occurred. The solid was filtered and dried
1
to afford 14 (0.28 g, 15%): mp 139-145 °C; H NMR (DMSO-
d6) δ 12.32 (s, NH), 8.17 (s, NH), 7.30 (m, 9H), 2.39 (s, 3H).
Anal. (C17H14F3N3) C, H, N.
[4-(1-Hyd r a zon oeth yl)-5-m eth yl-1H-p yr a zol-3-yl](4-tr i-
flu or om et h yl-p h en yl)a m in e, 15, a n d 3-((4-Tr iflu or o-
m eth yl-p h en yl)a m in o)-1H-p yr a zole-4-ca r bon itr ile, 16. A
solution of 13 (0.31 g, 1.0 mmol) in EtOH (12 mL) containing
hydrazine hydrate (0.24 mL, 4.9 mmol) was refluxed for 2 h
and allowed to cool to room temperature. After removal of
the solvent and chromatography of the crude (eluent: hexane/
EtOAc, 1/3) pyrazole 16 was isolated (82 mg, 29%): mp 151-
2-Am in o-6-oxocycloh ex-1-en ylt h ioca r b on yl-4-t r iflu o-
r om eth yl-p h en yla n ilid e, 9. To a stirred solution of 2-ami-
nocyclohexenone (3.0 g, 27.0 mmol) in CH3CN (50 mL) was
dropwise added (p-CF3)PhNCS (27.6 mmol). The solution was
refluxed overnight, cooled, and concentrated to one-half its
volume. The resulting crystals were filtered and dried to
afford 9 as a tautomeric mixture (3.96 g, 47%): mp 201-203
1
153; H NMR (DMSO-d6) δ 11.35 (s, NH), 9.58 (s, NH), 7.55
(m, 5H), 2.30 (s, 1H). Anal. (C11H10F3N4) C, H, N. Hydrazone
15 was isolated as well (85 mg, 29%): mp 196-198 °C; 1H NMR
(DMSO-d6) δ 12.62 (s, NH), 11.45 (s, NH), 8.05 (d, J ) 7.8,
2H), 7.75 (d, J ) 7.8, 2H), 2.08 (s, 3H), 1.97 (s, 3H). Anal.
(C13H14F3N5) C, H, N.
1
°C; H NMR (DMSO-d6) δ 14.91 (s, NH), 12.46 (s, NH2), 9.31
(s, NH2), 7.21 (s, 4H), 2.72 (t, J ) 6.0, 2H), 2.47 (t, J ) 6.0,
2H), 1.78 (m, 2H). Anal. (C14H13F3N2OS) C, H, N.
5-Meth yl-3-((4-tr iflu or om eth yl-ph en yl)am in o)-1H-pyr a-
zole-4-ca r boxylic Acid Eth yl Ester , 17. To a suspension
of p-trifluoromethyl-phenylthiosemicarbazide (3.4 g, 14.4 mmol)
in dry EtOH (40 mL) was slowly added concentrated HCl (4.3
mL) at room temperature. After the mixture stirred for 10
3-((4-Tr iflu or om et h yl-p h en yl)a m in o)-1,5,6,7-t et r a h y-
d r oin d a zol-4-on e, 10. To a solution of 9 (3.9 g, 12.6 mmol)
in EtOH (100 mL) was added hydrazine hydrate (0.73 mL, 15.0
mmol). The solution was refluxed overnight and cooled to

Pyrazole Leflunomides as B-Cell Immunosuppressants
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 18 3537
min, a solution of ethyl 2-chloroacetate (2.12 mL, 14.5 mmol)
in EtOH (10 mL) was added dropwise resulting in a gradual
dissolution of the suspension. Further refluxing for 4 h
resulted in the formation of elemental sulfur which was
removed by filtration. The filtrate was concentrated in a
vacuum, and the solid obtained was recrystallized from EtOH
to yield 17 (2.8 g, 62%): mp 214-217 °C; 1H NMR (DMSO-d6)
δ 12.5 (s, NH), 8.55 (s, NH), 7.74 (d, J ) 7.8, 2H), 7.58 (d, J )
7.8, 2H), 4.23 (q, J ) 6.4, 2H), 2.42 (s, 3H), 1.32 (t, J ) 6.4,
3H). Anal. (C14H14F3N3O2) C, H, N.
[5-Meth yl-3-((4-tr iflu or om eth yl-ph en yl)am in o)-1H-pyr a-
zol-4-yl]m eth a n ol, 18. A solution of H2SO4 (0.17 mL, 3.2
mmol) in dry THF (1.7 mL) was dropwise added to a suspen-
sion of LiAlH4 (0.25 g, 6.4 mmol) in THF at -78 °C. The
resulting solution was allowed to reach room temperature and
was further stirred for 15 min. A solution of the ester 17 (0.5
g, 1.6 mmol) in THF (2.0 mL) was slowly added, and the
reaction was stirred until no more ester was present (ap-
proximately 15 min) as judged by TLC analysis. The reaction
was then quenched first by the careful addition of saturated
aqueous Na2SO4 (1.1 mL) and 10 min later by a 30% aqueous
solution of NaOH (0.5 mL). Diethyl ether was added, and the
resulting precipitate was removed by filtration. The filtrate
was dried (MgSO4), evaporated to dryness, and chromato-
graphed (eluent: hexane/EtOAc, 1/2) to give 18 (0.50 g, 12%):
mp 187-190 °C; 1H NMR (DMSO-d6) δ 11.87 (s, NH), 8.18 (s,
NH), 7.47 (m, 4H), 4.52 (t, J ) 6.0, OH), 4.31 (d, J ) 6, 2H),
2.19 (s, 3H). Anal. (C12H12F3N3O) C, H, N.
(4,5-Dim eth yl-1H-p yr a zol-3-yl)(4-tr iflu or om eth yl-p h e-
n yl)a m in e, 19. A solution of 17 (0.82 g, 2.6 mmol) in THF
was dropwise added to a refluxing suspension of LiAlH4 (0.5
g, 12.8 mmol) in dry THF (20 mL). After further refluxing for
2 h, TLC analysis showed the absence of starting material.
The reaction was allowed to cool to room temperature and was
carefully quenched first with a slow addition of EtOH (5 mL)
and then with an aqueous saturated solution of Na2SO4.
Extraction with EtOAc followed by chromatography of the
crude (eluent: hexane/EtOAc, 1/2) afforded dimethylpyrazole
19 (80 mg, 11%): mp 129-132 °C; 1H NMR (DMSO-d6) δ 7.43
(d, J ) 7.8, 2H), 7.02 (d, J ) 7.8, 2H), 6.12 (s, NH), 2.23 (s,
3H), 1.85 (s, 3H). Anal. (C12H12F3N3) C, H, N.
5-Meth yl-3-[m eth yl(4-tr iflu or om eth yl-p h en yl)a m in o]-
1H-p yr a zole-4-ca r bon itr ile, 20. To a cooled (-78 °C) solu-
tion of 3 (0.50 g, 1.9 mmol) in dry THF (60 mL) was dropwise
added BuLi (3.6 mL of a 1.6 M solution in hexane, 5.7 mmol).
After the mixture stirred at this temperature for 30 min, MeI
(0.35 mL, 5.7 mmol) was added and the reaction was allowed
to reach room temperature and quenched with water. Extrac-
tion with EtOAc followed by chromatography of the crude
(eluent: EtOAc/hexane, 2/1) gave 20 (17 mg, 34%): mp 146-
147 °C; 1H NMR (DMSO-d6) δ 7.55 (d, J ) 7.2, 2H), 7.03 (d, J
) 7.2, 2H), 3.37 (s, 3H), 2.32 (s, 3H).
(trinitrophenol). ELISA plates were coated with BSA-DNP by
overnight incubation at 4 °C, and after removal of BSA-DNP
solution residual protein-binding sites were blocked with 2%
BSA or 5% human albumin in PBS. Plates were washed three
times with PBS containing 0.05% Tween 20 and 0.02% NaN3
between each step. Serial dilutions of the supernatants in 2%
BSA-PBS were incubated overnight at 4 °C. Bound IgM Abs
was revealed using biotin-labeled affinity-purified goat anti-
mouse IgM antibody (1:15000; 2 h at 37 °C) and, subsequently,
streptavidin conjugated to alkaline phosphatase (1:10000 for
1 h at 37 °C). Enzyme activity was determined after addition
of p-nitrophenyl phosphate (1 mg/mL) in 1 M diethanolamine,
pH 9.8, by measuring the absorbency at 405/490 nm with a
Spectra Max reader usually after 30-45 min of incubation at
room temperature. Absorbance values obtained in the absence
of stimulus were used as low control; absorbency values
obtained in the presence of the stimulus were taken as high
control. Percent inhibition of the signal by the samples was
calculated according to the equation: inhibition(%) ) [(high -
low) - (sample - low)]/(high - low) × 100.
Mou se Mixed Lym p h ocyte Rea ction (MLR-M).31 Equal
amounts of spleen cells from C57/Bl6 mice (H-2b) and CBA/2
mice (H-2k) were mixed; 4 × 105 cells/well were incubated with
appropriate serial dilutions (DMSO/water) of drug samples in
200 µL of complete RPMI medium in flat bottom tissue culture
microtiter plates for 4 days at 37 °C in 5% CO2. Proliferation
was assessed by [3H]thymidine incorporation.
P r olifer a tion of th e Hu m a n T-Cell Lin e J u r k a t. The
cell line was maintained and propagated in fetal calf serum-
supplemented RPMI medium; 5 × 104 cells/well were incu-
bated in 200 µL of RPMI medium for 2 days. [3H]Thymidine
was measured at the end of the culture period.
(3) P h a r m a cok in etics. Male mice (OF1, ∼25 g) either
were administered a solution (ethanol + water + olive oil) of
0.612 mg of [3H]-3 together with 69.4 mg of unlabeled 3 orally
or were injected in the femoral vein with a solution (EtOH +
poly(ethylene glycol) + 5% glucose) of 1.08 mg of [3H]-3
together with 10.92 mg of nonlabeled 3. Blood was collected
from the vena cava (3 mice/time at 0.08, 0.5, 1, 2, 4, 8, 24 h
after intravenous bolus and at 0.5, 1, 2, 4, 8, 24 h after oral
administration). All collected samples were assayed for total
radioactivity by LSC and for parent compound by LC-RID.
An analogous protocol was applied for the assessment of the
plasma concentration of 3o as function of time. Electrospray
mass spectroscopy was used for the detection of the parent
compound, and quantitation was based on integration of the
peaks corresponding to the ion m/z 156.
Ack n ow led gm en t. We express our warmest thanks
to Dr. H. Andres for the labeling of 3 and Ms. Corinne
Simeon and Mr. Armin Bru¨lisauer, Hellmut Knecht,
Xaver Borer, and Andrea Florineth for their skillfull
technical assistance.
1,3-Dim eth yl-5-((4-tr iflu or om eth yl-p h en yl)a m in o)-1H-
p yr a zole-4-ca r bon itr ile, 21. Same procedure as for com-
pound 3, but instead of hydrazine hydrate as reagent N-me-
thylhydrazine was used (0.90 g, 56%): amorphous; 1H NMR
(DMSO-d6) δ 9.15 (s, NH), 7.58 (d, J ) 8.3, 2H), 6.93 (d, J )
8.3, 2H), 3.62 (s, 3H). Anal. (C13H11F3N4) C, H, N.
Su p p or tin g In for m a tion Ava ila ble: Detailed crystal-
lographic data concerning the two forms of 3 (8 pages).
Ordering information is given on any current masthead page.
(2) Biology. In h ibition of T-Cell-In d ep en d en t B-Cell
An tigen Resp on ses to TNP -LP S. Cu ltu r in g of Sp leen
Cells To Elicit in Vitr o An tibod y Resp on ses. Spleens from
C57Bl/6 nu/nu and C57Bl/6 mice were cultured in duplicate
in flat bottomed microtiter plates (2 × 105 cells/well). Serial
dilutions of drug solutions in serum-supplemented culture
medium (IMDM-ATL + 10% FCS)34 were added to a final
volume of 0.2 mL in the presence of optimal concentration of
TNP-LPS. The stimulated spleen cells were cultured for 4
days at 37 °C in a humidified CO2 incubator (5.5%), and then
the antigen supernatant from each of the two replicate cultures
were pooled and diluted 4-fold in PBS/BSA or PBS/HSA and
assayed in an ELISA.
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